**Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors**

Yadollah Omidi and Jaleh Barar

*Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran* 

#### **1. Introduction**

The challenge to comprehend the physiology as well as cell biology of the blood-brain barrier (BBB) began with Ehrlich and Goldman's experimental observations that the central nervous system (CNS) is not stained by intravascular vital dyes. These studies provided the first evidence of the presence of an obstructing barrier between blood and brain. Later on, researchers like Friedemann (1942) used basic highly lipid soluble dyes to cross the BBB in order to show the brain penetration of the dyes by direct transport across the cerebral microvasculature. In 1941, Broman presented his observations upon the existence of two different barrier systems within the brain: the BBB at the cerebral microvasculature, and the blood-CSF barrier at the choroid plexus. It is now clear that in fact three main barrier layers at the interface between blood and tissue protect the CNS: the endothelium of brain capillaries, and the epithelia of the choroid plexus (CP) and the arachnoid (Abbott, 2005; Engelhardt, 2006).

In 1941, Broman proposed that it was the cerebral capillary endothelial cells that contribute the physical barrier function of the BBB, and not the astrocytic end feet. The argument concerning whether the astrocytes or the capillary endothelium constitute the BBB was supported by electron microscopic cytochemical studies performed in 1967 by Reese and Karnovsky. They used horseradish peroxidase (HRP), ~40 kDa, to visualize the BBB by systemic injections of HRP which failed to reach the brain extracellular fluid, whereas intracerebroventricular injection into the CSF stained the brain extracellular fluid positive for HRP (Reese & Karnovsky, 1967).

It is now evident that the BBB is a unique membranous barrier, which restrictively isolates the brain parenchyma from the circulating molecules/compounds within the blood. The permeability of BBB is regulated by transport machineries of the brain capillary endothelial cells that are modulated by autocrine and paracrine secretions from several types of cells, such as the pericyte, the astrocyte, and neurons (Rubin & Staddon, 1999).

The pericyte cells share the capillary basement membrane with the endothelium and physically supports endothelial cells (Allt & Lawrenson, 2001). It has been revealed that there is approximately one pericyte for every three endothelial cells (Pardridge, 1999). It is deemed that the pericyte cells play a regulatory role in brain angiogenesis, endothelial cell

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 113

complex that includes both tight and adherens junctions. In terms of morphology, TJs form a continuous network of parallel, interconnected, intra-membrane strand of various proteins

Fig. 1. Schematic demonstration of part of cortex and brain capillary endothelial cells. Star shape astrocytes are in communication with both brain capillaries and neurons via end-foot. The ultrastructural characteristics (in particular presence of tight junction) of the brain capillary endothelial cells differ them from other peripheral capillaries. Some of important specialized transporters are illustrated on luminal section which represents vesicular trafficking machineries for traverse necessary macromolecules from blood to brain, adapted

Using freeze–fracture technique, it has been shown that the tight junctions of the BBB are characterized by the highest complexity found in the vasculature, so that protoplasmic fracture face (P-face) association of the BBB compare to the peripheral endothelial cells is 55% and 10%, respectively. The altered particles distribution implies the presence of a strong interaction between TJs of the BBB and cytoskeleton. TJs of the BBB, morphologically, are more comparable to TJs of the epithelial cells than to TJs of the endothelial cells in

arranged as a series of multiple barriers.

with permission (Omidi & Barar, 2012).

**2.1 Tight junctions of BBB** 

tight junction formation, BBB differentiation, and also contribute to the microvascular vasodynamic capacity and structural stability.

The astrocyte cells invest approximately 99% of the abluminal surface of the brain capillary and induce endothelial cells to differentiate directly through cell to cell communication or indirectly by secreting astrocytic factors (Pardridge, 1999). Brain capillary endothelial cells display different features in comparison with peripheral endothelial cells. The BBB can be thought of brain capillary endothelial cells (BCECs) with the physical and paracrine interactions between the endothelial cells (ECs), the pericyte, and the astrocyte (Abbott et al., 2006; Pardridge, 1999).

The ability of BCECs to form a restrictive barrier between blood and brain is not completely intrinsic to the brain microvascular endothelial cells, but instead is induced by the brain environment itself. Induction of BBB may be categorized as "directive" and "impremissive" events. The latter term means that the inductor functions upon a tissue that is already determined toward its final fate but still needs an exogenous stimulus for the expression of its full phenotype. Of note, with the lack of a brain neuronal environment, the selective restrictiveness characteristics of the BCECs disappear, and as a result appropriate incitement(s) ought to be continuous at the BBB microenvironment to maintain its functionalities (Abbott, 2005).

The astrocyte inductive effects upon endothelial cell differentiation have been examined by Stewart & Wiley (1981). They transplanted avascular tissue from 3-day-old quail brain into the coelomic cavity of chick embryos, the chick endothelial cells then vascularized the quail brain grafts formed a competent BBB. In contrast preformed microvessels growing in embryonic quail muscle, which were implanted in chick brain were leaky and lacked BBB enzymes (Stewart & Wiley, 1981).

With regard to the complexity of the BBB, basically, other differentiating factors apart from astrocytes may play a role on the formation of the BBB. However, we discuss the most important features of BBB in relation to drug delivery and targeting for brain tumors. Fig. 1 represents the schematic illustration of BCECs.

### **2. BBB junctional complexes and cell-to-cell interactions**

Stable cell-to-cell interactions are required to keep the structural integrity of tissues. Dynamic changes in cell-to-cell adhesion will participate in the morphogenesis of developing tissues. Adhesion mechanisms are highly regulated during tissue morphogenesis and related to the processes of cell motility and cell migration. Cell junctions, basically, can be classified into three functional groups, including: 1) tight junctions (TJs), 2) anchoring (adherent) junctions (AJs), and 3) gap (communication) junctions (GJs). Of these junctions, the TJs seal cells together in cell sheet, the AJs attach cells to their neighbors or to the extra-cellular matrix mechanically, and the GJs mediate the passage of chemicals or electrical signals from one interacting cell to its partner (Engelhardt, 2006; Omidi & Gumbleton, 2005). Because of crucial role of TJs in restrictive function of BBB, they are briefly discussed.

Fig. 2 shows the diagrammatic representation of TJs and its complexity with other proteins at the BBB. TJs of the BBB generate a rate-limiting restrictive barrier to paracellular diffusion of solutes between endothelial cells. They are the most apical element of the junctional

tight junction formation, BBB differentiation, and also contribute to the microvascular

The astrocyte cells invest approximately 99% of the abluminal surface of the brain capillary and induce endothelial cells to differentiate directly through cell to cell communication or indirectly by secreting astrocytic factors (Pardridge, 1999). Brain capillary endothelial cells display different features in comparison with peripheral endothelial cells. The BBB can be thought of brain capillary endothelial cells (BCECs) with the physical and paracrine interactions between the endothelial cells (ECs), the pericyte, and the astrocyte (Abbott et al.,

The ability of BCECs to form a restrictive barrier between blood and brain is not completely intrinsic to the brain microvascular endothelial cells, but instead is induced by the brain environment itself. Induction of BBB may be categorized as "directive" and "impremissive" events. The latter term means that the inductor functions upon a tissue that is already determined toward its final fate but still needs an exogenous stimulus for the expression of its full phenotype. Of note, with the lack of a brain neuronal environment, the selective restrictiveness characteristics of the BCECs disappear, and as a result appropriate incitement(s) ought to be continuous at the BBB microenvironment to maintain its

The astrocyte inductive effects upon endothelial cell differentiation have been examined by Stewart & Wiley (1981). They transplanted avascular tissue from 3-day-old quail brain into the coelomic cavity of chick embryos, the chick endothelial cells then vascularized the quail brain grafts formed a competent BBB. In contrast preformed microvessels growing in embryonic quail muscle, which were implanted in chick brain were leaky and lacked BBB

With regard to the complexity of the BBB, basically, other differentiating factors apart from astrocytes may play a role on the formation of the BBB. However, we discuss the most important features of BBB in relation to drug delivery and targeting for brain tumors. Fig. 1

Stable cell-to-cell interactions are required to keep the structural integrity of tissues. Dynamic changes in cell-to-cell adhesion will participate in the morphogenesis of developing tissues. Adhesion mechanisms are highly regulated during tissue morphogenesis and related to the processes of cell motility and cell migration. Cell junctions, basically, can be classified into three functional groups, including: 1) tight junctions (TJs), 2) anchoring (adherent) junctions (AJs), and 3) gap (communication) junctions (GJs). Of these junctions, the TJs seal cells together in cell sheet, the AJs attach cells to their neighbors or to the extra-cellular matrix mechanically, and the GJs mediate the passage of chemicals or electrical signals from one interacting cell to its partner (Engelhardt, 2006; Omidi & Gumbleton, 2005). Because of crucial role of TJs in

Fig. 2 shows the diagrammatic representation of TJs and its complexity with other proteins at the BBB. TJs of the BBB generate a rate-limiting restrictive barrier to paracellular diffusion of solutes between endothelial cells. They are the most apical element of the junctional

vasodynamic capacity and structural stability.

2006; Pardridge, 1999).

functionalities (Abbott, 2005).

enzymes (Stewart & Wiley, 1981).

represents the schematic illustration of BCECs.

restrictive function of BBB, they are briefly discussed.

**2. BBB junctional complexes and cell-to-cell interactions** 

complex that includes both tight and adherens junctions. In terms of morphology, TJs form a continuous network of parallel, interconnected, intra-membrane strand of various proteins arranged as a series of multiple barriers.

Fig. 1. Schematic demonstration of part of cortex and brain capillary endothelial cells. Star shape astrocytes are in communication with both brain capillaries and neurons via end-foot. The ultrastructural characteristics (in particular presence of tight junction) of the brain capillary endothelial cells differ them from other peripheral capillaries. Some of important specialized transporters are illustrated on luminal section which represents vesicular trafficking machineries for traverse necessary macromolecules from blood to brain, adapted with permission (Omidi & Barar, 2012).

#### **2.1 Tight junctions of BBB**

Using freeze–fracture technique, it has been shown that the tight junctions of the BBB are characterized by the highest complexity found in the vasculature, so that protoplasmic fracture face (P-face) association of the BBB compare to the peripheral endothelial cells is 55% and 10%, respectively. The altered particles distribution implies the presence of a strong interaction between TJs of the BBB and cytoskeleton. TJs of the BBB, morphologically, are more comparable to TJs of the epithelial cells than to TJs of the endothelial cells in

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 115

paracellular permeability. BBB endothelial cells in vivo reveal a P-face/E-face ratio of about 55%/45%, and as claudin-3 and claudin-5 are well expressed, it can be suggested that the degree of association with one or the other leaflet roughly reflects the stoichiometry of claudin expression in the TJs of BBB. However, in the non-BBB endothelial cells, tight junctions are almost completely associated with the E-face and claudin-3 is rarely or not

Many identified ubiquitous molecular components of junctional complexes in the epithelia such as claudins, occludins, zonula (ZO-1, ZO-2, ZO-3) , junctional adhesion molecules (JAMs), cingulin, and 7H6 have been detected at the BBB. Both tight and adherent junctions are composed of multiple protein complexes, which communicate with the actin

The TJ contains various proteins, which are necessary to form structural support for the tight junction such as zonula occludens proteins that belong to a family of membrane associated guanylate kinase-like proteins and serve as recognition proteins for tight junctional placement and as a support structure for signal transduction proteins. Junctional adherent molecules (JAMs) are localized at the TJ and are members of the immunoglobin superfamily, which can function in association with platelet-endothelial cellular adhesion molecule 1 (PECAM1) to regulate leukocyte migration. Cingulin is a double-stranded myosin-like protein that binds preferentially to ZO proteins at the globular head and to other cingulin molecules at the globular tail. The primary cytoskeletal protein, actin, has

In terms of TJ regulation, phosphorylation of both transmembrane and accessory proteins plays an important role in establishing and regulating the TJs. The Occludin and ZO1, as the primary regulatory proteins of the TJs, are phosphorylated on serine, threonine and tyrosine residues. The increased phosphorylation of serine/threonine correlates with decreased extractability of occludin and TJs assembly. Regulation of TJs is also dependent on tyrosine phosphorylation of proteins at cell to cell contacts. Development of TJ barrier functions has been correlated with decreased tyrosine phosphorylation of proteins at the TJ complexes. Protein kinase C (PKC) also is a major regulator of TJs formation and regulation. It plays an important role in ZO1 migration to the plasma membrane and there are PKC phosphorylation consensus sequences in the ZO1 protein, suggesting that ZO proteins serve as scaffolding for PKC signal transduction pathways on the cytoplasmic surface of intercellular junctions. It has also been shown that TJs are generally localized at cholesterolenriched regions or rafts within the plasma membrane. Caveolin-1, an integral protein within caveolae membrane domains may associate with TJ components. Caveolin-1 itself interacts with and regulates the activity of several signal transduction pathways and downstream targets. Several cytoplasmic signaling molecules are concentrated at TJ complexes and are involved in signaling cascades that control assembly and disassembly of

The structure and function of TJs in primary cultures of bovine brain endothelial cells were directly analyzed, using quantitative freeze-fracture electron microscopy, and ion and inulin permeability (Wolburg et al., 1994), and it was shown that the cultured brain endothelial

known binding sites on all of the ZO proteins, and on claudin and occludin.

Claudin 1, 3 and 5 are present at the BBB. Occludin functions as a dynamic regulatory protein causing increased electrical resistance across the membrane and decreased

expressed, adapted with permission (Omidi & Barar, 2012).

cytoskeleton of the cells (Kniesel & Wolburg, 2000).

TJs (Krizbai & Deli, 2003).

peripheral blood vessels. Although the tight junction of BBB discloses many characteristics of epithelial TJs, there are distinctive differences between them, e.g. in terms of particle density and distribution, in relation to response to ambient factors.

Fig. 2. Illustration of the brain capillary endothelial cells (BCECs) morphology and architecture. A) Schematic elongated two brain capillary endothelial cells with some apical transporters. B) Vesicular membrane of BCECs showing coated and uncoated vesicles. C) Transmission electron microscopy micrograph of BCECs showing tight junction (TJ). D) Tight junctional interactions of two endothelial cells. The TJ is embedded in a cholesterolenriched region of the plasma membrane. Claudins comprise a multigene family with 20 isoforms currently identified and form the backbone of TJ strands by making dimers and binding homotypically to claudins on adjacent cells to produce the primary seal of the TJ.

peripheral blood vessels. Although the tight junction of BBB discloses many characteristics of epithelial TJs, there are distinctive differences between them, e.g. in terms of particle

Fig. 2. Illustration of the brain capillary endothelial cells (BCECs) morphology and

architecture. A) Schematic elongated two brain capillary endothelial cells with some apical transporters. B) Vesicular membrane of BCECs showing coated and uncoated vesicles. C) Transmission electron microscopy micrograph of BCECs showing tight junction (TJ). D) Tight junctional interactions of two endothelial cells. The TJ is embedded in a cholesterolenriched region of the plasma membrane. Claudins comprise a multigene family with 20 isoforms currently identified and form the backbone of TJ strands by making dimers and binding homotypically to claudins on adjacent cells to produce the primary seal of the TJ.

density and distribution, in relation to response to ambient factors.

Claudin 1, 3 and 5 are present at the BBB. Occludin functions as a dynamic regulatory protein causing increased electrical resistance across the membrane and decreased paracellular permeability. BBB endothelial cells in vivo reveal a P-face/E-face ratio of about 55%/45%, and as claudin-3 and claudin-5 are well expressed, it can be suggested that the degree of association with one or the other leaflet roughly reflects the stoichiometry of claudin expression in the TJs of BBB. However, in the non-BBB endothelial cells, tight junctions are almost completely associated with the E-face and claudin-3 is rarely or not expressed, adapted with permission (Omidi & Barar, 2012).

Many identified ubiquitous molecular components of junctional complexes in the epithelia such as claudins, occludins, zonula (ZO-1, ZO-2, ZO-3) , junctional adhesion molecules (JAMs), cingulin, and 7H6 have been detected at the BBB. Both tight and adherent junctions are composed of multiple protein complexes, which communicate with the actin cytoskeleton of the cells (Kniesel & Wolburg, 2000).

The TJ contains various proteins, which are necessary to form structural support for the tight junction such as zonula occludens proteins that belong to a family of membrane associated guanylate kinase-like proteins and serve as recognition proteins for tight junctional placement and as a support structure for signal transduction proteins. Junctional adherent molecules (JAMs) are localized at the TJ and are members of the immunoglobin superfamily, which can function in association with platelet-endothelial cellular adhesion molecule 1 (PECAM1) to regulate leukocyte migration. Cingulin is a double-stranded myosin-like protein that binds preferentially to ZO proteins at the globular head and to other cingulin molecules at the globular tail. The primary cytoskeletal protein, actin, has known binding sites on all of the ZO proteins, and on claudin and occludin.

In terms of TJ regulation, phosphorylation of both transmembrane and accessory proteins plays an important role in establishing and regulating the TJs. The Occludin and ZO1, as the primary regulatory proteins of the TJs, are phosphorylated on serine, threonine and tyrosine residues. The increased phosphorylation of serine/threonine correlates with decreased extractability of occludin and TJs assembly. Regulation of TJs is also dependent on tyrosine phosphorylation of proteins at cell to cell contacts. Development of TJ barrier functions has been correlated with decreased tyrosine phosphorylation of proteins at the TJ complexes. Protein kinase C (PKC) also is a major regulator of TJs formation and regulation. It plays an important role in ZO1 migration to the plasma membrane and there are PKC phosphorylation consensus sequences in the ZO1 protein, suggesting that ZO proteins serve as scaffolding for PKC signal transduction pathways on the cytoplasmic surface of intercellular junctions. It has also been shown that TJs are generally localized at cholesterolenriched regions or rafts within the plasma membrane. Caveolin-1, an integral protein within caveolae membrane domains may associate with TJ components. Caveolin-1 itself interacts with and regulates the activity of several signal transduction pathways and downstream targets. Several cytoplasmic signaling molecules are concentrated at TJ complexes and are involved in signaling cascades that control assembly and disassembly of TJs (Krizbai & Deli, 2003).

The structure and function of TJs in primary cultures of bovine brain endothelial cells were directly analyzed, using quantitative freeze-fracture electron microscopy, and ion and inulin permeability (Wolburg et al., 1994), and it was shown that the cultured brain endothelial

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 117

ACM showed no tight or gap junctions. This finding directed them to conclude that CNS astrocytes generate soluble factor(s), which can provoke formation of tight junction components in non-CNS endothelial cells. Astrocyte inductive effect(s) on the expression of the BBB characteristic enzymes including γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase have been well presented (Allt & Lawrenson, 2001; Haseloff et al., 2005). Using an astrocyte coculture system with endothelial cells Rauh et al (1992) showed that a possible direct cell-to-cell contact between BCECs and astroglial cells is probably needed for astrocyte inductive effects. However this is a controversial issue as secreted ADF effects on endothelial cells are well recognized. Shivers et al (1988) and Tio et al. (1990) also reported that both ACC and ACM (isolated rat brain astrocyte cells) can induce the alkaline phosphatase activity and tight junction generation in the human umbilical cord vein

In 1999, Igarashi *et al.* examined the relationship of glial-derived neurotrophic factor (GDNF), which maintains the dopaminergic system and motor neurons *in vivo*, with BBB using isolated primary porcine brain capillary endothelial cells (PBCECs) and reported that GDNF at concentrations of 0.1 and 1 ng per ml can significantly enhance the barrier functionality (tight junction integrity) with increases in TEER values and decreases in mannitol permeability (Igarashi *et al.* 1999). It can be deduced that GDNF is able to seal tightly the paracellular pathway in addition to its homeostasis role on the CNS. Moreover, it appears that factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) can induce astrocyte differentiation. ADFs also influence the functionality of BBB carrier-mediated transport systems (Abbott, 2005). The use of BBB transports will be

Pericytes are an imperative cellular constituent of the BBB, which also play a regulatory role in terms of brain angiogenesis and tight junction formation within BCECs. These cells also contribute to the microvascular vasodynamic capacity and structural stability. They are actively involved in the neuroimmune network operating at the BBB and confer macrophage functions. The pericyte and endothelial cell interaction occurs via cytoplasmic processes of the pericyte indenting the EC and vice versa. This contact process is called "peg and socket"-an interdigitation process (Wakui et al., 1989). Larson *et al.* (1987) studied intercellular relationship in the microvasculature using fluorescent Lucifer yellow CH dye/radiolabeled nucleotide and freeze-fracture methodology, and showed that cultured pericytes presented gap junctions in freeze-fracture replicas and extensive nucleotide and dye transfer. Observing low dye transfer and high nucleotide transfer between bovine brain microvessel endothelial cells and pericytes, they proposed a possible junctional contact

Basically, some biomolecules including adhesive glycoprotein and fibronectin have been found localized at the BCECs and pericytes junctional sites adjacent to "adhesion plaques" at the plasma membrane. The adhesion plagues implied the existence of a mechanical linkage between pericytes and ECs, i.e. a linkage that allowed mechanical contraction or relaxation of the pericyte to influence vessel diameter. This later process may assist the endothelial cells to reduce their size. Pericytes have specific and localized distribution in different tissues displaying a granular morphology reflecting lysosomal enrichment. Using an *in vivo*

between these cells that promotes their differentiation (Larson et al., 1987).

endothelial cells (Shivers et al., 1988; Tio et al., 1990).

discussed later in this chapter.

**2.3 Pericytes** 

cells tend to lose the TJ-dependent BBB characteristics such as macromolecular impermeability and high electrical resistance with conditioned culture. The tight junction complexity index (CI), as the number of branch points per unit length of tight junctional strands, was decreased 5 hr after culturing the primary bovine brain microvessel endothelial cells. However, the association of TJ particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased steadily during culture with a major drop between 16 hr and 24 hr. Wolburg et al. showed that the CI could be increased by elevation of intracellular cyclic adenosine monophosphate (cAMP) levels, while phorbol esters had the opposite effect - the endothelial cells P-face association of TJ particles was enhanced by elevation of cAMP levels and by astrocyte coculture (ACC) or exposure to astrocyte conditioned-medium (ACM). The authors also highlighted that astrocytes induced the latter effect on P-face association; and also showed that elevation of cAMP levels together with ACM increased trans-endothelial/epithelial electrical resistance (TEER) synergistically and decreased inulin permeability of primary cultures. They, thus, concluded that P-face association of TJ particles in brain ECs may be a critical feature of the blood-brain barrier functionality that can be specifically modulated by astrocytes and cAMP levels.

#### **2.2 Astrocytes**

Astrocytes are glial cells that envelop >99% of the BBB endothelium (Hawkins & Davis, 2005). Astrocytes and endothelial cells reciprocally affect each other's structure and/functions - their interactions induce and modulate the development of the BBB. Such interaction enhances the TJs and reduces the gap junctional area of BCECs, while increases the number of astrocytic membrane particle assemblies and astrocyte density. Astrocytes are essential for proper neuronal activities, for which the close proximity of astrocytes and BCECs appear to be essential for a functional neurovascular unit (Abbott et al., 2006). Based on *in vitro* investigations, the coculture of BCECs with astrocytes can improve the BBB functionality such as permeability and cellular transport functions (Omidi et al., 2008).

The nature of the astrocyte-derived factors (ADFs) is not fully understood, inductive effect of ADFs on brain microvascular endothelial cell differentiation and BBB formation has been well reported. Investigation upon the modulatory effects of astrocyte on BCECs have revealed that the rat astrocyte cells are able to modulate the chick peripheral ECs to make them less permeable to large molecules. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) has so far been documented in the presence of ADFs. Many studies have shown the improvement of physiological parameters (e.g., increased TEER and decreased paracellular permeability) in different in vitro models of the BBB treated with ADFs. Moreover, it should be evoked that the interaction of BCECs and astrocytes is bidirectional and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction. From many kinds of different experimental designs, it is quite clear that astrocyte factors are able to modulate TJ restrictiveness under perhaps certain defined conditions. To test the astrocytic factors effect on endothelial cells, Shivers et al. (1988) aimed to find out whether these factors can initiate development of non-CNS microvessel endothelial cells by culturing and passaging bovine aorta and pulmonary artery endothelial cells in the presence of 50% ACM. The investigators reported the endothelial cells maintained in ACM displayed complex tight junctions as well as gap junctions, but the cells plated onto plastic or fibronectin-coated substrates without ACM showed no tight or gap junctions. This finding directed them to conclude that CNS astrocytes generate soluble factor(s), which can provoke formation of tight junction components in non-CNS endothelial cells. Astrocyte inductive effect(s) on the expression of the BBB characteristic enzymes including γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase have been well presented (Allt & Lawrenson, 2001; Haseloff et al., 2005). Using an astrocyte coculture system with endothelial cells Rauh et al (1992) showed that a possible direct cell-to-cell contact between BCECs and astroglial cells is probably needed for astrocyte inductive effects. However this is a controversial issue as secreted ADF effects on endothelial cells are well recognized. Shivers et al (1988) and Tio et al. (1990) also reported that both ACC and ACM (isolated rat brain astrocyte cells) can induce the alkaline phosphatase activity and tight junction generation in the human umbilical cord vein endothelial cells (Shivers et al., 1988; Tio et al., 1990).

In 1999, Igarashi *et al.* examined the relationship of glial-derived neurotrophic factor (GDNF), which maintains the dopaminergic system and motor neurons *in vivo*, with BBB using isolated primary porcine brain capillary endothelial cells (PBCECs) and reported that GDNF at concentrations of 0.1 and 1 ng per ml can significantly enhance the barrier functionality (tight junction integrity) with increases in TEER values and decreases in mannitol permeability (Igarashi *et al.* 1999). It can be deduced that GDNF is able to seal tightly the paracellular pathway in addition to its homeostasis role on the CNS. Moreover, it appears that factors secreted by brain endothelial cells including leukaemia inhibitory factor (LIF) can induce astrocyte differentiation. ADFs also influence the functionality of BBB carrier-mediated transport systems (Abbott, 2005). The use of BBB transports will be discussed later in this chapter.

#### **2.3 Pericytes**

116 Novel Therapeutic Concepts in Targeting Glioma

cells tend to lose the TJ-dependent BBB characteristics such as macromolecular impermeability and high electrical resistance with conditioned culture. The tight junction complexity index (CI), as the number of branch points per unit length of tight junctional strands, was decreased 5 hr after culturing the primary bovine brain microvessel endothelial cells. However, the association of TJ particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased steadily during culture with a major drop between 16 hr and 24 hr. Wolburg et al. showed that the CI could be increased by elevation of intracellular cyclic adenosine monophosphate (cAMP) levels, while phorbol esters had the opposite effect - the endothelial cells P-face association of TJ particles was enhanced by elevation of cAMP levels and by astrocyte coculture (ACC) or exposure to astrocyte conditioned-medium (ACM). The authors also highlighted that astrocytes induced the latter effect on P-face association; and also showed that elevation of cAMP levels together with ACM increased trans-endothelial/epithelial electrical resistance (TEER) synergistically and decreased inulin permeability of primary cultures. They, thus, concluded that P-face association of TJ particles in brain ECs may be a critical feature of the blood-brain barrier

functionality that can be specifically modulated by astrocytes and cAMP levels.

Astrocytes are glial cells that envelop >99% of the BBB endothelium (Hawkins & Davis, 2005). Astrocytes and endothelial cells reciprocally affect each other's structure and/functions - their interactions induce and modulate the development of the BBB. Such interaction enhances the TJs and reduces the gap junctional area of BCECs, while increases the number of astrocytic membrane particle assemblies and astrocyte density. Astrocytes are essential for proper neuronal activities, for which the close proximity of astrocytes and BCECs appear to be essential for a functional neurovascular unit (Abbott et al., 2006). Based on *in vitro* investigations, the coculture of BCECs with astrocytes can improve the BBB functionality such as permeability and cellular transport functions (Omidi et al., 2008).

The nature of the astrocyte-derived factors (ADFs) is not fully understood, inductive effect of ADFs on brain microvascular endothelial cell differentiation and BBB formation has been well reported. Investigation upon the modulatory effects of astrocyte on BCECs have revealed that the rat astrocyte cells are able to modulate the chick peripheral ECs to make them less permeable to large molecules. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) has so far been documented in the presence of ADFs. Many studies have shown the improvement of physiological parameters (e.g., increased TEER and decreased paracellular permeability) in different in vitro models of the BBB treated with ADFs. Moreover, it should be evoked that the interaction of BCECs and astrocytes is bidirectional and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction. From many kinds of different experimental designs, it is quite clear that astrocyte factors are able to modulate TJ restrictiveness under perhaps certain defined conditions. To test the astrocytic factors effect on endothelial cells, Shivers et al. (1988) aimed to find out whether these factors can initiate development of non-CNS microvessel endothelial cells by culturing and passaging bovine aorta and pulmonary artery endothelial cells in the presence of 50% ACM. The investigators reported the endothelial cells maintained in ACM displayed complex tight junctions as well as gap junctions, but the cells plated onto plastic or fibronectin-coated substrates without

**2.2 Astrocytes** 

Pericytes are an imperative cellular constituent of the BBB, which also play a regulatory role in terms of brain angiogenesis and tight junction formation within BCECs. These cells also contribute to the microvascular vasodynamic capacity and structural stability. They are actively involved in the neuroimmune network operating at the BBB and confer macrophage functions. The pericyte and endothelial cell interaction occurs via cytoplasmic processes of the pericyte indenting the EC and vice versa. This contact process is called "peg and socket"-an interdigitation process (Wakui et al., 1989). Larson *et al.* (1987) studied intercellular relationship in the microvasculature using fluorescent Lucifer yellow CH dye/radiolabeled nucleotide and freeze-fracture methodology, and showed that cultured pericytes presented gap junctions in freeze-fracture replicas and extensive nucleotide and dye transfer. Observing low dye transfer and high nucleotide transfer between bovine brain microvessel endothelial cells and pericytes, they proposed a possible junctional contact between these cells that promotes their differentiation (Larson et al., 1987).

Basically, some biomolecules including adhesive glycoprotein and fibronectin have been found localized at the BCECs and pericytes junctional sites adjacent to "adhesion plaques" at the plasma membrane. The adhesion plagues implied the existence of a mechanical linkage between pericytes and ECs, i.e. a linkage that allowed mechanical contraction or relaxation of the pericyte to influence vessel diameter. This later process may assist the endothelial cells to reduce their size. Pericytes have specific and localized distribution in different tissues displaying a granular morphology reflecting lysosomal enrichment. Using an *in vivo*

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 119

Efforts to generate an *in vitro* BBB model, in fact, have been based upon measurement of the TEER, assessment of the sucrose permeability and expression of the specific enzymes and markers of the BBB. The higher TEER and the lower sucrose permeability confer the better characteristics. To achieve this aim different techniques have been recruited, e.g. utilizing of the hydrocortisone and serum free medium in order to increase the TEER (up to 1000 cm2)

The influence of extracellular matrix on the BBB properties has been investigated by several researchers using cell lines and primary isolated BCECs. Shivers *et al.* (1988) showed that the local control of tight junction biogenesis in brain capillary endothelial cells depends on astrocyte-produced factors and extracellular matrix. The ECs in general do not express their final destination-specific differentiated features until those features are induced by local environment-produced conditions including extracellular matrix. Using primary cultures of PBCECs, Tilling *et al*. (1998) examined the effect of collagen IV, fibronectin, laminin and a secreted acidic protein and rich in cysteine alone or one-to-one mixtures of them. They showed that these proteins are involved in tight junction formation between cerebral capillary endothelial cells by presenting increased TEER (Robert & Robert, 1998; Tilling et

The effect of cAMP on BBB function has been studied by several researchers. Using combination of astrocyte conditioned-medium and cAMP elevators, Rubin et al (1991) reported a cell culture *in vitro* BBB model that generated high resistance tight junctions and exhibited low rates of paracellular permeability. Hurst et al (1996) showed that a coculture BBB model of the immortalized human umbilical vein endothelial cells ECV304 ( reassigned later as T24 bladder epithelial carcinoma cell) with rat C6 glioma cells can generate a BBB model with high TEER (400-600 .cm2). They demonstrated bioelectrical alterations by vasoactive agonists and cAMP elevators (i.e. decreased TEER by histamine, bradykinin, and serotonin and increased TEER by cAMP, such as forskolin elevators). The researchers also showed formation of inositol triphosphates (IPs) that can induce the release of calcium ions from cellular storage sites and a subsequent rise in intracellular calcium which can activate diacylglycerol (DAG) and accordingly the PKC that could increase the permeability of the endothelial cells (Hurst & Clark, 1998). Investigation on the effects of elevated intracellular cAMP and astrocyte derived factors on the F-actin cytoskeleton and paracellular permeability of RBE4 cell monolayers have revealed that the cAMP effects on the TEER

appear likely to be independent of new gene transcription (Rist et al., 1997).

The protein GDNF can activate the barrier functions of the BCECs in the presence of cAMP. It has been reported that GDNF not only can promote the barrier restrictiveness but also support the survival of neurons in the presence of cAMP. The role of other factors on brain ECs signaling and the BBB formation is uncertain (Igarashi et al., 1999). However, some other factors such as vascular endothelial growth factor (VEGF) appear to increase the permeability of BCECs because of loss of occludin and ZO-1 from the endothelial cell

by stimulating the formation of barrier properties (Hoheisel *et al.* 1998).

**4. Modulation of BBB permeability** 

**4.2 The role of cyclic AMP (cAMP)** 

**4.1 Extracellular matrix** 

al., 1998).

rat model, it has been reported that blood-brain barrier disruption, e.g. by hyperosmotic mannitol with adriamycin, causes an increase of pericyte lysosomes (Kondo et al., 1987). Pericytes are rich in plasmalemmal and cytoplasmic vesicles as well as microfilament bundles, but interestingly only about 10% of vesicles locate to the surface facing ECs. Most importantly pericytes modulate endothelial cells phenotype not only by physical action but also via secreting epidermal growth factor (EGF). Indeed, it has already been shown that EGF is an effective endothelial cell mitogen that enhances angiogenesis, and is concentrated at and during pericytes-ECs cross-talk and interdigitation processes. The expression of γ-GTP has been shown in the pericyte as well as the brain microvessel endothelial cells. Using γ-GTP as the sole marker for the primary isolated brain capillary endothelial cells seems not to be right and proper due to possible pericytic contamination. The transforming growth factor-beta (TGF-b), produced by pericytes, plays an important role in reducing lymphocyte infiltration into the CNS in inflammatory demyelinating diseases. Pericytes can stabilize capillary-like structures formed by endothelial cells in coculture with astrocytes. This latter process can be driven by TGF-b1, which is one of the TGF-b isoforms (Allt & Lawrenson, 2001; Ramsauer et al., 2002).

The cultured pericytes in the endothelial cell conditioned-medium (ECCM) allowed the cerebral pericyte aminopeptidase N (pAPN) to be re-expressed, while purified pericytes deprived of endothelial cells even in the presence of ACM showed no reexpression. This indicates that endothelial cells constitute an essential requirement for the in vitro reexpression of pAPN, but not astrocytes. Pericytes are involved in amino acid and peptide catabolism of the brain. This suggests that pericytes play a key metabolic role aside from structural role in relation to the BBB maintenance and homeostasis. In addition, based on the reciprocal pericytes-ECs interactions and cross talk, it can be suggested that the two-way interdigitations may stimulate not only the BBB endothelial cell activities but also promote pericyte functions. Pericytes may have a role in the possible cross-talk between pericytes, astrocyte and endothelial cells, a triple intercommunication process that may bring us to consider the notion of pericyte positive contribution in the physiopathology of the BBB as well as certain diseases such as Alzheimer. For example, β-amyloid peptide 42 can be uptaken by the phagocytic pericytes and astrocytes. This process helps to clear the exogenous peptide from the brain extracellular space and deliver it to the blood circulation system that shows also the contribution of pericytes in the BBB transport functionality (Allt & Lawrenson, 2001; Balabanov & Dore-Duffy, 1998; Pluta et al., 2000).

#### **3. Bioelectrical properties and permeability**

Due to the association of the TEER with permeability, TEER values commonly are exploited to describe the permeability of the BBB. The [14C] sucrose permeability is used to assess the restrictiveness of BBB in relation to *in vivo* (1.2 10-7 cm.sec-1). *In vivo* TEER values vary in the epithelial and endothelial cells. For instance human placental endothelium shows TEER 22–52 Ω.cm2 that permits rapid paracellular exchange of nutrients and waste between the mother and fetus (Jinga *et al.* 2000), whereas urinary bladder epithelium has a very high TEER of 6000–30000 Ω.cm2, necessary for preserving urine composition (Powell 1981). The BBB possesses TEER vales of ~2000 Ω.cm2, which helps to maintain brain homeostasis (Engelhardt, 2006).

Efforts to generate an *in vitro* BBB model, in fact, have been based upon measurement of the TEER, assessment of the sucrose permeability and expression of the specific enzymes and markers of the BBB. The higher TEER and the lower sucrose permeability confer the better characteristics. To achieve this aim different techniques have been recruited, e.g. utilizing of the hydrocortisone and serum free medium in order to increase the TEER (up to 1000 cm2) by stimulating the formation of barrier properties (Hoheisel *et al.* 1998).

#### **4. Modulation of BBB permeability**

#### **4.1 Extracellular matrix**

118 Novel Therapeutic Concepts in Targeting Glioma

rat model, it has been reported that blood-brain barrier disruption, e.g. by hyperosmotic mannitol with adriamycin, causes an increase of pericyte lysosomes (Kondo et al., 1987). Pericytes are rich in plasmalemmal and cytoplasmic vesicles as well as microfilament bundles, but interestingly only about 10% of vesicles locate to the surface facing ECs. Most importantly pericytes modulate endothelial cells phenotype not only by physical action but also via secreting epidermal growth factor (EGF). Indeed, it has already been shown that EGF is an effective endothelial cell mitogen that enhances angiogenesis, and is concentrated at and during pericytes-ECs cross-talk and interdigitation processes. The expression of γ-GTP has been shown in the pericyte as well as the brain microvessel endothelial cells. Using γ-GTP as the sole marker for the primary isolated brain capillary endothelial cells seems not to be right and proper due to possible pericytic contamination. The transforming growth factor-beta (TGF-b), produced by pericytes, plays an important role in reducing lymphocyte infiltration into the CNS in inflammatory demyelinating diseases. Pericytes can stabilize capillary-like structures formed by endothelial cells in coculture with astrocytes. This latter process can be driven by TGF-b1, which is one of the TGF-b isoforms (Allt & Lawrenson,

The cultured pericytes in the endothelial cell conditioned-medium (ECCM) allowed the cerebral pericyte aminopeptidase N (pAPN) to be re-expressed, while purified pericytes deprived of endothelial cells even in the presence of ACM showed no reexpression. This indicates that endothelial cells constitute an essential requirement for the in vitro reexpression of pAPN, but not astrocytes. Pericytes are involved in amino acid and peptide catabolism of the brain. This suggests that pericytes play a key metabolic role aside from structural role in relation to the BBB maintenance and homeostasis. In addition, based on the reciprocal pericytes-ECs interactions and cross talk, it can be suggested that the two-way interdigitations may stimulate not only the BBB endothelial cell activities but also promote pericyte functions. Pericytes may have a role in the possible cross-talk between pericytes, astrocyte and endothelial cells, a triple intercommunication process that may bring us to consider the notion of pericyte positive contribution in the physiopathology of the BBB as well as certain diseases such as Alzheimer. For example, β-amyloid peptide 42 can be uptaken by the phagocytic pericytes and astrocytes. This process helps to clear the exogenous peptide from the brain extracellular space and deliver it to the blood circulation system that shows also the contribution of pericytes in the BBB transport functionality (Allt

Due to the association of the TEER with permeability, TEER values commonly are exploited to describe the permeability of the BBB. The [14C] sucrose permeability is used to assess the restrictiveness of BBB in relation to *in vivo* (1.2 10-7 cm.sec-1). *In vivo* TEER values vary in the epithelial and endothelial cells. For instance human placental endothelium shows TEER 22–52 Ω.cm2 that permits rapid paracellular exchange of nutrients and waste between the mother and fetus (Jinga *et al.* 2000), whereas urinary bladder epithelium has a very high TEER of 6000–30000 Ω.cm2, necessary for preserving urine composition (Powell 1981). The BBB possesses TEER vales of ~2000 Ω.cm2, which helps to maintain brain homeostasis

& Lawrenson, 2001; Balabanov & Dore-Duffy, 1998; Pluta et al., 2000).

**3. Bioelectrical properties and permeability** 

(Engelhardt, 2006).

2001; Ramsauer et al., 2002).

The influence of extracellular matrix on the BBB properties has been investigated by several researchers using cell lines and primary isolated BCECs. Shivers *et al.* (1988) showed that the local control of tight junction biogenesis in brain capillary endothelial cells depends on astrocyte-produced factors and extracellular matrix. The ECs in general do not express their final destination-specific differentiated features until those features are induced by local environment-produced conditions including extracellular matrix. Using primary cultures of PBCECs, Tilling *et al*. (1998) examined the effect of collagen IV, fibronectin, laminin and a secreted acidic protein and rich in cysteine alone or one-to-one mixtures of them. They showed that these proteins are involved in tight junction formation between cerebral capillary endothelial cells by presenting increased TEER (Robert & Robert, 1998; Tilling et al., 1998).

#### **4.2 The role of cyclic AMP (cAMP)**

The effect of cAMP on BBB function has been studied by several researchers. Using combination of astrocyte conditioned-medium and cAMP elevators, Rubin et al (1991) reported a cell culture *in vitro* BBB model that generated high resistance tight junctions and exhibited low rates of paracellular permeability. Hurst et al (1996) showed that a coculture BBB model of the immortalized human umbilical vein endothelial cells ECV304 ( reassigned later as T24 bladder epithelial carcinoma cell) with rat C6 glioma cells can generate a BBB model with high TEER (400-600 .cm2). They demonstrated bioelectrical alterations by vasoactive agonists and cAMP elevators (i.e. decreased TEER by histamine, bradykinin, and serotonin and increased TEER by cAMP, such as forskolin elevators). The researchers also showed formation of inositol triphosphates (IPs) that can induce the release of calcium ions from cellular storage sites and a subsequent rise in intracellular calcium which can activate diacylglycerol (DAG) and accordingly the PKC that could increase the permeability of the endothelial cells (Hurst & Clark, 1998). Investigation on the effects of elevated intracellular cAMP and astrocyte derived factors on the F-actin cytoskeleton and paracellular permeability of RBE4 cell monolayers have revealed that the cAMP effects on the TEER appear likely to be independent of new gene transcription (Rist et al., 1997).

The protein GDNF can activate the barrier functions of the BCECs in the presence of cAMP. It has been reported that GDNF not only can promote the barrier restrictiveness but also support the survival of neurons in the presence of cAMP. The role of other factors on brain ECs signaling and the BBB formation is uncertain (Igarashi et al., 1999). However, some other factors such as vascular endothelial growth factor (VEGF) appear to increase the permeability of BCECs because of loss of occludin and ZO-1 from the endothelial cell

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 121

metabolism of neuropeptides is mediated by membrane peptidases at the BBB. Three members of the membrane peptidases (dipeptidyl peptidase A; aminopeptidase N; dipeptidyl peptidase IV) have been found in the BCECs, but many more vessels are positive for them, in particular more positive for dipeptidyl peptidase A than other two. The expression of the BBB enzymes depends on the different parameters such as direct cell-tocell communications or cell-derived factors. Enzymatic activities of the -GTP and ALP are taken as indicators for the expression of the BBB phenotype; reader is directed to see (Allt &

**6. Transportation of exogenous/endogenous compounds across BBB** 

The capability of a particular substance to cross the BBB and enter the CNS is dependent upon a number of parameters, including physicochemical properties such as molecular weight (MW), lipophilicity, pKa, hydrogen bonding as well as biological factors. The BBB transportation, nevertheless, may generally be classified into different categories, including: 1) passive diffusion that depends on physicochemical properties and mainly on the lipophilicity of the molecule, 2) facilitated transport via carrier-mediated transporters (Glut1, LAT1), 3) paracellular pathway (small hydrophilic componds), 4) receptor-mediated endocytosis/transcytosis, and 4) Liquid-phase (adsorptive) endocytosis/transcytosis. Fig. 3

Brain drug delivery and targeting requires overcoming the limited access of drugs to the brain. Different methods have been developed to achieve BBB penetration, including: opening of BBB TJs by means of osmotic or biologically active agents (such as bradykinin and histamine); exploiting of various specific transport mechanisms. The last methodology includes: conjugation of a drug with a targeting protein, or to a monoclonal antibody that gains access to the brain by receptor-mediated transcytosis, or to a small peptide-vectors to enhance brain uptake of several therapeutic drugs. Further the use of drug delivery devices such as liposomes has also been reported. As shown in Fig. 4, the BBB carrier-mediated transporters comprise two different classes, including the efflux and the influx pump

The BBB represents a major hindrance to the entry of many therapeutic drugs into the brain and efflux pumps are part of this protection. P-glycoprotein (P-gp; MDR1/ABCB1) is an ATP-binding cassette (ABC) drug transport protein that is predominantly found in the apical membranes of a number of epithelial cell types in the body as well as the brain microvessel endothelial cells. The putative transmembrane structural organization of human MDR1 P-gp is primarily found in the cell plasma membrane as 12 transmembrane segments that are thought to fold together and form a three-dimensional barrier like structure in the cell plasma membrane. The latter polypeptide chain consists of two similar halves. Each half contains of six putative transmembrane segments and intracellular ATP-binding site. The hydrolysis of ATP provides the energy for active drug export. Schinkel et al (1995) showed that mouse MDR1a and the human MDR1 P-glycoprotein actively transport ivermectin, dexamethasone, digoxin, and cyclosporin A and, to a lesser extent, morphine across a polarized kidney epithelial cell layer in vitro. The investigator reported that injection of the radiolabeled substrates of P-gp in MDR1a knockout and wild-type mice resulted in 20- to

Lawrenson, 2000; Allt & Lawrenson, 2001; Orte et al., 1999).

represents schematic illustration of BBB transport systems.

transport systems.

**6.1 Efflux transporters** 

junctions (Wang et al., 2001), while cAMP acts against such phenomenon. All of these processes, somehow, play a role in modulating the full BBB characteristics. By means of an unpassaged primary culture of rat BCECs, it has been shown that certain cell-surface receptors may fulfill a role in BBB regulation. For example, brain endothelial regulation was shown by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1-like (2MeSATP-sensitive) receptors that are linked to Ca2+ mobilization. It should be stated that differential MAPK coupling of these receptors appear to exert fundamentally distinct influences over brain endothelial function in terms of cAMP modulation (Albert et al., 1997).

#### **4.3 Inflammatory mediators**

Basically, the brain endothelium forming the BBB can be modulated by a range of inflammatory mediators. Given that the main routes for penetration of polar solutes across the BBB include the paracellular tight junctional pathway and vesicular machineries, inflammatory mediators appear to influence both pathways, while such impacts can also be seen in other closely associated cells such as pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. Various inflammatory agents are able to increase both endothelial permeability and vessel diameter, and these together can result in significant leakiness of BBB and accordingly cerebral edema. Of these agents, the bradykinin (Bk) is able to increases the permeability of BBB by acting on B2 receptors, perhaps via elevation of Ca2+, activation of phospholipase A2, release of arachidonic acid, and production of free radicals. Serotonin (5HT) can also increase the permeability of BBB through a calcium-dependent mechanism. Histamine as nervous system neurotransmitters possesses capability of consistent blood-brain barrier opening mediated by H2 receptors and elevation of Ca2+, but the H1 receptor coupled to an elevation of cAMP can decrease the permeability of BBB. Elevation of arachidonic acid may also cause gross opening of the BBB to large molecules such as peptides/proteins. There exist a number of studies showing purposed recruitment of such mechanisms for deliberate opening of the BBB for drug delivery to the brain; readers are directed to see (Abbott, 2000).

#### **5. BBB enzymes and other differentiation markers**

Several markers are most commonly exploited and include γ-GTP and alkalin phosphatase (ALP) enzymes expression, or antigenic endothelial cell markers such as: Factor VIII , von Willebrand Factor (vWF). Additionally the uptake of acetylated low density lipoprotein (LDL) i.e. dioctadecyl-indocarbocyanine (Di-I) labeled acetylated LDL (Di-I-Ac-LDL) and the binding of lectin, i.e. biotinylated lectins (BL) *Ultex europaeus I* (UE I) and *Bandeiraea simplicifolia* isolation B4 *(* BSA IB4) have been exploited. Further, a high glucose transporter (GLUT-1) density has been shown as a marker of brain microvascular endothelia. The - GTP, ALP and membrane peptidases (aminopeptidase N, peptidyle dipeptidase and dipeptidyl peptidase IV) have been used as marker for the BBB. The -GTP is of special importance due to its high expression in the BCECs, therefore it is used as a marker for ECs of the BBB. By contrast, this marker is absent from brain ECs of paraventricular nucleus, where the BBB characteristics and properties are lacking. The induction of -GTP by astrocyte has been reported, even though the high expression of this enzyme in the pericytes makes it a non-specific marker of the BBB. The ALP is highly expressed in the BCECs and used as a marker. Like γ-GTP, its expression can be modulated by astrocytes. The metabolism of neuropeptides is mediated by membrane peptidases at the BBB. Three members of the membrane peptidases (dipeptidyl peptidase A; aminopeptidase N; dipeptidyl peptidase IV) have been found in the BCECs, but many more vessels are positive for them, in particular more positive for dipeptidyl peptidase A than other two. The expression of the BBB enzymes depends on the different parameters such as direct cell-tocell communications or cell-derived factors. Enzymatic activities of the -GTP and ALP are taken as indicators for the expression of the BBB phenotype; reader is directed to see (Allt & Lawrenson, 2000; Allt & Lawrenson, 2001; Orte et al., 1999).

#### **6. Transportation of exogenous/endogenous compounds across BBB**

The capability of a particular substance to cross the BBB and enter the CNS is dependent upon a number of parameters, including physicochemical properties such as molecular weight (MW), lipophilicity, pKa, hydrogen bonding as well as biological factors. The BBB transportation, nevertheless, may generally be classified into different categories, including: 1) passive diffusion that depends on physicochemical properties and mainly on the lipophilicity of the molecule, 2) facilitated transport via carrier-mediated transporters (Glut1, LAT1), 3) paracellular pathway (small hydrophilic componds), 4) receptor-mediated endocytosis/transcytosis, and 4) Liquid-phase (adsorptive) endocytosis/transcytosis. Fig. 3 represents schematic illustration of BBB transport systems.

Brain drug delivery and targeting requires overcoming the limited access of drugs to the brain. Different methods have been developed to achieve BBB penetration, including: opening of BBB TJs by means of osmotic or biologically active agents (such as bradykinin and histamine); exploiting of various specific transport mechanisms. The last methodology includes: conjugation of a drug with a targeting protein, or to a monoclonal antibody that gains access to the brain by receptor-mediated transcytosis, or to a small peptide-vectors to enhance brain uptake of several therapeutic drugs. Further the use of drug delivery devices such as liposomes has also been reported. As shown in Fig. 4, the BBB carrier-mediated transporters comprise two different classes, including the efflux and the influx pump transport systems.

#### **6.1 Efflux transporters**

120 Novel Therapeutic Concepts in Targeting Glioma

junctions (Wang et al., 2001), while cAMP acts against such phenomenon. All of these processes, somehow, play a role in modulating the full BBB characteristics. By means of an unpassaged primary culture of rat BCECs, it has been shown that certain cell-surface receptors may fulfill a role in BBB regulation. For example, brain endothelial regulation was shown by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1-like (2MeSATP-sensitive) receptors that are linked to Ca2+ mobilization. It should be stated that differential MAPK coupling of these receptors appear to exert fundamentally distinct influences over brain endothelial function in terms of cAMP modulation (Albert et al., 1997).

Basically, the brain endothelium forming the BBB can be modulated by a range of inflammatory mediators. Given that the main routes for penetration of polar solutes across the BBB include the paracellular tight junctional pathway and vesicular machineries, inflammatory mediators appear to influence both pathways, while such impacts can also be seen in other closely associated cells such as pericytes, astrocytes, smooth muscle, microglia, mast cells, and neurons. Various inflammatory agents are able to increase both endothelial permeability and vessel diameter, and these together can result in significant leakiness of BBB and accordingly cerebral edema. Of these agents, the bradykinin (Bk) is able to increases the permeability of BBB by acting on B2 receptors, perhaps via elevation of Ca2+, activation of phospholipase A2, release of arachidonic acid, and production of free radicals. Serotonin (5HT) can also increase the permeability of BBB through a calcium-dependent mechanism. Histamine as nervous system neurotransmitters possesses capability of consistent blood-brain barrier opening mediated by H2 receptors and elevation of Ca2+, but the H1 receptor coupled to an elevation of cAMP can decrease the permeability of BBB. Elevation of arachidonic acid may also cause gross opening of the BBB to large molecules such as peptides/proteins. There exist a number of studies showing purposed recruitment of such mechanisms for deliberate opening of the BBB for drug delivery to the brain; readers

Several markers are most commonly exploited and include γ-GTP and alkalin phosphatase (ALP) enzymes expression, or antigenic endothelial cell markers such as: Factor VIII , von Willebrand Factor (vWF). Additionally the uptake of acetylated low density lipoprotein (LDL) i.e. dioctadecyl-indocarbocyanine (Di-I) labeled acetylated LDL (Di-I-Ac-LDL) and the binding of lectin, i.e. biotinylated lectins (BL) *Ultex europaeus I* (UE I) and *Bandeiraea simplicifolia* isolation B4 *(* BSA IB4) have been exploited. Further, a high glucose transporter (GLUT-1) density has been shown as a marker of brain microvascular endothelia. The - GTP, ALP and membrane peptidases (aminopeptidase N, peptidyle dipeptidase and dipeptidyl peptidase IV) have been used as marker for the BBB. The -GTP is of special importance due to its high expression in the BCECs, therefore it is used as a marker for ECs of the BBB. By contrast, this marker is absent from brain ECs of paraventricular nucleus, where the BBB characteristics and properties are lacking. The induction of -GTP by astrocyte has been reported, even though the high expression of this enzyme in the pericytes makes it a non-specific marker of the BBB. The ALP is highly expressed in the BCECs and used as a marker. Like γ-GTP, its expression can be modulated by astrocytes. The

**4.3 Inflammatory mediators** 

are directed to see (Abbott, 2000).

**5. BBB enzymes and other differentiation markers** 

The BBB represents a major hindrance to the entry of many therapeutic drugs into the brain and efflux pumps are part of this protection. P-glycoprotein (P-gp; MDR1/ABCB1) is an ATP-binding cassette (ABC) drug transport protein that is predominantly found in the apical membranes of a number of epithelial cell types in the body as well as the brain microvessel endothelial cells. The putative transmembrane structural organization of human MDR1 P-gp is primarily found in the cell plasma membrane as 12 transmembrane segments that are thought to fold together and form a three-dimensional barrier like structure in the cell plasma membrane. The latter polypeptide chain consists of two similar halves. Each half contains of six putative transmembrane segments and intracellular ATP-binding site. The hydrolysis of ATP provides the energy for active drug export. Schinkel et al (1995) showed that mouse MDR1a and the human MDR1 P-glycoprotein actively transport ivermectin, dexamethasone, digoxin, and cyclosporin A and, to a lesser extent, morphine across a polarized kidney epithelial cell layer in vitro. The investigator reported that injection of the radiolabeled substrates of P-gp in MDR1a knockout and wild-type mice resulted in 20- to

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 123

Multidrug-resistance associated protein (MRP) (Zhang *et al.* 2000) actively transports a broad range of anionic compounds out of the cell in the BBB and is known as another member of the ABC superfamily of transport proteins. It has approximately 15% amino acid sequence homology to P-gp, and the characteristics ATP binding sites that allow for the active transport of a diverse array of compounds out of the cell. In contrast to P-gp, MRP transports organic anions. Zhang et al (2000) reported that the MRP1, MRP4, MRP5, and MRP6 were consistently expressed in both the capillary-enriched fraction of the brain homogenate and the BBMEC monolayers. The expression of MRP2 has also been shown in the isolated primary porcine microvessel endothelial cells (Fricker *et al.* 2002). The presence of several different MRP homologues at the BBB highlights the MRP role in controlling the permeability of the BBB to organic anions. In the brain, MRP4 was shown to be expressed on the luminal membrane of BCECs as well as the basolateral membrane of the choroid plexus. The chemotherapeutic drug topotecan was shown to be accumulated in the brain and the CSF in an MRP4 knockout mouse model. This clearly highlights the important role that MRP4 plays in determining the CNS distribution of this drug (Leggas et al., 2004). It is likely that multiple efflux drug transporters including MRP4 govern the brain penetration and activity of this anticancer agent. Readers are directed to see (Tsuji, 2005; Urquhart & Kim,

It should be stated that most of anticancer agents are substrate to ABC transporters. Of these, the MDR1/ABCB1 can pump out a wide range of compounds (e.g., Acebutolol, Actinomycin D, Amprenavir, Azidopine, Betamethasone, Calcein-AM, Cepharanthin, Cerivastatin, Chloroquine, Cimetidine, Clarithromycin, Colchicine, Cortisol, Cyclosporin A, Daunorubicin, Dexamethasone, Digitoxin, Digoxin, Dipyridamole, Docetaxel, Domperidone, Doxorubicin, Eletriptan, Emetine, Epinastine, Erythromycin, Estradiol-17b-D-glucuronide, Estrone, Ethynyl estradiol, Etoposide, Fexofenadine, Grepafloxacin, Imatinib, Indinavir, Irinotecan, Ivermectin, Lansoprazole, Levofloxacin, Loperamide, Losartan, Lovastatin, Methylprednisolone, Mitoxantrone, Morphine, Neostigmine, Omeprazole, Pantoprazole, Prazosin, Prednisolone, Puromycin, Quinidine2, Ramosetron, Ranitidine, Reserpine, Ritonavir, Saquinavir, Somatostain, Sparfloxacin, Talinolol, Taxol, Terfenadine, Trimethoprim, Vecuronium, Verapamil, Vinblastine, Vincristine), while the MRP4/ABCC4 is able to efflux a narrower spectrum (e.g., cAMP, cGMP, Dehydroepiandrosterone-3-sulfate, Estradiol-17b-Dglucuronide, Folate, Methotrexate, Prostaglandin E1, Prostaglandin E2). The breast cancer resistance protein (BCRP/ABCG2) have been reported to be expressed as active efflux drug transporters at the BBB that can display efflux functionality similar to that of P-gp for a wide range of componds (e.g., Azidodeoxythymidine, Bisantrene, Cerivastatin, Doxorubicin, Daunorubicin, Dehydroepiandrosterone-3-sulfate, Etoposide, Estrone-3-sulfate, Estradiol-17b-D-glucuronide, Folate, Flavopiridol, Imatinib mesylate, Mitoxantrone, Methotrexate, Prazosin, Pantoprazole, Pravastatin, Rhodamine 123, Topotecan), for more details readers are directed to

Many of these chemicals are important anticancer agents (e.g., Doxorubicin, Vinblastine, Vincristine, Methotrexate, Docetaxel, Etoposide, Idarubicin, and Taxol), thus it is vital to inhibit these efflux machineries to reach suitable concentration in brain during brain tumors chemotherapy. Various compounds were reported as inhibitors for these efflux

transportsers such as colchicine, phenothiazines and quinacrine.

2009).

see (Ohtsuki & Terasaki, 2007).

50-fold higher levels of radioactivity in the MDR1a knockout mice brain for digoxin and cyclosporin A (Schinkel et al., 1995b). These researchers generated mice with a genetic disruption of the drug-transporting MDR1a P-gp and showed that the P-gp knockout mice were overall healthy but they accumulated much higher levels of substrate drugs in the brain with markedly slower elimination. For the drugs (e.g., anticancer agents) that are P-gp substrates, this can lead to dramatically increased toxicity (Schinkel et al., 1995a). Thus, drugs inhibiting the MDR1 P-gp activity should be co-administered during chemotherapy of the brain tumors. The authors concluded that P-gp is the major determinant for the pharmacology of several medically important drugs apart from anti-cancer agents, especially in the BBB.

Fig. 3. Schematic illustration of transportation systems for shuttling of endogenous and/or exogenous substrates at the BBB. 1) Lipid-soluble small substrates (<500 Da) are able to diffuse across the membrane – they may be effluxed back into the blood circulation through efflux transporters (e.g., P-gp, MRP4). 2) Carrier-mediated transport machineries (e.g., Glut1, LAT1) are responsible for transport of small endogenous molecules (e.g., amino acids, nucleosides, and glucose). 3) Some small hydrophilic molecules can be transported via paracellular route. 4) Larger molecules (e.g., insulin, transferrin) are transported through receptor-mediated endpcytosis/transcytosis using vesicular trafficking towards the brain parenchyma. 5) Some large proteins (e.g., albumin) are transported across the BBB by adsorptive-mediated endocytosis/transcytosis. Of the carrier-mediated transporters, glucose transporters (Gluts) are responsible for traverse of glucose from blood to brain and btween different cells within the brain parenchyma. Adherens junctions provide a path for cell-to-cell intercommunication within endothelial cells of the BBB, adapted with permission (Omidi & Barar, 2012).

50-fold higher levels of radioactivity in the MDR1a knockout mice brain for digoxin and cyclosporin A (Schinkel et al., 1995b). These researchers generated mice with a genetic disruption of the drug-transporting MDR1a P-gp and showed that the P-gp knockout mice were overall healthy but they accumulated much higher levels of substrate drugs in the brain with markedly slower elimination. For the drugs (e.g., anticancer agents) that are P-gp substrates, this can lead to dramatically increased toxicity (Schinkel et al., 1995a). Thus, drugs inhibiting the MDR1 P-gp activity should be co-administered during chemotherapy of the brain tumors. The authors concluded that P-gp is the major determinant for the pharmacology of several medically important drugs apart from anti-cancer agents,

Fig. 3. Schematic illustration of transportation systems for shuttling of endogenous and/or exogenous substrates at the BBB. 1) Lipid-soluble small substrates (<500 Da) are able to diffuse across the membrane – they may be effluxed back into the blood circulation through efflux transporters (e.g., P-gp, MRP4). 2) Carrier-mediated transport machineries (e.g., Glut1, LAT1) are responsible for transport of small endogenous molecules (e.g., amino acids, nucleosides, and glucose). 3) Some small hydrophilic molecules can be transported via paracellular route. 4) Larger molecules (e.g., insulin, transferrin) are transported through receptor-mediated endpcytosis/transcytosis using vesicular trafficking towards the brain parenchyma. 5) Some large proteins (e.g., albumin) are transported across the BBB by adsorptive-mediated endocytosis/transcytosis. Of the carrier-mediated transporters, glucose transporters (Gluts) are responsible for traverse of glucose from blood to brain and btween different cells within the brain parenchyma. Adherens junctions provide a path for cell-to-cell intercommunication within endothelial cells of the BBB, adapted with permission

especially in the BBB.

(Omidi & Barar, 2012).

Multidrug-resistance associated protein (MRP) (Zhang *et al.* 2000) actively transports a broad range of anionic compounds out of the cell in the BBB and is known as another member of the ABC superfamily of transport proteins. It has approximately 15% amino acid sequence homology to P-gp, and the characteristics ATP binding sites that allow for the active transport of a diverse array of compounds out of the cell. In contrast to P-gp, MRP transports organic anions. Zhang et al (2000) reported that the MRP1, MRP4, MRP5, and MRP6 were consistently expressed in both the capillary-enriched fraction of the brain homogenate and the BBMEC monolayers. The expression of MRP2 has also been shown in the isolated primary porcine microvessel endothelial cells (Fricker *et al.* 2002). The presence of several different MRP homologues at the BBB highlights the MRP role in controlling the permeability of the BBB to organic anions. In the brain, MRP4 was shown to be expressed on the luminal membrane of BCECs as well as the basolateral membrane of the choroid plexus. The chemotherapeutic drug topotecan was shown to be accumulated in the brain and the CSF in an MRP4 knockout mouse model. This clearly highlights the important role that MRP4 plays in determining the CNS distribution of this drug (Leggas et al., 2004). It is likely that multiple efflux drug transporters including MRP4 govern the brain penetration and activity of this anticancer agent. Readers are directed to see (Tsuji, 2005; Urquhart & Kim, 2009).

It should be stated that most of anticancer agents are substrate to ABC transporters. Of these, the MDR1/ABCB1 can pump out a wide range of compounds (e.g., Acebutolol, Actinomycin D, Amprenavir, Azidopine, Betamethasone, Calcein-AM, Cepharanthin, Cerivastatin, Chloroquine, Cimetidine, Clarithromycin, Colchicine, Cortisol, Cyclosporin A, Daunorubicin, Dexamethasone, Digitoxin, Digoxin, Dipyridamole, Docetaxel, Domperidone, Doxorubicin, Eletriptan, Emetine, Epinastine, Erythromycin, Estradiol-17b-D-glucuronide, Estrone, Ethynyl estradiol, Etoposide, Fexofenadine, Grepafloxacin, Imatinib, Indinavir, Irinotecan, Ivermectin, Lansoprazole, Levofloxacin, Loperamide, Losartan, Lovastatin, Methylprednisolone, Mitoxantrone, Morphine, Neostigmine, Omeprazole, Pantoprazole, Prazosin, Prednisolone, Puromycin, Quinidine2, Ramosetron, Ranitidine, Reserpine, Ritonavir, Saquinavir, Somatostain, Sparfloxacin, Talinolol, Taxol, Terfenadine, Trimethoprim, Vecuronium, Verapamil, Vinblastine, Vincristine), while the MRP4/ABCC4 is able to efflux a narrower spectrum (e.g., cAMP, cGMP, Dehydroepiandrosterone-3-sulfate, Estradiol-17b-Dglucuronide, Folate, Methotrexate, Prostaglandin E1, Prostaglandin E2). The breast cancer resistance protein (BCRP/ABCG2) have been reported to be expressed as active efflux drug transporters at the BBB that can display efflux functionality similar to that of P-gp for a wide range of componds (e.g., Azidodeoxythymidine, Bisantrene, Cerivastatin, Doxorubicin, Daunorubicin, Dehydroepiandrosterone-3-sulfate, Etoposide, Estrone-3-sulfate, Estradiol-17b-D-glucuronide, Folate, Flavopiridol, Imatinib mesylate, Mitoxantrone, Methotrexate, Prazosin, Pantoprazole, Pravastatin, Rhodamine 123, Topotecan), for more details readers are directed to see (Ohtsuki & Terasaki, 2007).

Many of these chemicals are important anticancer agents (e.g., Doxorubicin, Vinblastine, Vincristine, Methotrexate, Docetaxel, Etoposide, Idarubicin, and Taxol), thus it is vital to inhibit these efflux machineries to reach suitable concentration in brain during brain tumors chemotherapy. Various compounds were reported as inhibitors for these efflux transportsers such as colchicine, phenothiazines and quinacrine.

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 125

Fig. 4. Carrier-mediated transporters of the BBB (efflux and the influx transport systems) at luminal and basolateral membranes, adapted with permission from (Omidi & Barar, 2012). Astrocytes, neurons and microglial cells are intercommunication with brain capillary endotheilal cells. P-gp: P-glycoprotein. MRP1: multidrug resistance associated protein 1. MRP4: multidrug resistance associated protein 4. MRP5: multidrug resistance associated

protein 5. BCRP: breast cancer resistance protein. Glut1: glucose transporter 1.

anion transporter 2.

**6.3 Endocytosis, transcytosis and exocytosis** 

and probably transcytosis of macromolecules.

MCT1: monocarboxylate transporter 1. LAT1: large neutral amino acids transporter 1. ASCT2: neutral amino acid transporter 2. EAAT: excitatory amino acid transporters. CNT2: centrative nucleoside transport 2. ENT: equilibrative nucleoside transport 2. SERT: serotonin transporter. NET: norepinephrine transporter. CRT: creatine transporter. TAUT: taurine transporter. OATP2: organic anion-transporting polypeptide. OAT3: organic anion transporter 3. OATP1A2: organic anion-transporting polypeptide. OAT2: organic

Cell membranous vesicular machinery domains comprise numerous components including lipid rafts, caveolae and clathrin-coated pits. All of them appear to participate in endocytosis

Caveolae are flask-shaped invaginations of the plasma membrane coated by a 22 kDa structural protein caveolin. Initially detected in endothelial cells, caveolae tend to mediate

#### **6.2 Influx transporters**

BBB influx transporters can be divided into different groups as follows:


Given that the main properties of the BBB that differ the brain capillary endothelia from other blood microvessel because of the presence of restrictive high-resistance tight junctions between blood and brain parenchyma, the BBB forming BCECs almost completely prevents the uptake of potential CNS drugs via the paracellular pathway. Thus compounds passing the BBB almost exclusively have to exploit the transcellular pathway, but this is not always the case since there are increasing evidences that a broad variety of transport machineries are involved, including both carrier-mediated transport and receptor-mediated transcytosis for transporting compounds into the brain (the so called active influx) and multidrug transport pumps for actively effluxing unwanted chemicals out of brain (the so called active efflux). In the case of transport into the brain, numerous systems have been discovered, including transport proteins for amino acids, monocarboxylic acids, organic cations, hexoses, nucleotides and peptides. Several of these proteins have successfully been used in prodrug strategies to enable or at least enhance brain uptake of neurotherapeutic agents. Classic examples are l-dopa and progabide. Of these influx transport machineries, both pyrimidine and purine nucleoside analogs are currently used clinically as anti-metabolite drugs. Cytarabine, an analog of deoxycytidine (1--d-arabinofuranosylcytosine, araC, Cytosar-Us), is used as combination chemotherapy in the treatment of chronic myelogenous, leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphomas; Gemcitabine (dFdC, 2',2'-di.uorodeoxycytidine, Gemzars), a broad spectrum agent, which is used for treatment of a variety of cancers including pancreatic and bladder cancers. Capecitabine (5'-deoxy-5-N-[(pentoxy) carbonyl]-cytidine, Xelodas) is used, as a prodrug, in treatment of metastatic colorectal cancer. Two purine nucleoside anti-metabolite drugs, Fludarabine (9--d-arabinofuranosyl-2-.uoroadenine), and Cladribine (2-chloro-2' deoxyadenosine, CdA, Leustatins) are used for treatment of low-grade lymphomas and chronic lymphocytic leukemia (Omidi & Gumbleton, 2005).

1. the energy transport systems for transport of glucose and mannose (Glut1); lactate, short-chain fatty acids, biotin, salicylic acid and valproic acid (MCT) and creatine

2. the amino acid transport systems such as small (LAT2/4F2hc) and large (LAT1/4F2hc) neutral amino acid transporter systems for transport of neutral amino acids and L-dopa; acidic amino acid transporter for aspartate and glutamate (ASCT2); basic amino acid transporter (BAAT) for arginine and lysine; the -amino acid transporter for -alanine and taurine (TAUT); System A (ATA2) for small neutral amino acids; System

3. the organic anion transport system such as OATP2 for digoxin and organic anions,

5. the peptide transport systems such as oligopeptide transporters (PepT1, PepT2), polypeptid transport systems such as OAT3 for PAH, HVA, indoxyl sulfate; OATP14

6. the neurotransmitter transport systems such as GAT2/BGT1, SERT and NET respectively for transport of -aminobutyric acid, serotonin and norepinephrine, and

Given that the main properties of the BBB that differ the brain capillary endothelia from other blood microvessel because of the presence of restrictive high-resistance tight junctions between blood and brain parenchyma, the BBB forming BCECs almost completely prevents the uptake of potential CNS drugs via the paracellular pathway. Thus compounds passing the BBB almost exclusively have to exploit the transcellular pathway, but this is not always the case since there are increasing evidences that a broad variety of transport machineries are involved, including both carrier-mediated transport and receptor-mediated transcytosis for transporting compounds into the brain (the so called active influx) and multidrug transport pumps for actively effluxing unwanted chemicals out of brain (the so called active efflux). In the case of transport into the brain, numerous systems have been discovered, including transport proteins for amino acids, monocarboxylic acids, organic cations, hexoses, nucleotides and peptides. Several of these proteins have successfully been used in prodrug strategies to enable or at least enhance brain uptake of neurotherapeutic agents. Classic examples are l-dopa and progabide. Of these influx transport machineries, both pyrimidine and purine nucleoside analogs are currently used clinically as anti-metabolite drugs. Cytarabine, an analog of deoxycytidine (1--d-arabinofuranosylcytosine, araC, Cytosar-Us), is used as combination chemotherapy in the treatment of chronic myelogenous, leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphomas; Gemcitabine (dFdC, 2',2'-di.uorodeoxycytidine, Gemzars), a broad spectrum agent, which is used for treatment of a variety of cancers including pancreatic and bladder cancers. Capecitabine (5'-deoxy-5-N-[(pentoxy) carbonyl]-cytidine, Xelodas) is used, as a prodrug, in treatment of metastatic colorectal cancer. Two purine nucleoside anti-metabolite drugs, Fludarabine (9--d-arabinofuranosyl-2-.uoroadenine), and Cladribine (2-chloro-2' deoxyadenosine, CdA, Leustatins) are used for treatment of low-grade lymphomas and

7. the choline transport system for choline and thiamine (Ohtsuki & Terasaki, 2007).

BBB influx transporters can be divided into different groups as follows:

4. the nucleoside transport systems such as ENT2 and CNT2,

chronic lymphocytic leukemia (Omidi & Gumbleton, 2005).

**6.2 Influx transporters** 

ASC/system B0+,

for thyroid hormones,

(CRT),

Fig. 4. Carrier-mediated transporters of the BBB (efflux and the influx transport systems) at luminal and basolateral membranes, adapted with permission from (Omidi & Barar, 2012). Astrocytes, neurons and microglial cells are intercommunication with brain capillary endotheilal cells. P-gp: P-glycoprotein. MRP1: multidrug resistance associated protein 1. MRP4: multidrug resistance associated protein 4. MRP5: multidrug resistance associated protein 5. BCRP: breast cancer resistance protein. Glut1: glucose transporter 1. MCT1: monocarboxylate transporter 1. LAT1: large neutral amino acids transporter 1. ASCT2: neutral amino acid transporter 2. EAAT: excitatory amino acid transporters. CNT2: centrative nucleoside transport 2. ENT: equilibrative nucleoside transport 2. SERT: serotonin transporter. NET: norepinephrine transporter. CRT: creatine transporter. TAUT: taurine transporter. OATP2: organic anion-transporting polypeptide. OAT3: organic anion transporter 3. OATP1A2: organic anion-transporting polypeptide. OAT2: organic anion transporter 2.

#### **6.3 Endocytosis, transcytosis and exocytosis**

Cell membranous vesicular machinery domains comprise numerous components including lipid rafts, caveolae and clathrin-coated pits. All of them appear to participate in endocytosis and probably transcytosis of macromolecules.

Caveolae are flask-shaped invaginations of the plasma membrane coated by a 22 kDa structural protein caveolin. Initially detected in endothelial cells, caveolae tend to mediate

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 127

Basically, an appropriate cell culture model confers a useful platform for not only transcellular and paracellular drug diffusional processes, but also metabolism and active transport processes. Such model can be used for investigating the nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Thus, ideally the *in vitro* BBB cell model should represent a restrictive paracellular barrier functionality, physiologically realistic cell architecture and functional expression of key transporter mechanisms. Such model should also confer ease of culture to meet the technical and time constraints of a screening program. Based upon these facts,

Despite many benefits of immortalized cell based BBB models, the lack of restrictive barrier function has limited their use for permeability screenings and therefore the primary BCECs have been isolated to be used as a cell culture BBB *in vitro* model. The main advantage from pharmaceutical perspective is that these primary models, practically the bovine and porcine models, possess a restrictive paracellular pathway. Nevertheless, BCECs grown in tissue cultures probably lack many of the characteristics of the BBB *in vivo*. In an attempt to improve such models, endothelial/astrocyte and endothelial/pericyte cocultures have been examined, even attempts to generate a 3 dimensionals (3D) model have been made. For instance aortic cells cocultured with astrocytes (C6 cells) in the presence of flow within hollow fiber tube develop a selective barrier with an estimated electrical resistance of 2,900 .cm2 (Stanness et al., 1996). The same group later reported that the previously introduced 3D dynamic *in vitro* BBB model can be successfully used for the coculture of differentiated serotonergic neurons in the presence of a BBB (Stanness et al., 1999). A number of lesscomplicated *in vitro* coculture BBB models have also been established and developed. To produce continuous BBB *in vitro* models, several academic groups have reported the generation of immortalized brain microvessel endothelial cell lines. An immortalized human brain capillary endothelial BB19 cell line was transformed with the E6E7 genes of human papilloma virus and retained their endothelial nature. It was used to study the cytoadherence of Plasmodium falciparum-infected erythrocytes (Prudhomme et al., 1996). Human cerebral microvascular endothelial cells SV- HCEC, transfected with the plasmid pSV3-neo coding for the SV40 large T antigen, was utilized to serve as a human BBB *in vitro* model showing the expression of factor VIII-related antigen, the uptake of acetylated lowdensity lipoprotein, the binding of fluorescently labeled lectins, the expression of transferrin receptor, and high activities of the ALP and γ-GTP (Muruganandam et al., 1997). Immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene displaying the expression of Glut-1 and P-gp. The TR-BBB cell line has also been established using same approach from a transgenic rat (Hosoya et al., 2000). An immortalized rat brain endothelial cell line, RBE4, was immortalized by transfection with a plasmid containing the E1A adenovirus gene (Roux et al., 1994). The RBE4 cell line appears to be most commonly used cell line to be exploited for investigation of different features of BBB, e.g. the transport of insulin, the expression of P-gp, the uptake of L-dopa and the expression of transferrin receptor (Balbuena et al., 2011; Hulsermann et al., 2009; Yu et al., 2007). The MBEC4 cells, established from BALB/c mouse cerebral microvessel endothelial cells, were used to investigate the high-affinity efflux transport system for glutathione conjugates (Homma et al., 1999). The CR3 cells, established by genomic introduction of the immortalizing SV40 large T gene under the control of the human vimentin promoter, displayed endothelial

immortalized cell based BBB models often fail to meet all these essentialities.

the selective uptake of molecules as small as folate to full size proteins such as albumin and lipoproteins. These micro-domains are highly enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins. Caveolae have been implicated in a wide range of cellular functions including transcytosis, receptor-mediated uptake, stabilization of lipid rafts and compartmentalization of a number of signaling events at the cell surface. Several studies have also shown that caveolae-mediated uptake of materials is not limited to macromolecules; in certain cell-types, viruses (e.g. simian virus 40) and even entire bacteria (e.g. specific strains of *E. Coli*) are engulfed and transferred to intracellular compartments in a caveolae-dependent fashion. Clathrin-mediated endocytosis is the most widely studied vesicular membrane internalizing system, and participation of clathrincoated vesicles has also been investigated in terms of receptor-mediated transport in the BBB. Clathrin forms a non-covalently bound triskelion structure composed of three heavy chains (192 kDa each) and three light chains (Omidi & Gumbleton, 2005; Smith & Gumbleton, 2006). Fig. 5 represents schematic illustration of the clathrin coated vesicles (CCV) and its main protein "triskelion".

Fig. 5. TEM micrograph (A) and schematic representation of molecules involved in assembly of the clathrin coated pit (B) and its main protein triskelion (C). TEM: transmission electron microscopy; CCV: clathrin coated vesicle (Omidi & Barar, 2012).

#### **7. BBB cell culture models**

A simple, applicable, and robust *in vitro* cell based BBB model can provide a useful tool for screening of permeability to central nervous system-acting drugs. Thus, in the 1970s, techniques for isolation of brain microvessels were introduced using mechanical or enzymatic homogenization. In the late 1970's, BCECs were plated in tissue culture and this led to the early *in vitro* BBB model establishment (DeBault et al., 1979). The primary BCECs cultures are very well-characterized systems used as *in vitro* models; nevertheless they have the disadvantage of being very laborious and variable and must be repeated often to provide an adequate stock of cells. Therefore various immortalized endothelial cell lines have been developed and examined such as CR3, EC219, RBE4 b.End3, ECV304, and even epithelial cell line MDCK.

the selective uptake of molecules as small as folate to full size proteins such as albumin and lipoproteins. These micro-domains are highly enriched in glycosphingolipids, cholesterol, sphingomyelin, and lipid-anchored membrane proteins. Caveolae have been implicated in a wide range of cellular functions including transcytosis, receptor-mediated uptake, stabilization of lipid rafts and compartmentalization of a number of signaling events at the cell surface. Several studies have also shown that caveolae-mediated uptake of materials is not limited to macromolecules; in certain cell-types, viruses (e.g. simian virus 40) and even entire bacteria (e.g. specific strains of *E. Coli*) are engulfed and transferred to intracellular compartments in a caveolae-dependent fashion. Clathrin-mediated endocytosis is the most widely studied vesicular membrane internalizing system, and participation of clathrincoated vesicles has also been investigated in terms of receptor-mediated transport in the BBB. Clathrin forms a non-covalently bound triskelion structure composed of three heavy chains (192 kDa each) and three light chains (Omidi & Gumbleton, 2005; Smith & Gumbleton, 2006). Fig. 5 represents schematic illustration of the clathrin coated vesicles

Fig. 5. TEM micrograph (A) and schematic representation of molecules involved in assembly of the clathrin coated pit (B) and its main protein triskelion (C). TEM: transmission electron

A simple, applicable, and robust *in vitro* cell based BBB model can provide a useful tool for screening of permeability to central nervous system-acting drugs. Thus, in the 1970s, techniques for isolation of brain microvessels were introduced using mechanical or enzymatic homogenization. In the late 1970's, BCECs were plated in tissue culture and this led to the early *in vitro* BBB model establishment (DeBault et al., 1979). The primary BCECs cultures are very well-characterized systems used as *in vitro* models; nevertheless they have the disadvantage of being very laborious and variable and must be repeated often to provide an adequate stock of cells. Therefore various immortalized endothelial cell lines have been developed and examined such as CR3, EC219, RBE4 b.End3, ECV304, and even

microscopy; CCV: clathrin coated vesicle (Omidi & Barar, 2012).

(CCV) and its main protein "triskelion".

**7. BBB cell culture models** 

epithelial cell line MDCK.

Basically, an appropriate cell culture model confers a useful platform for not only transcellular and paracellular drug diffusional processes, but also metabolism and active transport processes. Such model can be used for investigating the nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Thus, ideally the *in vitro* BBB cell model should represent a restrictive paracellular barrier functionality, physiologically realistic cell architecture and functional expression of key transporter mechanisms. Such model should also confer ease of culture to meet the technical and time constraints of a screening program. Based upon these facts, immortalized cell based BBB models often fail to meet all these essentialities.

Despite many benefits of immortalized cell based BBB models, the lack of restrictive barrier function has limited their use for permeability screenings and therefore the primary BCECs have been isolated to be used as a cell culture BBB *in vitro* model. The main advantage from pharmaceutical perspective is that these primary models, practically the bovine and porcine models, possess a restrictive paracellular pathway. Nevertheless, BCECs grown in tissue cultures probably lack many of the characteristics of the BBB *in vivo*. In an attempt to improve such models, endothelial/astrocyte and endothelial/pericyte cocultures have been examined, even attempts to generate a 3 dimensionals (3D) model have been made. For instance aortic cells cocultured with astrocytes (C6 cells) in the presence of flow within hollow fiber tube develop a selective barrier with an estimated electrical resistance of 2,900 .cm2 (Stanness et al., 1996). The same group later reported that the previously introduced 3D dynamic *in vitro* BBB model can be successfully used for the coculture of differentiated serotonergic neurons in the presence of a BBB (Stanness et al., 1999). A number of lesscomplicated *in vitro* coculture BBB models have also been established and developed. To produce continuous BBB *in vitro* models, several academic groups have reported the generation of immortalized brain microvessel endothelial cell lines. An immortalized human brain capillary endothelial BB19 cell line was transformed with the E6E7 genes of human papilloma virus and retained their endothelial nature. It was used to study the cytoadherence of Plasmodium falciparum-infected erythrocytes (Prudhomme et al., 1996). Human cerebral microvascular endothelial cells SV- HCEC, transfected with the plasmid pSV3-neo coding for the SV40 large T antigen, was utilized to serve as a human BBB *in vitro* model showing the expression of factor VIII-related antigen, the uptake of acetylated lowdensity lipoprotein, the binding of fluorescently labeled lectins, the expression of transferrin receptor, and high activities of the ALP and γ-GTP (Muruganandam et al., 1997). Immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene displaying the expression of Glut-1 and P-gp. The TR-BBB cell line has also been established using same approach from a transgenic rat (Hosoya et al., 2000). An immortalized rat brain endothelial cell line, RBE4, was immortalized by transfection with a plasmid containing the E1A adenovirus gene (Roux et al., 1994). The RBE4 cell line appears to be most commonly used cell line to be exploited for investigation of different features of BBB, e.g. the transport of insulin, the expression of P-gp, the uptake of L-dopa and the expression of transferrin receptor (Balbuena et al., 2011; Hulsermann et al., 2009; Yu et al., 2007). The MBEC4 cells, established from BALB/c mouse cerebral microvessel endothelial cells, were used to investigate the high-affinity efflux transport system for glutathione conjugates (Homma et al., 1999). The CR3 cells, established by genomic introduction of the immortalizing SV40 large T gene under the control of the human vimentin promoter, displayed endothelial

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 129

Passive diffusion involves the movement of drug molecules down a concentration or electrochemical gradient without the expenditure of energy. Firstly, if we consider only the transport of drug molecules across BBB via passive diffusional processes then the overall flux (J) of a drug in one dimension (i.e. the net mass of drug that diffuses through a unit area

*J DK A dC*

Where J is the flux of drug; D is the diffusion coefficient of drug across the cellular barrier; Kp is a global partition coefficient (cell membrane/aqueous fluid); A is the surface area of the barrier available for absorption; x is the thickness of the absorption barrier, and (dC/dx)t is the concentration gradient of drug across the absorption barrier. The negative sign in Equation (2) indicates that diffusion proceeds from high to low concentration and hence the flux is a positive quantity. In fact, the greater this concentration gradient, the greater the rate

drug approximate D.Kp. The processes of drug partitioning with the biomembrane (including partitioning between extracellular fluid and plasma membrane, partitioning between plasma membrane and cell cytosol, and other organelle interactions etc.) and of drug diffusion across the biomembranes (including a range of organelle and macromolecule interactions that will influence the diffusion process) are largely dependent upon the molecular properties of the drug, i.e. molecular size/shape, ionic properties (hydrogen bonding potential, pKa), and hydrophobic properties. Basically, such molecular properties will determine if passive diffusional transport across an epithelial/endothelial barrier involves either a predominantly paracellular (between cells) pathway negotiating a tortuous intercellular route via the aqueous channels formed by the anastomosing tight junctional fibrils between adjacent cells, or predominantly (but not exclusively) transcellular (across the cell) pathway requiring partitioning of drug into the plasma membrane bilayer. In fact, it should be stated that if a drug's molecular properties afford partitioning into biomembranes (i.e. nonionized form of the drug predominates and is of a sufficient hydrophobic nature) then the membrane surface area available for transcellular diffusion will be considerably greater, by many orders of magnitude, than the surface area available for diffusion via the paracellular route. As a corollary, the transcellular diffusion can potentially result in a higher permeability and a higher rate and extent of absorption. For detailed information, reader is directed to see (Omidi &

With respect to in-silico methods, the passive diffusion seems to be well described by physicochemical parameters of the respective solutes (e.g., lipohilicity, H-bond acceptor/donor features, molecular weight, polar surface area, number of rotatable bonds). These descriptors can be simply calculated and are thus a versatile tool for high-throughput in-silico screening of large compound databases. Basically, small hydrophilic molecules

exploit the paracellular pathway to diffuse into organs (Fig. 3).

*dx* (2)

) of an epithelial/endothelial barrier to a given

per unit time) can be described by Equation (2).

of diffusion of a drug across the cell membrane.

The apparent permeability coefficient (

Gumbleton, 2005; Wolburg, 2006)

**8.2 Macromolecular permeation** 

*<sup>p</sup> <sup>t</sup>*

morphological and biochemical characteristics for up to 30 passages. The b.End3 cells was used to study the uptake and efflux of transferrin (Tf) and Fe showing the expression of Tf receptor and suggesting a receptor-mediated endocytosis for the uptake (Lechardeur et al., 1995; Lechardeur & Scherman, 1995). Given that the immortalized continuous cell lines tend not to generate a restrictive barrier property, isolated primary BCECs have been used from a variety of sources including human, bovine, porcine, monkey, rat, canine, and murine. For more details, readers are directed to see following citations (Gumbleton & Audus, 2001; Lacombe et al., 2011; Ribeiro et al., 2010).

#### **8. BBB permeation and in-slico prediction models**

Undoubtedly, the BBB is designed to protect the brain from entering of toxic compounds. As outlined above, the main underlying concept seems to be to force the compounds to take the transcellular route, where nutrients are actively transported into the brain and possibly toxic compounds are expelled via active efflux pumps. Thus, BBB permeation is a multifactorial, complex issue which requires advanced computational methods for proper modeling.

Computational models, in general, exploit two different approaches. The passive diffusioncontrolled permeability is dependent upon the inherent physicochemical characteristics (e.g., logP, solubility and surface area) of the compounds, and basically molecular descriptor based methods are used to generate predictive models. The ligand-receptor (i.e., influx/efflux transporters, or receptor mediated endocytosis) active/facilitated transport can be considered for carrier/receptor mediated trafficking. Given these, predictive in-silico models suitable for both the lead identification and the lead optimization processes should include both categories. The most commonly used type of data appear to be the logBB values that are described as the ratio of the steady-state concentration of a designated compound in the brain to that of the blood which can describe by Equation (1).

$$\text{LogBB} = \text{Log}(\{\text{C}\_{\text{Brain}}\}/\{\text{C}\_{\text{Rload}}\}) \tag{1}$$

The most commonly used in vitro model for BCECs culture is based on Transwell™ system, which consists of a porous membrane support submerged in culture media. This system is normally characterized by two direction diffusion, i.e. apical to basal (A to B) or basal to apical (B to A). Given existence of large number of drug-like compounds (e.g., ChemNavigator), a very small number of molecules have been drawn to carefully monitor the main permeation driving/limiting force (i.e. passive diffusion, active influx or active efflux), however the data available mostly represent both passive and active transport phenomena. For detailed information, reader is directed to see (Wolburg, 2006).

#### **8.1 Passive diffusion**

Having assumed that the rate of drug release from the formulation is not rate-limiting, the absorption of drug molecules across BBB depend upon: 1) the rate of drug dosing which takes into account the administered dose (mass) and the dosing interval (τ ; time), 2) the interactions of drug molecules with circulating biomolecules in blood (protein binding), 3) drug biostability and clearance, 4) the apparent absorption rate constant for the drug (Ka; time-1). Clearly the stability of drug during the absorption process and importantly the intrinsic permeability of the BBB to the drug are critical factors in determining logBB.

morphological and biochemical characteristics for up to 30 passages. The b.End3 cells was used to study the uptake and efflux of transferrin (Tf) and Fe showing the expression of Tf receptor and suggesting a receptor-mediated endocytosis for the uptake (Lechardeur et al., 1995; Lechardeur & Scherman, 1995). Given that the immortalized continuous cell lines tend not to generate a restrictive barrier property, isolated primary BCECs have been used from a variety of sources including human, bovine, porcine, monkey, rat, canine, and murine. For more details, readers are directed to see following citations (Gumbleton & Audus, 2001;

Undoubtedly, the BBB is designed to protect the brain from entering of toxic compounds. As outlined above, the main underlying concept seems to be to force the compounds to take the transcellular route, where nutrients are actively transported into the brain and possibly toxic compounds are expelled via active efflux pumps. Thus, BBB permeation is a multifactorial, complex issue which requires advanced computational methods for proper modeling.

Computational models, in general, exploit two different approaches. The passive diffusioncontrolled permeability is dependent upon the inherent physicochemical characteristics (e.g., logP, solubility and surface area) of the compounds, and basically molecular descriptor based methods are used to generate predictive models. The ligand-receptor (i.e., influx/efflux transporters, or receptor mediated endocytosis) active/facilitated transport can be considered for carrier/receptor mediated trafficking. Given these, predictive in-silico models suitable for both the lead identification and the lead optimization processes should include both categories. The most commonly used type of data appear to be the logBB values that are described as the ratio of the steady-state concentration of a designated

 LogBB=Log([CBrain]/[CBlood]) (1) The most commonly used in vitro model for BCECs culture is based on Transwell™ system, which consists of a porous membrane support submerged in culture media. This system is normally characterized by two direction diffusion, i.e. apical to basal (A to B) or basal to apical (B to A). Given existence of large number of drug-like compounds (e.g., ChemNavigator), a very small number of molecules have been drawn to carefully monitor the main permeation driving/limiting force (i.e. passive diffusion, active influx or active efflux), however the data available mostly represent both passive and active transport

Having assumed that the rate of drug release from the formulation is not rate-limiting, the absorption of drug molecules across BBB depend upon: 1) the rate of drug dosing which takes into account the administered dose (mass) and the dosing interval (τ ; time), 2) the interactions of drug molecules with circulating biomolecules in blood (protein binding), 3) drug biostability and clearance, 4) the apparent absorption rate constant for the drug (Ka; time-1). Clearly the stability of drug during the absorption process and importantly the

intrinsic permeability of the BBB to the drug are critical factors in determining logBB.

compound in the brain to that of the blood which can describe by Equation (1).

phenomena. For detailed information, reader is directed to see (Wolburg, 2006).

Lacombe et al., 2011; Ribeiro et al., 2010).

**8.1 Passive diffusion** 

**8. BBB permeation and in-slico prediction models** 

Passive diffusion involves the movement of drug molecules down a concentration or electrochemical gradient without the expenditure of energy. Firstly, if we consider only the transport of drug molecules across BBB via passive diffusional processes then the overall flux (J) of a drug in one dimension (i.e. the net mass of drug that diffuses through a unit area per unit time) can be described by Equation (2).

$$\mathbf{J} = -\mathbf{D} \cdot \mathbf{K}\_p \cdot \mathbf{A} \cdot \left(\overset{\text{def}}{\bigvee} \mathbf{dx}\right)\_t \tag{2}$$

Where J is the flux of drug; D is the diffusion coefficient of drug across the cellular barrier; Kp is a global partition coefficient (cell membrane/aqueous fluid); A is the surface area of the barrier available for absorption; x is the thickness of the absorption barrier, and (dC/dx)t is the concentration gradient of drug across the absorption barrier. The negative sign in Equation (2) indicates that diffusion proceeds from high to low concentration and hence the flux is a positive quantity. In fact, the greater this concentration gradient, the greater the rate of diffusion of a drug across the cell membrane.

The apparent permeability coefficient () of an epithelial/endothelial barrier to a given drug approximate D.Kp. The processes of drug partitioning with the biomembrane (including partitioning between extracellular fluid and plasma membrane, partitioning between plasma membrane and cell cytosol, and other organelle interactions etc.) and of drug diffusion across the biomembranes (including a range of organelle and macromolecule interactions that will influence the diffusion process) are largely dependent upon the molecular properties of the drug, i.e. molecular size/shape, ionic properties (hydrogen bonding potential, pKa), and hydrophobic properties. Basically, such molecular properties will determine if passive diffusional transport across an epithelial/endothelial barrier involves either a predominantly paracellular (between cells) pathway negotiating a tortuous intercellular route via the aqueous channels formed by the anastomosing tight junctional fibrils between adjacent cells, or predominantly (but not exclusively) transcellular (across the cell) pathway requiring partitioning of drug into the plasma membrane bilayer. In fact, it should be stated that if a drug's molecular properties afford partitioning into biomembranes (i.e. nonionized form of the drug predominates and is of a sufficient hydrophobic nature) then the membrane surface area available for transcellular diffusion will be considerably greater, by many orders of magnitude, than the surface area available for diffusion via the paracellular route. As a corollary, the transcellular diffusion can potentially result in a higher permeability and a higher rate and extent of absorption. For detailed information, reader is directed to see (Omidi & Gumbleton, 2005; Wolburg, 2006)

#### **8.2 Macromolecular permeation**

With respect to in-silico methods, the passive diffusion seems to be well described by physicochemical parameters of the respective solutes (e.g., lipohilicity, H-bond acceptor/donor features, molecular weight, polar surface area, number of rotatable bonds). These descriptors can be simply calculated and are thus a versatile tool for high-throughput in-silico screening of large compound databases. Basically, small hydrophilic molecules exploit the paracellular pathway to diffuse into organs (Fig. 3).

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 131

diffusion coefficient varies little between drugs. Further, the epithelial permeability of biotechnology products, like that of traditional low molecular drugs, are subjected to the extent of degradation occurring within the barrier itself, in which the role of proteases and nucleases will be important for biotechnology drugs, readers are directed to see (Wolburg,

The integrity of the BBB in metastatic cancerous tumors appears to be different from the normal ones. In addition to the various morphological alterations of BBB in metastatic cancerous tumors (e.g., compromised tight junction structure and increases in the perivascular space, fenestrated BCECs, increased number and activity of pinocytic vacuole), the expression of transporters has also been reported to be altered in the microvasculature of the brain tumors. This implies that the BBB is less intact in primary and metastatic brain tumors compared with the normal brain vasculature, and accordingly the pharmacotherapy of brain tumors demands specific strategies. So far, to enhance the amount of therapeutic agent to a brain tumor, a number of strategies have been exploited including: 1) increasing drug plasma concentration (e.g., intraarterial infusion), 2) physicochemical modification to increase drug permeability, 3) design of inactive drug precursors (the so-called prodrugs) that could more easily cross the blood–brain barrier before conversion to a drug with active formulation, and 4) osmotic disruption of the blood–brain barrier using osmotic-disruptive agents such as mannitol (Provenzale et al., 2005). Of these methodologies, liposomal formulations seem to be promising since they can passively target tumors in which there is disorganized vasculature. This appears to be also related to higher permeability than to disorganization *per se*; nonetheless specific features (e.g., vesicle size, chemical affinity, and thermal/pH sensitiveness) may affect the targeting potential of liposomes. The hyperthermia looms to be an important means for modifying the target environment and increasing liposome delivery to tumors. For example, in animal models, it was observed that the heating to temperatures up to 41–43°C can increase the tumor microvascular pore size and accordingly increases its permeability to various substances (e.g., ferritin, antibodies, and liposomes), perhaps hyperthermia can disaggregate the endothelial cell cytoskeleton. For instance, in a human tumor xenograft murine model (SKOV-3), the extravasation of 100 nm liposomes was not observed at 34°C, but it was seen after heating to 40°C (Kong et al., 2001). In 2010, Bellavance et al reported on development of a novel cationic liposome formulation composed of DPPC:DC-Chol:DOPE:DHPE Oregon Green, which was shown to possesses efficient internalization and intracellular delivery to F98 and U-118 glioblastoma (GBM) cells in pH-sensitive manner. At which point, they suggested such liposomal formulation as a novel potent and efficient vehicle for cytosolic delivery of intracellular therapeutics such as chemotherapy agents to the glioblastoma (Bellavance et al., 2010). So far, various liposomal and polymeric formulations have been introduced as smart (thermosensitive and/or pH sensitive) nanomedicines that can provide a controlled drug release in target tumors. In particular, the pH-sensitive nanosystems have been given greater attention since the pH targeting approach is regarded as a more general strategy than conventional specific tumor cell surface targeting approaches. In fact, the nanosystems display greater potential to overcome multidrug resistance of various tumors when they are combined with triggered release mechanisms by endosomal or lysosomal acidity plus endosomolytic capability – this important domain has been well reviewed recently, see (Lee et al., 2008).

2006).

**9. Targeting brain tumours** 

The impact of the molecule's steric, ionic and hydrophobic properties upon passive membrane transport and epithelial/endothelial permeability are equally applicable for peptides, proteins and nucleic acids, as they are for traditional low molecular weight drugs. Nevertheless, their permeation across biological barriers will be limited by the presence of a significant number of hydrogen bond acceptor and donor groups, i.e. requiring considerable desolvation energy for the molecule to leave the aqueous environment and partition into a biological membrane. The diffusion of macromolecules (peptides, proteins and nucleic acids) across biological membranes appears to be through endocytosis/transcytosis route or in some cases via the paracellular pathway (Fig. 3). For example, to pursue the binding, uptake and transcytosis of 60 nm porous nanoparticles (NPs) differing in their surface charge and inner composition, Jallouli et al studied their trafficking at the BBB. Having used maltodextrins with/without a cationic ligand, they showed that the cationic NPs were accumulated mainly around the paracellular area, while neutral NPs were mainly on the cell surface and the dipalmitoyl phosphatidyl glycerol (DPPG) NPs were at both paracellular areas and on the surface of the cells. It was shown that filipin can increase the binding and uptake (sterols may entail in their efflux), while decrease the transcytosis of neutral NPs. They concluded that the neutral NPs, like LDL, exploit the caveolae pathway and suggested the neutral and cationic 60 nm porous NPs as potential candidates for drug delivery to the brain (Jallouli et al., 2007). It is believed that Tf receptor, as a molecular part of vesicular trafficking, can facilitate brain delivery of NPs *in vivo*. To explore the attributed mechanism of this process, Chang et al evaluated the endocytosis of poly(lactic-co-glycolic acid) (PLGA) NPs coated with transferrin using an *in vitro* coculture of BCECs and astrocytes. Using solvent diffusion method, they prepared PLGA NPs by means of DiI as a fluorescent marker and coated with Tween 20, BSA and Tf. Depending upon DiI incorporation and surface coating, the size of NPs varied from 63 to 90 nm. In comparison with BSA NPs, the Tf-NPs were found to be highly adsorbed BCECs through an energy-dependent process. Having used specific inhibition, these researchers showed that the Tf-NPs can interact with BCECs in a specific manner and enter the cells via the caveolae endocytic pathway (Chang et al., 2009). Having used a cross-reacting material 197 (CRM197) which is a non-toxic mutant of diphtheria toxin, Wang et al reported that the apical-to-basal transcytosis of CRM197 can involve the caveolae-mediated pathway in the hCMEC/D3 endothelial cells as the caveolin-1 mRNA and protein expression levels were significantly increased by CRM197. These researchers speculated that the upregulation of caveolin-1 may be mediated via a PI3K/Akt dependent pathway and reduction of the phospho-FOXO1A (forkhead box O) transcription factor. Based upon such findings, it was proposed that carrier protein CRM197-mediated delivery across the BBB is involved in the induction of FOXO1A transcriptional activity and upregulation of caveolin-1 expression (Wang et al., 2010). In fact, the BCECs exploit a variety of endocytic pathways (i.e., clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis) for the internalization of exogenous materials. It is deemed that the properties of drug delivery vehicles can direct the intracellular processing in brain endothelial cells. Using fixed-size NPs, it has been shown that surface modifications of nanoparticles (e.g., charge and protein ligands) can affect their mode of internalization by BCECs and thereby the subcellular fate (Georgieva et al., 2011).

It should be also evoked that the diffusion coefficient of a drug is inversely proportional to its molecular weight, and while for traditional low molecular weight drugs (100-500 Da) the diffusion coefficient varies little between drugs. Further, the epithelial permeability of biotechnology products, like that of traditional low molecular drugs, are subjected to the extent of degradation occurring within the barrier itself, in which the role of proteases and nucleases will be important for biotechnology drugs, readers are directed to see (Wolburg, 2006).

#### **9. Targeting brain tumours**

130 Novel Therapeutic Concepts in Targeting Glioma

The impact of the molecule's steric, ionic and hydrophobic properties upon passive membrane transport and epithelial/endothelial permeability are equally applicable for peptides, proteins and nucleic acids, as they are for traditional low molecular weight drugs. Nevertheless, their permeation across biological barriers will be limited by the presence of a significant number of hydrogen bond acceptor and donor groups, i.e. requiring considerable desolvation energy for the molecule to leave the aqueous environment and partition into a biological membrane. The diffusion of macromolecules (peptides, proteins and nucleic acids) across biological membranes appears to be through endocytosis/transcytosis route or in some cases via the paracellular pathway (Fig. 3). For example, to pursue the binding, uptake and transcytosis of 60 nm porous nanoparticles (NPs) differing in their surface charge and inner composition, Jallouli et al studied their trafficking at the BBB. Having used maltodextrins with/without a cationic ligand, they showed that the cationic NPs were accumulated mainly around the paracellular area, while neutral NPs were mainly on the cell surface and the dipalmitoyl phosphatidyl glycerol (DPPG) NPs were at both paracellular areas and on the surface of the cells. It was shown that filipin can increase the binding and uptake (sterols may entail in their efflux), while decrease the transcytosis of neutral NPs. They concluded that the neutral NPs, like LDL, exploit the caveolae pathway and suggested the neutral and cationic 60 nm porous NPs as potential candidates for drug delivery to the brain (Jallouli et al., 2007). It is believed that Tf receptor, as a molecular part of vesicular trafficking, can facilitate brain delivery of NPs *in vivo*. To explore the attributed mechanism of this process, Chang et al evaluated the endocytosis of poly(lactic-co-glycolic acid) (PLGA) NPs coated with transferrin using an *in vitro* coculture of BCECs and astrocytes. Using solvent diffusion method, they prepared PLGA NPs by means of DiI as a fluorescent marker and coated with Tween 20, BSA and Tf. Depending upon DiI incorporation and surface coating, the size of NPs varied from 63 to 90 nm. In comparison with BSA NPs, the Tf-NPs were found to be highly adsorbed BCECs through an energy-dependent process. Having used specific inhibition, these researchers showed that the Tf-NPs can interact with BCECs in a specific manner and enter the cells via the caveolae endocytic pathway (Chang et al., 2009). Having used a cross-reacting material 197 (CRM197) which is a non-toxic mutant of diphtheria toxin, Wang et al reported that the apical-to-basal transcytosis of CRM197 can involve the caveolae-mediated pathway in the hCMEC/D3 endothelial cells as the caveolin-1 mRNA and protein expression levels were significantly increased by CRM197. These researchers speculated that the upregulation of caveolin-1 may be mediated via a PI3K/Akt dependent pathway and reduction of the phospho-FOXO1A (forkhead box O) transcription factor. Based upon such findings, it was proposed that carrier protein CRM197-mediated delivery across the BBB is involved in the induction of FOXO1A transcriptional activity and upregulation of caveolin-1 expression (Wang et al., 2010). In fact, the BCECs exploit a variety of endocytic pathways (i.e., clathrin-mediated endocytosis, caveolar endocytosis, and macropinocytosis) for the internalization of exogenous materials. It is deemed that the properties of drug delivery vehicles can direct the intracellular processing in brain endothelial cells. Using fixed-size NPs, it has been shown that surface modifications of nanoparticles (e.g., charge and protein ligands) can affect their mode of internalization by

BCECs and thereby the subcellular fate (Georgieva et al., 2011).

It should be also evoked that the diffusion coefficient of a drug is inversely proportional to its molecular weight, and while for traditional low molecular weight drugs (100-500 Da) the The integrity of the BBB in metastatic cancerous tumors appears to be different from the normal ones. In addition to the various morphological alterations of BBB in metastatic cancerous tumors (e.g., compromised tight junction structure and increases in the perivascular space, fenestrated BCECs, increased number and activity of pinocytic vacuole), the expression of transporters has also been reported to be altered in the microvasculature of the brain tumors. This implies that the BBB is less intact in primary and metastatic brain tumors compared with the normal brain vasculature, and accordingly the pharmacotherapy of brain tumors demands specific strategies. So far, to enhance the amount of therapeutic agent to a brain tumor, a number of strategies have been exploited including: 1) increasing drug plasma concentration (e.g., intraarterial infusion), 2) physicochemical modification to increase drug permeability, 3) design of inactive drug precursors (the so-called prodrugs) that could more easily cross the blood–brain barrier before conversion to a drug with active formulation, and 4) osmotic disruption of the blood–brain barrier using osmotic-disruptive agents such as mannitol (Provenzale et al., 2005). Of these methodologies, liposomal formulations seem to be promising since they can passively target tumors in which there is disorganized vasculature. This appears to be also related to higher permeability than to disorganization *per se*; nonetheless specific features (e.g., vesicle size, chemical affinity, and thermal/pH sensitiveness) may affect the targeting potential of liposomes. The hyperthermia looms to be an important means for modifying the target environment and increasing liposome delivery to tumors. For example, in animal models, it was observed that the heating to temperatures up to 41–43°C can increase the tumor microvascular pore size and accordingly increases its permeability to various substances (e.g., ferritin, antibodies, and liposomes), perhaps hyperthermia can disaggregate the endothelial cell cytoskeleton. For instance, in a human tumor xenograft murine model (SKOV-3), the extravasation of 100 nm liposomes was not observed at 34°C, but it was seen after heating to 40°C (Kong et al., 2001). In 2010, Bellavance et al reported on development of a novel cationic liposome formulation composed of DPPC:DC-Chol:DOPE:DHPE Oregon Green, which was shown to possesses efficient internalization and intracellular delivery to F98 and U-118 glioblastoma (GBM) cells in pH-sensitive manner. At which point, they suggested such liposomal formulation as a novel potent and efficient vehicle for cytosolic delivery of intracellular therapeutics such as chemotherapy agents to the glioblastoma (Bellavance et al., 2010). So far, various liposomal and polymeric formulations have been introduced as smart (thermosensitive and/or pH sensitive) nanomedicines that can provide a controlled drug release in target tumors. In particular, the pH-sensitive nanosystems have been given greater attention since the pH targeting approach is regarded as a more general strategy than conventional specific tumor cell surface targeting approaches. In fact, the nanosystems display greater potential to overcome multidrug resistance of various tumors when they are combined with triggered release mechanisms by endosomal or lysosomal acidity plus endosomolytic capability – this important domain has been well reviewed recently, see (Lee et al., 2008).

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 133

the EGFR-expressing rat glioma cell line F98(EGFR), they showed that the bioconjugate retained its affinity for F98(EGFR) cells and the IC50 of the bioconjugate was 220 nmol/L. The bioconjugate in rats bearing i.c. implants of either F98(EGFR) or F98(WT) gliomas was determined 24 hr following convection enhanced delivery of (125)I-labeled complex, showing specific molecular targeting of the tumor. Based on such findings, they concluded that the antibody-drug bioconjugate is therapeutically useful approach in brain tumors (Wu et al., 2006). In 2009, Veiseh et al reported on development of an iron oxide nanoparticle coated with polyethylene glycol-grafted chitosan with ability to cross the BBB and target brain tumors in a genetically engineered mouse model. The nanoprobe was conjugated to a tumor-targeting agent, chlorotoxin, and a near-IR fluorophore. Using *in vivo* magnetic resonance, biophotonic imaging, and histologic and biodistribution analyses, they showed an innocuous toxicity profile induced by the nanoprobe, while it showed a sustained retention in tumors and suggested its application for the diagnosis and treatment of a variety of tumor types in brain (Veiseh et al., 2009). Using novel quaternary ammonium beta-cyclodextrin (QAbetaCD) NPs (with 65-88 nm diameter and controllable cationic properties), Gil et al reported successful delivery of doxorubicin (DOX) across the BBB. They showed that QAbetaCD NPs are not toxic to bovine brain microvessel endothelial cells (BBMVECs) at concentrations up to 500 g/mL. They also showed that the DOX/QAbetaCD complexes can kill U87 cells as effectively as DOX alone, while the QAbetaCD NPs completely protect BBMVECs from cytotoxicity of DOX. And as a result, it was suggested that the QAbetaCD NPs as safe and effective delivery system for anticancer agents such as DOX for brain tumors (Gil et al., 2009). Upon the of note tropism of mesenchymal stem cells (MSCs) for brain tumors, Roger et al exploited the MSCs as NP delivery vehicles, in which they used two types of NPs loaded with coumarin-6, i.e. poly-lactic acid NPs (PLA-NPs) and lipid nanocapsules (LNCs). They showed efficient internalization of the NPs into MSCs that were able to migrate toward an experimental human glioma model. They suggested MSCs as potential cellular carriers for delivery of NPs into brain tumors (Roger et al., 2010). In 2011, A dual-targeting drug carrier (PAMAM-PEG-WGA-Tf) was developed based on the PEGylated fourth generation PAMAM dendrimer with Tf and wheat germ agglutinin (WGA) on the periphery and DOX loaded in the interior (He et al., 2011). Having nanoscaled size (~ 20 nm), the PAMAM-PEG-WGA-Tf efficiently inhibited the growth rate of the C6 glioma cells, while it reduced the cytotoxicity of DOX to the normal cells. These researchers reported significantly increase and accumulation of DOX in the tumor site (due to the targeting effects of both Tf and WGA) and suggested that it could be used as a BBB

penetrating agent with tumor targeting properties (He et al., 2011).

Entry of blood circulating agents into brain is highly controlled by selectively functional presence of BBB. This makes brain drug delivery and targeting very intricate. Owing to unique biology of brain capillary endothelial cells, carrier and/or receptor mediated transport machineries of BBB can be exploited using smart pharmaceuticals. In the case of tumors such as glioma, use of intelligent molecular Trajan horses appears to provide a combined imaging-therapy as "theranostic" to ease brain drug delivery and targeting by simultaneous imaging techniques such as positron emission tomography (PET). In near future, it is expected new multifunctional "all in one" therapeutic to be translated into clinic to cure brain tumors in much more efficient manner. Such therapeutics may consist of homing device for targeting, imaging moiety for sensing/imaging, and therapeutic itself in a

**11. Chapter summary** 

In addition, little is known about impacts of metastatic disease on the BBB. Although the brain metastases respond to chemotherapy modalities, such responses are largely dependent upon condition of patients. For example, 14 patients with brain metastases from small cell lung cancer (SCLC) were treated with combination therapy of cyclophosphamide, doxorubicin, vincristine, and etoposide. Of the treated patients, 9 of 11 patients (82%) showed responses in their brain lesions, whereas 9 of 12 evaluated patients (75%) had responses in their extracranial lesions (Lee et al., 1989). In another study, patients with SCLC and brain metastases were treated with cisplatin, ifosfamide, and irinotecan with rhG-CSF support. The response rate was 50% in brain lesions and 62% in extracranial primary or metastatic lesions (Fujita et al., 2000). The most studies for assessing leakiness of the BBB in human brain tumors appear to be the T1-weighted techniques (in particular, T1-weighted dynamic contrast-enhanced imaging). In fact, researchers implement this methodology using a 3D spoiled gradient acquisition steady-state technique that monitors contrast material accumulation over a few minutes rather than observing the first-pass phenomenon. The main advantages of this technique seem to be the relatively short imaging time, a need for only a single dose of contrast material, and availability of a large number of user-friendly analysis programs (Provenzale et al., 2005).

#### **10. Recent advancements for crossing BBB**

Selective functional presence of BBB makes brain delivery and targeting very challenging issue, for which various strategies have been developed, including different classes of nanomedicines. These novel strategies are largely dependent upon biological characteristics of BBB, and accordingly nanomedicines exploit endocytic pathways. Unfortunately, patients with primary brain tumors and brain metastases have a very poor prognosis. This is often exacerbated with low responses to chemotherapy attributed to BBB selective control on permeation of cytotoxic agents. Nanomedicines represent great promise in glioma therapy as they protect therapeutic agent and allow its sustained release even though tumor specific targeting paradigms with extensive intratumoral distribution must be developed for efficient delivery.

Paclitaxel as an active agent against gliomas and various brain metastases is substrate of Pgp efflux transporter, and thus it is pumped out of brain parenchyma. To tackle this issue, Koziara et al. developed novel cetyl alcohol/polysorbate NPs for encapsulation and delivery of anticancer agents such as paclitaxel (PX) to brain. They showed significant increase of paclitaxel in brain using such NPs because of the limited binding of PX to p-gp (Koziara et al., 2004). With an effort to evaluate the characteristics of an ultrasmall superparamagnetic iron oxides (USPIO) agent in patients with brain tumors and to correlate changes on MRI with histopathologic data collected systematically in all patients, Taschner et al. examined 9 patients with brain tumors before and 24 hr after administration of a USPIO at a dose of 2.6 mg Fe/kg. They witnessed USPIO-related changes of signal intensity in gadolinium-enhancing brain tumors in 7 patients. Upon such findings, they suggested that USPIO agents can offer complementary information useful to differentiate between brain tumors and areas of radiation necrosis (Taschner et al., 2005). Interestingly, in 2006, Wu et al. reported construction of a drug delivery vehicle with ability to target the epidermal growth factor receptor (EGFR) and its mutant isoform EGFRvIII. In their work, the EGFR targeting monoclonal antibody, cetuximab, was covalently linked to a Polyamidoamine (PAMAM) dendrimer containing the cytotoxic drug methotrexate. Using

In addition, little is known about impacts of metastatic disease on the BBB. Although the brain metastases respond to chemotherapy modalities, such responses are largely dependent upon condition of patients. For example, 14 patients with brain metastases from small cell lung cancer (SCLC) were treated with combination therapy of cyclophosphamide, doxorubicin, vincristine, and etoposide. Of the treated patients, 9 of 11 patients (82%) showed responses in their brain lesions, whereas 9 of 12 evaluated patients (75%) had responses in their extracranial lesions (Lee et al., 1989). In another study, patients with SCLC and brain metastases were treated with cisplatin, ifosfamide, and irinotecan with rhG-CSF support. The response rate was 50% in brain lesions and 62% in extracranial primary or metastatic lesions (Fujita et al., 2000). The most studies for assessing leakiness of the BBB in human brain tumors appear to be the T1-weighted techniques (in particular, T1-weighted dynamic contrast-enhanced imaging). In fact, researchers implement this methodology using a 3D spoiled gradient acquisition steady-state technique that monitors contrast material accumulation over a few minutes rather than observing the first-pass phenomenon. The main advantages of this technique seem to be the relatively short imaging time, a need for only a single dose of contrast material, and availability of a large number of user-friendly

Selective functional presence of BBB makes brain delivery and targeting very challenging issue, for which various strategies have been developed, including different classes of nanomedicines. These novel strategies are largely dependent upon biological characteristics of BBB, and accordingly nanomedicines exploit endocytic pathways. Unfortunately, patients with primary brain tumors and brain metastases have a very poor prognosis. This is often exacerbated with low responses to chemotherapy attributed to BBB selective control on permeation of cytotoxic agents. Nanomedicines represent great promise in glioma therapy as they protect therapeutic agent and allow its sustained release even though tumor specific targeting paradigms with extensive intratumoral distribution must be developed for

Paclitaxel as an active agent against gliomas and various brain metastases is substrate of Pgp efflux transporter, and thus it is pumped out of brain parenchyma. To tackle this issue, Koziara et al. developed novel cetyl alcohol/polysorbate NPs for encapsulation and delivery of anticancer agents such as paclitaxel (PX) to brain. They showed significant increase of paclitaxel in brain using such NPs because of the limited binding of PX to p-gp (Koziara et al., 2004). With an effort to evaluate the characteristics of an ultrasmall superparamagnetic iron oxides (USPIO) agent in patients with brain tumors and to correlate changes on MRI with histopathologic data collected systematically in all patients, Taschner et al. examined 9 patients with brain tumors before and 24 hr after administration of a USPIO at a dose of 2.6 mg Fe/kg. They witnessed USPIO-related changes of signal intensity in gadolinium-enhancing brain tumors in 7 patients. Upon such findings, they suggested that USPIO agents can offer complementary information useful to differentiate between brain tumors and areas of radiation necrosis (Taschner et al., 2005). Interestingly, in 2006, Wu et al. reported construction of a drug delivery vehicle with ability to target the epidermal growth factor receptor (EGFR) and its mutant isoform EGFRvIII. In their work, the EGFR targeting monoclonal antibody, cetuximab, was covalently linked to a Polyamidoamine (PAMAM) dendrimer containing the cytotoxic drug methotrexate. Using

analysis programs (Provenzale et al., 2005).

efficient delivery.

**10. Recent advancements for crossing BBB** 

the EGFR-expressing rat glioma cell line F98(EGFR), they showed that the bioconjugate retained its affinity for F98(EGFR) cells and the IC50 of the bioconjugate was 220 nmol/L. The bioconjugate in rats bearing i.c. implants of either F98(EGFR) or F98(WT) gliomas was determined 24 hr following convection enhanced delivery of (125)I-labeled complex, showing specific molecular targeting of the tumor. Based on such findings, they concluded that the antibody-drug bioconjugate is therapeutically useful approach in brain tumors (Wu et al., 2006). In 2009, Veiseh et al reported on development of an iron oxide nanoparticle coated with polyethylene glycol-grafted chitosan with ability to cross the BBB and target brain tumors in a genetically engineered mouse model. The nanoprobe was conjugated to a tumor-targeting agent, chlorotoxin, and a near-IR fluorophore. Using *in vivo* magnetic resonance, biophotonic imaging, and histologic and biodistribution analyses, they showed an innocuous toxicity profile induced by the nanoprobe, while it showed a sustained retention in tumors and suggested its application for the diagnosis and treatment of a variety of tumor types in brain (Veiseh et al., 2009). Using novel quaternary ammonium beta-cyclodextrin (QAbetaCD) NPs (with 65-88 nm diameter and controllable cationic properties), Gil et al reported successful delivery of doxorubicin (DOX) across the BBB. They showed that QAbetaCD NPs are not toxic to bovine brain microvessel endothelial cells (BBMVECs) at concentrations up to 500 g/mL. They also showed that the DOX/QAbetaCD complexes can kill U87 cells as effectively as DOX alone, while the QAbetaCD NPs completely protect BBMVECs from cytotoxicity of DOX. And as a result, it was suggested that the QAbetaCD NPs as safe and effective delivery system for anticancer agents such as DOX for brain tumors (Gil et al., 2009). Upon the of note tropism of mesenchymal stem cells (MSCs) for brain tumors, Roger et al exploited the MSCs as NP delivery vehicles, in which they used two types of NPs loaded with coumarin-6, i.e. poly-lactic acid NPs (PLA-NPs) and lipid nanocapsules (LNCs). They showed efficient internalization of the NPs into MSCs that were able to migrate toward an experimental human glioma model. They suggested MSCs as potential cellular carriers for delivery of NPs into brain tumors (Roger et al., 2010). In 2011, A dual-targeting drug carrier (PAMAM-PEG-WGA-Tf) was developed based on the PEGylated fourth generation PAMAM dendrimer with Tf and wheat germ agglutinin (WGA) on the periphery and DOX loaded in the interior (He et al., 2011). Having nanoscaled size (~ 20 nm), the PAMAM-PEG-WGA-Tf efficiently inhibited the growth rate of the C6 glioma cells, while it reduced the cytotoxicity of DOX to the normal cells. These researchers reported significantly increase and accumulation of DOX in the tumor site (due to the targeting effects of both Tf and WGA) and suggested that it could be used as a BBB penetrating agent with tumor targeting properties (He et al., 2011).

#### **11. Chapter summary**

Entry of blood circulating agents into brain is highly controlled by selectively functional presence of BBB. This makes brain drug delivery and targeting very intricate. Owing to unique biology of brain capillary endothelial cells, carrier and/or receptor mediated transport machineries of BBB can be exploited using smart pharmaceuticals. In the case of tumors such as glioma, use of intelligent molecular Trajan horses appears to provide a combined imaging-therapy as "theranostic" to ease brain drug delivery and targeting by simultaneous imaging techniques such as positron emission tomography (PET). In near future, it is expected new multifunctional "all in one" therapeutic to be translated into clinic to cure brain tumors in much more efficient manner. Such therapeutics may consist of homing device for targeting, imaging moiety for sensing/imaging, and therapeutic itself in a

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 135

Engelhardt, B. (2006). Development of the Blood-Brain Interface, In: *Blood-Brain Barriers,* 

Fujita, A.; Fukuoka, S.; Takabatake, H.; Tagaki, S. & Sekine, K. (2000). Combination

Georgieva, J.V.; Kalicharan, D.; Couraud, P.O.; Romero, I.A.; Weksler, B.; Hoekstra, D. &

Gumbleton, M. & Audus, K.L. (2001). Progress and limitations in the use of in vitro cell

Haseloff, R.F.; Blasig, I.E.; Bauer, H.C. & Bauer, H. (2005). In search of the astrocytic

Hawkins, B.T. & Davis, T.P. (2005). The blood-brain barrier/neurovascular unit in health

He, H.; Li, Y.; Jia, X.R.; Du, J.; Ying, X.; Lu, W.L.; Lou, J.N. & Wei, Y. (2011). PEGylated

Vol.90, No.11, (November 2001), pp. 1681-1698, ISSN 0022-3549

GmbH & Co. KGaA, ISBN 3-527-31088-6, Weinheim

No.4, (November 2000), pp. 291-295, ISSN 0030-2414

1525-7797

0272-4340

(2000), pp. 357-370, ISSN 1061-186X

2009), pp. 610-618, ISSN 1061-186X

6997

Dermietzel, R., Spray, D. C., and Nedergaard, M., pp. 11-40, WILEY-VCH Verlag

chemotherapy of cisplatin, ifosfamide, and irinotecan with rhG-CSF support in patients with brain metastases from non-small cell lung cancer. *Oncology*, Vol.59,

Zuhorn, I.S. (2011). Surface characteristics of nanoparticles determine their intracellular fate in and processing by human blood-brain barrier endothelial cells in vitro. *Mol.Ther.*, Vol.19, No.2, (February 2011), pp. 318-325, ISSN 1525-0016 Gil, E.S.; Li, J.; Xiao, H. & Lowe, T.L. (2009). Quaternary ammonium beta-cyclodextrin

nanoparticles for enhancing doxorubicin permeability across the in vitro bloodbrain barrier. *Biomacromolecules.*, Vol.10, No.3, (March 2009), pp. 505-516, ISSN

cultures to serve as a permeability screen for the blood-brain barrier. *J.Pharm.Sci.*,

factor(s) modulating blood-brain barrier functions in brain capillary endothelial cells in vitro. *Cell Mol.Neurobiol.*, Vol.25, No.1, (February 2005), pp. 25-39, ISSN

and disease. *Pharmacol.Rev.*, Vol.57, No.2, (June 2005), pp. 173-185, ISSN 0031-

Poly(amidoamine) dendrimer-based dual-targeting carrier for treating brain tumors. *Biomaterials*, Vol.32, No.2, (January 2011), pp. 478-487, ISSN 0142-9612 Homma, M.; Suzuki, H.; Kusuhara, H.; Naito, M.; Tsuruo, T. & Sugiyama, Y. (1999). High-

affinity efflux transport system for glutathione conjugates on the luminal membrane of a mouse brain capillary endothelial cell line (MBEC4). *J.Pharmacol.Exp.Ther.*, Vol.288, No.1, (January 1999), pp. 198-203, ISSN 0022-3565 Hosoya, K.I.; Takashima, T.; Tetsuka, K.; Nagura, T.; Ohtsuki, S.; Takanaga, H.; Ueda, M.;

Yanai, N.; Obinata, M. & Terasaki, T. (2000). mRna expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting. *J.Drug Target*, Vol.8, No.6,

apolipoprotein E fragment coupled liposomes by cultured brain microvessel endothelial cells and intact brain capillaries. *J.Drug Target*, Vol.17, No.8, (September

vasoactive agonists and cyclic AMP in a blood-brain barrier model system.

Hulsermann, U.; Hoffmann, M.M.; Massing, U. & Fricker, G. (2009). Uptake of

Hurst, R.D. & Clark, J.B. (1998). Alterations in transendothelial electrical resistance by

*Neurochem.Res.*, Vol.23, No.2, (February 1998), pp. 149-154, ISSN 0364-3190

vehicle that could be activated by outside/inside stimulation (pH, temperature, enzyme). To translate such fascinating molecular therapy into clinical use, however, we need to recruit several disciplines such as nanotechnology, biotechnology, biophotonics, engineering, biopharmaceutics and clinical expertise.

#### **12. References**


vehicle that could be activated by outside/inside stimulation (pH, temperature, enzyme). To translate such fascinating molecular therapy into clinical use, however, we need to recruit several disciplines such as nanotechnology, biotechnology, biophotonics, engineering,

Abbott, N.J. (2000). Inflammatory mediators and modulation of blood-brain barrier

Abbott, N.J. (2005). Dynamics of CNS barriers: evolution, differentiation, and

Abbott, N.J.; Ronnback, L. & Hansson, E. (2006). Astrocyte-endothelial interactions at the

Albert, J.L.; Boyle, J.P.; Roberts, J.A.; Challiss, R.A.; Gubby, S.E. & Boarder, M.R. (1997).

Allt, G. & Lawrenson, J.G. (2000). The blood-nerve barrier: enzymes, transporters and

Allt, G. & Lawrenson, J.G. (2001). Pericytes: cell biology and pathology. *Cells Tissues.Organs*,

Balabanov, R. & Dore-Duffy, P. (1998). Role of the CNS microvascular pericyte in the blood-

Balbuena, P.; Li, W. & Ehrich, M. (2011). Assessments of tight junction proteins

Bellavance, M.A.; Poirier, M.B. & Fortin, D. (2010). Uptake and intracellular release kinetics

Chang, J.; Jallouli, Y.; Kroubi, M.; Yuan, X.B.; Feng, W.; Kang, C.S.; Pu, P.Y. & Betbeder, D.

DeBault, L.E.; Kahn, L.E.; Frommes, S.P. & Cancilla, P.A. (1979). Cerebral microvessels and

Vol.15, No.7, (July 1979), pp. 473-487, ISSN 0073-5655

No.5, (November 1997), pp. 935-941, ISSN 0007-1188

(May 2000), pp. 1-12, ISSN 0361-9230

2010), pp. 251-259, ISSN 0378-5173

292, ISSN 0378-5173

Vol.169, No.1, (2001), pp. 1-11, ISSN 1422-6405

permeability. *Cell Mol.Neurobiol.*, Vol.20, No.2, (April 2000), pp. 131-147, ISSN 0272-

modulation. *Cell Mol.Neurobiol.*, Vol.25, No.1, (February 2005), pp. 5-23, ISSN

blood-brain barrier. *Nat.Rev.Neurosci.*, Vol.7, No.1, (January 2006), pp. 41-53, ISSN

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase. *Br.J.Pharmacol.*, Vol.122,

receptors--a comparison with the blood-brain barrier. *Brain Res.Bull.*, Vol.52, No.1,

brain barrier. *J.Neurosci.Res.*, Vol.53, No.6, (September 1998), pp. 637-644, ISSN

occludin, claudin 5 and scaffold proteins ZO1 and ZO2 in endothelial cells of the rat blood-brain barrier: cellular responses to neurotoxicants malathion and lead acetate. *Neurotoxicology*, Vol.32, No.1, (January 2011), pp. 58-67, ISSN 0161-

of liposome formulations in glioma cells. *Int.J.Pharm.*, Vol.395, No.1-2, (August

(2009). Characterization of endocytosis of transferrin-coated PLGA nanoparticles by the blood-brain barrier. *Int.J.Pharm.*, Vol.379, No.2, (September 2009), pp. 285-

derived cells in tissue culture: isolation and preliminary characterization. *In Vitro*,

biopharmaceutics and clinical expertise.

**12. References** 

4340

0272-4340

1471-003X

0360-4012

813X


Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 137

Leggas, M.; Adachi, M.; Scheffer, G.L.; Sun, D.; Wielinga, P.; Du, G.; Mercer, K.E.; Zhuang,

Ohtsuki, S. & Terasaki, T. (2007). Contribution of carrier-mediated transport systems to the

Omidi, Y. & Barar, J. (2012). Impacts of blood-brain barrier in drug delivery and targeting of brain tumours. *BioImpacts*, Vol.2, No.1, (2012), pp. in press, ISSN 2228-5652 Omidi, Y.; Barar, J.; Ahmadian, S.; Heidari, H.R. & Gumbleton, M. (2008). Characterisation

Omidi, Y. and Gumbleton, M. (2005). Biological Membranes and Barriers, In: *Biomaterials for* 

Orte, C.; Lawrenson, J.G.; Finn, T.M.; Reid, A.R. & Allt, G. (1999). A comparison of blood-

Pardridge, W.M. (1999). Blood-brain barrier biology and methodology. *J.Neurovirol.*, Vol.5,

Pluta, R.; Misicka, A.; Barcikowska, M.; Spisacka, S.; Lipkowski, A.W. & Januszewski, S.

Prudhomme, J.G.; Sherman, I.W.; Land, K.M.; Moses, A.V.; Stenglein, S. & Nelson, J.A.

Ramsauer, M.; Krause, D. & Dermietzel, R. (2002). Angiogenesis of the blood-brain barrier in

Reese, T.S. & Karnovsky, M.J. (1967). Fine structural localization of a blood-brain barrier to

barrier. *Acta Neurochir.Suppl*, Vol.76 (2000), pp. 73-77, ISSN 0001-6268 Provenzale, J.M.; Mukundan, S. & Dewhirst, M. (2005). The role of blood-brain barrier

Vol.185, No.3, (September 2005), pp. 763-767, ISSN 0361-803X

(November 1997), pp. 1187-1197, ISSN 0892-6638

(September 2007), pp. 1745-1758, ISSN 0253-6269

Press, ISBN 9-780-84932334-8, New York

647-655, ISSN 0020-7519

9525

pp. 1274-1276, ISSN 0892-6638

Vol.199, No.6, (June 1999), pp. 509-517, ISSN 0340-2061

No.6, (December 1999), pp. 556-569, ISSN 1355-0284

6484

Y.; Panetta, J.C.; Johnston, B.; Scheper, R.J.; Stewart, C.F. & Schuetz, J.D. (2004). Mrp4 confers resistance to topotecan and protects the brain from chemotherapy. *Mol.Cell Biol.*, Vol.24, No.17, (September 2004), pp. 7612-7621, ISSN 0270-7306 Muruganandam, A.; Herx, L.M.; Monette, R.; Durkin, J.P. & Stanimirovic, D.B. (1997).

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier. *FASEB J.*, Vol.11, No.13,

blood-brain barrier as a supporting and protecting interface for the brain; importance for CNS drug discovery and development. *Pharm.Res.*, Vol.24, No.9,

and astrocytic modulation of system L transporters in brain microvasculature endothelial cells. *Cell Biochem.Funct.*, Vol.26, No.3, (2008), pp. 381-391, ISSN 0263-

*Delivery and Targeting of Proteins Nucleic Acids,* Mahato, R. I., pp. 232-274, CRC

brain barrier and blood-nerve barrier endothelial cell markers. *Anat.Embryol.(Berl)*,

(2000). Possible reverse transport of beta-amyloid peptide across the blood-brain

permeability in brain tumor imaging and therapeutics. *AJR Am.J.Roentgenol.*,

(1996). Studies of Plasmodium falciparum cytoadherence using immortalized human brain capillary endothelial cells. *Int.J.Parasitol.*, Vol.26, No.6, (June 1996), pp.

vitro and the function of cerebral pericytes. *FASEB J.*, Vol.16, No.10, (August 2002),

exogenous peroxidase. *J.Cell Biol.*, Vol.34, No.1, (July 1967), pp. 207-217, ISSN 0021-


Igarashi, Y.; Utsumi, H.; Chiba, H.; Yamada-Sasamori, Y.; Tobioka, H.; Kamimura, Y.;

Jallouli, Y.; Paillard, A.; Chang, J.; Sevin, E. & Betbeder, D. (2007). Influence of surface charge

*Int.J.Pharm.*, Vol.344, No.1-2, (November 2007), pp. 103-109, ISSN 0378-5173 Kniesel, U. & Wolburg, H. (2000). Tight junctions of the blood-brain barrier. *Cell Mol.Neurobiol.*, Vol.20, No.1, (February 2000), pp. 57-76, ISSN 0272-4340 Kondo, A.; Inoue, T.; Nagara, H.; Tateishi, J. & Fukui, M. (1987). Neurotoxicity of

Kong, G.; Braun, R.D. & Dewhirst, M.W. (2001). Characterization of the effect of

Koziara, J.M.; Lockman, P.R.; Allen, D.D. & Mumper, R.J. (2004). Paclitaxel nanoparticles for

Krizbai, I.A. & Deli, M.A. (2003). Signalling pathways regulating the tight junction

Lacombe, O.; Videau, O.; Chevillon, D.; Guyot, A.C.; Contreras, C.; Blondel, S.; Nicolas, L.;

Larson, D.M.; Carson, M.P. & Haudenschild, C.C. (1987). Junctional transfer of small

*Microvasc.Res.*, Vol.34, No.2, (September 1987), pp. 184-199, ISSN 0026-2862 Lechardeur, D. & Scherman, D. (1995). Functional expression of the P-glycoprotein mdr in

Lechardeur, D.; Schwartz, B.; Paulin, D. & Scherman, D. (1995). Induction of blood-brain

Lee, E.S.; Gao, Z. & Bae, Y.H. (2008). Recent progress in tumor pH targeting

Lee, J.S.; Murphy, W.K.; Glisson, B.S.; Dhingra, H.M.; Holoye, P.Y. & Hong, W.K. (1989).

Vol.61, No.7, (April 2001), pp. 3027-3032, ISSN 0008-5472

Vol.11, No.5, (October 1995), pp. 283-293, ISSN 0742-2091

Vol.220, No.1, (September 1995), pp. 161-170, ISSN 0014-4827

pp. 108-112, ISSN 0006-291X

2004), pp. 259-269, ISSN 0168-3659

(February 2003), pp. 23-31, ISSN 0145-5680

*Mol.Pharm.* (March 2011), ISSN 1543-8384

Vol.7, No.7, (July 1989), pp. 916-922,

0006-8993

ISSN 0168-3659

Furuuchi, K.; Kokai, Y.; Nakagawa, T.; Mori, M. & Sawada, N. (1999). Glial cell linederived neurotrophic factor induces barrier function of endothelial cells forming the blood-brain barrier. *Biochem.Biophys.Res.Commun.*, Vol.261, No.1, (July 1999),

and inner composition of porous nanoparticles to cross blood-brain barrier in vitro.

adriamycin passed through the transiently disrupted blood-brain barrier by mannitol in the rat brain. *Brain Res.*, Vol.412, No.1, (May 1987), pp. 73-83, ISSN

hyperthermia on nanoparticle extravasation from tumor vasculature. *Cancer Res.*,

the potential treatment of brain tumors. *J.Control Release*, Vol.99, No.2, (September

permeability in the blood-brain barrier. *Cell Mol.Biol.(Noisy.-le-grand)*, Vol.49, No.1,

Ghettas, A.; Benech, H.; Thevenot, E.; Pruvost, A.; Bolze, S.; Kraczkowski, L.; Prevost, C. & Mabondzo, A. (2011). In-Vitro Primary Human and Animal Cell-Based Blood-Brain Barrier Models as a Screening Tool in Drug Discovery.

molecules in cultured bovine brain microvascular endothelial cells and pericytes.

primary cultures of bovine cerebral capillary endothelial cells. *Cell Biol.Toxicol.*,

barrier differentiation in a rat brain-derived endothelial cell line. *Exp.Cell Res.*,

nanotechnology. *J.Control Release*, Vol.132, No.3, (December 2008), pp. 164-170,

Primary chemotherapy of brain metastasis in small-cell lung cancer. *J.Clin.Oncol.*,


Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors 139

Taschner, C.A.; Wetzel, S.G.; Tolnay, M.; Froehlich, J.; Merlo, A. & Radue, E.W. (2005).

Tilling, T.; Korte, D.; Hoheisel, D. & Galla, H.J. (1998). Basement membrane proteins

Tio, S.; Deenen, M. & Marani, E. (1990). Astrocyte-mediated induction of alkaline

Tsuji, A. (2005). Small molecular drug transfer across the blood-brain barrier via carrier-

Urquhart, B.L. & Kim, R.B. (2009). Blood-brain barrier transporters and response to CNS-

Veiseh, O.; Sun, C.; Fang, C.; Bhattarai, N.; Gunn, J.; Kievit, F.; Du, K.; Pullar, B.; Lee, D.;

Wakui, S.; Furusato, M.; Hasumura, M.; Hori, M.; Takahashi, H.; Kano, Y. & Ushigome, S.

Wang, P.; Xue, Y.; Shang, X. & Liu, Y. (2010). Diphtheria toxin mutant CRM197-mediated

Wang, W.; Dentler, W.L. & Borchardt, R.T. (2001). VEGF increases BMEC monolayer

Wolburg, H. (2006). The Endothelial Frontier, In: *Blood-Brain Barriers,* Dermietzel, R., Spray,

Wolburg, H.; Neuhaus, J.; Kniesel, U.; Krauss, B.; Schmid, E.M.; Ocalan, M.; Farrell, C. &

*Eur.J.Morphol.*, Vol.28, No.2-4, (1990), pp. 289-300, ISSN 0924-3860

No.3, (September 1998), pp. 1151-1157, ISSN 0022-3042

Vol.38, No.2, (1989), pp. 136-142, ISSN 0022-0744

Vol.5, No.1, (January 2006), pp. 52-59, ISSN 1535-7163

(July 2010), pp. 717-725, ISSN 1044-7431

ISBN 3-527-31088-6, Weinheim

0361-803X

1545-5343

ISSN 0031-6970

ISSN 0008-5472

0363-6135

Characteristics of ultrasmall superparamagnetic iron oxides in patients with brain tumors. *AJR Am.J.Roentgenol.*, Vol.185, No.6, (December 2005), pp. 1477-1486, ISSN

influence brain capillary endothelial barrier function in vitro. *J.Neurochem.*, Vol.71,

phosphatase activity in human umbilical cord vein endothelium: an in vitro model.

mediated transport systems. *NeuroRx.*, Vol.2, No.1, (January 2005), pp. 54-62, ISSN

active drugs. *Eur.J.Clin.Pharmacol.*, Vol.65, No.11, (November 2009), pp. 1063-1070,

Ellenbogen, R.G.; Olson, J. & Zhang, M. (2009). Specific targeting of brain tumors with an optical/magnetic resonance imaging nanoprobe across the blood-brain barrier. *Cancer Res.*, Vol.69, No.15, (August 2009), pp. 6200-6207,

(1989). Two- and three-dimensional ultrastructure of endothelium and pericyte interdigitations in capillary of human granulation tissue. *J.Electron Microsc.(Tokyo)*,

transcytosis across blood-brain barrier in vitro. *Cell Mol.Neurobiol.*, Vol.30, No.5,

permeability by affecting occludin expression and tight junction assembly. *Am.J.Physiol Heart Circ.Physiol*, Vol.280, No.1, (January 2001), pp. H434-H440, ISSN

D. C., and Nedergaard, M., pp. 77-108, WILEY-VCH Verlag GmbH & Co. KGaA,

Risau, W. (1994). Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes. *J.Cell Sci.*, Vol.107 ( Pt 5) (May 1994), pp. 1347-1357, ISSN 0021-9533 Wu, G.; Barth, R.F.; Yang, W.; Kawabata, S.; Zhang, L. & Green-Church, K. (2006). Targeted

delivery of methotrexate to epidermal growth factor receptor-positive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates. *Mol.Cancer Ther.*,


Ribeiro, M.M.; Castanho, M.A. & Serrano, I. (2010). In vitro blood-brain barrier models--

Robert, A.M. & Robert, L. (1998). Extracellular matrix and blood-brain barrier function. *Pathol.Biol.(Paris)*, Vol.46, No.7, (September 1998), pp. 535-542, ISSN 0369-8114 Roger, M.; Clavreul, A.; Venier-Julienne, M.C.; Passirani, C.; Sindji, L.; Schiller, P.; Montero-

Roux, F.; Durieu-Trautmann, O.; Chaverot, N.; Claire, M.; Mailly, P.; Bourre, J.M.;

Rubin, L.L. & Staddon, J.M. (1999). The cell biology of the blood-brain barrier.

Schinkel, A.H.; Mol, C.A.; Wagenaar, E.; van, D.L.; Smit, J.J. & Borst, P. (1995a). Multidrug

Schinkel, A.H.; Wagenaar, E.; van, D.L.; Mol, C.A. & Borst, P. (1995b). Absence of the mdr1a

Shivers, R.R.; Arthur, F.E. & Bowman, P.D. (1988). Induction of gap junctions and brain

Smith, M.W. & Gumbleton, M. (2006). Endocytosis at the blood-brain barrier: from basic

Stanness, K.A.; Guatteo, E. & Janigro, D. (1996). A dynamic model of the blood-brain barrier "in vitro". *Neurotoxicology*, Vol.17, No.2, (1996), pp. 481-496, ISSN 0161-813X Stanness, K.A.; Neumaier, J.F.; Sexton, T.J.; Grant, G.A.; Emmi, A.; Maris, D.O. & Janigro, D.

Stewart, P.A. & Wiley, M.J. (1981). Developing nervous tissue induces formation of blood-

*Annu.Rev.Neurosci.*, Vol.22 (1999), pp. 11-28, ISSN 0147-006X

No.7-8, (July 1995a), pp. 1295-1298, ISSN 0959-8049

1995b), pp. 1698-1705, ISSN 0021-9738

Vol.768, No.1-2, (September 1997), pp. 10-18, ISSN 0006-8993

2010), pp. 8393-8401, ISSN 0142-9612

113, ISSN 0021-9541

14, ISSN 1122-9497

0012-1606

pp. 191-214, ISSN 1061-186X

1999), pp. 3725-3731, ISSN 0959-4965

latest advances and therapeutic applications in a chronological perspective. *Mini.Rev.Med.Chem.*, Vol.10, No.3, (March 2010), pp. 262-270, ISSN 1389-5575 Rist, R.J.; Romero, I.A.; Chan, M.W.; Couraud, P.O.; Roux, F. & Abbott, N.J. (1997). F-actin

cytoskeleton and sucrose permeability of immortalised rat brain microvascular endothelial cell monolayers: effects of cyclic AMP and astrocytic factors. *Brain Res.*,

Menei, C. & Menei, P. (2010). Mesenchymal stem cells as cellular vehicles for delivery of nanoparticles to brain tumors. *Biomaterials*, Vol.31, No.32, (November

Strosberg, A.D. & Couraud, P.O. (1994). Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. *J.Cell Physiol*, Vol.159, No.1, (April 1994), pp. 101-

resistance and the role of P-glycoprotein knockout mice. *Eur.J.Cancer*, Vol.31A,

P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. *J.Clin.Invest*, Vol.96, No.4, (October

endothelium-like tight junctions in cultured bovine endothelial cells: local control of cell specialization. *J.Submicrosc.Cytol.Pathol.*, Vol.20, No.1, (January 1988), pp. 1-

understanding to drug delivery strategies. *J.Drug Target*, Vol.14, No.4, (May 2006),

(1999). A new model of the blood--brain barrier: co-culture of neuronal, endothelial and glial cells under dynamic conditions. *Neuroreport*, Vol.10, No.18, (December

brain barrier characteristics in invading endothelial cells: a study using quail--chick transplantation chimeras. *Dev.Biol.*, Vol.84, No.1, (May 1981), pp. 183-192, ISSN


**Part 6** 

**Gene Therapy of Glioma** 

Yu, C.; Kastin, A.J.; Tu, H.; Waters, S. & Pan, W. (2007). TNF activates P-glycoprotein in cerebral microvascular endothelial cells. *Cell Physiol Biochem.*, Vol.20, No.6, (2007), pp. 853-858, ISSN 1015-8987

**Part 6** 

**Gene Therapy of Glioma** 

140 Novel Therapeutic Concepts in Targeting Glioma

Yu, C.; Kastin, A.J.; Tu, H.; Waters, S. & Pan, W. (2007). TNF activates P-glycoprotein in

pp. 853-858, ISSN 1015-8987

cerebral microvascular endothelial cells. *Cell Physiol Biochem.*, Vol.20, No.6, (2007),

**8** 

*Spain* 

**Glioma-Parvovirus Interactions:** 

Jon Gil-Ranedo, Marina Mendiburu-Eliçabe, Marta Izquierdo and José M. Almendral *Centro de Biología Molecular "Severo Ochoa"* 

*Consejo Superior de Investigaciones Científicas (CSIC)* 

*Departamento de Biología Molecular, Cantoblanco, Madrid* 

*Universidad Autónoma de Madrid (UAM)* 

**Molecular Insights and Therapeutic Potential** 

Brain tumours remain one of the most devastating diseases of modern medicine. Although they only represent approximately 1.9% of primary tumours in Europe, their mortality is around 70% and they are within the group of the 10 cancer types causing the highest yearly mortality rate. Gliomas are malignancies of neuroepithelial origin and represent 40-60% of brain tumours. In particular, glioblastoma multiforme (GBM, astrocytic tumours of type IV) is the most aggressive and frequent of primary brain tumours, representing 60% of gliomas. Despite clinical practice advances, the mean survival time of GBM patients has not improved significantly within the last few decades, and it remains around 12-15 months. Current standard of care includes maximal safe surgical resection, and a combination of radio- and chemotherapy with concomitant and adjuvant temozolomide or carmustine wafers (Wen and Kesari 2008). At the moment, the clinical improvement reached is modest, with a 5-year survival rate of less than 5% (Mangiola et al. 2010). The poor results obtained with conventional therapies may be explained by their relatively unspecific nature (Newton 2010), the inefficient delivery of many drugs to the tumoral tissue due to the blood-brain and blood-tumour barriers, as well as by the intrinsic radio- and chemo-resistance of GBM

In light of the limitations of conventional treatment strategies, the necessity of new approaches that would be more effective against GBM became evident. The current understanding of the molecular biology of GBM has set researchers on the path of more targeted and specific therapies exploiting the molecular properties of the tumour. Most targeted agents are tyrosine kinase inhibitors, or monoclonal antibodies directed against either cell surface growth factor receptors or intercellular signaling molecules (angiogenesis) (Van Meir et al. 2010). The overall experience of the monotherapy with targeted agents has shown limited efficacy, with response rates of less than 10-15% and no prolongation of survival (Clarke et al. 2010; Van Meir et al. 2010). Other promising therapies for GBM are also currently being investigated, including combined therapy with targeted agents, immunotherapy, gene therapy, or oncolytic virotherapy (Clarke et al. 2010; Van Meir et al.

**1. Introduction** 

(Newton 2010).

2010).

## **Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential**

Jon Gil-Ranedo, Marina Mendiburu-Eliçabe,

Marta Izquierdo and José M. Almendral

*Centro de Biología Molecular "Severo Ochoa"* 

*Consejo Superior de Investigaciones Científicas (CSIC)* 

*Universidad Autónoma de Madrid (UAM)* 

*Departamento de Biología Molecular, Cantoblanco, Madrid* 

*Spain* 

#### **1. Introduction**

Brain tumours remain one of the most devastating diseases of modern medicine. Although they only represent approximately 1.9% of primary tumours in Europe, their mortality is around 70% and they are within the group of the 10 cancer types causing the highest yearly mortality rate. Gliomas are malignancies of neuroepithelial origin and represent 40-60% of brain tumours. In particular, glioblastoma multiforme (GBM, astrocytic tumours of type IV) is the most aggressive and frequent of primary brain tumours, representing 60% of gliomas. Despite clinical practice advances, the mean survival time of GBM patients has not improved significantly within the last few decades, and it remains around 12-15 months. Current standard of care includes maximal safe surgical resection, and a combination of radio- and chemotherapy with concomitant and adjuvant temozolomide or carmustine wafers (Wen and Kesari 2008). At the moment, the clinical improvement reached is modest, with a 5-year survival rate of less than 5% (Mangiola et al. 2010). The poor results obtained with conventional therapies may be explained by their relatively unspecific nature (Newton 2010), the inefficient delivery of many drugs to the tumoral tissue due to the blood-brain and blood-tumour barriers, as well as by the intrinsic radio- and chemo-resistance of GBM (Newton 2010).

In light of the limitations of conventional treatment strategies, the necessity of new approaches that would be more effective against GBM became evident. The current understanding of the molecular biology of GBM has set researchers on the path of more targeted and specific therapies exploiting the molecular properties of the tumour. Most targeted agents are tyrosine kinase inhibitors, or monoclonal antibodies directed against either cell surface growth factor receptors or intercellular signaling molecules (angiogenesis) (Van Meir et al. 2010). The overall experience of the monotherapy with targeted agents has shown limited efficacy, with response rates of less than 10-15% and no prolongation of survival (Clarke et al. 2010; Van Meir et al. 2010). Other promising therapies for GBM are also currently being investigated, including combined therapy with targeted agents, immunotherapy, gene therapy, or oncolytic virotherapy (Clarke et al. 2010; Van Meir et al. 2010).

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 145

(only 5 Kb), the use of alternate splicing, extensive postranslational modifications and proteolytic processings, maximize the coding capacity of the parvovirus genome. The two NS polypeptides play multiple roles in virus life cycle. The smaller NS2 protein (28 kDa) contains three isoforms arising from alternate splicings that can bind several cellular proteins and shuttle from the nucleus to the cytoplasm via the CRM1 export pathway. Functions assigned to NS2 include assisting capsid assembly, messenger translation, DNA replication, and virus production in a cell type specific manner. The larger NS1 (82 kDa), is a multifunctional nuclear phosphoprotein, highly toxic for most cells, and performing crucial

activities in the MVM unique rolling-hairpin mode of DNA synthesis (see below).

Fig. 1. Outline of the *Parvoviridae*. (A) Taxonomic structure of the *Parvoviridae* (from Tijssen

Many of the members of the *Parvoviridae* were initially isolated from tumours or from transformed cell lines in culture, corresponding with the requirement of these viruses for multiple functions provided by proliferative cells. This unique biological feature, together with requirements for diverse factors that are linked to the neoplastic growth, account for the capacity of the parvoviruses to infect and lyse preferentially cells transformed by a high diversity of physico-chemical and biological agents (Mousset and Rommelaere 1982; Cornelis et al. 1988), and to interfere with tumour formation in animal models (Toolan and Ledinko 1965; Dupressoir et al. 1989; reviewed in Rommelaere et al. 2010). These studies validated, at least for some parvoviruses, many of the requirements that oncolytic viruses should fulfill to be used in the clinic, such as oncotropism, no genomic integration, low

This review is focused on the interactions of glioma cells with two parvoviruses which oncolytic properties have been best evaluated, the rat parvovirus H-1, and the p and i *wildtype* strains of the mouse parvovirus Minute Virus of Mice (MVM). Over the past decade, the characteristics of the infection of these viruses in rodent and human glioma cell lines, primary human glioblastoma cultures, and preclinical xenotransplanted animal models,

et al. 2011, *in press*). (B) Organization of the MVM genome. The position of the two promoters (P4, P38) are designated by arrows. Splicing sites, and virus coded main nonstructural (NS1, NS2), and structural (VP1, VP2) polypeptides are illustrated in their

respective reading frame (based on Cotmore and Tattersall 1987).

toxicity of structural components, or apathogenicity for humans.

**2.2 Parvoviruses as oncolytic agents** 

have been extensively studied (see Table 1).

Virus-mediated therapy, or virotherapy, is emerging as a promising biological approach to complement or potentiate physical and chemical anti-cancer conventional treatments (reviewed in Eager and Nemunaitis 2011). The increasing knowledge on molecular mechanisms underlying cancer development, and on the host-virus interphase regulating viral infections, is allowing the rational design of virotherapies against some human tumours. Ideally, the infection of a clinically competent oncolytic virus candidate should specifically target, replicate in, and destroy the tumour cells, but sparing surrounding nontransformed cells. Multiple restrictions and uncertainties operating at different levels, such as at cellular (specificity of tumour markers in non-transformed cells, tumour microenvironment), anatomical (accessibility, vascular barriers), or physiological (innate and specific immune responses) levels, constitute important challenges against effective virotherapy. In spite of them, intensive research efforts being conducted in many laboratories are developing or using RNA and DNA oncolytic viruses for glioma therapy (see other chapters of this book). Indeed, clinical trials were performed (Haseley et al. 2009; Kroeger et al. 2010) or are currently in progress against glioblastoma using different virotherapeutic approaches. For the first time H-1, a member of the *Parvoviridae*, is within the ongoing clinical trial protocols (http://clinicaltrials.gov/). Some general features of this virus family, as well as molecular bases of the anti-glioma activities of the parvoviruses, are reviewed below.

#### **2. The Parvoviruses: General features and anti-cancer properties**

#### **2.1 Parvovirus capsid structure and genome organization**

The *Parvoviridae* is a family of spherical, non-enveloped icosahedral viruses (Berns and Parrish 2007). The number of viruses being identified as members of this family is rapidly increasing in the recent years. Current classification from the International Committee of Taxonomy of Viruses (ICTV) for the *Parvoviridae* includes two subfamilies and nine geni (see Figure 1A). The parvoviral capsids are ~260 Å in diameter, and encapsidate a ssDNA genome of ~5000 nucleotides. The number of capsid protein species per virion varies among parvoviruses, but are generally composed by three polypeptides, VP1, VP2 and VP3 (molecular weights in the range of 60-84 kDa). Capsids contain 60 copies (in total) of VP protein subunits in a T=1 icosahedral capsid arrangement. The three-dimensional structure of the capsid, which has been determined to high resolution by X-ray crystallography for many members (Tsao et al. 1991; reviewed in Chapman and Agbandje-McKenna 2006), shows a conserved overall topology of eight-stranded antiparallel beta-barrel core motif that forms the contiguous capsid shell, and large loop insertions between the beta-strands. The surface features include small protrusions (spike-like) commonly at the icosahedral threefold axes, and depressions that may be located at the icosahedral two-fold (dimple-like) and/or around the five-fold (canyon-like) axes.

Virus members of the autonomously replicating Parvovirus genus infect mammals and have a wide range of natural hosts, including humans, monkeys, dogs, livestock, felines and rodents. A main molecular model of this genus is the mouse parvovirus Minute Virus of Mice (MVM), which genome is organized as two overlapping transcription units (see Figure 1B) timely regulated (Clemens and Pintel 1988). The left-hand gene driven by the P4 promoter encodes the NS1 and NS2 nonstructural proteins, and the right-hand gene driven by the P38 promoter encodes the VP1 and VP2 structural proteins. In spite of its small size

Virus-mediated therapy, or virotherapy, is emerging as a promising biological approach to complement or potentiate physical and chemical anti-cancer conventional treatments (reviewed in Eager and Nemunaitis 2011). The increasing knowledge on molecular mechanisms underlying cancer development, and on the host-virus interphase regulating viral infections, is allowing the rational design of virotherapies against some human tumours. Ideally, the infection of a clinically competent oncolytic virus candidate should specifically target, replicate in, and destroy the tumour cells, but sparing surrounding nontransformed cells. Multiple restrictions and uncertainties operating at different levels, such as at cellular (specificity of tumour markers in non-transformed cells, tumour microenvironment), anatomical (accessibility, vascular barriers), or physiological (innate and specific immune responses) levels, constitute important challenges against effective virotherapy. In spite of them, intensive research efforts being conducted in many laboratories are developing or using RNA and DNA oncolytic viruses for glioma therapy (see other chapters of this book). Indeed, clinical trials were performed (Haseley et al. 2009; Kroeger et al. 2010) or are currently in progress against glioblastoma using different virotherapeutic approaches. For the first time H-1, a member of the *Parvoviridae*, is within the ongoing clinical trial protocols (http://clinicaltrials.gov/). Some general features of this virus family, as well as molecular bases of the anti-glioma activities of the parvoviruses, are

**2. The Parvoviruses: General features and anti-cancer properties** 

The *Parvoviridae* is a family of spherical, non-enveloped icosahedral viruses (Berns and Parrish 2007). The number of viruses being identified as members of this family is rapidly increasing in the recent years. Current classification from the International Committee of Taxonomy of Viruses (ICTV) for the *Parvoviridae* includes two subfamilies and nine geni (see Figure 1A). The parvoviral capsids are ~260 Å in diameter, and encapsidate a ssDNA genome of ~5000 nucleotides. The number of capsid protein species per virion varies among parvoviruses, but are generally composed by three polypeptides, VP1, VP2 and VP3 (molecular weights in the range of 60-84 kDa). Capsids contain 60 copies (in total) of VP protein subunits in a T=1 icosahedral capsid arrangement. The three-dimensional structure of the capsid, which has been determined to high resolution by X-ray crystallography for many members (Tsao et al. 1991; reviewed in Chapman and Agbandje-McKenna 2006), shows a conserved overall topology of eight-stranded antiparallel beta-barrel core motif that forms the contiguous capsid shell, and large loop insertions between the beta-strands. The surface features include small protrusions (spike-like) commonly at the icosahedral threefold axes, and depressions that may be located at the icosahedral two-fold (dimple-like)

Virus members of the autonomously replicating Parvovirus genus infect mammals and have a wide range of natural hosts, including humans, monkeys, dogs, livestock, felines and rodents. A main molecular model of this genus is the mouse parvovirus Minute Virus of Mice (MVM), which genome is organized as two overlapping transcription units (see Figure 1B) timely regulated (Clemens and Pintel 1988). The left-hand gene driven by the P4 promoter encodes the NS1 and NS2 nonstructural proteins, and the right-hand gene driven by the P38 promoter encodes the VP1 and VP2 structural proteins. In spite of its small size

**2.1 Parvovirus capsid structure and genome organization** 

and/or around the five-fold (canyon-like) axes.

reviewed below.

(only 5 Kb), the use of alternate splicing, extensive postranslational modifications and proteolytic processings, maximize the coding capacity of the parvovirus genome. The two NS polypeptides play multiple roles in virus life cycle. The smaller NS2 protein (28 kDa) contains three isoforms arising from alternate splicings that can bind several cellular proteins and shuttle from the nucleus to the cytoplasm via the CRM1 export pathway. Functions assigned to NS2 include assisting capsid assembly, messenger translation, DNA replication, and virus production in a cell type specific manner. The larger NS1 (82 kDa), is a multifunctional nuclear phosphoprotein, highly toxic for most cells, and performing crucial activities in the MVM unique rolling-hairpin mode of DNA synthesis (see below).

Fig. 1. Outline of the *Parvoviridae*. (A) Taxonomic structure of the *Parvoviridae* (from Tijssen et al. 2011, *in press*). (B) Organization of the MVM genome. The position of the two promoters (P4, P38) are designated by arrows. Splicing sites, and virus coded main nonstructural (NS1, NS2), and structural (VP1, VP2) polypeptides are illustrated in their respective reading frame (based on Cotmore and Tattersall 1987).

#### **2.2 Parvoviruses as oncolytic agents**

Many of the members of the *Parvoviridae* were initially isolated from tumours or from transformed cell lines in culture, corresponding with the requirement of these viruses for multiple functions provided by proliferative cells. This unique biological feature, together with requirements for diverse factors that are linked to the neoplastic growth, account for the capacity of the parvoviruses to infect and lyse preferentially cells transformed by a high diversity of physico-chemical and biological agents (Mousset and Rommelaere 1982; Cornelis et al. 1988), and to interfere with tumour formation in animal models (Toolan and Ledinko 1965; Dupressoir et al. 1989; reviewed in Rommelaere et al. 2010). These studies validated, at least for some parvoviruses, many of the requirements that oncolytic viruses should fulfill to be used in the clinic, such as oncotropism, no genomic integration, low toxicity of structural components, or apathogenicity for humans.

This review is focused on the interactions of glioma cells with two parvoviruses which oncolytic properties have been best evaluated, the rat parvovirus H-1, and the p and i *wildtype* strains of the mouse parvovirus Minute Virus of Mice (MVM). Over the past decade, the characteristics of the infection of these viruses in rodent and human glioma cell lines, primary human glioblastoma cultures, and preclinical xenotransplanted animal models, have been extensively studied (see Table 1).

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 147

whereas U87MG and SW1088 astrocytoma cells were resistant to this virus. The MVMi infections were more interesting, as they uncovered novel insights on parvovirus-tumour cells interactions. Although this virus did not complete its life cycle, it efficiently killed the U373MG, U87MG, and SW1088 astrocytic tumour cells. The abortive infection was not restricted at the transcription or gene expression steps, as the viral messenger RNAs, as well as both non-structural (NS1) and structural (VP1, VP2) proteins, accumulated to normal levels. However, in the U87MG glioblastoma, MVMi failed to amplify its DNA genome. All these analyses consistently showed that only the non-availability of multimeric replicative DNA forms to be encapsidated hampered MVMi virions maturation in the U87MG cells.

Fig. 2. Distinct genome replication capacity of parvovirus MVM in human glioblastomas. (A) Model of MVM genome replication (based on Cotmore and Tattersall 1995). The ssDNA virus genome is illustrated showing the particular structure of the 3´ left and 5´ right termini and their base mispairing. Only the initial steps of the model leading to the configuration of the dsDNA monomeric (mRF) and dimeric (dRF) intermediates, are outlined. (B) DNA replication of MVMi in human U87MG and U373MG cells. The glioblastomas were infected by MVMi, fixed and proceeded for immunofluorescence at 48hpi. Staining was with an anti-

NS1 antibody and FISH using MVM specific TxR-oligonucleotides. NS1 protein

only in U373MG, are shown (scale bar, 10 µm).

accumulated into the nucleus in both glioblastoma cells, but MVM replication occurring


Table 1. Contributions on human Glioma-Parvovirus interactions.

The first report about cytotoxicity of a parvovirus against transformed neural cells was on the MVMp and MVMi infection of rat C6 glioma and several human glioblastoma and astrocytoma cells (Rubio et al. 2001). The MVM infections of human cells were cytotoxic but poorly productive, as found some years afterwards by an independent study (Wollmann et al. 2005). Although a parallel study with the MVM and H-1 viruses has not being carefully addressed, several subsequent reports suggested that H-1 is a more powerful oncolytic agent against human glioblastoma, regarding the levels of both cytotoxicity and virus yield in culture (Herrero et al. 2004; Di Piazza et al. 2007). Further recent preclinical studies have convincingly supported H-1 as anti-glioblastoma agent for clinical purposes, as its infection synergized with radiation (Geletneky et al. 2010a), and moreover it improved survival and remission of advanced intracranial U87MG human glioblastoma in rat models (Geletneky et al. 2010b; Kiprianova et al. 2011). However, as discussed below for MVM, there are particularly interesting aspects in the non-productive glioma-parvovirus interaction, as they may uncover molecular processes altered in glioma and helping to identify cellular targets for cancer treatments.

#### **3. Parvovirus genome replication may be restricted in human glioblastoma**

The two best characterized strains (p and i) of the MVM, exhibiting distinct tropism in spite of their sequence and capsid structure similarity (reviewed in Cotmore and Tattersall 2007), conform a valuable system to explore molecular aspects of virus-host interactions in human gliomas (Rubio et al. 2001). MVMp infection of U373MG was cytotoxic and productive,

Table 1. Contributions on human Glioma-Parvovirus interactions.

for cancer treatments.

The first report about cytotoxicity of a parvovirus against transformed neural cells was on the MVMp and MVMi infection of rat C6 glioma and several human glioblastoma and astrocytoma cells (Rubio et al. 2001). The MVM infections of human cells were cytotoxic but poorly productive, as found some years afterwards by an independent study (Wollmann et al. 2005). Although a parallel study with the MVM and H-1 viruses has not being carefully addressed, several subsequent reports suggested that H-1 is a more powerful oncolytic agent against human glioblastoma, regarding the levels of both cytotoxicity and virus yield in culture (Herrero et al. 2004; Di Piazza et al. 2007). Further recent preclinical studies have convincingly supported H-1 as anti-glioblastoma agent for clinical purposes, as its infection synergized with radiation (Geletneky et al. 2010a), and moreover it improved survival and remission of advanced intracranial U87MG human glioblastoma in rat models (Geletneky et al. 2010b; Kiprianova et al. 2011). However, as discussed below for MVM, there are particularly interesting aspects in the non-productive glioma-parvovirus interaction, as they may uncover molecular processes altered in glioma and helping to identify cellular targets

**3. Parvovirus genome replication may be restricted in human glioblastoma**  The two best characterized strains (p and i) of the MVM, exhibiting distinct tropism in spite of their sequence and capsid structure similarity (reviewed in Cotmore and Tattersall 2007), conform a valuable system to explore molecular aspects of virus-host interactions in human gliomas (Rubio et al. 2001). MVMp infection of U373MG was cytotoxic and productive, whereas U87MG and SW1088 astrocytoma cells were resistant to this virus. The MVMi infections were more interesting, as they uncovered novel insights on parvovirus-tumour cells interactions. Although this virus did not complete its life cycle, it efficiently killed the U373MG, U87MG, and SW1088 astrocytic tumour cells. The abortive infection was not restricted at the transcription or gene expression steps, as the viral messenger RNAs, as well as both non-structural (NS1) and structural (VP1, VP2) proteins, accumulated to normal levels. However, in the U87MG glioblastoma, MVMi failed to amplify its DNA genome. All these analyses consistently showed that only the non-availability of multimeric replicative DNA forms to be encapsidated hampered MVMi virions maturation in the U87MG cells.

Fig. 2. Distinct genome replication capacity of parvovirus MVM in human glioblastomas. (A) Model of MVM genome replication (based on Cotmore and Tattersall 1995). The ssDNA virus genome is illustrated showing the particular structure of the 3´ left and 5´ right termini and their base mispairing. Only the initial steps of the model leading to the configuration of the dsDNA monomeric (mRF) and dimeric (dRF) intermediates, are outlined. (B) DNA replication of MVMi in human U87MG and U373MG cells. The glioblastomas were infected by MVMi, fixed and proceeded for immunofluorescence at 48hpi. Staining was with an anti-NS1 antibody and FISH using MVM specific TxR-oligonucleotides. NS1 protein accumulated into the nucleus in both glioblastoma cells, but MVM replication occurring only in U373MG, are shown (scale bar, 10 µm).

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 149

Fig. 3. Translational control of MVM gene expression in GBM based on PKR activity. Virus entry and traffic into the nucleus is followed by transcription of the early NS gene. Folding of viral messenger RNAs produces regions of dsRNA secondary structure with variable length and complexity. In most normal cells (left), the presence of dsRNA in the cytoplasm leads to PKR activation and eIF2α phosphorylation, which inhibits cap-dependent mRNA translation. In GBM (right), the failure of PKR activation in response to infection allows viral

cell lines have lost the PKR gene (Beretta et al. 1996), whereas PKR activation, but not expression, was hampered in H-Ras transformed fibroblasts (Mundschau and Faller 1992), and PKR plays a role in the tumour-suppressor function of p53 (Yoon et al. 2009). Other transformed cells however, as some human glioblastoma cells, harbor functional PKR that can be activated by specific dsRNA to promote selective killing (Shir and Levitzki 2002; Friedrich et al. 2005). Therefore, in most cell transformation processes viruses find a favoured environment for messenger translation and gene expression. The defective

mRNA translation and replication to proceed (based on Ventoso et al. 2010).

The molecular mechanisms underlying the failed DNA amplification of MVMi in U87MG glioblastoma remain unclear, although some clues can be drawn. As shown in Figure 2B, the U87MG, as the fully permissive U373MG cells, expressed high amounts of NS1 protein (the major viral replicative factor) that translocated normally into the nucleus, although viral DNA was not synthesized to detectable levels (Gil-Ranedo et al., in preparation). When the viral DNA replicative intermediates accumulated in U87MG were resolved in agarose gels (Rubio et al. 2001), a conversion of the incoming viral genome (ssDNA) to the monomeric replicative form (mRF) occurred. However, the subsequent synthesis of the dimeric form (dRF) was not observed. These findings are next interpreted under a current MVM replication model (Figure 2A). The conversion reaction (ssDNA to mRF) is exclusively accomplished by cellular factors S-phase dependent, involving elongation by the δ and other cellular polymerases (Bashir et al. 2000), which are apparently functional in U87MG cells. For several of the following steps of the replication model, the NS1 endonuclease and helicase activities are essential to provide 3´OH ends for the replication fork to proceed (Nuesch et al. 1995). It seems therefore likely that these NS1 activities, or the interaction with cellular factor recruited to the MVM origin of replication to assist NS1 activities (Christensen et al. 1997; Christensen et al. 2001), are not functional in infected U87MG cells.

It is worth mentioning that the infection of human glioblastomas by other parvoviruses may be not restricted at the DNA replication level. The also autonomous H-1 rat parvovirus, closely related to MVM, productively infected the U373MG human glioblastoma cell line, as well as human short-term cultures derived from histologically and immunologically confirmed glioblastomas (NCH82, NCH89, NCH125, NCH149) and gliosarcoma (NCH37). Cell killing, viral DNA amplification, protein expressions and infectious virus particles production was demonstrated in all of these cultures (Herrero et al. 2004). The broader host range of H-1 than MVM toward human glioblastomas may suggest that subtle genetic changes between parvovirus genomes may result in drastically different infection outcomes.

#### **4. Translational control of parvovirus gene expression in glioblastoma**

The degree of permissiveness of many host cells to a particular virus infection is regulated, to a great extent, at the level of the accessibility of the translational machinery to the viral messenger RNAs. The complexity of virus-host interaction at this level is exemplified by the multiple mechanisms evolved by viruses (e.g. affinity of viral messengers for the ribosome, or dependence on initiation factors) to overcome the also evolving cellular barriers (Bushell and Sarnow 2002; Schneider and Mohr 2003). A major role at this interface is played by the Protein Kinase R (PKR), which is activated by the dsRNA species generated by the replication of RNA and some DNA viruses (Balachandran and Barber 2000; Elde et al. 2009). Upon activation, PKR phosphorylates the Ser51 residue of the alpha subunit of the initiation factor 2 (eIF2α), preventing eIF2 from forming the ternary complex with GTP and the initiator Met-tRNA (Dever 2002; Dey et al. 2005), leading to the inhibition of translation initiation and thus aborting virus multiplication. The multiple strategies evolved by viruses to inhibit or bypass PKR activation include for example binding to PKR (Kitajewski et al. 1986; Gale et al. 1998), sequestering the dsRNA (Lu et al. 1995), or recruiting a host phosphatase that dephosphorylates eIF2α (He et al. 1997).

Cell transformation is often associated to reduction or complete suppression of the antiviral defense mechanisms, including PKR responsiveness. For example, some leukemia-derived

The molecular mechanisms underlying the failed DNA amplification of MVMi in U87MG glioblastoma remain unclear, although some clues can be drawn. As shown in Figure 2B, the U87MG, as the fully permissive U373MG cells, expressed high amounts of NS1 protein (the major viral replicative factor) that translocated normally into the nucleus, although viral DNA was not synthesized to detectable levels (Gil-Ranedo et al., in preparation). When the viral DNA replicative intermediates accumulated in U87MG were resolved in agarose gels (Rubio et al. 2001), a conversion of the incoming viral genome (ssDNA) to the monomeric replicative form (mRF) occurred. However, the subsequent synthesis of the dimeric form (dRF) was not observed. These findings are next interpreted under a current MVM replication model (Figure 2A). The conversion reaction (ssDNA to mRF) is exclusively accomplished by cellular factors S-phase dependent, involving elongation by the δ and other cellular polymerases (Bashir et al. 2000), which are apparently functional in U87MG cells. For several of the following steps of the replication model, the NS1 endonuclease and helicase activities are essential to provide 3´OH ends for the replication fork to proceed (Nuesch et al. 1995). It seems therefore likely that these NS1 activities, or the interaction with cellular factor recruited to the MVM origin of replication to assist NS1 activities (Christensen

et al. 1997; Christensen et al. 2001), are not functional in infected U87MG cells.

**4. Translational control of parvovirus gene expression in glioblastoma** 

phosphatase that dephosphorylates eIF2α (He et al. 1997).

The degree of permissiveness of many host cells to a particular virus infection is regulated, to a great extent, at the level of the accessibility of the translational machinery to the viral messenger RNAs. The complexity of virus-host interaction at this level is exemplified by the multiple mechanisms evolved by viruses (e.g. affinity of viral messengers for the ribosome, or dependence on initiation factors) to overcome the also evolving cellular barriers (Bushell and Sarnow 2002; Schneider and Mohr 2003). A major role at this interface is played by the Protein Kinase R (PKR), which is activated by the dsRNA species generated by the replication of RNA and some DNA viruses (Balachandran and Barber 2000; Elde et al. 2009). Upon activation, PKR phosphorylates the Ser51 residue of the alpha subunit of the initiation factor 2 (eIF2α), preventing eIF2 from forming the ternary complex with GTP and the initiator Met-tRNA (Dever 2002; Dey et al. 2005), leading to the inhibition of translation initiation and thus aborting virus multiplication. The multiple strategies evolved by viruses to inhibit or bypass PKR activation include for example binding to PKR (Kitajewski et al. 1986; Gale et al. 1998), sequestering the dsRNA (Lu et al. 1995), or recruiting a host

Cell transformation is often associated to reduction or complete suppression of the antiviral defense mechanisms, including PKR responsiveness. For example, some leukemia-derived

It is worth mentioning that the infection of human glioblastomas by other parvoviruses may be not restricted at the DNA replication level. The also autonomous H-1 rat parvovirus, closely related to MVM, productively infected the U373MG human glioblastoma cell line, as well as human short-term cultures derived from histologically and immunologically confirmed glioblastomas (NCH82, NCH89, NCH125, NCH149) and gliosarcoma (NCH37). Cell killing, viral DNA amplification, protein expressions and infectious virus particles production was demonstrated in all of these cultures (Herrero et al. 2004). The broader host range of H-1 than MVM toward human glioblastomas may suggest that subtle genetic changes between parvovirus genomes may result in drastically different infection outcomes.

Fig. 3. Translational control of MVM gene expression in GBM based on PKR activity. Virus entry and traffic into the nucleus is followed by transcription of the early NS gene. Folding of viral messenger RNAs produces regions of dsRNA secondary structure with variable length and complexity. In most normal cells (left), the presence of dsRNA in the cytoplasm leads to PKR activation and eIF2α phosphorylation, which inhibits cap-dependent mRNA translation. In GBM (right), the failure of PKR activation in response to infection allows viral mRNA translation and replication to proceed (based on Ventoso et al. 2010).

cell lines have lost the PKR gene (Beretta et al. 1996), whereas PKR activation, but not expression, was hampered in H-Ras transformed fibroblasts (Mundschau and Faller 1992), and PKR plays a role in the tumour-suppressor function of p53 (Yoon et al. 2009). Other transformed cells however, as some human glioblastoma cells, harbor functional PKR that can be activated by specific dsRNA to promote selective killing (Shir and Levitzki 2002; Friedrich et al. 2005). Therefore, in most cell transformation processes viruses find a favoured environment for messenger translation and gene expression. The defective

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 151

Fig. 4. Principal protein effectors of the signaling pathways altered in GBM. The two major pathways altered in GBM, the PI3K/AKT/mTOR and Ras/RAF/MEK/MAPK, may be activated from the membrane-coupled receptor. Arrows designate the activation cascades mainly due to phosphorylation. Two inhibitors, PTEN and NF1 (Van Meir et al. 2010), relevant for GBM development, are also outlined. Note the key role of RAF proteins

The MEK1/2 proteins are the main downstream effectors of the activated RAF protein kinases, and subsequently they phosphorylate ERK, which dissociates from the complex and translocates into the nucleus (Khokhlatchev et al. 1998). Active ERK induces many transcription factors including ATF5, which expression inversely correlates with malignant glioma prognosis (Sheng et al. 2010). In glioma, the RAF kinase isoforms are constitutively activated, overexpressed, or mutated (Wellbrock et al. 2004; Lyustikman et al. 2008), and although most mutations map to BRAF (Davies et al. 2002), the activity of the complex may be regulated by CRAF, as it also acts as an effector of BRAF (Wan et al. 2004). Therefore, finding viral proteins that become specific substrate of the RAF kinases in infected cells may

complex as regulator of ERK nuclear translocation (references in the text).

support anti-glioma therapeutic virotherapies.

translational control in cancer cells have been exploited by different virotherapy systems (reviewed in Parato et al. 2005), as those conducted with naturally oncotropic VSV and Reovirus (Strong et al. 1998; Balachandran and Barber 2004), or with genetically engineered oncolytic Adenovirus and Herpesvirus (Farassati et al. 2001; Cascallo et al. 2003), which replicate inefficiently in cells with intact PKR pathways, but may complete gene expression and replication in malignant gliomas (Shah et al. 2007).

In recent years, the important role that translational control plays in the gene expression and natural oncotropism of the ssDNA virus members of the *Parvoviridae* has been recognized. In the Adeno-Associated Virus type 5 (AAV5), a member of the Dependovirus genus, viral replication and protein synthesis was highly enhanced by the co-expression of an adenovirus VA I RNA (Nayak and Pintel 2007a) that, by binding to PKR, prevents its otherwise activation by a short RNA sequence of AAV5 messengers (Nayak and Pintel 2007b). The infection of untransformed fibroblast cells by MVM of the Parvovirus genus, activated PKR and subsequently phosphorylated eIF2α (Ventoso et al. 2010). This mechanism drastically inhibited the synthesis of the NS1 protein, which controls the expression of the viral late messengers and genome replication, leading to a drastic inhibition of virus gene expression and multiplication in culture (see Figure 3, left). In support of this phenomenon observed in cells, purified PKR was highly activated by the R1 genomic messenger of MVM *in vitro*, leading to phosphorylation of the eIF2α translation initiation factor. In contrast, the human glioblastoma U373MG cells showed basal levels of eIF2α phosphorylation, and moreover failed to increase PKR-mediated eIF2α phosphorylation in response to MVM infection, thereby allowing viral gene expression to proceed (Figure 3, right). Therefore, the oncolytic capacity of MVM, H-1, and other parvoviruses against glioblastoma may be largely related to the failure of PKR activation. This conclusion is consistent with the widely studied permissiveness to parvovirus gene expression and toxicity of multiple human cells transformed by oncogenes and tumour suppressors genes (Mousset and Rommelaere 1982; Salome et al. 1990; Telerman et al. 1993), which disturb PKR-based antiviral innate immunity.

#### **5. Parvovirus capsid assembly targets deregulated Raf signaling in glioblastoma**

A virotherapy of glioma with potential clinical benefit should exploit synergy interactions between molecular targets upregulated in glioma cells, and viral factors involved in the replication or maturation of the therapeutic candidate. The genetic alterations undergoing malignant gliomas are multiple and complex, although some signaling pathways play major roles. Alterations in tyrosine kinase growth factor receptors (EGFR, PDGFR, MET and ERBB2), as those found in almost all World Health Organization (WHO) grade II, III and IV astrocytomas, result in constitutive downstream signaling of the RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT pathways. The MAPK pathway is also important for glioma development as it is activated in most WHO grade I tumours. These epidemiological studies are consistent with experimental evidences in mouse models showing that the expression of activated KRAS, CRAF or BRAF in neural progenitor cells combined with either AKT activation, or Ink4aArf loss, leads to the development of high-grade gliomas in vivo (Robinson et al. 2010). In the MAPK signal cascade (see Figure 4), assembled membraneassociated complex formed by kinases and scaffold proteins transduces mitogenic and other stimuli from the cell surface to the nucleus (Marais and Marshall 1996).

translational control in cancer cells have been exploited by different virotherapy systems (reviewed in Parato et al. 2005), as those conducted with naturally oncotropic VSV and Reovirus (Strong et al. 1998; Balachandran and Barber 2004), or with genetically engineered oncolytic Adenovirus and Herpesvirus (Farassati et al. 2001; Cascallo et al. 2003), which replicate inefficiently in cells with intact PKR pathways, but may complete gene expression

In recent years, the important role that translational control plays in the gene expression and natural oncotropism of the ssDNA virus members of the *Parvoviridae* has been recognized. In the Adeno-Associated Virus type 5 (AAV5), a member of the Dependovirus genus, viral replication and protein synthesis was highly enhanced by the co-expression of an adenovirus VA I RNA (Nayak and Pintel 2007a) that, by binding to PKR, prevents its otherwise activation by a short RNA sequence of AAV5 messengers (Nayak and Pintel 2007b). The infection of untransformed fibroblast cells by MVM of the Parvovirus genus, activated PKR and subsequently phosphorylated eIF2α (Ventoso et al. 2010). This mechanism drastically inhibited the synthesis of the NS1 protein, which controls the expression of the viral late messengers and genome replication, leading to a drastic inhibition of virus gene expression and multiplication in culture (see Figure 3, left). In support of this phenomenon observed in cells, purified PKR was highly activated by the R1 genomic messenger of MVM *in vitro*, leading to phosphorylation of the eIF2α translation initiation factor. In contrast, the human glioblastoma U373MG cells showed basal levels of eIF2α phosphorylation, and moreover failed to increase PKR-mediated eIF2α phosphorylation in response to MVM infection, thereby allowing viral gene expression to proceed (Figure 3, right). Therefore, the oncolytic capacity of MVM, H-1, and other parvoviruses against glioblastoma may be largely related to the failure of PKR activation. This conclusion is consistent with the widely studied permissiveness to parvovirus gene expression and toxicity of multiple human cells transformed by oncogenes and tumour suppressors genes (Mousset and Rommelaere 1982; Salome et al. 1990; Telerman et al. 1993),

and replication in malignant gliomas (Shah et al. 2007).

which disturb PKR-based antiviral innate immunity.

**glioblastoma** 

**5. Parvovirus capsid assembly targets deregulated Raf signaling in** 

stimuli from the cell surface to the nucleus (Marais and Marshall 1996).

A virotherapy of glioma with potential clinical benefit should exploit synergy interactions between molecular targets upregulated in glioma cells, and viral factors involved in the replication or maturation of the therapeutic candidate. The genetic alterations undergoing malignant gliomas are multiple and complex, although some signaling pathways play major roles. Alterations in tyrosine kinase growth factor receptors (EGFR, PDGFR, MET and ERBB2), as those found in almost all World Health Organization (WHO) grade II, III and IV astrocytomas, result in constitutive downstream signaling of the RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT pathways. The MAPK pathway is also important for glioma development as it is activated in most WHO grade I tumours. These epidemiological studies are consistent with experimental evidences in mouse models showing that the expression of activated KRAS, CRAF or BRAF in neural progenitor cells combined with either AKT activation, or Ink4aArf loss, leads to the development of high-grade gliomas in vivo (Robinson et al. 2010). In the MAPK signal cascade (see Figure 4), assembled membraneassociated complex formed by kinases and scaffold proteins transduces mitogenic and other

Fig. 4. Principal protein effectors of the signaling pathways altered in GBM. The two major pathways altered in GBM, the PI3K/AKT/mTOR and Ras/RAF/MEK/MAPK, may be activated from the membrane-coupled receptor. Arrows designate the activation cascades mainly due to phosphorylation. Two inhibitors, PTEN and NF1 (Van Meir et al. 2010), relevant for GBM development, are also outlined. Note the key role of RAF proteins complex as regulator of ERK nuclear translocation (references in the text).

The MEK1/2 proteins are the main downstream effectors of the activated RAF protein kinases, and subsequently they phosphorylate ERK, which dissociates from the complex and translocates into the nucleus (Khokhlatchev et al. 1998). Active ERK induces many transcription factors including ATF5, which expression inversely correlates with malignant glioma prognosis (Sheng et al. 2010). In glioma, the RAF kinase isoforms are constitutively activated, overexpressed, or mutated (Wellbrock et al. 2004; Lyustikman et al. 2008), and although most mutations map to BRAF (Davies et al. 2002), the activity of the complex may be regulated by CRAF, as it also acts as an effector of BRAF (Wan et al. 2004). Therefore, finding viral proteins that become specific substrate of the RAF kinases in infected cells may support anti-glioma therapeutic virotherapies.

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 153

al. 2000). The important role of Raf-1 phosphorylation in VP nuclear transport is best illustrated in heterologous cell systems devoid of Raf-1. For example, the expression of parvovirus VP proteins in insect cells resulted in non-phosphorylated VP trimers and aggregates (Riolobos et al. 2010) that, although at low efficiency, self-assembly into viruslike particles (VLPs) accumulating in the perinuclear region of the cytoplasm (Hernando et al. 2000; Yuan and Parrish 2001). However the co-expression of a constitutively active Raf-1 kinase in the insect cells, as the truncated mutant Raf-22W with transforming activity (Stanton et al. 1989), restored the phosphorylation and nuclear transport competence of the

Fig. 6. Molecular overview of parvovirus MVM life cycle steps in glioma cells. **Virus Entry**: VP2 cleavage, Phospholipase activity and VP1 NLS externalization are required to deliver the genome across the NPC. **Capsid Assembly**: VP synthesis, phosphorylation and assembly into trimers lead to translocation into the nucleus by the NLM and NLS signals.

presumably occurs in pre-assembled empty capsids. DNA-filled virions actively egress from the nucleus by the NES at VP2 n-terminus and CRM1-NS2-Nucleoporins mediation. For three processes (I, II, III), the cellular and viral factors found involved in the regulation of glioma-parvovirus interactions, are highlighted. NLS, nuclear localization sequence; NLM, nuclear localization motif; S, cell cycle dependent DNA synthesis; NES, nuclear export sequence; NPC, nuclear pore complex. CRM1, nuclear export factor (adapted with

**Maturation and Egress:** Viral DNA is amplified in the S phase and encapsidation

modifications from Maroto et al. 2004, and Valle et al. 2006).

VP trimer (Riolobos et al. 2010).

The parvovirus capsid is composed of 60 protein subunits (named VP) folded in an eightstranded antiparallel β-barrel motif (Tsao et al. 1991). From their synthesis in the cytoplasm, the VP proteins undergo a well-regulated assembly process that leads to the maturation of the infectious particle in the nucleus of permissive cells. In MVM, the VP1/VP2 capsid proteins are synthesized at an approximate 1/5 ratio, and rapidly assemble into two types of trimers in the cytoplasm at stoichiometric amounts (Riolobos et al. 2006). As outlined in Figure 5, in mammalian permissive cells these trimers are translocated into the nucleus driven by two nuclear-targeting sequences: (i) a non-conventional structured nuclear localization motif (NLM) evolutionary conserved in the parvovirus β-barrel (Lombardo et al. 2000); and (ii) a conventional nuclear localization sequence (NLS) found in the VP1 Nterminus (Lombardo et al. 2002; Vihinen-Ranta et al. 2002).

Fig. 5. Role of Raf-1 (C-Raf) in the nuclear transport of MVM capsid assembly intermediates. MVM capsid proteins (VP1 and VP2) assemble into two types of trimers at their 1/5 stoichiometry of synthesis. Trimers gain nuclear transport competence upon cytoplasmic Raf-1 phosphorylation (VP2 N-terminus is a major phosphorylated domain), and are driven into the nucleus by the NLM and NLS sequences exposed to the transport machinery. Inside the nucleus the fully assembled icosahedral capsid harbors the NLS and NLM facing to the particle inward. Abbreviations: NPC, nuclear pore complex; NLM, nuclear localization motif; NLS, nuclear localization sequence (based on Lombardo et al. 2000; Maroto et al. 2000; Riolobos et al. 2006; Riolobos et al. 2010).

The VP proteins of MVM undergo cytoplasmic phosphorylation by the Raf-1 kinase (Riolobos et al. 2010). Raf-1 (or C-RAF) (72-74 kDa) is a cytoplasmic major protein isoform of the conserved MAPK signaling module with intrinsic serine-threonine kinase activity. The phosphorylation of the VP capsid subunits by Raf-1 occurs at specific Ser/Thr sites *in vitro* yielding a characteristic 2-D phosphopeptides map found in the MVM infections (Maroto et

The parvovirus capsid is composed of 60 protein subunits (named VP) folded in an eightstranded antiparallel β-barrel motif (Tsao et al. 1991). From their synthesis in the cytoplasm, the VP proteins undergo a well-regulated assembly process that leads to the maturation of the infectious particle in the nucleus of permissive cells. In MVM, the VP1/VP2 capsid proteins are synthesized at an approximate 1/5 ratio, and rapidly assemble into two types of trimers in the cytoplasm at stoichiometric amounts (Riolobos et al. 2006). As outlined in Figure 5, in mammalian permissive cells these trimers are translocated into the nucleus driven by two nuclear-targeting sequences: (i) a non-conventional structured nuclear localization motif (NLM) evolutionary conserved in the parvovirus β-barrel (Lombardo et al. 2000); and (ii) a conventional nuclear localization sequence (NLS) found in the VP1 N-

Fig. 5. Role of Raf-1 (C-Raf) in the nuclear transport of MVM capsid assembly intermediates.

The VP proteins of MVM undergo cytoplasmic phosphorylation by the Raf-1 kinase (Riolobos et al. 2010). Raf-1 (or C-RAF) (72-74 kDa) is a cytoplasmic major protein isoform of the conserved MAPK signaling module with intrinsic serine-threonine kinase activity. The phosphorylation of the VP capsid subunits by Raf-1 occurs at specific Ser/Thr sites *in vitro* yielding a characteristic 2-D phosphopeptides map found in the MVM infections (Maroto et

MVM capsid proteins (VP1 and VP2) assemble into two types of trimers at their 1/5 stoichiometry of synthesis. Trimers gain nuclear transport competence upon cytoplasmic Raf-1 phosphorylation (VP2 N-terminus is a major phosphorylated domain), and are driven into the nucleus by the NLM and NLS sequences exposed to the transport machinery. Inside the nucleus the fully assembled icosahedral capsid harbors the NLS and NLM facing to the particle inward. Abbreviations: NPC, nuclear pore complex; NLM, nuclear localization motif; NLS, nuclear localization sequence (based on Lombardo et al. 2000; Maroto et al. 2000;

Riolobos et al. 2006; Riolobos et al. 2010).

terminus (Lombardo et al. 2002; Vihinen-Ranta et al. 2002).

al. 2000). The important role of Raf-1 phosphorylation in VP nuclear transport is best illustrated in heterologous cell systems devoid of Raf-1. For example, the expression of parvovirus VP proteins in insect cells resulted in non-phosphorylated VP trimers and aggregates (Riolobos et al. 2010) that, although at low efficiency, self-assembly into viruslike particles (VLPs) accumulating in the perinuclear region of the cytoplasm (Hernando et al. 2000; Yuan and Parrish 2001). However the co-expression of a constitutively active Raf-1 kinase in the insect cells, as the truncated mutant Raf-22W with transforming activity (Stanton et al. 1989), restored the phosphorylation and nuclear transport competence of the VP trimer (Riolobos et al. 2010).

Fig. 6. Molecular overview of parvovirus MVM life cycle steps in glioma cells. **Virus Entry**: VP2 cleavage, Phospholipase activity and VP1 NLS externalization are required to deliver the genome across the NPC. **Capsid Assembly**: VP synthesis, phosphorylation and assembly into trimers lead to translocation into the nucleus by the NLM and NLS signals. **Maturation and Egress:** Viral DNA is amplified in the S phase and encapsidation presumably occurs in pre-assembled empty capsids. DNA-filled virions actively egress from the nucleus by the NES at VP2 n-terminus and CRM1-NS2-Nucleoporins mediation. For three processes (I, II, III), the cellular and viral factors found involved in the regulation of glioma-parvovirus interactions, are highlighted. NLS, nuclear localization sequence; NLM, nuclear localization motif; S, cell cycle dependent DNA synthesis; NES, nuclear export sequence; NPC, nuclear pore complex. CRM1, nuclear export factor (adapted with modifications from Maroto et al. 2004, and Valle et al. 2006).

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 155

The Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) is in part supported by an

Balachandran, S. and Barber, G.N. 2000. Vesicular stomatitis virus (VSV) therapy of tumors.

Balachandran, S. and Barber, G.N. 2004. Defective translational control facilitates vesicular

Bashir, T., Horlein, R., Rommelaere, J., and Willwand, K. 2000. Cyclin A activates the DNA

Beretta, L., Gabbay, M., Berger, R., Hanash, S.M., and Sonenberg, N. 1996. Expression of the

Berns, N. and Parrish, C.R. 2007. in Parvoviridae. Lippincott Willians and Wilkins,

Bushell, M. and Sarnow, P. 2002. Hijacking the translation apparatus by RNA viruses. J Cell

Cascallo, M., Capella, G., Mazo, A., and Alemany, R. 2003. Ras-dependent oncolysis with an

Chapman, S.M. and Agbandje-McKenna, M. 2006. Atomic structure of viral particles. in

Christensen, J., Cotmore, S.F., and Tattersall, P. 1997. Parvovirus initiation factor PIF: a novel

Christensen, J., Cotmore, S.F., and Tattersall, P. 2001. Minute virus of mice initiator protein

Clemens, K.E. and Pintel, D.J. 1988. The two transcription units of the autonomous

Cornelis, J.J., Becquart, P., Duponchel, N., Salome, N., Avalosse, B.L., Namba, M., and

Cotmore, S.F. and Tattersall, P. 1987. The autonomously replicating parvoviruses of

Cotmore, S.F. and Tattersall, P. 1995. DNA replication in the autonomous parvovirus. Semin

complex with origin DNA for nicking to occur. J Virol 75(15): 7009-7017. Clarke, J., Butowski, N., and Chang, S. 2010. Recent advances in therapy for glioblastoma.

Parvoviruses (ed. J.R. Kerr, S.F. Cotmore, M.E. Bloom, R.M. Linden, and C.R.

human DNA-binding factor which coordinately recognizes two ACGT motifs. J

NS1 and a host KDWK family transcription factor must form a precise ternary

parvovirus minute virus of mice are transcribed in a temporal order. J Virol 62(4):

Rommelaere, J. 1988. Transformation of human fibroblasts by ionizing radiation, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H-1 virus and minute virus of mice. J Virol 62(5):

replication model. Proc Natl Acad Sci U S A 97(10): 5522-5527.

adenovirus VAI mutant. Cancer research 63(17): 5544-5550.

Parrish), pp. 107-123. Hodder A., London, UK.

polymerase delta -dependent elongation machinery in vitro: A parvovirus DNA

protein kinase PKR in modulated by IRF-1 and is reduced in 5q- associated

institutional grant from Fundación Ramón Areces.

stomatitis virus oncolysis. Cancer cell 5(1): 51-65.

leukemias. Oncogene 12(7): 1593-1596.

IUBMB Life 50(2): 135-138.

Philadelphia, PA.

Biol 158(3): 395-399.

Virol 71(8): 5733-5741.

Arch Neurol 67(3): 279-283.

vertebrates. Adv Virus Res 33: 91-174.

1448-1451.

1679-1686.

Virol 6(11): 271-281.

**8. References** 

The enhanced VP nuclear transport and MVM capsid assembly by oncogenic deregulated Raf-1 should facilitate parvovirus maturation in cancer cells. Indeed, in rat C6 and human U373MG glioblastoma cells infected with MVM, the characteristic pattern of VP phosphorylation by Raf-1 was conserved, correlating with a high activity of the MAPK signaling pathway (Riolobos et al. 2010). Moreover these glioblastomas were efficiently killed by MVM and the virus underwent productive maturation to high yields (Rubio et al. 2001). Given the difficulties encountered in searching effective chemotherapies against the Raf signaling cascade for cancer treatment (Madhunapantula and Robertson 2008; Newton 2010), the above mentioned findings may support MVM and related parvoviruses as replicative inhibitors specifically targeting glioblastomas with deregulated Raf signaling.

#### **6. Conclusions**

We have summarized in this chapter some molecular mechanisms involved in glioma-MVM interactions. Figure 6 illustrates some major events of MVM life cycle, highlighting those three steps at which the interaction of this parvovirus with glioma cells was found to be modulated. Final comments related to these findings are in brief : (i) Identifying the relevance of PKR translational control for MVM cytotoxic infection in U373MG glioblastoma, connects the parvoviral oncolysis with a common mechanism exhibited by other oncolytic viruses to preferentially infect tumour cells with deregulated IFN and other innate antiviral responses; (ii) Raf-1 requirement for MVM nuclear capsid assembly not only assignes parvoviral oncolysis to an important kinase upregulated in human cancer, but also underlies a novel general mechanism to be exploited in oncolytic virotherapy; (iii) the lack of correlation between NS1-mediated MVM replication and gene expression may be important to better understand the cellular machinery regulating theses processes in normal vs. tumour cells. It is likely that other signals and mechanisms tightly regulated during MVM life cycle steps (see Figure 6) be also found perturbed in cancer cells, what could provide additional targets for anti-glioma intervention.

Finally, the dependence of parvovirus infection on cellular factors which are functionally dysregulated in a cancer-specific manner may bring two important benefits: (i) the use of parvoviruses as markers of host-cell perturbations (e.g. signaling) coupled to transformation, and (ii) it may allow to apply these simple viruses as specific therapeutic agents against precise types of glioblastomas showing permissive genetic profiles, toward a personalized parvoviral anti-cancer virotherapy.

#### **7. Acknowledgments**

The experimental contributions of the following past members of the laboratory is gratefully acknowledged: S. Guerra, E. Hernando, E. Lombardo, A. López-Bueno, B. Maroto, J. C. Ramirez, L. Riolobos, M.- P. Rubio, N. Valle, and I. Ventoso. We are indebted to Michael Kann (Bourdeaux) for collaborative support on nuclear transport studies, and to Peter Tattersall (Yale, CT) and Jean Rommelaere (DKFZ, Heidelberg) for generous thoughtful advices along the years.

This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (SAF2008-03238) and Comunidad de Madrid (S-SAL/0185/2006) to the laboratory of J.M.A. The Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) is in part supported by an institutional grant from Fundación Ramón Areces.

#### **8. References**

154 Novel Therapeutic Concepts in Targeting Glioma

The enhanced VP nuclear transport and MVM capsid assembly by oncogenic deregulated Raf-1 should facilitate parvovirus maturation in cancer cells. Indeed, in rat C6 and human U373MG glioblastoma cells infected with MVM, the characteristic pattern of VP phosphorylation by Raf-1 was conserved, correlating with a high activity of the MAPK signaling pathway (Riolobos et al. 2010). Moreover these glioblastomas were efficiently killed by MVM and the virus underwent productive maturation to high yields (Rubio et al. 2001). Given the difficulties encountered in searching effective chemotherapies against the Raf signaling cascade for cancer treatment (Madhunapantula and Robertson 2008; Newton 2010), the above mentioned findings may support MVM and related parvoviruses as replicative inhibitors specifically targeting glioblastomas with deregulated Raf signaling.

We have summarized in this chapter some molecular mechanisms involved in glioma-MVM interactions. Figure 6 illustrates some major events of MVM life cycle, highlighting those three steps at which the interaction of this parvovirus with glioma cells was found to be modulated. Final comments related to these findings are in brief : (i) Identifying the relevance of PKR translational control for MVM cytotoxic infection in U373MG glioblastoma, connects the parvoviral oncolysis with a common mechanism exhibited by other oncolytic viruses to preferentially infect tumour cells with deregulated IFN and other innate antiviral responses; (ii) Raf-1 requirement for MVM nuclear capsid assembly not only assignes parvoviral oncolysis to an important kinase upregulated in human cancer, but also underlies a novel general mechanism to be exploited in oncolytic virotherapy; (iii) the lack of correlation between NS1-mediated MVM replication and gene expression may be important to better understand the cellular machinery regulating theses processes in normal vs. tumour cells. It is likely that other signals and mechanisms tightly regulated during MVM life cycle steps (see Figure 6) be also found perturbed in cancer cells, what could

Finally, the dependence of parvovirus infection on cellular factors which are functionally dysregulated in a cancer-specific manner may bring two important benefits: (i) the use of parvoviruses as markers of host-cell perturbations (e.g. signaling) coupled to transformation, and (ii) it may allow to apply these simple viruses as specific therapeutic agents against precise types of glioblastomas showing permissive genetic profiles, toward a

The experimental contributions of the following past members of the laboratory is gratefully acknowledged: S. Guerra, E. Hernando, E. Lombardo, A. López-Bueno, B. Maroto, J. C. Ramirez, L. Riolobos, M.- P. Rubio, N. Valle, and I. Ventoso. We are indebted to Michael Kann (Bourdeaux) for collaborative support on nuclear transport studies, and to Peter Tattersall (Yale, CT) and Jean Rommelaere (DKFZ, Heidelberg) for generous thoughtful

This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (SAF2008-03238) and Comunidad de Madrid (S-SAL/0185/2006) to the laboratory of J.M.A.

provide additional targets for anti-glioma intervention.

personalized parvoviral anti-cancer virotherapy.

**7. Acknowledgments** 

advices along the years.

**6. Conclusions** 


Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 157

Haseley, A., Alvarez-Breckenridge, C., Chaudhury, A.R., and Kaur, B. 2009. Advances in oncolytic virus therapy for glioma. *Recent Pat CNS Drug Discov* 4(1): 1-13. He, B., Gross, M., and Roizman, B. 1997. The gamma(1)34.5 protein of herpes simplex virus 1

Hernando, E., Llamas-Saiz, A.L., Foces-Foces, C., McKenna, R., Portman, I., Agbandje-

Khokhlatchev, A.V., Canagarajah, B., Wilsbacher, J., Robinson, M., Atkinson, M., Goldsmith,

Kiprianova, I., Thomas, N., Ayache, A., Fischer, M., Leuchs, B., Klein, M., Rommelaere, J.,

Kitajewski, J., Schneider, R.J., Safer, B., Munemitsu, S.M., Samuel, C.E., Thimmappaya, B.,

Kroeger, K.M., Muhammad, A.K., Baker, G.J., Assi, H., Wibowo, M.K., Xiong, W., Yagiz, K.,

Lombardo, E., Ramirez, J.C., Agbandje-McKenna, M., and Almendral, J.M. 2000. A beta-

Lombardo, E., Ramirez, J.C., Garcia, J., and Almendral, J.M. 2002. Complementary roles of

Mangiola, A., Anile, C., Pompucci, A., Capone, G., Rigante, L., and De Bonis, P. 2010.

mice into the nucleus for viral assembly. J Virol 74(8): 3804-3814.

melanoma and other malignancies? Cancer research 68(1): 5-8.

homodimerization and nuclear translocation. Cell 93(4): 605-615.

application of parvovirus H-1. Clin Cancer Res. 17: 5333-5342.

S A 94(3): 843-848.

109(1): 76-84.

Cell 45(2): 195-200.

10(4): 507-514.

304.

complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U

McKenna, M., and Almendral, J.M. 2000. Biochemical and physical characterization of parvovirus minute virus of mice virus-like particles. Virology 267(2): 299-309. Herrero, Y.C.M., Cornelis, J.J., Herold-Mende, C., Rommelaere, J., Schlehofer, J.R., and

Geletneky, K. 2004. Parvovirus H-1 infection of human glioma cells leads to complete viral replication and efficient cell killing. International journal of cancer

E., and Cobb, M.H. 1998. Phosphorylation of the MAP kinase ERK2 promotes its

and Schlehofer, J.R. 2011. Regression of glioma in rat models by intranasal

and Shenk, T. 1986. Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase.

Candolfi, M., Lowenstein, P.R., and Castro, M.G. 2010. Gene therapy and virotherapy: novel therapeutic approaches for brain tumors. *Discov Med* 10(53): 293-

stranded motif drives capsid protein oligomers of the parvovirus minute virus of

multiple nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice during assembly and onset of infection. J Virol 76(14): 7049-7059. Lu, Y., Wambach, M., Katze, M.G., and Krug, R.M. 1995. Binding of the influenza virus NS1

protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology 214(1): 222-228. Lyustikman, Y., Momota, H., Pao, W., and Holland, E.C. 2008. Constitutive activation of Raf-1 induces glioma formation in mice. Neoplasia (New York, NY 10(5): 501-510. Madhunapantula, S.V. and Robertson, G.P. 2008. Is B-Raf a good therapeutic target for

Glioblastoma therapy: going beyond Hercules Columns. Expert Rev Neurother


Cotmore, S.F. and Tattersall, P. 2007. Parvoviral host range and cell entry mechanisms. Adv

Davies, H., Bignell, G.R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin,

Mutations of the BRAF gene in human cancer. Nature 417(6892): 949-954. Dever, T.E. 2002. Gene-specific regulation by general translation factors. Cell 108(4): 545-556. Dey, M., Cao, C., Dar, A.C., Tamura, T., Ozato, K., Sicheri, F., and Dever, T.E. 2005.

Di Piazza, M., Mader, C., Geletneky, K., Herrero, Y.C.M., Weber, E., Schlehofer, J., Deleu, L.,

Dupressoir, T., Vanacker, J.M., Cornelis, J.J., Duponchel, N., and Rommelaere, J. 1989.

Eager, R.M. and Nemunaitis, J. 2011. Clinical development directions in oncolytic viral

Elde, N.C., Child, S.J., Geballe, A.P., and Malik, H.S. 2009. Protein kinase R reveals an evolutionary model for defeating viral mimicry. Nature 457(7228): 485-489. Farassati, F., Yang, A.D., and Lee, P.W. 2001. Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1. Nat Cell Biol 3(8): 745-750. Friedrich, I., Eizenbach, M., Sajman, J., Ben-Bassat, H., and Levitzki, A. 2005. A cellular

Gale, M., Jr., Blakely, C.M., Kwieciszewski, B., Tan, S.L., Dossett, M., Tang, N.M., Korth,

Geletneky, K., Hartkopf, A.D., Krempien, R., Rommelaere, J., and Schlehofer, J.R. 2010a.

Geletneky, K., Kiprianova, I., Ayache, A., Koch, R., Herrero, Y.C.M., Deleu, L., Sommer, C.,

regulation. Molecular and cellular biology 18(9): 5208-5218.

substrate recognition. Cell 122(6): 901-913.

therapy. Cancer Gene Ther 18(5): 305-317.

in rat models. Neuro Oncol. 12: 804-814.

H., Garnett, M.J., Bottomley, W., Davis, N., Dicks, E., Ewing, R., Floyd, Y., Gray, K., Hall, S., Hawes, R., Hughes, J., Kosmidou, V., Menzies, A., Mould, C., Parker, A., Stevens, C., Watt, S., Hooper, S., Wilson, R., Jayatilake, H., Gusterson, B.A., Cooper, C., Shipley, J., Hargrave, D., Pritchard-Jones, K., Maitland, N., Chenevix-Trench, G., Riggins, G.J., Bigner, D.D., Palmieri, G., Cossu, A., Flanagan, A., Nicholson, A., Ho, J.W., Leung, S.Y., Yuen, S.T., Weber, B.L., Seigler, H.F., Darrow, T.L., Paterson, H., Marais, R., Marshall, C.J., Wooster, R., Stratton, M.R., and Futreal, P.A. 2002.

Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha

and Rommelaere, J. 2007. Cytosolic activation of cathepsins mediates parvovirus H-1-induced killing of cisplatin and TRAIL-resistant glioma cells. J Virol 81(8): 4186-

Inhibition by parvovirus H-1 of the formation of tumors in nude mice and colonies in vitro by transformed human mammary epithelial cells. Cancer research 49(12):

screening assay to test the ability of PKR to induce cell death in mammalian cells.

M.J., Polyak, S.J., Gretch, D.R., and Katze, M.G. 1998. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase

Improved killing of human high-grade glioma cells by combining ionizing radiation with oncolytic parvovirus H-1 infection. J Biomed Biotechnol 2010:

Thomas, N., Rommelaere, J., and Schlehofer, J.R. 2010b. Regression of advanced rat and human gliomas by local or systemic treatment with oncolytic parvovirus H-1

Virus Res 70: 183-232.

4198.

3203-3208.

350748.

Mol Ther 12(5): 969-975.


Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential 159

Rommelaere, J., Geletneky, K., Angelova, A.L., Daeffler, L., Dinsart, C., Kiprianova, I.,

Rubio, M.P., Guerra, S., and Almendral, J.M. 2001. Genome replication and

Schneider, R.J. and Mohr, I. 2003. Translation initiation and viral tricks. Trends Biochem Sci

Shah, A.C., Parker, J.N., Gillespie, G.Y., Lakeman, F.D., Meleth, S., Markert, J.M., and

Sheng, Z., Li, L., Zhu, L.J., Smith, T.W., Demers, A., Ross, A.H., Moser, R.P., and Green, M.R.

Shir, A. and Levitzki, A. 2002. Inhibition of glioma growth by tumor-specific activation of

Stanton, V.P., Jr., Nichols, D.W., Laudano, A.P., and Cooper, G.M. 1989. Definition of the

Strong, J.E., Coffey, M.C., Tang, D., Sabinin, P., and Lee, P.W. 1998. The molecular basis of

Telerman, A., Tuynder, M., Dupressoir, T., Robaye, B., Sigaux, F., Shaulian, E., Oren, M.,

Tijssen, P., Agbandje-McKenna, M., Almendral, J.M., Bergoin, M., Flegel, T.W., Hedman, K.,

Toolan, H. and Ledinko, N. 1965. Growth and cytopathogenicity of H-viruses in human and

Tsao, J., Chapman, M.S., Agbandje, M., Keller, W., Smith, K., Wu, H., Luo, M., Smith, T.J.,

Valle, N., Riolobos, L., and Almendral, J.M. 2006. Synthesis, post-translational modification

parvovirus. Proc Natl Acad Sci U S A 90(18): 8702-8706.

simian cell cultures. Nature 208(5012): 812-813.

range of a murine parvovirus in human cells. J Virol 75(23): 11573-11582. Salome, N., van Hille, B., Duponchel, N., Meneguzzi, G., Cuzin, F., Rommelaere, J., and

therapeutics. Cytokine Growth Factor Rev 21(2-3): 185-195.

restricted to specific oncogenes. Oncogene 5(1): 123-130.

oncolytic viruses. Gene Ther 14(13): 1045-1054.

28(3): 130-136.

Med 16(6): 671-677.

17(12): 3351-3362.

251(5000): 1456-1464.

London, UK.

and cellular biology 9(2): 639-647.

900.

press.

Schlehofer, J.R., and Raykov, Z. 2010. Oncolytic parvoviruses as cancer

postencapsidation functions mapping to the nonstructural gene restrict the host

Cornelis, J.J. 1990. Sensitization of transformed rat cells to parvovirus MVMp is

Cassady, K.A. 2007. Enhanced antiglioma activity of chimeric HCMV/HSV-1

2010. A genome-wide RNA interference screen reveals an essential CREB3L2-ATF5- MCL1 survival pathway in malignant glioma with therapeutic implications. Nat

double-stranded RNA-dependent protein kinase PKR. Nat Biotechnol 20(9): 895-

human raf amino-terminal regulatory region by deletion mutagenesis. Molecular

viral oncolysis: usurpation of the Ras signaling pathway by reovirus. EMBO J

Rommelaere, J., and Amson, R. 1993. A model for tumor suppression using H-1

Kleinschmidt, J., Li, Y., Pintel, D.J., and Tattersall, P. 2011. ICTV Ninth Report in

Rossmann, M.G., Compans, R.W., and et al. 1991. The three-dimensional structure of canine parvovirus and its functional implications. Science (New York, NY

and trafficking of the parvovirus structural polypeptides. in Parvoviruses (ed. J.R. Kerr, S.F. Cotmore, M.E. Bloom, R.M. Linden, and C.R. Parrish). Hodder A.,


Marais, R. and Marshall, C.J. 1996. Control of the ERK MAP kinase cascade by Ras and Raf.

Markert, J.M., Medlock, M.D., Rabkin, S.D., Gillespie, G.Y., Todo, T., Hunter, W.D., Palmer,

Maroto, B., Valle, N., Saffrich, R., and Almendral, J.M. 2004. Nuclear export of the

Mousset, S. and Rommelaere, J. 1982. Minute virus of mice inhibits cell transformation by

Mousset, S. and Rommelaere, J. 1982. Minute virus of mice inhibits cell transformation by

Mundschau, L.J. and Faller, D.V. 1992. Oncogenic ras induces an inhibitor of double-

Nayak, R. and Pintel, D.J. 2007a. Adeno-associated viruses can induce phosphorylation of

Nayak, R. and Pintel, D.J. 2007b. Positive and negative effects of adenovirus type 5 helper

Newton, H.B. 2010. Overview of the Molecular Genetics and Molecular Chemotherapy of

Nuesch, J.P., Cotmore, S.F., and Tattersall, P. 1995. Sequence motifs in the replicator protein

Riolobos, L., Valle, N., Hernando, E., Maroto, B., Kann, M., and Almendral, J.M. 2010. Viral

Robinson, J.P., VanBrocklin, M.W., Guilbeault, A.R., Signorelli, D.L., Brandner, S., and

origin: identification of the linking tyrosine. Virology 209(1): 122-135. Parato, K.A., Senger, D., Forsyth, P.A., and Bell, J.C. 2005. Recent progress in the battle between oncolytic viruses and tumours. Nature reviews 5(12): 965-976. Riolobos, L., Reguera, J., Mateu, M.G., and Almendral, J.M. 2006. Nuclear transport of

malignant glioma: results of a phase I trial. Gene Ther 7(10): 867-874. Maroto, B., Ramirez, J.C., and Almendral, J.M. 2000. Phosphorylation status of the

major phosphorylation sites. J Virol 74(23): 10892-10902.

exposed on the capsid surface. J Virol 78(19): 10685-10694.

simian virus 40. Nature 300(5892): 537-539.

simian virus 40. Nature 300(5892): 537-539.

Journal of biological chemistry 267(32): 23092-23098.

virus-associated RNA. J Virol 81(21): 11908-11916.

AAV5 virus production. J Virol 81(5): 2205-2212.

parvovirus capsid. J Mol Biol 357(3): 1026-1038.

Ink4a/Arf loss or Akt activation. Oncogene 29(3): 335-344.

Therapeutic Strategies (ed. S.K. Ray).

2090-2099.

C.A., Feigenbaum, F., Tornatore, C., Tufaro, F., and Martuza, R.L. 2000. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of

parvovirus minute virus of mice particle: mapping and biological relevance of the

nonenveloped parvovirus virion is directed by an unordered protein signal

stranded RNA-dependent eukaryotic initiation factor 2 alpha-kinase activation. The

eIF2alpha via PKR activation, which can be overcome by helper adenovirus type 5

functions on adeno-associated virus type 5 (AAV5) protein accumulation govern

GBM. in Glioblastoma Molecular Mechanisms of Pathogenesis and Current

of parvovirus MVM essential for nicking and covalent attachment to the viral

trimeric assembly intermediates exerts a morphogenetic control on the icosahedral

oncolysis that targets Raf-1 signaling control of nuclear transport. J Virol 84(4):

Holmen, S.L. 2010. Activated BRAF induces gliomas in mice when combined with

Cancer Surv 27: 101-125.


**9** 

*Canada* 

**The Potential and Challenges of** 

**siRNA-Based Targeted Therapy** 

*3Department of Pathology and Laboratory Medicine,* 

*4Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver,* 

*University of British Columbia, Vancouver,* 

M. Verreault1,3,\*, S. Yip3,4, B. Toyota5 and M.B. Bally1,2,3

**for Treatment of Patients with Glioblastoma** 

*1Experimental Therapeutics, British Columbia Cancer Agency, Vancouver, BC 2Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver,* 

*5Division of Neurosurgery, British Columbia Cancer Agency,Vancouver, BC* 

Brain tumor is the second leading cause of cancer-related death in children under age of 20. An estimated total of 62,930 new cases of primary brain tumors in United States (CBTRUS, 2010) and 2,600 in Canada (CCS, 2010) were expected to be diagnosed in 2010 in adults and children. This includes both malignant (23,720 in US) and non-malignant (39,210 in US) brain tumors (CBTRUS, 2010). The most common (80%) form of malignant primary brain tumor originates from the neuroglial cells and is referred to as glioma (CBTRUS, 2010). Tumors originating from the astrocytes constitute 76% of cases of gliomas, and glioblastoma (GBM) is the most common malignant form of glioma (53.8%) (CBTRUS, 2010). Despite aggressive therapeutic interventions, 90% of patients are expected to succumb to the disease within five years after diagnosis (Stupp et al., 2005). Research is desperately needed to improve our understanding of the disease and to define strategies that will increase the

GBM is a highly malignant type of primary brain cancer which mainly affects patients in their fifties and older. It is classified as a grade IV tumor using the World Health Organization (WHO) grading system and is associated with a median survival of approximately 12-15 months compared to 48 months for diffuse astrocytoma (WHO grade II), its lower grade counterpart (Louis et al., 2007). Secondary GBMs progress after a period of growth from low-grade gliomas (LGGs), while *de novo* GBMs arise rapidly into the most

**1. Introduction** 

\* Corresponding Author

efficacy of our treatment options.

**2. Clinical and molecular features of GBM** 


*Canada* 

## **The Potential and Challenges of siRNA-Based Targeted Therapy for Treatment of Patients with Glioblastoma**

M. Verreault1,3,\*, S. Yip3,4, B. Toyota5 and M.B. Bally1,2,3 *1Experimental Therapeutics, British Columbia Cancer Agency, Vancouver, BC 2Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, 3Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, 4Department of Pathology and Laboratory Medicine, British Columbia Cancer Agency, Vancouver, 5Division of Neurosurgery, British Columbia Cancer Agency,Vancouver, BC* 

#### **1. Introduction**

160 Novel Therapeutic Concepts in Targeting Glioma

Van Meir, E.G., Hadjipanayis, C.G., Norden, A.D., Shu, H.K., Wen, P.Y., and Olson, J.J. 2010.

Ventoso, I., Berlanga, J.J., and Almendral, J.M. 2010. Translation control by protein kinase R

Vihinen-Ranta, M., Wang, D., Weichert, W.S., and Parrish, C.R. 2002. The VP1 N-terminal

Wan, P.T., Garnett, M.J., Roe, S.M., Lee, S., Niculescu-Duvaz, D., Good, V.M., Jones, C.M.,

Wellbrock, C., Karasarides, M., and Marais, R. 2004. The RAF proteins take centre stage. Nat

Yoon, C.H., Lee, E.S., Lim, D.S., and Bae, Y.S. 2009. PKR, a p53 target gene, plays a crucial

Yuan, W. and Parrish, C.R. 2001. Canine parvovirus capsid assembly and differences in

mammalian and insect cells. Virology 279(2): 546-557.

Wen, P.Y. and Kesari, S. 2008. Malignant gliomas in adults. N Engl J Med 359(5): 492-507. Wollmann, G., Tattersall, P., and van den Pol, A.N. 2005. Targeting human glioblastoma

glioma. CA Cancer J Clin 60(3): 166-193.

infection. J Virol 76(4): 1884-1891.

Rev Mol Cell Biol 5(11): 875-885.

5043-5051.

7852-7857.

Cell 116(6): 855-867.

Exciting new advances in neuro-oncology: the avenue to a cure for malignant

restricts minute virus of mice infection: role in parvovirus oncolysis. J Virol 84(10):

sequence of canine parvovirus affects nuclear transport of capsids and efficient cell

Marshall, C.J., Springer, C.J., Barford, D., and Marais, R. 2004. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.

cells: comparison of nine viruses with oncolytic potential. J Virol 79(10): 6005-6022.

role in the tumor-suppressor function of p53. Proc Natl Acad Sci U S A 106(19):

Brain tumor is the second leading cause of cancer-related death in children under age of 20. An estimated total of 62,930 new cases of primary brain tumors in United States (CBTRUS, 2010) and 2,600 in Canada (CCS, 2010) were expected to be diagnosed in 2010 in adults and children. This includes both malignant (23,720 in US) and non-malignant (39,210 in US) brain tumors (CBTRUS, 2010). The most common (80%) form of malignant primary brain tumor originates from the neuroglial cells and is referred to as glioma (CBTRUS, 2010). Tumors originating from the astrocytes constitute 76% of cases of gliomas, and glioblastoma (GBM) is the most common malignant form of glioma (53.8%) (CBTRUS, 2010). Despite aggressive therapeutic interventions, 90% of patients are expected to succumb to the disease within five years after diagnosis (Stupp et al., 2005). Research is desperately needed to improve our understanding of the disease and to define strategies that will increase the efficacy of our treatment options.

#### **2. Clinical and molecular features of GBM**

GBM is a highly malignant type of primary brain cancer which mainly affects patients in their fifties and older. It is classified as a grade IV tumor using the World Health Organization (WHO) grading system and is associated with a median survival of approximately 12-15 months compared to 48 months for diffuse astrocytoma (WHO grade II), its lower grade counterpart (Louis et al., 2007). Secondary GBMs progress after a period of growth from low-grade gliomas (LGGs), while *de novo* GBMs arise rapidly into the most

<sup>\*</sup> Corresponding Author

siRNA-Based Therapy for Glioblastoma Patients 163

molecular biomarkers are making inroads into the clinic diagnostic workup of brain tumors

Fig. 1. Micro- and macroscopic view of a GBM tumor. a) Astrocytomas, both low and high grade, consist of neoplastic cells with elongated nuclei in association with variable amount of eosinophilic cytoplasm often extended into coarse processes. GBM often display an exaggerated degree of morphologic heterogeneity as seen in this figure, with tumor cells with bizarre and giant nuclei. b) Pseudopalisading necrosis is a cardinal feature of GBM and is characterized by an area of tissue necrosis surrounded immediately by a rim of viable tumor cells. The microenvironment in this region is often characterized by low oxygen tension (hence the necrosis) and activation of various hypoxia-mediated molecular pathways such as HIF-1α in the tumor cells (Kaur et al., 2005; Rong et al., 2006). c) Low magnification survey of a GBM showing multiple foci of microvascular proliferation (MVP) characterized by abnormal proliferation and hypertrophy of endothelial cells as a result of an overly pro-angiogenic environment. The presence of MVP automatically "upgrades" diagnosis from an astrocytoma to a WHO grade IV GBM. Foci of MVP are often found in areas with other malignant histological features including florid mitotic activity and tumor necrosis. d) Intraoperative picture of a GBM showing significant distortion of the gross morphology of the cerebral cortex resulting in expansion and discoloration of the gyri. e) and f) Magnetic resonance imaging (MRI) of a GBM using T1 sequence in conjunction with intravenous administration of gadolinium (GAD) dye. The tumor is represented in the axial (e) and coronal (f) planes, which is essential for localization of the lesion and surgical planning. The surfeit of tumor-associated and abnormally developed blood vessels (i.e. the MVP) with a defective blood-brain barrier (BBB) allows for penetration of GAD into the tissue, and accentuates areas of BBB breakdown. This so-called "contrast- enhancing"

In addition to changes in nucleotide sequences within the genetic makeup of a cancer cell, the epigenome, defined by the profile of selective methylation of CpG-islands and complex

(Jansen et al., 2010; Yip et al., 2008).

appearance is typical of high grade gliomas.

malignant form without the typical period of indolent growth associated with secondary GBMs. The latter preferentially affects older patients and is frequently associated with amplification of the epidermal growth factor receptor (*EGFR)* locus and inactivation of the phosphatase and tensin homolog *(PTEN)* tumor suppressor gene. Recent discovery of the recurrent R132H mutation in the isocitrate dehydrogenase 1 (*IDH1*) gene in a vast majority of secondary GBMs and a large percentage of LGGs further contributed to differentiate histologically similar GBM at the molecular level (Ichimura et al., 2009; Yan et al., 2009). In addition, the R132H *IDH1* genotype and the mutation of *TP53* are found to be frequently associated in secondary GBMs. Due to the aggressive biological phenotype of GBM, most patients present with acute headache, vomiting, occasional transient or partial blindness due to raised intracranial pressure, and local or general brain dysfunction, possibly leading to altered behavior or seizures (Brada et al., 2001; Chandana et al., 2008).

Current diagnosis and grading of gliomas are dependent on the histological analysis of biopsied or resected tumor tissue, together with immunohistochemistry and molecular testing on selected markers (Louis et al., 2007). Morphologic criteria are often arbitrary and based on the histological profile of tumor cells including 1) a high density of small and pleomorphic tumor cells that are characterized by large and elongated nuclei (Figure 1a), 2) the frequency of mitotic figures, 3) the presence of tumor necrosis that can be surrounded by dense accumulations of tumor cells (pseudopalisading necrosis, Figure 1b) and 4) the presence of microvascular proliferation (Figure 1c) (Gray, 2005; Prados M., 2002; Scherer, 1940). The presence of all these histological entities indicates an astrocytoma of WHO grade IV. Anaplastic astrocytoma, a WHO grade III tumor, does not have tumor necrosis or foci of microvascular proliferation. A feature that is common to all gliomas, regardless of grade and cell type, is the extent of local invasion and distant infiltration of the normal neuropil by the neoplastic cells. Histological correlations of this phenomenon were described by Scherer in 1940 (Scherer, 1940). Scherer described the aggregation and migration of glioma cells along normal blood vessels (perivascular satellitosis), neurons (perineuronal satellitosis), below the pial surface (subpial spread), and finally along large white matter tracts (intrafascicular growth). These "Scherer's secondary structures" are easily identified and constitute microscopic landmarks of glioma malignancy (Holland, 2000). However, accurate pathological classification of a tumor is often confounded by an undersampling bias, especially in cases of needle biopsies of large lesions. In addition, there is significant heterogeneity among gliomas at the genetic, epigenetic, and histological level (Walker C. et al., 2003).

There is little doubt that molecular heterogeneity contributes to the clonal evolution (Gerlinger & Swanton, 2010) within a tumor, which directly impacts clinical behavior such as treatment failure and disease recurrence (Yip et al., 2009). The example of the selection of GBM tumor cells carrying inactivating mutations in the mismatch repair gene mutS homolog 6 (*MSH6*) during treatment illustrates well this phenomenon and will be discussed further in section 5. Moreover, one microscopic field of GBM sample taken from the same patient can appear vastly different from another area. In addition, low and high-grade gliomas from different patients, with similar morphological appearance and of the same histological grade, can behave quite differently (Frazier et al., 2009; Krex et al., 2007; Yamanaka et al., 2006). Therefore, traditional histopathology alone is not sufficient to provide information with respect to prognostic and predictive substratification. Fortunately,

malignant form without the typical period of indolent growth associated with secondary GBMs. The latter preferentially affects older patients and is frequently associated with amplification of the epidermal growth factor receptor (*EGFR)* locus and inactivation of the phosphatase and tensin homolog *(PTEN)* tumor suppressor gene. Recent discovery of the recurrent R132H mutation in the isocitrate dehydrogenase 1 (*IDH1*) gene in a vast majority of secondary GBMs and a large percentage of LGGs further contributed to differentiate histologically similar GBM at the molecular level (Ichimura et al., 2009; Yan et al., 2009). In addition, the R132H *IDH1* genotype and the mutation of *TP53* are found to be frequently associated in secondary GBMs. Due to the aggressive biological phenotype of GBM, most patients present with acute headache, vomiting, occasional transient or partial blindness due to raised intracranial pressure, and local or general brain dysfunction, possibly leading to

Current diagnosis and grading of gliomas are dependent on the histological analysis of biopsied or resected tumor tissue, together with immunohistochemistry and molecular testing on selected markers (Louis et al., 2007). Morphologic criteria are often arbitrary and based on the histological profile of tumor cells including 1) a high density of small and pleomorphic tumor cells that are characterized by large and elongated nuclei (Figure 1a), 2) the frequency of mitotic figures, 3) the presence of tumor necrosis that can be surrounded by dense accumulations of tumor cells (pseudopalisading necrosis, Figure 1b) and 4) the presence of microvascular proliferation (Figure 1c) (Gray, 2005; Prados M., 2002; Scherer, 1940). The presence of all these histological entities indicates an astrocytoma of WHO grade IV. Anaplastic astrocytoma, a WHO grade III tumor, does not have tumor necrosis or foci of microvascular proliferation. A feature that is common to all gliomas, regardless of grade and cell type, is the extent of local invasion and distant infiltration of the normal neuropil by the neoplastic cells. Histological correlations of this phenomenon were described by Scherer in 1940 (Scherer, 1940). Scherer described the aggregation and migration of glioma cells along normal blood vessels (perivascular satellitosis), neurons (perineuronal satellitosis), below the pial surface (subpial spread), and finally along large white matter tracts (intrafascicular growth). These "Scherer's secondary structures" are easily identified and constitute microscopic landmarks of glioma malignancy (Holland, 2000). However, accurate pathological classification of a tumor is often confounded by an undersampling bias, especially in cases of needle biopsies of large lesions. In addition, there is significant heterogeneity among gliomas at the genetic, epigenetic, and histological level (Walker C. et

There is little doubt that molecular heterogeneity contributes to the clonal evolution (Gerlinger & Swanton, 2010) within a tumor, which directly impacts clinical behavior such as treatment failure and disease recurrence (Yip et al., 2009). The example of the selection of GBM tumor cells carrying inactivating mutations in the mismatch repair gene mutS homolog 6 (*MSH6*) during treatment illustrates well this phenomenon and will be discussed further in section 5. Moreover, one microscopic field of GBM sample taken from the same patient can appear vastly different from another area. In addition, low and high-grade gliomas from different patients, with similar morphological appearance and of the same histological grade, can behave quite differently (Frazier et al., 2009; Krex et al., 2007; Yamanaka et al., 2006). Therefore, traditional histopathology alone is not sufficient to provide information with respect to prognostic and predictive substratification. Fortunately,

altered behavior or seizures (Brada et al., 2001; Chandana et al., 2008).

al., 2003).

molecular biomarkers are making inroads into the clinic diagnostic workup of brain tumors (Jansen et al., 2010; Yip et al., 2008).

Fig. 1. Micro- and macroscopic view of a GBM tumor. a) Astrocytomas, both low and high grade, consist of neoplastic cells with elongated nuclei in association with variable amount of eosinophilic cytoplasm often extended into coarse processes. GBM often display an exaggerated degree of morphologic heterogeneity as seen in this figure, with tumor cells with bizarre and giant nuclei. b) Pseudopalisading necrosis is a cardinal feature of GBM and is characterized by an area of tissue necrosis surrounded immediately by a rim of viable tumor cells. The microenvironment in this region is often characterized by low oxygen tension (hence the necrosis) and activation of various hypoxia-mediated molecular pathways such as HIF-1α in the tumor cells (Kaur et al., 2005; Rong et al., 2006). c) Low magnification survey of a GBM showing multiple foci of microvascular proliferation (MVP) characterized by abnormal proliferation and hypertrophy of endothelial cells as a result of an overly pro-angiogenic environment. The presence of MVP automatically "upgrades" diagnosis from an astrocytoma to a WHO grade IV GBM. Foci of MVP are often found in areas with other malignant histological features including florid mitotic activity and tumor necrosis. d) Intraoperative picture of a GBM showing significant distortion of the gross morphology of the cerebral cortex resulting in expansion and discoloration of the gyri. e) and f) Magnetic resonance imaging (MRI) of a GBM using T1 sequence in conjunction with intravenous administration of gadolinium (GAD) dye. The tumor is represented in the axial (e) and coronal (f) planes, which is essential for localization of the lesion and surgical planning. The surfeit of tumor-associated and abnormally developed blood vessels (i.e. the MVP) with a defective blood-brain barrier (BBB) allows for penetration of GAD into the tissue, and accentuates areas of BBB breakdown. This so-called "contrast- enhancing" appearance is typical of high grade gliomas.

In addition to changes in nucleotide sequences within the genetic makeup of a cancer cell, the epigenome, defined by the profile of selective methylation of CpG-islands and complex

siRNA-Based Therapy for Glioblastoma Patients 165

Severe pulmonary toxicity and limited efficacy of the drug due to insufficient drug delivery motivated the development of carmustine wafers (Gliadel®, Guilford Pharmaceuticals; Figure 2). Administration of carmustine using biodegradable polymer wafers provides a controlled release of the drug in the brain micro-environment while minimizing systemic toxicity (Lin & Kleinberg, 2008). However, recent trials showed that the system provides no additional benefit compared to TMZ (Adamson et al., 2009). A combination regimen consisting of the alkylating agents lomustine and procarbazine, and the microtubules destabilizer vincristine (PCV), has also been used extensively for the treatment of GBM (Kappelle et al., 2001). Yet, high incidence of hematological toxicities and inferior response rates in comparison to TMZ (Kappelle et al., 2001; Stupp et al., 2006) favored the establishment of TMZ as the current standard of care. The topoisomerase I inhibitor irinotecan (CPT-11; Camptosar®, Pharmacia & Upjohn) is a FDA-approved drug for colorectal cancer. Promising results for GBM patients in phase II trials were demonstrated with this compound, especially when used in combination with other agents such as TMZ or carmustine (Brandes et al., 2004b; Friedman et al., 2009; Turner et al., 2002; Vredenburgh et al., 2009), and it may soon be integrated among the Food and Drug Administration (FDA)-approved options for GBM patients. This compound is now recommended by the National Comprehensive Cancer Network for use in combination with bevacizumab

Fig. 2. Intraoperative picture of the placement of Gliadel® wafers into the resection cavity of a recurrent anaplastic oligoastrocytoma. The wafers, impregnated with the alkylating agent carmustine, deliver a locally concentrated dose of chemotherapeutic agent, reducing

More recently, targeted agents have been developed and showed some activity in GBM patients. Among them, a monoclonal antibody raised against the vascular endothelial growth factor (VEGF), bevacizumab, was recently approved by the FDA for the treatment of recurrent GBM, as several clinical trials demonstrated its efficacy as a single agent to prolong the progression-free survival of patients (Friedman et al., 2009; Kreisl et al., 2009).

(Avastin, Genentech/Roche) for GBM treatment (NCCN, 2011).

systemic toxicity.

covalent modifications of histone proteins, is equally important in defining malignant behavior (Ting et al., 2006). Epigenetic modifications result in transcriptional silencing or activation of affected gene or genes, and can have widespread consequences in the expression of genes important in survival, development, and growth regulation. Aberrant epigenetic changes in brain tumors are well documented (Fouse & Costello, 2009; Kim et al., 2006; Wu et al., 2010) and recently, The Cancer Genome Atlas (TCGA) consortium undertook a systematic large scale profiling of the GBM epigenomes (CGARN, 2008). The best-known brain tumor epigenetic biomarker is the hypermethylation of the O-6 methylguanine methyltransferase (*MGMT)* promoter. *MGMT* promoter hypermethylation correlates with an enhanced response to combined temozolomide (TMZ; Temodar®, Merck) chemotherapy and radiotherapy in patients with GBMs (Hegi et al., 2005), and predicts a better prognosis in elderly GBM patients (Gerstner et al., 2009). The discovery of the glioma-CpG Island Methylator Phenotype from epigenome-wide analysis of TCGA glioblastoma has also highlighted the potentials of the epigenome-wide approach (Noushmehr et al., 2010). The information obtained from genetic and epigenetic analyses of a tumor sample can be used to guide the decision of the treatment to offer the patient.

#### **3. Standard of care and other therapeutic options**

The current standard of care for GBM patients starts with maximal safe surgical resection. Patients might undergo repeated surgical resections to treat recurrent tumor growth (Wen & Kesari, 2008). As mentioned in the previous section, neoplastic cells from both low and highgrade glioma do share some common characteristics including the innate ability to infiltrate and invade surrounding normal brain tissue, which results in the distal spread of tumor cells at an early stage of the disease and the difficulty in clearly delineating the tumor margin. Surgical resection is followed by radiation therapy and concurrent TMZ, followed by adjuvant TMZ therapy (Clarke et al., 2010). TMZ is an alkylating agent, triggering cell death by the addition of DNA damaging alkyl groups on guanine bases (Newlands et al., 1997). The inclusion of TMZ in GBM standard of care improved the 2-year survival times from 10.4% to 26.5% (Stupp et al., 2005). However, the 5-year survival of GBM patients still remains less than 10% (CBTRUS, 2010; Stupp et al., 2009). As mentioned earlier, an important predictive factor in response to TMZ is the level of methylation of the *MGMT* promoter region found in the tumor. Promoter methylation of the *MGMT* gene prevents the expression of the MGMT enzyme capable of removing alkyl groups (Jacinto & Esteller, 2007). GBM tumors carrying a non-methylated promoter (40-55% of cases (Hegi et al., 2005; Sadones et al., 2009)) respond poorly to TMZ and there is currently no alternative treatment for these patients. At present, all patients with GBM are treated with TMZ with concurrent radiation therapy. Recurrent GBM tumors are often chemoresistant and exhibit accelerated growth rate (Cahill et al., 2007; Yip et al., 2009). Interestingly, a subset of patients with GBM who recurred after the initial treatment (surgery, radiation, TMZ) does respond to metronomic doses of TMZ. This treatment option consists of a dose-intensive daily intake of the drugs to maintain tumor suppression (Perry et al., 2010).

Before the introduction of TMZ as the standard of care for GBM patients, nitrosoureas were the foundation of GBM treatment for more than 30 years (Stupp et al., 2006). Among them, the alkylating agent carmustine (BiCNU®, Bristol-Myers Squibb) was used in conjunction with radiotherapy (Brandes et al., 2004a; Walker M. D. et al., 1978; Walker M. D. et al., 1980).

covalent modifications of histone proteins, is equally important in defining malignant behavior (Ting et al., 2006). Epigenetic modifications result in transcriptional silencing or activation of affected gene or genes, and can have widespread consequences in the expression of genes important in survival, development, and growth regulation. Aberrant epigenetic changes in brain tumors are well documented (Fouse & Costello, 2009; Kim et al., 2006; Wu et al., 2010) and recently, The Cancer Genome Atlas (TCGA) consortium undertook a systematic large scale profiling of the GBM epigenomes (CGARN, 2008). The best-known brain tumor epigenetic biomarker is the hypermethylation of the O-6 methylguanine methyltransferase (*MGMT)* promoter. *MGMT* promoter hypermethylation correlates with an enhanced response to combined temozolomide (TMZ; Temodar®, Merck) chemotherapy and radiotherapy in patients with GBMs (Hegi et al., 2005), and predicts a better prognosis in elderly GBM patients (Gerstner et al., 2009). The discovery of the glioma-CpG Island Methylator Phenotype from epigenome-wide analysis of TCGA glioblastoma has also highlighted the potentials of the epigenome-wide approach (Noushmehr et al., 2010). The information obtained from genetic and epigenetic analyses of a tumor sample can

The current standard of care for GBM patients starts with maximal safe surgical resection. Patients might undergo repeated surgical resections to treat recurrent tumor growth (Wen & Kesari, 2008). As mentioned in the previous section, neoplastic cells from both low and highgrade glioma do share some common characteristics including the innate ability to infiltrate and invade surrounding normal brain tissue, which results in the distal spread of tumor cells at an early stage of the disease and the difficulty in clearly delineating the tumor margin. Surgical resection is followed by radiation therapy and concurrent TMZ, followed by adjuvant TMZ therapy (Clarke et al., 2010). TMZ is an alkylating agent, triggering cell death by the addition of DNA damaging alkyl groups on guanine bases (Newlands et al., 1997). The inclusion of TMZ in GBM standard of care improved the 2-year survival times from 10.4% to 26.5% (Stupp et al., 2005). However, the 5-year survival of GBM patients still remains less than 10% (CBTRUS, 2010; Stupp et al., 2009). As mentioned earlier, an important predictive factor in response to TMZ is the level of methylation of the *MGMT* promoter region found in the tumor. Promoter methylation of the *MGMT* gene prevents the expression of the MGMT enzyme capable of removing alkyl groups (Jacinto & Esteller, 2007). GBM tumors carrying a non-methylated promoter (40-55% of cases (Hegi et al., 2005; Sadones et al., 2009)) respond poorly to TMZ and there is currently no alternative treatment for these patients. At present, all patients with GBM are treated with TMZ with concurrent radiation therapy. Recurrent GBM tumors are often chemoresistant and exhibit accelerated growth rate (Cahill et al., 2007; Yip et al., 2009). Interestingly, a subset of patients with GBM who recurred after the initial treatment (surgery, radiation, TMZ) does respond to metronomic doses of TMZ. This treatment option consists of a dose-intensive daily intake of

Before the introduction of TMZ as the standard of care for GBM patients, nitrosoureas were the foundation of GBM treatment for more than 30 years (Stupp et al., 2006). Among them, the alkylating agent carmustine (BiCNU®, Bristol-Myers Squibb) was used in conjunction with radiotherapy (Brandes et al., 2004a; Walker M. D. et al., 1978; Walker M. D. et al., 1980).

be used to guide the decision of the treatment to offer the patient.

**3. Standard of care and other therapeutic options** 

the drugs to maintain tumor suppression (Perry et al., 2010).

Severe pulmonary toxicity and limited efficacy of the drug due to insufficient drug delivery motivated the development of carmustine wafers (Gliadel®, Guilford Pharmaceuticals; Figure 2). Administration of carmustine using biodegradable polymer wafers provides a controlled release of the drug in the brain micro-environment while minimizing systemic toxicity (Lin & Kleinberg, 2008). However, recent trials showed that the system provides no additional benefit compared to TMZ (Adamson et al., 2009). A combination regimen consisting of the alkylating agents lomustine and procarbazine, and the microtubules destabilizer vincristine (PCV), has also been used extensively for the treatment of GBM (Kappelle et al., 2001). Yet, high incidence of hematological toxicities and inferior response rates in comparison to TMZ (Kappelle et al., 2001; Stupp et al., 2006) favored the establishment of TMZ as the current standard of care. The topoisomerase I inhibitor irinotecan (CPT-11; Camptosar®, Pharmacia & Upjohn) is a FDA-approved drug for colorectal cancer. Promising results for GBM patients in phase II trials were demonstrated with this compound, especially when used in combination with other agents such as TMZ or carmustine (Brandes et al., 2004b; Friedman et al., 2009; Turner et al., 2002; Vredenburgh et al., 2009), and it may soon be integrated among the Food and Drug Administration (FDA)-approved options for GBM patients. This compound is now recommended by the National Comprehensive Cancer Network for use in combination with bevacizumab (Avastin, Genentech/Roche) for GBM treatment (NCCN, 2011).

Fig. 2. Intraoperative picture of the placement of Gliadel® wafers into the resection cavity of a recurrent anaplastic oligoastrocytoma. The wafers, impregnated with the alkylating agent carmustine, deliver a locally concentrated dose of chemotherapeutic agent, reducing systemic toxicity.

More recently, targeted agents have been developed and showed some activity in GBM patients. Among them, a monoclonal antibody raised against the vascular endothelial growth factor (VEGF), bevacizumab, was recently approved by the FDA for the treatment of recurrent GBM, as several clinical trials demonstrated its efficacy as a single agent to prolong the progression-free survival of patients (Friedman et al., 2009; Kreisl et al., 2009).

siRNA-Based Therapy for Glioblastoma Patients 167

uptake or routing of the hybrid compound to lysosome compartments for degradation

Fig. 3. Electron micrograph showing the structure of brain capillaries from normal and tumor tissue. a) This normal brain capillary is made of two endothelial cells bound together with tight junctions (J) and forming a thin and regular layer around the lumen. Processes of pericytes (P) can be seen embedded in the basement membrane. b, c, d) Capillaries from astrocytoma tumors are lined by immature endothelial cells (E) of irregular thickness and with hyperplastic nuclei (N), discontinuous basement membrane (BM) and a decreased presence of pericytes or astrocytic processes. Abnormal intercellular junctions (AJ),

fenestrations (F) and irregular slit-like lumen (L) can be seen in these micrographs. Adapted

In the case where therapeutic agents capable of crossing the BBB, such as TMZ or irinotecan, are available and do exhibit considerable anticancer activity, the most significant problem for GBM cancer patients is repopulation of malignant cells following treatment, causing inevitable relapse (CBTRUS, 2010; Stupp et al., 2005). For example, recent identification of the somatic inactivation of the mismatch repair gene *MSH6* in a subgroup of recurrent GBM has highlighted the constant evolution of malignant glioma cells in the presence of selective pressure, in this case alkylator chemotherapy and concurrent radiotherapy. This initial discovery, achieved through whole kinome sequencing of two recurrent GBM, was bolstered by corroborative findings from the TCGA project studying a much larger number of GBM samples (CGARN, 2008; Hunter et al., 2006). Subsequent studies have also highlighted the importance of somatic *MSH6* integrity on the *in vivo* and *in vitro* growth of

from (Deane & Lantos, 1981; Rojiani & Dorovini-Zis, 1996).

**5. The targets and therapeutic effects** 

(Banks, 2009).

However, results have been disappointing in terms of improvements in median survival, and a clinical trial is currently ongoing and was designed to investigate the value of combining bevacizumab with TMZ (Chinot et al., 2011). It is hoped that bevacizumab therapy can induce a normalization of the tumor vasculature (see section 10) and thus improve the homogenous delivery of the cytotoxic agent TMZ. The EGFR inhibitors gefitinib (Iressa®, AstraZeneca Canada Inc.) and erlotinib (Tarceva®, Roche) have also shown some efficacy in the treatment of malignant glioma (Raizer et al., 2010; Rich et al., 2004), but response rates have been variable and unpredictable (Stupp et al., 2006). Imatinib mesylate (Gleevec®, Novartis) was developed to specifically inhibit Bcr-Abl signal transduction in chronic myeloid leukemia, and was later shown to also inhibit c-Kit and platelet-derived growth factor receptor (PDGFR) activity. The latter finding supported clinical testing of imatinib for GBM treatment, and similarly to the EGFR inhibitors, response rates were variable but promising (Razis et al., 2009; Wen et al., 2006). Other targeted therapy compounds currently evaluated for use in GBM treatment are described in section 5.

#### **4. Challenges**

One of the major challenges of GBM chemotherapy is the achievement of adequate drug concentration within the tumor itself, and this obstacle can largely be attributed to the presence of the BBB (Clarke et al., 2010). Unlike capillaries elsewhere in the body, endothelial cells of brain capillaries have tight junctions that are highly resistant to the passage of ions or small molecules and do not exhibit trans-endothelial transport (Fawcett, 1994). Moreover, the astrocytes and pericytes play an important role in regulating this barrier through molecular cross-talk involving adhesion and tight-junction molecules, the integrins and other extra-cellular matrix (ECM) molecules (reviewed in (Wolburg et al., 2009)). The BBB in GBM tumors exhibits more frequent fenestrations, a loss of tight intercellular junctions and less developed astrocytic pericapillary sheath, all factors contributing to increasing its permeability (Engerhard et al., 1999) (Figure 3). However, this compromised BBB still acts as an obstacle for many drugs (Pardridge, 2007). Therefore, the main limitation for drug choice in the treatment of GBM is the capacity of the compound to penetrate the BBB. Two main mechanisms by which a synthetic drug molecule can cross the BBB are: 1) by transmembrane diffusion or 2) through transporters (Banks, 2009). Most drugs used for management of GBM (e.g. TMZ, carmustine) are small and lipophilic molecules that cross the BBB by transmembrane diffusion. This mechanism is a nonsaturable process that depends on molecular diffusion through cell membranes. Factors influencing this process include a good balance between liposolubility (penetration through cell membrane) and hydrosolubility (to improve drug circulation and presentation to the BBB) which are influenced by chemical structure, size and charge (Banks, 2009). The capacity to escape the ATP-Binding Cassette (ABC) transporters, such as the P-glycoprotein (Begley, 2004a) responsible for brain-to-blood efflux, is also important. Saturable transport systems (i.e. ligand transporters) can be used to improve the pharmacokinetic profile of a substance and to target uptake into specific regions of the central nervous system (CNS) (Banks, 2009). Analogs of transporter ligands have been developed for various CNS pathologies (Begley, 2004b). Alternatively, influx transporters can be targeted in a "Trojanhorse" like strategy (i.e. molecules that do not cross the BBB are coupled to ligand molecules that do) but unfortunately, these chemical modifications often result in a decrease in ligand

However, results have been disappointing in terms of improvements in median survival, and a clinical trial is currently ongoing and was designed to investigate the value of combining bevacizumab with TMZ (Chinot et al., 2011). It is hoped that bevacizumab therapy can induce a normalization of the tumor vasculature (see section 10) and thus improve the homogenous delivery of the cytotoxic agent TMZ. The EGFR inhibitors gefitinib (Iressa®, AstraZeneca Canada Inc.) and erlotinib (Tarceva®, Roche) have also shown some efficacy in the treatment of malignant glioma (Raizer et al., 2010; Rich et al., 2004), but response rates have been variable and unpredictable (Stupp et al., 2006). Imatinib mesylate (Gleevec®, Novartis) was developed to specifically inhibit Bcr-Abl signal transduction in chronic myeloid leukemia, and was later shown to also inhibit c-Kit and platelet-derived growth factor receptor (PDGFR) activity. The latter finding supported clinical testing of imatinib for GBM treatment, and similarly to the EGFR inhibitors, response rates were variable but promising (Razis et al., 2009; Wen et al., 2006). Other targeted therapy

compounds currently evaluated for use in GBM treatment are described in section 5.

One of the major challenges of GBM chemotherapy is the achievement of adequate drug concentration within the tumor itself, and this obstacle can largely be attributed to the presence of the BBB (Clarke et al., 2010). Unlike capillaries elsewhere in the body, endothelial cells of brain capillaries have tight junctions that are highly resistant to the passage of ions or small molecules and do not exhibit trans-endothelial transport (Fawcett, 1994). Moreover, the astrocytes and pericytes play an important role in regulating this barrier through molecular cross-talk involving adhesion and tight-junction molecules, the integrins and other extra-cellular matrix (ECM) molecules (reviewed in (Wolburg et al., 2009)). The BBB in GBM tumors exhibits more frequent fenestrations, a loss of tight intercellular junctions and less developed astrocytic pericapillary sheath, all factors contributing to increasing its permeability (Engerhard et al., 1999) (Figure 3). However, this compromised BBB still acts as an obstacle for many drugs (Pardridge, 2007). Therefore, the main limitation for drug choice in the treatment of GBM is the capacity of the compound to penetrate the BBB. Two main mechanisms by which a synthetic drug molecule can cross the BBB are: 1) by transmembrane diffusion or 2) through transporters (Banks, 2009). Most drugs used for management of GBM (e.g. TMZ, carmustine) are small and lipophilic molecules that cross the BBB by transmembrane diffusion. This mechanism is a nonsaturable process that depends on molecular diffusion through cell membranes. Factors influencing this process include a good balance between liposolubility (penetration through cell membrane) and hydrosolubility (to improve drug circulation and presentation to the BBB) which are influenced by chemical structure, size and charge (Banks, 2009). The capacity to escape the ATP-Binding Cassette (ABC) transporters, such as the P-glycoprotein (Begley, 2004a) responsible for brain-to-blood efflux, is also important. Saturable transport systems (i.e. ligand transporters) can be used to improve the pharmacokinetic profile of a substance and to target uptake into specific regions of the central nervous system (CNS) (Banks, 2009). Analogs of transporter ligands have been developed for various CNS pathologies (Begley, 2004b). Alternatively, influx transporters can be targeted in a "Trojanhorse" like strategy (i.e. molecules that do not cross the BBB are coupled to ligand molecules that do) but unfortunately, these chemical modifications often result in a decrease in ligand

**4. Challenges** 

uptake or routing of the hybrid compound to lysosome compartments for degradation (Banks, 2009).

Fig. 3. Electron micrograph showing the structure of brain capillaries from normal and tumor tissue. a) This normal brain capillary is made of two endothelial cells bound together with tight junctions (J) and forming a thin and regular layer around the lumen. Processes of pericytes (P) can be seen embedded in the basement membrane. b, c, d) Capillaries from astrocytoma tumors are lined by immature endothelial cells (E) of irregular thickness and with hyperplastic nuclei (N), discontinuous basement membrane (BM) and a decreased presence of pericytes or astrocytic processes. Abnormal intercellular junctions (AJ), fenestrations (F) and irregular slit-like lumen (L) can be seen in these micrographs. Adapted from (Deane & Lantos, 1981; Rojiani & Dorovini-Zis, 1996).

#### **5. The targets and therapeutic effects**

In the case where therapeutic agents capable of crossing the BBB, such as TMZ or irinotecan, are available and do exhibit considerable anticancer activity, the most significant problem for GBM cancer patients is repopulation of malignant cells following treatment, causing inevitable relapse (CBTRUS, 2010; Stupp et al., 2005). For example, recent identification of the somatic inactivation of the mismatch repair gene *MSH6* in a subgroup of recurrent GBM has highlighted the constant evolution of malignant glioma cells in the presence of selective pressure, in this case alkylator chemotherapy and concurrent radiotherapy. This initial discovery, achieved through whole kinome sequencing of two recurrent GBM, was bolstered by corroborative findings from the TCGA project studying a much larger number of GBM samples (CGARN, 2008; Hunter et al., 2006). Subsequent studies have also highlighted the importance of somatic *MSH6* integrity on the *in vivo* and *in vitro* growth of

siRNA-Based Therapy for Glioblastoma Patients 169

**Self-sufficiency in growth signals and insensitivity to growth suppressors** 

shRNA in PEGylated immunolip. (h-Insuline R and m-Transferrin R) i.v.

ASO plasmid in PEGylated immunolip. (h-Insuline R and m-Transferrin R) i.v.

(Aprinocarsen)

PEI-complexed siRNA i.t.

Agent Benefit Ref

shRNA i.p. Tumor growth inhibition

siRNA cDNA i.p. Enhanced efficacy of taxol

shRNA i.t. Tumor growth inhibition

ASO in cat. lip. Tumor growth inhibition.

GRN183 ASO i.n. Tumor growth inhibition

Table 1. Protein targets that have been inhibited by means of non-viral gene silencing agents in orthotopic GBM pre-clinical studies or clinical trials (CTr) (definition of abbreviations is

radiation

Tumor growth inhibition and increased survival

Tumor growth inhibition and increased survival

and increased survival

on tumor growth inhibition

PEI-siRNA i.t. Tumor growth inhibition (Grzelinski et al.,

shRNA i.t. Tumor growth inhibition (Gondi et al., 2004)

and enhanced sensitivity to

More active when combined with IFN-γ injections

ASO i.t. Increased survival (Mukai et al., 2000)

and increased survival

Increased survival when given with immunization

Minimal benefit (Grossman et al.,

Increased survival (Hendruschk et al.,

Increased survival (Wyszko et al.,

2005)

2011)

2006)

2006)

2008)

2008)

(Zhang et al., 2004)

(Zhang et al., 2002)

(Gondi et al., 2007)

(Badiga et al., 2011; Lakka et al., 2005)

2008; Zukiel et al.,

(Hashizume et al.,

(Schneider et al.,

(George et al., 2009)

(George et al., 2008)

Target Experimental design

EGFR I.cr. U87 line in mice

mice

UPA/UPAR 4910 line i.cr. in mice

Bcl-2 U251 line i.cr. in mice

Survivin U87 line i.cr. in mice

**Induction of angiogenesis** Pleitrophin U87 line i.cr. in mice

**Invasion and metastasis** Cathepsin B SNB19 line i.cr. in mice

**Replicative immortality** hTERT SNB-19 and LN-

**Other function** 

MMPs SNB19 or U251

line i.cr. in mice

18 lines i.cr. in

U373 line i.cr. in

U251MG i.cr. in

given at the beginning of this chapter)

mice

mice

rats

TGF-β F98 line i.cr. in rats

Tenascin-C Phase I CTr Long RNA molecule

i.t.

Nanoparticlecomplexed ASO i.p.

**Evasion of cell death**

PKC family Phase II CTr ASO i.v.

I.cr U87 line in

GBM with direct impact on patient survival (Cahill et al., 2007; Yip et al., 2009). In fact, a careful evaluation of the TCGA data of the recurrent GBM cohort shows that a majority of the recurrent tumors with the *MSH6* hypermutation phenotype, a genetic signature of defects in the cellular mismatch repair (MMR) system, also have somatic mutations and epigenetic silencing affecting other members of the MMR family. This highlights a novel mechanism of tumor survival during alkylator therapy and is especially relevant in a cancer for which alkylators, such as TMZ, are used as the therapeutic mainstay. One possible explanation for this phenomenon is that a small number of GBM cells, prior to therapy, harbor mutations in the MMR genes and these cells are positively selected during therapy, leading to clonal expansion. Alternatively, and equally plausible, random mutations inactivating MMR genes during therapy could lead to outgrowth of a resistant clone of tumor cells which exhibits therapeutic resistance.

Although it is acknowledged that advances in GBM treatment will continue to rely on conventional treatment approaches (surgery, radiation and/or chemotherapy), the value of combining standard chemotherapy with targeted agents that increase tumor drug sensitivity is now being recognized (Krakstad & Chekenya, 2010). The goal of this effort is to target survival and/or proliferation-promoting proteins which are overexpressed, or have upregulated activity, in cancer cells. This approach has the additional benefit of overcoming resistance mechanisms due to the activation of single pathways (e.g. *MGMT*). Moreover, many trials have indicated that groups of cancer patients treated similarly on the basis of a histopathology classification (e.g. WHO II astrocytoma, WHO III anaplastic astrocytoma, and WHO IV glioblastoma) exhibit very different responses to the same treatment (Stupp et al., 2006), implying that various gene expression patterns underlying the disease phenotype may play a more important role in treatment outcomes. This observation highlights the necessity of developing tumor-targeted gene therapy treatments that would be personalized for the specific molecular lesions present in a patient's tumor.

Cancer is believed to be a genetic disease, arising as a result of genetic mutations that endow the cell with many specific functional capabilities (Vogelstein & Kinzler, 1993) such as cell proliferation, survival, invasion and metastasis (Hanahan & Weinberg, 2000). Recent advances in molecular genetics have enabled the development of methods to specifically target gene expression (small interfering RNA; siRNA or antisense oligonucleotides; ASO) or the activity of proteins (small molecule inhibitors; SMI). Our current understanding of cancer biology has made it clear that gene targeting therapies will provide an effective strategy to treat cancer. The next paragraphs will present an overview of the approaches used in targeted therapy for the treatment of malignant glioma. The targets and potential therapeutic effects will be discussed based on the six essential alterations in cell physiology that dictate malignant growth as described elsewhere (Hanahan & Weinberg, 2000,2011) and consist of the capacity for (I) sustaining proliferative signals and II) evading growth suppressors; (III) resisting cell death; (IV) inducing angiogenesis; (V) activating invasion and metastasis and (VI) enabling replicative immortality. Table 1 and 2 present a list of genes or associated protein targets that have been inhibited by means of non-viral gene silencing agents in orthotopic GBM pre-clinical or clinical studies (Table 1), or by SMI in GBM clinical trials phase II/III (Table 2). It is interesting to note that very few of these studies have assessed the efficacy of the targeted therapy agent in combination with standard chemotherapy or radiation.

GBM with direct impact on patient survival (Cahill et al., 2007; Yip et al., 2009). In fact, a careful evaluation of the TCGA data of the recurrent GBM cohort shows that a majority of the recurrent tumors with the *MSH6* hypermutation phenotype, a genetic signature of defects in the cellular mismatch repair (MMR) system, also have somatic mutations and epigenetic silencing affecting other members of the MMR family. This highlights a novel mechanism of tumor survival during alkylator therapy and is especially relevant in a cancer for which alkylators, such as TMZ, are used as the therapeutic mainstay. One possible explanation for this phenomenon is that a small number of GBM cells, prior to therapy, harbor mutations in the MMR genes and these cells are positively selected during therapy, leading to clonal expansion. Alternatively, and equally plausible, random mutations inactivating MMR genes during therapy could lead to outgrowth of a resistant clone of

Although it is acknowledged that advances in GBM treatment will continue to rely on conventional treatment approaches (surgery, radiation and/or chemotherapy), the value of combining standard chemotherapy with targeted agents that increase tumor drug sensitivity is now being recognized (Krakstad & Chekenya, 2010). The goal of this effort is to target survival and/or proliferation-promoting proteins which are overexpressed, or have upregulated activity, in cancer cells. This approach has the additional benefit of overcoming resistance mechanisms due to the activation of single pathways (e.g. *MGMT*). Moreover, many trials have indicated that groups of cancer patients treated similarly on the basis of a histopathology classification (e.g. WHO II astrocytoma, WHO III anaplastic astrocytoma, and WHO IV glioblastoma) exhibit very different responses to the same treatment (Stupp et al., 2006), implying that various gene expression patterns underlying the disease phenotype may play a more important role in treatment outcomes. This observation highlights the necessity of developing tumor-targeted gene therapy treatments that would be personalized

Cancer is believed to be a genetic disease, arising as a result of genetic mutations that endow the cell with many specific functional capabilities (Vogelstein & Kinzler, 1993) such as cell proliferation, survival, invasion and metastasis (Hanahan & Weinberg, 2000). Recent advances in molecular genetics have enabled the development of methods to specifically target gene expression (small interfering RNA; siRNA or antisense oligonucleotides; ASO) or the activity of proteins (small molecule inhibitors; SMI). Our current understanding of cancer biology has made it clear that gene targeting therapies will provide an effective strategy to treat cancer. The next paragraphs will present an overview of the approaches used in targeted therapy for the treatment of malignant glioma. The targets and potential therapeutic effects will be discussed based on the six essential alterations in cell physiology that dictate malignant growth as described elsewhere (Hanahan & Weinberg, 2000,2011) and consist of the capacity for (I) sustaining proliferative signals and II) evading growth suppressors; (III) resisting cell death; (IV) inducing angiogenesis; (V) activating invasion and metastasis and (VI) enabling replicative immortality. Table 1 and 2 present a list of genes or associated protein targets that have been inhibited by means of non-viral gene silencing agents in orthotopic GBM pre-clinical or clinical studies (Table 1), or by SMI in GBM clinical trials phase II/III (Table 2). It is interesting to note that very few of these studies have assessed the efficacy of the targeted therapy agent in combination with standard

tumor cells which exhibits therapeutic resistance.

for the specific molecular lesions present in a patient's tumor.

chemotherapy or radiation.


Table 1. Protein targets that have been inhibited by means of non-viral gene silencing agents in orthotopic GBM pre-clinical studies or clinical trials (CTr) (definition of abbreviations is given at the beginning of this chapter)

siRNA-Based Therapy for Glioblastoma Patients 171

GBM cases; EGFR gene amplification was reported in 40% of cases; EGFR truncated transcript encoding for a constitutive activity of the receptor was reported in 20% of cases and mutations of EGFR extracellular domain was reported in 15% of cases (Nicholas et al., 2006; Ohgaki et al., 2004). These mutations are quite often combined in the same tumor cell, leading to overactivation of EGFR pathways (Ekstrand et al., 1991; Idbaih et al., 2008). ASO- (Zhang et al., 2002) and RNA interference- (Zhang et al., 2004) mediated inhibition of EGFR has been shown to induce a strong reduction in tumor growth and increased the survival of orthotopic GBM tumor bearing mice. However, the inhibition of EGFR using SMI in clinical phase II trials was not representative of this success. As discussed earlier, erlotinib (Prados M. D. et al., 2009; Raizer et al., 2010) or gefitinib (Franceschi et al., 2007; Rich et al., 2004) SMI showed variable activity in GBM patients although better efficacy was seen when used in combination with TMZ. The constitutive activation of the PI3K/AKT pathway associated with the mutation of PTEN (60% of GBM cases) (Haas-Kogan et al., 1998; Knobbe et al., 2002) has also received considerable attention as it is generally associated with aggressive disease and poor prognosis (Ermoian et al., 2002; Rasheed et al., 1997; Schmidt et al., 1999). The mammalian target of rapamycin (mTOR) was identified as a major downstream effector in this pathway (Manning & Cantley, 2003). Phase II clinical trials using the mTOR SMI Temsirolimus (CCI-779) induced disease stabilization and an increase in survival in some

GBM patients of phase II clinical trials (Chang et al., 2005; Galanis et al., 2005).

in a pre-clinical orthotopic model of GBM (George et al., 2008).

The ability of tumor cell populations to expand in number is determined not only by the rate of cell proliferation but also by the rate of cell death. Acquired resistance toward programmed cell death, apoptosis, is a hallmark of most and perhaps all types of cancer (Hanahan & Weinberg, 2000,2011), causing the tumor to resist conditions that would normally kill cells. In this context, Bcl-2 targeted therapy has attracted the most attention with studies both in pre-clinical cancer models and patients (ASO Oblimersen, SMI AT-101). Although the potential of Bcl-2 targeting has not yet been demonstrated in GBM patients, one report showed that siRNA-mediated inhibition of Bcl-2 can increase the efficacy of taxol

Strong evidence indicates that the growth of GBM depends on angiogenesis (Jain et al., 2007; Norden et al., 2009). However, experimental models of GBM have shown that the resulting vessels are poorly organized and poorly functional, and it is believed that high levels of angiogenesis in GBM are associated with increased hypoxia and interstitial fluid pressure, which contribute to the disease malignancy and resistance to treatments (Blasberg et al., 1983; Groothuis et al., 1983). This aspect of GBM vasculature will be discussed further in section 10. Members of the VEGF family have emerged as prime mediators of angiogenesis in GBM (Jain et al., 2007). As already noted, several clinical trials have demonstrated the efficacy of bevacizumab, a monoclonal antibody (mAb) against VEGF as a single agent and in combination with TMZ or irinotecan to prolong the survival of patients (Friedman et al., 2009; Kreisl et al., 2009; Lai et al., 2011). Bevacizumab was recently approved by the FDA for the treatment of recurrent GBM and is showing promising efficacy in clinical trials for newly

**5.2 Evasion of cell death** 

**5.3 Induction of angiogenesis** 


Table 2. Protein targets that have been inhibited by means of SMI or mAb in GBM clinical trials phase II/III (definition of abbreviations is given at the beginning of this chapter)

#### **5.1 Self-sufficiency in growth signals and insensitivity to growth suppressors**

Tumor cells generate many of their own growth signals, thereby reducing their dependence on stimulation from the normal tissue microenvironment (Hanahan & Weinberg, 2000,2011). Molecular strategies for achieving autonomy involve: I) alterations in extracellular growth signals, II) alterations in transducers of those signals, III) alterations in components enabling or preventing the cell to enter cell cycle (Hanahan & Weinberg, 2000). For example, protein overexpression of EGFR, involved in growth signal transduction, was reported in 60% of

PKC family Phase II/III CTr SMI Enzastaurin Minimal benefit (Kreisl et al., 2010;

**Self-sufficiency in growth signals and insensitivity to growth suppressors** 

(Tarceva®), Gefitinib (Iressa®)

Phase II CTr SMI Suramin i.v No benefit with

(Gleevec®) i.v

(Avastin) i.v.

Phase II CTr Thalidomide i.v. Minimal benefit as a

Integrins Phase I/II CTr SMI Cilengitide i.v. Some activity with TMZ (Stupp et al.,

Table 2. Protein targets that have been inhibited by means of SMI or mAb in GBM clinical trials phase II/III (definition of abbreviations is given at the beginning of this chapter)

Tumor cells generate many of their own growth signals, thereby reducing their dependence on stimulation from the normal tissue microenvironment (Hanahan & Weinberg, 2000,2011). Molecular strategies for achieving autonomy involve: I) alterations in extracellular growth signals, II) alterations in transducers of those signals, III) alterations in components enabling or preventing the cell to enter cell cycle (Hanahan & Weinberg, 2000). For example, protein overexpression of EGFR, involved in growth signal transduction, was reported in 60% of

**5.1 Self-sufficiency in growth signals and insensitivity to growth suppressors** 

HDAC Phase II CTr SMI Vorinostat i.v Modest activity (Galanis et al.,

i.v.

i.v

Agent Benefit Ref

Minimal as single-agent. Some efficacy with TMZ

Disease stabilization and increased survival

Active, with and w/o irinotecan or TMZ and FDA-approved for recurrent disease

single agent, some benefit with carmustine

or irinotecan

radiotherapy

Phase II CTr SMI Tipifarnib i.v No benefit (Cloughesy et al.,

(Franceschi et al., 2007; Prados M. D. et al., 2009; Raizer et al., 2010)

Wick et al., 2010)

(Chang et al., 2005; Galanis et al., 2005)

(Laterra et al.,

2006; Lustig et al.,

Wen et al., 2006)

(Friedman et al., 2009; Kreisl et al., 2009; Lai et al.,

(Fadul et al., 2008; Fine et al., 2003; Marx et al., 2001)

2004)

2008)

2009)

2011)

2010)

Variable response (Razis et al., 2009;

Target Experimental

Growth factors

Farnesyltransferase

Fibroblast growth factors

design

EGFR Phase II CTr SMI Erlotinib

mTOR Phase II CTr SMI Temsirolimus

PDGF R Phase II CTr SMI Imatinib

VEGF Phase II/III CTr mAb Bevacizumab

**Induction of angiogenesis**

**Invasion and metastasis**

GBM cases; EGFR gene amplification was reported in 40% of cases; EGFR truncated transcript encoding for a constitutive activity of the receptor was reported in 20% of cases and mutations of EGFR extracellular domain was reported in 15% of cases (Nicholas et al., 2006; Ohgaki et al., 2004). These mutations are quite often combined in the same tumor cell, leading to overactivation of EGFR pathways (Ekstrand et al., 1991; Idbaih et al., 2008). ASO- (Zhang et al., 2002) and RNA interference- (Zhang et al., 2004) mediated inhibition of EGFR has been shown to induce a strong reduction in tumor growth and increased the survival of orthotopic GBM tumor bearing mice. However, the inhibition of EGFR using SMI in clinical phase II trials was not representative of this success. As discussed earlier, erlotinib (Prados M. D. et al., 2009; Raizer et al., 2010) or gefitinib (Franceschi et al., 2007; Rich et al., 2004) SMI showed variable activity in GBM patients although better efficacy was seen when used in combination with TMZ. The constitutive activation of the PI3K/AKT pathway associated with the mutation of PTEN (60% of GBM cases) (Haas-Kogan et al., 1998; Knobbe et al., 2002) has also received considerable attention as it is generally associated with aggressive disease and poor prognosis (Ermoian et al., 2002; Rasheed et al., 1997; Schmidt et al., 1999). The mammalian target of rapamycin (mTOR) was identified as a major downstream effector in this pathway (Manning & Cantley, 2003). Phase II clinical trials using the mTOR SMI Temsirolimus (CCI-779) induced disease stabilization and an increase in survival in some GBM patients of phase II clinical trials (Chang et al., 2005; Galanis et al., 2005).

#### **5.2 Evasion of cell death**

The ability of tumor cell populations to expand in number is determined not only by the rate of cell proliferation but also by the rate of cell death. Acquired resistance toward programmed cell death, apoptosis, is a hallmark of most and perhaps all types of cancer (Hanahan & Weinberg, 2000,2011), causing the tumor to resist conditions that would normally kill cells. In this context, Bcl-2 targeted therapy has attracted the most attention with studies both in pre-clinical cancer models and patients (ASO Oblimersen, SMI AT-101). Although the potential of Bcl-2 targeting has not yet been demonstrated in GBM patients, one report showed that siRNA-mediated inhibition of Bcl-2 can increase the efficacy of taxol in a pre-clinical orthotopic model of GBM (George et al., 2008).

#### **5.3 Induction of angiogenesis**

Strong evidence indicates that the growth of GBM depends on angiogenesis (Jain et al., 2007; Norden et al., 2009). However, experimental models of GBM have shown that the resulting vessels are poorly organized and poorly functional, and it is believed that high levels of angiogenesis in GBM are associated with increased hypoxia and interstitial fluid pressure, which contribute to the disease malignancy and resistance to treatments (Blasberg et al., 1983; Groothuis et al., 1983). This aspect of GBM vasculature will be discussed further in section 10. Members of the VEGF family have emerged as prime mediators of angiogenesis in GBM (Jain et al., 2007). As already noted, several clinical trials have demonstrated the efficacy of bevacizumab, a monoclonal antibody (mAb) against VEGF as a single agent and in combination with TMZ or irinotecan to prolong the survival of patients (Friedman et al., 2009; Kreisl et al., 2009; Lai et al., 2011). Bevacizumab was recently approved by the FDA for the treatment of recurrent GBM and is showing promising efficacy in clinical trials for newly

siRNA-Based Therapy for Glioblastoma Patients 173

more efficacious than single agent chemotherapy in the treatment of aggressive cancers, clinicians and scientists are now beginning to realize the benefits of combining agents targeting different biological pathways in order to effectively silence as many cancer

The therapeutic value of targeting two different pathways is exemplified by some research data obtained by our laboratory (Verreault et al., 2011a). One of the most commonly reported molecular defects in GBM is the aberrant activation of the PI3K/AKT pathway, which is associated with increased proliferation rate, invasion, metastasis and poor prognosis (Ermoian et al., 2002; Haas-Kogan et al., 1998; Li X. Y. et al., 2010). Rictor, the rapamycin-insensitive companion of mTOR, is a protein member of the mTOR Complex 2 (mTORC2), and can activate AKT through direct phosphorylation at its serine 473 site (Sarbassov et al., 2004; Sarbassov et al., 2005; Sparks & Guertin, 2010). Elevated levels of Rictor were found in human GBM tumor tissue samples and cell lines when compared to normal brain tissue (Masri et al., 2007). Rictor and EGFR proteins were silenced alone and in combination by siRNA *in vitro* transfection in a panel of three human GBM lines (U251MG, U118MG and LN229). It was found that the co-silencing of Rictor and EGFR exerted effects on cell migration and sensitivity to chemotherapeutic drugs that were not observed by the single silencing of either target (Verreault et al., 2011a). The most striking evidence of the validity of this combined silencing came from the *in vivo* aspect of this study, which was done by intracranial inoculation in mice brains of U251MG cells expressing small hairpin RNA (shRNA) specific to each target. Silencing of EGFR or Rictor alone had no significant effect on tumor growth, but the dual silencing resulted in the eradication of the tumor (Verreault et al., 2011a). Also, tumor growth block in response to the combined suppression of EGFR and PI3K/AKT pathway was reported previously using the SMIs gefitinib and LY294002 in a GBM xenograft model (Fan et al., 2003), while monotherapy of each inhibitor had no impact on tumor burden. Taken together, these studies strongly support the value of inhibiting both EGFR and PI3K/mTORC2/Rictor/AKT pathways to achieve therapeutic effects that may not be observed by the single inhibition of either pathway, and provide compelling evidences of the potential of targeting multiple pro-oncogenic pathways in

The idea of targeting gene expression at the level of transcription or translation has been mirrored by the emergence of gene therapy as a strategy to specifically silence the activity of any defective or overactive gene without the limiting step of SMI availability (Bumcrot et al., 2006). Since tumor cells have a different pattern of gene expression in comparison with normal cells, gene silencing can theoretically be used to specifically target tumor-associated genes or mutated genes without altering gene expression of normal cells (Helene, 1994). The most commonly used strategies employed to achieve gene silencing involve administration of ASOs or siRNAs that can inhibit the expression of specific proteins. However, the therapeutic value of this technology is proving difficult to establish, in part because of the lack of pharmaceutically viable products that can be administered orally, intravenously or intraperitoneally, and that can deliver the gene silencing agent to tumor cell populations in sufficient quantity to achieve target knockdown. Despite the fact that we are still learning to navigate the technology of RNAi in order to achieve optimal benefits with minimal side-

phenotypes as possible.

GBM.

**7. Drug delivery to brain tumors** 

diagnosed GBM patients (Lai et al., 2008). Many other VEGF or VEGFR inhibitors are currently being tested in the clinic (Norden et al., 2009).

#### **5.4 Invasion and metastasis**

Proteins responsible for tissue invasion and metastatic behavior are often effectors allowing the cell to grow in the absence of ECM adhesion signals. The most obvious example is the integrins family, which is involved in ECM anchorage-independent growth of tumor cells, and provides the traction necessary for cell motility and invasion (reviewed in (Desgrosellier & Cheresh, 2010)). An integrin SMI, cilengitide, has shown some promising activity in GBM clinical trial phase I/II in combination with TMZ (Stupp et al., 2010), and is now moving to phase III (Carter, 2010). In addition, enzymes that are involved in the degradation of the ECM will allow cancer cells to invade surrounding brain tissue. Matrix-metalloproteinases (MMPs) were shown to play a central role in the proteolysis necessary for this process (Nakada et al., 2003). Intratumoral administration of a shRNA against MMP-9 inhibited tumor growth in an orthotopic GBM mouse model (Lakka et al., 2005). To date, no MMP inhibitors have made their way to a phase II clinical trial for GBM treatment. Moreover, the clinical evaluation of MMP inhibitors as single agents in cancers other than GBM has not been associated with significant anti-tumor responses (Brinker et al., 2008; Chu et al., 2007) and they will most likely show better efficacy in a combination setting.

#### **5.5 Replicative immortality**

Some studies suggest that at a given point during the course of tumor progression, evolving premalignant cell populations acquire the capacity to breach the mortality barrier (Hanahan & Weinberg, 2000); they become capable of unlimited replicative cycles. Overexpression of telomerase reverse transcriptase (hTERT), a unique ribonucleoprotein enzyme responsible for adding telomeric repeats onto 3' ends of chromosomes (Holt & Shay, 1999), could play an important role in the development of cellular immortality and oncogenesis. Telomerase activity has been detected in 89% of GBM cases and correlates with tumor grade (Le et al., 1998), whereas low expression of hTERT was shown to be associated with a better prognosis (Wager et al., 2008). Pre-clinical investigation of hTERT targeted therapy illustrates that downregulation of this gene results in tumor regression and increased survival in orthotopic GBM murine models (George et al., 2009; Mukai et al., 2000).

#### **6. The potential of targeting multiple pathways**

Hallmarks of cancer cell malignancy include upregulation or dysregulation of multiple pathways, with deregulations increasing in number as the cancer progresses. In contrast to this observation, the vast majority of clinical trials to date have focused on a single agent that targets a single molecular aberration. It is expected that a therapeutic modality targeting one of these dysregulated pathways will only result in modest benefits to patients in terms of disease-free survival time. Cellular proliferation, growth and death are regulated by an intricate network of cellular functions, and it is very likely that disturbances in the balance between these pathways will lead to the activation of compensating mechanisms in normal cells as well as cancer cells. While it is well understood that a combination of chemotherapeutic agents inclusive of drugs with differing mechanisms of action is generally

diagnosed GBM patients (Lai et al., 2008). Many other VEGF or VEGFR inhibitors are

Proteins responsible for tissue invasion and metastatic behavior are often effectors allowing the cell to grow in the absence of ECM adhesion signals. The most obvious example is the integrins family, which is involved in ECM anchorage-independent growth of tumor cells, and provides the traction necessary for cell motility and invasion (reviewed in (Desgrosellier & Cheresh, 2010)). An integrin SMI, cilengitide, has shown some promising activity in GBM clinical trial phase I/II in combination with TMZ (Stupp et al., 2010), and is now moving to phase III (Carter, 2010). In addition, enzymes that are involved in the degradation of the ECM will allow cancer cells to invade surrounding brain tissue. Matrix-metalloproteinases (MMPs) were shown to play a central role in the proteolysis necessary for this process (Nakada et al., 2003). Intratumoral administration of a shRNA against MMP-9 inhibited tumor growth in an orthotopic GBM mouse model (Lakka et al., 2005). To date, no MMP inhibitors have made their way to a phase II clinical trial for GBM treatment. Moreover, the clinical evaluation of MMP inhibitors as single agents in cancers other than GBM has not been associated with significant anti-tumor responses (Brinker et al., 2008; Chu et al., 2007)

Some studies suggest that at a given point during the course of tumor progression, evolving premalignant cell populations acquire the capacity to breach the mortality barrier (Hanahan & Weinberg, 2000); they become capable of unlimited replicative cycles. Overexpression of telomerase reverse transcriptase (hTERT), a unique ribonucleoprotein enzyme responsible for adding telomeric repeats onto 3' ends of chromosomes (Holt & Shay, 1999), could play an important role in the development of cellular immortality and oncogenesis. Telomerase activity has been detected in 89% of GBM cases and correlates with tumor grade (Le et al., 1998), whereas low expression of hTERT was shown to be associated with a better prognosis (Wager et al., 2008). Pre-clinical investigation of hTERT targeted therapy illustrates that downregulation of this gene results in tumor regression and increased survival in orthotopic

Hallmarks of cancer cell malignancy include upregulation or dysregulation of multiple pathways, with deregulations increasing in number as the cancer progresses. In contrast to this observation, the vast majority of clinical trials to date have focused on a single agent that targets a single molecular aberration. It is expected that a therapeutic modality targeting one of these dysregulated pathways will only result in modest benefits to patients in terms of disease-free survival time. Cellular proliferation, growth and death are regulated by an intricate network of cellular functions, and it is very likely that disturbances in the balance between these pathways will lead to the activation of compensating mechanisms in normal cells as well as cancer cells. While it is well understood that a combination of chemotherapeutic agents inclusive of drugs with differing mechanisms of action is generally

currently being tested in the clinic (Norden et al., 2009).

and they will most likely show better efficacy in a combination setting.

GBM murine models (George et al., 2009; Mukai et al., 2000).

**6. The potential of targeting multiple pathways** 

**5.4 Invasion and metastasis** 

**5.5 Replicative immortality** 

more efficacious than single agent chemotherapy in the treatment of aggressive cancers, clinicians and scientists are now beginning to realize the benefits of combining agents targeting different biological pathways in order to effectively silence as many cancer phenotypes as possible.

The therapeutic value of targeting two different pathways is exemplified by some research data obtained by our laboratory (Verreault et al., 2011a). One of the most commonly reported molecular defects in GBM is the aberrant activation of the PI3K/AKT pathway, which is associated with increased proliferation rate, invasion, metastasis and poor prognosis (Ermoian et al., 2002; Haas-Kogan et al., 1998; Li X. Y. et al., 2010). Rictor, the rapamycin-insensitive companion of mTOR, is a protein member of the mTOR Complex 2 (mTORC2), and can activate AKT through direct phosphorylation at its serine 473 site (Sarbassov et al., 2004; Sarbassov et al., 2005; Sparks & Guertin, 2010). Elevated levels of Rictor were found in human GBM tumor tissue samples and cell lines when compared to normal brain tissue (Masri et al., 2007). Rictor and EGFR proteins were silenced alone and in combination by siRNA *in vitro* transfection in a panel of three human GBM lines (U251MG, U118MG and LN229). It was found that the co-silencing of Rictor and EGFR exerted effects on cell migration and sensitivity to chemotherapeutic drugs that were not observed by the single silencing of either target (Verreault et al., 2011a). The most striking evidence of the validity of this combined silencing came from the *in vivo* aspect of this study, which was done by intracranial inoculation in mice brains of U251MG cells expressing small hairpin RNA (shRNA) specific to each target. Silencing of EGFR or Rictor alone had no significant effect on tumor growth, but the dual silencing resulted in the eradication of the tumor (Verreault et al., 2011a). Also, tumor growth block in response to the combined suppression of EGFR and PI3K/AKT pathway was reported previously using the SMIs gefitinib and LY294002 in a GBM xenograft model (Fan et al., 2003), while monotherapy of each inhibitor had no impact on tumor burden. Taken together, these studies strongly support the value of inhibiting both EGFR and PI3K/mTORC2/Rictor/AKT pathways to achieve therapeutic effects that may not be observed by the single inhibition of either pathway, and provide compelling evidences of the potential of targeting multiple pro-oncogenic pathways in GBM.

#### **7. Drug delivery to brain tumors**

The idea of targeting gene expression at the level of transcription or translation has been mirrored by the emergence of gene therapy as a strategy to specifically silence the activity of any defective or overactive gene without the limiting step of SMI availability (Bumcrot et al., 2006). Since tumor cells have a different pattern of gene expression in comparison with normal cells, gene silencing can theoretically be used to specifically target tumor-associated genes or mutated genes without altering gene expression of normal cells (Helene, 1994). The most commonly used strategies employed to achieve gene silencing involve administration of ASOs or siRNAs that can inhibit the expression of specific proteins. However, the therapeutic value of this technology is proving difficult to establish, in part because of the lack of pharmaceutically viable products that can be administered orally, intravenously or intraperitoneally, and that can deliver the gene silencing agent to tumor cell populations in sufficient quantity to achieve target knockdown. Despite the fact that we are still learning to navigate the technology of RNAi in order to achieve optimal benefits with minimal side-

siRNA-Based Therapy for Glioblastoma Patients 175

1995). The i.n. technique allows for a noninvasive bypass of the BBB via the nasal mucosa, through the olfactory and trigeminal nerves, directly to the brain and cerebrospinal fluid (Thorne et al., 2004). The ASO GRN163 specific to hTERT was successfully delivered to preclinical orthotopic tumors using this technique (Hashizume et al., 2008). The i.n. technique was also used in a phase I/II clinical trial to administer perillyl alcohol, a Ras inhibitor, and results suggested some antitumor activity without any toxicity in GBM patients (da Fonseca et al., 2008). These studies, together with other pre-clinical reports (Sakane et al., 1999; Thorne et al., 2004; Wang D. et al., 2006; Wang F. et al., 2003), suggest that the i.n. route of administration may be of great therapeutic value for treatment of brain tumors and could be part of the solution to the issue of polynucleotide therapeutics delivery. It should be noted, however, that material delivery in tissues other than the brain (liver, kidney, heart, muscles) was also detected following i.n. administration (Thorne et al., 2004), suggesting the need for combining this administration route with delivery systems that would improve specificity to the tumor. The currently accepted mechanisms of transport following intranasal administration are the intraneuronal transport following endocytosis or an extracellular diffusion along the nerves (Thorne et al., 1995). Thus it is clear that the size limitation imposed by these routes may restrict the possible delivery systems that could be used.

Strategies used to administer agents through systemic administration include simple infusion as well as more sophisticated delivery systems designed to promote intracellular delivery. In this context, the success of gene silencing therapy for cancer depends in large part on stable and tumor-specific delivery, which can be achieved only if therapeutic molecules can survive as active agents as they cross various biological barriers. These barriers are i) degradation in the blood or uptake by the liver, ii) passage from the circulation across the BBB and into the extravascular space within the tumor, ii) passage into cytoplasm of target cells, iv) release from the carrier and/or the endosomes if associated with a carrier system or internalized via endocytosis, v) escape from nucleases in tumor cell's cytoplasm and vi) binding to target mRNA. As described earlier, the BBB consists of endothelial cells, pericytes, astrocytes endfeet and neuronal cells that are organized in such way to confer a unique selective permeability to the CNS vascular network (Rubin & Staddon, 1999), restricting the passive transport of most therapeutic molecules (Pardridge, 2007). Some success in delivering molecules across the BBB has been made with long circulating carrier systems (see section 8) that can take advantage of the fact that tumorassociated BBB consists of poorly formed vascular endothelium that is more permeable to circulating macromolecules than the normal BBB (Patel et al., 2009). Strategies to open the BBB have also been explored and include osmotic disruption (Bellavance et al., 2008), the use of vasomodulators to increase permeability (Ningaraj et al., 2003), or the use of potassium channel agonists to increase the formation of transport vesicles (Ningaraj, 2006). Although it has been shown that siRNAs are more stable in cells than a single-stranded antisense molecule (Bertrand et al., 2002), naked RNA sequences injected *in vivo* are rapidly eliminated and have a short duration of effect (Khan et al., 2004). Pre-clinical studies suggest that this can be overcome by use of multiple i.v. or i.p. injections of naked siRNAs (Filleur et al., 2003), and can lead to successful downregulation of the target in intracranial tumor (George et al., 2008). However, other studies reported very little accumulation of siRNA in

**7.2 Limitation to systemic administration** 

effects, the high specificity and potency of siRNAs, together with the unlimited possibility of designs for siRNAs against any genes, make this technology an attractive option for targeted therapy. Once a viable option for safe and effective delivery to the tumor is defined, RNAi will be regarded as the most powerful tool for designing personalized treatment strategies. Some of the strategies that have been developed and explored pre-clinically for gene silencing agent delivery to brain tumors are discussed below, with references to successful achievements in the clinic for chemotherapeutic agent delivery.

#### **7.1 Bypassing the blood-brain barrier**

As discussed earlier, the BBB constitutes one of the main barriers to the development of new therapeutics for GBM treatment. Hence, a great deal of research has been focused on defining strategies aimed at bypassing the BBB and increasing delivery of therapeutics. One of these strategies consists of a direct intratumoral (i.t.) injection, and has been successfully used in pre-clinical brain tumor models for delivery of gene silencing agents (George et al., 2009; Gondi et al., 2004; Lakka et al., 2005; Thakker et al., 2005), and in the clinic for chemotherapeutic agents (Boiardi et al., 2005; Patchell et al., 2002). The only clinical trial testing the efficacy of RNAi in GBM was done by local delivery of a 146 nucleotides long RNA molecule (ATN-RNA) targeting Tenascin-C mRNA (Wyszko et al., 2008; Zukiel et al., 2006). Although its role in GBM pathology is still unclear, the expression of the ECM glycoprotein Tenascin-C was found to correlate with tumor grade (Pas et al., 2006). Treatment with ATN-RNA was associated with increased survival in GBM patients and these results constitute the first demonstration of a potential clinical application for RNAi in the treatment of GBM. Convention-enhanced delivery (CED) is another technique tested in the clinic for local delivery, and consists of placing catheters into the surgical cavity after the resection procedure and to deliver antineoplastic agents through the catheters using positive pressure (0.5 to 15.0μl/min). This technique was shown to increase the anti-tumor efficacy of paclitaxel (Lidar et al., 2004) and of the recombinant protein Cintredekin besudotox (Kunwar et al., 2007) in GBM patients. CED was also used to deliver siRNAs to the CNS of non-human primates, and resulted in a durable and specific silencing of the selected target (Querbes et al., 2009). Carmustine wafers (Gliadel) are currently used in the clinic and represent a good example of local delivery (Figure 2). In this system, carmustine is incorporated in a hydrophobic matrix made of a polyanhydride polymer that protects the agent from hydrolysis (Brem et al., 1994; Grossman et al., 1992). After tumor resection, the wafer discs are implanted at the surface of the resection cavity and the drug is slowly released for a period of three weeks (Brem et al., 1991). Although wafers have shown some promising efficacy when combined with TMZ (Gururangan et al., 2001), it is not indicated for patients with infiltrative or multifocal tumors. It is believed that local administration does not allow access to infiltrative cancer cells that are a predominant hallmark of GBM, and this is especially true for macro-molecules such as ASOs or siRNAs.

In order to overcome the limitations of direct i.t. administration of agents, other routes of administration have been explored, including systemic administration (intraperitoneal (i.p.) and intravenous (i.v.)), some of which integrating the use of delivery systems (Table 1). Challenges and opportunities for these strategies will be discussed in the following section. Interestingly, the intranasal (i.n.) route of delivery was recently shown to promote a rapid and efficient delivery of molecules that do not cross the BBB to the brain (Thorne et al.,

effects, the high specificity and potency of siRNAs, together with the unlimited possibility of designs for siRNAs against any genes, make this technology an attractive option for targeted therapy. Once a viable option for safe and effective delivery to the tumor is defined, RNAi will be regarded as the most powerful tool for designing personalized treatment strategies. Some of the strategies that have been developed and explored pre-clinically for gene silencing agent delivery to brain tumors are discussed below, with references to successful

As discussed earlier, the BBB constitutes one of the main barriers to the development of new therapeutics for GBM treatment. Hence, a great deal of research has been focused on defining strategies aimed at bypassing the BBB and increasing delivery of therapeutics. One of these strategies consists of a direct intratumoral (i.t.) injection, and has been successfully used in pre-clinical brain tumor models for delivery of gene silencing agents (George et al., 2009; Gondi et al., 2004; Lakka et al., 2005; Thakker et al., 2005), and in the clinic for chemotherapeutic agents (Boiardi et al., 2005; Patchell et al., 2002). The only clinical trial testing the efficacy of RNAi in GBM was done by local delivery of a 146 nucleotides long RNA molecule (ATN-RNA) targeting Tenascin-C mRNA (Wyszko et al., 2008; Zukiel et al., 2006). Although its role in GBM pathology is still unclear, the expression of the ECM glycoprotein Tenascin-C was found to correlate with tumor grade (Pas et al., 2006). Treatment with ATN-RNA was associated with increased survival in GBM patients and these results constitute the first demonstration of a potential clinical application for RNAi in the treatment of GBM. Convention-enhanced delivery (CED) is another technique tested in the clinic for local delivery, and consists of placing catheters into the surgical cavity after the resection procedure and to deliver antineoplastic agents through the catheters using positive pressure (0.5 to 15.0μl/min). This technique was shown to increase the anti-tumor efficacy of paclitaxel (Lidar et al., 2004) and of the recombinant protein Cintredekin besudotox (Kunwar et al., 2007) in GBM patients. CED was also used to deliver siRNAs to the CNS of non-human primates, and resulted in a durable and specific silencing of the selected target (Querbes et al., 2009). Carmustine wafers (Gliadel) are currently used in the clinic and represent a good example of local delivery (Figure 2). In this system, carmustine is incorporated in a hydrophobic matrix made of a polyanhydride polymer that protects the agent from hydrolysis (Brem et al., 1994; Grossman et al., 1992). After tumor resection, the wafer discs are implanted at the surface of the resection cavity and the drug is slowly released for a period of three weeks (Brem et al., 1991). Although wafers have shown some promising efficacy when combined with TMZ (Gururangan et al., 2001), it is not indicated for patients with infiltrative or multifocal tumors. It is believed that local administration does not allow access to infiltrative cancer cells that are a predominant hallmark of GBM,

achievements in the clinic for chemotherapeutic agent delivery.

and this is especially true for macro-molecules such as ASOs or siRNAs.

In order to overcome the limitations of direct i.t. administration of agents, other routes of administration have been explored, including systemic administration (intraperitoneal (i.p.) and intravenous (i.v.)), some of which integrating the use of delivery systems (Table 1). Challenges and opportunities for these strategies will be discussed in the following section. Interestingly, the intranasal (i.n.) route of delivery was recently shown to promote a rapid and efficient delivery of molecules that do not cross the BBB to the brain (Thorne et al.,

**7.1 Bypassing the blood-brain barrier** 

1995). The i.n. technique allows for a noninvasive bypass of the BBB via the nasal mucosa, through the olfactory and trigeminal nerves, directly to the brain and cerebrospinal fluid (Thorne et al., 2004). The ASO GRN163 specific to hTERT was successfully delivered to preclinical orthotopic tumors using this technique (Hashizume et al., 2008). The i.n. technique was also used in a phase I/II clinical trial to administer perillyl alcohol, a Ras inhibitor, and results suggested some antitumor activity without any toxicity in GBM patients (da Fonseca et al., 2008). These studies, together with other pre-clinical reports (Sakane et al., 1999; Thorne et al., 2004; Wang D. et al., 2006; Wang F. et al., 2003), suggest that the i.n. route of administration may be of great therapeutic value for treatment of brain tumors and could be part of the solution to the issue of polynucleotide therapeutics delivery. It should be noted, however, that material delivery in tissues other than the brain (liver, kidney, heart, muscles) was also detected following i.n. administration (Thorne et al., 2004), suggesting the need for combining this administration route with delivery systems that would improve specificity to the tumor. The currently accepted mechanisms of transport following intranasal administration are the intraneuronal transport following endocytosis or an extracellular diffusion along the nerves (Thorne et al., 1995). Thus it is clear that the size limitation imposed by these routes may restrict the possible delivery systems that could be used.

#### **7.2 Limitation to systemic administration**

Strategies used to administer agents through systemic administration include simple infusion as well as more sophisticated delivery systems designed to promote intracellular delivery. In this context, the success of gene silencing therapy for cancer depends in large part on stable and tumor-specific delivery, which can be achieved only if therapeutic molecules can survive as active agents as they cross various biological barriers. These barriers are i) degradation in the blood or uptake by the liver, ii) passage from the circulation across the BBB and into the extravascular space within the tumor, ii) passage into cytoplasm of target cells, iv) release from the carrier and/or the endosomes if associated with a carrier system or internalized via endocytosis, v) escape from nucleases in tumor cell's cytoplasm and vi) binding to target mRNA. As described earlier, the BBB consists of endothelial cells, pericytes, astrocytes endfeet and neuronal cells that are organized in such way to confer a unique selective permeability to the CNS vascular network (Rubin & Staddon, 1999), restricting the passive transport of most therapeutic molecules (Pardridge, 2007). Some success in delivering molecules across the BBB has been made with long circulating carrier systems (see section 8) that can take advantage of the fact that tumorassociated BBB consists of poorly formed vascular endothelium that is more permeable to circulating macromolecules than the normal BBB (Patel et al., 2009). Strategies to open the BBB have also been explored and include osmotic disruption (Bellavance et al., 2008), the use of vasomodulators to increase permeability (Ningaraj et al., 2003), or the use of potassium channel agonists to increase the formation of transport vesicles (Ningaraj, 2006).

Although it has been shown that siRNAs are more stable in cells than a single-stranded antisense molecule (Bertrand et al., 2002), naked RNA sequences injected *in vivo* are rapidly eliminated and have a short duration of effect (Khan et al., 2004). Pre-clinical studies suggest that this can be overcome by use of multiple i.v. or i.p. injections of naked siRNAs (Filleur et al., 2003), and can lead to successful downregulation of the target in intracranial tumor (George et al., 2008). However, other studies reported very little accumulation of siRNA in

siRNA-Based Therapy for Glioblastoma Patients 177

that IrC™ is much better tolerated than the free drug. This increase in tolerability permitted the administration of 100mg/kg IrCTM doses, which provided an increase in average survival of 83% compared to the untreated group. These studies demonstrate the potential of LNP delivery systems to improve chemotherapeutic drug delivery to brain tumors and consequently, to increase drug therapeutic effects. Interestingly, very little effort has been made in the development of SMI that could be administered in LNPs. It appears that if the expertise and knowledge that was gained over the last decades in the field of lipid-based delivery systems was directed towards improving SMI delivery to brain tumors, potential

In developing lipid-based delivery systems for gene silencing agents, the goal is to design a system that simultaneously achieves high efficiency (defined by delivery and release of the agent to the disease site), prolonged effects and low toxicity (Lundstrom & Boulikas, 2003). Small cationic LNPs can interact with negatively charged DNA or RNA, leading to the formation of complexes with a prolonged half-life in the circulation (Cattel et al., 2004) and capable of promoting cellular internalization (Storm & Crommelin, 1998). A report showed that hTERT-targeted ASO delivery using cationic LNPs resulted in increased survival of intracranial tumor bearing mice (Mukai et al., 2000). Numerous other studies have been done using i.v. or i.p. injections of siRNAs (Aigner, 2008; Sioud & Sorensen, 2003; Sorensen D. R. et al., 2003) or ASOs (Shoji & Nakashima, 2004) complexed with cationic LNPs in cancer models other than brain tumor. These techniques led to good silencing efficiency with no significant signs of toxicity. It is important to mention that some studies have demonstrated immune system activation induced by common cationic lipids following systemic administration (Freimark et al., 1998; Li S. et al., 1999; Scheule et al., 1997). Furthermore, another limitation to the therapeutic use of positively charged complexes is that they are cleared rapidly following intravenous administration (Nishikawa et al., 1998) as they bind to proteins in the plasma and form aggregates which are eliminated by nontarget cells (Ogris et al., 1999). Since neutralized complexes have proven to be less efficient, cationic complexes possessing hydrophilic steric barriers, achieved through the use of surface-grafted polymers like PEG, have been pursued to address the problem of plasma protein binding and rapid elimination (Allen et al., 2002). PEG-immunoliposomes (made by adding antibodies at the surface of LNPs, see section 9) are able to efficiently deliver ASO (Zhang et al., 2002) and siRNAs (Zhang et al., 2004) to orthotopic brain tumors following systemic administration. Stable nucleic-acid-lipid particles (SNALP) consist of lipid bilayer particles prepared with a mixture of cationic and fusogenic lipids, and have been shown to exhibit the stability, small size, low surface charge and low toxicity required for *in vivo* administration (Morrissey et al., 2005), and to promote efficient siRNA cellular uptake (Heyes et al., 2005). The lipid particles are coated with PEG molecules which dissociates from the SNALP rapidly after administration, thus transforming the carrier into a transfection-competent entity (Ambegia et al., 2005). SNALP-formulated siRNA have shown improved circulation time and increased downregulation efficacy in mice and nonhuman primates liver (Judge et al., 2009; Morrissey et al., 2005; Zimmermann et al., 2006), and recent modifications to the lipid composition have engendered a substantial 10-fold improvement in activity *in vivo* (Semple et al., 2010). Alternatives to cationic lipids complexed with therapeutic polynucleotides are also being explored to overcome the limitations observed with current formulations. In particular, reductions in *in vivo* toxicity and targeting efficiency for a certain cell population are a main focus. These options are

discussed further in a previously published review (Verreault et al., 2006).

therapeutic success could have already been achieved.

the brain following i.v. administration, and preferential accumulation was observed in the liver and the kidneys (Braasch et al., 2004; De Paula et al., 2007; Santel et al., 2006). Other reports show that a high-pressure delivery technique could increase delivery of siRNAs given i.v. (Lewis et al., 2002; McCaffrey et al., 2002; Song et al., 2003) or i.p (Heidel et al., 2004), but no evidence of delivery to the brain was shown (Lewis et al., 2002). Moreover, this technique is not relevant to human therapies as it involves high pressure and massive volume delivery schemes to generate transiently high local intravascular pressure (Lieberman et al., 2003; Shuey et al., 2002). It is now widely recognized that if siRNAs are to be used in the clinic for GBM patients, they will have to be formulated with a delivery system strategy in order to increase the agent's half-life and tumor specific delivery.

#### **8. Lipid nanoparticle delivery systems**

Studies over the last few decades have established that liposomal nanoparticle (LNP) formulations of selected antineoplastic agents can be more effective than a drug administered in its free form, due to their capacity to increase drug circulation time. Further, increased tumor delivery is observed due to the increased permeability of blood vessels in the tumor environment, a process referred to as the "enhanced permeability and retention effect" (EPR) (Maeda et al., 2009). Conventional LNPs, which consist of bilayer lipid vesicles, are prepared with phospholipids (e.g. phosphatidylcholine or phosphatidylglycerol) (Storm & Crommelin, 1998). The incorporation of cholesterol in these formulations influences the mechanical strength and permeability of LNP membranes (Ohvo-Rekila et al., 2002). Stealth LNP can also be made by coating the LNP surface with the hydrophilic polymer polyethylene glycol (PEG), which provides a barrier against interactions with molecular and cellular components in the plasma compartment (Storm & Crommelin, 1998) and can engender remarkable increases in plasma longevity of the carrier (Park et al., 2004). The FDA-approved and commercially available doxorubicin LNP formulation (Caelyx®, Schering-Plough or Doxil®, Centocor Ortho Biotech Inc) is an example of a PEG-coated formulation (Barenholz, 2007).

Liposomal formulations have shown some success in the delivery of drugs to brain tumors. Caelyx® was reported to be less toxic in the clinic than the unencapsulated form (Judson et al., 2001; O'Brien et al., 2004; Porter & Rifkin, 2007); when used to treat malignant glioma, stabilization of the disease (reduction of tumor volume of < 50% or a < 25% increase in tumor volume for more than 8 weeks) was observed (Fabel et al., 2001; Hau et al., 2004). An orthotopic GBM pre-clinical study showed anti-vascular activity of doxorubicin when encapsulated in LNPs, and these effects were not observed with the free form of the drug or in normal brain tissue (Zhou et al., 2002). Other pre-clinical studies showed that liposomal formulations of irinotecan are more efficacious than the unencapsulated form in brain tumors (Krauze et al., 2007; Noble et al., 2006; Verreault et al., 2011b) and in colorectal and adenocarcinoma tumors (Hattori et al., 2009; Messerer et al., 2004; Ramsay et al., 2008). More specifically, our laboratory has established that Irinophore CTM (IrCTM), a LPN formulation of irinotecan, exhibits improved anti-cancer efficacy compared to the free drug in a GBM orthotopic model (Verreault et al., 2011b). We demonstrated that the presence in the brain of irinotecan and its active metabolite SN-38 is extended following administration of IrC™ compared to irinotecan. At equivalent doses (50 mg/kg), the average survival of GBMtumor bearing animals was improved of 17% compared to the free drug-treated group (49.1% compared to untreated animals). Further, a repeated dose tolerability study showed

the brain following i.v. administration, and preferential accumulation was observed in the liver and the kidneys (Braasch et al., 2004; De Paula et al., 2007; Santel et al., 2006). Other reports show that a high-pressure delivery technique could increase delivery of siRNAs given i.v. (Lewis et al., 2002; McCaffrey et al., 2002; Song et al., 2003) or i.p (Heidel et al., 2004), but no evidence of delivery to the brain was shown (Lewis et al., 2002). Moreover, this technique is not relevant to human therapies as it involves high pressure and massive volume delivery schemes to generate transiently high local intravascular pressure (Lieberman et al., 2003; Shuey et al., 2002). It is now widely recognized that if siRNAs are to be used in the clinic for GBM patients, they will have to be formulated with a delivery

system strategy in order to increase the agent's half-life and tumor specific delivery.

Studies over the last few decades have established that liposomal nanoparticle (LNP) formulations of selected antineoplastic agents can be more effective than a drug administered in its free form, due to their capacity to increase drug circulation time. Further, increased tumor delivery is observed due to the increased permeability of blood vessels in the tumor environment, a process referred to as the "enhanced permeability and retention effect" (EPR) (Maeda et al., 2009). Conventional LNPs, which consist of bilayer lipid vesicles, are prepared with phospholipids (e.g. phosphatidylcholine or phosphatidylglycerol) (Storm & Crommelin, 1998). The incorporation of cholesterol in these formulations influences the mechanical strength and permeability of LNP membranes (Ohvo-Rekila et al., 2002). Stealth LNP can also be made by coating the LNP surface with the hydrophilic polymer polyethylene glycol (PEG), which provides a barrier against interactions with molecular and cellular components in the plasma compartment (Storm & Crommelin, 1998) and can engender remarkable increases in plasma longevity of the carrier (Park et al., 2004). The FDA-approved and commercially available doxorubicin LNP formulation (Caelyx®, Schering-Plough or Doxil®, Centocor Ortho Biotech Inc) is an example of a PEG-coated

Liposomal formulations have shown some success in the delivery of drugs to brain tumors. Caelyx® was reported to be less toxic in the clinic than the unencapsulated form (Judson et al., 2001; O'Brien et al., 2004; Porter & Rifkin, 2007); when used to treat malignant glioma, stabilization of the disease (reduction of tumor volume of < 50% or a < 25% increase in tumor volume for more than 8 weeks) was observed (Fabel et al., 2001; Hau et al., 2004). An orthotopic GBM pre-clinical study showed anti-vascular activity of doxorubicin when encapsulated in LNPs, and these effects were not observed with the free form of the drug or in normal brain tissue (Zhou et al., 2002). Other pre-clinical studies showed that liposomal formulations of irinotecan are more efficacious than the unencapsulated form in brain tumors (Krauze et al., 2007; Noble et al., 2006; Verreault et al., 2011b) and in colorectal and adenocarcinoma tumors (Hattori et al., 2009; Messerer et al., 2004; Ramsay et al., 2008). More specifically, our laboratory has established that Irinophore CTM (IrCTM), a LPN formulation of irinotecan, exhibits improved anti-cancer efficacy compared to the free drug in a GBM orthotopic model (Verreault et al., 2011b). We demonstrated that the presence in the brain of irinotecan and its active metabolite SN-38 is extended following administration of IrC™ compared to irinotecan. At equivalent doses (50 mg/kg), the average survival of GBMtumor bearing animals was improved of 17% compared to the free drug-treated group (49.1% compared to untreated animals). Further, a repeated dose tolerability study showed

**8. Lipid nanoparticle delivery systems** 

formulation (Barenholz, 2007).

that IrC™ is much better tolerated than the free drug. This increase in tolerability permitted the administration of 100mg/kg IrCTM doses, which provided an increase in average survival of 83% compared to the untreated group. These studies demonstrate the potential of LNP delivery systems to improve chemotherapeutic drug delivery to brain tumors and consequently, to increase drug therapeutic effects. Interestingly, very little effort has been made in the development of SMI that could be administered in LNPs. It appears that if the expertise and knowledge that was gained over the last decades in the field of lipid-based delivery systems was directed towards improving SMI delivery to brain tumors, potential therapeutic success could have already been achieved.

In developing lipid-based delivery systems for gene silencing agents, the goal is to design a system that simultaneously achieves high efficiency (defined by delivery and release of the agent to the disease site), prolonged effects and low toxicity (Lundstrom & Boulikas, 2003). Small cationic LNPs can interact with negatively charged DNA or RNA, leading to the formation of complexes with a prolonged half-life in the circulation (Cattel et al., 2004) and capable of promoting cellular internalization (Storm & Crommelin, 1998). A report showed that hTERT-targeted ASO delivery using cationic LNPs resulted in increased survival of intracranial tumor bearing mice (Mukai et al., 2000). Numerous other studies have been done using i.v. or i.p. injections of siRNAs (Aigner, 2008; Sioud & Sorensen, 2003; Sorensen D. R. et al., 2003) or ASOs (Shoji & Nakashima, 2004) complexed with cationic LNPs in cancer models other than brain tumor. These techniques led to good silencing efficiency with no significant signs of toxicity. It is important to mention that some studies have demonstrated immune system activation induced by common cationic lipids following systemic administration (Freimark et al., 1998; Li S. et al., 1999; Scheule et al., 1997). Furthermore, another limitation to the therapeutic use of positively charged complexes is that they are cleared rapidly following intravenous administration (Nishikawa et al., 1998) as they bind to proteins in the plasma and form aggregates which are eliminated by nontarget cells (Ogris et al., 1999). Since neutralized complexes have proven to be less efficient, cationic complexes possessing hydrophilic steric barriers, achieved through the use of surface-grafted polymers like PEG, have been pursued to address the problem of plasma protein binding and rapid elimination (Allen et al., 2002). PEG-immunoliposomes (made by adding antibodies at the surface of LNPs, see section 9) are able to efficiently deliver ASO (Zhang et al., 2002) and siRNAs (Zhang et al., 2004) to orthotopic brain tumors following systemic administration. Stable nucleic-acid-lipid particles (SNALP) consist of lipid bilayer particles prepared with a mixture of cationic and fusogenic lipids, and have been shown to exhibit the stability, small size, low surface charge and low toxicity required for *in vivo* administration (Morrissey et al., 2005), and to promote efficient siRNA cellular uptake (Heyes et al., 2005). The lipid particles are coated with PEG molecules which dissociates from the SNALP rapidly after administration, thus transforming the carrier into a transfection-competent entity (Ambegia et al., 2005). SNALP-formulated siRNA have shown improved circulation time and increased downregulation efficacy in mice and nonhuman primates liver (Judge et al., 2009; Morrissey et al., 2005; Zimmermann et al., 2006), and recent modifications to the lipid composition have engendered a substantial 10-fold improvement in activity *in vivo* (Semple et al., 2010). Alternatives to cationic lipids complexed with therapeutic polynucleotides are also being explored to overcome the limitations observed with current formulations. In particular, reductions in *in vivo* toxicity and targeting efficiency for a certain cell population are a main focus. These options are discussed further in a previously published review (Verreault et al., 2006).

siRNA-Based Therapy for Glioblastoma Patients 179

carrier specific to mouse transferrin receptor only in a human xenograft murine model (Zhang et al., 2003). This dual receptor targeting strategy was used to deliver ASOs or shRNA plasmids to orthotopic brain tumors (Zhang et al., 2004; Zhang et al., 2002) and resulted in 88-100% increase in animals' lifespan when compared to untreated animals. Thus, a treatment aimed at targeting BTIC could include a proportion of transferrin and insulin targeting immunoliposomes that would also incorporate antibodies against the CD133 marker. Such system could increase the likelihood that all targeted CD133+ cells are part of the tumor tissue. The complex and heterogeneous nature of brain tumors seems to require multivalency of delivery systems in order to achieve highly specific and efficient siRNA delivery. Targeted delivery systems will also have to be versatile, allowing the encapsulation of diverse combinations of siRNA (e.g. against EGFR and Rictor) selected

Interestingly, the capacity of exogenous neural stem cells (NSC) administered directly into the brain parenchyma, the cerebral ventricles or even in the systemic circulation to migrate great distances into sites of intracranial pathology has triggered the interest of using these cells as anti-cancer therapeutics vehicles (reviewed in (Yip et al., 2006)). Indeed, exogenous NSCs were shown to be able to accumulate around brain tumors and to track tumor cells migrating into the surrounding tissue (Aboody et al., 2000). This unique tropism of NSCs for gliomas has motivated the development "genetically-armed" NSC to target cancer cells through the delivery of a variety of therapeutic gene products. NSCs have been produced to express cytokines to enhance the immune response against the tumor (Benedetti et al., 2000; Ehtesham et al., 2002b), the proapoptotic protein TRAIL (Ehtesham et al., 2002a) or the prodrug converting enzyme cytosine deaminase (Aboody et al., 2000). Given the potential of NSCs to deliver therapeutic agents in a specific and sustained manner to brain tumors, it will be interesting to evaluate whether shRNA-expressing NSCs could be used to secrete and deliver siRNAs in the vicinity of tumors and invading tumor cells. These therapeutic NSCs can also be designed to express bioluminescence or red fluorescence (Shah et al., 2005; Yip & Shah, 2008). Hence, it is hoped that therapeutic NSCs could be used as biological, motile and dynamic diagnostic tools as well as specific delivery systems for therapeutic agents in gliomas, especially for infiltrating tumor cells in the close vicinity to normal CNS structures and therefore not remediable by traditional therapy (Shah et al., 2005; Yip & Shah, 2008). However, it is not sure whether therapeutic NSCs have the ability to transgress the abnormal tumor-associated vasculature, and research into the underlying molecular

**10. Opportunity for improving tumor drug delivery by vascular normalization**  Pre-clinical models showed that GBM tumors are poorly perfused (Blasberg et al., 1983; Groothuis et al., 1983) due to factors such as reduced blood flow rates, elevated hematocrit and interstitial fluid pressure, and an increase in geometric resistance (Baish et al., 1996; Vajkoczy & Menger, 2000; Vajkoczy et al., 1998; Yuan et al., 1994). The microvasculature of GBM was characterized as tortuous and fenestrated vessels with diameters that are larger than normal (Vajkoczy & Menger, 2004) and discontinuous basement membrane which rarely encloses pericytes (Deane & Lantos, 1981). The poorly organized architecture of GBM vessels, illustrated in figure 3, impedes vascular function and reduces drug delivery to the tumor tissue. In glioma (Kamoun et al., 2009; Sorensen A. G. et al., 2009; Winkler et al., 2004), tumor vascular normalization has been described as the specific activity of an agent

based on the patient's tumor genetic profile.

mechanism and clinical utility of these cells is active and ongoing.

#### **9. Targeted delivery**

The concept of targeted delivery has been suggested by many to be the solution to the obstacle of siRNA delivery to brain tumors (Lichota et al., 2009; Prakash et al., 2010). An efficient targeted delivery strategy should promote specific crossing of the therapeutic material across tumor-associated BBB, passage through cancer cell membranes, and prevention of accumulation in healthy tissue. Antibody-coupled liposomes (immunoliposomes) combine the capacity of LNPs to increase nucleic acid half-life in the blood compartment with specific targeting to tumor sites. One of the first attempts to deliver material to the brain using immunoliposomes was done by coupling the monoclonal antibody OX26 specific against the transferrin receptor (Huwyler et al., 1996) to PEGylated liposomes made with DSPE lipids. The transferrin receptor is present at the surface of normal brain capillary endothelial cells and is upregulated in brain tumor tissue (Recht et al., 1990). Following i.v. injection of OX26 coupled-DSPE-PEG immunoliposomes encapsulating the chemotherapeutic agent daunomycin, an average of 0.03% of the injected dose of daunomycin was measured in the brain of rats after 60 min, while only 0.008% of injected daunomycin dose was measured following administration of free daunomycin or non-OX26-conjugated daunomycin DSPE-PEG carrier (Huwyler et al., 1996). The use of mouse transferrin receptor-targeted immunoliposomes has also shown success in the delivery of bigger molecules such as DNA plasmids (Shi et al., 2001) or siRNAs (Pirollo et al., 2006) to brain tumors.

It can be speculated that many other types of antibodies specific against GBM cells or microenvironment antigens could also be used to produce immunoliposomes that would increase delivery of nucleic acid sequences to the tumor tissue. For example, the arginine-glycineaspartic acid (RGD) motif of fibronectin has been used to target delivery of siRNAs in a s.c. model of neuroblastoma (Schiffelers et al., 2004). RGD binds to integrins that are expressed on activated endothelial cells found in tumor vasculature of many advanced cancers including GBM (Gladson & Cheresh, 1991). *In vivo* studies demonstrated the accumulation of CY5.5-RGD in cells and vessels of orthotopic GBM tumors following i.v. injection (Hsu et al., 2006), supporting its potential use for siRNA targeted delivery to GBM tumors. Tumorassociated endothelial cells in GBM have higher levels of VEGFR2 than normal endothelial cells (Charalambous et al., 2006). Targeted delivery specific to the VEGFR2 receptor could also be used to specifically deliver material across the brain tumor-associated BBB. CD44 is a surface receptor overexpressed in GBM tumor cells (Axelsen et al., 2007) and is another example of GBM-specific marker that could be used for immunoliposome targeting. Further, antigens found at the surface of brain tumor initiating cells (BTIC) (e.g. CD133) (Altaner, 2008; Guo et al., 2006) could potentially allow for specific delivery of silencing agents against defective genes in these cells. BTIC are a sub-population of cells that have the ability to reconstitute the overall tumor cell population and are typically more resistant to chemotherapy and radiation than the rest of the tumor cell population (Hadjipanayis & Van Meir, 2009). It is now believed that treatment resistance and eventual relapse result in part from a failure to eliminate BTICs (Xie & Chin, 2008). It is important to note that CD133 is also expressed in normal stem cells (Tarnok et al., 2009) and use of this antigen for targeted delivery could negatively impact healthy CD133+ cells. Therefore, it appears necessary to combine several targeted delivery strategies to achieve both efficacy and specificity. For example, use of immunoliposomes specific to human insulin receptor and mouse transferrin receptor was more effective at delivering nucleic acid molecules to brain tumors cells than a

The concept of targeted delivery has been suggested by many to be the solution to the obstacle of siRNA delivery to brain tumors (Lichota et al., 2009; Prakash et al., 2010). An efficient targeted delivery strategy should promote specific crossing of the therapeutic material across tumor-associated BBB, passage through cancer cell membranes, and prevention of accumulation in healthy tissue. Antibody-coupled liposomes (immunoliposomes) combine the capacity of LNPs to increase nucleic acid half-life in the blood compartment with specific targeting to tumor sites. One of the first attempts to deliver material to the brain using immunoliposomes was done by coupling the monoclonal antibody OX26 specific against the transferrin receptor (Huwyler et al., 1996) to PEGylated liposomes made with DSPE lipids. The transferrin receptor is present at the surface of normal brain capillary endothelial cells and is upregulated in brain tumor tissue (Recht et al., 1990). Following i.v. injection of OX26 coupled-DSPE-PEG immunoliposomes encapsulating the chemotherapeutic agent daunomycin, an average of 0.03% of the injected dose of daunomycin was measured in the brain of rats after 60 min, while only 0.008% of injected daunomycin dose was measured following administration of free daunomycin or non-OX26-conjugated daunomycin DSPE-PEG carrier (Huwyler et al., 1996). The use of mouse transferrin receptor-targeted immunoliposomes has also shown success in the delivery of bigger molecules such as DNA plasmids (Shi et al., 2001) or siRNAs (Pirollo et

It can be speculated that many other types of antibodies specific against GBM cells or microenvironment antigens could also be used to produce immunoliposomes that would increase delivery of nucleic acid sequences to the tumor tissue. For example, the arginine-glycineaspartic acid (RGD) motif of fibronectin has been used to target delivery of siRNAs in a s.c. model of neuroblastoma (Schiffelers et al., 2004). RGD binds to integrins that are expressed on activated endothelial cells found in tumor vasculature of many advanced cancers including GBM (Gladson & Cheresh, 1991). *In vivo* studies demonstrated the accumulation of CY5.5-RGD in cells and vessels of orthotopic GBM tumors following i.v. injection (Hsu et al., 2006), supporting its potential use for siRNA targeted delivery to GBM tumors. Tumorassociated endothelial cells in GBM have higher levels of VEGFR2 than normal endothelial cells (Charalambous et al., 2006). Targeted delivery specific to the VEGFR2 receptor could also be used to specifically deliver material across the brain tumor-associated BBB. CD44 is a surface receptor overexpressed in GBM tumor cells (Axelsen et al., 2007) and is another example of GBM-specific marker that could be used for immunoliposome targeting. Further, antigens found at the surface of brain tumor initiating cells (BTIC) (e.g. CD133) (Altaner, 2008; Guo et al., 2006) could potentially allow for specific delivery of silencing agents against defective genes in these cells. BTIC are a sub-population of cells that have the ability to reconstitute the overall tumor cell population and are typically more resistant to chemotherapy and radiation than the rest of the tumor cell population (Hadjipanayis & Van Meir, 2009). It is now believed that treatment resistance and eventual relapse result in part from a failure to eliminate BTICs (Xie & Chin, 2008). It is important to note that CD133 is also expressed in normal stem cells (Tarnok et al., 2009) and use of this antigen for targeted delivery could negatively impact healthy CD133+ cells. Therefore, it appears necessary to combine several targeted delivery strategies to achieve both efficacy and specificity. For example, use of immunoliposomes specific to human insulin receptor and mouse transferrin receptor was more effective at delivering nucleic acid molecules to brain tumors cells than a

**9. Targeted delivery** 

al., 2006) to brain tumors.

carrier specific to mouse transferrin receptor only in a human xenograft murine model (Zhang et al., 2003). This dual receptor targeting strategy was used to deliver ASOs or shRNA plasmids to orthotopic brain tumors (Zhang et al., 2004; Zhang et al., 2002) and resulted in 88-100% increase in animals' lifespan when compared to untreated animals. Thus, a treatment aimed at targeting BTIC could include a proportion of transferrin and insulin targeting immunoliposomes that would also incorporate antibodies against the CD133 marker. Such system could increase the likelihood that all targeted CD133+ cells are part of the tumor tissue. The complex and heterogeneous nature of brain tumors seems to require multivalency of delivery systems in order to achieve highly specific and efficient siRNA delivery. Targeted delivery systems will also have to be versatile, allowing the encapsulation of diverse combinations of siRNA (e.g. against EGFR and Rictor) selected based on the patient's tumor genetic profile.

Interestingly, the capacity of exogenous neural stem cells (NSC) administered directly into the brain parenchyma, the cerebral ventricles or even in the systemic circulation to migrate great distances into sites of intracranial pathology has triggered the interest of using these cells as anti-cancer therapeutics vehicles (reviewed in (Yip et al., 2006)). Indeed, exogenous NSCs were shown to be able to accumulate around brain tumors and to track tumor cells migrating into the surrounding tissue (Aboody et al., 2000). This unique tropism of NSCs for gliomas has motivated the development "genetically-armed" NSC to target cancer cells through the delivery of a variety of therapeutic gene products. NSCs have been produced to express cytokines to enhance the immune response against the tumor (Benedetti et al., 2000; Ehtesham et al., 2002b), the proapoptotic protein TRAIL (Ehtesham et al., 2002a) or the prodrug converting enzyme cytosine deaminase (Aboody et al., 2000). Given the potential of NSCs to deliver therapeutic agents in a specific and sustained manner to brain tumors, it will be interesting to evaluate whether shRNA-expressing NSCs could be used to secrete and deliver siRNAs in the vicinity of tumors and invading tumor cells. These therapeutic NSCs can also be designed to express bioluminescence or red fluorescence (Shah et al., 2005; Yip & Shah, 2008). Hence, it is hoped that therapeutic NSCs could be used as biological, motile and dynamic diagnostic tools as well as specific delivery systems for therapeutic agents in gliomas, especially for infiltrating tumor cells in the close vicinity to normal CNS structures and therefore not remediable by traditional therapy (Shah et al., 2005; Yip & Shah, 2008). However, it is not sure whether therapeutic NSCs have the ability to transgress the abnormal tumor-associated vasculature, and research into the underlying molecular mechanism and clinical utility of these cells is active and ongoing.

#### **10. Opportunity for improving tumor drug delivery by vascular normalization**

Pre-clinical models showed that GBM tumors are poorly perfused (Blasberg et al., 1983; Groothuis et al., 1983) due to factors such as reduced blood flow rates, elevated hematocrit and interstitial fluid pressure, and an increase in geometric resistance (Baish et al., 1996; Vajkoczy & Menger, 2000; Vajkoczy et al., 1998; Yuan et al., 1994). The microvasculature of GBM was characterized as tortuous and fenestrated vessels with diameters that are larger than normal (Vajkoczy & Menger, 2004) and discontinuous basement membrane which rarely encloses pericytes (Deane & Lantos, 1981). The poorly organized architecture of GBM vessels, illustrated in figure 3, impedes vascular function and reduces drug delivery to the tumor tissue. In glioma (Kamoun et al., 2009; Sorensen A. G. et al., 2009; Winkler et al., 2004), tumor vascular normalization has been described as the specific activity of an agent

siRNA-Based Therapy for Glioblastoma Patients 181

GBM orthotopic models and in clinical studies. (ii) Use of targeted agents customized therapy: the anti-tumor efficacy of SMIs or siRNAs should be tested using GBM tumors arising from orthotopic inoculation of tumor cells isolated from patient tumor biopsies for which a list of genetic defects (e.g. EGFR amplification, PTEN mutation) is available. (iii) Use of delivery systems to circumvent the obstacles of delivery to brain tumors and improve efficacy of targeted agents and chemotherapeutics: antibody-coupled carriers could be designed to improve delivery to the tumor, and the ligand specificity of such a carrier could

 The most significant problem for GBM cancer patients is repopulation of malignant cells following treatment, causing inevitable relapse. This is thought to be the result of genetic mutations that endow the cell with many specific functional capabilities such as cell proliferation, survival, invasion and metastasis. Although it is acknowledged that advances in GBM treatment will continue to rely on conventional treatment approaches (surgery, radiation and/or chemotherapy), the value of combining standard chemotherapy with targeted agents that increase tumor drug

 Several targeted therapy approaches are currently being evaluated in GBM preclinical and clinical studies using SMI, ASOs and RNAi-based compounds (siRNA or shRNA), and the targets include proteins involved in sustaining proliferative signals, evading growth suppressors, resisting cell death, inducing angiogenesis, activating

 The potential benefits of combining agents targeting different biological pathways in order to effectively silence as many cancer phenotypes as possible is also being

 Another major challenge of GBM treatment is the achievement of adequate concentration of the therapeutic agent within the tumor itself, and this obstacle can largely be attributed to the presence of the BBB. Strategies that are currently being tested to overcome this obstacle include the bypass of the BBB using alternative delivery routes (i.t., CED, i.n., "genetically-armed" NSCs) or the use of delivery systems (LNPs, immunoliposomes) which have been shown to promote a more efficient and specific delivery of therapeutics to brain tumors following intravenous

It has become clear that delivery systems, whether they are lipid-based, polymer-based or antibody-conjugated, can have a significant benefit in enhancing stability of drugs, facilitating delivery to tumor sites and perhaps delivery to the intracellular compartments containing the molecular targets. Moreover, the reduced toxicity profile associated with many liposomal drug formulations compared to the free form of the drug (Mayer et al., 1995; O'Brien et al., 2004) could be used to administer higher doses that would lead to

 The poorly organized architecture of GBM vessels is thought to impede vascular function and to reduce drug delivery to the tumor tissue, and the normalization of GBM vasculature has been approached in an attempt to improve drug delivery to

be made based on immunohistopathology analysis of each individual tumor.

invasion and metastasis and enabling replicative immortality.

**Chapter summary**

recognized.

administration.

brain tumors.

sensitivity is now being recognized.

(e.g. antiangiogenic therapy) against proliferating vasculature, which results in the growth inhibition of new vessels, the pruning of immature and inefficient tumor vessels, and the normalization of surviving vasculature by increasing the fraction of pericyte-covered vessels, restoring the abnormally thick and irregular basement membrane and reducing the high vascular permeability of these vessels (Baffert et al., 2006; Jain, 2001). The normalization of tumor vessels appears to be transient in nature, but was suggested to create a window where blood flow is improved, leading to an opportunity to improve delivery of other drugs (Jain, 2005). In GBM patients, a "vascular normalization index", defined by changes in vascular permeability (Ktrans values), microvessel volume and circulating collagen IV, was found to be closely associated with overall survival and progression-free survival in response to Cediranib, a pan-VEGFR inhibitor (Sorensen A. G. et al., 2009). Pre-clinically, the delivery of TMZ in an intracranial model of glioma was increased after treatment with the angiogenesis inhibitor SU5416. It was suggested that SU5416 restored capillary architecture and decreased interstitial fluid pressure (Ma et al., 2003), allowing for an increase in TMZ delivery to the tumor tissue. Our laboratory has recently reported that IrCTM therapy (once weekly for three weeks) can lead to normalization of GBM blood vessel structure and function (Verreault et al., 2011c). IrCTM treatment restored the basement membrane architecture of the tumor vasculature, reduced blood vessel diameters and reduced vessel permeability to the fluorescent dye Hoechst 33342, suggesting a restoration of the vessel architecture and function to a more normal state (Verreault et al., 2011c). Treatment also increased the quantity of vessel in the center of tumors, suggesting a more homogeneous distribution of blood across the entire tumor (Verreault et al., 2011c). Further, IrCTM significantly reduced Ktrans values calculated from Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) studies (Verreault et al., 2011c), which was also suggestive of a decrease in vessel permeability (O'Connor et al., 2007). Taken together, these observations strongly suggested an improvement in vascular function: the tumor blood vessels in tumors from animals treated with IrCTM were behaving more like vessels in the normal brain. Thus, IrC™ exerts a dual mechanism of action in GBM tumors. As described in section 8, the therapeutic activity of irinotecan is improved by the extended exposure of tumor cells to the drug provided by the drug carrier (Verreault et al., 2011b). Moreover, the effects of the formulation on the tumor micro-environment may increase the delivery and efficacy of a second agent (Verreault et al., 2011c). This dual mechanism may provide an opportunity for designing a therapy which would encompass the cytotoxic activity of an optimized chemotherapeutic agent together with the increase in tumor delivery of an antibody-coupled carrier encapsulating siRNAs specific to promalignancy genes. It can be expected that such therapy could lead to significant therapeutic benefits for GBM patients. Studies designed to evaluate the capacity of IrCTM therapy to increase delivery and efficacy of TMZ in GBM are currently ongoing.

#### **11. Conclusion**

It is widely recognized that there is a tremendous potential in the use of targeted therapy agents to treat GBM and, importantly, to become the therapeutic modality of choice when developing target-specific personalized treatment options. The following three main areas of investigation could lead to improved treatment outcome in GBM: (i) Use of targeted agents in combination with conventional treatment options: the capacity of SMIs or siRNAs to enhance the activity of chemotherapeutic agents or radiation should be tested in established GBM orthotopic models and in clinical studies. (ii) Use of targeted agents customized therapy: the anti-tumor efficacy of SMIs or siRNAs should be tested using GBM tumors arising from orthotopic inoculation of tumor cells isolated from patient tumor biopsies for which a list of genetic defects (e.g. EGFR amplification, PTEN mutation) is available. (iii) Use of delivery systems to circumvent the obstacles of delivery to brain tumors and improve efficacy of targeted agents and chemotherapeutics: antibody-coupled carriers could be designed to improve delivery to the tumor, and the ligand specificity of such a carrier could be made based on immunohistopathology analysis of each individual tumor.

#### **Chapter summary**

180 Novel Therapeutic Concepts in Targeting Glioma

(e.g. antiangiogenic therapy) against proliferating vasculature, which results in the growth inhibition of new vessels, the pruning of immature and inefficient tumor vessels, and the normalization of surviving vasculature by increasing the fraction of pericyte-covered vessels, restoring the abnormally thick and irregular basement membrane and reducing the high vascular permeability of these vessels (Baffert et al., 2006; Jain, 2001). The normalization of tumor vessels appears to be transient in nature, but was suggested to create a window where blood flow is improved, leading to an opportunity to improve delivery of other drugs (Jain, 2005). In GBM patients, a "vascular normalization index", defined by changes in vascular permeability (Ktrans values), microvessel volume and circulating collagen IV, was found to be closely associated with overall survival and progression-free survival in response to Cediranib, a pan-VEGFR inhibitor (Sorensen A. G. et al., 2009). Pre-clinically, the delivery of TMZ in an intracranial model of glioma was increased after treatment with the angiogenesis inhibitor SU5416. It was suggested that SU5416 restored capillary architecture and decreased interstitial fluid pressure (Ma et al., 2003), allowing for an increase in TMZ delivery to the tumor tissue. Our laboratory has recently reported that IrCTM therapy (once weekly for three weeks) can lead to normalization of GBM blood vessel structure and function (Verreault et al., 2011c). IrCTM treatment restored the basement membrane architecture of the tumor vasculature, reduced blood vessel diameters and reduced vessel permeability to the fluorescent dye Hoechst 33342, suggesting a restoration of the vessel architecture and function to a more normal state (Verreault et al., 2011c). Treatment also increased the quantity of vessel in the center of tumors, suggesting a more homogeneous distribution of blood across the entire tumor (Verreault et al., 2011c). Further, IrCTM significantly reduced Ktrans values calculated from Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) studies (Verreault et al., 2011c), which was also suggestive of a decrease in vessel permeability (O'Connor et al., 2007). Taken together, these observations strongly suggested an improvement in vascular function: the tumor blood vessels in tumors from animals treated with IrCTM were behaving more like vessels in the normal brain. Thus, IrC™ exerts a dual mechanism of action in GBM tumors. As described in section 8, the therapeutic activity of irinotecan is improved by the extended exposure of tumor cells to the drug provided by the drug carrier (Verreault et al., 2011b). Moreover, the effects of the formulation on the tumor micro-environment may increase the delivery and efficacy of a second agent (Verreault et al., 2011c). This dual mechanism may provide an opportunity for designing a therapy which would encompass the cytotoxic activity of an optimized chemotherapeutic agent together with the increase in tumor delivery of an antibody-coupled carrier encapsulating siRNAs specific to promalignancy genes. It can be expected that such therapy could lead to significant therapeutic benefits for GBM patients. Studies designed to evaluate the capacity of IrCTM therapy to

increase delivery and efficacy of TMZ in GBM are currently ongoing.

It is widely recognized that there is a tremendous potential in the use of targeted therapy agents to treat GBM and, importantly, to become the therapeutic modality of choice when developing target-specific personalized treatment options. The following three main areas of investigation could lead to improved treatment outcome in GBM: (i) Use of targeted agents in combination with conventional treatment options: the capacity of SMIs or siRNAs to enhance the activity of chemotherapeutic agents or radiation should be tested in established

**11. Conclusion** 


It has become clear that delivery systems, whether they are lipid-based, polymer-based or antibody-conjugated, can have a significant benefit in enhancing stability of drugs, facilitating delivery to tumor sites and perhaps delivery to the intracellular compartments containing the molecular targets. Moreover, the reduced toxicity profile associated with many liposomal drug formulations compared to the free form of the drug (Mayer et al., 1995; O'Brien et al., 2004) could be used to administer higher doses that would lead to

siRNA-Based Therapy for Glioblastoma Patients 183

Badiga, A.V., Chetty, C., Kesanakurti, D. et al. (2011). MMP-2 siRNA inhibits radiation-

Baffert, F., Le, T., Sennino, B. et al. (2006). Cellular changes in normal blood capillaries

Baish, J.W., Gazit, Y., Berk, D.A., Nozue, M., Baxter, L.T. & Jain, R.K. (1996). Role of tumor

network model. *Microvasc Res*, Vol. 51, No. 3, pp. (327-46), ISBN: 0026-2862. Banks, W.A. (2009). Characteristics of compounds that cross the blood-brain barrier. *BMC* 

Barenholz, Y. (2007). Amphipathic weak base loading into preformed liposomes having a

Begley, D.J. (2004a). ABC transporters and the blood-brain barrier. *Curr Pharm Des*, Vol. 10,

Begley, D.J. (2004b). Delivery of therapeutic agents to the central nervous system: the

Bellavance, M.A., Blanchette, M. & Fortin, D. (2008). Recent advances in blood-brain barrier

Benedetti, S., Pirola, B., Pollo, B. et al. (2000). Gene therapy of experimental brain tumors

Bertrand, J.R., Pottier, M., Vekris, A., Opolon, P., Maksimenko, A. & Malvy, C. (2002).

*Biochem Biophys Res Commun*, Vol. 296, No. 4, pp. (1000-4), ISBN: 0006-291X. Blasberg, R.G., Kobayashi, T., Horowitz, M. et al. (1983). Regional blood flow in

Boiardi, A., Bartolomei, M., Silvani, A. et al. (2005). Intratumoral delivery of mitoxantrone in

Braasch, D.A., Paroo, Z., Constantinescu, A. et al. (2004). Biodistribution of phosphodiester

Brada, M., Collins, V.P., Dorward, N.L. & Thomas, D.G.T. (2001). Tumours of the brain and

Brandes, A.A., Tosoni, A., Amista, P. et al. (2004a). How effective is BCNU in recurrent

Brandes, A.A., Tosoni, A., Basso, U. et al. (2004b). Second-line chemotherapy with irinotecan

*Neurooncol*, Vol. 72, No. 2, pp. (125-31), ISBN: 0167-594X.

*Physiol*, Vol. 290, No. 2, pp. (H547-59), ISBN: 0363-6135.

*Neurol*, Vol. 9 Suppl 1, No. pp. (S3), ISBN: 1471-2377.

3rd edition, pp. (1-25), ISBN: 978-0-8493-8828-6.

No. 12, pp. (1295-312), ISBN: 1381-6128.

1932-6203.

0163-7258.

1550-7416.

1078-8956.

ISBN: 0364-5134.

ISBN: 0960-894X.

2753), ISBN: 0-19-262926-3.

(1281-4), ISBN: 1526-632X.

enhanced invasiveness in glioma cells. *PloS one*, Vol. 6, No. 6, pp. (e20614), ISBN:

undergoing regression after inhibition of VEGF signaling. *Am J Physiol Heart Circ* 

vascular architecture in nutrient and drug delivery: an invasion percolation-based

transmembrane ammonium ion gradient: from the bench to approved Doxil. *Liposome Technology: Entrapment of drugs and other materials into liposomes*, Vol. 2, No.

problems and the possibilities. *Pharmacol Ther*, Vol. 104, No. 1, pp. (29-45), ISBN:

disruption as a CNS delivery strategy. *AAPS J*, Vol. 10, No. 1, pp. (166-77), ISBN:

using neural progenitor cells. *Nature medicine*, Vol. 6, No. 4, pp. (447-50), ISBN:

Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo.

ethylnitrosourea-induced brain tumors. *Ann Neurol*, Vol. 14, No. 2, pp. (189-201),

association with 90-Y radioimmunotherapy (RIT) in recurrent glioblastoma. *J* 

and phosphorothioate siRNA. *Bioorg Med Chem Lett*, Vol. 14, No. 5, pp. (1139-43),

spinal cord in adults. *Oxford Textbook of Oncology* Vol. 2, No. 2nd edition, pp. (2707-

glioblastoma in the modern era? A phase II trial. *Neurology*, Vol. 63, No. 7, pp.

plus carmustine in glioblastoma recurrent or progressive after first-line temozolomide chemotherapy: a phase II study of the Gruppo Italiano Cooperativo

increased drug delivery to brain tumors. To date, the full potential of this technology has not been explored in GBM and may constitute a significant opportunity for delivery of targeted therapeutic approaches that encompass the use of multiple therapeutics all designed to inhibit phenotypes of GBM that contribute to its aggressive behavior.

It is now obvious that conventional treatment approaches for patients affected by GBM must change if improved treatment outcomes are going to be achieved. One important avenue would be to determine how many treatment agents must be included in order to achieve GBM cure. While most combination clinical trials will typically test 2 or 3 agents, it may be necessary to consider using 5 or even 10 different compounds that will block or eradicate all tumorigenic phenotypes of a cancer. Obviously, the design complexity of these trials may be a limiting factor. However, pre-clinical approaches where animals are inoculated orthotopically with tumor cells from patient samples could allow for testing several combination therapy options in a model that is more representative of the clinical reality than conventional models using commercially available cell lines. Moreover, non-invasive imaging using cancer cell lines expressing fluorescent proteins (e.g. mKate2 or mCherry proteins (Verreault et al., 2011d)) or bioluminescence (Maes et al., 2009) will facilitate the use of such models by providing immediate information on treatment response. It can be expected that the future of GBM treatment will incorporate information acquired from preclinical models obtained from orthotopic inoculation of patients' tumor samples to guide treatment decisions for these particular patients. We can be hopeful that the personalization of therapy options will improve treatment outcomes for individuals diagnosed with this devastating cancer.

#### **12. References**


increased drug delivery to brain tumors. To date, the full potential of this technology has not been explored in GBM and may constitute a significant opportunity for delivery of targeted therapeutic approaches that encompass the use of multiple therapeutics all designed to

It is now obvious that conventional treatment approaches for patients affected by GBM must change if improved treatment outcomes are going to be achieved. One important avenue would be to determine how many treatment agents must be included in order to achieve GBM cure. While most combination clinical trials will typically test 2 or 3 agents, it may be necessary to consider using 5 or even 10 different compounds that will block or eradicate all tumorigenic phenotypes of a cancer. Obviously, the design complexity of these trials may be a limiting factor. However, pre-clinical approaches where animals are inoculated orthotopically with tumor cells from patient samples could allow for testing several combination therapy options in a model that is more representative of the clinical reality than conventional models using commercially available cell lines. Moreover, non-invasive imaging using cancer cell lines expressing fluorescent proteins (e.g. mKate2 or mCherry proteins (Verreault et al., 2011d)) or bioluminescence (Maes et al., 2009) will facilitate the use of such models by providing immediate information on treatment response. It can be expected that the future of GBM treatment will incorporate information acquired from preclinical models obtained from orthotopic inoculation of patients' tumor samples to guide treatment decisions for these particular patients. We can be hopeful that the personalization of therapy options will improve treatment outcomes for individuals diagnosed with this

Aboody, K.S., Brown, A., Rainov, N.G. et al. (2000). Neural stem cells display extensive

Adamson, C., Kanu, O.O., Mehta, A.I. et al. (2009). Glioblastoma multiforme: a review of

Aigner, A. (2008). Cellular delivery in vivo of siRNA-based therapeutics. *Curr Pharm Des*,

Allen, C., Dos Santos, N., Gallagher, R. et al. (2002). Controlling the physical behavior and

Ambegia, E., Ansell, S., Cullis, P., Heyes, J., Palmer, L. & MacLachlan, I. (2005). Stabilized

Axelsen, J.B., Lotem, J., Sachs, L. & Domany, E. (2007). Genes overexpressed in different

*Acad Sci USA*, Vol. 104, No. 32, pp. (13122-13127), ISBN: 0027-8424.

No. 23, pp. (12846-51), ISBN: 0027-8424.

No. 8, pp. (1061-83), ISBN: 1744-7658.

Vol. 14, No. 34, pp. (3603-19), ISBN: 1873-4286.

1669, No. 2, pp. (155-63), ISBN: 0006-3002.

tropism for pathology in adult brain: evidence from intracranial gliomas. *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 97,

where we have been and where we are going. *Expert Opin Investig Drugs*, Vol. 18,

biological performance of liposome formulations through use of surface grafted poly(ethylene glycol). *Biosci Rep*, Vol. 22, No. 2, pp. (225-50), ISBN: 0144-8463. Altaner, C. (2008). Glioblastoma and stem cells. *Neoplasma*, Vol. 55, No. 5, pp. (369-74), ISBN:

plasmid-lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression. *Biochimica et biophysica acta*, Vol.

human solid cancers exhibit different tissue-specific expression profiles. *Proc Natl* 

inhibit phenotypes of GBM that contribute to its aggressive behavior.

devastating cancer.

**12. References** 

0028-2685.


siRNA-Based Therapy for Glioblastoma Patients 185

Clarke, J., Butowski, N. & Chang, S. (2010). Recent advances in therapy for glioblastoma.

Cloughesy, T.F., Wen, P.Y., Robins, H.I. et al. (2006). Phase II trial of tipifarnib in patients

da Fonseca, C.O., Linden, R., Futuro, D., Gattass, C.R. & Quirico-Santos, T. (2008). Ras

De Paula, D., Bentley, M.V. & Mahato, R.I. (2007). Hydrophobization and bioconjugation for

Deane, B.R. & Lantos, P.L. (1981). The vasculature of experimental brain tumours. Part 1. A

Desgrosellier, J.S. & Cheresh, D.A. (2010). Integrins in cancer: biological implications and

Ehtesham, M., Kabos, P., Gutierrez, M.A. et al. (2002a). Induction of glioblastoma apoptosis

Ehtesham, M., Kabos, P., Kabosova, A., Neuman, T., Black, K.L. & Yu, J.S. (2002b). The use

Ekstrand, A.J., James, C.D., Cavenee, W.K., Seliger, B., Pettersson, R.F. & Collins, V.P. (1991).

Engerhard, H.H., Groothuis, D.G. & Coons, S.W. (1999). Chapter 11: The Blood-Brain

Ermoian, R.P., Furniss, C.S., Lamborn, K.R. et al. (2002). Dysregulation of PTEN and protein

Fabel, K., Dietrich, J., Hau, P. et al. (2001). Long-term stabilization in patients with malignant

Fadul, C.E., Kingman, L.S., Meyer, L.P. et al. (2008). A phase II study of thalidomide and

Fan, Q.W., Specht, K.M., Zhang, C., Goldenberg, D.D., Shokat, K.M. & Weiss, W.A. (2003).

*Cancer research*, Vol. 62, No. 20, pp. (5657-63), ISBN: 0008-5472.

Vol. 51, No. 8, pp. (2164-72), ISBN: 0008-5472.

Vol. 8, No. 5, pp. (1100-6), ISBN: 1078-0432.

with recurrent malignant glioma either receiving or not receiving enzyme-inducing antiepileptic drugs: a North American Brain Tumor Consortium Study. *J Clin* 

pathway activation in gliomas: a strategic target for intranasal administration of perillyl alcohol. *Arch Immunol Ther Exp (Warsz)*, Vol. 56, No. 4, pp. (267-76), ISBN:

enhanced siRNA delivery and targeting. *RNA*, Vol. 13, No. 4, pp. (431-56), ISBN:

sequential light and electron microscope study of angiogenesis. *J Neurol Sci*, Vol. 49,

therapeutic opportunities. *Nat Rev Cancer*, Vol. 10, No. 1, pp. (9-22), ISBN: 1474-

using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand. *Cancer research*, Vol. 62, No. 24, pp. (7170-4), ISBN: 0008-

of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma.

Genes for epidermal growth factor receptor, transforming growth factor alpha, and epidermal growth factor and their expression in human gliomas in vivo. *Cancer Res*,

barrier: Structure, Function, and Response to Neoplasia & Chapter 20: Anatomy and Growth Patterns of Diffuse Gliomas. *The Gliomas*, Vol. 1, No. 1, pp. (115-121),

kinase B is associated with glioma histology and patient survival. *Clin Cancer Res*,

glioma after treatment with liposomal doxorubicin. *Cancer*, Vol. 92, No. 7, pp.

irinotecan for treatment of glioblastoma multiforme. *J Neurooncol*, Vol. 90, No. 2,

Combinatorial efficacy achieved through two-point blockade within a signaling pathway-a chemical genetic approach. *Cancer Res*, Vol. 63, No. 24, pp. (8930-8),

*Arch Neurol*, Vol. 67, No. 3, pp. (279-283), ISBN: 1538-3687.

*Oncol*, Vol. 24, No. 22, pp. (3651-6), ISBN: 1527-7755.

No. 1, pp. (55-66), ISBN: 0022-510X.

0004-069X.

1355-8382.

1768.

5472.

ISBN: 0721648258.

ISBN: 0008-5472.

(1936-42), ISBN: 0008-543X.

pp. (229-35), ISBN: 0167-594X.

di Neuro-Oncologia (GICNO). *J Clin Oncol*, Vol. 22, No. 23, pp. (4779-4786), ISBN: 0732-183X.


Brem, H., Mahaley, M.S., Jr., Vick, N.A. et al. (1991). Interstitial chemotherapy with drug

Brem, H., Tamargo, R.J., Olivi, A. et al. (1994). Biodegradable polymers for controlled

Brinker, B.T., Krown, S.E., Lee, J.Y. et al. (2008). Phase 1/2 trial of BMS-275291 in patients

Bumcrot, D., Manoharan, M., Koteliansky, V. & Sah, D.W. (2006). RNAi therapeutics: a

Cahill, D.P., Levine, K.K., Betensky, R.A. et al. (2007). Loss of the mismatch repair protein

*Association for Cancer Research*, Vol. 13, No. 7, pp. (2038-45), ISBN: 1078-0432. Carter, A. (2010). Integrins as target: first phase III trial launches, but questions remain. *J* 

Cattel, L., Ceruti, M. & Dosio, F. (2004). From conventional to stealth liposomes: a new

CBTRUS. (2010). Statistical Report: Primary Brain and Central Nervous System Tumors

CCS. (2010). Statistics for brain cancer. *In: Canadian Cancer Society*, Vol. Accessed in February

CGARN. (2008). Comprehensive genomic characterization defines human glioblastoma

Chandana, S.R., Movva, S., Arora, M. & Singh, T. (2008). Primary brain tumors in adults. *Am* 

Chang, S.M., Wen, P., Cloughesy, T. et al. (2005). Phase II study of CCI-779 in patients with

Charalambous, C., Chen, T.C. & Hofman, F.M. (2006). Characteristics of tumor-associated

Chinot, O.L., de La Motte Rouge, T., Moore, N. et al. (2011). AVAglio: Phase 3 trial of

Chu, Q.S., Forouzesh, B., Syed, S. et al. (2007). A phase II and pharmacological study of the

*Natl Cancer Inst*, Vol. 102, No. 10, pp. (675-7), ISBN: 1460-2105.

2011, No. pp. (Available from: www.cancer.ca), ISBN:

*Fam Physician*, Vol. 77, No. 10, pp. (1423-30), ISBN: 0002-838X.

*J Neurosurg*, Vol. 80, No. 2, pp. (283-90), ISBN: 0022-3085.

0732-183X.

543X.

1120-009X.

www.cbtrus.org ), ISBN:

ISBN: 0167-6997.

1865-8652.

No. 4, pp. (E22), ISBN: 1092-0684.

9), ISBN: 1552-4450.

pp. (441-6), ISBN: 0022-3085.

di Neuro-Oncologia (GICNO). *J Clin Oncol*, Vol. 22, No. 23, pp. (4779-4786), ISBN:

polymer implants for the treatment of recurrent gliomas. *J Neurosurg*, Vol. 74, No. 3,

delivery of chemotherapy with and without radiation therapy in the monkey brain.

with human immunodeficiency virus-related Kaposi sarcoma: a multicenter trial of the AIDS Malignancy Consortium. *Cancer*, Vol. 112, No. 5, pp. (1083-8), ISBN: 0008-

potential new class of pharmaceutical drugs. *Nat Chem Biol*, Vol. 2, No. 12, pp. (711-

MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment. *Clinical cancer research : an official journal of the American* 

Frontier in cancer chemotherapy. *J Chemother*, Vol. 16 Suppl 4, No. pp. (94-7), ISBN:

Diagnosed in the United States in 2004-2006. *In: Central Brain Tumor Registry of the United States, Hinsdale, IL.*, Vol. Accessed in February 2011, No. pp. (Available from:

genes and core pathways. *Nature*, Vol. 455, No. 7216, pp. (1061-8), ISBN: 1476-4687.

recurrent glioblastoma multiforme. *Invest New Drugs*, Vol. 23, No. 4, pp. (357-61),

endothelial cells derived from glioblastoma multiforme. *Neurosurg Focus*, Vol. 20,

bevacizumab plus temozolomide and radiotherapy in newly diagnosed glioblastoma multiforme. *Advances in therapy*, Vol. 28, No. 4, pp. (334-40), ISBN:

matrix metalloproteinase inhibitor (MMPI) COL-3 in patients with advanced soft tissue sarcomas. *Invest New Drugs*, Vol. 25, No. 4, pp. (359-67), ISBN: 0167-6997.


siRNA-Based Therapy for Glioblastoma Patients 187

Gondi, C.S., Lakka, S.S., Dinh, D.H., Olivero, W.C., Gujrati, M. & Rao, J.S. (2007).

Gray, H. (2005). Chapter 22: Cerebral Hemisphere & Chapter 23: Basal Ganglia. *Gray's* 

Groothuis, D.R., Pasternak, J.F., Fischer, J.M., Blasberg, R.G., Bigner, D.D. & Vick, N.A.

Grossman, S.A., Alavi, J.B., Supko, J.G. et al. (2005). Efficacy and toxicity of the antisense

Grzelinski, M., Urban-Klein, B., Martens, T. et al. (2006). RNA interference-mediated gene

Guo, W., Lasky, J.L., 3rd & Wu, H. (2006). Cancer stem cells. *Pediatr Res*, Vol. 59, No. 4 Pt 2,

Gururangan, S., Cokgor, L., Rich, J.N. et al. (2001). Phase I study of Gliadel wafers plus

Haas-Kogan, D., Shalev, N., Wong, M., Mills, G., Yount, G. & Stokoe, D. (1998). Protein

Hadjipanayis, C.G. & Van Meir, E.G. (2009). Tumor initiating cells in malignant gliomas:

Hanahan, D. & Weinberg, R.A. (2000). The hallmarks of cancer. *Cell*, Vol. 100, No. 1, pp. (57-

Hanahan, D. & Weinberg, R.A. (2011). Hallmarks of cancer: the next generation. *Cell*, Vol.

Hashizume, R., Ozawa, T., Gryaznov, S.M. et al. (2008). New therapeutic approach for brain

Hattori, Y., Shi, L., Ding, W. et al. (2009). Novel irinotecan-loaded liposome using phytic

astrocytomas. *Neuro Oncol*, Vol. 7, No. 1, pp. (32-40), ISBN: 1522-8517. Grossman, S.A., Reinhard, C., Colvin, O.M. et al. (1992). The intracerebral distribution of

*Cancer Res*, Vol. 43, No. 7, pp. (3362-7), ISBN: 0008-5472.

ISBN: 0950-9232.

pp. (4051-60), ISBN: 1078-0432.

76, No. 4, pp. (640-7), ISBN: 0022-3085.

pp. (59R-64R), ISBN: 0031-3998.

9822.

1432-1440.

70), ISBN: 0092-8674.

Vol. 17, No. 7, pp. (751-66), ISBN: 1043-0342.

*Oncol*, Vol. 3, No. 4, pp. (246-50), ISBN: 1522-8517.

144, No. 5, pp. (646-74), ISBN: 1097-4172.

No. 2, pp. (112-20), ISBN: 1522-8517.

1, pp. (30-7), ISBN: 1873-4995.

430), ISBN: 0443071683.

angiogenesis and tumor growth in gliomas. *Oncogene*, Vol. 23, No. 52, pp. (8486-96),

Intraperitoneal injection of a hairpin RNA-expressing plasmid targeting urokinasetype plasminogen activator (uPA) receptor and uPA retards angiogenesis and inhibits intracranial tumor growth in nude mice. *Clin Cancer Res*, Vol. 13, No. 14,

*Anatomy: The anatomical basis of Clinical Practice* Vol. 1, No. 39th edition, pp. (387-

(1983). Regional measurements of blood flow in experimental RG-2 rat gliomas.

oligonucleotide aprinocarsen directed against protein kinase C-alpha delivered as a 21-day continuous intravenous infusion in patients with recurrent high-grade

BCNU delivered by surgically implanted biodegradable polymers. *J Neurosurg*, Vol.

silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts. *Hum Gene Ther*,

temozolomide in adults with recurrent supratentorial high-grade gliomas. *Neuro* 

kinase B (PKB/Akt) activity is elevated in glioblastoma cells due to mutation of the tumor suppressor PTEN/MMAC. *Curr Biol*, Vol. 8, No. 21, pp. (1195-8), ISBN: 0960-

biology and implications for therapy. *J Mol Med*, Vol. 87, No. 4, pp. (363-74), ISBN:

tumors: Intranasal delivery of telomerase inhibitor GRN163. *Neuro Oncol*, Vol. 10,

acid with high therapeutic efficacy for colon tumors. *J Control Release*, Vol. 136, No.


Fawcett, D.W. (1994). Section 11: The nervous tissue. *A textbook of histology*, Vol. 1, No. 12th

Filleur, S., Courtin, A., Ait-Si-Ali, S. et al. (2003). SiRNA-mediated inhibition of vascular

Fine, H.A., Wen, P.Y., Maher, E.A. et al. (2003). Phase II trial of thalidomide and carmustine

Fouse, S.D. & Costello, J.F. (2009). Epigenetics of neurological cancers. *Future oncology*, Vol.

Franceschi, E., Cavallo, G., Lonardi, S. et al. (2007). Gefitinib in patients with progressive

Frazier, J.L., Johnson, M.W., Burger, P.C., Weingart, J.D. & Quinones-Hinojosa, A. (2009).

Galanis, E., Buckner, J.C., Maurer, M.J. et al. (2005). Phase II trial of temsirolimus (CCI-779)

George, J., Banik, N.L. & Ray, S.K. (2008). Combination of taxol and Bcl-2 siRNA induces

George, J., Banik, N.L. & Ray, S.K. (2009). Combination of hTERT knockdown and IFN-

Gerlinger, M. & Swanton, C. (2010). How Darwinian models inform therapeutic failure

Gerstner, E.R., Yip, S., Wang, D.L., Louis, D.N., Iafrate, A.J. & Batchelor, T.T. (2009). Mgmt

glioblastoma. *Neurology*, Vol. 73, No. 18, pp. (1509-10), ISBN: 1526-632X. Gladson, C.L. & Cheresh, D.A. (1991). Glioblastoma expression of vitronectin and the alpha

Gondi, C.S., Lakka, S.S., Dinh, D.H., Olivero, W.C., Gujrati, M. & Rao, J.S. (2004). RNAi-

complexes. *J Immunol*, Vol. 160, No. 9, pp. (4580-6), ISBN: 0022-1767. Friedman, H.S., Prados, M.D., Wen, P.Y. et al. (2009). Bevacizumab alone and in

Study. *J Clin Oncol*, Vol. 23, No. 23, pp. (5294-304), ISBN: 0732-183X. Galanis, E., Jaeckle, K.A., Maurer, M.J. et al. (2009). Phase II trial of vorinostat in recurrent

*Oncol*, Vol. 27, No. 12, pp. (2052-8), ISBN: 1527-7755.

103, No. 8, pp. (1139-43), ISBN: 1532-1827.

88, No. 6, pp. (1924-32), ISBN: 0021-9738.

tumor growth. *J Cell Mol Med*, Vol. No. pp. ISBN: 1582-4934.

*Clin Cancer Res*, Vol. 15, No. 23, pp. (7186-95), ISBN: 1078-0432.

endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. *Cancer Res*, Vol.

for patients with recurrent high-grade gliomas. *J Clin Oncol*, Vol. 21, No. 12, pp.

high-grade gliomas: a multicentre phase II study by Gruppo Italiano Cooperativo di Neuro-Oncologia (GICNO). *Br J Cancer*, Vol. 96, No. 7, pp. (1047-51), ISBN: 0007-

Rapid malignant transformation of low-grade astrocytomas: report of 2 cases and review of the literature. *Surgical neurology*, Vol. Aug 6, No. pp. ISBN: 1879-3339. Freimark, B.D., Blezinger, H.P., Florack, V.J. et al. (1998). Cationic lipids enhance cytokine

and cell influx levels in the lung following administration of plasmid: cationic lipid

combination with irinotecan in recurrent glioblastoma. *J Clin Oncol*, Vol. 27, No. 28,

in recurrent glioblastoma multiforme: a North Central Cancer Treatment Group

glioblastoma multiforme: a north central cancer treatment group study. *J Clin* 

apoptosis in human glioblastoma cells and inhibits invasion, angiogenesis, and

gamma treatment inhibited angiogenesis and tumor progression in glioblastoma.

initiated by clonal heterogeneity in cancer medicine. *British journal of cancer*, Vol.

methylation is a prognostic biomarker in elderly patients with newly diagnosed

v beta 3 integrin. Adhesion mechanism for transformed glial cells. *J Clin Invest*, Vol.

mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion,

edition, pp. (336-339), ISBN: 0412046911

63, No. 14, pp. (3919-22), ISBN: 0008-5472.

5, No. 10, pp. (1615-29), ISBN: 1744-8301.

(2299-304), ISBN: 0732-183X.

pp. (4733-40), ISBN: 1527-7755.

0920.

angiogenesis and tumor growth in gliomas. *Oncogene*, Vol. 23, No. 52, pp. (8486-96), ISBN: 0950-9232.


siRNA-Based Therapy for Glioblastoma Patients 189

Jain, R.K., di Tomaso, E., Duda, D.G., Loeffler, J.S., Sorensen, A.G. & Batchelor, T.T. (2007).

Jansen, M., Yip, S. & Louis, D.N. (2010). Molecular pathology in adult gliomas: diagnostic,

Judge, A.D., Robbins, M., Tavakoli, I. et al. (2009). Confirming the RNAi-mediated

Judson, I., Radford, J.A., Harris, M. et al. (2001). Randomised phase II trial of pegylated

Kappelle, A.C., Postma, T.J., Taphoorn, M.J. et al. (2001). PCV chemotherapy for recurrent glioblastoma multiforme. *Neurology*, Vol. 56, No. 1, pp. (118-20), ISBN: 0028-3878. Kaur, B., Khwaja, F.W., Severson, E.A., Matheny, S.L., Brat, D.J. & Van Meir, E.G. (2005).

angiogenesis. *Neuro-oncology*, Vol. 7, No. 2, pp. (134-53), ISBN: 1522-8517. Khan, A., Benboubetra, M., Sayyed, P.Z. et al. (2004). Sustained polymeric delivery of gene

Knobbe, C.B., Merlo, A. & Reifenberger, G. (2002). Pten signaling in gliomas. *Neuro Oncol*,

Krakstad, C. & Chekenya, M. (2010). Survival signalling and apoptosis resistance in

Krauze, M.T., Noble, C.O., Kawaguchi, T. et al. (2007). Convection-enhanced delivery of

Kreisl, T.N., Kim, L., Moore, K. et al. (2009). Phase II trial of single-agent bevacizumab

Krex, D., Klink, B., Hartmann, C. et al. (2007). Long-term survival with glioblastoma

glioblastoma. *J Clin Oncol*, Vol. 27, No. 5, pp. (740-5), ISBN: 1527-7755. Kreisl, T.N., Kotliarova, S., Butman, J.A. et al. (2010). A phase I/II trial of enzastaurin in

studies. *J Drug Target*, Vol. 12, No. 6, pp. (393-404), ISBN: 1061-186X. Kim, T.Y., Zhong, S., Fields, C.R., Kim, J.H. & Robertson, K.D. (2006). Epigenomic profiling

Vol. 4, No. 3, pp. (196-211), ISBN: 1522-8517.

*clinical investigation*, Vol. 119, No. 3, pp. (661-73), ISBN: 1558-8238.

1471-003X.

0008-5472.

(135), ISBN: 1476-4598.

403), ISBN: 1522-8517.

ISBN: 1523-5866.

1460-2156.

ISBN: 1474-4465.

(2542-52), ISBN: 1527-7755.

Angiogenesis in brain tumours. *Nat Rev Neurosci*, Vol. 8, No. 8, pp. (610-22), ISBN:

prognostic, and predictive markers. *Lancet neurology*, Vol. 9, No. 7, pp. (717-26),

mechanism of action of siRNA-based cancer therapeutics in mice. *The Journal of* 

liposomal doxorubicin (DOXIL/CAELYX) versus doxorubicin in the treatment of advanced or metastatic soft tissue sarcoma: a study by the EORTC Soft Tissue and Bone Sarcoma Group. *Eur J Cancer*, Vol. 37, No. 7, pp. (870-7), ISBN: 0959-8049. Kamoun, W.S., Ley, C.D., Farrar, C.T. et al. (2009). Edema control by cediranib, a vascular

endothelial growth factor receptor-targeted kinase inhibitor, prolongs survival despite persistent brain tumor growth in mice. *J Clin Oncol*, Vol. 27, No. 15, pp.

Hypoxia and the hypoxia-inducible-factor pathway in glioma growth and

silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo

reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma. *Cancer research*, Vol. 66, No. 15, pp. (7490-501), ISBN:

glioblastomas: opportunities for targeted therapeutics. *Mol Cancer*, Vol. 9, No. pp.

nanoliposomal CPT-11 (irinotecan) and PEGylated liposomal doxorubicin (Doxil) in rodent intracranial brain tumor xenografts. *Neuro Oncol*, Vol. 9, No. 4, pp. (393-

followed by bevacizumab plus irinotecan at tumor progression in recurrent

patients with recurrent high-grade gliomas. *Neuro Oncol*, Vol. 12, No. 2, pp. (181-9),

multiforme. *Brain : a journal of neurology*, Vol. 130, No. 10, pp. (2596-606), ISBN:


Hau, P., Fabel, K., Baumgart, U. et al. (2004). Pegylated liposomal doxorubicin-efficacy in

Hegi, M.E., Diserens, A.C., Gorlia, T. et al. (2005). MGMT gene silencing and benefit from

Heidel, J.D., Hu, S., Liu, X.F., Triche, T.J. & Davis, M.E. (2004). Lack of interferon response in

Helene, C. (1994). Control of oncogene expression by antisense nucleic acids. *Eur J Cancer*,

Hendruschk, S., Wiedemuth, R., Aigner, A. et al. (2011). RNA interference targeting survivin

Heyes, J., Palmer, L., Bremner, K. & MacLachlan, I. (2005). Cationic lipid saturation

Holland, E.C. (2000). Glioblastoma multiforme: the terminator. *Proceedings of the National* 

Holt, S.E. & Shay, J.W. (1999). Role of telomerase in cellular proliferation and cancer. *J Cell* 

Hsu, A.R., Hou, L.C., Veeravagu, A. et al. (2006). In vivo near-infrared fluorescence imaging

Hunter, C., Smith, R., Cahill, D.P. et al. (2006). A hypermutation phenotype and somatic

Ichimura, K., Pearson, D.M., Kocialkowski, S. et al. (2009). IDH1 mutations are present in the

Idbaih, A., Ducray, F., Sierra Del Rio, M., Hoang-Xuan, K. & Delattre, J.Y. (2008).

Jacinto, F.V. & Esteller, M. (2007). MGMT hypermethylation: a prognostic foe, a predictive friend. *DNA Repair (Amst)*, Vol. 6, No. 8, pp. (1155-60), ISBN: 1568-7864. Jain, R.K. (2001). Normalizing tumor vasculature with anti-angiogenic therapy: a new

Jain, R.K. (2005). Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. *Science*, Vol. 307, No. 5706, pp. (58-62), ISBN: 1095-9203.

Vol. 30A, No. 11, pp. (1721-6), ISBN: 0959-8049.

*Neuro-oncology*, Vol. No. pp. ISBN: 1523-5866.

*release*, Vol. 107, No. 2, pp. (276-87), ISBN: 0168-3659.

*Physiol*, Vol. 180, No. 1, pp. (10-8), ISBN: 0021-9541.

*oncology*, Vol. 11, No. 4, pp. (341-7), ISBN: 1522-8517.

8, No. 6, pp. (315-23), ISBN: 1536-1632.

ISBN: 0008-543X.

1533-4406.

0027-8424.

0027-8424.

8956.

(978-92), ISBN: 1549-490X.

0156.

patients with recurrent high-grade glioma. *Cancer*, Vol. 100, No. 6, pp. (1199-207),

temozolomide in glioblastoma. *N Engl J Med*, Vol. 352, No. 10, pp. (997-1003), ISBN:

animals to naked siRNAs. *Nat Biotechnol*, Vol. 22, No. 12, pp. (1579-82), ISBN: 1087-

exerts antitumoral effects in vitro and in established glioma xenografts in vivo.

influences intracellular delivery of encapsulated nucleic acids. *Journal of controlled* 

*Academy of Sciences of the United States of America*, Vol. 97, No. 12, pp. (6242-4), ISBN:

of integrin alphavbeta3 in an orthotopic glioblastoma model. *Mol Imaging Biol*, Vol.

MSH6 mutations in recurrent human malignant gliomas after alkylator chemotherapy. *Cancer research*, Vol. 66, No. 8, pp. (3987-91), ISBN: 0008-5472. Huwyler, J., Wu, D. & Pardridge, W.M. (1996). Brain drug delivery of small molecules using

immunoliposomes. *Proc Natl Acad Sci U S A*, Vol. 93, No. 24, pp. (14164-9), ISBN:

majority of common adult gliomas but rare in primary glioblastomas. *Neuro-*

Therapeutic application of noncytotoxic molecular targeted therapy in gliomas: growth factor receptors and angiogenesis inhibitors. *Oncologist*, Vol. 13, No. 9, pp.

paradigm for combination therapy. *Nat Med*, Vol. 7, No. 9, pp. (987-9), ISBN: 1078-


siRNA-Based Therapy for Glioblastoma Patients 191

Louis, D.N., Ohgaki, H., Wiestler, O.D. et al. (2007). The 2007 WHO classification of tumours

Lundstrom, K. & Boulikas, T. (2003). Viral and non-viral vectors in gene therapy: technology

Lustig, R., Mikkelsen, T., Lesser, G. et al. (2008). Phase II preradiation R115777 (tipifarnib) in

Ma, J., Li, S., Reed, K., Guo, P. & Gallo, J.M. (2003). Pharmacodynamic-mediated effects of

Maeda, H., Bharate, G.Y. & Daruwalla, J. (2009). Polymeric drugs for efficient tumor-

Maes, W., Deroose, C., Reumers, V. et al. (2009). In vivo bioluminescence imaging in an

Marx, G.M., Pavlakis, N., McCowatt, S. et al. (2001). Phase II study of thalidomide in the

Masri, J., Bernath, A., Martin, J. et al. (2007). mTORC2 activity is elevated in gliomas and

Mayer, L.D., Masin, D., Nayar, R., Boman, N.L. & Bally, M.B. (1995). Pharmacology of

McCaffrey, A.P., Meuse, L., Pham, T.T., Conklin, D.S., Hannon, G.J. & Kay, M.A. (2002).

Messerer, C.L., Ramsay, E.C., Waterhouse, D. et al. (2004). Liposomal irinotecan:

Morrissey, D.V., Lockridge, J.A., Shaw, L. et al. (2005). Potent and persistent in vivo anti-

Mukai, S., Kondo, Y., Koga, S., Komata, T., Barna, B.P. & Kondo, S. (2000). 2-5A antisense

Nakada, M., Okada, Y. & Yamashita, J. (2003). The role of matrix metalloproteinases in glioma invasion. *Front Biosci*, Vol. 8, No. 1, pp. (e261-9), ISBN: 1093-4715.

ISBN: 0001-6322.

ISBN: 1533-0346.

6, pp. (1004-9), ISBN: 1522-8517.

pp. (409-19), ISBN: 1873-3441.

(573-8), ISBN: 0300-5127.

(31-8), ISBN: 0167-594X.

pp. (1002-7), ISBN: 1087-0156.

No. 16, pp. (4461-7), ISBN: 0008-5472.

0836.

305, No. 3, pp. (833-9), ISBN: 0022-3565.

No. 24, pp. (11712-20), ISBN: 1538-7445.

*Cancer*, Vol. 71, No. 3, pp. (482-8), ISBN: 0007-0920.

of the central nervous system. *Acta neuropathologica*, Vol. 114, No. 2, pp. (97-109),

development and clinical trials. *Technol Cancer Res Treat*, Vol. 2, No. 5, pp. (471-86),

newly diagnosed GBM with residual enhancing disease. *Neuro Oncol*, Vol. 10, No.

the angiogenesis inhibitor SU5416 on the tumor disposition of temozolomide in subcutaneous and intracerebral glioma xenograft models. *J Pharmacol Exp Ther*, Vol.

targeted drug delivery based on EPR-effect. *Eur J Pharm Biopharm*, Vol. 71, No. 3,

experimental mouse model for dendritic cell based immunotherapy against malignant glioma. *J Neurooncol*, Vol. 91, No. 2, pp. (127-39), ISBN: 0167-594X. Manning, B.D. & Cantley, L.C. (2003). United at last: the tuberous sclerosis complex gene

products connect the phosphoinositide 3-kinase/Akt pathway to mammalian target of rapamycin (mTOR) signalling. *Biochem Soc Trans*, Vol. 31, No. Pt 3, pp.

treatment of recurrent glioblastoma multiforme. *J Neurooncol*, Vol. 54, No. 1, pp.

promotes growth and cell motility via overexpression of rictor. *Cancer Res*, Vol. 67,

liposomal vincristine in mice bearing L1210 ascitic and B16/BL6 solid tumours. *Br J* 

RNA interference in adult mice. *Nature*, Vol. 418, No. 6893, pp. (38-9), ISBN: 0028-

formulation development and therapeutic assessment in murine xenograft models of colorectal cancer. *Clin Cancer Res*, Vol. 10, No. 19, pp. (6638-49), ISBN: 1078-0432.

HBV activity of chemically modified siRNAs. *Nature biotechnology*, Vol. 23, No. 8,

telomerase RNA therapy for intracranial malignant gliomas. *Cancer Res*, Vol. 60,


Kunwar, S., Prados, M.D., Chang, S.M. et al. (2007). Direct intracerebral delivery of

Lai, A., Filka, E., McGibbon, B. et al. (2008). Phase II pilot study of bevacizumab in

Lai, A., Tran, A., Nghiemphu, P.L. et al. (2011). Phase II study of bevacizumab plus

Lakka, S.S., Gondi, C.S., Dinh, D.H. et al. (2005). Specific interference of urokinase-type

Laterra, J.J., Grossman, S.A., Carson, K.A., Lesser, G.J., Hochberg, F.H. & Gilbert, M.R.

Le, S., Zhu, J.J., Anthony, D.C., Greider, C.W. & Black, P.M. (1998). Telomerase activity in human gliomas. *Neurosurgery*, Vol. 42, No. 5, pp. (1120-5), ISBN: 0148-396X. Lewis, D.L., Hagstrom, J.E., Loomis, A.G., Wolff, J.A. & Herweijer, H. (2002). Efficient

Li, S., Wu, S.P., Whitmore, M. et al. (1999). Effect of immune response on gene transfer to the

Li, X.Y., Zhang, L.Q., Zhang, X.G. et al. (2010). Association between AKT/mTOR signalling

Lichota, J., Skjorringe, T., Thomsen, L.B. & Moos, T. (2009). Macromolecular drug transport

Lidar, Z., Mardor, Y., Jonas, T. et al. (2004). Convection-enhanced delivery of paclitaxel for

Lieberman, J., Song, E., Lee, S.K. & Shankar, P. (2003). Interfering with disease: opportunities

Lin, S.H. & Kleinberg, L.R. (2008). Carmustine wafers: localized delivery of

*Neurosurg*, Vol. 100, No. 3, pp. (472-9), ISBN: 0022-3085.

No. 7, pp. (837-44), ISBN: 1527-7755.

Vol. 32, No. 1, pp. (107-8), ISBN: 1061-4036.

No. 5 Pt 1, pp. (L796-804), ISBN: 0002-9513.

(Epub), ISBN: 1573-7373.

(397-403), ISBN: 1471-4914.

No. 3, pp. (343-59), ISBN: 1744-8328.

1471-4159.

(1372-80), ISBN: 0360-3016.

1527-7755.

0021-9258.

8517.

cintredekin besudotox (IL13-PE38QQR) in recurrent malignant glioma: a report by the Cintredekin Besudotox Intraparenchymal Study Group. *J Clin Oncol*, Vol. 25,

combination with temozolomide and regional radiation therapy for up-front treatment of patients with newly diagnosed glioblastoma multiforme: interim analysis of safety and tolerability. *Int J Radiat Oncol Biol Phys*, Vol. 71, No. 5, pp.

temozolomide during and after radiation therapy for patients with newly diagnosed glioblastoma multiforme. *J Clin Oncol*, Vol. 29, No. 2, pp. (142-8), ISBN:

plasminogen activator receptor and matrix metalloproteinase-9 gene expression induced by double-stranded RNA results in decreased invasion, tumor growth, and angiogenesis in gliomas. *J Biol Chem*, Vol. 280, No. 23, pp. (21882-92), ISBN:

(2004). Suramin and radiotherapy in newly diagnosed glioblastoma: phase 2 NABTT CNS Consortium study. *Neuro Oncol*, Vol. 6, No. 1, pp. (15-20), ISBN: 1522-

delivery of siRNA for inhibition of gene expression in postnatal mice. *Nat Genet*,

lung via systemic administration of cationic lipidic vectors. *Am J Physiol*, Vol. 276,

pathway and malignancy grade of human gliomas. *J Neurooncol*, Vol. No. pp.

into the brain using targeted therapy. *J Neurochem*, Vol. 113, No. 1, pp. (1-13), ISBN:

the treatment of recurrent malignant glioma: a phase I/II clinical study. *J* 

and roadblocks to harnessing RNA interference. *Trends Mol Med*, Vol. 9, No. 9, pp.

chemotherapeutic agents in CNS malignancies. *Expert Rev Anticancer Ther*, Vol. 8,


siRNA-Based Therapy for Glioblastoma Patients 193

Park, J.W., Benz, C.C. & Martin, F.J. (2004). Future directions of liposome- and

Pas, J., Wyszko, E., Rolle, K. et al. (2006). Analysis of structure and function of tenascin-C. *Int J Biochem Cell Biol*, Vol. 38, No. 9, pp. (1594-602), ISBN: 1357-2725. Patchell, R.A., Regine, W.F., Ashton, P. et al. (2002). A phase I trial of continuously infused

Patel, M.M., Goyal, B.R., Bhadada, S.V., Bhatt, J.S. & Amin, A.F. (2009). Getting into the

Perry, J.R., Belanger, K., Mason, W.P. et al. (2010). Phase II trial of continuous dose-intense

Pirollo, K.F., Zon, G., Rait, A. et al. (2006). Tumor-targeting nanoimmunoliposome complex

Porter, C.A. & Rifkin, R.M. (2007). Clinical benefits and economic analysis of pegylated

Prados, M. (2002). Histology of Primary Tumors of the Central Nervous System. *American* 

Prados, M.D., Chang, S.M., Butowski, N. et al. (2009). Phase II study of erlotinib plus

Prakash, S., Malhotra, M. & Rengaswamy, V. (2010). Nonviral siRNA delivery for gene

Querbes, W., Ge, P., Zhang, W. et al. (2009). Direct CNS delivery of siRNA mediates robust

Raizer, J.J., Abrey, L.E., Lassman, A.B. et al. (2010). A phase II trial of erlotinib in patients

Rasheed, B.K., Stenzel, T.T., McLendon, R.E. et al. (1997). PTEN gene mutations are seen in

*Neurooncol*, Vol. 60, No. 1, pp. (37-42), ISBN: 0167-594X.

pp. (196-205), ISBN: 0093-7754.

(35-58), ISBN: 1172-7047.

ISBN: 1043-0342.

ISBN: 1550090984

ISBN: 1527-7755.

1557-8526.

ISBN: 0008-5472.

29), ISBN: 1940-6029.

Vol. 68, No. 3, pp. (607-17), ISBN: 0939-6411.

1557-9190.

pp. (2051-7), ISBN: 1527-7755.

immunoliposome-based cancer therapeutics. *Semin Oncol*, Vol. 31, No. 6 Suppl 13,

intratumoral bleomycin for the treatment of recurrent glioblastoma multiforme. *J* 

brain: approaches to enhance brain drug delivery. *CNS Drugs*, Vol. 23, No. 1, pp.

temozolomide in recurrent malignant glioma: RESCUE study. *Journal of clinical oncology : official journal of the American Society of Clinical Oncology*, Vol. 28, No. 12,

for short interfering RNA delivery. *Hum Gene Ther*, Vol. 17, No. 1, pp. (117-24),

liposomal doxorubicin/vincristine/dexamethasone versus doxorubicin/vincristine/dexamethasone in patients with newly diagnosed multiple myeloma. *Clin Lymphoma Myeloma*, Vol. 7 No. Suppl 4, pp. (S150-5), ISBN:

*Cancer Society Atlas of Clinical Oncology: Brain Cancer*, Vol. 1, No. 1, pp. (29-103),

temozolomide during and after radiation therapy in patients with newly diagnosed glioblastoma multiforme or gliosarcoma. *J Clin Oncol*, Vol. 27, No. 4, pp. (579-84),

silencing in neurodegenerative diseases. *Methods Mol Biol*, Vol. 623, No. pp. (211-

silencing in oligodendrocytes. *Oligonucleotides*, Vol. 19, No. 1, pp. (23-29), ISBN:

with recurrent malignant gliomas and nonprogressive glioblastoma multiforme postradiation therapy. *Neuro Oncol*, Vol. 12, No. 1, pp. (95-103), ISBN: 1523-5866. Ramsay, E., Alnajim, J., Anantha, M. et al. (2008). A novel liposomal irinotecan formulation

with significant anti-tumour activity: use of the divalent cation ionophore A23187 and copper-containing liposomes to improve drug retention. *Eur J Pharm Biopharm*,

high-grade but not in low-grade gliomas. *Cancer Res*, Vol. 57, No. 19, pp. (4187-90),


NCCN. (2011). National Comprehensive Cancer Network Clinical Practice Guidelines in

Newlands, E.S., Stevens, M.F., Wedge, S.R., Wheelhouse, R.T. & Brock, C. (1997).

Nicholas, M.K., Lukas, R.V., Jafri, N.F., Faoro, L. & Salgia, R. (2006). Epidermal growth

Ningaraj, N.S., Rao, M. & Black, K.L. (2003). Calcium-dependent potassium channels as a

Nishikawa, M., Takemura, S., Takakura, Y. & Hashida, M. (1998). Targeted delivery of

Noble, C.O., Krauze, M.T., Drummond, D.C. et al. (2006). Novel nanoliposomal CPT-11

Norden, A.D., Drappatz, J. & Wen, P.Y. (2009). Antiangiogenic therapies for high-grade glioma. *Nat Rev Neurol*, Vol. 5, No. 11, pp. (610-20), ISBN: 1759-4766. Noushmehr, H., Weisenberger, D.J., Diefes, K. et al. (2010). Identification of a CpG island

O'Brien, M.E., Wigler, N., Inbar, M. et al. (2004). Reduced cardiotoxicity and comparable

Ogris, M., Brunner, S., Schuller, S., Kircheis, R. & Wagner, E. (1999). PEGylated

Ohgaki, H., Dessen, P., Jourde, B. et al. (2004). Genetic pathways to glioblastoma: a population-based study. *Cancer Res*, Vol. 64, No. 19, pp. (6892-9), ISBN: 0008-5472. Ohvo-Rekila, H., Ramstedt, B., Leppimaki, P. & Slotte, J.P. (2002). Cholesterol interactions

Pardridge, W.M. (2007). Blood-brain barrier delivery. *Drug Discov Today*, Vol. 12, No. 1-2,

efficacy. *Cancer Res*, Vol. 66, No. 5, pp. (2801-6), ISBN: 0008-5472.

gliomas. *Clin Cancer Res*, Vol. 12, No. 24, pp. (7261-70), ISBN: 1078-0432. Ningaraj, N.S. (2006). Drug delivery to brain tumours: challenges and progress. *Expert Opin* 

*Drug Deliv*, Vol. 3, No. 4, pp. (499-509), ISBN: 1742-5247.

pp. (Available from: www.nccn.org), ISBN:

Vol. 16, No. 5, pp. (291-8), ISBN: 0214-0934.

17, No. 5, pp. (510-22), ISBN: 1878-3686.

Vol. 96, No. 2, pp. (189-95), ISBN: 0007-0920.

Vol. 6, No. 4, pp. (595-605), ISBN: 0969-7128.

0305-7372.

ISBN: 0022-3565.

0163-7827.

pp. (54-61), ISBN: 1359-6446.

Oncology. *In: Anaplastic Glioma/Glioblastoma*, Vol. Accessed in February 2011, No.

Temozolomide: a review of its discovery, chemical properties, pre-clinical development and clinical trials. *Cancer Treat Rev*, Vol. 23, No. 1, pp. (35-61), ISBN:

factor receptor - mediated signal transduction in the development and therapy of

target protein for modulation of the blood-brain tumor barrier. *Drug News Perspect*,

plasmid DNA to hepatocytes in vivo: optimization of the pharmacokinetics of plasmid DNA/galactosylated poly(L-lysine) complexes by controlling their physicochemical properties. *J Pharmacol Exp Ther*, Vol. 287, No. 1, pp. (408-15),

infused by convection-enhanced delivery in intracranial tumors: pharmacology and

methylator phenotype that defines a distinct subgroup of glioma. *Cancer Cell*, Vol.

efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. *Ann Oncol*, Vol. 15, No. 3, pp. (440-9), ISBN: 0923-7534. O'Connor, J.P., Jackson, A., Parker, G.J. & Jayson, G.C. (2007). DCE-MRI biomarkers in the

clinical evaluation of antiangiogenic and vascular disrupting agents. *Br J Cancer*,

DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. *Gene Ther*,

with phospholipids in membranes. *Prog Lipid Res*, Vol. 41, No. 1, pp. (66-97), ISBN:


siRNA-Based Therapy for Glioblastoma Patients 195

Semple, S.C., Akinc, A., Chen, J. et al. (2010). Rational design of cationic lipids for siRNA delivery. *Nature biotechnology*, Vol. 28, No. 2, pp. (172-6), ISBN: 1546-1696. Shah, K., Bureau, E., Kim, D.E. et al. (2005). Glioma therapy and real-time imaging of neural

Shi, N., Zhang, Y., Zhu, C., Boado, R.J. & Pardridge, W.M. (2001). Brain-specific expression

Shoji, Y. & Nakashima, H. (2004). Current status of delivery systems to improve target

Shuey, D.J., McCallus, D.E. & Giordano, T. (2002). RNAi: gene-silencing in therapeutic intervention. *Drug Discov Today*, Vol. 7, No. 20, pp. (1040-6), ISBN: 1359-6446. Sioud, M. & Sorensen, D.R. (2003). Cationic liposome-mediated delivery of siRNAs in adult mice. *Biochem Biophys Res Commun*, Vol. 312, No. 4, pp. (1220-5), ISBN: 0006-291X. Song, E., Lee, S.K., Wang, J. et al. (2003). RNA interference targeting Fas protects mice from fulminant hepatitis. *Nat Med*, Vol. 9, No. 3, pp. (347-51), ISBN: 1078-8956. Sorensen, A.G., Batchelor, T.T., Zhang, W.T. et al. (2009). A "vascular normalization index"

Sorensen, D.R., Leirdal, M. & Sioud, M. (2003). Gene silencing by systemic delivery of

Sparks, C.A. & Guertin, D.A. (2010). Targeting mTOR: prospects for mTOR complex 2

Storm, G. & Crommelin, D.J.A. (1998). Liposomes: quo vadis? *Pharmaceutical Science &* 

Stupp, R., Hegi, M.E., Mason, W.P. et al. (2009). Effects of radiotherapy with concomitant

Stupp, R., Hegi, M.E., Neyns, B. et al. (2010). Phase I/IIa study of cilengitide and

glioblastoma. *J Clin Oncol*, Vol. 28, No. 16, pp. (2712-8), ISBN: 1527-7755. Stupp, R., Hegi, M.E., van den Bent, M.J. et al. (2006). Changing paradigms--an update on

Stupp, R., Mason, W.P., van den Bent, M.J. et al. (2005). Radiotherapy plus concomitant and

Tarnok, A., Ulrich, H. & Bocsi, J. (2009). Phenotypes of stem cells from diverse origin.

*Technology Today*, Vol. 1, No. 1, pp. (19-31), ISBN: 1461-5347.

*Oncol*, Vol. 10, No. 5, pp. (459-66), ISBN: 1474-5488.

*Cytometry A*, Vol. 77, No. 1, pp. (6-10), ISBN: 1552-4930.

0165-5728.

1381-6128.

2836.

5594.

300), ISBN: 1538-7445.

pp. (165-80), ISBN: 1083-7159.

96), ISBN: 1533-4406.

pp. (34-41), ISBN: 0364-5134.

22, pp. (12754-9), ISBN: 0027-8424.

with nanoparticles. *Journal of neuroimmunology*, Vol. 195, No. 1-2, pp. (21-7), ISBN:

precursor cell migration and tumor regression. *Annals of neurology*, Vol. 57, No. 1,

of an exogenous gene after i.v. administration. *Proc Natl Acad Sci U S A*, Vol. 98, No.

efficacy of oligonucleotides. *Curr Pharm Des*, Vol. 10, No. 7, pp. (785-96), ISBN:

as potential mechanistic biomarker to predict survival after a single dose of cediranib in recurrent glioblastoma patients. *Cancer Res*, Vol. 69, No. 13, pp. (5296-

synthetic siRNAs in adult mice. *J Mol Biol*, Vol. 327, No. 4, pp. (761-6), ISBN: 0022-

inhibitors in cancer therapy. *Oncogene*, Vol. 29, No. 26, pp. (3733-44), ISBN: 1476-

and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. *Lancet* 

temozolomide with concomitant radiotherapy followed by cilengitide and temozolomide maintenance therapy in patients with newly diagnosed

the multidisciplinary management of malignant glioma. *Oncologist*, Vol. 11, No. 2,

adjuvant temozolomide for glioblastoma. *N Engl J Med*, Vol. 352, No. 10, pp. (987-


Razis, E., Selviaridis, P., Labropoulos, S. et al. (2009). Phase II study of neoadjuvant imatinib

Recht, L., Torres, C.O., Smith, T.W., Raso, V. & Griffin, T.W. (1990). Transferrin receptor in

Rich, J.N., Reardon, D.A., Peery, T. et al. (2004). Phase II trial of gefitinib in recurrent glioblastoma. *J Clin Oncol*, Vol. 22, No. 1, pp. (133-42), ISBN: 0732-183X. Rojiani, A.M. & Dorovini-Zis, K. (1996). Glomeruloid vascular structures in glioblastoma

Rong, Y., Durden, D.L., Van Meir, E.G. & Brat, D.J. (2006). 'Pseudopalisading' necrosis in

Rubin, L.L. & Staddon, J.M. (1999). The cell biology of the blood-brain barrier. *Annu Rev* 

Sadones, J., Michotte, A., Veld, P. et al. (2009). MGMT promoter hypermethylation correlates

Sakane, T., Yamashita, S., Yata, N. & Sezaki, H. (1999). Transnasal delivery of 5-fluorouracil to the brain in the rat. *J Drug Target*, Vol. 7, No. 3, pp. (233-40), ISBN: 1061-186X. Santel, A., Aleku, M., Keil, O. et al. (2006). A novel siRNA-lipoplex technology for RNA

Sarbassov, D.D., Ali, S.M., Kim, D.H. et al. (2004). Rictor, a novel binding partner of mTOR,

Scherer, H.J. (1940). The forms of growth in gliomas and their practical significance. *Brain*,

Scheule, R.K., St George, J.A., Bagley, R.G. et al. (1997). Basis of pulmonary toxicity

Schiffelers, R.M., Ansari, A., Xu, J. et al. (2004). Cancer siRNA therapy by tumor selective

Schmidt, E.E., Ichimura, K., Goike, H.M., Moshref, A., Liu, L. & Collins, V.P. (1999).

Schneider, T., Becker, A., Ringe, K., Reinhold, A., Firsching, R. & Sabel, B.A. (2008). Brain

*Gene Ther*, Vol. 8, No. 6, pp. (689-707), ISBN: 1043-0342.

cytoskeleton. *Curr Biol*, Vol. 14, No. 14, pp. (1296-302), ISBN: 0960-9822. Sarbassov, D.D., Guertin, D.A., Ali, S.M. & Sabatini, D.M. (2005). Phosphorylation and

*Cancer Res*, Vol. 15, No. 19, pp. (6258-66), ISBN: 1078-0432.

*Neurosurg*, Vol. 72, No. 6, pp. (941-5), ISBN: 0022-3085.

85, No. 6, pp. (1078-84), ISBN: 0022-3085.

65, No. 6, pp. (529-39), ISBN: 0022-3069.

1879-0852.

0022-3069.

(1222-34), ISBN: 0969-7128.

(1098-101), ISBN: 1095-9203.

Vol. 1, No. 63, pp. (1-35), ISBN: NA.

Vol. 32, No. 19, pp. (e149), ISBN: 1362-4962.

*Neurosci*, Vol. 22, No. pp. (11-28), ISBN: 0147-006X.

in glioblastoma: evaluation of clinical and molecular effects of the treatment. *Clin* 

normal and neoplastic brain tissue: implications for brain-tumor immunotherapy. *J* 

multiforme: an immunohistochemical and ultrastructural study. *J Neurosurg*, Vol.

glioblastoma: a familiar morphologic feature that links vascular pathology, hypoxia, and angiogenesis. *Journal of neuropathology and experimental neurology*, Vol.

with a survival benefit from temozolomide in patients with recurrent anaplastic astrocytoma but not glioblastoma. *Eur J Cancer*, Vol. 45, No. 1, pp. (146-53), ISBN:

interference in the mouse vascular endothelium. *Gene Ther*, Vol. 13, No. 16, pp.

defines a rapamycin-insensitive and raptor-independent pathway that regulates the

regulation of Akt/PKB by the rictor-mTOR complex. *Science*, Vol. 307, No. 5712, pp.

associated with cationic lipid-mediated gene transfer to the mammalian lung. *Hum* 

delivery with ligand-targeted sterically stabilized nanoparticle. *Nucleic Acids Res*,

Mutational profile of the PTEN gene in primary human astrocytic tumors and cultivated xenografts. *J Neuropathol Exp Neurol*, Vol. 58, No. 11, pp. (1170-83), ISBN:

tumor therapy by combined vaccination and antisense oligonucleotide delivery

with nanoparticles. *Journal of neuroimmunology*, Vol. 195, No. 1-2, pp. (21-7), ISBN: 0165-5728.


siRNA-Based Therapy for Glioblastoma Patients 197

Wager, M., Menei, P., Guilhot, J. et al. (2008). Prognostic molecular markers with no impact

Walker, C., du Plessis, D.G., Joyce, K.A. et al. (2003). Phenotype versus genotype in gliomas

Walker, M.D., Alexander, E., Jr., Hunt, W.E. et al. (1978). Evaluation of BCNU and/or

Walker, M.D., Green, S.B., Byar, D.P. et al. (1980). Randomized comparisons of radiotherapy

Wang, D., Gao, Y. & Yun, L. (2006). Study on brain targeting of raltitrexed following

Wang, F., Jiang, X. & Lu, W. (2003). Profiles of methotrexate in blood and CSF following

Wen, P.Y. & Kesari, S. (2008). Malignant gliomas in adults. *The New England journal of* 

Wen, P.Y., Yung, W.K., Lamborn, K.R. et al. (2006). Phase I/II study of imatinib mesylate for

Wick, W., Puduvalli, V.K., Chamberlain, M.C. et al. (2010). Phase III study of enzastaurin

Winkler, F., Kozin, S.V., Tong, R.T. et al. (2004). Kinetics of vascular normalization by

Wolburg, H., Noell, S., Mack, A., Wolburg-Buchholz, K. & Fallier-Becker, P. (2009). Brain

Wu, X., Rauch, T.A., Zhong, X. et al. (2010). CpG island hypermethylation in human astrocytomas. *Cancer research*, Vol. 70, No. 7, pp. (2718-27), ISBN: 1538-7445. Wyszko, E., Rolle, K., Nowak, S. et al. (2008). A multivariate analysis of patients with brain

Xie, Z. & Chin, L.S. (2008). Molecular and cell biology of brain tumor stem cells: lessons from

Yamanaka, R., Arao, T., Yajima, N. et al. (2006). Identification of expressed genes

Yan, H., Parsons, D.W., Jin, G. et al. (2009). IDH1 and IDH2 mutations in gliomas. *The New England journal of medicine*, Vol. 360, No. 8, pp. (765-73), ISBN: 1533-4406.

08. *Clin Cancer Res*, Vol. 12, No. 16, pp. (4899-907), ISBN: 1078-0432.

*Cancer*, Vol. 98, No. 11, pp. (1830-8), ISBN: 1532-1827.

*Neurosurg*, Vol. 49, No. 3, pp. (333-43), ISBN: 0022-3085.

*medicine*, Vol. 359, No. 5, pp. (492-507), ISBN: 1533-4406.

*Clin Oncol*, Vol. 28, No. 7, pp. (1168-74), ISBN: 1527-7755.

Vol. 303, No. 23, pp. (1323-9), ISBN: 0028-4793.

(4841-51), ISBN: 1078-0432.

(97-104), ISBN: 0344-5704.

(1-7), ISBN: 0378-5173.

63), ISBN: 1535-6108.

0001-6837.

1092-0684.

(75-96), ISBN: 1432-0878.

No. 44, pp. (5994-6002), ISBN: 0950-9232.

on decision-making: the paradox of gliomas based on a prospective study. *Br J* 

displaying inter- or intratumoral histological heterogeneity. *Clinical cancer research : an official journal of the American Association for Cancer Research*, Vol. 9, No. 13, pp.

radiotherapy in the treatment of anaplastic gliomas. A cooperative clinical trial. *J* 

and nitrosoureas for the treatment of malignant glioma after surgery. *N Engl J Med*,

intranasal administration in rats. *Cancer Chemother Pharmacol*, Vol. 57, No. 1, pp.

intranasal and intravenous administration to rats. *Int J Pharm*, Vol. 263, No. 1-2, pp.

recurrent malignant gliomas: North American Brain Tumor Consortium Study 99-

compared with lomustine in the treatment of recurrent intracranial glioblastoma. *J* 

VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases. *Cancer Cell*, Vol. 6, No. 6, pp. (553-

endothelial cells and the glio-vascular complex. *Cell Tissue Res*, Vol. 335, No. 1, pp.

tumors treated with ATN-RNA. *Acta Pol Pharm*, Vol. 65, No. 6, pp. (677-84), ISBN:

neural progenitor/stem cells. *Neurosurg Focus*, Vol. 24, No. 3-4, pp. (E25), ISBN:

characterizing long-term survival in malignant glioma patients. *Oncogene*, Vol. 25,


Thakker, D.R., Natt, F., Husken, D. et al. (2005). siRNA-mediated knockdown of the

Thorne, R.G., Emory, C.R., Ala, T.A. & Frey, W.H., 2nd. (1995). Quantitative analysis of the

Thorne, R.G., Pronk, G.J., Padmanabhan, V. & Frey, W.H., 2nd. (2004). Delivery of insulin-

Ting, A.H., McGarvey, K.M. & Baylin, S.B. (2006). The cancer epigenome--components and

Turner, C.D., Gururangan, S., Eastwood, J. et al. (2002). Phase II study of irinotecan (CPT-11)

Vajkoczy, P. & Menger, M.D. (2000). Vascular microenvironment in gliomas. *J Neurooncol*,

Vajkoczy, P. & Menger, M.D. (2004). Vascular microenvironment in gliomas. *Cancer Treat* 

Vajkoczy, P., Schilling, L., Ullrich, A., Schmiedek, P. & Menger, M.D. (1998).

Verreault, M., Strutt, D., Masin, D. et al. (2011b). Irinophore CTM, a lipid-based

Verreault, M., Strutt, D., Masin, D. et al. (2011c). Vascular normalization in orthotopic

Verreault, M., Strutt, D., Masin, D., Fink, D., Gill, R. & Bally, M. (2011d). Development of

Vredenburgh, J.J., Desjardins, A., Reardon, D.A. & Friedman, H.S. (2009). Experience with

intracranial tumors *Anticancer Research*, Vol. 31, No. 6, pp. (2161-71). Verreault, M., Webb, M.S., Ramsay, E.C. & Bally, M.B. (2006). Gene silencing in the

*Cereb Blood Flow Metab*, Vol. 18, No. 5, pp. (510-20), ISBN: 0271-678X. Verreault, M., Stegeman, A., Warburton, C., Strutt, D., Masin, D. & Bally, M. (2011a).

in an orthotopic glioblastoma tumor model. *In revision for Plos One*.

*Oncol*, Vol. 4, No. 2, pp. (102-108), ISBN: 1522-8517.

Vol. 50, No. 1-2, pp. (99-108), ISBN: 0167-594X.

*Res*, Vol. 117, No. pp. (249-62), ISBN: 0927-3042.

*BMC Cancer*, Vol. Apr 8, No. 11, pp. (124).

No. 4, pp. (138-41), ISBN: 0168-9525.

(80-91), ISBN: 1522-8517.

(782-9, 714), ISBN: 1359-4184.

(278-82), ISBN: 0006-8993.

(481-96), ISBN: 0306-4522.

0890-9369.

ahead of print]

serotonin transporter in the adult mouse brain. *Mol Psychiatry*, Vol. 10, No. 8, pp.

olfactory pathway for drug delivery to the brain. *Brain Res*, Vol. 692, No. 1-2, pp.

like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. *Neuroscience*, Vol. 127, No. 2, pp.

functional correlates. *Genes & development*, Vol. 20, No. 23, pp. (3215-31), ISBN:

in children with high-risk malignant brain tumors: the Duke experience. *Neuro* 

Characterization of angiogenesis and microcirculation of high-grade glioma: an intravital multifluorescence microscopic approach in the athymic nude mouse. *J* 

Combined RNAi mediated suppression of Rictor and EGFR inhibits tumor growth

nanoparticulate formulation of irinotecan, is more effective than free irinotecan when used to treat an orthotopic glioblastoma model. *J Control Release*, Oct 5, [Epub

glioblastoma following intravenous treatment with lipid-based nanoparticulate formulations of irinotecan (Irinophore CTM), doxorubicin (Caelyx®) or vincristine.

glioblastoma cell lines expressing red fluorescence for non-invasive live imaging of

development of personalized cancer treatment: the targets, the agents and the delivery systems. *Curr Gene Ther*, Vol. 6, No. 4, pp. (505-33), ISBN: 1566-5232. Vogelstein, B. & Kinzler, K.W. (1993). The multistep nature of cancer. *Trends Genet*, Vol. 9,

irinotecan for the treatment of malignant glioma. *Neuro Oncol*, Vol. 11, No. 1, pp.


**10** 

*USA* 

**Hypoxia Responsive Vectors** 

*2Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Fl* 

**Targeting Astrocytes in Glioma** 

Manas R. Biswal1, Howard M. Prentice2 and Janet C. Blanks1,2\*

*1Center for Complex Systems and Brain Sciences, Charles E. Schmidt College of Science,* 

Gliomas are the most common brain tumor in the central nervous system (CNS). The majority of malignant gliomas arise from neoplastic transformation of resident astrocytes. Gliomas are very aggressive tumors since they are characterized by widespread invasion of brain tissue. The exact pathogenesis and underlying mechanisms for glioma cell infiltration are currently unclear. Cell-cell interaction and tissue microenvironment play an important role in tumor progression leading to modification and infiltration of surrounding tissue. The hypoxic microenvironment contributes to abnormal neovascularization of the glioma.

Uncontrolled cellular proliferation, abnormal angiogenesis and invasion of surrounding tissue make these tumors difficult to treat. Poor prognosis and ineffective treatments for glioma point to the necessity of developing new therapeutic strategies. The use of gene therapy to deliver a therapeutic gene may successfully overcome the failure of conventional therapies. Although non-cell specific and unregulated promoters have been used to target gliomas, it is possible that unregulated vectors elicit damage in cells that do not require the therapeutic protein. Thus it may be of value to design a regulated, tissue-specific vector to express a therapeutic protein in a specific cell type and to regulate it according to the local

The hypoxic microenvironment is known to play a major role in several conditions including cerebral ischemia, retinal angiogenesis, gliomas, and other cancers. Hypoxia inducible factor 1 alpha (HIF-1α), the most common transcription factor induced during hypoxia, binds to a hypoxia regulated enhancer (HRE) region of oxygen sensitive genes leading to their transcription. By taking advantage of hypoxia for regulating foreign gene expression, a hypoxia regulated vector could be exploited to modify the cells that reside within the target tissue. Use of a cell-specific promoter would be an added advantage to

Our recently developed vector platform works in a manner such that in an hypoxic environment, HIF-1α will bind to the HRE region and express the foreign protein in a cell-

**1. Introduction** 

tissue environment.

\* Corresponding Author

restrict gene expression only in certain cells.

