**4.2 Liposomal carrier systems for rheumatoid arthritis treatment**

Several liposome systems have been tried for improved efficacy of rheumatoid arthritis treatments. This work was pioneered by Williams, et. al., who demonstrated that

The Development of Targeted Drug Delivery Systems for Rheumatoid Arthritis Treatment 121

Cyclosporine A is indicated for several different conditions; therefore, micelle-based methods for improving the solubility of of this DMARD have been more thoroughly researched. Cyclosporine has been successfully encapsulated by block copolymers of PEG and poly(caprolactone) (Aliabadi et al., 2005), PEG phospholipids (Lee et al., 1999), and hydrophobically modified polysaccharides, specifically dextran and hydroxypropylcellulose (Francis et al., 2005). Recently, polysialic acid, a relatively uninvestigated polysaccharide with propertiers similar to those of PEG in regards to minimizing uptake by the reticuloendotheial system, was used to encapsulate cyclopsorine for future use in rheumatoid arthritis treatment (Bader et al., 2011). The utility of the above cyclosporine

Nanoparticle systems for delivery of rheumatoid arthritis therapeutics have primarily been based upon polymers. Numerous researchers have explored the use of poly(D,Llactic/glycolic acid) (PLGA) nanoparticles based upon their capacity to extend the circulation time and control the release of encapsulated drugs. In the realm of rheumatoid arthritis drug delivery, a glutocorticoid, betamethasone, was incorporated into PLGA nanoparticles with a size of 100-200 nm. Intravenous administration to arthritic rats and mice showed that the PLGA-betamethasone system was more effective at reducing the inflammatory response than the free glutocorticoid (Higaki et al., 2005). Targeting ability and, consequently, efficacy of the betamethasone was further improved by modifying the PLGA nanoparticles with PEG,

Other polymeric nanoparticle systems involve covalently linking the drug molecule to one of the components so as to slow down therapeutic release, as occurs for polymer-drug conjugates, while still protecting the drug via encapsulation. For example, methylprednisolone was conjugated to cyclodextrin, and the resultant compound selfassembled to yield nanoparticles with a size of approximately 27 nm. When administered intravenously at a frequency of one dose per week to arthritic mice, a significantly greater reduction in synovitis and pannus formation was acheieved than that obtained for free methylprednisolone administered daily at an equivalent cumulative dose (Hwang et al., 2008). Another example is the ionic complexation of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) conjugated to PEG (PEG-TRAIL), which bears a positive charge, with negatively charged hyaluronic acid (HA) with sizes that range from 100 to 270 nm, dependent upon the relative concentration of the two components. One formulation of the HA-PEG-TRAIL complex was capable of signficantly reducing the secretion of proinflammatory mediators relative to PEG-TRAIL alone when administered subcutaneously to arthritic mice (Kim et al., 2010), thereby emphasizing the importance of nanoparticulate carrier systems. The HA may also serve as an active targeting (Section 5). Despite the demonstrated efficacy of gold as a DMARD (Section 2), and the approval of gold nanoparticles for the treatment of rheumatoid arthritis by the FDA, little research has been conducted specifically directed towards the use of metallic nanoparticles for passive targeting in rheumatoid arthritis. The decline in the use of gold as a rheumatoid arthritis therapeutic was largely based on adverse side effects caused by non-specific toxicity. However, the synthesis of gold core-gold(I)-thiomalate nanoparticles (Au@AU(I)-TM) was recently completed. Au@Au(I)-TM bears surface carboxylate groups which may be further modified for specific drug delivery applications (Corthey et al., 2010); therefore, these nanoparticles may pave the way to a renewed interest in the use of gold in the treatment of rheumatoid arthritis.

micelles *in vivo* has not yet been demonstrated.

**4.4 Nanoparticle carrier systems for rheumatoid arthritis treatment** 

forming so-called "stealth nanosteroids" (Ishihara et al., 2009).

technecium labelled liposomes accumulated within the synovial tissue of rheumatoid arthritis patients upon intravenous administration (Williams et al., 1986, 1987). A similar phenomenon was observed for phosphatidylcholine and cholesterol based liposomes given to arthritic rats (Love et al., 1989), and encapsulation of clondronate, an anti-inflammatory therapeutic that reduces bone resorption, resulted in a halt in disease progression and a reversal in inflammation (Camilleri et al., 1995; Love et al., 1992). The efficacy of the liposomal clondronate was attributed to depletion of the synovial macrophages (Love et al., 1992; Richards et al., 1999). Comparable results were obtained with liposomal methotrexate that was prepared by conjugation of the the γ-carboxylic acid residue to the phospholipid. The resultant liposomes yielded a significant reduction of established joint inflammation, in combination with a decrease in toxicity, relative to comparable doses of free drug (Williams et al., 1994b. Furthermore, peritoneal macrophages isolated from the liposomal methotrexate treated arthritic rats were shown to express less TNF- and prostaglandin (PGE2) (Williams et al., 1994a). Conventional (i.e. not PEGylated) liposomes have also been used by other investigators to improve the efficacy of aurothiomalate (Konigsberg et al., 1999).

Stealth liposomes have been used to improve the therapeutic efficacy of glucocorticoids, the use of which is often hindered by the necessity for frequent intravenous administration and by non-specific organ toxicity. Following on studies that showed an accumulation of PEG liposomes within the inflamed tissue of arthritic rats (Gabizon et al., 1994; Hong & Tseng, 2003), Metselaar et al. (2002; 2003; 2004) investigated the efficacy of prednisolone loaded liposomes. Upon administration to arthritic rats and mice, the prednisolone liposomes reversed the inflammatory response, while free prednisolone had little effect on the course of the disease. The results were attributed purely to the passive targeting capacity of the stealth liposomes (Metselaar et al., 2004; Metselaar et al., 2002; Metselaar et al., 2003). Current research is focused upon extending the use of the stealth liposomes to other glucocorticoids, including dexamethasone and budesonide, and optimizing the therapeutic index (van den Hoven et al., 2011). PEG modified liposomes have also been used to improve the efficacy of superoxide dismutase (SOD), a free radical scavenger which suffers from a short half life. When encapsulated in small, stealth liposomes, the circulation of SOD was significantly extended, and the drug passively accumulated within the joint tissue (Corvo et al., 1999).

#### **4.3 Micelle carrier systems for rheumatoid arthritis treatment**

Despite the low aqueous solubility of a number of DMARDs and glucocorticoids, micelles are a relatively unexplored drug delivery system for rheumatoid arthritis treatment. Camptothecin, originally developed as an anti-cancer drug, has recently been proposed as a new method of controlling pannus formation and reducing cartilage degradation. To circumvent problems with solubility and stability, micelles prepared from PEGphospholipids (Ashok et al., 2004) were used for drug encapsulation. The camptothecin micelles proved to be more effective than free camptothecin at abrogating inflammation when administered to arthritic mice. The efficacy of the micelles was further increased by modification with vasoactive intenstinal peptide (VIP) due to the over expression of VIP receptor by activated synovial macrophages (Koo et al., 2011). Similarly, micelles generated from a phospholipid preparation (Phosphogliv) and loaded with methotrexate were better able to reduce inflammation when administered to arthritic rats than the drug alone (Zykova et al., 2007).

technecium labelled liposomes accumulated within the synovial tissue of rheumatoid arthritis patients upon intravenous administration (Williams et al., 1986, 1987). A similar phenomenon was observed for phosphatidylcholine and cholesterol based liposomes given to arthritic rats (Love et al., 1989), and encapsulation of clondronate, an anti-inflammatory therapeutic that reduces bone resorption, resulted in a halt in disease progression and a reversal in inflammation (Camilleri et al., 1995; Love et al., 1992). The efficacy of the liposomal clondronate was attributed to depletion of the synovial macrophages (Love et al., 1992; Richards et al., 1999). Comparable results were obtained with liposomal methotrexate that was prepared by conjugation of the the γ-carboxylic acid residue to the phospholipid. The resultant liposomes yielded a significant reduction of established joint inflammation, in combination with a decrease in toxicity, relative to comparable doses of free drug (Williams et al., 1994b. Furthermore, peritoneal macrophages isolated from the liposomal methotrexate treated arthritic rats were shown to express less TNF- and prostaglandin (PGE2) (Williams et al., 1994a). Conventional (i.e. not PEGylated) liposomes have also been used by other

investigators to improve the efficacy of aurothiomalate (Konigsberg et al., 1999).

**4.3 Micelle carrier systems for rheumatoid arthritis treatment** 

al., 1999).

(Zykova et al., 2007).

Stealth liposomes have been used to improve the therapeutic efficacy of glucocorticoids, the use of which is often hindered by the necessity for frequent intravenous administration and by non-specific organ toxicity. Following on studies that showed an accumulation of PEG liposomes within the inflamed tissue of arthritic rats (Gabizon et al., 1994; Hong & Tseng, 2003), Metselaar et al. (2002; 2003; 2004) investigated the efficacy of prednisolone loaded liposomes. Upon administration to arthritic rats and mice, the prednisolone liposomes reversed the inflammatory response, while free prednisolone had little effect on the course of the disease. The results were attributed purely to the passive targeting capacity of the stealth liposomes (Metselaar et al., 2004; Metselaar et al., 2002; Metselaar et al., 2003). Current research is focused upon extending the use of the stealth liposomes to other glucocorticoids, including dexamethasone and budesonide, and optimizing the therapeutic index (van den Hoven et al., 2011). PEG modified liposomes have also been used to improve the efficacy of superoxide dismutase (SOD), a free radical scavenger which suffers from a short half life. When encapsulated in small, stealth liposomes, the circulation of SOD was significantly extended, and the drug passively accumulated within the joint tissue (Corvo et

Despite the low aqueous solubility of a number of DMARDs and glucocorticoids, micelles are a relatively unexplored drug delivery system for rheumatoid arthritis treatment. Camptothecin, originally developed as an anti-cancer drug, has recently been proposed as a new method of controlling pannus formation and reducing cartilage degradation. To circumvent problems with solubility and stability, micelles prepared from PEGphospholipids (Ashok et al., 2004) were used for drug encapsulation. The camptothecin micelles proved to be more effective than free camptothecin at abrogating inflammation when administered to arthritic mice. The efficacy of the micelles was further increased by modification with vasoactive intenstinal peptide (VIP) due to the over expression of VIP receptor by activated synovial macrophages (Koo et al., 2011). Similarly, micelles generated from a phospholipid preparation (Phosphogliv) and loaded with methotrexate were better able to reduce inflammation when administered to arthritic rats than the drug alone Cyclosporine A is indicated for several different conditions; therefore, micelle-based methods for improving the solubility of of this DMARD have been more thoroughly researched. Cyclosporine has been successfully encapsulated by block copolymers of PEG and poly(caprolactone) (Aliabadi et al., 2005), PEG phospholipids (Lee et al., 1999), and hydrophobically modified polysaccharides, specifically dextran and hydroxypropylcellulose (Francis et al., 2005). Recently, polysialic acid, a relatively uninvestigated polysaccharide with propertiers similar to those of PEG in regards to minimizing uptake by the reticuloendotheial system, was used to encapsulate cyclopsorine for future use in rheumatoid arthritis treatment (Bader et al., 2011). The utility of the above cyclosporine micelles *in vivo* has not yet been demonstrated.

#### **4.4 Nanoparticle carrier systems for rheumatoid arthritis treatment**

Nanoparticle systems for delivery of rheumatoid arthritis therapeutics have primarily been based upon polymers. Numerous researchers have explored the use of poly(D,Llactic/glycolic acid) (PLGA) nanoparticles based upon their capacity to extend the circulation time and control the release of encapsulated drugs. In the realm of rheumatoid arthritis drug delivery, a glutocorticoid, betamethasone, was incorporated into PLGA nanoparticles with a size of 100-200 nm. Intravenous administration to arthritic rats and mice showed that the PLGA-betamethasone system was more effective at reducing the inflammatory response than the free glutocorticoid (Higaki et al., 2005). Targeting ability and, consequently, efficacy of the betamethasone was further improved by modifying the PLGA nanoparticles with PEG, forming so-called "stealth nanosteroids" (Ishihara et al., 2009).

Other polymeric nanoparticle systems involve covalently linking the drug molecule to one of the components so as to slow down therapeutic release, as occurs for polymer-drug conjugates, while still protecting the drug via encapsulation. For example, methylprednisolone was conjugated to cyclodextrin, and the resultant compound selfassembled to yield nanoparticles with a size of approximately 27 nm. When administered intravenously at a frequency of one dose per week to arthritic mice, a significantly greater reduction in synovitis and pannus formation was acheieved than that obtained for free methylprednisolone administered daily at an equivalent cumulative dose (Hwang et al., 2008). Another example is the ionic complexation of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) conjugated to PEG (PEG-TRAIL), which bears a positive charge, with negatively charged hyaluronic acid (HA) with sizes that range from 100 to 270 nm, dependent upon the relative concentration of the two components. One formulation of the HA-PEG-TRAIL complex was capable of signficantly reducing the secretion of proinflammatory mediators relative to PEG-TRAIL alone when administered subcutaneously to arthritic mice (Kim et al., 2010), thereby emphasizing the importance of nanoparticulate carrier systems. The HA may also serve as an active targeting (Section 5).

Despite the demonstrated efficacy of gold as a DMARD (Section 2), and the approval of gold nanoparticles for the treatment of rheumatoid arthritis by the FDA, little research has been conducted specifically directed towards the use of metallic nanoparticles for passive targeting in rheumatoid arthritis. The decline in the use of gold as a rheumatoid arthritis therapeutic was largely based on adverse side effects caused by non-specific toxicity. However, the synthesis of gold core-gold(I)-thiomalate nanoparticles (Au@AU(I)-TM) was recently completed. Au@Au(I)-TM bears surface carboxylate groups which may be further modified for specific drug delivery applications (Corthey et al., 2010); therefore, these nanoparticles may pave the way to a renewed interest in the use of gold in the treatment of rheumatoid arthritis.

The Development of Targeted Drug Delivery Systems for Rheumatoid Arthritis Treatment 123

another possibility for active targeting (Banquy et al., 2008; Zhang et al., 2008). Preliminary studies demonstrated that a large number of PLGA-PEG nanoparticles surface modified with a peptide specific for ICAM-1 were endocytosed by vascular endothelial cells as compared to unmodified particles (Zhang et al., 2008). A similar phenomenon was observed for polyester-based polymeric nanoparticles labeled with ligand specific for E-selectin (Banquy et al., 2008). Likewise, liposomes surface modified with the polysaccharide Sialyl Lewis X, which is known to bind selectively to E-selectin, were shown to accumulate within inflamed regions when administered intravenously to arthritic mice (Hirai et al., 2007). The vitronectin receptor, overexpressed by both angiogenic endothelial cells and RASMs (Tandon et al., 2005; Wilder, 2002), is an attractive target based upon the extensive use of Arg-Glyc-Asp (RGD) tripeptide labeled carrier systems for drug delivery to invasive tumor tissue (Hsu et al., 2007). An antibody to V3, Vitaxin (ME-522), is currently being clinically developed for rheumatoid arthritis (Wilder, 2002); however, the RGD peptide sequence has not yeen been applied towards increasing the targeting ability and efficacy of existing

A recent push in the realm of drug delivery for cancer treatment has been the development of multifunctional carrier systems whereby a single platform can be used for the release of multiple therapeutics in a controlled fashion or for both therapeutic release and diagnostic imaging (Jabr-Milane et al., 2008; van Vlerken & Amiji, 2006). Some of the success achieved in this "theranostic" approach to cancer treatment has spilled over into the study of improved treatment methods for rheumatoid arthritis. For example, folate-conjugated radiopharmaceuticals designed to target malignant tissue showed significant accumulation in the joints of patients who also had rheumatoid arthritis (Paulos et al., 2004). Radiolabelled biologics have provided an additional example of the potential for theranostics to improve rheumatoid arthritis treatment. 99m-technetium labelled infliximab (99mTc-infliximab) was used to show a strong correlation between the amount of infliximab taken up by inflamed

As discussed in brief above, in order to be optimally effective, the drug delivery systems must release their payloads at the desired site of action, i.e. the pannus tissue. In the realm of cancer drug delivery, numerous pH- and temperature-responsive carrier systems, in the form of conjugates, dendrimers, liposomes, and micelles, have been developed (Ganta et al., 2008). Although the same potential also exists for rheumatoid arthritis drug delivery, particularly in light of the lower pH of the pannus tissue relative to native tissue, to the authors' knowledge, only two pH-responsive systems, dexamethasone-HPMA conjugates (P-Dex) and dexamethasone-PEG conjugates (PEG-DEX), have been reported. Drug release was shown to increase as the pH decreased. *In vitro* and *in vivo* tests were used to demonstrate that P-Dex and PEG-DEX are more effective than free dexamethasone in regards to reducing the production of proinflammatory mediators, preventing joint damage, and targeting inflamed tissue (Liu et al., 2010; Quan et al., 2010). In addition to temperature and pH, other environmental triggers may be used. For example, elevated elastace levels have been closely linked with inflammation and the increased enzymatic activity maybe be used for cleavage. The upregulation in enzymes may additionally serve as a means of active

tissue and a reduction in symptoms and swelling (Malviya et al., 2010).

DMARDs.

**5.2 Multifunctional carrier systems** 

**5.3 Stimuli-responsive carrier systems** 

targeting (Meers, 2001).
