**2. Alkylphospholipids in clinical trials**

In the late 1970's and early 1980's systemic investigations of structure – activity relationship were performed to screen lysophospholipids, alkylphospholipids and etherlipids to identify new candidates for cancer treatment. Among them, especially Miltefosine (Fig. 1), basically a simple phosphorus acid diester, displayed high inhibitory activity against chemically induced mammary carcinomas in rats (Eibl & Unger, 1990). It became the first drug based on a phospholipid structure demonstrating the high potential of this simple structured molecule class. The main advantage of this class of drugs is the target. In contrast to most anti-cancer drugs, which interfere at the DNA level with cell proliferation, alkylphospholipids act at the cell membrane, where they disturb the PI3K/Akt/mTOR signal transduction pathway (Fig. 2). Initial preclinical tests were promising, indicating a good anti-cancer activity against several human tumour xenograft models in the mouse (Arndt et al., 1997; Fichtner et al., 1994), including different breast cancer cell lines like: MT-3 (Zeisig et al., 1998), MDA-MB 435 and MDA-MB 231 (Sobottka & Berger, 1992), MaTu (Arndt et al., 1999), MT-1 (Naundorf et al., 1992), C3H, Ca 755 (Zeisig et al., 1991) and also syngeneic models like murine P388 leukemia, and B 16 melanoma (Zeisig et al., 1991). Preclinical experiments further demonstrated that alkylphospholipids, if used in liposomal form, are able to abolish multi drug resistance in human breast cancer xenografts (Zeisig et al., 2004) and inhibit metastasis if combined with an aggregation inhibitor inside liposomes in murine syngene (Wenzel et al., 2010) and human xenograft breast cancer models (Wenzel et al., 2009). Perifosine in combination with dioleylphosphoethanolamine, as a component of the liposome bilayer, also enhances transport of drugs across the blood brain barrier and in this way improves the treatment of intracerebral tumours and metastases (Orthmann et al., 2010). Miltefosine was also tested as an alternative approach for the treatment of patients with progressive cutaneous lesions from breast cancer in Phase I and II studies, which indicated that Miltefosine (either used alone or in conjunction with other therapies for distant metastases) is an effective and tolerable local treatment for cutaneous breast cancer (Clive et al., 1999; Unger & Eibl, 1991).

Only small changes in the molecular structure (slightly longer alkyl chain, a modified head group) while maintaining molecular size and shape resulted in Perifosine (OPP). Gills et al. (Gills & Dennis, 2009) summarised the clinical trials with Perifosine as single agent until 2009. Seven Phase 1 single agent studies of Perifosine have been completed. The trials demonstrated that Perifosine can be safely given to humans with a manageable toxicity profile. The dose limiting toxicity in the Phase I studies was, similar to Miltefosine, gastrointestinal: nausea, vomiting and diarrhea. Perifosine as single agent has been further evaluated in Phase II studies for the treatment of most common cancers, including breast, prostate, head and neck, pancreatic cancers, melanoma, renal cell carcinoma, advanced brain tumours, soft-tissue sarcomas, hepatocellular carcinoma, as well as in haematological malignancies including multiple myeloma and Waldenstrom's macroglobulinemia (WM). Potent activity with Perifosine, given as single-agent, has been observed so far in sarcoma and WM patients.

OPP resistant MCF7 cells as compared to OPP sensitive MT3 breast cancer cells. On the other hand the properties of an efficient OPP formulation are mainly determined by

In the late 1970's and early 1980's systemic investigations of structure – activity relationship were performed to screen lysophospholipids, alkylphospholipids and etherlipids to identify new candidates for cancer treatment. Among them, especially Miltefosine (Fig. 1), basically a simple phosphorus acid diester, displayed high inhibitory activity against chemically induced mammary carcinomas in rats (Eibl & Unger, 1990). It became the first drug based on a phospholipid structure demonstrating the high potential of this simple structured molecule class. The main advantage of this class of drugs is the target. In contrast to most anti-cancer drugs, which interfere at the DNA level with cell proliferation, alkylphospholipids act at the cell membrane, where they disturb the PI3K/Akt/mTOR signal transduction pathway (Fig. 2). Initial preclinical tests were promising, indicating a good anti-cancer activity against several human tumour xenograft models in the mouse (Arndt et al., 1997; Fichtner et al., 1994), including different breast cancer cell lines like: MT-3 (Zeisig et al., 1998), MDA-MB 435 and MDA-MB 231 (Sobottka & Berger, 1992), MaTu (Arndt et al., 1999), MT-1 (Naundorf et al., 1992), C3H, Ca 755 (Zeisig et al., 1991) and also syngeneic models like murine P388 leukemia, and B 16 melanoma (Zeisig et al., 1991). Preclinical experiments further demonstrated that alkylphospholipids, if used in liposomal form, are able to abolish multi drug resistance in human breast cancer xenografts (Zeisig et al., 2004) and inhibit metastasis if combined with an aggregation inhibitor inside liposomes in murine syngene (Wenzel et al., 2010) and human xenograft breast cancer models (Wenzel et al., 2009). Perifosine in combination with dioleylphosphoethanolamine, as a component of the liposome bilayer, also enhances transport of drugs across the blood brain barrier and in this way improves the treatment of intracerebral tumours and metastases (Orthmann et al., 2010). Miltefosine was also tested as an alternative approach for the treatment of patients with progressive cutaneous lesions from breast cancer in Phase I and II studies, which indicated that Miltefosine (either used alone or in conjunction with other therapies for distant metastases) is an effective and tolerable local treatment for cutaneous breast cancer

Only small changes in the molecular structure (slightly longer alkyl chain, a modified head group) while maintaining molecular size and shape resulted in Perifosine (OPP). Gills et al. (Gills & Dennis, 2009) summarised the clinical trials with Perifosine as single agent until 2009. Seven Phase 1 single agent studies of Perifosine have been completed. The trials demonstrated that Perifosine can be safely given to humans with a manageable toxicity profile. The dose limiting toxicity in the Phase I studies was, similar to Miltefosine, gastrointestinal: nausea, vomiting and diarrhea. Perifosine as single agent has been further evaluated in Phase II studies for the treatment of most common cancers, including breast, prostate, head and neck, pancreatic cancers, melanoma, renal cell carcinoma, advanced brain tumours, soft-tissue sarcomas, hepatocellular carcinoma, as well as in haematological malignancies including multiple myeloma and Waldenstrom's macroglobulinemia (WM). Potent activity with Perifosine, given as single-agent, has been observed so far in sarcoma

cholesterol concentration, which should be below 50 mol%.

**2. Alkylphospholipids in clinical trials** 

(Clive et al., 1999; Unger & Eibl, 1991).

and WM patients.

Fig. 1. Structural formula of pharmaceutically tested alkylphospholipids.


Table 1. Names, abbreviation, IUPAC names, formula, molecular weights and references of most common alkylphospholipids

Erucylphosphocholine is an alkylphospholipids derivative with a 22 carbon atom chain and a cis-13,14 double bond. Although it differs from miltefosine only in alkyl chain length and the presence of a double bond (Fig. 1), significant differences were found in pharmacological properties. This structural modification increases hydrophobicity resulting in the formation of lamellar supramolecular structures, which abolished hemolytic side effects and allows Erucylphosphocholine to be administrated intravenously (Erdlenbruch et al., 1999; Kaufmann-Kolle et al., 1996; van Blitterswijk & Verheij, 2008). It is a potent inducer of apoptosis (Jendrossek et al., 2003) that exerts more potent antineoplastic effects *in vitro* and *in vivo* than Miltefosine.

Interaction of Alkylphospholipid Formulations with Breast Cancer

al., 1993).

Cells in the Context of Anticancer Drug Development 365

sphingomyelin (SM) in membrane lipid rafts. Inhibition of PC biosynthesis blocks the downstream sphingomyeline synthase (SMS) that catalyzes synthesis of sphingomyelin and diacylglycerol in the trans-Golgi (van Blitterswijk et al., 2003). Possible consequence is the accumulation of the ceramide, which is a second SMS substrate and can trigger apoptosis (Wieder et al., 1998). Another consequence of the PC shortage is the oxidative stress with reactive oxygen species (ROS) formation (Smets et al., 1999; Vrablic et al., 2001; Wagner et

Fig. 2. Alkylphospholipid targets in lipid metabolism and signalling pathways summarized

Alkylphospholipids interfere with phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB)/Akt survival pathway, which is important for proliferation, differentiation, survival and intracellular trafficking (Fig. 2). They inhibit phosphorylation and recruitment of PKB/Akt to the membrane, which is essential for its activation (Elrod et al., 2007; Kondapaka et al., 2003; Rahmani et al., 2005; Tazzari et al., 2008) probably by decreased

Alkylphospholipids inhibit PC hydrolysis to phosphatidic acid (PA) by phospholipase D and further to diacylgycerol (DAG) (Kiss & Crilly, 1997; Lucas et al., 2001). PA and DAG are second messengers, essential for the mitogen-activated protein kinase (MAPK) pathways, which regulate mitosis, metabolism, survival, apoptosis and differentiation (Chen et al., 2001; Kyriakis & Avruch, 2001; Pearson et al., 2001). PA is also involved in the activation of protein kinase C-ζ (Limatola et al., 1994), mTOR (Fang et al., 2001) and c-Raf (Rizzo et al., 2000). DAG activates proteins with the C1 domain, such as protein kinases C and D, Ras guanine-releasing protein (RasGRP) and indirectly MAPK/ERK pathway (Carrasco &

after van Blitterswijk et al. (van Blitterswijk & Verheij, 2008).

**3.3 Influence of alkylphospholipids on major signaling pathways** 

production of PIP3 (Gills & Dennis, 2009; van Blitterswijk & Verheij, 2008).
