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Feb 8, 2012 - Key-words: Camptothecins, Drug Delivery, Liposomes, Micelles, Block-copolymers, Graft copolymers, Irinotecan, Topotecan,. Lurtotecan.
Cancer Therapy Vol 8, page 90 Cancer Therapy Vol 8, 90-104, 2012

Nano-particulate Drug Delivery Systems for Camptothecins Research Article

Nilesh Patankar1,2 and Dawn Waterhouse1,2 1

Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada Experimental Therapeutics, B.C. Cancer Agency, Vancouver, BC, V5Z 1L3, Canada

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*Correspondence: Dr. Dawn Waterhouse, Cancer Specialist, Experimental Therapeutics, B C Cancer Agency, 675 W 10th Avenue, Vancouver, BC V5Z 1L3, Canada, Phone: +1-604-675-8022, Fax: +1-604-675-8183, E-mail: [email protected] Key-words: Camptothecins, Drug Delivery, Liposomes, Micelles, Block-copolymers, Graft copolymers, Irinotecan, Topotecan, Lurtotecan Received: 22 January 2012; Revised: 5 February 2012 Accepted: 6 February 2012 ; electronically published: 8 February 2012

Summary Camptothecin was initially discovered in 1966 and since its discovery there have been a number of analogs developed giving us a group of molecules that is simply referred to as the camptothecins. The parent camptothecin was shown to have promising activity in an anti-cancer setting when it was discovered and there has been much interest and research into the various camptothecins in the years following, specifically with aim to increase the deliverable dose and to limit toxicities associated with use. This article reviews a number of approaches to formulate the camptothecins using nanopaticulate approaches to allow for enhanced delivery, uptake and activity while potentially also reducing toxicity.

I. Introduction

limitations of clinical use. Substitutions at the 9 or 10 positions have shown to produce compounds with greater water solubility and enhanced TOP1 inhibitory activity (Pommier, 2006; Srivastava et al., 2005; Wall et al., 1993) with irinotecan which is a prodrug of SN38 and topotecan being two successful candidates from this class. In 1996 the FDA approved irinotecan (marketed by Pfizer under the name of Camptosar®) and topotecan (marketed by Glaxo-Smithkline under the name of Hycamtin®) for the treatment of colon, lung, breast and ovarian cancers (Gore et al., 2001; Saltz et al., 2000).

A. The Camptothecins Camptothecin (CPT) is a quinoline alkaloid derived from the bark, wood and fruit of the Asian tree Camptotheca acuminata (Wall and Wani, 1995). It was first discovered in 1966 by Drs. Wall and Wani and developed at National Cancer Institute (NCI) to demonstrate potential antitumor activity (Wall and Wani, 1995). Initial preclinical testing in mouse L1210 leukemia and rat Walker carcinosarcoma models showed promising results in terms of tumor inhibition (ME Wall, 1966). This was followed by early clinical trials carried out in mid 1970s that demonstrated partial success, but which were subsequently discontinued due to serious toxicity concerns. The water insolubility of the active form was one of the major issues in further clinical development at that time. Research continued to provide information about the structure and biological activity of this molecule however it was not until the mid 1980s when the mechanism of action of CPT was fully understood and interest in use of camptothecin as a therapeutic was rekindled. Renewed research led to the development of water soluble analogs of CPT that also retained the anticancer activity, thus overcoming one of the major

II. Mechanism of action In 1985, topoisomerase 1 (TOP1) was discovered to be the cellular target of CPT (Hsiang et al., 1985). TOP1 is an essential enzyme present in higher eukaryotes that plays an important role during DNA replication process. During replication, DNA, which exists as a supercoiled double helix, unwinds to generate single strands that act as a template for synthesis of new strands. TOP1 forms a transient cleavable complex with DNA and introduces a nick in the DNA to relieve the torsional stress during this process. This complex is extremely transient and enzyme is released thus allowing re-ligation after release of the new strand. 90

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CPT binds to the TOP1 nicked DNA complex and stabilizes it. This prevents re-ligation of the nicked strand to result in the formation of irreversible double stranded breaks (Hsiang et al., 1985; Hsiang et al., 1989; Hsiang and Liu, 1988). Thus the cytotoxic effects of camptothecins are sphase specific. In vitro studies have shown that cells in the s-phase are 100-1000 times more sensitive to camptothecins than those in G1 or G2 (Li et al., 1972). Camptothecin has a five ring heterocyclic structure with α-hydroxylactone within its E-ring which is essential for its anti-tumor activity (Figure 1). However this E-ring is highly unstable at physiological pH and is readily converted to a clinically inactive carboxylate form. Lurtotecan, (10,11-(methyl ethylenedioxy)-7-((Nmethylpiperazino) methyl) camptothecin) and 9-nitro camptothecins are some of the other CPT derivatives

under investigation (Figure 1). The S conformation at C-20 is very important for the activity as the 20(R) isomer of CPT was found to be 100-fold less active than that of 20(S) (Srivastava et al., 2005). Modifications in rings C and D have generally resulted in the loss or reduction in the cytotoxic activity (Srivastava et al., 2005). The CPT E-ring tolerates minor modifications; e.g. ring enlargement to form β-hydroxy lactone or acylation of hydroxyl group stabilizes the lactone (Simanek). Apart from those mentioned above, other CPT modifications that have been investigated include 10,11-methylenedioxy camptothecins, morpholino camptothecins, exatecan, belotecan, silatecans, chimmitecans, as well as E-ring modifications such as diflomotecan, homocamptothecins, 20-hydroxy-linked modifications, hydrophobic and esters of CPT and amino acid esters of CPT.

Figure 1: Structure and chemistry of camptothecin and analogs B. Camptothecin Delivery Challenges The discovery of the anti-tumor potential of CPT and subsequently that of taxol were two major breakthroughs in the field of cancer chemotherapeutics. Various CPT derivatives have been evaluated in several pre-clinical and clinical trials since then. However, in spite of possessing potent anti-tumor activity, a full realization of this potential has not yet been realized in the clinic. One of the major obstacles in achieving this goal as mentioned previously is that of severe toxicities

or side effects associated with these compounds. A critical second issue is the challenge in terms of delivering the optimum concentration of the required form of the drug to the tumor site and ultimately inside the tumor cells, a challenge associated with both the insolubility of the parent compound in addition to rapid clearance. To combat the insolubility, early clinical trials were conducted using sodium salt of CPT (Gottlieb et al., 1970; Muggia et al., 1972).

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Cancer Therapy Vol 8, page 92 The successful generation of several modifications of CPT with the aim of delivering more soluble form of CPT to tumors led to renewed interest and ultimately in two clinically approved forms of CPT, irinotecan and topotecan currently in clinical use for the treatment of various cancers. The structurally labile nature of the Ering at physiological pH poses separate and distinct challenge for all CPT analogs in terms of delivering an active form of the drug to the tumor site in that pH dependent opening of the lactone ring renders carboxylate form that is inactive as a TOP1 inhibitor. Although reversible, re-lactonization of ring open form at physiological pH is seldom possible. At neutral pH half-life of this conversion is few minutes irrespective of the type of CPT, hence, shortly after administration very small quantities of CPT and its analogs are available in the active lactone form. This plasma inactivation of CPT is further exacerbated by preferential albumin binding of carboxylate form which is observed to be species dependent (Burke et al., 1995; Mi et al., 1995). Thus, inherent physico-chemical characteristics, unstable lactone ring and toxicities due to non-tumor specific actions pose a daunting task of delivering sufficient amounts of active form of CPT to the site of action. Synthesizing a stable analog that possesses adequate biological activity till it reaches the site of action without possessing significant toxicity is a difficult task. Hence, designing a delivery vehicle that can be use to achieve this goal can be a more practical approach.

has emerged as a very promising platform in the field of drug delivery. The success of nanoparticulate drug delivery systems (NDDS) has been reflected in the regulatory approval of number of nanopharmaceuticals in the last decade (Table 1). Lipid based carriers and biopharmaceutical polymers have emerged as two of the more promising options following impressive achievements liposomal and micellar formulations. With the advent of clinically approved liposomal doxorubicin (Batist et al., 2001; Gordon et al., 2001), (Messerer et al., 2004) and daunorubicin (Messerer et al., 2004; Saltz, 2000; Saltz et al., 2000), the potential of liposomal carriers to improve the therapeutic activity of anticancer drugs has become increasingly established. The early success of micellar paclitaxel (NK-105) (Hamaguchi et al., 2007) and cisplatin (NC-6004) (Nishiyama et al., 2003; Uchino et al., 2005) in reducing the undesirable side effects associated with these potent anticancer drugs have demonstrated the huge potential of micelles as carriers for highly water insoluble compounds. These carrier formulations paired with the drug delivery challenges associated with CPTs rendered them natural candidates to be explored with the help of NDDS. Several nanoparticulate formulations of CPTs are currently being developed and evaluated with the aim of improving therapeutic index of these compounds. A brief overview of few that have shown promising results either pre-clinically or clinically has been presented in this article. We have tried to cover diverse types of NDDS that are being developed around CPTs including liposomes (Table 1) and micelles (Table 2) with additional discussion of structurally similar blockcopolymers.

III. Nanoparticulate drug delivery systems of camptothecins Following success from numerous pre-clinical as well as early clinical studies, pharmaceutical nanotechnology

Table 1: Selected approved lipid based and liposomal drug formulations

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Table 2: Lipid based camptothecin formulations - Liposomes NDDS are known to produce their action by different mechanisms, however the two most accepted explanations behind the success of passively targeted NDDS developed for the cancer treatment are; a) enhanced permeation and retention or more commonly known as the EPR effect and b) possessing altered pharmacokinetic (PK) characteristics that are distinct

from the encapsulated drug. For effective delivery of drugs to the target tumor site, nanoparticles should remain in the circulation for extended time without getting eliminated by either renal or hepatic mechanisms. EPR based targeting relies on exploiting the leaky vasculature present at the tumor site (Figure 2).

Figure 2: Passive tumor targeting with enhanced permeation and retention (EPR) effect. (Adapted from Nature Reviews Drug Discovery 2, (May 2003) 93

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Neovasculature developed around tumor masses typically has defective vascular architecture with large gaps (typically between 100-600 nm diameter) in normally tightly packed endothelial cell junctions and is accompanied with impaired lymphatic clearance. Thus macromolecules in the circulation with particle diameter below 600 nm can be selectively extravasated to the tumor interstitial spaces through this leaky tumor vasculature. The absence of an active lymphatic network prevents clearance of extravasated macromolecules from tumor interstitium. However, for the EPR effect to take place the size of macromolecules should also be large enough to bypass renal clearance (>40 kDa) (Li et al., 1993; Maeda, 2001). The size of macromolecules also plays an important role in extending the circulation time and avoiding rapid clearance by macrophages and cells of mononuclear phagocyte system (MPS) as the size of sinusoids in the spleen and kupffer cells in liver varies between 150-200 nm. Small molecule drugs can thus be optimized for extended circulation and tumor delivery by virtue of encapsulation or conjugation in custom designed nanoparticles that are approximately 100 nm in size. While this method of passive targeting is effective, issues of specificity and intracellular drug delivery still exist. Active targeting by incorporating ligands or antibodies specific to antigens or growth factors overexpressed by tumor cells either directly on the surface of the drug molecules or nanoparticles carrier is currently being pursued to get around these issues with varying degree of success (Blanco et al., 2009; Drummond et al., 1999).

D. Liposomal camptothecin (parent compound) Burke et al., first showed that highly water insoluble camptothecin was readily soluble in lipids (Burke et al., 1993; Burke, 1992). These studies were conducted using small unilamellar liposomal vesicles made from the neutral lipid dimyristoylphosphatidylcholine (DMPC) or electronegative dimyristoylphosphatidylglycerol (DMPG). They demonstrated using different CPT analogs that these compounds can bind to phospholipids with different affinities. Further, binding of CPTs to lipids was observed to shift the hydrolysis equilibrium (in phosphate buffered saline) of CPTs toward the lactone form. The amount of lactone form of camptothecin existing at equilibrium increased from 13% to 100% while maintaining the original biological activity against cancer cells when complexed with either DMPC or DMPG lipids (Burke, 1992). Encouraged by these observations, various lipids were screened for the CPT loading capacity. Sugarman and group were able to develop a lipid complex of CPT (LC-CPT) using Nglutaryl phosphatidyl ethanolamine (NGPE) with 95% CPT loading efficiency at a drug to lipid (D/L) weight ratio of 1:12.5 (Sugarman et al., 1996). LC-CPT showed greater potency than CPT when tested for cytotoxicity against MDA-Panc3 and DIFI cells particularly at lower concentrations (20% weight loss. A biodistribution study conducted using these liposomes in tumor-bearing mice showed 9.6-fold higher tumor accumulation over free CPT 24 h post dose. Thus, encapsulation of CPT into lipid bilayers of liposomal vesicles did help in overcoming solubility issues to certain extent. However, effective drug retention in vivo still remained a major challenge as in most studies the highly hydrophobic CPT tended to

C. Nanoliposomal camptothecins Liposomes are amphiphilic structures made up of phospholipids (Figure 3). These are bilayered vesicles encompassing a central aqueous core. An ability to encapsulate hydrophobic (partitioning in the lipid bilayer) as well as hydrophilic (encapsulation in the aqueous core) molecules makes them unique and versatile drug carriers. Along with the potential for targeted delivery and reduced toxicity, improved lactone stability has been shown to be an added advantage when liposomes were used as carriers for various CPTs.

Figure 3 Schematic representation of a liposome vesicle. 94

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diffuse out from the lipid bilayers quite easily. Additionally, the small size and unilamellarity of liposomes limits the amount of CPT that can be loaded into the bilayers (due to small area of curvature). These problems limited the development of formulations for the parent compound CPT leading to research into the potential for the relatively larger aqueous core of liposomes as a better site for encapsulation of water soluble analogs of CPT including topotecan, irinotecan, lurtotecan etc.

plasma t1/2 of 7 and 11 h, respectively and AUC increases of 200 and 300-fold respectively over that of free irinotecan. The authors have utilized the convection-enhanced delivery (CED) technique to overcome blood brain barrier limitations to drug delivery to successfully increase levels of formulated irinotecan and doxorubicin delivered to rat brain tissue and further, demonstrated a significant survival advantage in rats bearing orthotopic brain tumor xenografts (Krauze et al., 2007). In recognition of the majority of cancer therapy relying on combinations of two or more drugs, particularly when considering regimens containing irinotecan, Celator Pharmaceuticals has developed a single liposomal formulation with both irinotecan and floxuridine encapsulated within the same DSPC/DSPG/Cholesterol (7:2:1, mol:mol:mol) based vesicles at levels designed to optimize a synergistic ratio as defined in vitro (Tardi et al., 2007). This formulation has progressed through Phase I testing and was found to be well tolerated, with the desired plasma drug ratio of 1:1 (molar) being maintained for up to 24 hours post administration (Batist et al., 2009).

E. Liposomal Irinotecan Irinotecan (CPT-11) is currently used clinically in the treatment of colorectal and lung cancers. It is an active pro-drug that undergoes carboxylesterasemediated conversion to the more active SN-38 metabolite. Both irinotecan and SN-38 are sensitive to the hydrolytic conversion of the lactone form to inactive carboxylate as that of other CPTs. Several liposomal formulations of irinotecan have been prepared with varying degree of success and are being evaluated preclinically or clinically (Drummond et al., 2006; Hattori et al., 2009; Messerer et al., 2004; Ramsay et al., 2008b; Tardi et al., 2007). Our laboratory has developed a novel distearoylphosphatidylcholine/cholesterol(55:45mol:mol ) liposomal formulation of irinotecan using divalent metal copper and transmembrane ionophore A23187 (termed Irinophore C™) in order to trap irinotecan inside the aqueous core (Baker et al., 2008; Messerer et al., 2004; Patankar et al., 2011; Ramsay et al., 2006; Ramsay et al., 2008b; Verreault et al., 2011). This formulation showed greater than 95% irinotecan loading efficiency at a D/L of 0.2 (mole ratio). When evaluated pharmacokinetically, Irinophore C™ showed 8-fold increase in t1/2, a 100-fold increase in Cmax, a 1,000-fold increase in AUC, and a 1,000-fold decrease in clearance of the active lactone form of irinotecan. The efficacy of Irinophore C™ has been evaluated in multiple different xenograft tumor models following single-dose treatment (LS 180), three doses administered at 4-day intervals (H460), or three doses administered at 7-day intervals (HT-29, Capan-1 and PC-3) (Ramsay et al., 2008b). Delay in the time required for tumors to reach four times their original size (T-C) was used as a marker of activity. Irinophore C™ showed significantly greater delay in tumor progression than free irinotecan at equivalent dose of 40 mg/kg in all five models tested with most striking (T-C) values observed in HT-29 and Capan-1 models (10 to 14-fold improvement). Drummond et al., utilized polyphosphate or sucrose octasulfate (SOS) co-encapsulation inside liposomes as a means to load and retain irinotecan inside DSPC/cholesterol made liposomes (Drummond et al., 2006). These liposomes achieved loading efficiency of >800 g irinotecan per mole of phospholipids and high drug retention ability as observed from in vivo drug release t1/2 of 56.8 h. Further pharmacokinetic analysis following i.v. administration of polyphosphate or SOS in Sprague-Dawley rats showed impressive results with

F. Liposomal SN-38 SN-38 is an active metabolite of CPT-11 and is known to be approximately 1000-fold more cytotoxic than the parent compound CPT-11. SN-38 also exists in ring open and ring closed forms depending on the pH. However, as with camptothecin itself, formulation of liposomal SN-38 delivery systems was hampered by its intrinsic aqueous insolubility and further, low affinity to lipid membranes. A novel SN-38 liposomal formulation (LE-SN-38) was developed using an approach wherein more soluble ring opened carboxylate form of SN-38 was encapsulated into liposomes that were freeze dried following drug encapsulation. Reconstitution of lyophilized material was carried out in an acidic buffer to promote conversion of SN-38 to the more lipophilic lactone form which then was able to partition into lipid membranes (Zhang et al., 2004). This formulation showed >95% encapsulation efficiency. When tested in multiple murine and human xenograft tumor models (P388, HT-29, Capan1 and MX1) the liposomal form showed better efficacy in reducing tumor growth than free irinotecan treated mice. A pharmacokinetic evaluation of this formulation following a single i.v. dose of 10 mg/kg showed a t1/2 of 6.4 h and plasma AUC of 3.92 µg.h/mL (Pal et al., 2005). Biodistribution showed significant uptake by liver and spleen which could be a concern given the potential for SN-38 mediated toxicity at these organ sites. G. Liposomal Topotecan Like irinotecan, topotecan has low affinity for lipid bilayers. Burke et al., first showed that topotecan can be effectively encapsulated in the aqueous core of liposomal vesicles made with distearoylphosphatidylcholine (DSPC) (Burke and Gao, 1994). 95

Cancer Therapy Vol 8, page 96 Topotecan encapsulated into sphingomyelin/cholesterol liposomes with the help of manganese sulfate and transmembrane ionophore A23187 showed 90-100% trapping efficiency at a D/L of 1/8 (mole ratio) (Tardi et al., 2000). Pharmacokinetically, this formulation showed extended blood circulation time with a 400-fold increase in plasma AUC over that of free drug and enhanced lactone stability (84% after 24 h of injection compared to 50% after 5 min of injection for free topotecan). Efficacy studies in the L1210 murine tumor model showed a significant improvement in median survival of more than 60 days over that of 15 days as observed for free topotecan. Similar observations were noted in the human breast carcinoma model (MDA 435/LCC6). Another formulation prepared using ammonium sulfate gradient in DSPC/cholesterol liposomes showed 90% encapsulation efficiency at a D/L of 1/5.4 (mole ratio) (Liu et al., 2002). More than 60% of topotecan in this formulation existed in the lactone form when incubated in PBS at 37◦C. A pharmacokinetic study examining a single i.v. dose of 5 mg/kg free or liposomal topotecan showed 14-fold and 40-fold increases in plasma concentration and AUC respectively. Efficacy assessments in syngeneic C-26 and HTB-9 murine models showed a therapeutic advantage in terms of tumor volume inhibition by liposomal form over free topotecan. Vali et al., compared the effect of PEG coating on the pharmacokinetic characteristics of topotecan loaded DMPC/Cholestereol and DSPC/cholesterol liposomes and observed 2-fold increase in AUC by pegylated liposomes over conventional (Dadashzadeh et al., 2008; Vali et al., 2008). However, the interference of PEG coating on the intracellular delivery of topotecan is still debatable. Most of the liposomal formulations described in the studies above were able to encapsulate topotecan effectively and also showed good activity against different tumor models; however, these formulations were not able to retain the encapsulated topotecan as effectively as that of liposomal formulations of anthracyclines (Drummond et al., 2008; Gabizon et al., 2003) or vicristine (Boehlke and Winter, 2006) as observed in these formulations. Drummond and coworkers sought to address this deficiency by developing a nanoliposomal formulation of topotecan using transmembrane gradients of triethylammonium salts of polyphosphate or SOS (Drummond et al., 2010). This formulation not only showed an encapsulation efficiency of >90% but also proved to superior in terms of drug retention as observed from the plasma half lives of 6 h and 8.4 h (following i.v. administration of 5 mg/kg topotecan to mice) for liposomes prepared using TEA-polyphosphate and TEASOS respectively. Anti-tumor activity assessments carried out in human prostate (DU-145) and breast (BT-474) xenograft models showed better activity by the liposomal form over that of free topotecan as measured by delay in tumor size increase over time. Further modification of

these liposomes with surface conjugated antibodies specific against HER2 and EGFR receptors to produce immunoliposomes showed increased tumor uptake as well as greater tumor inhibition as compared to non targeted liposomes when tested in tumor models inoculated with HER2 and EGFR over-expressing cell lines (Drummond et al., 2010). H. Liposomal Lurtotecan In addition to topotecan and irinotecan, formulated lurtotecan is another CPT analog that has shown significant clinical activity. Although toxicity concerns were observed with the free drug, liposomal lurtotecan showed promising outcomes in the preclinical setting and has reached phase II clinical trials. Lurtotecan has been encapsulated and tested in both pegylated (SPI-355) and non-pegylated (NX211/OSI211) liposomal formulations (Colbern et al., 1998; Dark et al., 2005; Desjardins et al., 2001; Duffaud et al., 2004; Emerson et al., 2000). Pre-clinical pharmacokinetic evaluation of SPI-355 in Sprague Dawley rats showed extended blood circulation with 1250- and 35-fold increases in Cmax and AUC respectively over that of free drug. Efficacy assessment in HT-29 colon carcinoma xenografts demonstrated that SPI-355 was 20 times more effective in inhibiting tumor growth than free lurtotecan. Although the liposomal form increased the toxicity of lurtotecan by 4-fold (as demonstrated by weight loss) the increased efficacy allowed administration of several fold lower dose of SPI-355 than free Lurtotecan to achieve similar or greater response. Similar to that of SPI-355, NX211 also improved the pharmacokinetic profile of lurtotecan and most importantly showed significantly higher (70-fold) distribution of lurtotecan into tumor tissues when compared to that observed with free lurtotecan. Both formulations significantly improved the lactone stability of lurtotecan over extended period. The anti-tumor activity of NX211 was assessed in KB and ES-2 ovarian xenograft tumor models. NX211 showed improved therapeutic index than free lurtotecan as observed from LD50/ED60 values of 2.9 (KB sarcoma) and 14.4 (ES-2 ovarian cancer) for NX211 compared to 1 and 1.1 for free lurtotecan. LD50 here as referred to indicates lethal dose for 50% of animals whereas ED60 and ED80, are the effective doses at which 60% and 80% tumor growth inhibition occurred respectively (Emerson et al., 2000). Biodistribution studies with NX211 showed major accumulation in the spleen whereas the liver was the major uptake site for the free drug. A Phase I clinical study of NX211 was carried out to determined the maximum tolerated dose (MTD), toxicity profile and PK (Kehrer et al., 2002). Dose escalation was carried out using doses ranging between 0.4 – 4.3 mg/m2 by administering NX211 as a 30 minute i.v. infusion every 3 weeks. Common toxicities observed following peripheral infusion were neutropenia and thrombocytopenia which were non-cumulative. The dose recommended for phase II studies was 3.8 mg/m2. 96

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Interestingly it was noted that the amount of lurtotecan excreted in the urine was directly related to the decrease in neutrophil and platelet count. Another phase I study was conducted using this formulation in order to define MTD and recommended dose for phase II when given as short i.v. infusion on days 1, 2, and 3 after every three weeks (Gelmon et al., 2004). This study also had the objective of determining MTD in either minimally pretreated or heavily pretreated patients. Dose escalation was carried out using a dose range of 0.15 mg/m2/d to 2.1 mg/m2/d. PK parameters were also noted and compared with that of free lurtotecan. As noted with other camptothecin formulations, the liposomal form showed significantly higher AUC (100-fold), Cmax and t1/2 over free form which increased with the dose. MTDs noted for minimally and heavily pretreated patients were 2.1 and 1.8 mg/m2/d respectively and recommended doses for phase II were 1.8 and 1.5 mg/m2/d for minimally and heavily pretreated patients respectively. These studies led to a randomized phase II trial for patients with recurrent ovarian cancer who were previously treated with at least one platinum-containing regimen liposomal lurtotecan using two different treatment schedules (Calvert AH, 2002): i.v. infusion of liposomal lurtotecan using either schedule A (1.8 mg/m2/d on days 1-3 every 3 weeks) or schedule B (2.4 mg/m2/d on days 1 and 8 every 3 weeks). Patients who had received prior treatment with topotecan or any other topoisomerase I inhibitor were excluded from the study. Although schedule A showed greater hematologic toxicity than schedule B, a higher number of patients responded to schedule A and therefore was declared as superior. Surprisingly the AUC observed in schedule A was lower than that of schedule B. A second Phase II study was conducted in topotecan resistant epithelial ovarian cancer patients who were pre-treated with topotecan either as a single agent or in a combination setting (Seiden et al., 2004). Liposomal lurtotecan was administered using a dose of 2.4 mg/m2 on days 1 and 8 every 3 weeks (21 days). Results showed moderate hematological toxicity with liposomal lurtotecan but no significant clinical activity other than stable disease in eight out of twenty-two patients evaluated. Hence, it was concluded that this schedule was not appropriate for topotecan refractory or resistant ovarian cancer patients. However, positive response observed in topotecan naïve population with an alternate schedule suggested further evaluation of this formulation in different conditions. Apart from the above mentioned, other liposomal CPT analogs that have showed promising activity in pre-clinical studies and which are currently under investigation are belotecan (Yu et al., 2007; Zamboni et al., 2009) and silatecan (Zamboni et al., 2008). I. Micellar Camptothecins Liposomal carrier systems have wide applicability in delivery of hydrophilic and water soluble compounds such as doxorubicin (Cheung et al., 1998; Gabizon et al., 1982; Johnston et al., 2008), vincristine (Boehlke and

Winter, 2006), mitoxantrone (Cui et al., 2009) and amphotericin B (Taylor et al., 1982),. As mentioned above there is a propensity however, for highly active pharmaceuticals to be poorly water-soluble which leads to poor drug absorption and low bioavailability in free form, and limits formulation within liposomes to the lipid bilayers which leads to difficulty in maintenance of targeting properties. Polymeric micelles are nanoscopic drug delivery systems composed of block or graft copolymers that form into core/shell colloidal carriers of diameter from 10 – 100 nm (Mahmud et al., 2007). The hydrophobic core is thought to stabilize micellar structure while providing sequestration for a variety of compounds including small molecule, protein or DNA/RNA based drugs. This is by means of hydrogen bond formation with the aqueous surroundings which form a tight shell around the micellar core. As a result, the contents of the hydrophobic core are effectively protected against hydrolysis and enzymatic degradation while the hydrophilic shell allows avoidance of opsonization, escape from the MPS in the liver and also avoidance of the filtration of interendothelial cells in the spleen. Block copolymers are made up of blocks of polymerized monomers that differ in physico-chemical properties such as charge and/or polarity. Graft copolymers are a special type of branched copolymer in which the side chains are structurally distinct from and grafted to the main chain. When combined in water, hydrophilic block or graft copolymers may form stable complexes with oppositely charged molecules. Conversely, self-assembly occurs when amphiphilic block copolymers are combined in solvent selective for one of the polymeric segments. It is these selfassemblies in aqueous solutions that are of interest for drug delivery. Due to the evasion of MPS and small size, polymeric micellar formulations are also able to take advantage of the EPR effect in similar fashion as liposomes, and in fact, are ‘hallmark examples’ of the applicability of the EPR effect (Maeda et al., 2006). The full potential of this technology has not yet been realized however, perhaps due to premature leakage of drugs from carrier or inefficient or insufficient intracellular delivery provided by relying solely on EPR effect and passive delivery. Studies have shown that increasing hydrophobic chain length leads to enhanced retention of drug within the micelles and afford sustained drug release following accumulation in target tissue (Huh et al., 2005; Zhang et al., 2005). To this end, increasingly sophisticated polymeric micellar formulations are in development including use of polymers beyond the typical poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL) and those for active targeting and multifunctional polymeric micelles (reviewed in (Mahmud et al., 2007)) which include attachment of targeting ligands to the surface of micelles and development of micelles with stimulusresponsive properties such as light, electrolyte, redox

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Cancer Therapy Vol 8, page 98 potential or pH sensitivity to allow targeting and triggered release of drug payload. Polymeric micelle/drug formulations are derived by one of three principle methods: solution dilution, direct dissolution or solvent evaporation. For hydrophobic compounds such as camptothecin, the solution dilution method is most typically employed, consisting of dissolution of copolymers and drug in water miscible organic solvent followed by aqueous dilution. By use of different combinations of copolymers, researchers are able to adjust many of the parameters of micelles such as critical micelle concentration, association number, core radius, shell thickness and hydrodynamic radius.

Initial use of micelles for drug formulation was with doxorubicin (Yokoyama et al., 1990) in which Yokoyama et al., (1990) conjugated doxorubicin itself to aspartic cid residues of poly(ethylene glycol)poly(aspartic acid) block copolymers. Use of this formulation resulted in selective delivery to (Yokoyama et al., 1999) murine solid tumor colon adenocarcinoma 26 (C 26) as well as significantly increased efficacy. Since then, formulations have been developed for cisplatin (Mizumura et al., 2001), methotrexate (Li and Kwon, 2000), paclitaxel (Huh et al., 2005; Wang et al., 2005; Zhang et al., 1997), and several forms of camptothecins (see Table 3 for reference).

Table 3 Lipid based camptothecin formulations - Micellar Chitosan is of interest due to its biocompatibility, The only micellar anticancer drug currently biodegradability and non-toxicty, however it is poorly marketed or undergoing clinical trials is Genexol-PM® soluble in organic solvents and in aqueous media at (paclitaxel loaded polymeric micelle). This product is physiological pH which has previously limited currently in phase IV testing for recurrent breast cancer applicability for drug delivery systems. patients with prior taxane treatment as well as a phase Duan et al., have overcome the limitations of I/II trial in patients with advanced ovarian cancer, in using pure chitosan by graft copolymerization of combination with carboplatin, phase II in combination chitosan with ε-caprolactone at different ratios (Duan et with cisplatin in advanced non-small cell lung cancer al., 2009) retaining the amino groups of the CS forming and phase III in patients with recurrent or metastatic CS-g-PCL copolymers. They have encapsulated SN-38 breast cancer. Genexol-PM® has also been tested in the within these micelles by co-dissolution in DMSO, phase II setting in patients with unresectable locally removal of solvent then hydration in dH2O and found advanced or metastatic pancreatic cancer in which that a graft ratio of CS-g-PCL (1:24) was optimal in results showed good tolerability but progression free terms of critical aggregation concentration (CAC) as survival only equivalent to that of gemcitabine treated well as in vitro drug release properties (51.2% release patients (Opanasopit et al., 2004). over 80 hours). There has been substantial effort placed in examining A group out of Pakistan has also recently various cationic polymers for micelle formation, successfully solubilized camptothecin using chitosan including use of poly(ethyleneimine) (PEI) (Jeong et al., based polymeric micelles (Ranjha, 2009) by attaching 2005), poly(4-vinyl pyridine) (Wang et al., 2002), long chain alkyl groups to the chitosan thereby creating poly(N-methyldietheneamine sebacate) (Wang et al., hydrophobic moieties as the core and making quaternary 2007) and chitosan (Duan et al., 2009). ammonium as hydrophilic moieties as the shell. The resulting ammonium palmitoyl glycol chitosan polymeric micelles were added to camptothecin. The 98

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authors found highest CPT solubility with the less hydrophobic polymers.

reduced drug release. As with other PEG containing formulations, increasing PEG chain length resulted in increased blood residence times for the micelles, with 24 fold more camptothecin present in circulation at 4 hours for PEG10,000 containing micelles than for free camptothecin with a half life of 16.5 hours. Tumour accumulation was also significantly higher for PEG10,000 containing micelles with ~ 2% administered dose being located in tumour tissue at 4 hours post injection, compared to ~0.1% free drug (Shi et al., 2005). Cortesi et al., (1997) performed a direct comparison of liposomes, micelles and microemulsions in the encapsulation/association and in vitro cytotoxicity of camptothecin with the carrier formulation (Cortesi et al., 1997). The authors compared polyoxyethylenepolyoxypropylene block copolymers with egg phosphatidyl/cholesterol (EPC/CH; 8:2 mol:mol) liposomes and labrasol/plurol isostearate/isosteaylisostearate/water microemulsion. Camptothecin was loaded into micelles by addition to colpolymers in Tween 80:Tween 85 (1:1) in water (5% w/v). In this study, the use of microemulsion resulted in solubilization of five-fold higher levels of CPT than did the micelles however none of the formulations tested exhibited markedly enhanced cytotoxicity as compared to free drug alone (Cortesi et al., 1997). The most advanced micellar camptothecin formulation is that of NK012, initially developed as means of encapsulation of cisplatin (Nishiyama et al., 1999) and oxaliplatin (Cabral et al., 2005), using platinum metal complex formation with glutamic acid and which is now being developed as a platform formulation for a number of poorly soluble anticancer drugs by Nippon Kayaku Co., Ltd., including SN-38. In this formulation, silver nitrate was complexed with platinums to increase aqueous solubility. The block copolymer utilized for all formulations including SN-38 was poly(ethylene glycol)-poly(glutamic acid) (PEGP(Glu)), however there were appreciable differences. For example, average particle diameter for oxaliplatin micelles was 40 nm (Cabral et al., 2005) whereas for SN-38, average diameter was 20 nm (Koizumi et al., 2006). Results from Phase I testing of the micellar SN38 have indicated antitumor activity including partial responses and several occurrences of prolonged stable disease across a variety of advanced refractory cancers (Hamaguchi et al., 2010).

J. Block Copolymers As mentioned above, block copolymers are made up of blocks of polymerized monomers that differ in physico-chemical properties. Several groups have used block copolymers for formulation of camptothecins including Gao et al., (2008) (Gao et al., 2008) who used Methoxy poly(ethylene glycol)-poly(D,L-lactide) (mPEG-PDLLA, PELA) copolymers for encapsulation of 9-nitro-20(S)-camptothecin (9-NC) with encapsulation rate success of 80 – 90% and maintenance of 80% 9-NC in lactone conformation after 160 minutes incubation in PBS. The authors also demonstrated that larger hydrophobic cores resulted in encapsulation of greater amounts of drug and slower in vitro release rate (Gao et al., 2008). This reliance on the hydrophobic core component was also demonstrated by Opanasopit et al., (2004) (Opanasopit et al., 2004) who used poly(ethylene glycol)-poly(benzyl L-aspartate) block copolymers (PEG-PBLA) micelles to encapsulate camptothecin by means of evaporation method. The authors showed that over 92% encapsulation could be achieved when using benzyl esterification for the copolymers. Opanasopit however showed that shorter hydrophobic segments actually decreased the release rate of the camptothecin (Opanasopit et al., 2004) as well as increasing overall micelle stability. The authors note that the use of benzyl ester substituted groups may be advantageous over that of purely aspartate due to provision of space to accommodate CPT molecules. Aspartate has been used with varying success by Yokoyama et al., (2004) (Yokoyama et al., 2004) in incorporation of camptothecin into poly(ethylene glycol)-poly(aspartate) block copolymers by three methods: 1) dialysis, resulting in low camptothecin incorporation; 2) emulsion, unsuccessful due to large aggregate formation; or 3) evaporation in which CPT and block copolymers were dissolved in chloroform then solvent evaporated which resulted in 60% encapsulation efficiency. In this paper the authors point to not the hydrophobicity of the inner core, but rather the rigidity, pointing out that longer acyl chains are more flexible (Yokoyama et al., 2004). The use of poly(ethylene glycol) (PEG) has been widely applied in liposomal formulations and also for micelles and PEG is in fact, the most commonly used corona-forming polymer, as highlighted in the poly (methoxy-polyethylene- glycopolycaprolactone) (Me-PEGPCL) camptothecin micelles developed by Shi et al., (2005) (Shi et al., 2005). In this study PEGs of differing chain lengths were tested (2,000, 5,000 or 10,000) for in vitro release, pharmacokinetics in rats and biodistribution in S180 tumour bearing mice. The authors found that while increasing PEG chain length increased the micelle stability for drug loading, it also increased rate of drug release in vitro, while longer PCL chains

K. Antibody targeted micelles Following on the observation that agonistic Fas antibodies potentiated camptothecin cytotoxicity (Shao et al., 2001), McCarron et al., (2008) sought to target camptothecin loaded poly(lactic-co-glycolic acid) (PLGA) micelles to tumour cells by means of Fas antibody ligation (McCarron et al., 2008). CPT and PLGA were co-dissolved in dichloromethane and DMSO which was then diluted in acetone. 99

Cancer Therapy Vol 8, page 100 Organic solvents were then evaporated off in MES buffer and further washes performed in MES buffer. The combination of Fas antibody and camptothecin in these micelles were shown to have synergistic effect on the % cell survival of HCT116 cells in culture with uptake of micelles into the lysosomes of cells. The authors point to the maintenance of CPT in lactone form as a feature of this system, however the data supporting this was not presented. Sawant et al., (2008) (Sawant et al., 2008) used a conjugated anticancer nucleosome specific monoclonal abtibody 2C5 (mAb 2C5) to the outer shell of PEG-phosphatidyl ethanolamine (PEG-PE)/vitamin E micelles as a means of targeting a broad variety of tumour cells via tumour cell surface bound nucleosomes. They then used this system to load camptothecin by co-dissolution in chloroform, removal of solvent and hydration in 5 mM Na-citrate buffered saline. mAb in PBS was added to the micelle-drug solution with vortexing prior to pH adjustment and dialysis against 10 mM acetate buffer. The use of vitamin E in this system was to increase the content of hydrophobic fragments therefore increasing drug solubility by 40% over non-vitamin E containing micelles (Sawant et al., 2008). The targeted camptothecin loaded micelles were shown to have greater cytotoxicity against two cancer cell lines (4T1 and B16F1) in vitro which was reasoned to be due to enhanced endocytotic uptake of drug containing micelles as well as targeting by the mAb 2C5.

III. Conclusion: Due to the versatility of lipid-based formulations for drug delivery, these systems are being actively researched and applied to the use of camptothecins in oncology therapy. While liposomal systems are certainly more advanced in general terms, with several formulations marketed for various indications, including cancer, research in micellar and other polymer based formulations is rapidly advancing and it is likely that one or more of these formulations will progress successfully through clinical testing in the near future. This paper has provided an overview of research into lipid-based formulations of camptothecins, both liposomal and micellar/polymer designed for parenteral administration in the treatment of cancers, primarily ovarian and colorectal. These types of formulations enable the delivery of higher amounts of drugs to target tissues by virtue of the pharmacokinetic properties of the carrier and may also reduce toxicity to non-target tissues. Further enhancements to targeting are possible with conjugation of antibodies to tumor-specific epitopes such as anti-HER-2 imunoliposomes being developed by Kirpotin et al., (Kirpotin et al., 1997) or the anti-Fas micelles being developed by McCarron et al., (McCarron et al., 2008) and as more anti-cancer drugs come off patent, we may expect to see further growth in this general research area.

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Dr. Dawn Waterhouse

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