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Liposomal encapsulation of irinotecan and potential for the use of liposomal drug in the treatment of… Messerer, Corrie Lynn 2002

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L I P O S O M A L E N C A P S U L A T I O N OF IRINOTECAN A N D POTENTIAL FOR THE USE OF L I P O S O M A L D R U G IN THE T R E A T M E N T OF L I V E R M E T A S T A S E S ASSOCIATED WITH A D V A N C E D C O L O R E C T A L C A N C E R  by CORRIE L Y N N M E S S E R E R B . S c , University of British Columbia, 2000 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENT FOR THE D E G R E E OF M A S T E R  OF SCIENCE in  THE F A C U L T Y OF G R A D U A T E STUDIES D E P A R T M E N T OF P A T H O L O G Y A N D L A B O R A T O R Y MEDICINE We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A August, 2002 © Corrie Lynn Messerer, 2002  In  presenting  degree  at  this  the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  freely available for copying  of  department publication  this or  reference  thesis by  for  his thesis  and study. scholarly  or for  her  of  this  Department  of  PgUinlnqr  The University of British Vancouver, Canada  Date  DE-6 (2/88)  | b - D f - 0 2 -  Columbia  purposes  requirements that  agree  may  representatives.  financial  permission.  I further  the  It  gain shall not  be is  that  the  Library  permission  granted  by  understood be  for  an  advanced  shall for  the that  allowed without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  Colorectal cancer is the second leading cause o f cancer mortality in North America, primarily because o f a high incidence o f hepatic metastases, which are relatively unresponsive to the systemic chemotherapy.  Irinotecan, a camptothecin analogue recently approved for used in  conjunction with 5-fluorouricil/leucovorin, is a marginal improvement but toxic and by no means curative. Liposomal drug formulations are argued to be more effective at treating liver-localised carcinomas when compared with their free drug counterparts, because o f their intrinsic affinity for the liver and extended lifespan. This work examined the suitability o f a liposomal irinotecan formulation i n the treatment o f colorectal liver metastases. DSPC/cholesterol liposomes using an ionophore-generated  Irinotecan was encapsulated i n transmembrane  proton gradient.  After i.v. injection, liposomal drug was eliminated from the plasma much more slowly than free drag, and after 1 h circulating levels o f liposome-associated drug were 10 fold greater.  In  addition, high-performance liquid chromography analysis o f plasma samples revealed that liposome-associated irinotecan is protected from inactivating hydrolysis with > 80 % remaining in the active lactone form up to 24 h after administration. These improved pharmacokinetics observed for the liposomal drug were associated with increased efficacy in both solid tumour and orthotopic human models o f colorectal metastases.  Using a model ( L S I 8 0 ) o f colorectal  metastases in S C I D / R A G 2 M mice it was demonstrated that liposome-encapsulated drug was more effective at arresting tumour growth than was free drug. Further, i n the human model o f colorectal liver metastases (LS174T), liposomal irinotecan substantially increase life span relative to free drug with all members surviving long-term (75 days) as compared to a survival time o f 30 and 50 days for the control and free drug treated groups. These results illustrate that liposomal encapsulation can substantially enhance the therapeutic activity o f irinotecan, and emphasize the potential for liposomal irinotecan in the treatment o f liver metastases. ii  T A B L E OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF T A B L E S  vii  ABBREVIATIONS  viii  ACKNOWLEDGEMENTS  ix  DEDICATION  x  C H A P T E R 1 INTRODUCTION  1  1.1 Foreword  1  1.2 Liposomes 1.2.1 Phospholipids  2 6  1.2.2 Cholesterol  8  1.3 Preparation of SUVs and L U V s  10  1.4 Encapsulation 1.4.1 Passive encapsulation 1.4.2 Active encapsulation 1.5 Requirements for effective drug delivery 1.5.1 Liposome serum protein interactions and clearance 1.5.2 Extravasation through blood vessels 1.5.3 Liposome accumulation in the liver 1.5.4 Drug release  11 13 13 16 16 19 21 24  1.6 Liposomes as drug carriers 1.6.1 Selecting a drug for encapsulation  24 25  1.7 Irinotecan 1.7.1 Mechanism of action 1.7.2 Activation/Metabolism 1.7.3 Mechanisms of resistance  26 28 28 29  1.8 Advanced colorectal cancer and liver metastases 1.8.1 Current treatment strategies 1.8.2.1 Systemic chemotherapy 1.8.2.2 Liver resection  30 31 31 32  iii  1.9 Specific Aims  32  1.10 Hypothesis  32  C H A P T E R 2 M A T E R I A L S A N D METHODS  35  2.1 Materials  35  2.2 Preparation of liposomes  36  2.3 MnS04 pH gradient loading of irinotecan  36  2.4 In vitro characteristics of liposomal irinotecan  37  2.5 Pharmacokinetic studies in SCID/RAG-2M mice  38  2.6 Establishing a maximum tolerated drug dose  40  2.7 Liposomal irinotecan anti-tumour efficacy in the human LSI80 solid tumour model  40  2.8 Liposomal irinotecan anti-tumour efficacy in the human LS174T orthotopic tumour model  41  2.9 Liposome mediated drug delivery to LS174T liver metastases  41  2.10 Statistical analysis  42  C H A P T E R 3 L I P O S O M A L IRINOTECAN A N D ITS THERAPEUTIC A C T I V I T Y AGAINST COLORECTAL METASTASES  43  3.1 In vitro irinotecan uptake and release characteristics  43  3.2 In vivo plasma elimination of free and liposomal irinotecan  47  3.3 Acute toxicity of free and liposomal irinotecan  51  3.4 Efficacy of single and multiple dose administration of liposomal and free irinotecan in established LSI80 human solid tumours  53  3.5 Efficacy of single and multiple dose administration of liposomal and free irinotecan in established LS174T human orthotopic tumours 53 3.6 Liposome mediated delivery to liver metastases  55  C H A P T E R 4 DISCUSSION  59  4.1 Summary of results  59  4.2 Discussion  60 iv  REFERENCES APPENDIX  LIST O F FIGURES  Figure 1.1 Liposome structure  4  Figure 1.2 Camptothecin ring hydrolysis  5  Figure 1.3 Phospholipid structure  7  Figure 1.4  9  Structure of cholesterol and insertion into the lipid bilayer  Figure 1.5 Active and passive encapsulation  12  Figure 1.6  15  Active drug entrapment in liposomes  Figure 1.7 Liposome target site accumulation  17  Figure 1.8 Structure o f a normal capillary  20  Figure 1.9 Anatomy o f the liver sinusoid  23  Figure 1.10 Irinotecan metabolism  27  Figure 3.1 Effect of the A23187 ionophore in p H gradient-mediated loading o f irinotecan  44  Figure 3.2 The effect of drug to lipid ratio on irinotecan loading and the transmembrane p H gradient  45  Figure 3.3 Efficiency of irinotecan encapsulation at different drug to lipid ratios  46  Figure 3.4 Release of irinotecan from DSPC/cholesterol liposomes  48  Figure 3.5 Plasma elimination of free and liposomal irinotecan  49  Figure 3.6 Toxicity of free and liposomal irinotecan  52  Figure 3.7 Antitumour efficacy of free and liposomal irinotecan as in L S I 80 tumour models. 54 Figure 3.8 Antitumour efficacy of free and liposomal irinotecan in L S 1 7 4 T tumour models.... 56 Figure 3.9 Liposome-mediated drug delivery to the tumour  57  Figure A - l H P L C calibration curve, aqueous standards  77  Figure A - 2 H P L C calibration curve, plasma standards  78  Figure A - 3 Representative chromatogram  79 vi  LIST OF T A B L E S  Table 3.1  Analysis o f plasma irinotecan following i.v. administration o f free or liposomal drug  vii  50  ABBREVIATIONS  H-CHE 5-FU APC CRC CYP3A Dil DMSO DSPC EDTA HDL HEPES HPLC IC i.v. LUV MLV MPS MTD MTT NPC PEG s.c. s.d. s.e. SHE SUV UGT 3  5 0  tritiated [ H] cholesteryl hexadeecyl ether 5-fluorouracil 7-ethyl-10-[4-N-(5-aminopentanoic acid)-l-piperidino] carbonyloxycamptothecin colorectal cancer cytochrome P450 3 A family 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate dimethyl sulfoxide l,2-disteroyl-sn-glycero-3-phosphocholine ethylenediaminetetra-acetic acid high-density lipoproteins A -2-hydroxyethylpiperazine-A -2-ethane-sulphonic acid high-performance liquid chromatography concentration necessary for 50% inhibition o f growth intravenous large unilamellar vesicles multilamellar vesicles mononuclear phagocytic system maximum tolerated dose 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide 7-ethyl-10-(4-amino-1 -piperidino) carbonyloxycamptothecine poly(ethylene glycol) subcutaneous standard deviation standard error buffer composed o f 300 m M sucrose, 20 m M H E P E S and 15 m M E D T A small unilamellar vesicles uridine diphosphate glucuronsyltransferases 3  r  A  viii  ACKNOWLEDGEMENTS  I would like to thank everyone at Advanced Therapeutics and Celator Technologies, Inc. who were o f great assistance through answering m y many questions or offering their advice.  I must  mention, in particular, the animal technicians Dana Masin, Rebecca N g , H o n g Y a n and Sophia Tan as well as Natashia Harasym for their expertise and tremendous help with the animal studies. I would especially like to thank Marcel B a l l y for inviting me to j o i n his lab and for his support, advice and ideas during my time there.  Corrie Messerer June 2002  ix  To my mum , for her unfailing love and support  and  in loving memory of my dad.  x  CHAPTER 1  INTRODUCTION  1.1 Foreword  Cancer is a general term that encompasses a myriad o f discrete diseases, which although sharing the characteristic o f inappropriate cell growth and differentiation, otherwise exhibit distinctive behaviours.  These behaviours along with a classification, derived from the site o f origin and  histological appearance, dictate the individual treatment regime prescribed for "each" cancer.  Anticancer treatment strategies fall under one o f two modalities, being either localised or systemic treatments.  Surgical resection, alone or i n combination with radiation therapy, is the  leader i n achieving cures i n localised disease, but is not suitable for treatment o f metastatic or disseminated cancers.  Chemotherapy, as a single modality, is successful i n curing a number o f  disseminated haematologic  and solid tumour malignancies including certain  leukaemias,  lymphomas, and testicular cancers.  While significant advances have been made i n chemotherapeutic strategies over the past thirty years to the extent that some cancers, as previously mentioned, are potentially curable, for many of the most common and fatal cancers such as those originating in the breast, lung and colon, little progress has been made (National Cancer Institute o f Canada: Canadian Cancer Statistics 2002,  Toronto, Canada,  2002).  Short o f early detection and intervention by surgical removal o f  diseased tissues before metastasis, treatment for these cancers relies heavily on chemotherapy 1  and long term prognosis for the patient is not encouraging.  Acknowledgment that current  chemotherapeutic standards are unacceptable is reflected in the vast amounts o f time, energy and money currently being invested in the arduous process o f development and testing o f new chemotherapeutic agents. problematic, often  Unfortunately, many chemotherapeutic drugs appear to be inherently  exhibiting properties  far  from ideal including poor  solubility, rapid  metabolism, instability and unfavourable biodistribution most often resulting in dose-limiting toxicities and suboptimal therapy. Ideally drugs would localise directly and specifically with a high therapeutic index (ratio o f effective dose to toxic dose) to the disease tissue.  A novel approach for improving cancer chemotherapy, instigated more than three decades ago and recently gaining ground, is based on the principle that chemotherapy's unreliable success is not a consequence o f poor drug design, but at least partly caused by ineffective drug delivery to the cancerous cells.  Marginally effective or severely toxic drugs can be converted into useful  agents simply through improvements in drag delivery.  Although several methods have been  explored with the aim o f improving delivery, including polymer particles, and microspheres, that which is most rapidly gaining popularity and is most advanced i n development are lipid-based carriers, and i n particular, liposomes.  Liposomes are establishing a reputation for converting  traditional chemotherapeutic drugs such as vinicristine, doxorubicin and daunorubicin into much more active and efficient anti-cancer agents (see Mayer et al., 2001 for review).  The present  work investigates the potential for encapsulation o f the novel drag irinotecan as an alternative treatment for advanced colorectal cancer.  1.2 Liposomes  Liposomes are among the most commonly employed lipid-based drug delivery systems. 2  A wide  range o f potential non-toxic lipid constituents, whose manipulation controls such physical properties as trapping efficacies and release rates (Gregoriadis, 1991), impart tremendous versatility over other carriers.  Bangham et al. in 1965 were the first to investigate liposomes in  the form o f multilamellar vesicles ( M L V s ) , which generate spontaneously upon lipid hydration and are characterised by concentric lipid bilayers separated by aqueous channels (Figure 1.1). The distinctive bilayer formation by lipids is due to their amphipathic nature, which drives their hydrophilic head to face outside and their hydrophobic tails to be sheltered on the inside. Liposome technology has not been limited to drug carrier systems, and liposomes were originally described as a tool for studying membrane structure and biophysics (Sessa and Weissmann, 1968), but have since come to play a significant role in drug delivery.  A t their most basic level, liposomes serve as solubilising agents and can act as carriers for either water-soluble or water-insoluble drugs.  Agents associated with liposomes, particularly those in  the aqueous core, exhibit enhanced stability and receive substantial protection from enzymemediated degradation and deterioration, other unfavourable physiological processes or pHs.  For  instance, camptothecin and related analogues require a closed a-hydroxy (lactone) ring moiety for activity, but at physiological pHs rapid hydrolysis to the inactive ring-open compound is observed (Figure 1.2) (reviewed in Burke and B o m , 1996).  Liposome entrapment o f these drugs  in an acidic environment favours the closed-ring version thereby significantly increasing their half-lives in vivo (Burke and Gao, 1994). chemotherapy,  however,  liposome  Perhaps  encapsulation  often  o f greatest advantage for use in alters  the  pharmacokinetics  and  biodistribution properties o f the associated drugs (Hwang, 1987; A l l e n et al., 1995; Scherphof et al., 1997), diminishing toxicities associated with non-specific drug accumulation in healthy tissues while enhancing drug delivery to the disease site, resulting in improved efficacy by increasing selective delivery. 3  Figure 1.1 Liposome structure. Freeze-fracture electron microscopy of (A) multilamellar vesicles, (B) large unilamellar vesicles, and (C) small unilamellar vesicles composed of phosphatidylcholine.  4  Figure 1.2 Camptothecin ring hydrolysis. A n intact lactone ring is necessary for camptothecin-mediated cytotoxicity. p H mediated hydrolysis o f camptothecin drives the equation to the right, and the ring-open inactive carboxylate species at physiological p H . Preferential binding o f the carboxylate species to serum proteins further drives the reaction in this direction.  5  1.2.1  Phospholipids  Phospholipids, consisting o f a polar headgroup coupled by glycerol-phosphate backbone to an apolar tail (Figure 1.3), comprise one o f the major classes o f membrane-forming lipids. versatility o f phospholipids as liposome components  is attributable  The  to various possible  combinations o f headgroup and acyl chain tail which together dictate bilayer properties including surface charge and rate of elimination.  For example, anionic lipids, such as phosphatidylserine,  generally promote serum binding and rapid liposome elimination from the blood stream (Moghimi and Patel, 1989), but this is dependent on chemical properties o f the particular anionic lipid used.  It has been reported (Gabizon and Papahadjopoulos, 1988) that incorporation o f  phosphotidylglycerol or phosphotidylionositol can actually enhance circulation lifetime o f an infected liposome. In the design o f an effective lipid based drug formulation, liposomes must be maintained in the blood stream for a sufficient period o f time and concentration to move from the blood compartment to diseased tissue in an extravascular site; consequently, certain charged lipids are not typically used and the focus has been on the zwitterionic phosphatidylcholine (PC).  A key phospholipid characteristic that can dramatically affect its properties as a liposomal carrier is the temperature o f its gel to liquid-crystalline phase transition (T ), which is primarily c  influenced by its acyl chain structure (McElhaney, 1982; Huang et al., 1993; Wang et a l , 1997). A t temperatures below the transition temperature, the bilayer is i n the highly ordered gel phase in which the tight packing is largely due to the restricted motion o f the acyl chains.  At  temperatures above the phase transition temperature, membrane order is decreased, acyl chains have much greater freedom o f movement and membranes exhibit much enhanced permeability to both solvents and solutes.  Membrane permeability increases with increased chain unsaturation  and decreased acyl chain length (Papahadjopoulos et al., 1973b). 6  The liposomes used in this  'Neutral phospholipids Choline  0=P—o"  i C H j r C H — CH  -Headgroup  2  —  Glycerol backbone  A A o=c CH  CH  2  •«—CH CH-N H  CH  Phosphatidylserlne (PS)  Z  I I CH2  CH2  T  CH2  CH  CH  2  1CH CH  CH CH  OH  OH  2  CH2  II CH3 C H CC H H  3 2  2  H  H  OH  Saturated fatty acids  2  1 1 CHo  2  OH/  2  II CH? CH2 2  «—CH2CH(0H)CH 0H  Inositol Phosphatldyllnositol (PI)  CH2  2  3  CH2  II  2  3  COO"  Glycerol Phosphatidylglycerol (PG)  CH5 CH2  +  2  11  CH 1 CH CH CH  3  2  CH  2  +  Negative phospholipids Phosphatidic acid (PA) Serine  CHo  2  CHzCHjN^Ha  Ethanolamine  CHj  I  2  Phosphatidylcholine (PC)  CHj 2  CH CH N (CH )  Phosphatidylethanolamine (PE)  c=o  f l  •*  Acyl chain  Laurie (12:0)  CH (CH )i COOH  Myristic (14:0)  CH (CH2) COOH  Palmitic (16:0)  CH (CH2) COOH  Stearic (18:0)  CH (CH ) COOH  3  2  H  0  12  3  14  3  3  6H  2  16  Unsaturated fatty acids Palmitoleic (16:1 A^)  CHafCH^sCH^HfCH^TCOOH  Oleic (18:1 A9)  CH^CH^TCH^HfCH^TCOOH  Unoleic ( 1 8 : 2 A 9 , 1 2 )  CH (CH )4CH=CHCH CH=CH(CH ) COOH 3  Figure 1.3 Phospholipid structure, (reproduced from Parr, 1995)  7  2  2  2  7  thesis were prepared with disteroylphosphatidylcholine ( D S P C ) , a lipid known to exhibit improved drug retention properties.  Phospholipids are not limited to forming lipid bilayers, but display lipid polymorphism, whereby following hydration they form one o f a variety o f structures (Cullis et al., 1986). The specific structure a group o f phospholipids has an affinity to form is greatly influenced by the size o f its headgroup i n relation to its tail.  For instance, lipids with particularly large headgroups, such as  those used in emulsifying detergents, are prone to form micelles; whereas, unsaturated P E molecules aggregate into hexagonal structures (Cullis and de Kruijff, 1979).  Although the use  of non-bilayer forming lipids has been explored for the preparation o f liposomal drug carriers that can fuse with cell membranes, the formulations used here have been prepared with the bilayer-forming P C lipids and cholesterol  1.2.2  Cholesterol  Cholesterol, an amphipathic molecule composed o f a rigid steroid ring and aliphatic tail (Figure 1.4), is a major neutral lipid component in eukaryotic cell membranes.  In the lipid bilayer, its  steroid ring associates with the phospholipid acyl chains, while the hydroxyl group is oriented toward the lipid/water interface and positioned next to the carbonyl ester o f the neighbouring lipid headgroup, but there is no interaction.  Incorporation o f cholesterol into the membrane  decreases membrane order o f phospholipids in the gel phase while increasing order in the liquidcrystalline phase (de K r u y f f et al., 1973; Demel and De Kruyff, 1976).  A t amounts greater than  7 mol %, cholesterol decreases the enthalpy o f the gel to liquid-crystalline phase transition, until at 33 mol % it is no longer detected (Hubbell and M c C o n n e l l , 1971).  Cholesterol also  moderates lipid bilayer permeability, decreasing permeability o f membranes o f saturated or 8  o  Figure 1.4 Structure of cholesterol (A) and insertion into the lipid bilayer (B).  9  unsaturated lipids below the transition temperature and increasing permeability o f saturated lipids above the transition temperature (Bittman and Blau, 1972).  In the design o f lipid-based carriers for drug delivery, incorporation o f cholesterol into the liposome membrane  has  shown the  additional benefit  o f increasing liposome stability  (Papahadjopoulos et al., 1973a), primarily by decreasing lipid exchange with lipoproteins ( K i r b y et al., 1980; Hunt, 1982). Moreover, cholesterol levels in excess o f 30 mol % decrease serum protein binding (Patel et al., 1983; Senrple et a l , 1996), which increases the circulation lifespan of the liposome (Kirby et al., 1980; Patel et al., 1983) and slows release o f entrapped contents (Fielding and Abra, 1992).  It has recently been suggested (Dos Santos et al., 2002), however,  that under certain conditions where P C liposomes are stabilised through the use o f polyethylene glycol ( P E G ) , cholesterol may be unnecessary.  1.3 Preparation SUVs and L U V s  L i p i d hydration results in the spontaneous formation o f M L V s (Figure 1.1 A ) ranging from 1 to 10 um in diameter.  These vesicles are o f little clinical value as carriers, however because their  large size causes them to be rapidly eliminated from the circulation.  Moreover, the tight  packing o f their concentric lipid bilayers results in a low trapped volume, which limits the drug loading capacity for these carriers. M L V s are however, useful in making more practical lipid formulations, notably small and large unilamellar vesicles ( S U V s and L U V s , respectively) characterised not only by their size, but also by their single lipid bilayer.  Sonication or French press shearing (Barenholtz et al., 1979) o f M L V s results in uniformly sized S U V s (Figure 1.1C) with a small diameter (25-50 um) and low trapped volume (0.5-1.0 u L / u m o l 10  lipid).  Their small size bestows a high degree o f surface curvature, causing them to be prone to  fusion and particularly unstable when administered intravenously.  L U V s (50-500nm diameter) (Figure L I B ) are made by many procedures including detergent dialysis, infusion and reverse phase evaporation, but the most versatile and frequently used is high pressure extrusion (Szoka et al., 1980; Hope et al., 1985) with inert gas polycarbonate filters o f known pore sizes.  through  This has proven to be both a convenient and  reproducible method resulting in the formation o f vesicles resistant to fusion, having a large trapped volume 1.5-10 u L / u m o l lipid and being o f an ideal size for circulation and extravasation (see section 1.5).  1.4 Encapsulation  Drug loading into lipid vesicles can be accomplished through passive or active measures (Figure 1.5).  Passively encapsulated drugs are added during the lipid hydration process whereby the  liposomes are prepared in a solution containing the agent o f interest. This typically results in poor drug entrapment but varies with the individual compound.  For example, encapsulation  efficiency o f aqueous drugs vary with trapped volume and those o f lipophilic agents dependent on the capacity o f the bilayer to incorporate the agent while maintaining structure.  Passive  encapsulation is generally used to load hydrophobic drugs that have a propensity to partition into the lipid bilayer, or highly water-soluble drugs that can be retained in the aqueous core o f the structure.  Other agents, which may possess hydrophobic attributes,  as well as charged  hydrophilic moieties, may be actively loaded into preformed liposomes through the use o f transmembrane ion gradients (Cullis et al., 1983).  11  =3«  Passive entrapment Drug added to hydrated lipid mixture before extrusion.  A**%  Mf-  Drug is trapped in lipid bilayer <n or aqueous core when \_ liposomes are formed.  hydrophobic drug  hydrophilic drug  Active entrapment Hydrophilic drug added to preformed liposomes. no gradient  ionophore  pH7.5  Drug loaded into aqueous core under power of a proton gradient that may be maintained by an ionophore. 1  phospholipid ionophore hydrophobic hydrophilic  Figure 1.5 Active and passive drug encapsulation. 12  1.4.1  Passive encapsulation  Passive encapsulation is a method normally used with hydrophobic drugs such as amphotericin B (Madden et al., 1990b) and cyclosporin A (Ouyang et al., 1995) that partition into the lipid bilayer, and is generally  characterised  by  inefficient  loading.  For such agents trapping  efficiency varies with packing constraints o f the membrane and the lipid characteristics and while high entrapment efficiency can be achieved it is with a low drug to lipid ratio. Furthermore, hydrophobic drug partitioned into the liposome bilayer can rapidly exchange into other membranes and in vivo the drug quickly leaves the carrier (Choice et al., 1995).  It is important to highlight that passive trapping plays a role in preparing liposomes for active drug loading v i a transmembrane ion gradient-based trapping methods.  In this case liposomes  are extruded in the presence o f a specific buffer that is trapped in the aqueous core, the external buffer is then' exchanged in order to generate an ion gradient across the bilayer.  This ion  gradient can then be used to actively load selected drugs, such as irinotecan, as described in this thesis.  1.4.2  Active encapsulation  Active encapsulation refers to drug loading based on methods whereby the agent is added to preformed liposomes.  It is most commonly employed with agents containing protonizable  amine or carboxyl functions, and weak bases such as vincristine, and doxorubicin, can accumulate inside in response to a p H gradient (Mayer et a l , 1985; Madden et al., 1990a; Mayer et al., 1993).  A proton gradient generated across the lipid bilayer (acidic interior) aids  entrapment, as the neutral weak base is able to cross the bilayer whereupon it is protonated and 13  unable to permeate back across it (Addanki et al., 1968; Rottenberg, 1979).  Assuming that the  p K a o f the protonizable function is the same on both sides o f the bilayer, at equilibrium intravesicular and external drug concentrations can be derived from the Henderson-Hasselbach equation (Figure 1.6): [HA ] /[HA ] +  +  i n  = [H ] /[H ] +  o u t  +  in  out  Thus a p H difference o f 3 can result in drug accumulation up to a maximum gradient o f 10  3  higher inside the liposome compared with outside.  Active loading is generally the preferred mode o f drug entrapment as it offers several advantages over passive methods.  It is applicable to any lipid formulation capable o f maintaining an ion  gradient, and typically results in encapsulation efficiencies range o f 90-100 % .  So long as the  internal buffering capacity is maintained, drug trapping is independent o f the initial drug to lipid ratio, thereby maximizing the amount o f drug loaded into each vesicle.  Furthermore, the  solubility o f drugs o f intermediate solubility can be enhanced by manipulation o f the internal p H or the addition o f counter-ions for the drug to form molecular complexes with and be trapped internally.  Finally, because drug is trapped in the liposome core rather than the bilayer, the rate  o f drug loss can be reduced up to 30 fold (Mayer et a l , 1986a).  Conventional p H gradient-  based loading methods use an internal 300 m M citrate buffer o f p H 4.0; however, a more recent variation, which has shown success with agents previously unloadable by this method, uses a proton gradient, MnSCU and a divalent cation ionophore that maintains the  transmembrane  gradient (Fenske et al., 1998).  Generation o f the transmembrane p H gradient is straightforward and involves the exchange o f the external buffer.  This can be achieved by one o f several methods including size exclusion  chromatography, titration to a different p H , or dialysis. 14  The transmembrane p H gradient for  K  Outside  Inside  pH 7.5  pH4.0  = [B]„[H ] [BH ]  B  K  =  [B],[H], [BH ]  +  +  0  At equillibrium, if:  [B]„ = [B],  Then:  [BHl [BH ]  =  +  H-l) [H ] +  0  0  Figure 1 . 6 Active drug entrapment in liposomes. Equilibrium distribution o f a lipophilic amine (weak base) in response to a liposome transmembrane p H gradient. Only the neutral species is able to cross the lipid bilayer in significant amounts. (Reproduced from (Parr, 1995)  15  several PC-based liposome species are stable for hours or days even at elevated temperatures (Harrigan et al., 1992) and cholesterol further reinforces stability (Madden et al., 1990a).  1.5 Requirements for effective drug delivery  Following intravenous administration, liposomes are exposed to a variety o f cells, circulating proteins and other soluble factors resident in the circulating blood volume. Through interactions with the vesicle's lipid bilayer, these factors can instigate bilayer destabilization and/or trigger biological processes that increase membrane mononuclear phagocytic system (MPS).  permeability or liposome clearance via the  O n the most basic level, an effective carrier must  withstand these potential deleterious interactions in the blood compartment and be capable o f gaining access to an extravascular disease site; consequently, effective delivery o f a liposomeassociated drug is dictated by the rate o f carrier elimination from the plasma compartment; effective drug retention by the carrier en route to the target; and finally, adequate access to, and preferential uptake at the target site enabling direct delivery o f drug to diseased cells (Figure 1.7).  1.5.1  Liposome serum protein interactions and clearance  Although pharmacodynamic and pharmacokinetic behaviours o f liposomes are dictated primarily by lipid composition and size, they are significantly influenced by liposomal interactions in the blood compartment.  Intravenous  administration immediately exposes liposomes to cells,  lipoproteins and other circulating soluble factors including proteins, carbohydrates and small ions that can compromise drug delivery.  To maintain their therapeutic potential in this  menacing environment, liposomes must first avoid the bilayer- destabilizing effects o f circulating 16  Figure 1.7 Liposome target site accumulation. A, in order to effectively reach the target site, i.v. administered liposomes must be retained in the bloodstream for an extended period during which they encounter many different cell types and other soluble factors with which they may interact (B and C); D, passive carrier targeting exploits the characteristic altered leaky vasculature at the disease site which enables liposomes to extravasate into the interstitial space; liposome-associated drug may be released into the intercellular space (E) or liposomes may be phagocytosed by tumour-associated macrophages; G, surface-bound ligands can increase specificity o f liposome targeting.  17  lipoproteins (Williams et al., 1998).  Overcoming this hurdle, their fate is dictated primarily by  liposomal surface interactions with serum protein components, which can increase membrane permeability, or immune activation, both o f which flag liposomes for recognition and clearance by phagocytic cells o f the M P S .  High-density lipoproteins ( H D L s ) , such as A p o A - l , which inserts into the lipid bilayer (Klausner et al., 1985), are able to attack liposomes and "steal" phospholipids, disrupting membrane integrity and initiating liposome disintegration and premature release o f their contents into the circulation (Kirby et al., 1980).  Opsonizing plasma proteins may also interact with the  lipid bilayer, adsorbing to the liposome surface and tagging carriers for recognition phagocytosis by members o f the M P S (Aragnol and Leserman, 1986; A l v i n g et al., 1992; Chonn et al., 1992).  The M P S is a part o f the host defence system designed to help protect against foreign invaders and is a primary site o f liposome and associated drug clearance from the blood (Senior et al., 1985; Hwang, 1987; Woodle and D . D . , 1992; A l l e n et al., 1995).  In the absence o f a  compromised M P S , foreign particulates including bacteria and defined particles such as latex beads (Pretten and L l o y d , 1986) and conventional liposomes (Roerdink et al., 1981), are efficiently removed from the circulation by Kupffer cells in the liver and fixed macrophages in the spleen, lung and bone marrow, leaving little chance o f their accumulation in cells other than the phagocytic ones comprising the M P S .  Importantly, the rate o f liposome elimination from  the circulation can decrease through use o f formulations designed to minimise their interactions with the cells o f the M P S .  For example, large liposomes are removed more quickly than small  ones, surface charge can promote elimination o f liposomes, and this, in turn, affects the degree o f opsonin adsorption also play significant roles (Gregoriadis, 1988).  18  Complement proteins act as primary opsonizing proteins (Coleman, 1986; M o g h i m i and Patel, 1989), actively flagging objects for recognition and uptake by the M P S (Chonn et al., 1991; Chonn et al., 1992).  Proteins coupled to the surface o f liposome-based carriers are particularly  prone to attacks and can stimulate immune responses by attracting and interacting with complement proteins, resulting in activation o f the complement cascade (Devine et a l , 1994) and the consequent formation o f the membrane attack complex which has pore forming abilities (Silverman et al., 1984).  Although lipoprotein and opsonizing protein interactions with liposomes posed significant obstacles for development o f liposomal drug delivery systems, current understanding o f the chemical and physical attributes has provided numerous solutions to minimise liposome removal by the M P S and liposome destabilisation by serum protein incorporation.  Cholesterol  incorporation into the lipid bilayer, for example, enables liposomes to avoid the bilayer destabilizing effects o f lipoproteins (Kirby et a l , 1980), and inert hydrophobic polymers, such as polyethyleneglycol (PEG), at the liposome surface attracts a water shell to the surface, reducing interactions with circulating proteins (Lasic et al., 1991; Senior et a l , 1991; Torchilin et al., 1995), reducing both the rate and extent o f liposome uptake into the M P S .  1.5.2  Extravasation through blood vessels  The next hurdle for delivery o f lipid vesicles is escape from the confines o f the circulatory system and access to the disease site in an extravascular compartment.  This entails liposomes  crossing the vascular endothelium lining the blood vessels, composed o f endothelial cells, a basement membrane and associated smooth muscle cells, into the interstitial space (Figure 1.8).  19  Figure 1.8 Structure of a normal capillary. The capillary is formed from a continuous sheet o f endothelial cells, shown with a nucleus (Nu), that encircle the capillary lumen and are connected by tight junctions (TJ). The capillary is supported by a basement membrane (BM).  20  Liposomes extravasate into the interstitial space through gaps or fenestrae in the vascular endothelial lining (Figure 1.7).  In normal tissues, vascular endothelial cells are coupled by tight  junctions, which impede rapid and widespread distribution o f liposome carriers.  While  liposomes do not readily accumulate in healthy tissues, an altered vasculature observed in certain disease states enables increased liposome-associated drug levels over extended period o f time. Rapidly growing neovasculature and permeabilizing signals from disease cells during tumour growth (Richardson et al., 1979; Proffitt et a l , 1983; Papahadjopoulos, 1988), infection (BakkerWoudenberg et al., 1992) and inflammation (O'Sullivan et al., 1988) enables the filtration o f large molecules through fenestrae o f 200-500 nm and preferential liposome extravasation at the disease site into the interstitial compartment where the carriers can then act as local drug infusion reservoirs (passive targeting). absent lymphatic drainage  This selective accumulation is further promoted by impaired or causing slow diffusion through the  interstitial space.  Some  investigators have argued that additional liposome delivery specificity can be engineered by incorporation o f ligands, such as specific antibodies, that can target diseased cells directly (active targeting).  This has only been demonstrated successfully when the target cell population is  located in the blood compartment (Lopes de Menezes et al., 1998) or lung (Ahmad et al., 1993).  1.5.3  Liposome Accumulation in the Liver  Even i n the absence o f deliberate targeting, liposomes display an affinity for accumulation within the liver, often to such an extent that it can become a significant obstacle when attempting to target cells beyond this organ. manipulating  liposome  While uptake in the liver can be reduced to some extent by  size and  composition ( W u and  Zern,  1996),  underpinning  this  phenomenon are: (1) Kupffer cell activity; and (2) the distinctive nature o f hepatic circulation.  21  The liver is uniquely endowed with a dual blood supply from both the hepatic artery and the portal vein.  A s a result, the liver has an impressive blood flow (25 % o f cardiac output) and any  intravenously injected substance is assured to pass through this organ (reviewed in Gumucio et al., 1996).  This high throughput o f blood flow is reflective o f its functions in blood  detoxification and filtering which is mediated by Kupffer cells and fenestrae in the vascular endothelium.  The structure o f the liver is such that blood flows through sinusoids lined by  endothelial and Kupffer cells (Figure 1.9) (Gumucio et al., 1996).  This vascular endothelial  lining is discontinuous, enabling molecules and other substances < 100-150 nm to extravasate through fenestrae into the space o f Disse where they may have access to hepatocytes, or in theory, other non-native cell populations that may have established. Thus, together with the high volume o f blood, the leaky vasculature can act like a sieve, favouring extravasation and accumulation o f liposomes not taken up by Kupffer cells in the space o f Disse.  Given the natural tendency for liposomes to accumulate in the liver, it has been postulated that liposomal drugs targeting liver localised disease would be highly effective. this has not proven to be true.  T o date, however,  Although reports have demonstrated, using liver-localised cancer  cells, that liposomal anticancer drugs can be effective, these studies were completed using highly phagocytic cancer cell lines.  L i m and colleagues ( L i m et al., 2000) attempted to address the  issue associated with treatment o f liver-localised cancer and concluded that critical attributes governing the activity o f such liposomal drug formulations were the rate o f drug release as well as the regional distribution o f the carriers following localisation.  This thesis explores those  topics further, with a focus on a drug better suited for treatment o f liver cancer.  22  bile canaliculi  Figure 1.9 Anatomy of the liver sinusoid. Branches of the hepatic artery (HA) from the heart, and the portal vein (PV), from the gastrointestinal tract, supply the blood that percolates through hepatic sinusoids (S) to the central vein (CV). Endothelial cells lining the sinusoids are discontinuous enabling small molecules to escape into the space of Disse where they are in contact with hepatocytes. Hepatocytes are arranged in plates radiating out from the central vein and border bile canaliculi, which receive bile from surrounding hepatocytes and convey it to bile ducts. (Adapted from Paulsen, 1996)  23  1.5.4  Drug Release  Regardless o f liposomal drug dosage, only drug released from the liposome is considered bioavailable and able to mediate a therapeutic effect.  Drug release from circulating liposomes is  influenced simultaneously by: passive diffusion o f the drug across the lipid bilayer; disruption o f the liposomal membrane through interactions with circulating factors; and enzymatic degradation o f the lipid bilayer, causing premature release o f its contents.  Although the extent to which  enzymatic activity is involved in drug release is specific to the particular enzymes involved, the degree to which passive diffusion or membrane disruption affect drug release can be manipulated by altering the chemical composition o f the liposome, the site o f liposome administration, for example  intraperitoneal  administration  leads  to  slower  degradation  than  intravenous  administration (Hwang, 1987), and influenced by the tissues involved.  In optimised liposome-drug formulations, lipid-based carriers release their contents gradually, influenced primarily by passive diffusion, and the diseased cells take up the free drug.  There  does not appear to be significant uptake o f discrete lipid-dmg packages, however, liposomal drug can become associated with cells through direct liposome interactions with certain cell populations ( Szoka and Papahadjopoulos, 1980; Straubinger et al., 1983).  1.6 Liposomes as Drug Carriers  The significance o f liposomes as drug delivery vehicles is now well established.  Not only does  this apply to their ability to buffer the toxicity o f encapsulated drugs, but also to protect against degrading factors in the physiological environment as well as acting as a slow release drug reservoir at the disease site, often increasing efficacy o f the entrapped drug.  24  Liposomes have  shown promise as carriers in many areas including those for antifungal drugs (Madden et al., 1990b), antibacterials (Nacucchio et al., 1988; Bakker-Woudenberg et al., 1989; Cordeiro et al., 2000), antiparasitics (Madden et al., 1990b), and anticancer agents (Mayer et al., 1998).  Among  those lipid-based drug carrier-associated anticancer drugs approved for clinical therapy or in latestage clinical development are: liposomal vincristine (Inex Pharmaceuticals Inc.); liposomal doxil and doxorubicin (Johnson & Johnson); liposomal myocet (Elan); and daunorubicin (NeXstar Pharmaceuticals Inc.).  While liposome encapsulation can enhance delivery o f a drug,  in selecting a drug for encapsulation, it is critical to consider the target cell population and choose a drug that w i l l be therapeutically active in those cells.  For example, liver-localised  disease has only been poorly managed by drugs such as doxorubicin and vincristine, irrespective of whether they are administered as free or liposomal formulations ( L i m et al., 2000).  1.6.1  Selecting a drug for encapsulation  Camptothecin is a natural anti-neoplastic agent derived from the Asian Camptotheca  acuminata  plant (Figure 1.2), which exhibited a significant range o f anti-tumour activity against both ascitic and solid animal tumour models.  Despite the development o f a water-soluble sodium salt and  progression through phase I and phase II clinical trials, its further development as a therapeutic agent was hampered by low effectiveness and severe systemic toxicities.  Interest in camptothecin was renewed in the late 1980s motivated by the identification o f topoisomerase I as its cellular target (Hsiang and L i u , 1988), the identification o f its structureactivity relationship (Jaxel et al., 1989) which spurred the development o f water-soluble derivatives (Wall et al., 1993) with lower toxicities than the parent compound, and the finding  25  that in some tumour tissues, topoisomerase I levels are higher than in their normal tissue counterparts (Giovanella et al., 1989; van der Zee et al., 1991).  Camptothecins are cell cycle specific agents, targeting cells that are actively replicating D N A . Cytotoxicity o f all members is dependent on a closed lactone ring, which at physiological p H undergoes  rapid hydrolysis to an inactive carboxylate or ring-opened form (Figure  (reviewed in Burke and B o m , 2000).  1.2)  The poor water solubility o f camptothecins and their  inactivation at physiological p H have led to their classification as suitable candidates for encapsulation in a carrier that would facilitate intravenous administration, while providing protection from inactivating factors in the biological milieu.  Liposome structure bestows both a hydrophilic and hydrophobic compartment upon the carrier. The ability for both o f these compartments to facilitate stabilization o f the lactone form o f camptothecins was demonstrated in the early 1990s (Burke et al., 1993; Burke and Gao, 1994; Burke, 1996).  These studies suggested that camptothecins are capable o f associating with  membranes, and that bilayer-localized camptothecins preferentially partition as the active lactone form, thereby promoting and stabilizing the active lactone structure.  Some o f the newer  members o f the camptothecin family are water soluble, and when encapsulated w i l l associate to a large degree in the aqueous core which can be manipulated to a low p H , thereby also contributing to the stability o f the lactone ring (Burke and Gao, 1994; Tardi et a l , 2000).  1.7 Irinotecan  Irinotecan (Figure 1.10), a semisynthetic derivative o f the natural alkaloid camptothecan and the first of the water-soluble analogues, was introduced into the clinic in the late 1980s and now has 26  Figure 1.10 Irinotecan metabolism. Irinotecan can undergo activating or deactivating metabolism i n the liver. Carboxylesterases mediate activating hydrolysis to form the active drug, SN-38. SN-38 can be further metabolised to S N - 3 8 G by uridine diphosphate glucuronosyltransferases (UGTs). Both SN-38 and S N 3 8 G are significantly excreted in the bile and may undergo enterohepatic recirculation stimulated by intestinal P-glucuronidase activity. Irinotecan may also undergo detoxifying oxidation by members o f the cytochrome p450 3 A family o f hepatic enzymes resulting primarily i n an aminopentanocarboxylic metabolite ( A P C ) and an amino-piperidine derivative (NPC).  27  become a mainstay o f 2  n d  line treatment o f colorectal cancer, non-small cell lung cancer, brain  cancer and esophageal cancer.  1.7.1  Mechanism of Action  Camptothecins inhibit topoisomerase I activity, and are active primarily during the S phase o f the cell cycle ( L i et al., 1972; Liehr et al., 1996). Topoisomerase I is critical for cell growth and proliferation as it makes a transient single-stranded break in the D N A duplex, which allows unwinding o f the supercoiled D N A , enabling the replication fork to proceed in duplicating the DNA.  After topoisomerase-mediated D N A cleavage, camptothecins bind to and stabilize the  topoisomerase  I-DNA  cleavable  complex, thereby  preventing  religation  o f the D N A .  Subsequent accumulation o f cleavable complexes ( Hsiang et al., 1988; Hsiang et a l , 1989) inside the cell, arrests D N A replication and causes double-stranded D N A breaks, which lead to eventual cell apoptosis'(Zhang et al., 1990). consequently  a function o f topoisomerase  The magnitude o f camptothecin cytotoxicity is I expression and D N A replication in a cell.  Topoisomerase I is overexpressed in several tumour types including breast, lung and colorectal tumours (Potmesil, 1994).  Although it is expressed throughout the cell cycle, cells in the S-  phase are 1000 times more sensitive to camptothecins than they are during the G phases (Del Bino et al., 1991).  1.7.2 Activation/Metabolism  Irinotecan is a prodrug o f limited activity, but which after conversion to its active form, SN-38 (Figure 1.10) by carboxylesterase-mediated de-esterfication in the plasma and the liver, is 1000 times more potent than the parent compound (Kawato et al., 1991). 28  SN-38 can undergo further  conjugation by hepatic undine diphosphate glucuronosyltransferases ( U G T s ) to S N - 3 8 G , which although inactive, has been found to be the predominant plasma form o f SN-38 in vivo (Rivory and Robert, 1995a; Lokiec et al., 1996; Rivory et al., 1997;).  SN-38 and S N - 3 8 G are  significantly excreted in the bile, and can undergo enterohepatic recirculation with SN-38 being reconstituted by intestinal p-glucuronidase (Figure 1.10) (Takasuna et al., 1995).  Irinotecan can  also undergo degrading oxidation reactions mediated by the cytochrome p450 3 A (Haaz et al., 1998a; Haaz et al., 1998b) family o f hepatic enzymes to form the inactive metabolites, 7-ethyl10-[4-N-(5-aminopentanoic acid)-l-piperidino] carbonyloxycamptothecin ( A P C ) (Rivory et al., 1996; R i v o r y et al., 1997; Sparreboom et al., 1998) and 7-ethyl-10-(4-amino-l-piperidino) carbonyloxycamptothecine ( N P C ) , the primary amino-piperidine metabolite (Dodds et a l , 1998). Irinotecan and its metabolites are primarily excreted in feces, with irinotecan as the major excretion product (Slatter et al., 2000).  1.7.3  Cellular mechanisms of resistance  Cellular resistance to chemotherapeutic  agents can pose a major complication in patient  treatment, even so far as resulting in treatment failure.  A common mechanism responsible for  such resistance is the reduction o f the intracellular concentration o f the drug via efflux mediated by pumps o f the multi-drug resistance family such as P-glycoprotein.  Camptothecins do not  appear to be good substrates for P-glycoprotein which is commonly overexpressed in colorectal adenocarcinoma (reviewed in Rivory and Robert, 1995b; Jung and Zamboni, 2001), although both irinotecan and SN-38 are transported by the multidrug resistance protein (Chen et al., 1999) and there is further evidence for their efflux by a yet unidentified anionic pump (reviewed in (Jung and Zamboni, 2001).  29  Modulation o f topoisomerase I activity and expression may also mediate drug resistance. Irinotecan cytotoxicity has been linked to cellular levels o f topoisomerase I activity (Jansen et al., 1997), thus reductions in topoisomerase I protein levels can directly affect activity.  Also,  alterations in topoisomerase I m R N A levels or mutations in the gene, causing reduced expression or activity or engendering camptothecin resistance (reviewed in R i v o r y and Robert, 1995b).  1.8 Advanced Colorectal Cancer A n d Liver Metastases  Colorectal cancer ( C R C ) is the second leading cause o f cancer mortality in North America, and the most frequent cause o f cancer deaths not provoked by a chemical carcinogen (ie. nicotine and lung cancer).  Approximately 17,600 Canadians w i l l be diagnosed with colorectal cancer in  2002 and 6,600 w i l l succumb to the disease (National Cancer Institute o f Canada: Canadian Cancer Statistics 2002, Toronto, Canada, 2002).  If left untreated, only 30% o f the patients w i l l  survive 3 years and few w i l l survive 5 years beyond the detection o f their liver metastasis (Bengmark and Hafstrom, 1969; Adson et al., 1984).  The liver is the primary site o f blood borne metastases from cancers o f several different origins, including carcinomas o f the lung and breast, but colorectal adenocarcinomas, which drain into the portal circulation, are particularly prone to spread to the liver.  In contrast to gastric and  pancreatic neoplastic spread to the liver, which also reveals spread to other organs, the liver is often the sole site o f metastases for C R C .  Hepatic metastases are normally asymptomatic until  quite advanced and are detected either at the time o f primary cancer diagnosis or during a routine follow-up (Choti and Bulkley, 1999). A s many as 70% o f patients with colorectal cancer w i l l present with (synchronous) liver metastases at the time o f their primary diagnosis or develop liver  metastases  (metachronous)  as  their  disease 30  progresses  (Kemeny  et  al.,  1980).  Complications that arise due to neoplasia in the liver include erythrocytosis, hypoglycemia and eventual liver failure.  1.8.1  Current treatment strategies  Neoplasia in the liver are challenging cancers to manage, and despite the development o f novel treatment strategies such as intrahepatic arterial chemotherapy and thermal ablation, surgical resection remains the treatment o f choice for appropriately selected patients.  Systemic  chemotherapy is the most common treatment for patients with colorectal hepatic metastases.  1.8.2.1 Systemic chemotherapy  Although systemic chemotherapy is the predominant modality o f care for hepatic metastases, it is currently palliative in intent, and at best may induce a transient reduction in tumour size, temporarily increasing the patient's  quality o f life.  Since the  1960s  chemotherapeutic  intervention o f liver metastases has rested almost exclusively on 5-fluorouracil-based treatment regimes, which yield responses in the range o f 20-30%. patients develop resistance to these cytotoxic agents.  Moreover, most o f these responsive  Irinotecan was recently introduced as a  novel agent for the treatment o f advanced colorectal cancer and initially approved for use as a second line treatment for refractory disease.  Its remarkable therapeutic ability, eliciting  responses in patients resistant to the conventional treatments, lead to its rapid promotion to a first line treatment for advanced C R C administered in conjunction with the conventional therapy o f 5fluorouricil and leucovorin.  31  1.8.2.2 Liver resection  Surgical resection is the only known potentially curative treatment for liver metastases. However strict qualification criteria including a maximum o f three neoplasia in a single lobule restrict this treatment to only 10% o f the patient population, and still overall 5-year survival rates o f only 25%-40% are obtained (Scheele et a l , 1991; Kemeny et al., 1993).  1.9 Specific Aims  The aims o f this thesis were to: 1) characterise the DSPC/cholesterol liposomal irinotecan formulation 2) evaluate the potential o f liposomal irinotecan to treat liver metastases in a xenograft model o f advanced human colorectal cancer.  1.10  Hypothesis  It is argued that liposomal drug formulations should be more effective at treating liverlocalised carcinomas, including liver metastases when compared with their free drug counterparts, because of their intrinsic affinity for the liver and extended lifespan.  Although colorectal cancer is the second leading cause o f cancer mortality in North America, primarily because o f a high incidence o f hepatic metastases, the development o f new treatment options for these patients is relatively unsuccessful compared to that for several other cancers. Liposomal drugs are currently establishing themselves as powerful chemotherapeutic agents, as liposomes are able to alter the pharmacokinetics o f traditional agents, converting marginally 32  effective or severely toxic drugs into potentially useful agents simply through improvements in drug delivery.  Furthermore, liposomes' natural tendency to accumulate in the liver and  evidence for increased therapeutic efficacy for liposomal drug formulations in treating liver disease, suggests that encapsulation o f the appropriate  agent(s) w i l l provide an effective  alternative treatment for patients with advanced colorectal cancer. The camptothecin family o f anti-neoplastic agents is one group that have been proposed as suitable candidates encapsulation, primarily due to the importance o f their lactone ring, which  for  undergoes  inactivating hydrolysis at physiological p H , but which could be stabilized i f effectively encapsulated.  Recent advancement o f the camptothecin analogue irinotecan through clinical  trials into the clinic where it continues to perform admirably and has quickly been promoted to first line treatment for advanced colorectal cancer in conjunction with 5-fluorouricil/leucovorin, suggests that it may have further potential which may be exploited by encapsulation.  The results presented in this thesis lend support to the thesis statement.  First this work  documents that irinotecan is particularly well suited for liposome encapsulation.  The results  illustrate that under an ionophore-mediated proton gradient, irinotecan can be efficiently trapped even at relatively high drug to lipid ratios. Using improvements in pharmacokinetic properties as a measure o f an effective liposome-drug combination, it is demonstrated that DSPC/cholesterol encapsulation o f irinotecan significantly improves circulation longevity o f the drug as compared to the free agent, which to a large degree is maintained as the active lactone species. Next, the question o f a possible role for liposomal drug in the treatment o f liver cancer was investigated. Tumour size and growth rate and/or increase in lifespan were used as indicators in efficacy studies o f liposomal and free irinotecan conducted in the L S I 80 solid tumour model o f colorectal metastasis, and the L S 1 7 4 T orthotopic model o f human colorectal liver metastasis.  33  It is  concluded that DSPC/cholesterol encapsulation o f irinotecan offers a significant advantage in the treatment o f liver-localised metastases.  {  34  CHAPTER 2  M A T E R I A L S AND METHODS  2.1 Materials  Irinotecan hydrochloride (Camptosar®, Pharmacia) was purchased from the British Columbia Cancer Agency Pharmacy.  1,2 Disteroyl-s«-glycero-3-phosphocholine ( D S P C ) was purchased  from Avanti Polar Lipids (Alabaster, A L ) .  Cholesterol, the divalent cation ionophore A23187,  N-2-hydroxyethylpiperazine-N-2ethane-sulphonic acid ( H E P E S ) , and sephadex G-50 (medium) and were obtained  from the  Sigma Chemical  Company (St. Louis, M O ) .  Tritiated  [ H]cholesteryl hexadecyl ether ( H - C H E ) ( N E N , Boston, M A ) was used as a liposome marker, 3  3  and [ C ] methylamine hydrochloride, used in determining the p H gradient, was obtained from 14  Amersham  Biosciences  Corp.  (Baie  d'Urfe,  PQ).  Dil,  (1,1 '-dioctadecyl-3,3,3',3'-  tetramethylindocarbocyanine perchlorate) used as a fluorescent lipid probe, was purchased from Molecular Probes (distributed by Cederlane Laboratories Limited, Hornby, O N ) . chemicals used in this study were analytical or H P L C grade.  A l l other  The L S I 8 0 and L S 1 7 4 T tumour  cell lines were originally purchased from the A T C C (Manassas, V A ) and were maintained in tissue culture. twenty.  Cells were used for experiments when they were between passages three and  After the twentieth passage the cells were discarded and the new cell lines were  reverted back to frozen stock.  Male S C I D / R A G - 2 M mice (8-10 weeks old) were bred at the  British Columbia Cancer Agency Animal Breeding Facility.  35  2.2 Preparation of liposomes  Large unilamellar vesicles consisting o f l,2-disteroyl-3-^-phosphatidylcholine ( D S P C ) and cholesterol (55:45 mole percent) were prepared using well-established extrusion technology as previously described by Hope et al. (1985).  When D i l was used as a fluorescent lipid marker,  the D S P C , cholesterol, D i l ratio was 54.5:45:0.5 mole percent. Briefly, the indicated lipids were dissolved in chloroform in the presence o f H - C H E and dried to' a homogenous lipid film under 3  nitrogen gas, which was further chloroform.  dried under vacuum overnight to remove  any residual  The dried lipid films were subsequently hydrated in 300 m M manganese sulfate,  p H 3.4, to a final lipid concentration o f 50 or 100 mg/ml.  The resulting multilamelar vesicle  mixture was freeze-thawed five times in liquid nitrogen (Mayer et al., 1986b), and size-reduced using high-pressure extrusion through two-stacked polycarbonate filters, one each o f 100 nm and 80 nm pore size (Whatman Inc., Newton, M A ) using an extruder (Lipex Biomembranes Inc., Vancouver, B C ) .  The vesicle size o f the resulting large unilamellar vesicles ( L U V s ) was  typically in the range o f 115 ± 15 nm based on quasi-elastic light scattering (Nicomp Particle Sizer M o d e l 270, Santa Barbara, C A ) .  The external buffer was exchanged by running the  sample down a Sephadex G-50 column equilibrated in 300 m M sucrose, 20 m M H E P E S and 15 m M E D T A ( S H E buffer) at p H 7.4.  2.3 M n S O . p H gradient loading of irinotecan  Irinotecan was loaded into the liposomes by way o f an ionophore-mediated proton gradient (Fenske et al., 1998). buffer (pH 7.4).  Drug uptake was performed at 0.3 mg irinotecan per 1 mg lipid in S H E  The divalent cation ionophore A23187 (0.5 ug per 1 mg lipid) was first added  to the liposomes and the mixture was incubated at 37°C or 60°C for 10 minutes to enable 36  ionophore incorporation into the lipid bilayer.  Subsequently, irinotecan was added and the  mixture was incubated at 37°C or 60°C for 1 h.  During this process the mixture was protected  from the light, as irinotecan has been shown to undergo photodegradation exposure to light (Dodds et al., 1997).  upon extended  Encapsulation efficiency was determined at several  timepoints over a 2-hour period at both 37°C and 60°C. The extent o f irinotecan encapsulation was assessed by running an aliquot o f the mixture down a Sephadex G-50 mini spin column (Madden et al., 1990a) to remove unencapsulated irinotecan and lipid concentrations in the eluent.  drug and subsequent measurement o f  Irinotecan concentration was quantified at its  peak absorbance o f 370 nm using a Beckman DU®-64 Spectrophotometer (Beckman Coulter Canada Inc., Mississauga, O N ) . Radioactivity was assessed by counting a mixture o f the sample and 5 m l o f Pico-Fluor-15 (Packard, Meriden, C T ) scintillation cocktail on a Packard 1900 scintillation counter (Packard, Meriden, C T ) . The efficiency o f irinotecan loading was typically between 90 and 100%.  In preparation for intravenous administration, the A23187 ionophore  was removed from the liposomal drug preparation and the external buffer exchanged to 0.9 % saline through manual tangential flow as per instructions provided by the column (400 k D cutoff) supplier (Spectrum Laboratories Inc., Rancho Dominguez, C A ) , which was also employed to reduce volume to achieve an appropriate concentration, based on the desired drug dose, for i.v. administration.  2.4 In vitro characteristics of liposomal irinotecan  For drug release studies, drug loaded liposomes were prepared as described in sections 2.2 and 2.3.  These were then transferred to Fisherbrand dialysis tubing (10 m m wide, 12,000-14,000  molecular weight cut off, Fischer Scientific, Nepean, O N ) and the samples were dialysed against 4 litres o f S H E buffer for 72 hours at 37°C.  A t the indicated time points, 50 ul aliquots, in 37  triplicate, were removed from the dialysis bag and analysed for relative encapsulated drug and lipid concentrations as previously described (section 2.3).  To assess the effect o f the  transmembrane gradient on drug uptake and vice versa, the magnitude o f the proton gradient was measured following a methylamine-based procedure described by (Harrigan et al., 1992), with modifications.  Briefly, drug loaded liposomes were prepared as described in sections 2.2 and  2.3 to a total volume o f 500 p i .  After the loading incubation, 50 ul aliquots from each sample  were assayed for loading efficiency, and samples were cooled to room temperature.  An  appropriate amount o f C-methylamine (not to exceed a H / C ratio o f 1/10) was then added to 14  3  1 4  each sample and allowed to incubate for 1 h at room temperature at which time radioactivity was measured, using a dual label analysis by the Packard instrumentation, in 100 ul aliquots from the samples and the total eluents o f 100 ul aliquots, in triplicate, run down mini spin columns. A series o f calculations based on these measurements and the assumption that the liposomes used have a trapped volume o f 1.2 ul/umole lipid led to the determination o f the  transmembrane  proton gradients.  2.5 Pharmacokinetic studies in S C I D / R A G 2 M mice  Free irinotecan (50 mg/kg) and liposomal irinotecan (167 mg/kg lipid, 50 mg/kg irinotecan) were injected via the lateral tail vein into male S C I D / R A G - 2 M and the plasma elimination o f both the lipid carrier and the drug were determined over a 24-h time course.  Animals were terminated by  C O 2 asphyxiation at the indicated time points, and blood was retrieved v i a cardiac puncture and collected into E D T A coated tubes (Microtainers, Becton Dickinson). B l o o d plasma was isolated from hematocrit following centrifugation o f the whole blood at 2500 x g for 10 minutes. 25 p i aliquots o f plasma were combined with 5 ml Pico-Fluor 15, and scintillation counting o f the  38  marker H - C H E (Stein et al, 3  1980) was used to quantify the liposomal carrier in the plasma.  Total irinotecan and relative circulating lactone and carboxylate species concentrations were determined using H P L C analysis as described by (Chollet et al., 1998), with modifications (please refer to the Appendix for details).  Briefly, irinotecan was extracted from 100 ul o f  plasma by diluting the sample in ice-cold methanol to precipitate the proteins and solubilise the liposomes.  The samples were analysed immediately.  Standard curves for the two species o f  drug were generated by dissolving the drug in either D M S O for the lactone species or 50% acetonitrile: 50% 20 m M borate buffer (pH 9) for the carboxylate species, (although it was observed that this technique resulted in an incomplete conversion to the carboxylate form, resulting in an over- estimation of the carboxylate species).  Separation was achieved using a  Symmetry® Q s cartridge column (100 A , particle size 5 um; 250 x 4.6 m m I.D., Waters) with a Symmetry® Sentry™ C\% guard column (particle size 5 um; 20 x 3.9 I.D., Waters) with a runtime of 20 minutes at a flow rate o f 1.5 ml/min, and maintained at 35°C.  A two-solvent mobile phase  consisted  acetate,  of  mobile  phase  A  (75  mM  ammonioum  7.5  mM  tetra-  butylammoniumbromide, p H 6.4 adjusted with glacial acetic acid) and mobile phase B (100 % acetonitrile), with the elution gradient containing a mixture o f 76 % A : 24 % B .  For sample  analysis, 80 u l o f each sample and standard were loaded into 1 m l H P L C sample vials (Waters, Milford, M A ) with 200 ul inserts (Chromatographic Specialties Inc., Brockville, O N ) and 10 ul was injected into the column. Irinotecan was quantified using an H P L C system equipped with a Waters M o d e l 717 plus autosampler (set to 4°C), a M o d e l 600E pump and controller and a M o d e l 470 Scanning Fluorescence Detector (Waters, Milford, wavelength o f 362 nm and an emission wavelength o f 425 nm.  M A ) set at an excitation Data were acquired and  processed with the Millenium32® chromatography manager (Version 3.20, Waters, Milford, MA).  39  2.6 Establishing a maximum tolerated drug dose  Limited dose ranging studies were used in an effort to determine the maximum tolerated dose (MTD).  Male S C I D / R A G - 2 M mice in groups o f two were given free or encapsulated drug by  single or multiple (3 doses, each 4 days apart) i.v. injections into the lateral tail vein.  Animals  were monitored for weight loss and other signs o f stress/toxicity for a period o f 30 days. Individual animals that lost more than 20 % o f their original weight or those appearing severely stressed, as judged by appearance and/or behaviour and assessed by qualified animal care technicians, were terminated.  After 30 days, all remaining animals were terminated by C O 2  asphyxiation and necroscopies were conducted to identify any additional drug toxicities. Toxicity studies aimed at determining the exact L D . o dose o f a drug formulation are not sanctioned by the Canadian Council on A n i m a l Care nor the institutional A n i m a l Care Committee.  2.7 Liposomal irinotecan anti-tumour efficacy using the human LS180 solid tumour model  M a l e S C I D / R A G - 2 M mice (20-30 g, 6 per group) were inoculated with 1 x 10 subcutaneously on the right posterior dorsal side. a size accurately measurable  6  cells  Once the tumour had established and reached  with callipers (tumour volume > 0.04 cm ) (day 11 post  inoculation), free or encapsulated irinotecan dosing schedules o f single or multiple (day 11, 15, 19) injections o f 50 or 100 mg/kg irinotecan via the lateral tail vein were initiated.  Animals  were monitored daily for any signs o f stress or toxicity and were terminated when weight loss exceeded 25% or when animals displayed increasing health deterioration including lethargy, scruffy coats, or dehydration.  Survival time for terminated animals was recorded as the 40  following day.  Animals were monitored for sixty days or until all animals had been terminated.  Drug mediated off-set o f tumour growth was estimated by extrapolation o f a line tangential to rapid tumour growth down to the x axis, which indicated a time in days post inoculation.  2.8 Liposomal irinotecan anti-tumour efficacy using the human LS174T orthotopic tumour model  Male S C L D / R A G - 2 M mice (20-30 g, 6 per group) were inoculated intrasplenically with 5 x 10  6  LS174T cells. Following inoculation, these cells travel via the portal vein to the liver were they seed and establish widespread tumour growth.  O n day 7 post cell inoculation, animals began  dosing schedules o f single or multiple injections (day 7, 11, 15) o f 50 mg/kg free or liposomal irinotecan administered via the lateral tail vein.  Animals were monitored daily and terminated  upon signs o f severe weight loss (> 25%) and/or health deterioration as demonstrated lethargy, scruffy coats or dehydration.  by  Survival time for terminated animals was recorded as the  following day.  2.9 Liposome mediated drug delivery to LS174T liver metastases  To illustrate liposome mediated drug delivery to tumours in the liver, male S C I D / R A G - 2 M mice (20-30 g, 2 per group) were inoculated intrasplenically as described in the previous section. Once the tumours were well established in the liver (day 19 post inoculation), animals were given  a single  lipid  dose o f  167  mg/kg, o f irinotecan  (50  mg/kg)  or  mock-loaded  DSPC/Cholesterol/Dil (54.5:45:0.5, mole percent) liposomes injected v i a the lateral tail vein. 24 hours later animals were terminated by C O 2 asphyxiation, whole blood was collected via cardiac puncture as outlined in pharmacokinetic studies, and a portion o f a lobe o f the liver was 41  harvested for sectioning and histochemistry. visualisation using fluorescent microscopy.  D i l inclusion in the liposomes enabled their  L i v e r sections taken from untreated animals (n=2)  were H & E stained to reveal tumour-associated cells.  2.10  Statistical analysis  One-way A N O V A (analysis o f variance) was performed on the tumour volume results obtained after the administration o f free or liposomal irinotecan.  C o m m o n time points were compared  using the post hoc comparison o f means, Scheffe test.  Survival data was analysed using a  multiple-sample test in the Survival Analysis module o f Statistica™ (Statsoft Inc, Tulsa, O K ) . This test is an extension o f Gehan's generalized W i l c o x o n test, Peto and Peto's generalized W i l c o x o n test, and the log-rank test. Differences were considered significant at p < 0.05.  42  CHAPTER 3  L I P O S O M A L I R I N O T E C A N A N D ITS T H E R A P E U T I C A C T I V I T Y A G A I N S T COLORECTAL METASTASES  3.1 In vitro Irinotecan uptake and release characteristics  Studies illustrating the in vitro irinotecan loading in liposomes composed o f D S P C  and  cholesterol at 37°C or 60°C and the importance o f the A23187 ionophore are presented in Figure 3.1.  A t 37°C, less than 30% o f the drug was encapsulated over 2 hours, i n contrast to 100%  encapsulation at 60°C, where the time required to achieve maximum uptake was 20 minutes.  In  the absence o f the A23187 ionophore, drug accumulation in the liposomes was less than 20%, irrespective o f the incubation temperature.  Moreover, in the absence o f the ionophore the  transmembrane p H gradient was reduced to approximately 1, emphasizing the ionophore's significant role in maintaining the proton gradient, as well as the dependence o f efficient drug encapsulation on this transmembrane gradient.  The extent o f irinotecan loading was influenced  by the starting drug to lipid ratio (Figure 3.2A).  Irinotecan encapsulation increased with  increasing drug to lipid ratios, but above 0.3:1 (wt:wt) the gain in loading attributable to an increased initial drug to lipid ratio was reduced, and the efficiency o f drug encapsulation was also decreased.  A s illustrated in Figure 3.3, greater than 95% loading could routinely be  achieved when the starting drug to lipid ratio was 0.3:1 (wt:wt).  If the same experiment was  completed at a 0.5:1 (wt:wt) starting drug to lipid ratio, greater drug loading was achieved (final drug to lipid ratio o f 0.35:1 (wt:wt), but the efficiency was less than 80%.  The reduction in  loading efficiency at higher drug to lipid ratios is correlated with degradation o f p H gradient (Figure 3.2B).  This liposomal irinotecan formulation (prepared at 0.3:1 (wt:wt) drug to lipid 43  20  i 0  0.12  1  0.10  40  60 80 time (minutes)  100  120  B  C  ^ o  0.08 0.06 0.04 0.02  J  Q  0.00 20  40  60 80 time (minutes)  100  120  Figure 3.1 Effect of the A23187 ionophore in p H gradient-mediated loading of irinotecan into DSPC/cholesterol liposomes at 60°C (A) or 37°C (B). Loading was evaluated at two temperatures in the presence ( O ) or absence ( • ) of the ionophore at a drug to lipid ratio of 0.1:1 (wt:wt). Drug encapsulation was determined by the mini spin column-based procedure described in section 2.3.  44  0.40 0.35 _X_  5 0.30 -I °  _J_  0.25  TO X)  •Q.  0.20  _x_  g> 0.15  1  0.10 0.05 0.00 0.0  0.1  0.2 0.3 0.4 initial drug to lipid ratio (wt:wt)  3 -,  0.5  B  0) "g 'co  o '•a 'o  -2c CD T3  ro  X Q.  01 C  ro i E CP  E CO  c ro  0.0  0.1  0.2 0.3 drug to lipid ratio (wt:wt)  0.4  0.5  Figure 3.2 The effect of drug to lipid ratio on irinotecan loading (A) and the transmembrane p H gradient (B). A, DSPC/cholesterol liposomes were incubated at 60°C for 1 hour at different drug to lipid ratios, and the extent o f irinotecan encapsulation was determined (see section 2.3) and is expressed as the average final drug to lipid ratio + s.d.. B, the transmembrane p H gradient in samples with different drug to lipid ratios was determined, by a C-methylamine procedure described in section 2.4, following 1 hour irinotecan incubation with the liposomes. 14  45  Figure 3.3 Efficiency of irinotecan encapsulation at different drug to lipid ratios. Efficiency o f irinotecan loading into DSPC/cholesterol liposomes at 60°C was evaluated for initial drug to lipid ratios o f 0.3:1(* ) and 0.5:1 (O ) (wt:wt). Drug encapsulation was determined as per the procedure described in section 2.3. Aliquots were taken in triplicate over a 2 hour time course, and results are expressed as the average + s.d..  46  for up to 72 hours (Figure 3.4) with less than 2% o f the trapped irinotecan released, as measured by dialysis o f the loaded liposomes against a large volume (4 litres) o f S H E buffer.  3.2  In vivo plasma elimination of free and liposomal irinotecan  Pharmacokinetic studies investigated plasma elimination rates o f irinotecan and the liposomal carrier. M i c e were given an i.v. injection o f liposomal irinotecan (167 mg/kg lipid; 50 mg/kg drug); free irinotecan (50 mg/kg) or "empty" liposomes (167 mg/kg).  DSPC/cholesterol  liposomes exhibited an extended circulation lifespan (Figure 3.5A), with similar elimination rates for irinotecan- (filled  symbols)  and mock-loaded  (open  symbols)  liposomes  and  approximately 40% o f the injected lipid dose could still be found in the plasma compartment 24 h after i.v. injection.  Free irinotecan was eliminated rapidly (half-life o f less than 15 minutes)  from the plasma compartment (Figure 3.5B), while liposome-associated drug was maintained at high levels for up to 24 h, at which time circulating liposomal irinotecan was still 10 fold higher than the concentration o f free drug determined at 5 min following injection.  The relative  amount o f irinotecan retained in the circulating liposomes, and conversely the rate o f drug release, can be estimated using the data shown in Figure 3.5 A and B to define the irinotecan to lipid ratio (drug to lipid, wt:wt) at each o f the measured points (Figure 3.5C).  A caveat o f this  estimation is that it assumes the level o f free drug, in the plasma o f animals given liposomal irinotecan, is negligible. These data illustrate that DSPC/cholesterol liposomes provide gradual and sustained irinotecan release during the study's 24-hour timecourse, with 75% o f the irinotecan originally associated with the liposomes released within 24 hours.  A s indicated in sections 1.2 and 1.6.1, irinotecan undergoes reversible hydrolysis between a closed-ring lactone species and an open-ring carboxylate (Figure 1.2). 47  The extent and rate o f  100  —  q ro i_  •g  '.Or  80 - I .  60  3  40  ro o  20  12  24  36  48  —I—  60  72  time (hours)  Figure 3.4 Release of irinotecan from DSPC/cholesterol liposomes incubated at 37°C for 72 hours. Irinotecan-loaded liposomes (0.3:1 drug to lipid ratio) were dialized against 4 L o f S H E buffer (pH 7.4) over a 72 h period. A t the indicated timepoints, 50 ul aliquots, in triplicate, were removed from the dialysis bag and run down mini spin columns, as described in sections 2.3 and 2.4, to determine the amount o f drug still associated with lipsomes. Results are expressed as the average ± s.d..  48  E CO CD  0.00  1  '  0  5  '  ' 10  15  ' 20  ' 25  Time (hours)  Figure 3.5 Plasma elimination of free and liposomal irinotecan. v4,mice (n=6) were injected with lipsomal irinotecan ( • ) or mock-loaded liposomes (O ) at a lipid dose o f 167 mg/kg and a drug dose o f 50 mg/kg. A t the indicated times plasma was collected and the circulating lipid concentration was determined as described in section 2.6. B, plasma levels o f circulating liposomal irinotecan ( • ) or free drug. ( O ) were determined at various time points after i.v. injection (50 mg/kg free drug). C, irinotecan release rate from circulating liposomes was calculated from data presented in A and B. A l l data points represent the mean values obtained from 6 mice per time point and the error bars represent the standard deviation.  49  hydrolysis is p H dependent, with the carboxylate favoured at physiological p H ( M i and Burke, 1994).  Because only the lactone species is active, the relative concentrations o f both species are  significant from a therapeutic standpoint; hence, the relative proportions o f circulating lactone and carboxylate irinotecan species were quantified.  Five minutes after injection o f the free drug,  - 3 0 % o f the measured irinotecan was identified as the carboxylate form (Table 3.1), and this value increased to almost 60% at the 1 hr time point.  In contrast, liposome encapsulation  protected the lactone species, which up to 4 h after injection represented > 95% o f circulating irinotecan, and despite a decrease in relative plasma concentrations by 24 h postinfection to - 8 0 % lactone, the total drug concentration in the plasma remained 10 fold greater than the free drug concentration after only 5 min.  Table 3.1 Analysis of plasma irinotecan following i.v. administration of free or liposomal drug. Time Free irinotecan 5 min 15 m i n 1 h Liposomal irinotecan 1h 4h 24 h  Plasma concentration (ug/ml) Total Lactone Carboxylate*  Irinotecan (% o f total) Carboxylate* Lactone  8.7 5.4 3.8  3.3 4.4 5.5  12.0 9.8 9.3  73 55 41  27 45 59  1236.7 838.9 79.8  27.0 25.5 18.8  1263.7 864.4 98.6  98 97 81  2 3 19  * This is an approximation derived from standards at pH 9 to drive irinotecan towards the carboxylate species.  After establishing that liposome encapsulation o f irinotecan increases not only circulating total drug levels, but also offers significant protection in maintaining irinotecan as the active lactone species, the therapeutic benefits o f the formulations were assessed in two advanced colorectal tumour models.  50  3.3 Acute toxicity of free and liposomal irinotecan  A s the Canadian Council o f A n i m a l Care does not authorize formal L D i o and L D  5 u  studies, toxic  dose range finding studies in tumour free male S C I D / R A G - 2 M were conducted using only 2 mice per dose.  Unfortunately due to the nature o f commercially available irinotecan, (which is  supplied, prepared for injection, at 20 mg/ml dissolved with 45 mg/ml sorbitol, 0.9 mg/ml o f lactic acid and p H adjusted to 3.5) these limited dose escalation studies were inconclusive. They did, however, reveal an acute dose-limiting toxicity at the 100 mg/kg dose.  Because o f its rapid  onset (within minutes o f injection) it can be suggested that this toxicity may be related to drug formulation issues.  This toxicity appeared to be slightly reduced by dilution o f the drug in 5%  dextrose rather than 0.9% saline solution. A n evaluation o f drug-induced weight loss during the dose escalation studies, suggested DSPC/cholesterol irinotecan was as toxic as the free drug, and necroscopies revealed no gross abnormalities in any o f the tissues examined.  A more extensive  detennination o f drug induced weight loss was completed as part o f the efficacy studies described in section 3.4.  A n i m a l weight was monitored over the course o f the study and  suggested that liposomal irinotecan was more toxic than the free drug when the drug was administered 3 X at 100 mg/kg (Figure 3.6D).  The nadir in weight loss following this treatment  schedule in these tumour-bearing animals occurred between day 21 and day 25 post inoculation (10-15 days after the first drug dose), and at this time animals treated with liposomal drug had lost - 3 0 % (Figure 3.6) o f their original body weight and had to be terminated.  In contrast,  animals treated with other dosing schedules o f 50 or 100 mg/kg free or liposomal irinotecan exhibited a weight loss in the range o f 7-13 %.  51  Figure 3.6 Toxicity of free (A and C) and liposomal (B and D) irinotecan as assessed by weight loss in LS180 tumour models. Single injections o f free (A) or liposomal (B) irinotecan were administered i.v. on day 11 post cell inoculation, or three injections o f free ( Q or liposomal (D) were administered on day 11, 15 and 19 post cell inoculation. Groups shown are control, saline treated animals (O ); 50 mg/kg irinotecan/ dose ( • ); and 100 mg/kg irinotecan/ dose (T ). Arrows indicate drug administration.  52  3.4 Efficacy of single and multiple dose administration of liposomal and free irinotecan in established LS180 human solid tumours  The human L S I 80 solid tumour model was used to evaluate the anti-cancer activity o f liposomal irinotecan. Eleven days following s.c. inoculation o f 10 L S I 8 0 cells, an easily identifiable and 5  measurable tumour mass arises at the site o f injection. The L S I 80 anti-tumour studies are shown in Figure 3.7, which clearly illustrates that the DSPC/cholesterol irinotecan formulation was therapeutically more active than free drug.  These results show that while a single dose o f free  irinotecan (Figure 3.7A) slows the rate o f tumour growth such that the time to reach a 0.4 gm tumour is 19 days i f the animals are left untreated, 22 and 26 days i f treated with a single i.v. dose o f irinotecan at 50 and 100 mg/kg, respectively.  In contrast, a single i.v. dose o f 50 mg/kg  and 100 mg/kg o f liposomal irinotecan (Figure 3.7B) delayed the time to progression to a 0.4 gm tumour to 34 and 39 days, respectively.  Multiple doses o f the free drug provided for increased  efficacy (Figure 3.7C), and the tumours reached a mass o f 0.4 gm by day 30 and 40 when treated at the 50 and 100 mg/kg dose, respectively.  The tumour growth profile for animals treated 3 X  with 100 mg/kg free drug, closely matched that observed following a single injection o f 100 mg/kg liposomal drug.  Multiple doses o f the liposomal drug showed substantial delays in onset  o f tumour growth, where no increases in tumour size were noted over the 40 day evaluation period (Figure 3.7D).  3.5 Efficacy of single and multiple dose administration of liposomal and free irinotecan in established LS174T human orthotopic tumours  The results shown in Figure 3.7 suggest that the liposomal formulation is therapeutically more active than free drug, but these results are achieved using a s.c. tumour model.  The following  studies were initiated to determine whether similar gains in therapeutic activity can be achieved  53  Figure 3.7 Antitumour efficacy of free (A and Q and liposomal (B and D) irinotecan as in LS180 tumour models. Drug efficacy was assessed as a function of tumour volume following subcutaneous injuection of LSI80 tumour cells on day 0. Single injections of free (A) or liposomal (B) irinotecan were administered i.v. on day 11 post cell inoculation, or three injections of free (C) or liposomal (D) were administered on day 11, 15 and 19 post cell inoculation. Groups shown are control, saline treated animals (O ); 50 mg/kg irinotecan/ dose ( • ); and 100 mg/kg irinotecan/ dose ( • ). (Average ± s.e.). Arrows indicate drug administration. * Indicates termination of an animal.  54  using an animal model which restricts colorectal cancer metastasis to the liver.  The orthotopic  murine tumour model used to evaluate the anti-cancer activity o f liposomal irinotecan against liver metastases was based on intrasplenic injection o f L S 1 7 4 T cells.  When administered  intrasplenically, the primary site o f cell seeding is in the liver, although limited growth in the spleen is also common.  The LS174T is a relatively slow-growing model, and the effects o f a  large tumour burden do not become apparent until around day 30 post-inoculation, after which the host condition deteriorates quickly and the animals must be terminated.  The effects o f  multiple dosing o f free and liposomal drug in this model are illustrated i n Figure 3.8.  In the  absence o f treatment (saline control) the median survival time o f inoculated animals is 32 days, while animals treated with free drug exhibited a median survival o f 54 days and those treated with liposomal drug showed a median survival time o f 79 days  This represents a 69% increase  in median survival time.  3.6 Liposome mediated delivery to liver metastases  To determine whether the improved outcome observed in animals treated with liposomal drug is due in part to improved drug delivery through passive targeting liposome delivery to animals with L S 1 7 4 T liver tumours was assessed.  For these studies animals were dosed with a  liposomal irinotecan containing 0.5 mole percent o f D i l , a nonexchangable fluorescent marker. Representative photographs are shown in Figure 3.9.  Both empty (Figure 3.9B) and irinotecan-  loaded (Figure 3.9C) liposomes are seen predominantly in normal liver tissue, as opposed to the tumour mass.  These images suggest that peritumour delivery o f the liposomal drug is most  prominent and, in contrast to results obtained with s.c. tumours, direct delivery into the tumour is not evident 24 hours after i.v. injection o f the liposomal drug. vacuole-like  structures  seen only in the tumour tissue. 55  These images also illustrate large  As LS17T  cells are derived from a  100 4  ttt  20  30  40  50  60  70  80  90  Days post cell inoculation  Figure 3.8 Antitumour efficacy of free and liposomal irinotecan in LS174T tumour models. Mouse survival after intrasplenic injection of L S 1 7 4 T tumour cells on day 0. Three injections of 50 mg/kg free or liposomal drug were administered i.v. on days 7, 11, and 15 (indicated by arrows) post cell inoculation. Groups shown are control, untreated ( O ) ; free irinotecan ( • ) ; or liposomal irinotecan ( T ).  56  Figure 3.9 Liposome-mediated drug delivery to the tumour. D i l labeled DSPC/cholesterol liposomes, empty or irinotecan loaded, were injected intravenously. At 24 h the livers were harvested for histology. A, H & E staining showing healthy and tumour cells; B, empty and irinotecan-containing ( Q liposomes (fluorescent) are the tumour border. The thick arrows indicate tumour tissue, while the narrow arrows indicate normal liver tissue.  57  mucin-producing adenocarcinoma cell line (Niv et al., 1992), it was postulated that these may be mucous-containing vacuoles.  58  CHAPTER 4  DISCUSSION  4.1 Summary of results  The objective o f the studies presented in this thesis was to outline the potential for a liposomal drug formulation in the treatment o f human colorectal liver metastases.  A liposomal irinotecan  formulation was characterised in vitro, prior to evaluating its therapeutic activity in xenograft models o f advanced human colorectal cancer.  DSPC/cholesterol is an effective lipid formulation for liposomal irinotecan.  Liposome prepared  with this lipid combination could be efficiently loaded (>90%) with irinotecan to achieve drug to lipid ratios o f 0.3:1 (wt:wt).  The loading method relied on use o f a transmembrane p H gradient  to drive the accumulation o f the drug into preformed liposomes.  The p H gradient across these  liposomes was generated by preparing the liposomes in 300 m M M n S 0 4 and adding an 2+  ionophore ( A 2 3 1 8 7 ) capable o f transferring two protons into the liposome for every one M n transported out.  The resulting formulation is stable, with virtually no drug release in vitro for up  to 72 hours at 37°C.  When administered intravenously, irinotecan is gradually released from the  carrier, suggesting that the drug w i l l be bioavailable.  Liposome encapsulation significantly  increased and prolonged circulating irinotecan concentrations, where drug levels were more than 2 orders o f magnitude higher following i.v. injection o f the liposomal drug.  In terms o f  maintaining irinotecan in its active form, more than 80% o f the circulating drug remained in the active lactone form, following injection o f the liposomal drug, as compared to only 40% observed just 1 hour after injection o f free drug. 59  W h i l e free irinotecan was effective in slowing  growth o f L S I 80 solid tumours in S C I D / R A G - 2 M animals, the liposomal drug displayed a much more pronounced effect, arresting tumour growth for an extended time period.  Liposomal  irinotecan also increased survival in mice bearing the L S 1 7 4 T orthotopic tumours.  4.2 Discussion  The results presented in this thesis highlight the tremendous ability o f liposomal encapsulation to potentiate the therapeutic activity o f a drug and confirm previous speculation for a possible role o f liposomal anti-cancer drugs in treating liver-localised neoplasia.  The latter was based on  observations o f an inherent tendency for significant liposome accumulation in the liver and previous studies establishing a benefit for liposome-based treatments in liver-localized disease. Liposomes are a proven drug delivery system, but the mechanism o f action has consistently been defined as due to carrier-mediated increases in tumour delivery. However, the question must be raised regarding localisation within a tissue.  This could be best assessed using a tissue that has a  natural tendency to accumulate liposomes, such as a. liver, and attempts to differentiate between tumour delivery in the liver as opposed to tissue delivery.  Carrier-mediated alterations in drug pharmacokinetics and biodistribution grant liposomes an ability to reduce anticancer drug toxicity while maintaining or increasing efficacy (Boman et al., 1995; Mayer et al., 1998).  The former are optimised by manipulation o f lipid composition  which alters such critical parameters as: (1) drug retention and release; (2) circulation lifespan, and hence propensity to accumulate at the target site; and (3) carrier targeting. parameters,  the  application o f the  highly stable  Based on these  disteroylphosphatidylcholine/cholesterol  liposomes in irinotecan transport and delivery to metastatic colorectal cells was explored. DSPC/cholesterol (55:45, mole percent) is an optimised conventional liposome formulation that 60  exhibits increased blood residency lifetimes due to minimized interactions with opsonizing plasma proteins, reducing the rate o f MPS-based carrier elimination (Chonn et al., 1991).  Beyond those advantages o f improved pharmacokinetics and biodistribution, an additional benefit begot for camptothecin encapsulation is protection o f the active lactone Previous camptothecin  species.  loading into lipid-based carriers, including attempts at irinotecan  encapsulation(Burke et al., 1993; Sadzuka et al., 1999), have exploited their ability to partition into the lipid bilayer.  While this has been shown to confer protection o f the lactone species  (Burke et al., 1993; Sadzuka et al., 1999), therapeutically, this approach is limited by low drug loading efficiencies into the membrane  and the rapid exchange  o f membrane-localised  hydrophobic drugs from liposomes to other membranes after i.v. administration (Choice et al., 1995; Ouyang et al., 1995).  Tardi and colleagues (Tardi et a l , 2000) overcame this limitation  by successfully encapsulating topotecan in the aqueous interior o f large unilamellar vesicles using an ionophore-generated p H gradient.  Results o f the present work support their findings  that ionophore-mediated encapsulation o f a camptothecin analogue in a carrier's acidic interior is not only efficient (>90 % loading), but, as with topotecan, protected irinotecan in its lactone form for an extended length o f time, following i.v. administration.  In turn, it is logical to assume that  this protection w i l l substantially increase the propensity for the active species to be present at the site o f tumour growth.  In addition to improving the circulation lifespan o f irinotecan, DSPC/cholesterol liposomes provide sustained drug release over 24 h.  Together these pharmacokinetic changes are  anticipated to prolong tumour cell exposure o f this S-phase-specific drug.  This, in turn, should  increase tumour growth arrest and delays in tumour progression and increases in median survival.  It is worth noting that irinotecan cytotoxicity has the additional constraint o f requiring 61  carboxylesterase-mediated conversion to the vastly more potent SN-38.  These enzymes are  found in high concentrations in hepatic cells, as well as in serum and other tissues, including some tumour populations (Kawato et al., 1991; Guichard et al., 1999;).  The environment  responsible for liposomal irinotecan activation and the subsequent therapeutic activity observed against colorectal metastases is currently unclear, and depends to some extent on the specificity o f drug delivery, although it was likely activated within the vicinity o f the tumour, as there was no measurable circulating SN-38.  Initial animal studies evaluated the toxicity and efficacy o f liposomal irinotecan in the murine L S I 80 and L S 1 7 4 T models o f metastatic colorectal cancer.  W h i l e the activity o f camptothecins  has been evaluated, to a limited extent, in these cell lines in vitro (Jansen et al., 1997; te Poele and Joel, 1999; Kouniavsky et al., 2002), no studies involving irinotecan activity in these cells or their corresponding animal models had previously been conducted.  In both the L S I 8 0 and  L S 1 7 4 T models, liposomal irinotecan was considerably more efficacious than free drug. Improvements in tumour growth control and survival time were seen in all liposomal irinotecantreated groups, in contrast to groups treated with free drug.  A s shown in Figure 3.7, treatment  with liposomal irinotecan did not result in a decrease in tumour size, rather tumour growth was prevented for extended time periods.  It can be suggested that liposomal irinotecan provides  adequate control o f tumour expansion, but alone it is not sufficient to eradicate the disease.  This  is not surprising as it is generally accepted that tumours represent a heterogenous population o f cells and microenvironments.  A corollary o f this conjecture is that those cells sensitive to  irinotecan, are effectively killed, hence the arrest o f tumour growth, and those that overcome its cytotoxicity are responsible for reinitiation o f tumour growth.  62  There are numerous possible reasons as to why cells may evade the effects o f targeted irinotecan. Irinotecan must be activated, which in the L S I 8 0 solid tumour model is presumably mediated by tumour cell associated carboxylesterase, which may not be present at sufficient levels in all cells. Alternatively, it is understood that not all tumour cells are actively dividing within a tumour. Finally, the drug may not have access to all o f the tumour cells and at lower doses, not percolate as deeply into the tumour interstitial space.  It is presumed that those cells that, for whatever  reason, survived irinotecan treatment are responsible for reinstatement o f tumour growth.  In  turn this leads to speculation that, due to an altered cell population, the molecular characteristics of the delayed tumours are distinct from those with little or not disruption o f growth, and raises the possibility that they had developed new characteristics that could further challenge treatment. These are questions that have yet to be investigated, but their answers may lead to greater understanding o f tumour biology and maturation and perhaps even the development o f drug resistance.  Whether free or encapsulated, tumour cell responses to chemotherapeutic agents can vary with tumour physiology and tumour cell heterogeneity.  The ideal therapy would be effective in all  microenvironments o f a heterogeneous tumour population including cells o f varying growth rates, in different phases o f the cell cycle, originating from different tissues and those capable o f rapid adaptation to cytotoxic factors.  In practice effective chemotherapy is based on multiple  drugs whose cytotoxicity is derived from different mechanisms.  Platinum compounds such as  cisplatin and the next generation, oxaliplatin, are among those agents proposed to work well in combination with irinotecan and other camptothecins.  Evaluation o f cisplatin and irinotecan  activity in vitro confirmed that in the L S I 8 0 and L S 1 7 4 T cell lines they do function synergistically (Chew et al., unpublished observations).  Current protocols for advanced C R C  have incorporated irinotecan in conjunction with 5 - F U and leucovorin. It was also confirmed in 63  vitro that irinotecan and 5-FU function synergistically in these cell lines (Chew et al., unpublished observations).  These preliminary results, which further support the prevailing  understanding that optimal therapy is achieved with multiple drugs in combination, indicates that further studies towards a liposomal-based treatment for colorectal liver metastases should investigate the practicality and in vivo effect on efficacy o f dual loading o f either 5-FU or cisplatin with irinotecan.  In conclusion, liposome encapsulation o f irinotecan results in a potent drug formulation for the treatment o f liver metastases as a result o f increased drug longevity, protection o f the active lactone species, and rapid accumulation at the site o f tumour development i n the liver. 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Plasma-extracted standards  were prepared i n the same manner except by dilution o f working solutions in plasma.  100 p i o f  each standard was then transferred to eppendorf tubes containing 5 p i o f the internal standard, camptothecin, and 595 ul ice-cold methanol.  Standards were immediately frozen at -70°C. A l l  standards contained both the lactone and carboxylate species.  For sample analysis, 80 p i o f each standard was loaded into 1-ml H P L C sample vials (Waters, Milford, M A ) with 200 p i amber inserts (Chromatographic Specialties Inc., Brockville, O N ) , and 10 p i were injected into the column.  Calibration curves  Calibration curves were constructed using standard samples containing both the lactone and carboxylate forms o f irinotecan.  The calibration ranges were from 0.1 to 10 pg/ml for both the  75  lactone and carboxylate species.  The calibration curves were obtained by plotting the peak area  o f the analytes versus their respective concentrations (Figures A - l and A - 2 ) .  For plasma  extracted standards (A-2), the peak area o f the analytes was divided by that o f the internal standard to correct for deficiencies in extraction.  Sample preparation and measurements  Heparinized plasma samples chilled on ice were immediately centrifuged at 1500 g for 10 minutes at 4 ° C .  100 ul o f each sample was then transferred to eppendorf tubes containing 5 ul  of the internal standard, camptothecin, and 595 ul ice-cold methanol. performed as described for analysis o f the standards. Figure A - 3 .  76  Sample analysis was  A n example chromatogram is shown in  0.0  2.0  4.0  6.0  8.0  10.0  12.0  Irinotecan concentration (ng/mL)  Figure A - l H P L C calibration curve, aqueous standards. The calibration curves were obtained by plotting the peak area o f the analytes versus their respective concentrations. Standards contained both the lactone ( • )and carboxylate (o) forms o f the irinotecan.  77  / y=  1.8042X-0.2341 R  2  = 0.9972  /  -/-  /  y = 0.8744X-0.2165  0.0  2.0  4.0  6.0  8.0  10.0  12.0  Irinotecan concentration (ug/mL)  Figure A-2 H P L C obtained by plotting camptothecin, versus carboxylate (o) forms  calibration curve, plasma standards. The calibration curves were the peak area o f the analytes divided by that o f the internal standard, their respective concentrations. Standards contained both the ( • )and o f the irinotecan.  78  O.OOH  B.OO  20.00  10.00 Minutes  Identified Peaks Summary  BE  Height  Concentration  Units  1  carboxv  3.897  141930  8221  4.917e+000  ug/ml  2  lactone  6.206  194254  8585  3.055e+000  ug/ml  3  CPT  11.967  39480  2258  1.000e+000  Name  RT  Area  Figure A-3 Representative chromatogram illustrating irinotecan levels in plasma-extracted samples. Both the carboxylate and the lactone form, as well as the internal standard, camptothecin, are shown as distinct and separate peaks. The associated software (Millennium32® chromatograpy manager, Version 3.20, Waters, M i l f o r d , M A ) calculates the concentrations based on the area o f each peak in relation to standards.  79  

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