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Therapeutic applications of ceramide lipids for apoptosis induction Shabbits, Jennifer A. 2003

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THERAPEUTIC APPLICATIONS OF CERAMIDE LIPIDS FOR APOPTOSIS INDUCTION by JENNIFER A . SHABBITS B.Sc. (Biochemistry), Simon Fraser University, 1997  A THESIS SUBMITTED IN P A R T I A L F U L F U L M E N T OF T H E R E Q U I R E M E N T S FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY (Pharmaceutical Sciences) in THE F A C U L T Y OF G R A D U A T E STUDIES Faculty of Pharmaceutical Sciences Department of Pharmaceutics and Biopharmaceutics We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A April, 2003 © Jennifer A . Shabbits, 2003  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head of my department o r by h i s or her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .  Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada  ABSTRACT The emerging role of ceramide lipids in apoptosis and an increased understanding of their involvement in multidrug resistance (MDR) has revealed new opportunities for manipulating ceramide levels in order to achieve specific therapeutic objectives. The research presented in this thesis focused on the relationship between ceramide, M D R and apoptosis. Direct and indirect approaches for modulating intracellular ceramide levels were investigated in an attempt to chemosensitize M D R tumors and induce apoptosis. Inhibition  of  pro-apoptotic  ceramide  conversion  to  its  non-cytotoxic  glucosylceramide metabolite was shown to sensitize two human M D R breast cancer cell lines to the cytotoxic effects of tubulin-binding chemotherapy drugs.  Enhanced  sensitization was correlated with increased ceramide, suggesting that therapeutic manipulations aimed at increasing endogenous ceramide should promote apoptosis. On the basis of these results, the feasibility of delivering therapeutic amounts of exogenous ceramides to cells was then investigated. After evaluating different chain length ceramides it was determined that synthetic C6-ceramide was internalized and cytotoxic to cells whereas naturally occurring C]6-ceramide was neither internalized nor cytotoxic. This difference established the importance of intracellular delivery as a prerequisite to apoptosis induction by exogenous ceramides.  Liposome-based delivery  systems were then introduced in an attempt to overcome the limitations associated with intracellular delivery of natural ceramide. Physically stable liposomes containing up to 50 mole percent Ci6-ceramide in the lipid bilayer were successfully formulated. These liposomes were internalized by J774 macrophage cells in vitro and induced apoptosis with similar potency to free C6-ceramide.  ii  In order to translate this encouraging data to an in vivo model it was first necessary to evaluate the behavior of these liposomes in the circulation. Pharmacokinetic studies demonstrated in vivo stability over 24 hours following iv bolus administration. The antitumor activity of these liposomes was then evaluated in the J774 ascites tumor model. Optimal antitumor activity was observed following intraperitoneal administration of Ci6-ceramide liposomes on days 1, 5, and 9. This corresponded to a statistically significant increase in animal survival of 43.5% over non-ceramide control liposomes. Taken together, this research provides evidence for the rational design of ceramide-based liposomes as a novel approach for cancer chemotherapy.  iii  T A B L E OF CONTENTS  ABSTRACT T A B L E OF CONTENTS LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS ACKNOWLEDGEMENTS DEDICATION  ii iv viii xii xiii xvii xviii  CHAPTER 1: INTRODUCTION 1.1 THE M U L T I D R U G RESISTANCE P H E N O M E N O N 1.1.1 Overview of Multidrug Resistance in Cancer and Clinical Significance 1.1.2 Mechanisms of M D R 1.1.2.1 Transport-based M D R 1.1.2.2 Enzyme-related M D R 1.1.2.3 Tumor microenvironment 1.1.2.4 Alterations in apoptosis pathways 1.1.2.5 Alterations in ceramide metabolism  1 1 1 2 3 6 7 8 10  1.2  11 12 15  1.3  1.4  M O D U L A T I O N OF M U L T I D R U G RESISTANCE 1.2.1 M D R Modulation by Drug Transport Inhibitors 1.2.2 Apoptosis Pathways 1.2.2.1 Apoptosis induction and execution via the death receptor (extrinsic) pathway 1.2.2.2 Apoptosis induction and execution via the mitochondrial (intrinsic) pathway 1.2.2.3 The degradation phase of apoptosis: common effector caspases 1.2.3 M D R Modulation Strategies Related to Apoptosis Pathways 1.2.3.1 Downregulation of anti-apoptotic signals to chemosensitize tumors 1.2.3.2 Upregulation of pro-apoptotic signals 1.2.3.3 Modulation of ceramide metabolism  18 19 21 21 21 23 23  C E R A M I D E LIPIDS A S I N T R A C E L L U L A R SIGNALING MOLECULES 1.3.1 Structure and Metabolism of Ceramide Lipids 1.3.2 Biological Targets of Ceramide Lipids and their Role in Apoptosis 1.3.3 Ceramide Signaling via Lipid Rafts  24 25 28 32  L I P O S O M A L D R U G D E L I V E R Y SYSTEMS 1.4.1 Review of Liposomes 1.4.1.1 Liposome preparation and classification 1.4.1.2 Lipid composition 1.4.1.3 Lipid polymorphism  33 34 36 39 45  iv  1.4.2  1.4.3  1.5  Liposomes as Drag Carriers 1.4.2.1 Passive encapsulation 1.4.2.2 Active encapsulation Drag Delivery Approaches to M D R Modulation 1.4.3.1 Application of liposomes to M D R reversal 1.4.3.2 Application of liposomes to the delivery of bioactive lipids  THESIS OBJECTIVES A N D HYPOTHESIS  CHAPTER 2: T H E ROLE OF 1 -PHEN YL-2-DEC ANO Y L AMINO-3MORPHOLINO-1-PROPANOL (PDMP) AND P-GLYCOPROTEIN IN MODULATING CERAMIDE-MEDIATED SENSITIVITY OF HUMAN BREAST CANCER CELLS TO TUBULIN-BINDING ANTICANCER DRUGS 2.1 2.2 2.3  Introduction and Rationale Hypothesis Materials and Methods 2.3.1 Materials 2.3.2 Cell lines and culture 2.3.3 Evaluation of cell surface Pgp expression by flow cytometry 2.3.4 Evaluation of intracellular Pgp expression by fluorescence microscopy 2.3.5 M T T cytotoxicity assays 2.3.6 [ H]Taxol® uptake studies 2.3.7 Cell radiolabeling and lipid extraction 2.3.8 Lipid detection by thin layer chromatography (TLC) 2.3.9 Statistical analysis Results 2.4.1 Chemosensitization effects of PDMP in human breast cancer cells 2.4.2 Influence of exogenous C6-ceramide on PDMP-induced chemosensitization 2.4.3 Effect of Pgp inhibition on PDMP-induced chemosensitization 2.4.4 Correlation of chemosensitization effects with glucosylceramide levels Discussion 3  2.4  2.5  CHAPTER 3: INTRACELLULAR DELIVERY OF EXOGENOUS CERAMIDE LIPIDS INDUCES APOPTOSIS IN VITRO 3.1 3.2 3.3  Introduction and Rationale Hypothesis Materials and Methods 3.3.1 Materials 3.3.2 Cell lines and culture 3.3.3 M T T cytotoxicity assays 3.3.4 Lipid uptake studies 3.3.5 Spectrophotometric protein quantitation 3.3.6 Preparation of liposomes  47 47 49 50 50 53 53  55 55 56 57 57 58 58 59 60 60 61 62 62 62 62 72 72 73 77  82 82 83 83 83 84 84 84 85 86  v  3.3.7 3.3.8 3.4  3.5  Lactose trapping Cryo-transmisson electron microscopy  Results 3.4.1 The effect of ceramide acyl chain length on in vitro cytotoxicity in MDA435/LCC6 cells 3.4.2 Correlation of M T T cytotoxicity results with ceramide uptake 3.4.3 Formulation and cytotoxicity of C6-ceramide containing liposomes in MDA435/LCC6 cells 3.4.4 Formulation and cytotoxicity of Ci6-ceramide containing liposomes in MDA435/LCC6 cells 3.4.5 Cytotoxicity of free and liposomal ceramide in J774 murine macrophage cells Discussion  CHAPTER 4: DEVELOPMENT OF AN IN VITRO EXCHANGE ASSAY T O A C C U R A T E L Y PREDICT T H E LIPID AND DRUG RETENTION PROPERTIES OF LIPOSOME-BASED DELIVERY SYSTEMS 4.1 4.2 4.3  4.4  Introduction and Rationale Hypothesis Materials and Methods 4.3.1 Materials 4.3.2 Preparation of donor large unilamellar vesicles (LUVs) 4.3.3 Preparation of acceptor multilamellar vesicles (MLVs) 4.3.4 Separation of L U V and M L V populations 4.3.5 Liposomal encapsulation of doxorubicin 4.3.6 Liposomal encapsulation of verapamil 4.3.7 Ceramide lipid/drug release from liposomes using dialysis assays 4.3.8 Ceramide lipid/drug release from liposomes using the MLV-based exchange assay 4.3.9 In vivo ceramide lipid/drug release Results 4.4.1 Design of the MLV-based exchange assay procedure 4.4.2 Separation of control and ceramide-containing donor L U V and acceptor M L V populations 4.4.3 Evaluation of C6-ceramide retention using conventional in vitro dialysis assays 4.4.4 Evaluation of C6-ceramide retention following i.v. bolus administration 4.4.5 Evaluation of C6-ceramide retention using the MLV-based in vitro assay 4.4.6 Evaluation of Ci6-ceramide retention using the MLV-based in vitro assay 4.4.7 Evaluation of the MLV-based assay as a measure of liposomally encapsulated conventional drug release  87 87 88 88 90 92 95 105 112  118 118 121 121 121 122 122 123 124 124 125 125 126 126 126 128 130 130 133 136 136  vi  4.5  Discussion  CHAPTER 5: PHARMACOKINETIC EVALUATION AND ANTITUMOR ACTIVITY OF HIGH CERAMIDE CONTENT LIPOSOMES 5.1 5.2 5.3  5.4  Introduction and Rationale Hypothesis Materials and Methods 5.3.1 Materials 5.3.2 Cell line and culture 5.3.3 Preparation of liposomes 5.3.4 Pharmacokinetic analysis of control and ceramide liposomes 5.3.5 Establishment of the J774 ascites tumor model 5.3.6 Evaluation of antitumor activity 5.3.7 Statistical analysis Results 5.4.1 Pharmacokinetic analysis of control and ceramide liposomes following i.v. bolus administration 5.4.2 Evaluation of antitumor activity of ceramide liposomes in the J774 ascites tumor model  142  145 145 146 146 146 146 147 147 148 150 150 150 150 154  5.5 Discussion  159  CHAPTER 6: SUMMARY OF RESULTS AND FUTURE DIRECTIONS  163  REFERENCES  170  vii  LIST OF FIGURES Figure 1.1  A schematic illustration of apoptosis pathways highlighting the inter-relationships between key apoptotic proteins  17  Figure 1.2  The chemical structures of a natural and synthetic ceramide lipid  25  Figure 1.3  Sphingolipid metabolic pathways illustrating the various routes of ceramide synthesis and breakdown  27  Figure 1.4  A schematic illustration of a hypothetical multifunctional liposome  34  Figure 1.5  A n illustration of spontaneous bilayer formation upon hydration of amphipathic lipids in an aqueous buffer Schematic illustrations and scanning electron micrographs of the three main liposome classes  37  Figure 1.6 Figure 1.7  Figure 1.8  Figure 1.9  Figure 1.10  Figure 2.1  Figure 2.2  Figure 2.3  Figure 2.4  38  The general structure of a phospholipid depicting some commonly occurring headgroups and fatty acid moieties  40  The chemical structures of cholesterol and cholesteryl hemisuccinate lipids  43  A schematic illustration of a pegylated liposome showing the chemical structure of polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE)  44  Drug distribution following active and passive encapsulation for hydrophobic and hydrophilic drugs  48  Cell surface P-glycoprotein expression in wild-type and multidrug resistant MCF7 and MDA435/LCC6 breast cancer cells as shown by flow cytometry  65  Fluorescence microscopy images of wild-type and multidrug resistant MCF7 and MDA435/LCC6 cells following two-step staining with mouse anti-human Pgp C219 primary and goat antimouse IgG FITC secondary antibodies  66  The effect of 5 u M PDMP on Taxol® and vincristine cytotoxicity in wild-type and multidrug resistant MCF7 cells  68  The effect of 5 yM PDMP on Taxol® and vincristine cytotoxicity in wild-type and multidrug resistant MDA435/LCC6 cells  69  viii  Figure 2.5  Figure 2.6  Figure 3.1  Figure 3.2  Figure 3.3  Figure 3.4  Figure 3.5  Figure 3.6  Figure 3.7  Figure 3.8  Figure 3.9  Figure 3.10  Accumulation of [ H]Taxol® in wild-type and multidrug resistant M C F 7 and MDA43 5/LCC6 cells  71  A model to explain the influence of Pgp on ceramide metabolism in the context of chemosensitization  81  Cytotoxicity of various acyl chain length free ceramide lipids on wild-type and mdr-1 gene transfected MDA435/LCC6 human breast cancer cells  89  Cellular uptake of free C6- and Ci6-ceramide by wild-type and mdr1 gene transfected MDA43 5/LCC6 cells  91  Mean liposome vesicle diameter for C6-cer/DSPC/Chol (45:10:45) and DSPC/Chol (55:45) liposomes as determined by quasi-elastic light scattering  93  Cytotoxicity of control and C6-ceramide liposomes on wild-type and mdr-1 gene transfected MDA435/LCC6 human breast cancer cells  94  Cellular uptake of [ H]CHE liposome and [ C]C6-ceramide radiolabels by wild-type and mdr-1 gene transfected MDA435/LCC6 human breast cancer cells  96  Mean liposome vesicle diameter for Ci6-cer/DSPC/Chol (15:40:45) containing liposomes as determined by quasi-elastic light scattering  99  Cytotoxicity of Ci -cer/DSPC/Chol (15:40:45) and control DSPC/Chol (55:45) liposomes on wild-type and mdr-1 gene transfected MDA435/LCC6 human breast cancer cells  100  Cellular uptake of Ci -cer/DSPC/Chol (15:40:45) liposomes by wild-type and mdr-1 gene transfected MDA435/LCC6 cells  101  Mean liposome vesicle diameter for Ci6-cer/CHEMS (50:50) and control DPPC/CHEMS (50:50) liposomes as determined by quasielastic light scattering  103  Cryo-transmission electron micrographs of DPPC/CHEMS/PEG2000-DSPE (49.5:49.5:1) and C i cer/CHEMS/PEG oo-DSPE (49.5:49.5:1) liposomes  104  Cytotoxicity of C1 -cer/CHEMS and D P P C / C H E M S liposomes on wild-type and mdr-1 gene transfected MDA435/LCC6 human breast cancer cells  106  3  3  14  6  6  6  20  Figure 3.11  6  IX  Figure 3.12  Cellular uptake of Ci6-cer/CHEMS (50:50) liposomes by wild-type and mdr-1 gene transfected MDA435/LCC6 cells  107  Cytotoxicity of various acyl chain length free ceramide lipids on J774 murine macrophage cells  109  Cytotoxicity of Cie-cer/CHEMS and D P P C / C H E M S liposomes on J774 cells  110  Cellular uptake of [ H]CHE and [ C]Ci -ceramide labeled C i cer/CHEMS liposomes by J774 cells  111  Figure 4.1  A schematic illustration of the in vitro MLV-based assay  127  Figure 4.2  Profile of C6-ceramide release from C6-cer/DSPC/Chol/PEG2oooDSPE (15:10:40:5) donor L U V s following dialysis against HBS or HBS + 30% FBS for 24 hours at 37°C  131  Circulation profile of C6-ceramide and bulk liposomal lipid following i.v. bolus administration of C6-cer/DSPC/Chol/PEG2oooDSPE (15:10:40:5) liposomes to female Balb/c mice  132  Profile of C6-ceramide release from C6-cer/DSPC/Chol/PEG2oooDSPE (15:10:40:5) donor L U V s using the MLV-based exchange assay  134  Profile of Ci6-ceramide release from Ci6-cer/CHEMS (50:50) donor L U V s using the MLV-based exchange assay  137  Release profiles of doxorubicin and verapamil from liposomes as measured by dialysis assays, the in vitro MLV-based assay, or from plasma collected following i.v. bolus liposome administration  141  Correlation plots comparing observed and predicted log plasma concentration versus time graphs for control and ceramide liposomes modeled using a one-compartment, i.v. bolus dosing model in WinNonlin 1.1.  149  Plasma elimination profile of control (DPPC/CHEMS/PEG2000DSPE, 47.5/47.5/5) and ceramide-based (Ci -cer/CHEMS/PEG ooDSPE, 47.5/47.5/5) liposomes following i.v. bolus administration to female Balb/c mice  152  Figure 3.13  Figure 3.14  Figure 3.15  Figure 4.3  Figure 4.4  Figure 4.5  Figure 4.6  Figure 5.1  Figure 5.2  3  14  6  6  6  Figure 5.3  20  Evaluation of antitumor activity of Ci6-cer/CHEMS/PEG2ooo-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG oo-DSPE (47.5:47.5:5) 20  x  Figure 5.4  Figure 5.5  liposomes administered i.v. on days 1, 5, and 9 in the J774 ascites tumor model  156  Evaluation of antitumor activity of Ci -cer/CHEMS/PEG2ooo-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG2000-DSPE (47.5:47.5:5) liposomes administered i.p. on day 1 in the J774 ascites tumor model  157  Evaluation of antitumor activity of C] 6-cer/CHEMS/PEG2ooo-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG2000-DSPE (47.5:47.5:5) liposomes administered i.p. on days 1, 5, and 9 in the J774 ascites tumor model  158  6  xi  LIST OF TABLES Table 1.1  Anticancer drugs and drug classes typically associated with the M D R phenomenon  2  Table 1.2  Major mechanisms of multidrug resistance  3  Table 1.3  Third-generation M D R modulators  14  Table 1.4  Molecular shape of various lipids and their associated structures  45  Table 2.1  The effect of 5 u M PDMP, 1 ug/ml Valspodar and 2 u M C ceramide on anticancer drug cytotoxicity in wild-type and multidrug resistant MCF7 and MDA435/LCC6 breast cancer cells  64  The effect of Pgp blockade on the degree of PDMP-induced chemosensitization in MCF7/AdrR and MDA435/LCC6MDR I cells as measured by a sensitization ratio  74  Incorporation of [ H]palmitic acid into ceramide and glucosylceramide of treated and control MCF7 cells  76  Cytotoxicity of various acyl chain length free ceramide lipids in wild-type and resistant MDA435/LCC6 human breast cancer cells  90  Summary table describing various Ci6-ceramide containing liposome formulations attempted and their respective characteristics  98  Separation of empty [ H]-LUV donor and [ C ] - M L V acceptor vesicles by centrifugation  129  Table 2.2  Table 2.3  Table 3.1  Table 3.2  Table 4.1  Table 4.2  Table 4.3  6  3  J  l4  Correlation coefficient (r) and coefficient of determination (r ) for ceramide lipid, doxorubicin or verapamil release from liposomes as measured by the dialysis and MLV-based assays relative to actual in vivo results Separation of doxorubicin or verapamil-loaded [ HJ-LUV donor and [ C ] - M L V acceptor populations by centrifugation  140  Summary of plasma pharmacokinetic parameters for control and Ci6-ceramide containing liposomes following i.v. bolus administration at a dose of 100 mg/kg total lipid  153  14  Table 5.1  135  Xll  LIST OF ABBREVIATIONS 5FU  5-Fluorouracil  ABC  ATP-binding cassette  Abs  Absorbance  ADR  Adriamycin  AIC  Akaike Information Criterion  AIF  Apoptosis inducing factor  ANOVA  Analysis of variance  Apaf-1  Apoptotic protease activating factor-1  Ara-C  Cytosine arabinoside  AS-ODN  Antisense oligonucleotide  ATP  Adenosine triphosphate  AUC  Area under the curve  BSA  Bovine serum albumin  Cio-cer  N-decanoyl-D-erythrosphingosine  Cn-cer  N-myristoyl-D-erythrosphingosine  Ci6-cer  N-palmitoyl-D-erythrosphingosine  C2-cer  N-acetoyl-D-erythrosphingosine  C6-cer  N-hexanoyl-D-erythrosphingosine  Cg-cer  N-octanoyl-D-erythrosphingosine  Cmax  Peak plasma concentration achieved  CAD  Caspase-activated DNAse  CAPK  Ceramide activated protein kinase  CAPP  Ceramide activated protein phosphatase  Cer  Ceramide  CHE  Cholesteryl hexadecyl ether  CHEMS  Cholesteryl hemisuccinate  Choi  Cholesterol  CL  Plasma clearance  C0  2  Cryo-TEM  Carbon dioxide Cryo-transmission electron microscopy  CsA  Cyclosporin A  Cytc  Cytochrome c  Da  Daltons  DABCO  1,4-Diazobicyclo-(2,2,2)octane  DAPI  4',6-Diamidino-2-phenylindole  dATP  Deoxyadenosine triphosphate  DD  Death domain  DED  Death effector domain  DFF  D N A fragmentation factor  dH 0  Demineralized water  Diablo  Direct IAP binding protein with low pi  DISC  Death-inducing signaling complex  DMEM  Dulbecco's modified eagle's medium  DOPE  Dioleoylphosphatidylethanolamine  DOTAP  1,2-Dioleoyl-3-trimethylammonium propane  DPM  Disintegrations per minute  DPPC  Dipalmitoylphosphatidylcholine  DSPC  Disteroylphosphatidylcholine  EDTA  Ethylenediamminetetraacetic acid  EPC  Egg phosphatidylcholine  FBS  Fetal bovine serum  FITC  Fluorescein isothyocyanate  GCS  Glucosylceramide synthase  GlcCer  Glycosylceramide  GSH  Glutathione  GSL  Glycosphingolipid  GST  Glutathione-S-transferase  HBS  Hepes buffered saline  i.p.  Intraperitoneal  i.v.  Intravenous  IAP  Inhibitors of apoptosis  2  xiv  IC20  Concentration required to achieve 20% cell kill  IC50  Concentration required to achieve 50% cell kill  ILS  Increase in lifespan  J774  Murine-derived macrophage cell line  kDa  Kilodaltons  KSR  Kinase suppressor of Ras  LUV  Large unilamellar vesicle  MCF7  Human breast carcinoma, sensitive (wild-type)  MCF7/AdrR  Human breast carcinoma, M D R (selected for adriamycin resistance)  MDA435/LCC6 MDA435/LCC6  Human breast carcinoma, sensitive (wild-type) M O T /  Human breast carcinoma, M D R (transfected with mdr-1)  MDR  Multidrug resistance  mdr-1  Multidrug resistance gene encoding P-glycoprotein  MLV  Multilamellar vesicle  MRP  Multidrug resistance associated protein  MTT  3-(4,5-dimentylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide  NOE  N-oleoylethanolamine  PARP  Poly A D P ribose polymerase  PBS  Phosphate buffered saline  PBSB  PBS + 0.1%BSA  PBSBT  PBSB + 0.5% Tween-20  PC  Phosphatidylcholine  PDMP  1 -Phenyl-2-decanoylamino-3-morpholino-1 -propanol  PPMP  l-Phenyl-2-hexanoylamino-3-morpholino-l-propanol  PE  Phosphatidylethanolamine  PEG  Polyethylene glycol  Pgp  P-glycoprotein  PI  Phosphatidylinositol  PKB  Protein kinase B  PKC  Protein kinase C  xv  PP1  Protein phosphatase 1  PP2A  Protein phosphatase 2A  PPMP  1 -Phenyl-2-hexadecanoyIamino-3 -morpholino-1 -propanol  PPPP  1 -phenyl-2- hexadecanoylamino-3-pyrrolidino-1 -propanol  PS  Phosphatidylserine  PSC 833  Cyclosporin D analog (Valspodar)  PT  Permeability transition  RES  Reticuloendothelial system  ROS  Reactive oxygen species  SIP  Sphingosine-1 -phosphate  SAPK  Stress activated protein kinase  SD  Standard deviation  SEM  Standard error of the mean  SM  Sphingomyelin  Smac  Second mitochondria-derived activation of caspases  SMase  Sphingomyelinase  SR  Sensitization ratio  SUV  Small unilamellar vesicle Plasma elimination half-life  TNF  Tumor necrosis factor  TNF-R  TNF receptor  TRAIL  TNF-related apoptosis inducing ligand  TRAIL-R  TRAIL receptor  v  Volume of distribution  d  XIAP  X-linked inhibitor of apoptosis  xvi  ACKNOWLEDGEMENTS  First and foremost I am grateful to my supervisor Lawrence for his constant support and guidence, for helping me to become a scientist, and for also allowing me to become a teacher. I am also grateful to Marcel for creating such a unique and special lab environment. I feel very lucky to have been a part of such a wonderful research family. Thank you to the members of A.T. past for passing on your words of wisdom and for proving that there really is an end! Thank you to the members of A.T. present for creating such a great environment for work (and lots of play!). From lunches at the G G W to Drag Queen night at the Odessey, you made it fun to come to work. Thank you also to my family for giving me a lifetime of love and support, and for always encouraging me to do my best. And last but not least, thank you to Mike - my husband and best friend - for your unwavering love and understanding, and for always believing that one day this thesis would finally be complete.  xvii  DEDICATION  To my family.  xviii  CHAPTER 1 INTRODUCTION  1.1 T H E MULTIDRUG RESISTANCE PHENOMENON Multidrug resistance (MDR) is a major obstacle to the effective treatment of cancer. Despite vast improvements in our understanding of the mechanisms of drug resistance, the ability to modulate M D R has been complicated by the fact that many human tumors simultaneously exhibit multiple resistance mechanisms.  This section provides an  overview of M D R and its major mechanisms, and introduces the rationale underlying the studies described in this thesis to utilize ceramide manipulations to sensitize tumors to chemotherapy.  1.1.1  Overview of Multidrug Resistance in Cancer and Clinical Significance Each year approximately 1.5 million new cases of cancer are reported in North  America (1).  While many of these will respond favorably to initial treatment with  conventional chemotherapeutic regimens, the ultimate success in achieving long-term tumor regression or cures is frequently limited by the development of drug resistance. Drug resistant cancers are either inherently untreatable (intrinsic resistance), or they have progressed to develop resistance to a wide variety of anticancer agents over the course of treatment due to selection pressures caused by repeated drug administration (acquired resistance). The term multidrug resistance is used to describe the ability of tumor cells exposed to a single cytotoxic agent to develop resistance to a broad range of structurally and functionally unrelated drugs. Table 1.1 lists some anticancer drugs and their classes that are typically associated with M D R .  1  Table 1.1 Anticancer Drugs and Drug Classes Typically Associated with the MDR Phenomenon Anthracyclines | Taxanes Vinca Alkaloids Epipodophyllotoxins Doxorubicin Daunorubicin Epirubicin Idarubicin Mitoxantrone  j Paclitaxel 1 Docetaxel I | |  Vincristine Vinblastine Vinorelbine  Etoposide Teniposide  Other  Tamoxifen Mitomycin Dactinomycin  The clinical significance of this phenomenon is highlighted by estimations that up to one-third of all new cases of cancer each year in North America will eventually exhibit characteristics consistent with M D R .  Clearly then, the development of systems which  successfully modulate drug resistance in vivo will be invaluable in treating patients with previously non-responsive tumors.  1.1.2  Mechanisms of MDR There are numerous mechanisms that contribute to the M D R phenomenon. The  classical mechanism is decreased intracellular drug accumulation caused by drug efflux protein pumps that are often overexpressed in M D R tumors. Although drug transport is the best characterized resistance mechanism studied to date, several other mechanisms are also known to be involved. For example, increased D N A repair and alterations at the level of drug target enzymes have been implicated in drug resistance.  In addition,  specific features of the tumor microenvironment have been shown to contribute to resistance in many cell types. Since most chemotherapy approaches ultimately elicit their effects via apoptosis (programmed cell death), alterations at the level of apoptosis control provide yet another mechanism by which drug resistance may occur. The various classes  2  of cellular mechanisms that have been shown to contribute to M D R , which are summarized in Table 1.2, are briefly described below. Table 1.2 Major Mechanisms of Multidrug Resistance Drug Transport Based PgP MRP1  Enzyme Related t D N A Repair t GSH Conjugation Altered Topoisomerases  Tumor Microenvironment Vasculature-related Hypoxia ^pH  Apoptosis Control Bcl-2 family p53 IAP Ceramide  1.1.2.1 Transport-based MDR Two members of the ATP-Binding Cassette (ABC) superfamily of membrane transport ATPases, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1), have been shown to act as primary mediators of intracellular drug transportbased M D R .  When overexpressed in malignant cells they serve to actively efflux  anticancer drugs out of cells, resulting in reduced cellular drug accumulation and decreased cytotoxicity (2).  Their involvement in M D R has been corroborated by  observations that the extent of drug resistance correlates with the amount of Pgp and/or MRP1 expression (3), and introduction of these proteins into previously non-resistant tumor cells via gene transfection confers the M D R phenotype (4, 5). P-Glycoprotein The most well characterized mechanism of transport-based M D R involves the A B C family member Pgp, a 170 kDa transmembrane protein encoded by the mdr-1 gene (6, 7).  P-glycoprotein is tandemly duplicated with two homologous halves, each of  which spans the membrane 6 times and contains an A T P binding domain (8).  It is  3  believed that drags which bind to Pgp are actively effluxed out of the cell via the transmembrane portion of the protein, utilizing the energy supplied by Pgp-controlled A T P hydrolysis. Several mechanisms have been put forward to explain this transport function of Pgp. The "flippase" model postulates that Pgp encounters xenobiotics in the inner leaflet of the plasma membrane and flips the agents to the outer leaflet where they diffuse into the extracellular space (9). In the "hydrophobic vacuum cleaner" model proposed by Gottesman and Pastan (10) it is suggested that Pgp interacts directly with substrates in the plasma membrane via specific drug binding domains and pumps them out of the cell in an energy-dependent manner. Substrates for Pgp comprise a broad spectrum of agents including anthracyclines, vinca alkaloids, epipodophyllotoxins and taxanes. P-glycoprotein is expressed in many healthy tissues such as the liver, kidney, colon, small intestine and in specialized endothelial cells of the brain (reviewed in (11)). The identification of Pgp in normal tissues led to the establishment of its role as a protective mechanism in the transport and excretion of xenobiotics. In particular, the observation that Pgp is expressed in the brain suggests a role in the blood-brain barrier to prevent the permeation of drugs into the central nervous system. Whereas Pgp fulfills critical functions in transport processes involved in normal physiology, overexpression of this protein in tumor cells results in reduced intracellular accumulation of anticancer agents to sub-therapeutic levels through increased drug efflux. Direct evidence of Pgpmediated drag transport came from initial studies employing partially purified membrane vesicles in which it was demonstrated that vesicles prepared from resistant cells were more capable of binding and transporting radiolabeled vinblastine than those prepared  4  from sensitive cells (12).  Overexpression of Pgp in many tumors has since been  correlated with poor response to anticancer drug therapy and subsequent shorter survival in many clinical studies (13). In particular, a large study of patients with acute nonlymphoblastic leukemia revealed a strong correlation between Pgp detection at the time of diagnosis and a lower rate of complete regression after intensive chemotherapy, in addition to shorter patient survival (14).  Similar correlations have been made for  sarcomas (15) and neuroblastoma (16). Although Pgp is implicated in a number of multidrug resistant phenotypes, it does not completely explain the M D R phenomenon. Multidrug Resistance-Associated Protein (MRP) In the early 1990's another A B C family member, called the multidrug resistanceassociated protein (MRP1), was found to confer multidrug resistance through altered drug transport. This protein was first identified in a drug resistant lung cancer cell line that was found not to overexpress Pgp (17). Multidrug resistance-associated protein is a 190 kDa transmembrane glycoprotein that contains 17 potential membrane-spanning domains (18). It requires reduced cellular glutathione (GSH) for its function as a drug efflux pump (19), since it primarily transports drugs as glutathione or glucuronic acid conjugates (20). As with Pgp, MRP1 is expressed in normal human tissues such as muscle, lung, spleen and gall bladder (21), where it is believed to play a role in the excretion of organic anion drug conjugates. Overexpression of M R P 1 has been observed in a number of human tumors exhibiting the M D R phenotype, including lung cancer (22) and leukemias (23), and its overexpression is frequently correlated with poor clinical outcome (24, 25).  5  1.1.2.2 Enzyme-related MDR DNA Repair Enzymes Generally speaking, cell death via apoptosis (discussed in detail in Section 1.2.2) occurs when cellular damage exceeds the capacity of internal repair mechanisms. Since D N A is the primary target of many anticancer drugs, increased activity of D N A repair mechanisms reduces drug-induced damage and cytotoxicity. For example, upregulation of enzymes involved in the nucleotide excision repair pathway remove platinum-DNA adducts caused by cisplatin treatment and thus constitute a major cause of cisplatin resistance (26, 27).  Increased activity of the 0 -alkyl-guanine transferase enzyme 6  removes O -methylguanine, a common product of treatment with alkylating agents, s  which leads to resistance to this class of chemotherapeutic agents (28, 29).  Other  examples of M D R mediated by D N A repair enzymes include increased repair of single strand D N A breaks and early onset of D N A repair (reviewed in (30)). Glutathione S-Transferase (GST) Glutathione mediated detoxification pathways play an important role in eliminating chemotherapeutic agents from the body.  Glutathione S-transferase is an  enzyme system that conjugates reduced glutathione to organic molecules in order to produce polar compounds that are readily excretable. These enzymes are important in the biotransformation and detoxification of many anticancer agents. Overexpression of GST enzymes and increased intracellular levels of GSH, both of which contribute to the drug resistance by promoting anticancer drug inactivation and elimination, have been noted in many resistant cell lines (31, 32).  6  Topoisomerase Activity D N A topoisomerases are enzymes that catalyze changes in D N A topology required for replication to occur.  They act by causing either transient  single  (topoisomerase I) or double (topoisomerase II) strand breaks that facilitate D N A strand unwinding (33). Under physiological conditions these covalent enzyme-DNA cleavage complexes are short-lived intermediates that are well tolerated by the cell.  Many  anticancer agents (doxorubicin, etoposide, teniposide) exert their effects by stabilizing the DNA-enzyme complex, thus preventing D N A re-ligation from occurring. The resulting strand breaks then trigger apoptosis.  Resistance to chemotherapy drugs acting in this  manner can arise when the activity or quantity of these enzymes is reduced or when the enzyme is mutated to disfavor drug binding. 1.1.2.3 Tumor microenvironment Features of the tumor microenvironment can also contribute to M D R . Vasculature structure, vascular distribution and blood flow in malignant tumors are intrinsically different from those in normal tissues (34), and this can lead to decreased delivery of cytotoxic agents to the tumor site. The growth of blood vessels in tumors is irregular and the vasculature is often poorly formed and disorganized (35).  Furthermore, the  extracellular environment exhibits increased interstitial fluid pressure due to higher vascular permeability and the absence of a functional lymphatic system (36).  These  factors all contribute to decreased drug access to tumor cells and can lead to tumor regions which are oxygen deficient and nutrient deprived. The nutritional state of cells is known to directly influence the cellular uptake, metabolism and toxicity of some anticancer drugs (37). Nutrient deprived cells tend to proliferate slowly and the increased  7  presence of non-cycling cells renders tumors resistant to drugs that act via antiproliferative mechanisms. Hypoxic cells produce lactic acid, which creates an acidic tumor environment that further inhibits cell proliferation. This has been suggested to confer a resistance mechanism for drug- and radiation-based therapies that are most active against rapidly dividing cells (34). A n acidic tumor environment has also been suggested to confer a resistance mechanism for weak base drugs such as doxorubicin, whose cellular uptake is dependent on ionization state (38). 1.1.2.4 Alterations in apoptosis pathways Chemotherapeutic agents are generally believed to elicit their cytotoxic effects through the process of apoptosis, a form of programmed cell death (39, 40). The decision as to whether a cell undergoes apoptosis or continues to progress through the cell cycle is dependent upon the interplay between a complex set of genes and proteins that interact to regulate cell cycle progression. Drug resistance can emerge i f cells alter the expression of proteins that regulate the propagation of signals arising from cellular insults such as chemotherapy to protect against apoptosis.  While the apoptotic pathway is still not  completely understood, several proteins are known to be important regulators of this process. Bcl-2 Family The process of cellular apoptosis is regulated in part by the Bcl-2 family of proteins. Most notably, apoptosis is stimulated by expression of pro-apoptotic Bax and Bak, and is inhibited by expression of anti-apoptotic Bcl-2. Whether or not apoptosis occurs is regulated by hetero- and homodimerization of the Bax, Bak and Bcl-2 proteins. Overexpression of Bcl-2 or downregulation of Bax or Bak (all of which result in  8  increased Bcl-2 homodimer formation) has been correlated with decreased sensitivity to a wide range of chemotherapeutic agents and the emergence of resistance (41, 42). The mechanism by which this occurs will become apparent in Section 1.2.3 where mitochondria-regulated apoptosis is discussed. In addition, the apoptosis antagonists BclX  L  and Mcl-1 are also emerging as potential contributors to the M D R phenomenon.  Overexpression of B c l - X  L  has been demonstrated to confer a multidrug resistance  phenotype in some cancers (43, 44); however, the role of Mcl-1 in drug resistance is less clear. p53 Tumor Suppressor The  tumor suppressor gene p53 appears to play a significant role in  tumorigenesis, and several studies have found that p53 mutation correlates with aggressive tumor growth and recurrence (45). Its importance in cancer is highlighted by the fact that it is mutated in more than fifty percent of all human tumors (46). D N A damage, hypoxia, activation of oncogenes or loss of function of other tumor suppressor genes result in p53 accumulation (47), which either triggers cell cycle arrest in Gi so that the damage may be repaired or, if the extent of damage is deemed too great, it will direct the cell to undergo apoptosis (48). The integrity of this cellular defense mechanism is crucial for maintenance of an intact genome and, therefore, p53 is often referred to as the "guardian of the genome" (49, 50). Consequently, loss of p53 function, either through direct mutation of the p53 gene itself or via regulators of p53 function, results in disregulated cell growth, the accumulation of mutations and promotes genetic instabilityall of which may contribute to M D R .  9  Inhibitor of Apoptosis (IAP) Family Inhibitors of apoptosis proteins (IAPs) are a family of proteins that were first discovered in baculoviruses as apoptosis suppressors (51). Six IAP relatives have been identified in humans (reviewed in (52)). The mechanism used by IAPs to inhibit cell death is unclear, although there is evidence to suggest that several of the IAPs bind to pro-caspases and prevent them from being activated (53, 54) (described in Section 1.2.2). The ratio of caspases to IAPs likely determines whether a cell undergoes apoptosis, and alterations in this ratio may contribute to M D R . The LAP survivin is highly expressed in various malignant tissues and has been correlated with poor clinical outcome in some cases (reviewed in (55)). Overexpression of X-linked IAP (XIAP) has been associated with drug resistance in various pre-clinical animal models and was found to have adverse prognostic significance for patients with acute myeloid leukemias (56). 1.1.2.5 Alterations in Ceramide Metabolism The sphingolipid breakdown product ceramide has recently become the subject of considerable interest as an intracellular signaling molecule. The mechanisms of ceramide generation and its intracellular effects are discussed in detail in Section 1.3; the focus of this section will be on the specific relationship between ceramide lipids and M D R . Ceramide is recognized as a mediator of apoptosis and as a co-ordinator of cellular responses to stress (57).  Intracellular ceramide levels have been shown to  increase following exposure to many chemotherapy drugs (reviewed in (58)) and ionizing radiation (59), and cells subsequently exhibit morphologies typical of apoptosis (50, 60). Several studies have demonstrated that alterations in ceramide metabolism, whereby intracellular ceramide levels decrease and cells accumulate the non-apoptotic ceramide  10  metabolite glucosylceramide (GlcCer), contribute to the development of multidrug resistance (61-65).  In this scenario, although intracellular ceramide is generated by  chemotherapeutic agents or other stimuli, apoptosis-inducing threshold levels of ceramide are not achieved due to its rapid conversion to GlcCer.  Indeed, a number of drug  resistant cancer cell lines show higher levels of GlcCer than their drug sensitive counterparts (61), and analysis of clinical tumor specimens from patients who failed conventional chemotherapy revealed higher GlcCer levels than samples taken from those patients who responded well to treatment (66).  The level of activity of the  glucosylceramide synthase (GCS) enzyme, which converts ceramide to GlcCer, is believed to be responsible for this aspect of the M D R phenotype. This was demonstrated by retroviral transfection of GCS into drug sensitive M C F 7 human breast cancer cells. The resulting MCF7/GCS cell line expressed an 11-fold higher level of GCS, which correlated to an 11-fold increase in resistance to doxorubicin (67). Lipid metabolism studies further demonstrated that exposure of these cells to the cytokine TNF-ct, which is known to generate intracellular ceramide, caused an increase in ceramide in the drug sensitive M C F 7 cells and an increase in GlcCer in the MCF7/GCS cells. These findings suggest that increases in GCS activity and the resulting accumulation of intracellular GlcCer are important to the development of drug resistance in cancer cells.  1.2  MODULATION OF MULTIDRUG RESISTANCE The complex nature of the MDR-related drug transport, drug metabolism and  apoptosis pathways described above have presented researchers and clinicians with many  11  potential targets to pursue in an effort to promote and/or restore apoptosis signaling in resistant cells. Some of these strategies are described in the following section.  1.2.1  MDR Modulation by Drug Transport Inhibitors Identification of the molecular basis for M D R and studies of the effects of Pgp on  anticancer drugs resulted in the identification of the first generation of agents that could reverse or modulate M D R in vitro by directly inhibiting the drug efflux activity of Pgp. These modulators included pharmaceutical agents such as the calcium channel blocker verapamil  (68),  the  calmodulin  inhibitor  prochlorperazine  (69),  and  the  immunosuppressant cyclosporin A (70, 71). This chemosensitization approach involved co-administration of the M D R modulator with an anticancer drug in an attempt to block Pgp-mediated  anticancer  drug efflux and increase  intracellular anticancer  drug  accumulation. Although these first-generation modulating agents demonstrated potent M D R reversal activity when combined with anticancer agents in vitro, the doses required to achieve plasma concentrations adequate to reverse M D R in vivo (both pre-clinically and clinically) resulted in significant toxicities arising from the modulators' inherent pharmacological activities (72).  For example, whereas a 2-6 u M plasma verapamil  concentration is required to modulate M D R , the plasma concentration at which cardiovascular effects are observed is within the range of 0.4-1.2 u M (73).  These  complications highlighted the need to develop modulating agents with lower inherent toxicity and increased potency, and through these efforts a second-generation of M D R modulators was discovered.  12  Second-generation modulating agents largely comprised stereoisomers  or  structural analogs of the first generation drugs. These included dexverapamil and PSC 833 (Valspodar), a non-immunosuppressive analog of cyclosporin A . Dexverapamil was shown to reverse M D R to a degree equivalent to verapamil but without cardiovascular toxicities in several animal models (74).  The use of Valspodar has resulted in  considerable improvements in antitumor activity in ascites and solid M D R tumor models (reviewed in (75)), and is the modulating agent in the most advanced stage of development.  Phase III clinical trials combining Valspodar with various anticancer  agents have been conducted and improved responses to chemotherapy have been observed (76-79).  Generally speaking, however, co-administration of the second  generation modulators with anticancer drugs results in exacerbated anticancer drug toxicity due to altered anticancer drug pharmacokinetic and biodistribution properties (reviewed in (80)). This has been attributed to the blockade of Pgp and other membrane transporters in non-tumor tissues, and the resulting increase in anticancer drug exposure of healthy tissues has necessitated dose reduction in many studies (81), potentially compromising therapy via reduced tumor accumulation of the anticancer agent. Examples of such pharmacokinetic interactions include an 8-fold increase in plasma area under the curve (AUC) for the anticancer drug daunorubicin when combined with verapamil in rats (82), and an 8-fold increase in liver, kidney and intestinal vincristine accumulation was observed in mice following co-administration of verapamil and vincristine (83). Co-administration of Valspodar with doxorubicin in mice was shown to result in a 2-fold increase in C ax and a 10-fold increase in A U C , which was associated m  with increased toxicity (84). These complications have given rise to third-generation  13  modulating agents that have since been developed in an attempt to avoid such pharmacokinetic interactions. Third-generation modulating agents with increased Pgp specificity have been largely designed through combinatorial chemistry approaches.  This was done in  anticipation of improved therapeutic response and reduced toxicity compared to first- and second-generation modulators.  They are highly potent and can reverse M D R at  extremely low concentrations, typically in the nanomolar range. Some examples of thirdgeneration modulating agents are presented in Table 1.3. Although these newer agents appear to be active against their targets and well tolerated in combination with anticancer drugs, it remains to be seen whether they will be sufficient to achieve therapeutically effective M D R reversal in the clinic. The lack of specificity for action on tumor Pgp remains an inherent disadvantage, and alternative methods to improve the selectivity of M D R modulation at the tumor site may offer a significant advantage. Table 1.3 Third-Generation MDR Modulators Agent  Target  Company  Notes of Interest  Stage  LY335979  PGP  Eli Lilly  Phase I  • •  XR9576  PGP  Xenova  Phase I , II  • •  OC144093  PGP  Ontogen  Phase I  • • GF120918  PGP  Glaxo  Phase I • •  VX710 (Biricodar)  PGP & Vertex MRP  Phase I , II  latent modulating activity does not alter the PK of doxorubicin or etoposide does not alter the PK of Taxol® no increase in toxicity of coadministered anticancer drugs no increase in toxicity of coadministered doxorubicin or paclitaxel no increase in toxicity of coadministered doxorubicin increases doxorubicinol levels allows potentially efficacious plasma concentrations of paclitaxel to be achieved at lower doses  Refs  (85) (86, 87)  (88) (89, 90) (91-93)  14  1.2.2  Apoptosis Pathways Although M D R is a multifactorial phenomenon, a lack of cellular apoptosis is the  ultimate result regardless of the mechanism(s) involved. It will therefore be instructive to review the key pathways involved in the apoptotic response in order to understand how M D R modulation strategies may be designed to overcome biochemical alterations that lead to reduced apoptosis. Apoptosis is an important cellular process that serves to maintain normal tissue homeostasis by regulating a delicate balance between cell death and cell proliferation. Apoptosis is characterized by blebbing of the plasma membrane, externalization of the membrane lipid phosphatidylserine (PS), nuclear condensation, D N A cleavage into approximately 200 base-pair fragments and the formation of apoptotic bodies that contain self-enclosed fragments of the nucleus surrounded by cytoplasm and a cell membrane. Since the plasma membrane remains intact, the process of apoptosis does not trigger an inflammatory response and cells are rapidly engulfed by phagocytic cells such as macrophages.  These features stand in contrast to the characteristics of necrosis, the  prevailing form of cell death resulting from a non-specific injury such as blunt trauma, exposure to a toxin, or a loss of blood supply. In necrosis, cells undergo swelling and eventual rupture, and the release of cytoplasmic contents then triggers a pronounced inflammatory response (94). The apoptotic process can be divided into three major phases.  During the  induction phase cells receive and process diverse external stimuli which are integrated by the cell and a decision whether or not to commit to apoptosis is made. If the signals warrant apoptosis, cells enter the execution phase, during which the apoptotic pathways  15  become activated. Two basic pathways define the execution phase of apoptosis - the death receptor pathway (also known as the extrinsic pathway), and the mitochondrial pathway (often referred to as the intrinsic pathway), which are described in detail below. Apoptosis culminates with the degradation phase, in which the hallmarks of apoptosis become evident. Many of the biochemical and morphological features typical of apoptosis result from the selective proteolytic cleavage of cellular proteins mediated by a family of proteins known as caspases (cysteine proteases with specificity for aspartate residues: cysteine asp_artate-specific proteases). They are synthesized as dormant pro-enzymes that require proteolytic processing to become active (95-97).  Caspases may cleave and  activate other caspase family members or they may act on non-caspase targets, including proteins of the D N A repair system such as poly A D P ribose polymerase (PARP), and cytoskeletal and structural proteins such as lamanin B l and P-actin (94, 98). Although diverse stimuli can initiate apoptosis, common biochemical and morphological alterations are observed in the degradative phase, independent of the initial stimulus. This suggests that the apoptotic signals ultimately converge on common effector pathways prior to degradation.  A schematic illustration of the apoptosis  machinery and processes that will be discussed in this section is presented in Figure 1.1.  16  Death Receptor  ] Death Domain Caspase-8  Bax^  Bcl-2  •  Effector caspase (3/7)  Cyt C Apoptosome  A  P  a f  "  1  Caspase-9  Figure 1,1 A schematic illustration of apoptosis pathways highlighting the interrelationships between key apoptotic proteins (adapted from Herr et al, (99)). Abbreviations used: IAP, inhibitor of apoptosis; SMAC/Diablo, second mitochondriaderived activation of caspases/direct IAP binding protein with low pi; AIF, apoptosis inducing factor; cyt c, cytochrome c; Apaf-1, apoptotic protease activating factor-1.  17  1.2.2.1 Apoptosis induction and execution via the death receptor (extrinsic) pathway Apoptosis signaling through the death receptor pathway is typically initiated by cytokine binding to cell-surface receptors, of which members of the tumor necrosis factor (TNF) receptor superfamily are understood in the greatest detail. This family of receptors includes the Fas (CD95/Apo-l) receptor which binds Fas ligand (Fas-L), the T N F receptor (TNF-R, p55) which binds the TNFoc ligand, and the T R A I L (TNF-related apoptosis inducing ligand) receptor (TRAIL-R) which binds the T R A I L ligand (94). The Fas/Fas-L system is the most well characterized and will be described here to illustrate the intracellular cascade of events in the death receptor mediated pathway. Activation of death receptors leads to apoptosis through a cascade of proteinprotein interactions. The cytoplasmic domain of the Fas receptor contains an interaction motif known as the death domain (DD).  Binding of Fas-L to the Fas receptor causes  trimerization of the DDs, which in turn initiates the recruitment of adaptor molecules such as F A D D (Fas-associated protein with death domain; also known as MORT-1) (100). F A D D molecules interact with the receptor via common DDs at one end, while the other end of F A D D molecules contains death effector domains (DEDs) that interact with DEDs in pro-caspase-8 molecules. The complex of Fas-L/Fas-R, F A D D and procaspase-8 forms the death-inducing signaling complex (DISC).  Once assembled, the  DISC triggers the rapid self-activation of caspase-8. Activated caspase-8 can then act via two different mechanisms:  in so called Type I cells caspase-8 directly activates the  effector caspases-3, -6 and -9 (101). In Type II cells, however, the amount of caspase-8 that is generated is not sufficient to activate effector caspases and apoptosis instead proceeds through an amplification cascade involving mitochondrial dysfunction. This is  18  initiated by caspase-8 induced cleavage of the cytoplasmic protein Bid, whose C-terminal fragment (tBid) translocates to the mitochondria to activate the mitochondrial pathway of apoptosis (97). 1.2.2.2 Apoptosis induction and execution via the mitochondrial (intrinsic) pathway In contrast to the death receptor pathway, which is activated by a relatively small number of structurally related ligands, the mitochondrial pathway can be induced by a wide variety of unrelated agents, including U V and gamma irradiation, chemotherapeutic drugs, reactive oxygen species (ROS) and environmental stresses such as growth factor withdrawal or heat. Apoptosis signaling through the mitochondrial pathway is initiated by release of cytochrome c from the inner mitochondrial membrane space. Several models have been proposed to explain the process by which mitochondrial outer membrane permeability occurs and cytochrome c release occurs (95, 102). One theory depends on the phenomenon known as the mitochondrial permeability transition (PT) and the formation of a PT pore. The PT pore is a non-selective channel that forms at contact sites between the inner and outer mitochondrial membranes. Opening of the PT pore can be triggered by physiological effectors such as C a , ROS or 2+  changes in pH, and this results in a sudden increase in the permeability of the inner mitochondrial membrane to molecules with a molecular weight of less than 1.5 kDa. The PT event results in loss of mitochondrial membrane potential, osmotic swelling of the matrix and ultimate disruption of the outer membrane (103). A n alternative theory relies on the translocation of proteins/molecules to the mitochondria and the formation of pores large enough for the passage of soluble proteins such as cytochrome c. Proposed poreforming molecules include ceramide lipids (104-106) and the pro-apoptotic Bcl-2 family  19  members Bax and Bak, whose transcription is upregulated by activated p53 in response to D N A damage (94). Bax and Bak are believed to translocate from the cytoplasm to the outer mitochondrial membrane, possibly mediated by the tBid protein generated via the death receptor pathway, where they oligomerize to form pores that mediate cytochrome c release (reviewed in (95, 102)). To relate these events to the phenomenon of M D R , antiapoptotic Bcl-2 proteins have been demonstrated to inhibit Bax translocation to the mitochondria, thus preventing cytochrome c release and subsequent apoptosis activation. Whether or not apoptosis proceeds is regulated by a delicate balance between these proand anti-apoptotic Bcl-2 family members. Thus, overexpression of the Bcl-2 protein can tip the balance in favour of cell survival despite the presence of apoptotic initiating stimuli such as anticancer drugs. Once in the cytoplasm, cytochrome c facilitates assembly of the "apoptosome" by binding Apaf-1 (apoptotic protease activating factor-1) and A T P or dATP.  The  apoptosome activates pro-caspase-9 (107), which in turn activates caspase-3 (108). Other molecules that are released from the mitochondria include apoptosis inducing factor (AIF) (109), which is believed to translocate to the nucleus where it induces caspaseindependent D N A fragmentation (110) and may also directly activate caspase-3 (111), and SMAC/Diablo (second mitochondria-derived activator of caspases/direct IAP binding protein with low pi), which is believed to displace X I A P from pro-caspase-9, permitting its activation (112). In this scenario M D R can arise i f overexpression of X I A P renders SMAC/Diablo unable to facilitate the activation of caspase-9.  20  1.2.2.3 The degradation phase of apoptosis - common effector caspases Activation of both the death receptor and mitochondrial pathways culminate with activation of caspase-3 which, along with caspase-6 and -9, form the common effector caspases (113).  These caspases are responsible for cleavage of a variety of protein  substrates that disable critical cellular processes and break down structural components of the cell.  These include activation of a D N A fragmentation factor (DFF) which is  responsible for internucleosomal D N A cleavage (114), activation of C A D (caspaseactivated DNAse), cleavage of nuclear lamins involved in chromatin condensation and nuclear shrinkage, cleavage of cytoskeletal proteins such as actin, which leads to cell fragmentation and plasma membrane blebbing (111), and ultimate cell death.  1.2.3  MDR Modulation Strategies Related to Apoptotis Pathways A n increased understanding of the proteins and pathways involved in apoptosis  signaling, combined with evidence that many anticancer agents induce their cytotoxic effects via apoptosis (39, 40), has spawned great interest in developing approaches to chemosensitize tumors by altering apoptosis regulation. Some of these strategies are described below.  1.2.3.1 Downregulation of anti-apoptotic signals to chemosensitize tumors Pre-clinical and clinical trials are currently underway to evaluate the effectiveness of antisense oligonucleotide (AS-ODN) technologies directed against various antiapoptotic proteins in sensitizing M D R tumors. The most common approach to increase apoptotic susceptibility has been to down-regulate expression of apoptosis antagonists  21  using A S - O D N that block mRNA expression of the targeted protein in a nucleotide sequence specific manner. To date, the Bcl-2 family of proteins has received the most attention in this regard. Numerous studies have investigated the effect of Bcl-2 A S - O D N on drug sensitivity and several clear examples of true chemosensitization have been documented (115-117).  A pre-clinical study in mice bearing Bcl-2 overexpressing  human B-cell lymphoma showed complete cures in all mice following treatment with Bcl-2 antisense + low dose cyclophosphamide (118). Clinical trials with Genasense™ (Genta Inc.) Bcl-2 antisense are currently underway to investigate its effectiveness in combination with several different anticancer drugs. Preliminary phase I trial data in patients with relapsed and refractory lymphoma showed in vivo decreases in Bcl-2 protein levels and some antitumor activity (115). A phase I/II clinical study investigating Genasense™ + dacarbazine showed several antitumor responses in patients with resistant malignant melanoma (119). Although still in the pre-clinical stages of testing, similar approaches are aimed at downregulating LAP family members using antisense gene therapy strategies.  For example, downregulation of X I A P has been shown to induce  apoptosis in chemoresistant ovarian cancer cells (120). Anti-survivin antisense was able to downregulate survivin levels, which corresponded to complete chemosensitization of acute lymphoblastic leukemia cells to doxorubicin (121), and complete eradication of tumors derived from mouse thymic lymphoid tumors was observed in response to survivin antisense therapy (122).  22  1.2.3.2 Up regulation of pro-apoptotic signals Gene therapy approaches to introduce the normal p53 gene back into tumor cells in which it has been mutated have been investigated, and efficacy has been observed in tissue culture and animal models (123, 124). On the basis of observations that decreases in intracellular ceramide levels via conversion to GlcCer result in reduced apoptosis and the emergence of drug resistance, efforts to raise intracellular levels of pro-apoptotic ceramide by exogenous administration of the lipid or its sphingomyelin (SM) precursor have been explored.  Addition of  exogenous cell-permeable ceramide lipid enhanced the activity of paclitaxel in a human T-cell leukemia cell line (125). Co-administration of S M with the anticancer drug 5fluorouracil (5FU) resulted in significant tumor growth inhibition in an HT29 human colonic tumor xenograft mouse model (126).  Follow-up work by this group  demonstrated similar antitumor activity of S M in combination with both 5FU and irinotecan using other models of colon cancer (127). These results have been attributed to the enhancement of apoptotic cell death by increasing the intracellular pool of S M that is available for conversion to pro-apoptotic ceramide. 1.2.3.3 Modulation of ceramide metabolism The identification of GCS activity and GlcCer levels as markers for drug resistant tumors provided a new avenue for therapies directed at overcoming M D R . A number of studies have demonstrated that inhibition of GlcCer synthesis results in M D R circumvention.  L i u et al. demonstrated that downregulation of GCS activity in  doxorubicin resistant MCF7/AdrR cells by transfection with GCS antisense resulted in a 30% reduction in GCS activity, which was correlated with a 28-fold increase in  23  doxorubicin sensitivity (128). Other approaches have used GlcCer analogs such as 1phenyl-2-decanoylamino-3-morpholino-l-propanol inhibitor of GCS.  (PDMP)  (129-131),  a  specific  Combined treatment of sub-lethal concentrations of P D M P with  Taxol® or vincristine fully sensitized resistant neuroblastoma cells to the cytotoxic effects of these agents (132). Research presented in Chapter 2 extends this observation to human breast cancer cell lines and proposes an additional role for Pgp in this response (133). Both P D M P and its analog l-phenyl-2-hexadecanoylamino-3-pyrrolidino-l -propanol (PPPP) showed preferential killing of three M D R human carcinoma cell lines (134). In another approach, cytotoxicity of the novel synthetic retinoid fenretinide was found to be enhanced by modulators of ceramide metabolism such as dihydrosphingosine (safmgol) and l-phenyl-2-hexadecanoylamino-3-morpholino-l-propanol (PPMP), and synergistic relationships were observed in neuroblastoma, lung, melanoma, prostate, colon and pancreatic cancer cell lines (135).  Furthermore, pharmacologic suppression of acid  ceramidase by N-oleoylethanolamine (NOE) restored ceramide accumulation and sensitivity to cytokine-induced apoptosis in fibroblasts (136), and was demonstrated to increase endogenous ceramide levels and trigger apoptosis in resistant, metastatic colon cancer cells (137).  1.3  CERAMIDE LIPIDS AS INTRACELLULAR SIGNALING M O L E C U L E S From the preceeding sections it is apparent that ceramide lipids are of particular  therapeutic interest because of their role in apoptosis induction and their involvement in both the emergence and circumvention of M D R . These observations prompted the work discussed in this thesis, in which specific strategies to modulate ceramide levels in favour  24  of apoptosis were investigated.  Before describing this work, however, it will be  instructive to review the general biology of ceramide lipids.  1.3.1  Structure and Metabolism of Ceramide Lipids Ceramides comprise a group of cellular lipids characterized by a sphingosine base  linked to a variable length fatty acid by means of an amide linkage (Figure 1.2). Ceramides are typically classified based on the length of the fatty acid moiety. Natural ceramides isolated from mammalian membranes have acyl chain lengths that typically vary from 16 to 24 carbon atoms (Ci6-C 4-ceramide) (138, 139). These are generally 2  regarded as long-chain or endogenous ceramides and are among the most hydrophobic lipids in nature. They are found ubiquitously in the stratum corneum (140) where they have a central role in maintaining the water impermeability of the skin.  N-hexanoyl-D-erythrosphingosine (C6-ceramide) - a synthetic, short chain ceramide  Figure 1.2 The chemical structures of a natural and synthetic ceramide lipid. Both C6and Ci6-ceramide were extensively used in this thesis.  25  Ceramide constitutes the hydrophobic backbone of all the complex sphingolipids such as sphingomyelin, cerebrosides and gangliosides. Consequently, it has a central role in sphingolipid metabolism (Figure 1.3). Ceramide homeostasis is controlled by several inter-related pathways that regulate its synthesis and metabolism. Ceramide synthesis via the multi-step de novo synthetic pathway begins with the condensation of serine and palmitoyl-CoA by serine palmitoyltransferase to form 3-ketosphinganine, which is subsequently reduced to sphinganine.  The addition of an amide-linked fatty acid by  dihydroceramide synthase yields dihydroceramide. A desaturase enzyme introduces the A-trans double bond to produce ceramide. These reactions take place on the cytosolic surface of the endoplasmic reticulum. Alternatively, ceramide may be generated by activation of intracellular sphingomyelinase (SMase) enzymes that catalyze the hydrolysis of the membrane lipid sphingomyelin to ceramide and phosphocholine. Several isoforms of SMase have been identified to date and these are distinguished by pH optima and subcellular localization (141, 142). Acid SMase (aSMase) is localized in endosomes and lysosomes (143) and possibly caveolae (144), and displays a pH optimum of 4.5. A secreted form of aSMase has also been identified (145).  There also exist  several neutral SMases (nSMase) (144, 146) located in the plasma membrane, cytosol, endoplasmic reticulum and nuclear membranes.  Activation of aSMase versus nSMase  occurs differently in response to different stimuli (147). A n alkaline SMase has been identified in intestinal cells (148), although it is believed to function specifically in the metabolic degradation of dietary sphingomyelin and does not appear to have a major role in cell signaling.  26  Serine + Palmitoyl C o A ^  Serine  3 -Ketosphinganine ^ Sphingosine-1 -Phosphate  Sphinganine  SIP phosphatase | , ^ SPHkinase  \  Ceramidase  Dihydroceramide  Ceramide SM synthase •  Glucosylceramide  Dihydroceramide  Dihydroceramide  Sphingosine  Ceramide  Ketosphinganine  Ceramide kinase SMase  Sphingomyelin  Ceramide-1 Phosphate  Complex Glycosphingolipids  Figure 1.3 Sphingolipid metabolic pathways illustrating the various routes of ceramide synthesis and breakdown. Abbreviations used: S M , sphingomyelin; SMase, sphingomyelinase; GCS, glucosylceramide synthase; SPH, sphingosine; SIP, sphingosine-1 -phosphate  27  Ceramide breakdown proceeds primarily through the action of ceramidase enzymes that catabolize ceramide to sphingosine.  Three classes of mamalian  ceramidases have been identified and are again classified according to their subcellular localization and pH optima. Acid ceramidase is lysosomal (149), neutral ceramidase is mitochondrial (150), and alkaline ceramidase is found in the Golgi and endoplasmic reticulum (151). Sphingosine, in turn, can be converted to sphingosine-1-phosphate (SIP) via sphingosine-1-kinase.  Sphingosine-1-phosphate is also emerging as an important  regulator of apoptosis. It mediates proliferation and mitogenesis and tends to oppose the pro-apoptotic effects of ceramide.  In this regard the ceramide/SlP-rheostat model  proposes that the balance between intracellular ceramide and SIP levels determines cell fate (152).  Alternatively, ceramide may be metabolized to GlcCer, as described  previously, which serves as a precursor to numerous glycolipids and gangliosides.  1.3.2  Biological Targets of Ceramide Lipids and their Role in Apoptosis Although ceramide lipids were once believed to play a primarily structural role in  cellular membranes, it has become increasingly apparent that they also have important roles as regulators of cell function. In particular, the role for ceramide in mediating growth suppression and apoptosis has received significant attention. The production of ceramide is associated with numerous stress stimuli and thus, generation of ceramide has been suggested to be a universal feature of apoptosis. Several lines of evidence support this contention.  Among these are observations that many cytokines (153-155) and  environmental stresses (59, 156) known to initiate apoptosis induce rapid endogenous ceramide generation. Ionizing radiation and cytosine arabinoside (Ara-C) also elicit their  28  apoptotic effects through generation of intracellular ceramide via S M hydrolysis. The Fas receptor pathway may be responsible for aSMase-generated ceramide (157), and this mechanism has been shown to mediate doxorubicin-induced apoptosis (158).  Acid  SMase deficient mice are resistant to radiation (159) and Fas-induced apoptosis (160), the latter of which could be restored by addition of exogenous Ci6-ceramide. On the basis of correlations between apoptosis and changes in intracellular ceramide levels, it has been suggested that Ci6-ceramide in particular functions as the endogenous second messenger in apoptosis (161).  The chemotherapeutic agents paclitaxel, etoposide and the vinca  alkaloids also elicit their apoptotic effects in part by ceramide generated via the de novo pathway (58), which can be blocked by the ceramide synthesis inhibitor fumonisin B l (162).  Moreover, treatment of tumor cells with ceramidase inhibitors increases  endogenous ceramide levels and results in apoptosis (137). The addition of exogenous, cell-permeable ceramides has also been shown to induce apoptosis in many cell lines (153, 154, 157).  These effects are very specific, as the metabolic precursor  dihydroceramide, which differs only in the lack of 4-5 trans double bond, does not induce apoptosis although its uptake and metabolism are similar to that of ceramide (163). In addition, the examples of M D R reversal achieved by modulating endogenous ceramide levels previously described further support an important role for ceramide in apoptosis induction. The production of ceramide is thus emerging as an important component of apoptosis, and many mediators of apoptosis described in previous sections have been demonstrated to be both regulators of ceramide generation and downstream targets of ceramide action.  29  Ceramide presumably exerts its effects through direct molecular interactions with specific target molecules that, in turn, activate further signaling cascades. However, the exact nature of these targets and details of the downstream signaling events are not fully understood. Several direct targets have been identified, however, including phosphatases, proteases and kinases. Ceramide is known to activate serine/threonine phosphatases of the protein phosphatase-1 and -2A (PP1 and PP2A) families, which are collectively termed CAPP (ceramide activated protein phospatases).  Activation of CAPP promotes  growth suppression via dephosphorylation of the pro-growth cellular regulators P K C a , P K B , Bcl-2 and the retinoblastoma protein Rb (164, 165). Ceramide is also known to activate ceramide activated protein kinase (CAPK; also known as K S R - kinase suppressor of Ras) which phosphorylates Raf-1 and in turn activates the M A P kinase pathway.  This pathway culminates with inactivation of P K B , whose kinase activity  maintains the pro-apoptotic Bcl-2 family member Bad in the inactive form. Therefore, ceramide mediated inactivation of P K B promotes Bad-triggered cell death (166). Endosomal/lysosomal cathepsin D has also been identified as a ceramide binding protein, and it is believed that ceramide produced in this compartment induces release of the protease into the cytosol where it initiates a proteolytic cascade leading to apoptosis (167, 168). Although changes in endogenous levels of ceramide are observed in response to apoptosis-inducing agents and have been shown to preceed the onset of typical hallmarks of apoptosis (57), the exact mechanism(s) of ceramide-induced apoptosis remain enigmatic. Recent studies are pointing toward the mitochondria as an important mediator of ceramide-induced apoptosis. This is perhaps not surprising, given the central role of  30  mitochondria in regulating apoptosis signaling cascades. Caspase-8 activation has been shown to trigger the formation of ceramide (94, 169), which in turn activates the P A R P cleaving protease caspase-3 (170). The activation of aSMase by Fas and TNFct receptor ligation, both of which contain death domain regions that activate caspases, provide yet another link between caspases, ceramide and apoptosis (166). Ceramide has been shown to exert direct effects on isolated mitochondria and intact cells through the generation of ROS and induction of cytochrome c release (104), possibly via pore formation in mitochondrial membranes (105, 106).  Such pore formation is consistent with the  molecular geometry of natural ceramide lipids, which will be described in Section 1.4.1.3. The activation of caspases-9 and -3 by cytochrome c provides a link between ceramide signaling and execution caspases (171). The identification of components of sphingolipid signaling pathways such as sphingomyelin, ceramide and ceramide synthase within mitochondrial membranes further suggests the existence of a mitochondrial pathway of ceramide metabolism that may regulate apoptosis (172). Support for this comes from a study in which bacterial nSMase was targeted to different cellular compartments, including the plasma membrane, cytoplasm, mitochondria, Golgi, endoplasmic reticulum and nucleus. While ceramide levels were shown to increase in all compartments, only mitochondrial ceramide accumulation caused apoptosis (173), suggesting a role for endogenous mitochondrial ceramide in apoptosis. Overexpression of anti-apoptotic proteins such as Bcl-2 or Bcl-xL inhibit cell death but do not affect upstream ceramide generation, indicating that elevated ceramide is not simply a consequence of cell collapse (174).  31  1.3.3. Ceramide Signaling via Lipid Rafts In addition to the ceramide targets described above, emerging evidence has begun to suggest that some of the effects of ceramide are mediated by its unique biophysical properties. The polar head group, amide linkage and hydroxyl groups of the sphingosine and fatty acid chains enable ceramide lipids to act as both donors and acceptors of hydrogen bonds. This gives them the capacity to form extensive hydrogen bonds in the phospholipid bilayer.  Consequently, a relatively new focus of ceramide-mediated  apoptosis involves the formation of ceramide signaling platforms. The plasma membrane is largely composed of cholesterol and sphingomyelin, the latter of which is almost exclusively localized to the cytoplasmic leaflet. Hydrogen bond formation and hydrophobic van der Waals interactions mediate a tight association between the sterol ring of membrane associated cholesterol and the sphingomyelin pool. This results in a lateral segregation of these lipids into gel-like regions of tightly packed sphingolipids called rafts, which "float" in the liquid-crystalline phospholipid portion of the cell membrane (175). These membrane rafts are believed to act as specific sites for ceramide generation in response to various agonists and stress signals. For example, Fas receptor stimulation was shown to result in translocation of aSMase to the extracellular leaflet of the cell membrane (176, 177) where it mediates hydrolysis of sphingomyelin to ceramide within lipid rafts.  Ceramide itself has been shown to self-aggregate into  microdomains (178, 179) which are capable of fusing into larger macrodomains known as platforms (180). One possible mechanism for the biological action of ceramide is through changes in membrane structure and organization via formation of these platforms, which may serve to cluster and activate ligand-bound receptors in a process  32  known as capping (176, 181). In support of this theory, Cremesti et al. demonstrated that addition of exogenous Ci6-ceramide to soluble Fas ligand promoted receptor crosslinking (capping) and subsequent apoptosis (177).  The formation of ceramide signaling  platforms is likely involved in other signaling cascades, and may provide an explanation for ceramides' pleiotropic intracellular effects. Since ceramide lipids appear to be important in the context of apoptosis and multidrug resistance, the focus of this thesis was to better understand the role of ceramide in M D R and to identify ways in which these lipids could be directly introduced into tumor cells to induce apoptosis in a therapeutic setting.  Given that ceramides are  naturally occuring lipids, liposome-based delivery systems were investigated as delivery vehicles to efficiently introduce ceramides into tumor cells.  The following section  describes the preparation and properties of liposomes in this context.  1.4  LIPOSOMAL DRUG DELIVERY SYSTEMS Liposomes were first described by Bangham and colleagues who observed that  spherical membrane vesicles spontaneously formed upon hydration of dried lipids in aqueous solutions (182).  Liposomes were initially developed as model membrane  systems to evaluate the structural and functional roles of lipids in biological membranes (183). However, they were later developed for use as delivery vehicles for conventional drugs, and are more recently evolving as carriers of bioactive lipids.  33  1.4.1  Review of Liposomes Over the past several decades liposomes have progressed from conventional  phospholipid/cholesterol-based  first-generation  formulations,  to  sterically stabilized systems, to the more recent third-generation  second-generation multifunctional  liposomes that may incorporate multiple small molecule compounds, nucleic acids and bioactive lipid components (illustrated in Figure 1.4). These modifications have allowed for the production of liposomes with increased stability and longevity in the circulation, as well as enhanced  Figure 1.4 A schematic illustration of a hypothetical multifunctional liposome incorporating agents directed against multiple intracellular targets.  34  active targeting capabilities and controlled drug release properties triggered by p H (184, 185) or temperature (186). Many liposome formulations have been developed as effective carriers for the delivery of anticancer drugs (187-193), antimicrobial agents (194-196), genes (197) and antisense oligonucleotides (118, 198-200).  Some of these formulations have already  been approved for human use, and others are in various stages of clinical trials (reviewed in (201)). The therapeutic advantage of liposome-based drug delivery systems can be attributed to two main characteristics. First, liposome encapsulation mediates changes in drug circulation lifetimes and tissue distribution characteristics whereby these parameters are dictated by the liposomal carrier rather than the drug itself. For example, a drug such as doxorubicin, which exhibits rapid elimination from the plasma compartment following administration in its free form can remain in the circulation in excess of 24 hours when encapsulated in a sterically stabilized liposome. The second and perhaps most unique feature of liposome-based drug delivery systems stems from their use in enhancing drug accumulation at disease sites, which leads to a selective accumulation of drug in diseased versus healthy tissues. This process, termed passive targeting because it occurs in the absence of specific targeting ligands, is mediated by the natural process of extravasation and preferential accumulation of liposomes at sites of inflammation (202), infection (203) and tumor growth (204, 205). Whereas the permeability of normal vasculature structure is tightly controlled, the specific example of passive tumor accumulation is achieved because the tumor microvasculature is typically discontinuous and contains fenestrations varying between 100-780 nm in diameter (206). This allows liposomes to extravasate into the tumor tissue to provide locally concentrated drug delivery. Such tumor-specific  35  accumulation offers the additional advantage of decreasing drug accumulation in nontarget tissues, thereby reducing drug-associated toxicities in healthy tissues. 1.4.1.1 Liposome preparation and classification When most amphipathic phospholipids are dispersed in an aqueous buffer they spontaneously form bilayers (Figure 1.5).  Hydration of a dried lipid film produces  bilayer structures arranged in concentric rings called multilamellar vesicles (MLVs). Multilamellar vesicles are of limited value for pharmaceutical applications due to their heterogenous diameter range (1-10 microns) and rapid elimination from the plasma following in vivo administration (207). However, the size and lamellarity of M L V s can be modified by sonication, reverse-phase evaporation or extrusion techniques (208, 209). The most versatile and frequently utilized method involves extruding M L V s through polycarbonate filters under high pressures of an inert gas. This produces a homogenous population of large unilamellar vesicles (LUVs) of well-defined size that may be controlled by filter pore size (50-400 nm range). Large unilamellar vesicles are the most suitable for pharmaceutical applications due to their stability in the circulation and optimal passive targeting properties. Small unilamellar vesicles (SUV) in the 25-50 nm range can be prepared by sonication but are generally unstable due to the high radius of curvature of the membranes, making them unsuitable for pharmaceutical applications. Figure 1.6 depicts these three classes of liposomes. Both M L V s and L U V s were used in this research, and the extrusion procedure, which is described in detail in Chapter 3, was used for the preparation of all LUVs.  36  Figure 1.5 A n illustration of spontaneous bilayer formation upon hydration of amphipathic lipids in an aqueous buffer.  Multilamellar Vesicles (MLVs)  Large Unilamellar Vesicles (LUVs)  Small Unilamellar Vesicles (SUVs) Figure 1.6 Schematic illustrations and scanning electron micrographs of the three main liposome classes: unilamellar vesicles (MLVs, A and B); large unilamellar vesicles (LUVs, C and D); small unilamellar vesicles (SUVs, E and F). The bar in the lower left of image B represents 200 nm. Adapted from Ostro and Cullis, 1989 (210).  38  1.4.2.1 Lipid Composition Phospholipids Phospholipids are a class of amphipathic lipids that form one of the major components  of physiological  membranes  and synthetic  liposomes.  Although  phosphatidylcholine (PC) is the most abundant phospholipid in nature, other naturally occurring phospholipids include phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). Phosphatidylcholine is the phospholipid predominantly used in the research presented in this thesis. A generic phospholipid with commonly occurring headgroups and fatty acid moieties is illustrated in Figure 1.7. This illustration depicts the hydrophilic phosphate-containing head group and hydrophobic acyl chains esterified to a glycerol backbone.  The distinct properties of each phospholipid are  determined both by the nature of the head group and the composition of the acyl chains. Different head groups give rise to phospholipids which may be anionic (PS, PI) or neutral (PC, PE) at physiological pH. Naturally occurring cationic lipids are extremely rare. However, cationic lipids such as l,2-dioleoyl-3-trimethylammonium propane (DOTAP) have been synthesized for use in gene therapy studies in which the cationic lipids are complexed with D N A (211,212). Variability of the acyl chains arises from alterations in the hydrocarbon length and the degree of saturation.  The acyl chain length of  phospholipids comprising physiological membranes typically ranges from 12-24 carbon atoms, and both mono- and polyunsaturated acyl chains are common in nature.  39  'o  X p o V  •  S  O  ozH u o  ^  I §5.1 o O o go  2 o O u  o  I—I  V  ™s3 DI u u ^ ,. . V £C 2* S£Ho u ,, u u u l i U  £C  o  5  »ZC  ">  'ft. o  ns O  ft.  X I  O  M  £C  r»  t-C  *r w  <o  *X!  *X*  o o u  f-t  «  r*  If O  D.  «  o  y,  »*•*  £C  wC  CC  u o u o u  u  tX«  u  u u u  u  5  ' 'C  8  ft. 5» • g>  &  u  ^T*  1  »C  c  •9o ft.  _o -aa.  ,y to  ft.  > x: »  'ao  j2s  1 S  ,j  2  &. to  <  u  33  o  j.>. _»_  -a  J* c £  o  O  I  2 '8 .  >j • S  2  w  v\  Dl(  r—. MM  I "-e5 o  M  £C  u u u u u y  u  u  "S  *C  o o o o o o o o o o u u u u u  33  3  £C  o  o o '£  J  J  §  2  o  I £ - o — o — 0 o I 9 . ~ u — O — U — 0 — U — U — U — U — 0 — O — U = U — U — O — U — O — O — U — Q — u I o £ II £ £ £ £ a ? x £ £ a ? x x x ' - £ x £ £ £ u — q — o — o — u — u — o — u ~ - 0 — u — u — u — o — u — u — 0 — u — u — u — u 1  3  OX)  40  Cholesterol Cholesterol is a neutral, amphipathic physiological membranes.  lipid that is commonly found in  Its planar ring structure allows it to intercalate between  phospholipids in the bilayer, where the hydrophobic steroid ring can interact with phospholipid acyl chains via hydrophobic and van der Waals interactions. The hydroxyl group of cholesterol makes this otherwise hydrophobic molecule amphipathic and mediates hydrogen bond formation between the polar head groups of neighboring phospholipids and the aqueous environment.  The rigidifiying effect of incorporating  planar cholesterol into liposomes can be used to advantage in stabilizing liposome bilayers against lipoprotein-liposome interactions (213). The interaction of liposomes in vivo involves the binding of plasma proteins and lipoproteins, which dictates the subsequent interaction with the major elimination mechanism for liposomes - the reticuloendothelial system (RES). Liposome interactions with lipoproteins often result in extensive lipid exchange from the liposome bilayer into lipoproteins, which may result in the ultimate dissolution of the liposome. However, it was demonstrated that liposomes containing >30 mole percent cholesterol in the membrane were able to avoid lipoproteinmediated liposome destabilization.  The presence of cholesterol also makes it more  difficult for proteins to penetrate and disrupt the membrane structure (214-216) and results in reduced liposome opsonization and removal via phagocytic cells of the RES. The cholesterol derivative cholesteryl hemisuccinate  (CHEMS) contains a  succinate head group that is negatively charged at physiological pH and is therefore more polar than the single hydroxyl head group of cholesterol.  However, in an acidic  environment (pH<5.8) C H E M S becomes charge neutralized (protonated). This alters its  41  overall lipid geometry (described below), which has implications for liposome destabilization that can be exploited to achieve triggered drug release (217, 218). The chemical structures of cholesterol and C H E M S , both of which were used in this thesis, are shown in Figure 1.8. Polyethylene glycol-modified lipids Polyethylene glycol (PEG) is a flexible, hydrophilic polymer consisting of repeating units of ethylene glycol (CH -CH -0)n that is often chemically conjugated to 2  2  the headgroup of PE, which serves as a lipid anchor (Figure 1.9). Incorporation of PEGPE into liposome bilayers gives rise to "sterically stabilized" liposomes that demonstrate greatly improved liposome stability in the circulation (219, 220). Much like cholesterol, the ability of P E G to prolong liposome circulation longevity is believed to involve reduced interaction with lipoproteins and decreased serum protein binding to the liposome surface.  Whereas this was achieved through a rigidifying effect of the  membrane by cholesterol, P E G acts through reduced recognition by cells of the RES. Polyethylene glycol acts as a steric barrier that inhibits the close approach of plasma proteins that would otherwise mark non-PEGylated vesicles for elimination through the stimulation of phagocytic macrophages (221-224). It has also been speculated that the P E G coating may reduce cellular uptake directly, so that even in the presence of bound plasma proteins the P E G coating will inhibit receptor mediated binding at the level of the macrophage (225). Sterically stabilized liposomes containing PEG ooo-DSPE were used 2  for all in vivo studies in this thesis.  42  Cholesteryl hemisuccinate  Figure 1.8 Chemical structures of cholesterol and cholesteryl hemisuccinate lipids.  43  PEG P O L Y M E R  LINKER  LIPID A N C H O R  Figure 1.9 A schematic illustration of a pegylated liposome showing the chemical structure of polyethylene glycol derivatized phosphatidylethanolamine (PEG-PE).  44  1.4.1.2 Lipid polymorphism One factor to consider when designing liposomes is the molecular shape of the individual lipid components. In particular, the size and shape of the polar head group in relation to the hydrophobic acyl chains is important, as this largely dictates the type of lipid structure that is formed in an aqueous environment (226). The ability of lipids to adopt structures other than a bilayer has been described as lipid polymorphism. Table 1.4 illustrates the three main classes of lipid geometries and their respective phase behaviours upon dispersion in an aqueous environment. Table 1.4 Molecular Shape of Various Lipids and their Associated Structures (adapted from Cullis et al, 1996 (226)) Lipid  Lysophospholipids Detergents PEG-conjugated lipids Short-chain ceramides  Phosphatidylcholine Phosphatidylserine Sphingomyelin Cholesteryl hemisuccinate (neutral pH) Short-chain ceramides  Phosphatidylethanolamine Cholesteryl hemisuccinate (acidic pH) Long-chain ceramides  Molecular Shape  Structure  Inverted Cone  Micelle  Cylinder  Bilayer  Cone  Hexagonal (HII)  45  The large cross-sectional area of the polar head groups of lipids such as lysolipids and detergents relative to their single acyl chain gives rise to an inverted cone geometry that confers a tendency for these lipids to form micellar structures in an aqueous environment.  Many phospholipids have a cylindrical geometry in which the cross-  sectional area of the polar head group and non-polar acyl chains is approximately equal. These lipids spontaneously arrange into stable bilayers in an aqueous environment. When the cross-sectional area of the headgroup is small relative to that of the acyl chains the lipid takes on a cone-shaped geometry.  This confers a tendency to spontaneously  form hexagonal phase (Hn) or inverted micelle structures, and this particular organization is involved in membrane destabilization and fusion.  Natural (long-chain) ceramide  lipids, which are a primary focus of this thesis, fall into this latter category of molecular shape due to the small head group relative to the long fatty acyl tails (227). This limits the extent to which they can be stably incorporated into liposome bilayers. Cholesteryl hemisuccinate is a bilayer forming lipid at physiological pH (negatively charged polar head group) but charge neutralization of the head group upon protonation at low pH causes the molecular geometry to change from that of a cylinder to a cone, and the lipid therefore forms inverted micellar structures in an acidic environment.  This causes  liposome destabilization and forms the basis of pH sensitive liposomes, which have been described  for  the  intracellular delivery of liposome  encapsulated  drugs  and  macromolecules (218). Cholesteryl hemisuccinate can be used to stabilize the bilayer structure of non-bilayer forming lipids such as DOPE, and the work presented in Chapter 3 of this thesis extends this application to the stabilization of natural ceramide lipid in ceramide-containing liposome bilayers.  46  1.4.2  Liposomes as Drug Carriers Liposome-based drug delivery systems typically consist of a drug or small  molecule compound encapsulated either in the aqueous vesicle core or partitioned into the lipid bilayer of liposomes. There are two basic strategies for encapsulating drugs within liposomes. Entrapment of either hydrophilic or lipophilic drugs may be achieved during the vesicle formation process (passive trapping) or by loading amphipathic drugs into pre-formed liposomes in response to a transmembrane pH gradient (active trapping). The drug distribution patterns for these encapsulation procedures are illustrated in Figure 1.10. The passive loading procedure was used to encapsulate [ C]lactose for trapped 14  volume studies described in Chapter 3, and is the method by which the ceramidecontaining liposomes described in Chapters 3-5 were prepared.  Most conventional  anticancer drugs are currently encapsulated using procedures based on the active loading technique.  The active loading procedure was used to encapsulate doxorubicin and  verapamil for the drug release studies presented in Chapter 4. 1.4.2.1 Passive encapsulation Passive encapsulation of hydrophobic drugs involves including the compound in the original lipid mixture prior to hydration. The antifungal agent Amphoteracin B is one example of a drug that is encapsulated in this manner (228).  The encapsulation  efficiency of this technique depends on the capacity of the lipid bilayer to solubilize the drug while maintaining its vesicular structure.  Although trapping efficiencies  approaching 100% can be obtained, drugs of this class often exhibit appreciable exchange rates into other membranes and the drug rapidly leaves the liposome carrier in vivo (228).  47  This phenomenon is addressed by the development of a lipid/drug exchange assay that forms the basis of Chapter 4.  Passive Encapsulation — hydrophobic drug • Drug distributes into lipid bilayer  Passive Encapsulation — hydrophillic drug • Drug distributes into aqueous vesicle interior  Active Encapsulation — drug containing ionizable amine group • In absence of p H gradient, drug localized primarily in extra-liposomal buffer (pH 7.4)  • In presence of p H gradient, drug localized primarily in vesicle interior buffer (pH 4.0)  Figure 1.10 Drug distribution following active and passive encapsulation for hydrophobic and hydrophilic drugs  48  Passive encapsulation of hydrophilic agents involves dissolving the drag in the aqueous buffer used to hydrate the lipid film.  The encapsulation efficiency of this  method, therefore, depends on the internal aqueous volume of the liposome. The trapped volume of L U V s , which typically range from 1-10 u,l/umole lipid, can result in encapsulation efficiencies in excess of 50% (229). The retention of hydrophilic drugs trapped in this manner depends on the nature of both the drug and the lipid membrane, as well as the concentration of liposomes during hydration and extrusion.  Drugs that  possess some degree of lipophilic character, such as doxorubicin, exhibit more rapid release than membrane impermeable drugs (230).  Lipid bilayers composed of long-  chain, saturated phospholipids and cholesterol can also improve drug retention by decreasing membrane permeability. 1.4.2.2 Active encapsulation Active loading procedures are designed to load drug into the interior of preformed liposomes exhibiting a transmembrane proton gradient (inside acidic). Drags that are encapsulated by this method are typically lipophilic cations with ionizable amine groups, such as doxorubicin. In the neutral, extra-liposomal environment the drag is predominantly uncharged and can readily diffuse across the liposome bilayer when added to the liposome suspension. However, the drug is readily protonated upon reaching the acidic vesicle interior and becomes effectively trapped, as the charged species is significantly less membrane permeable.  Since many drugs are lipophilic weak base  amines, this mechanism of active loading has widespread applications (231). Although encapsulation efficiencies approaching 100% can be achieved with active loading procedures, the drag retention properties vary considerably and are dependent on the  49  membrane permeability properties of the drug and the ability of the liposome formulation to maintain the proton gradient (232). 1.4.3  Drug Delivery Approaches to MDR Modulation Despite the fact that we now know more than ever about the various mechanisms  that contribute to M D R , the fact remains that relatively small advances have been made to circumvent M D R and improve the clinical response of refractory tumors. Some of the growing evidence to suggest that liposomes are capable of providing many of the features that will likely be useful in improving the treatment of M D R tumors is presented below.  1.4.3.1 Application of liposomes to MDR reversal Liposomes can been applied to the treatment of M D R tumors using three basic approaches:  (1) using liposomes as carriers of anticancer drugs in order to increase  tumor-specific drug delivery, (2) combining potent conventional M D R modulators with liposomal anticancer drugs in order to reduce adverse drug/modulator pharmacokinetic interactions, and (3) incorporating M D R modulators into the liposome bilayer or encapsulated within the liposome core for tumor-specific M D R reversal. The advantages achieved by applying liposomes to M D R reversal strategies stem primarily from their ability to passively accumulate in solid tumors due to the increased permeability of tumor blood vessels relative to the vascular lining of healthy tissues (233). This allows for increased selectivity of anticancer drug delivery to the tumor site and reduced drug accumulation and toxicities in healthy tissues, which can provide tumor associated drug levels that are up to 10-fold higher than achievable with conventional aqueous anticancer drug formulations (205, 234). This increase in tumor drug exposure  50  has been shown to partially overcome drug resistance in M D R tumors, leading to enhanced antitumor activity independent of additional agents aimed at blocking molecular mediators of M D R (81, 235). This presumably reflects the effects of massaction driven improved intra-tumor drug accumulation that arises from elevated drug concentrations in the tumor interstitial space. While the increased dose intensity and selective tumor delivery afforded by liposomes improves the treatment of M D R tumors, this in itself is not sufficient to completely overcome resistance (236). A n exciting recent advance in liposomal anticancer drug delivery concerns the use of triggered drug release to dramatically increase the bioavailability of encapsulated agents specifically at the tumor site. This technology utilizes thermosensitive liposomes from which drug release can be dramatically increased by selective mild heating of tumors to 41-42°C (186).  This causes rapid exposure of tumor tissue to high  concentrations of anticancer drug and has been shown to cause complete regression of human xenograft solid tumors, which was not observed following treatment with conventional liposomal carriers (237, 238).  It will be particularly interesting to see  whether this technology can further enhance the treatment of M D R tumors by virtue of significant increases in tumor drug exposure. As an alternative approach, co-administration of M D R modulators with liposomal anticancer drugs was investigated in an attempt to alleviate the complications associated with altered Pgp function in healthy tissues by virtue of selective drug delivery to tumor tissue. It was found that co-administration of the Pgp inhibitor Valspodar with liposomal doxorubicin eliminated the adverse pharmacokinetic interactions observed upon Valspodar administration in combination with free doxorubicin (84).  This approach  51  allowed for the ability to deliver full dose chemotherapy in combination with Valspodar and resulted in full chemosensitization and marked tumor regression in M D R solid tumors (81).  This improved therapeutic index was attributed to the selective  accumulation of doxorubicin in tumor tissue so that anticancer drug toxicity associated with Pgp blockade in healthy tissues was minimized. In addition, the use of liposomes to selectively deliver anticancer drugs to solid tumors should minimize non-selective action of other chemosensitization approaches in healthy tissues, thus providing increased specificity of M D R modulation effects for tumor cells. Given the inability of current M D R modulating agents to differentiate between tumor and non-tumor Pgp, one particularly attractive application of liposome technology would be to encapsulate the modulating agent itself in a liposome-based delivery system. Since liposomes have a natural tendency to accumulate at tumor sites, such a system would enable the modulator to induce tumor-specific Pgp blockade, further minimizing the adverse toxicities of co-administered anticancer agents on healthy tissues.  This  would have the added benefit of increased versatility whereby a single liposome-based M D R modulator could be co-administered with any number of anticancer drugs in their free form, or perhaps even as co-encapsulated agents. Liposome encapsulation of M D R modulators has indeed been investigated, but to date the modulating agents have not proven to be amenable to formulation into stable, systemically viable liposomes. Although M D R modulating agents such as verapamil, prochlorperazine, cyclosporin A and the proprietary Pgp inhibitor OC144-093 can be efficiently encapsulated into liposomes, their high membrane permeabilities and rapid leakage following systemic administration has limited development in this area.  52  1.4.3.2 Application of liposomes to the delivery of bioactive lipids Although liposome-based drag delivery has primarily focused on delivery of encapsulated drugs as described above, they are more recently being explored as drug delivery vehicles for bioactive lipids that form part the liposome bilayer itself. For example, liposomes containing phosphatidylserine (PS) in the bilayer have been demonstrated to induce selective thrombosis after binding to V C A M - 1 in endothelial cells (239, 240).  The work contained in Chapters 3-5 of this thesis was aimed at  extending this application of liposomes to include liposome-based intracellular delivery of pro-apoptotic ceramide lipids in order to achieve chemosensitization and tumor cell death.  1.5  THESIS OBJECTIVES AND HYPOTHESIS The apparent involvement of ceramide lipids in both apoptosis and multidrug  resistance suggests that therapeutic approaches aimed at modulating ceramide, by increasing intracellular ceramide levels and/or by decreasing ceramide metabolism, should provide enhanced activity against both sensitive and M D R tumors. Consequently, the research contained in this thesis was designed to evaluate the therapeutic application of ceramide lipids within this context.  The following four specific objectives were  designed to test the overall hypothesis that therapeutic manipulations aimed at increasing intracellular ceramide levels will result in apoptosis:  Specific Objective 1: to investigate the effect of inhibiting ceramide metabolism on the sensitivity of multidrug resistant tumor cell lines to conventional chemotherapeutic agents  53  Specific Objective 2: to investigate the relationship between ceramide acyl chain length, intracellular delivery and apoptosis induction using exogenously applied ceramide lipids in vitro, and to determine whether these parameters could be enhanced by formulating ceramide into liposomal carriers Specific Objective 3: to develop an in vitro exchange assay that allows the in vivo drug/lipid retention properties of liposomal systems to be predicted Specific Objective 4: to characterize the in vivo pharmacokinetics and antitumor activity of ceramide-based liposomes  The experiments designed to test these specific objectives and the results that were obtained are presented in Chapters 2-5. The summarizing discussion presented in Chapter 6 outlines the overall conclusions drawn from these studies, and highlights areas of particular interest for potential future research.  54  CHAPTER 2  R O L E OF 1-PHENYL-2-DECANOYLAMINO-3-MORPHOLINO-l -PROPANOL (PDMP) AND P-GLYCOPROTEIN IN MODULATING CERAMIDE-MEDIATED SENSITIVITY OF HUMAN BREAST CANCER CELLS T O TUBULIN-BINDING ANTICANCER DRUGS*  2.1  INTRODUCTION AND RATIONALE As described in Chapter 1, overexpression of the drug efflux pump Pgp has long  been regarded as a major cause of M D R in a number of human malignancies. Multidrug resistance reversal agents often act by blocking Pgp's drug efflux activity, thereby increasing intracellular drug levels and inducing cell death. However, as more has been learned about Pgp its functional role has expanded to include activity as a drug flippase and phospholipid translocator (241), and it has been shown to transport short-chain fluorescent analogues of S M and GlcCer across membranes (242). In addition, Pgp may play a role in regulating some caspase-dependent apoptotic pathways, a function completely independent of its drug/lipid transport properties (243).  In light of these  expanding roles it is possible that Pgp may also be involved in regulating ceramide-based apoptosis and metabolism.  As discussed in Chapter 1, although ceramide is a pro-  apoptotic lipid its GlcCer metabolite is not. Thus, conversion of ceramide to GlcCer by the GCS enzyme is one mechanism by which cells can lower intracellular ceramide levels and avoid apoptosis. The correlation between ceramide, GlcCer and M D R suggests that inhibiting ceramide glycosylation should keep intracellular levels of the pro-apoptotic  'Adapted from: J A Shabbits and LD Mayer (2002). P-glycoprotein modulates sensitivity of human breast cancer cells to tubulin-binding anticancer drugs. Therapeutics, 1: 205-213.  ceramide-mediated Molecular Cancer  55  lipid elevated, making it a potential approach for inducing apoptosis and circumventing MDR. 1-phenyl-2-decanoylamino-3-morpholino-l -propanol (PDMP) is a well-known inhibitor of the GCS enzyme that has been shown to decrease GlcCer production and promote ceramide accumulation (130). Previous work has shown that P D M P sensitizes murine neuroblastoma cells to treatment with microtubule-affecting cytostatics (132). The research presented in this chapter describes experiments designed to evaluate P D M P mediated chemosensitization in two human breast cancer cell lines. Based on results obtained in early studies, the involvement of Pgp in ceramide-mediated cell death and chemosensitivity was also investigated. Cells which overexpress both Pgp and GCS, having developed resistance through drug selection, versus cells that were transfected with the mdr-1 gene and thus overexpress only Pgp, were specifically compared in order to investigate whether Pgp-based transport plays a specific role in ceramide-mediated chemosensitization and apoptosis.  2.2  HYPOTHESIS The hypothesis underlying the research presented in this chapter is that the  inhibition of ceramide metabolism to GlcCer by PDMP will increase endogenous ceramide levels and sensitize multidrug resistant cells to co-administered conventional chemotherapeutic agents.  56  2.3  MATERIALS AND METHODS  2.3.1  Materials Dulbeco's Modified Eagle's Medium (DMEM), RPMI 1640 Medium (RPMI) and  Hank's Balanced Salt Solution (Hank's) were obtained from Stem Cell Technologies (Vancouver, B C , Canada). Fetal bovine serum was obtained from Hyclone (Logan, UT). L-glutamine and trypsin-EDTA were purchased from Gibco B R L (Life Technologies, Burlington, O N , Canada).  Eppendorf tubes were obtained from V W R (West Chester,  PA). Formaldehyde was from Polysciences (Warrington, PA). Tween-20, D A B C O antifade, goat serum and all chemicals were obtained from the Sigma Chemical Company (St. Louis, M O ) , unless otherwise indicated. M T T reagent was purchased from SigmaAldrich  Canada  (Oakville,  ON,  Canada).  Human  IgG  was  from  ICN  (ImmunoBiologicals, Lisle, IL). Mouse isotype control-PE (Phycoerythrin), mouse antihuman Pgp-PE and goat anti-mouse IgG-FITC antibodies were obtained from B D Biosciences (Mississauga, O N , Canada).  DAPI stain was from Molecular Probes  (Eugene, OR). The C219 primary antibody was from Signet Laboratories (Dedham, M A ) . Microtitre (96-well) Falcon® plates and culture flasks were obtained from Becton Dickinson (Franklin Lakes, NJ). (Montreal, QC, Canada).  Taxol® was obtained from Bristol-Myers Squibb  Vincristine sulfate, cisplatin and doxorubicin-HCl were  obtained from Faulding (Canada)  Inc. (Vaudreuil, QC, Canada).  PDMP  and  glucosylceramide (Gaucher's spleen) were from Matreya Inc. (Pleasant Gap, PA). C^and Ci6-ceramide lipids were from Avanti Polar Lipids (Alabaster, A L ) . Valspodar was from Novartis (Dorval, QC, Canada). [ H]Taxol® was from Moravek Biochemicals Inc. 3  (Brea, CA). [ H]palmitic acid was from N E N (Boston, M A ) . Pico-fluor 40 scintillation 3  57  cocktail was purchased from Packard Biosciences (Groningen, The Netherlands). Silica gel G chromatography plates were from Analtech (Newark, DE).  2.3.2  Cell Lines and Culture The MDA435/LCC6 and M D A 4 3 5 / L C C 6 * human estrogen receptor negative MD  /  breast cancer cells were a generous gift from Dr. Robert Clarke, Georgetown University, Washington, D.C. The MCF7 human estrogen receptor positive breast adenocarcinoma cells were obtained from the American Type Tissue Collection (ATCC; Rockville, MD). The adriamycin resistant MCF7/AdrR cells were obtained from Dr. Gerald Batist at the Lady Davis Research Institute (Montreal, QC, Canada). A l l cell lines were grown as adherent monolayer cultures in 25-cm Falcon® flasks in D M E M (MDA435/LCC6 cells) 2  or R P M I 1640 (MCF7 cells) culture medium supplemented with 10% fetal bovine serum and 1% L-glutamine. Cells were maintained at 37°C in humidified air with 5% C 0 and 2  were sub-cultured weekly using 0.25% Trypsin with 1 m M E D T A .  2.3.3  Evaluation of Cell Surface Pgp Expression by Flow Cytometry Cells were harvested with 0.25% Trypsin + 1 m M E D T A , counted with trypan  blue and the concentration was adjusted to 10 cells/ml. Aliquots of 10 cells per cell line 7  6  were resuspended in 0.1 ml PBS + 0.1% B S A (PBSB) + 20% human serum and incubated on ice for 15 minutes.  Cells were incubated with anti-human Pgp-PE  (Phycoerythrin) or isotype control-PE for 45 minutes on ice, washed three times with PBSB and analyzed by flow cytometry using the EPICS E l i t e  ESP  flow cytometer  (Beckman-Coulter, Miami, FL).  58  2.3.4  Evaluation of Intracellular Pgp Expression by Fluorescence Microscopy Cells were harvested with 0.25% Trypsin + 1 m M E D T A , counted with trypan  blue, and 6 x l 0 cells of each cell type were placed in 1.7 ml Eppendorf tubes. 6  After  centrifugation at 7000 rpm for 15 seconds the pellets were resuspended in 0.3 ml 2% formaldehyde in PBS and left to incubate at room temperature for 30 minutes for fixation. In order to increase cell permeability, 1% Tween-20 was added for the last 15 minutes of fixation. Fixed cells were centrifuged at 11,000 rpm for 25 seconds and the pellets were resuspended in 0.1 ml PBSB followed by two washes with 1 ml PBS + 0.1 % B S A + 0.5%o Tween-20 (PBSBT). Pellets were resuspended in 0.75 ml blocking reagent containing PBSBT +100 ug/ml human IgG + 20% normal human serum. Samples were placed at 4°C overnight for blocking and then equally distributed to three Eppendorf tubes. Aliquot A was used as an autofluorescence control, aliquot B to control for nonspecific staining by exposure to secondary antibody only, and aliquot C for the two-step staining procedure.  Mouse anti-human Pgp (C219) primary antibody was added to  sample C at 1 p.g/ml and incubated at room temperature for 2 hours. A l l cells were washed twice with 1 ml PBSBT and once with PBSB, centrifuged at 11,000 rpm for 25 seconds and resuspended in 0.25 ml PBSBT + 20% goat serum. Goat anti-mouse IgGFITC secondary antibody was added to samples B and C, incubated at room temperature for 1 hour and washed twice with PBSBT. A l l samples were then washed with 1 ml PBSB, centrifuged and the pellets resuspended in 0.3 ml PBSB + 0.1 ug/ml DAPI. Cells were spun onto glass slides, dried for 10 minutes at room temperature and mounted in 10 ul of 2.5% D A B C O anti-fade in 90% glycerol. A l l images were acquired with a Leica DC 100 fluorescence microscope (Leica Microsystems (Canada), Richmond Hill, O N ,  59  Canada) and Image Database V4.01 software for 9 seconds. Images were later processed with Adobe Photoshop® 4.0 in an identical manner.  2.3.5  M T T Cytotoxicity Assays Cells were counted and seeded into 96-well micro titer Falcon® plates at 2 x l 0  3  cells/well in 0.1 ml complete medium. The perimeter wells of the 96-well plates were not used and contained 0.2 ml sterile water. After 24 hours at 37°C the medium was replaced with 0.2 ml of fresh medium containing Taxol®, vincristine sulfate, cisplatin or doxorubicin with or without PDMP, C6-ceramide or Valspodar at the concentrations specified for each experiment.  After 72 hours the cell viability was assessed using a  conventional 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction assay. Fifty microlitres of 5 mg/ml M T T reagent in PBS was added to each well.  Viable cells with active mitochondria reduce the M T T to an insoluble purple  formazan precipitate that is solubilized by the subsequent addition of 150 ul dimethyl sulfoxide. The formazan dye was measured spectrophotometrically (570 nm) using an M R X microplate reader (Dynex Technologies Inc., Chantilly, V A ) .  A l l assays were  performed in triplicate. The cytotoxic effect of each treatment was expressed as percent cell viability relative to untreated control cells (% control) and was defined as: [(Abss7o treated cells)/Abs o untreated cells)] x 100. 57  2.3.6  [ H]Taxol® Uptake Studies 3  Cells were seeded into 6-well Falcon® plates at 1.5xl0 cells/ml in 2 ml complete 6  medium. After 24 hours at 37°C the old medium was aspirated and cells were incubated  60  with 2 ml complete medium containing the indicated concentrations of Taxol  + 0.25  u-Ci/ml [ H]Taxol® in the absence or presence of 5 u M P D M P for the times indicated. 3  Culture medium was then removed and cells were washed twice with 0.2 ml ice-cold PBS. Cells were harvested by scraping into 0.5 ml PBS and radioactivity was measured by liquid scintillation counting. A n aliquot was obtained for cell counting by trypan blue exclusion. Uptake was expressed as picograms [ H]Taxol®/10 cells and was corrected 3  6  for binding using controls incubated at 4°C.  2.3.7  Cell Radiolabeling and Lipid Extraction Cells were seeded into 75-cm Falcon® flasks in complete medium and grown to 2  -70% confluence. Plating medium was replaced by 15 ml complete medium containing 1.0 uCi/ml [ H]palmitic acid with or without 5 u M PDMP and/or 1 p.g/ml Valspodar for 3  24 hours.  Cells were washed twice with ice-cold Hank's medium and harvested by  scraping into 2.5 ml ice-cold methanol containing 2% acetic acid. The collected cells were transferred to glass vials with teflon-lined screw caps. Lipids were extracted from the methanol layer by adding equal volumes of water and chloroform, vortexing and centrifuging at 3000 rpm for 5 minutes. The lipid-containing lower, organic phase was collected into pre-weighed tubes. Three 10 ul aliquots were removed for scintillation counting to determine the total amount of radiolabeled lipid.  The remaining organic  phase was dried under a stream of nitrogen and tubes were re-weighed to determine the total mass of extracted lipid.  61  2.3.8  Lipid Detection by Thin Layer Chromatography (TLC) The dried lipid film was dissolved in chloroform/methanol (2:1) and 300 ug total  lipid was spotted onto silica gel G T L C plates. chloroform/acetic  acid  (90:10)  chloroform/methanol/ammonium Commercial  Ci6-ceramide  and  for  hydroxide GlcCer  Plates were developed in  ceramide (70:20:4)  standards  for were  separation GlcCer  and  separation.  co-chromatographed.  Chromatography plates were visualized with iodine vapor and spots corresponding to ceramide or GlcCer were scraped into scintillation vials for quantitation by liquid scintillation counting.  The radioactivity of the selected spots was expressed as a  percentage of the total amount of radiolabeled lipid extracted from the cells.  2.3.9  Statistical Analysis Statistical analysis was performed using one way analysis of variance ( A N O V A )  followed by Student-Newman-Keuls analysis with InStat Version 3.0 for Windows (GraphPad Software, Inc., San Diego, CA). Mean differences with ap value <0.05 were considered statistically significant.  2.4  RESULTS  2.4.1  Chemosensitization Effects of PDMP in Human Breast Cancer Cells  M T T cytotoxicity studies comparing the chemosensitivity of wild-type cell lines to their drug resistant counterparts demonstrated  the presence of statistically significant  resistance to Taxol®, vincristine and doxorubicin in both the MCF7/AdrR and MDA435/LCC6  M D R 1  cell lines (Table 2.1). However, resistance to cisplatin did not reach  62  statistical significance for either cell line (p=0.66 and p=0.23 for the MCF7/AdrR and MDA435/LCC6  M D R 1  cell lines, respectively).  Flow cytometry studies using the  fluorescent Pgp-PE antibody demonstrated that cell-surface Pgp was overexpressed in both of the resistant cell lines relative to wild-type. The fluorescence intensity of the MCF7/AdrR cell line was 9.5 times greater than wild-type, and that of the MDA435/LCC6  M M /  cell line was 6.8 times greater than wild-type (Figure 2.1).  Immunofluorescence studies using a 2-step staining procedure employing the C219 antiPgp primary antibody and an IgG-FITC secondary antibody demonstrated that Pgp was intracellularly distributed and not solely localized to the plasma membrane (Figure 2.2). Low level background staining of the primary antibody was observed in the wild-type cells, as was low level non-specific staining with the FITC secondary antibody controls. However, the anti-Pgp staining in the M D R cells was increased and aggregates of high intensity fluorescence were observed, which is indicative of intracellularly distributed PgP-  63  Table 2.1 The Effect of 5 uM PDMP, 1 ug/ml Valspodar and 2 uM C -Ceramide on Anticancer Drug Cytotoxicity in Wild-Type and Multidrug Resistant MCF7 and MDA435/LCC6 Breast Cancer Cells 6  Cell Line  IC Vincristine  Taxol®  MCF7 MCF7 + PDMP MCF7/AdrR MCF7/AdrR + PDMP MCF7/AdrR + Valspodar MCF7/AdrR + Valspodar + PDMP MCF7/AdrR + PDMP + C -Cer  12.52 ± 3.64 nM 12.08 ±0.11 nM 3.24 ± 0.06 uM 0.50 ± 0.20 nM 60.77 ± 4.20 nM  MDA435/LCC6 MDA435/LCC6 + PDMP MDA435/LCC6 " *' MDA435/ LCC6 ' + PDMP MDA435/ LCC6*"-"" + Valspodar MDA435/ L C C 6 + Valspodar + PDMP MDA435/LCC6 """ + PDMP + C -Cer  1.81 ± 1.37 nM 1.27 ± 1.06 nM 30.01 ±4.13 nM  6  A  J  0.76 ± 0.18 nM 0.77 ± 0.16 nM 11.02 ± 2.16 uM 1.82 ± 0 . 1 6 \xM 33.93 ± 0.38 nM  b  c  30.40 ± 2.27 nM  a 50  b  c  d  0.68 ±0.08 nM  b  0.17 ± 0.06 uM 0.18 ± 0 . 0 7 uM 0.34 ± 0 . 1 2 uM 0.34 ± 0 . 1 2 uM N/D  b  e  17.26 ± 14.14 nM 1.48 ±0.01 uM 0.22 ± 0.02 nM 0.18 ± 0.02 nM 39.25 ± 7.90 nM  ± SEM Doxorubicin  d  b  Cisplatin 33.99 ± 3 . 2 9 uM 34.38 ± 4 . 5 5 uM 21.SI ± 9.08 uM 28.75 ± 12.52 uM N/D  N/D  N/D  N/D  N/D  0.25 ± 0.14 uM 0.23 ± 0.16 uM 3.87 ± 1.57 uM  b  10.17 ± 3 . 5 7 uM 9.95 ± 5.02 uM 14.25 ± 1.06 uM  MDR  2.21 ± 1.14 nM  c  1.04 ± 0.40 nM  c  3.41 ± 1.51uM  14.25 ± 1.06 uM  0.05 ± 0.04 nM  0.61 ± 0 . 4 7 nM  N/D  N/D  0.05 ± 0.04 nM  0.52 ± 0.37 nM  N/D  ' N/D  2.40 ± 1.97 nM  1.18 ± 0.38 nM  N/D  N/D  M M ;  A  6  Cells were incubated with increasing drug concentrations ± 5 u M P D M P , 1 fj.g/ml Valspodar and/or 2 p.M C6-ceramide over 72 hours, after which cell viability was analyzed by the M T T assay. The IC50 value was taken as the anticancer drug concentration that inhibits cell growth by 50% relative to untreated control cells. Statistically different from wild-type (p<0.05) Statistically different from resistant cells in the absence of PDMP (p<0.05) Statistically different from resistant cells + 1 p-g/ml Valspodar in the absence of PDMP (p<0.05) Not determined a  b  c  d  e  64  P-glycoprotein-PE Figure 2.1 Cell-surface P-glycoprotein expression as measured by flow cytometry, demonstrating 9.5 times greater Pgp expression in multidrug resistant M C F 7 cells compared to wild-type (A) and 6.8 times greater Pgp expression in multidrug resistant MDA435/LCC6 cells compared to wild-type (B) as shown by flow cytometry. Cells were stained with mouse isotype control-PE (Phycoerythrin) or mouse anti-human PgpPE antibody and analyzed by flow cytometry using the EPICS E l i t e flow cytometer (Beckman-Coulter, Miami, FL). ESP  65  M C F 7 » > -FITC c o n t r o l  M C F 7 / A d r R -FITC c o n t r o l  MCF7W-C219  MCF7/AdrR-C219  1  MDA435/LCC6<"-FITC c o n t r o l  MDA435/LCCSwt-C219  MDA435/LCC6<w°«>-FITC control  MDA435/LCC6«DR>-C219  Figure 2.2 Fluorescence microscopy images of intracellular Pgp staining in wild-type and multidrug resistant MCF7 and MDA435/LCC6 cells following two-step staining with mouse anti-human Pgp C219 primary and goat anti-mouse IgG FITC secondary antibodies. Images were acquired with a Leica D C 100 fluorescence microscope (Leica Microsystems (Canada), Richmond Hill, O N , Canada) and later processed with Adobe Photoshop 4.0 in an identical manner.  66  As described in Chapter 1, there are numerous mechanisms that are known to contribute to the M D R phenomenon. In order to investigate the specific involvement of ceramide production and metabolism, the ability of P D M P to sensitize the resistant cell lines to conventional chemotherapeutic agents by virtue of inhibition of ceramide metabolism to GlcCer was investigated. Cells were exposed to increasing concentrations of anticancer drug in the absence or presence of a non-toxic (5 uM) concentration of P D M P . Chemosensitization was evaluated by comparing the anticancer drug IC50 values (concentration of drug required to inhibit cell growth by 50% relative to untreated controls) in the presence and absence of PDMP, and a decrease in IC50 value toward that of the wild-type counterpart was indicative of chemosensitization.  P D M P induced  statistically significant chemosensitization of both resistant cell lines to the cytotoxic effects of Taxol® and vincristine. In the MCF7/AdrR cell line, P D M P decreased the Taxol® I C  50  from 3.24 ± 0.06 u M to 0.50 ± 0.20 u M and the vincristine I C  50  from 11.02  ± 2.16 u M to 1.82 + 0.16 u M (Figure 2.3 and Table 2.1). In the M D A 4 3 5 / L C C 6 line P D M P decreased the Taxol® I C vincristine I C  50  50  M M /  cell  from 30.01 ± 4.13 n M to 2.21 ± 1.14 n M and the  from 39.25 ± 7.90 n M to 1.04 ± 0.40 n M (Figure 2.4 and Table 2.1).  P D M P had no effect on the IC50 values for the wild-type counterparts of either cell line (Table 2.1). Interestingly, PDMP did not sensitize the resistant cells to the cytotoxic effects of the non-tubulin binding drugs doxorubicin or cisplatin (Table 2.1). presence of a PDMP-induced chemosensitization effect in the mdr-1 MDA435/LCC6™  cell  line  was  surprising  since  these  cells  The  transfected are  not  67  120  1E-12  1E-11  1E-10  1E-9  1E-8  1E-7  1E-6  1E-5  1E-4  Taxol Concentration (M)  Figure 2.3 Effect of 5 uJVI PDMP on Taxol (A) and vincristine (B) cytotoxicity in wild-type and multidrug resistant MCF7 cells. Cells were incubated with the indicated anticancer drug concentrations ± PDMP for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from at least three independent experiments; bars, SD.  68  120  A  TTTTTj  1 I I  1E-12  Mill)  I  I  I  IIHI[  ~T I  111111)'  ' I I MIHI)  1E-11 1E-10 1E-9  1  1E-8  1  1E-7  I I lllllj  rTTTIliq" "II Mill!) " I  1E-6  1E-5 1E-4  Taxol Concentration (M) 120  I •|  1E-12  i ri  IIIIIJ —I-TTIIIII) 1  "" i i i i m i )  1E-11 1E-10  1  i iiiini|  1E-9  i  1E-8  ITIIUI|—i  1E-7  ii  IIIIIJ  i "ii  1E-6  I'IIIIJ-"'!"  i rniii) 1 "I  1E-5 1E-4  Vincristine Concentration (M)  Figure 2.4 Effect of 5 u M PDMP on Taxol® (A) and vincristine (B) cytotoxicity in wild-type and multidrug resistant MDA435/LCC6 cells. Cells were incubated with the indicated anticancer drug concentrations + PDMP for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from at least three independent experiments; bars, SD.  69  known to overexpress GCS.  The observed PDMP effect in these cells therefore  suggested an additional involvement of Pgp in ceramide-mediated resistance. In order to demonstrate that the chemosensitization effect was not simply due to PDMP-induced alterations in cellular drug uptake, cells were incubated with 10 n M (MDA435/LCC6 cells) or 500 n M (MCF7 cells) Taxol® + 0.25 uCi/ml [ H]Taxol® for 1 3  and 4 hours in the absence and presence of 5 uJVl PDMP. The presence of P D M P did not affect cellular drug accumulation, since the percent accumulation of total [ H]Taxol® 3  after 1 and 4 hours was the same in the presence and absence of P D M P (Figure 2.5). Although the IC50 values for the wild-type and resistant counterparts of each cell line are different, cells were incubated with the same Taxol® concentration so that direct comparisons between uptake levels in the wild-type versus M D R cells could be made. A Taxol® concentration intermediate between the respective IC50 values was chosen. The results show increased Taxol  accumulation in both wild-type cell lines compared to  their M D R counterparts, which is consistent with Pgp overexpression. Taxol® accumulation was 2.5-3-fold greater in the M C F 7  w t  Specifically,  cells compared to  MCF7/AdrR cells, and was approximately 4-fold greater in M D A 4 3 5 / L C C 6 compared to M D A 4 3 5 / L C C 6  A/zw;  wt  cells  cells.  70  Figure 2.5 Accumulation of [ H]Taxol® in wild-type and multidrug resistant M C F 7 and MDA435/LCC6 cells. MCF7 cells were incubated with 500 n M Taxol® + 0.25 nCi/ml [ H]Taxol® and MDA435/LCC6 cells with 10 n M Taxol® + 0.25 u.Ci/ml [ H]Taxol® in the presence and absence of 5 u M PDMP for the times indicated. Culture medium was removed and cells were washed twice with ice-cold PBS. Cells were harvested by scraping into PBS and radioactivity was measured by liquid scintillation counting. Uptake is expressed as picograms Taxol®/10 cells and was corrected for binding using controls incubated at 4°C. Each value represents the mean from three experiments; bars, SD. 3  3  3  6  71  2.4.2  Influence of Exogenous C6-ceramide on PDMP-induced Chemosensitization Based on studies to date, PDMP-induced chemosensitization  effects  are  presumably attributed to increased intracellular ceramide accumulation as its metabolism to GlcCer is blocked. On this basis it was speculated that the addition of exogenous short-chain ceramide, which is itself cytotoxic (244), may further  enhance this  chemosensitization effect. In order to test this, cytotoxicity experiments with the various anticancer drugs were repeated in the presence of 2 u M C6-ceramide. This ceramide concentration reflected the approximate IC20 (20% reduction of cell growth), which would provide ceramide exposure without causing high cell death by itself. Combined treatment with Taxol® or vincristine plus PDMP and ceramide did not enhance chemosensitivity beyond that achieved by exposure to Taxol® or vincristine with PDMP in the absence of exogenous ceramide (Table 2.1).  2.4.3  Effect of Pgp Inhibition on PDMP-induced Chemosensitization In order to determine whether Pgp was involved in mediating the PDMP-induced  effect, the cytotoxicity experiments were repeated in the presence of 1 p.g/ml Valspodar, a potent Pgp inhibitor. Side-by-side cytotoxicity experiments were conducted in order to assess the degree of PDMP-induced shift in Taxol®/vincristine  IC50  values compared to  the degree of PDMP-induced shift in the Taxol®/vincristine + 1 p.g/ml Valspodar IC50 values. Although Valspodar itself induces Taxol® and vincristine chemosensitization by virtue of Pgp blockade, the focus of this experiment was to measure the degree of any further chemosensitization upon subsequent addition of PDMP. The P D M P effect in the  72  presence or absence of Valspodar was expressed as a sensitization ratio (SR), which was calculated as: SR = (Drug I C treatment - PDMP) / (Drug I C treatment + PDMP) 50  50  In the MCF7/AdrR cell line the presence of Pgp blockade induced by Valspodar treatment resulted in a 3-fold decrease in the SR for both Taxol® (SR = 6.5 without and SR = 2.0 with Valspodar) and vincristine (SR = 6.1 without and SR = 2.0 with Valspodar). In the M D A 4 3 5 / L C C 6  M/W/  cell line, treatment with Valspodar eliminated  the sensitization ratio completely (SR = 1.0) for both Taxol® and vincristine (Table 2.2). The diminished PDMP effect in the presence of Pgp blockade further supported a role for Pgp in the regulation of ceramide metabolism to GlcCer, and indicated that Pgp function was related to the PDMP response. Valspodar had no effect on the chemosensitivity of the wild-type, non-Pgp expressing cell lines.  2.4.4  Correlation of Chemosensitization Effects with Glucosylceramide Levels In order to correlate the observed chemosensitization effects with changes in  intracellular lipids, baseline and treatment-induced ceramide and GlcCer levels in wildtype and M D R cells were analyzed by T L C . GlcCer levels were approximately equal in the wild-type MDA435/LCC6 and the mdr-1 gene transfected M D A 4 3 5 / L C C 6 ™ cells (1.52 ± 0.21 and 1.54 ± 0.09 percent total [ H]lipid, respectively), which is consistent 3  with lack of GCS overexpression in this cell line. By contrast, GlcCer was elevated 2fold in the drug-selected MCF7/AdrR cells compared to wild-type cells (Table 2.3). Elevated GlcCer in this cell line is consistent with previous reports (61, 62), and confirmed that GCS is overexpressed in these cells. Treatment of the MCF7/AdrR cells  73  Table 2.2 The Effect of Pgp Blockade on the Degree of PDMP-induced Chemosensitization in MCF7/AdrR and MDA435/LCC6 Cells as Measured by a Sensitization Ratio MDR1  Sensitization Ratio (SR) Vincristine Taxol® 6.48 6.07 1.97 2.00 37.74 13.57 1.11 1.16 ab  MCF7/AdrR ± 5uM PDMP MCF7/AdrR + lug/ml Valspodar ± 5uM PDMP M D A 4 3 5 / L C C 6 ± 5uM PDMP M D A 4 3 5 / L C C 6 + lug/ml Valspodar ± 5uM PDMP MM/ MM/  Cells were incubated with increasing Taxol or vincristine concentrations ± 5 u M P D M P with and without 1 p,g/ml Valspodar for 72 hours. Cell viability was analyzed using the M T T assay. SR = (Drug I C treatment - PDMP)/(Drug I C treatment + PDMP) using the I C values presented in Table 2.1. a  b  50  50  50  74  with 5 u M P D M P reduced GlcCer to levels comparable to the wild-type counterpart, indicating that PDMP indeed inhibits GlcCer formation. This decrease in GlcCer was correlated with a 1.7-fold increase in pro-apoptotic ceramide, which provides the basis for PDMP-induced chemosensitization.  Treatment with P D M P did not alter GlcCer  levels in the M C F 7 wild-type cells. Treatment  with  1 \xg/m\ Valspodar increased  ceramide  production by  approximately 4-fold and GlcCer production by approximately 1.5 fold in the MCF7/AdrR cells. This was expected based on previous demonstration that Valspodar does elevate intracellular ceramide (245). Because these cells overexpress GCS, it would be expected that much of the ceramide formed in response to Valspodar would be converted to GlcCer.  Combined treatment with Valspodar + P D M P returned GlcCer  back to wild-type levels but increased ceramide a further 1.5-fold. This supports the finding that PDMP-induced chemosensitization was still observed in the presence of Pgp blockade, although to a lesser degree.  75  Table 2.3 Incorporation of [H] Palmitic Acid into Ceramide and Glucosylceramide of Treated and Control MCF7 Cells Lipid MCF7-wt  MCF7-wt + PDMP  Percent [ H]lipid ± SD MCF7/AdrR MCF7/AdrR + PDMP  Cer  0.54 ± 0.09  0.25 ± 0 . 0 1  0.21 ± 0 . 0 3  0.35 ± 0.05  GlcCer  1.19 ± 0 . 0 7  1.16 ± 0.18  2.19 ± 0.15  3  a  MCF7/AdrR + Valspodar  MCF7/AdrR + Valspodar + PDMP  b  0.86 + 0.01  1.33±0.08  1.32 ± 0.12  3.02 ± 0 . 3 5  1.16 ± 0.09  b  c  c  Cells were incubated with 1.0 uCi/ml [ H]palmitic acid in complete medium for 24 hours, washed and harvested by scraping. Total lipids were extracted using equal volumes of methanol-2% acetic acid/chloroform/water. The radioactivity of total extracted lipids was measured by scintillation counting. Lipids were spotted onto T L C plates and developed in chloroform/acetic acid (90:10) for ceramide or chloroform/methanol/ammonium hydroxide (70:20:4) for GlcCer. Lipid spots corresponding to co-chromatographed standards were scraped into scintillation vials for quantitation by liquid scintillation counting. The lipid amounts presented are expressed as a percentage of the total tritiated lipid extracted from the cells. Statistically different from MCF7/AdrR in the absence of 5uM P D M P (p<0.05) Statistically different from MCF7/AdrR + 1 u,g/ml Valspodar in the absence of 5 u M P D M P (p<0.05) a  3  b c  76  2.5  DISCUSSION The emerging role of ceramide as an intracellular signaling molecule involved in  mediating apoptosis and M D R suggests that the ability to modulate ceramide metabolism should provide a new avenue by which drug sensitivity in M D R cells may be increased. The small molecule P D M P is a competitive inhibitor of the GCS enzyme that blocks the conversion of pro-apoptotic ceramide to non-cytotoxic GlcCer at doses which are nontoxic to cells.  It has previously been demonstrated that P D M P sensitizes murine  neuroblastoma cells to Taxol® and vincristine (132). The results presented in this chapter extend the study of PDMP-induced chemosensitization to two human breast cancer cell lines. The chemosensitization effects of PDMP-altered ceramide metabolism in resistant MCF7/AdrR and M D A 4 3 5 / L C C 6  M M /  cell lines, which were developed for resistance by  drug selection and mdr-1 gene transfection, respectively, were compared. The rationale for choosing these cell lines was based on the fact that although both lines overexpress Pgp, the transfected M D A 4 3 5 / L C C 6 * cell line does not exhibit elevated GCS whereas MD  y  GCS expression in the MCF7/AdrR cells is increased. Consequently, these differences allowed for the differentiation of sensitization effects  associated with ceramide  metabolism versus Pgp transport. A non-toxic dose of P D M P was found to induce significant chemosensitization to Taxol® and vincristine in both of the resistant breast cancer cell lines under investigation. Interestingly, chemosensitization was not observed with the non-tubulin binding drugs doxorubicin or cisplatin. Since Taxol® and vincristine have both been demonstrated to induce ceramide formation in cancer cells (174, 246), it might initially seem that the P D M P effect was simply due to inhibition of the metabolism of pro-apoptotic ceramide  77  induced by the cytotoxic drugs to non-apoptotic GlcCer. However, i f this were the case one would expect to see PDMP-induced sensitization in the GCS overexpressing MCF7/AdrR cells only.  Similar to wild-type MDA435/LCC6 cells, which are not  affected by PDMP, the M D A 4 3 5 / L C C 6  M M /  cells do not display elevated GCS yet P D M P  still induced a chemosensitization response. The commonality between the MCF7/AdrR and M D A 4 3 5 / L C C 6  M£W/  cell lines, however, is elevated Pgp expression, and this led to  the investigation of a possible role for Pgp in ceramide signaling. This was further supported by the fact that PDMP had no effect on the chemosensitivity of non-Pgp expressing wild-type MCF7 or MDA435/LCC6 cells. The fact that the PDMP-induced effect is specific for tubulin-binding drugs is itself interesting, and suggests that intracellular transport mechanisms/microtubule assembly may be involved in the processes of ceramide signaling and/or metabolism. In order to rule out the possibility that P D M P modulated chemosensitivity via non-specific alterations in cellular drug uptake the accumulation of radiolabeled Taxol® in the presence and absence of PDMP was measured. P D M P had no effect on the cellular uptake of Taxol®.  Consequently, chemosensitization in the M D R cell lines was not  attributable to increased intracellular anticancer drug concentrations. In order to assess whether the combination of PDMP plus exogenous ceramide would further enhance the PDMP-induced chemosensitization effect the cytotoxicity assays were repeated in the presence of 2 u M C6-ceramide. Since this is a cell-permeable ceramide, it readily and rapidly distributes to various membranes within the cell. The observation that combination treatment with Taxol® or vincristine plus P D M P and ceramide did not further enhance chemosensitivity may be explained by the fact that  78  short-chain, cell-permeable ceramides can readily distribute to numerous intracellular locations due to their relatively hydrophilic nature. It has been reported that short-chain ceramides can be glycosylated (247).  However, GCS is specifically localized to the  cytoplasmic face of the Golgi (248, 249).  Therefore, a significant proportion of  exogenously added ceramide would be expected to localize in GCS-poor regions. Consequently, addition of PDMP may not significantly impact overall ceramide-induced apoptosis because the pool(s) of ceramide being modulated by P D M P are small relative to the overall amount of ceramide that is distributed throughout the cell. The specific involvement of Pgp in ceramide-mediated apoptosis was examined by evaluating the chemosensitization effect of P D M P in the presence of 1 u.g/ml Valspodar, a potent Pgp inhibitor. Interestingly, under conditions of Pgp blockade the P D M P effect was no longer observed in the M D A 4 3 5 / L C C 6 3-fold in the MCF7/AdrR cells.  M M y  cells, and was decreased  This suggested that Pgp function is involved in  regulating ceramide metabolism to GlcCer. Figure 2.6 provides an illustration of the proposed relationship between Pgp, GlcCer and P D M P that is consistent with these observations.  The presence of  intracellularly distributed Pgp has been demonstrated by a number of groups (250-254), and was confirmed in the two M D R cell lines under investigation by fluorescence microscopy studies.  Functional Pgp activity has been previously demonstrated in the  Golgi of multidrug resistant cells, including the same MCF7/AdrR cell line used in these studies (251, 252). It has also been shown that Pgp can mediate the translocation of GlcCer from the cytoplasmic face of the Golgi to the lumen where further processing to higher glycosphingolipid species is known to occur (255-257).  Wild-type (drug  79  sensitive) cells do not overexpress Pgp or GCS so exposure of these cells to anticancer agents results in increased intracellular ceramide and subsequent apoptosis.  The  negligible chemosensitizing effects of P D M P in these cells is attributed to the fact that relatively little ceramide is metabolized to GlcCer, and an equilibrium exists between ceramide and GlcCer that is generated. Resistant cells that overexpress Pgp are capable of translocating GlcCer across the Golgi membrane. In these cells the loss of GlcCer from the cytoplasmic face of the Golgi provides a driving force for further ceramide to GlcCer conversion.  Continuous removal of GlcCer by Pgp removes the negative  feedback control on the GCS enzyme and promotes further ceramide metabolism. This could explain why a PDMP-induced chemosensitization effect was observed in the M D A 4 3 5 / L C C 6 * cells even though they do not display elevated GlcCer. The loss of MD  /  sensitization in the presence of Valspodar further supports this theory. In the case of the MCF7/AdrR cells that additionally overexpress GCS, ceramide generated by anticancer drug exposure is rapidly converted to GlcCer before apoptosis pathways can be activated.  Chemosensitization was observed in these cells because  P D M P inhibits the conversion of ceramide to GlcCer. When P D M P is co-incubated with Valspodar the Pgp-mediated translocation of GlcCer across the Golgi should be blocked. This causes a relative accumulation of GlcCer on the cytoplasmic face of the Golgi, which in turn reduces the ceramide to GlcCer conversion via negative feedback. In this case P D M P is still acting as an inhibitor of the GCS enzyme, but the P D M P effect is diminished due to GlcCer product feedback inhibition. Taken together, the research contained in this chapter provides an increased understanding of the relationship between ceramide, ceramide metabolism and Pgp in the  80  context of multidrug resistance and chemosensitization. These observations suggest that therapeutic approaches aimed at increasing intracellular endogenous ceramide levels should enhance apoptosis. Based on these results, the focus of the research presented in Chapter 3 was to investigate the utility of exogenously applied ceramide lipids in achieving this goal.  CER  Golgi Apparatus  Figure 2.6 A model to explain the influence of Pgp on ceramide metabolism in the context of chemosensitization. Wild-type cells that do not overexpress Pgp or the GCS enzyme do not display PDMP-induced chemosensitization because very little ceramide is converted to glucosylceramide. Multidrug resistant cells that do overexpress Pgp, however, may translocate newly-formed GlcCer across the Golgi membrane. This provides a driving force for further Cer to GlcCer conversion and reduces negative feedback on the GCS enzyme. Inhibition of Pgp promotes GlcCer accumulation in the cytoplasm and reduces GlcCer formation via negative feedback, which in turn reduces PDMP-induced chemosensitization. Abbreviations used: cer, ceramide; GCS, glucosylceramide synthase; GlcCer, glucosylceramide; GSL, glycosphingolipid.  81  CHAPTER 3  INTRACELLULAR DELIVERY OF EXOGENOUS CERAMIDE LIPIDS INDUCES APOPTOSIS IN VITRO*  3.1  INTRODUCTION AND RATIONALE The role of ceramide as a mediator of apoptosis has attracted significant recent  attention. Although the specific mechanisms have not been fully defined, the evidence presented in Chapter 1 suggests its direct involvement in regulating cell death. Much of the research on ceramide to date has been aimed at elucidating the mechanism(s) by which it mediates apoptosis and other intracellular effects, and observations made during these studies suggest that stimuli which result in increased intracellular ceramide levels have pro-apoptotic effects.  Furthermore, the documented involvement of ceramide in  M D R , combined with the chemosensitization results presented in Chapter 2, suggest its therapeutic potential. Therefore, the objectives of the research presented in this chapter were to initially investigate the cellular uptake and cytotoxicity of various chain length synthetic ceramide lipids in order to identify therapeutically active exogenous ceramides, and subsequently to enhance these effects by formulating ceramide into liposome-based delivery systems in an effort to improve intracellular delivery and therefore pro-apoptotic activity.  *Adapted from: J A Shabbits and LD Mayer (2003). Intracellular delivery of ceramide lipids via liposomes enhances apoptosis in vitro. Biochemica et biophysica acta 78474: 1-9.  82  3.2  HYPOTHESIS The hypothesis  underlying the research presented in this chapter is that  intracellular delivery of exogenous ceramide lipids can be used to induce apoptosis in vitro.  3.3  MATERIALS AND METHODS  3.3.1  Materials A l l phospholipids and ceramides were obtained from Avanti Polar Lipids  (Alabaster, A L ) . Cholesterol, cholesteryl hemisuccinate and M T T reagent were obtained from Sigma-Aldrich Canada (Oakville, O N , Canada).  [ H]cholesteryl hexadecyl ether 3  (CHE) was purchased from Perkin Elmer (Boston, M A ) . [ C]C6- and [ C]Ci6-ceramide 14  14  were purchased from American Radiolabeled Chemicals (St. Louis, MO). Dulbeco's Modified Eagle's Medium (DMEM) and Hank's Balanced Salt Solution (without pH indicator; Hank's) were obtained from Stem Cell Technologies (Vancouver, B C , Canada). Fetal bovine serum was purchased from Hyclone (Logan, UT). L-glutamine and trypsin-EDTA were obtained from Gibco B R L (Burlington, O N , Canada).  The  Micro B C A Protein Assay kit was purchased from Pierce (Rockford, IL). Tissue culture flasks, incubation plates and cell scrapers were obtained from Falcon® (Becton Dickinson, Franklin Lakes, NJ).  Nucleopore® polycarbonate extrusion filters were  obtained from Whatman Inc. (Clifton, NJ). Liposomes were prepared using an extrusion apparatus (Lipex Biomembranes, Vancouver, B C , Canada).  [ C]lactose and Sephadex 14  G-50 chromatography beads were purchased from the Sigma Chemical Company (St.  83  Louis, MO). Pico-fluor 40 scintillation cocktail was purchased from Packard Biosciences (Groningen, The Netherlands).  3.3.2  Cell Lines and Culture The wild-type and mdr-1 gene transfected MDA435/LCC6 human breast cancer  cell lines described in Chapter 2 were used for these studies.  The J774 murine  macrophage cells also used in these studies were obtained from A T C C (Rockville, MD). Cells were cultured and prepared as described in Chapter 2 with the exception of J774 cells, which were harvested by gentle scraping rather than with trypsin-EDTA.  3.3.3  M T T Cytotoxicity Assays M T T cytotoxicity assays were performed as previously described in Chapter 2.  Free ceramide was diluted from ethanol stocks into complete medium and added to cells in 0.2 ml to achieve the desired final concentration. The Ci6-ceramide stock was kept warm, diluted into warm medium prior to addition to the cells and remained in solution at all times at the concentrations used in these studies. When the cytotoxicity of ceramidecontaining liposome formulations was investigated, calculations were based specifically on the concentration of ceramide added to each well.  Control (non-ceramide)  formulations were calculated on the basis of total lipid.  3.3.4  Lipid Uptake Studies Cell  suspensions  were diluted 1:1  with  trypan blue, counted  with  a  hemocytometer and seeded into 6-well Falcon® plates at 2.5xl0 cells/well in 2 ml 5  84  complete medium. The cells were allowed to adhere for 24 hours at 37°C, after which the medium was aspirated and replaced with 1 ml complete medium. Free ceramide was diluted from ethanol stocks into 1 ml complete medium as previously described and added to each well to give the desired final concentration of 1 u M C6-ceramide (corresponds to IC20 value) or 50 u M Ci6-ceramide. [ C]ceramide was incorporated at 14  0.1 uCi/nmole for each ceramide lipid to facilitate quantitation. Cells were incubated with the treatments for 1, 4 and 24 hours at 37°C. The incubation medium was then aspirated and cells were washed twice with 2 ml Hank's. Cells were gently scraped into 0.5 ml Hank's and collected into glass scintillation vials using glass pipettes. Each well was rinsed with an additional 0.5 ml Hank's to remove residual cells. A n aliquot of cells was removed for protein quantification and the remainder were counted for radioactivity by scintillation counting. Uptake was expressed as pmoles ceramide/p,g protein and was corrected for binding using controls incubated at 4°C.  3.3.5  Spectrophotometric Protein Quantitation The protein content of each cell aliquot was determined using the Pierce micro  B C A protein assay according to the method included with the assay kit. Briefly, a standard curve was prepared using the supplied purified bovine serum albumin (BSA) diluted in distilled water (dH20) to a final volume of 0.5 ml. Samples were prepared using 5 ul cell suspension + 495 ul dH20. Micro B C A reagents A , B and C were added in the specified ratios. A l l samples and standards were prepared in glass test tubes, which were heated in a 65°C waterbath for 1 hour and cooled to room temperature.  The  absorbance at 562 nm of each sample was read against a dH20 reference. The protein  85  concentration for each cell sample was determined using a standard curve prepared from the known B S A samples.  3.3.6  Preparation of Liposomes Lipids were weighed into individual test tubes and dissolved in 1 mL of  chloroform  (DPPC,  DSPC,  CHEMS,  Choi),  ethanol  (C -ceramide) 6  or  chloroform:methanol (2:1, v/v; Ci6-ceramide). Ci6-ceramide required brief heating at 65 °C to achieve complete dissolution.  Appropriate volumes of each lipid were  transferred to a single tube in order to achieve the desired ratio of each lipid component. A l l lipid ratios indicated in this research are on a mole:mole basis unless otherwise indicated. [ H]CHE was incorporated at 1 u.Ci/mg lipid as a non-exchangeable, non3  metabolizable lipid marker (258) to facilitate liposome quantitation. For the preparation of  ceramide-containing  liposomes,  [ C]C6-ceramide or 14  [ C]Ci6-ceramide was 14  incorporated into the formulation at 0.5 u,Ci/mg ceramide. The mixtures were evaporated with vortexing and heating under a stream of nitrogen gas and subjected to vacuum drying for a minimum of 4 hours to produce a homogenous lipid film. The lipid film was hydrated in 1 ml of warm Hepes buffered saline (HBS; 20 m M Hepes/150 m M NaCl; pH 7.4) with vortexing. Homogenously sized liposomes were then produced following a 10 cycle extrusion through three stacked 100 nm polycarbonate filters at 65 °C for nonceramide formulations and 95°C for ceramide formulations, using an extrusion apparatus. The resulting mean liposome diameter obtained following extrusion was approximately 100 nm as determined by quasi-elastic light scattering using the Nicomp 270 submicron  86  particle sizer model 370/270 (209, 229). Liposome and ceramide concentrations were determined by liquid scintillation counting.  3.3.7  Lactose Trapping In order to determine the liposome trapped volume, [ H]CHE labeled lipid films 3  were prepared as described above and hydrated in HBS (pH 7.4) containing [ C]lactose 14  at a concentration of 0.23 uCi [ C]lactose/mg lipid. Following extrusion and sizing, the 14  sample was passed down a Sephadex G-50 column equilibrated with HBS.  Trapped  volume was determined by the following equation: Trapped Volume = (A/B) I (C/D) where A = [ C]lactose dpm eluted from the Sephadex G-50 HBS column 14  B = [ C] lactose dpm of initial suspension prior to extrusion/ul 14  C = [ H]CHE dpm lipid eluted from the Sephadex G-50 HBS column 3  D — specific activity of lipid stock (dpm/umole total lipid)  3.3.8  Cryo-Transmission Electron Microscopy Control and ceramide-containing liposomes were analyzed by cryo-transmission  electron microscopy (cryo-TEM) performed in the laboratory of Dr. Katarina Edwards in Uppsala, Sweeden. The method employed and interpretation of liposome images have been previously described by that group (259). Briefly, liposome samples were diluted to 10 m M and transferred onto a copper grid containing a holey polymer film in a climate controlled chamber.  The samples were blotted to form a thin film, vitrified by  submersion in liquid ethane and transferred into the microscope at -165°C for analysis.  87  3.4  RESULTS  3.4.1  Effect of Ceramide Acyl Chain Length on In MDA435/LCC6 Human Breast Cancer Cells  Vitro Cytotoxicity in  The cytotoxic activity of free, exogenous ceramides with increasing acyl chain length was evaluated by incubating the M D A 4 3 5 / L C C 6 and M D A 4 3 5 / L C C 6 with free  M/)/  " cells  C2-, C6-, Cs-, C10-, C14-, or Ci6-ceramide over a range of 0-100 u M final  ceramide concentration. Both the wild-type and M D R cell lines were investigated in order to determine whether therapeutic strategies based on exogenous ceramide administration might be equally effective against sensitive and resistant tumor cells. The 72 hour M T T cytotoxicity results shown in Figure 3.1 demonstrate that cytotoxic activity is affected by ceramide acyl chain length, with the Ce- and Cs-ceramides being the most potent.  This may be explained by the cell-permeability characteristics of the various  ceramides. In order to be active, the exogenous ceramide must transfer from the tissue culture medium in which it is dispersed, cross the plasma membrane of the cell and exchange into cellular membranes from which it can interact with its intracellular target(s). The C2-ceramide, having the shortest acyl chain and therefore being the most hydrophilic, likely remains dispersed in the tissue culture media and does not readily exchange into cell membranes. This is consistent with the observed IC50 values of 74 u , M and 79 u M for the wild-type and M D R cell lines, respectively (Table 3.1). Cg-ceramides were the most cytotoxic, with  IC50  The C6- and  values in the 3-14 u M range, likely due  to their highly cell-permeable nature. As the chain length increased to C10-, C14- and Ci6ceramide the hydrophobic nature also increased. This would be expected to decrease the water solubility of the lipids and may reduce availability to intracellular membranes. Accordingly, IC50 values of 45 u M for Cio-ceramide and greater than 100 u . M for C14-  88  and Ci6-ceramides were obtained. These results led to the identification of C6-ceramide as the most potent exogenous ceramide form.  120  1E-8  1E-7  1E-6  1E-5  1E-4  Ceramide Concentration (M)  Figure 3.1 Cytotoxicity of various acyl chain length free ceramide lipids on wild-type (A) and mdr-1 gene transfected (B) MDA435/LCC6 human breast cancer cells. Cells were incubated with the indicated ceramide concentrations for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD.  89  Table 3.1 Cytotoxicity of Various Acyl Chain Length Free Ceramide Lipids in Wild-type and Resistant MDA435/LCC6 Human Breast Cancer Cells I C ± SEM (uM) C -Cer Cio-Cer 44.10±2.69 7.00±3.25 13.95±2.19 46.00±1.39 a  so  Cell Line Wild-type Resistant  C -Cer 73.60±7.92 79.20±6.04 2  C -Cer 2.71±0.69 5.67±0.70 6  8  Cn-Cer >100 >100  C -Cer >100 >100 16  Cells were incubated with increasing concentrations of free ceramide lipids of varying acyl chain length over 72 hours and cell viability was measured using the M T T assay. The IC50 value was taken as the ceramide concentration that inhibits cell growth by 50% relative to untreated control cells. a  3.4.2  Correlation of M T T Cytotoxicity Results with Ceramide Uptake In order to examine whether acyl chain length determines ceramide cytotoxicity  because of differences in cell-permeability, cellular uptake studies using radioactive C6and Ci6-ceramides were conducted. Figure 3.2 shows that in both the wild-type and resistant cell lines the [ C]C6-ceramide levels steadily increased to 7 pmol ceramide/p.g 14  protein over the 24 hour incubation period while [ C]Ci6-ceramide levels remained 14  under 2 pmol ceramide/p.g protein over 24 hours, which correlates with its lack of cytotoxic effect.  90  1 hour  4 hour  24 hour  Figure 3.2 Cellular uptake of free C6- and Ci6-ceramide by wild-type (A) and mdr-1 gene transfected MDA435/LCC6 cells (B). Cells were incubated with 1 u M C6-ceramide or 50 u.M Ci6-ceramide for the times indicated. [ C]C6- or [ C]Ci6-ceramide was added at 0.1 nCi/nmole ceramide for quantitation by scintillation counting. Cellular protein was measured spectrophotometrically (Abs 562 nm) using the micro B C A protein assay kit. Uptake is expressed as pmoles ceramide/ug protein and was corrected for binding using controls incubated at 4°C. Data are averaged means from two independent experiments conducted in triplicate; bars, SD. 14  l4  91  These results highlighted the importance of intracellular ceramide delivery in order to obtain the intended apoptotic response. Given its role as a bioactive lipid with dose-dependent cytotoxic activity, formulation of C6-ceramide into liposomes was investigated in order to facilitate the delivery of large amounts of this cytotoxic ceramide lipid to cells by virtue of the drug delivery characteristics of liposomes described in Chapter 1.  3.4.3  Formulation and Cytotoxicity of C6-Ceramide Containing Liposomes Liposomes containing up to 45 mole percent C6-ceramide were successfully  formulated into conventional DSPC/Chol liposomes with an overall composition of C6cer/DSPC/Chol (45:10:45). These liposomes displayed a mean diameter of 82.7 nm with a standard deviation of 31.8% (Figure 3.3) as measured by quasi-elastic light scattering using a Nicomp 270 submicron particle sizer model 370/270. Attempts to increase the level of ceramide incorporation beyond 45 mole percent resulted in difficulties hydrating the lipid film due to the formation of lipid aggregates. Control (non-ceramide) liposomes composed of DSPC/Chol (55:45), which have been extensively characterized in previous literature (208, 260), displayed a mean diameter of 101.2 nm with a standard deviation of 22% (Figure 3.3). Following successful formulation of C6-ceramide into liposomes, the in vitro cytotoxicity of this formulation was then evaluated.  Figure 3.4 demonstrates that CQ-  ceramide liposomes were cytotoxic to cells in vitro, with IC50 values of 15.5 ± 2.6 u M and 19.9 ± 4.72 u M for wild-type and mdr-1 gene transfected MDA435/LCC6 cells, respectively. The cytotoxicity was ceramide-specific, as control liposomes composed of  92  A: C -cer/DSPC/Chol (45:10:45) Liposomes 6  REL UOL  VOLUlffi-WT GAUSSIAN DISTRIBUTION ,  1  i i — i .  i  i  i  i |  I'  1 L.  i  SO so  tr =  E E  = -  E ao  tr =  nil Ii 1 r - H I Jill|iiiWHii"!j".fiil|' ' k ; !  o 20  8 lO  SO lOO <Vesicles>  S i z e (nn) —>  • =  ;  200  t 500  B: DSPC/Chol (55:45) Liposomes REL UOL  VOLUME—WT GAUSSIAN DISTRIBUTION I I ' •!. 1 I I  lOO so 60 40  tr  20  P  _i  ii!  xo S i z e (nn) —>  zo  SO  lOO (Vesicles)  200  SOO  1  i  I -1 .  XK  Figure 3.3 Mean liposome diameters of 82.7 ± 26.3 nm for C6-cer/DSPC/Chol (45:10:45; A ) and 101.2 ± 22.3 nm for DSPC/Chol (55:45; B) liposomes as determined by quasi-elastic light scattering using a Nicomp 270 submicron particle sizer model 370/270. Hepes buffered saline pH 7.4 was used for sample dilution.  93  Figure 3.4 Cytotoxicity of control and C6-ceramide liposomes on wild-type (A) and mdr-1 gene transfected (B) MDA435/LCC6 human breast cancer cells. Cells were incubated with the indicated ceramide or total lipid concentrations for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD.  94  DSPC/Chol (55:45) displayed no activity (IC  50  values >100 uM). The ceramide based  cytotoxicity results were then correlated with cellular lipid uptake, which was expressed as a percent of the total radioactivity added in order to allow for direct comparisons to be made between the ceramide lipid and bulk liposomal lipid. The results in Figure 3.5 indicate that the [ C]C6-ceramide component of the liposomes was taken up by the 14  tumor cells and 80% internalization was approached after 24 hours, whereas less than 10% of the [ H]CHE liposome label was cell-associated after 24 hours. These results 3  suggest that the C6-ceramide is being delivered not via liposomes, but rather by exchange of free ceramide lipid from the liposome bilayer into cellular membranes in a manner similar to that following addition of free C6-ceramide.  3.4.4  Formulation and Cytotoxicity of Ci6-Ceramide Containing Liposomes The uptake results presented in Figure 3.5 indicate that the incorporation of C6-  ceramide into liposomes did not afford any increased activity over free C6-ceramide. This lack of enhanced activity was attributed to the amphipathic nature of the short-chain ceramide, which appeared to have sufficient hydrophilicity to allow it to exchange from the liposomal carrier into the cellular membrane pool. Due to the reduced stability of C6ceramide in the liposome bilayer upon exposure to cellular membranes, the long-chain Ci6-ceramide, which showed no cytotoxic activity in its free form, became an attractive alternate ceramide lipid for liposomal formulation. This was investigated on the basis that the more hydrophobic Ci6-ceramide would be more likely to remain associated with its liposomal carrier during delivery.  Furthermore,  Ci6-ceramide is a more  physiologically relevant ceramide given that increases in endogenous Ci6-ceramide  95  100  [ H]CHE lipid ^i§] [ C]C.-ceramide  80-  O 60•  o ro o  ilBlllliii 40-  or vP  20-  Sill  4-  100  B  80CD O  60-  o CO o  40  H  20  h  T3 03  or ^  4 hour  1 hour  24 hour  Figure 3.5 Cellular uptake of [ H]CHE liposome and [ C]C6-ceramide radiolabels by wild-type (A) and mdr-1 gene transfected (B) MDA435/LCC6 human breast cancer cells. Cells were incubated with a liposome concentration corresponding to 1 u M C6-ceramide. Lipids were quantified by liquid scintillation counting. Uptake is expressed as a percentage of the total radioactivity added (corrected for binding with controls at 4°C) to allow for direct comparisons between liposomal and ceramide lipid to be made. Data are averaged means from two independent experiments conducted in triplicate; bars, SD. 3  14  96  accumulation have observed during apoptosis (161). Subsequent internalization of the ceramide-containing liposome should overcome the cell permeability limitations of free Ci6-ceramide, thereby improving its in vitro activity. The physico-chemical properties  of Ci6-ceramide described in Chapter 1  presented difficulties for liposome formulation. Several different lipid compositions and mole percentages were evaluated in an attempt to identify formulations that allowed Ci6ceramide to be successfully incorporated into the liposome bilayer, as well as to identify the maximum extent of ceramide incorporation (Table 3.2).  It was not possible to  incorporate Ci6-ceramide into stable, unilamellar vesicles at mole percentages greater than 15%. Liposomes composed of Ci6-cer/DSPC/Chol (15:40:45) were successfully prepared and displayed a mean diameter of 121.9 nm with a standard deviation of 43.6% (Figure 3.6). Evaluation of the in vitro cytotoxicity of Ci6-ceramide liposomes in the MDA435/LCC6 cells showed that these formulations displayed activity that was comparable to control (non-ceramide) DSPC/Chol (55:45) liposomes (Figure 3.7). This lack of cytotoxicity was correlated with cellular uptake studies, which indicated that neither the ceramide-lipid nor the liposomal carrier was internalized after 24 hours (Figure 3.8). Therefore, the inability of liposomes to enhance the cytotoxicity of Ci6ceramide appeared to be attributed to lack of internalization of the liposomes by the MDA435/LCC6 cells.  This was perhaps not surprising, since neutral (uncharged)  liposomes do not readily interact with tumor cells and are not internalized in the absence of a targeting ligand. Therefore, the liposome formulation was modified to include a  97  Table 3.2 Summary Table Describing Various Ci6-Ceramide Containing Liposome Formulations Attempted and their Respective Characteristics Lipid Composition" (molermole) C -cer/DSPC/Chol (15:40:45) C -cer/DSPC/Chol (20:35:45) C -cer/DSPC/Chol (20:50:30) Ci -cer/DSPC/Chol (20:50:30) C -cer/DPPC/Chol (15:40:45) C -cer/DPPC/Chol (20:35:45) C, -cer/Chol (50:50) C -cer/CHEMS (50:50) 16  16  16  6  16  16  6  16  C -cer/DPG/PEG o-DSPE (30:30:40) 16  35  Mole % C -Cer 15 20 20 20 15 20 50 50 I6  30  Formulation Characteristics hydrates well and extrudes with >80% efficiency lipid film difficult to hydrate; lipid aggregates lipid film difficult to hydrate; lipid aggregates lipid film difficult to hydrate; lipid aggregates hydrates well and extrudes with >80% efficiency lipid film difficult to hydrate; lipid aggregates lipid film difficult to hydrate; lipid aggregates hydrates well and extrudes with >80% efficiency at concentrations < 20 mg/ml total lipid hydrates well and extrudes but non-uniform, trimodal liposome size distribution with aggregates  Liposomes of all compositions were prepared in HBS pH 7.4 buffer using the extrusion method of vesicle formation with 100 nm polycarbonate filters. a  98  Ci -cer/DSPC/Chol (15:40:45) Liposomes 6  REL. U O L  YOLUME-IYT GAUSSIAN DISTRIBUTION -1—1  1  )  XOO  SO  60  20  O  '20 Size  Cnr<i>  so  100  200  500  IK  (Uesicles)  Figure 3.6 Mean liposome vesicle diameter of 121.9 ± 53.2 nm for Ci -cer/DSPC/Chol (15:40:45) liposomes as determined by quasi-elastic light scattering using a Nicomp 270 submicron particle sizer model 370/270. Hepes buffered saline pH 7.4 was used for sample dilution. 6  99  Figure 3.7 Cytotoxicity of Ci -cer/DSPC/Chol (15:40:45) and control DSPC/Chol (55:45) liposomes on wild-type (A) and mdr-1 gene transfected (B) MDA435/LCC6 human breast cancer cells. Cells were incubated with the indicated ceramide or total lipid concentrations for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD. 6  100  100-  |  [ H]CHE lipid 3  J [ C]C -ceramide 14  16  80-  o 60-  o  03 O  40  A  20  A  ro or  100  80  4H  0) o  60 -J  o  40-^  03 O  B  ~o 03  or  20  H  4 hour  1 hour  24 hour  Figure 3.8 Cellular uptake of Ci -cer/DSPC/Chol (15:40:45) liposomes by wild-type (A) and mdr-1 gene transfected (B) MDA435/LCC6 cells. Uptake of [ H]CHE bulk liposomal lipid and [ C]Ci6-ceramide are expressed as a percent of the total radioactivity added, normalized to 10 cells. Data are averaged means from two independent experiments conducted in triplicate; bars, SD. 6  3  ,4  5  101  negatively charged lipid in an attempt to increase the interaction of the liposomes with the target cells. To accomplish this goal the cholesterol component of the formulation was replaced by the acidic cholesterol ester cholesteryl hemisuccinate (CHEMS).  Using this  lipid, Ci6-ceramide could be incorporated into C H E M S containing liposomes at 50 mole percent, for a final lipid composition of Ci6-cer/CHEMS (50:50; Table 3.2). The increase in ceramide incorporation was attributed to the molecular geometry of C H E M S , as described in Chapter 1. These liposomes displayed a mean diameter of 95.2 nm with a standard deviation of 42.6% (Figure 3.9). Liposomes composed of high content Ci6-ceramide have not been previously described in the literature. Therefore, cryo-TEM images were obtained in order to further characterize the size and shape of these novel vesicles. Control liposomes composed of D P P C / C H E M S (50:50) were also evaluated.  The non-ceramide lipid of the control  liposomes in this case was changed from DSPC to DPPC to more closely match the 16 carbon acyl chain length of the ceramide. Control liposomes displayed a mean diameter of 97.2 nm with a standard deviation of 27.3% as measured by quasi-elastic light scattering (Figure 3.9). The representative cryo-EM micrographs in Figure 3.10 show that both the control and ceramide-based liposomes are spherical vesicles with an average diameter in the range of 100 nm, which confirms the quasi-elastic light scattering data. The images also indicate that the ceramide-based liposomes are uniform, primarily unilamellar vesicles. A small amount (1 mole percent) of the steric stabilizer PEG2000-  102  A: Cie-cer/CHEMS (50:50) Liposomes REU UOL  VOLUME-WT GAUSSIAN DISTRIBUTION ~i—1—1—1 1 1  lOO  1  r  t 1 frm  SO 60 40  Jfll 20 Ska.  50 lOO 200 <Solid P a r t i c l e s )  20 S i z e <nn) ->  500  IK  B: DPPC/CHEMS (50:50) Liposomes REL UOL  VOLUMESVT GAUSSIAN ]DISTRIBUTION 1  100  1  I I I  H -H  60  a  =  1  •  1  1 "1  •'..  E .'•  SO  1  1  =  •  ?. ' ':  —  = •  9 10 S i z e (nn) ->  11  I  H E  20  50  I  1  I I  E E  I 1 1 ii 11 11 1 1 1 m 1 I rJ ! Ii  r  AO 20  1  z •z —  =  z z. E  Ill'  m1  100 200 < Uesicles)  SOD  Figure 3.9 Mean liposome vesicle diameter of 95.2 + 40.5 nm for Ci6-cer/CHEMS (50:50) (A) and 97.2 ± 28.5 nm for control DPPC/CHEMS (50:50) (B) liposomes as determined by quasi-elastic light scattering using a Nicomp 270 submicron particle sizer model 370/270. Hepes buffered saline pH 7.4 was used for sample dilution.  103  Figure 3.10 Cryo-transmission electron micrographs of DPPC/CHEMS/PEG2000-DSPE (49.5:49.5:1; A ) and Ci -cer/CHEMS/PEG ooo-DSPE (49.5:49.5:1; B) liposomes. The bar in each image represents 100 nm. 6  2  104  DSPE was incorporated into these formulations to prevent liposome aggregation during transport. The DPPC/CHEMS and Ci6-cer/CHEMS formulations were also characterized on the basis of trapped volume, which provides a measure of the internal aqueous volume of the lipid vesicles.  Control (DPPC/CHEMS) and ceramide  (Ci -cer/CHEMS) 6  liposomes were determined to have trapped volumes of 1.81 ± 0.03 ul/umole total lipid and 1.64 ± 0.07 u.l/umole total lipid, respectively.  These results are consistent with  previously published results for other phospholipid/cholesterol liposome formulations (261). Evaluation of the in vitro cytotoxicity of Ci6-cer/CHEMS liposomes demonstrated a modest increase in activity over the Ci6-cer/DSPC/Chol formulation; however, the IC50 value remained greater than 100 u M (Figure 3.11). The slight improvement in activity was not attributable to the negative charge of the C H E M S lipid, as control liposomes composed of DPPC/CHEMS (50:50) showed no activity at all. The cytotoxicity was correlated with a modest increase in cellular uptake (-16% ceramide uptake at 24 hours for the C]6-cer/CHEMS formulation versus -4.5% ceramide uptake at 24 hours for the Ci6-cer/DSPC/Chol formulation; Figure 3.12). However, overall liposome internalization remained low.  3.4.5  Cytotoxicity of Free and Liposomal Ceramide in J774 Murine Macrophage Cells  The modest improvement in activity afforded by the Ci6-cer/CHEMS liposomes gave a preliminary indication that liposomal delivery of Ci6-ceramide may be effective i f efficient intracellular delivery could be achieved. In order to demonstrate this from a proof-of-principle perspective, the J774 murine macrophage cell line was investigated as a new target cell population.  This cell line has been demonstrated to internalize  105  120  A  TO 40-\ CD  °  20  • — D P P C / C H E M S (50:50) . — • — C - c e r a m i d e / C H E M S (50:50) 16  120  B  0 111 1  1E-7  i  i  i  i  i 111  j  1E-6  1  i — i  i  i  1111  "  1E-5  i ••  i"""  i  i  i  i ti  [  • "i  1E-4  Total Lipid/Ceramide Concentration (M)  Figure 3.11 Cytotoxicity of Cie-cer/CHEMS (50:50) and D P P C / C H E M S (50:50) liposomes on wild-type (A) and mdr-1 gene transfected (B) MDA435/LCC6 human breast cancer cells. Cells were incubated with the indicated ceramide or total lipid concentrations for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD.  106  Figure 3.12 Cellular uptake of Ci -cer/CHEMS (50:50) liposomes by wild-type (A) and mdr-1 gene transfected (B) MDA435/LCC6 cells. Uptake of [ H]CHE bulk liposomal lipid and [ C]Ci -ceramide are expressed as a percent of the total radioactivity added, normalized to 10 cells. Uptake is expressed as a percentage of the total radioactivity added (corrected for binding with controls at 4°C) in order to allow for direct comparisons between liposomal and ceramide lipid to be made. Data are averaged means from two independent experiments conducted in triplicate; bars, SD. 6  3  14  6  5  107  liposomes via the endocytic pathway (224), and should therefore facilitate intracellular delivery of the liposomes to an organelle (endosomes) where pro-apoptotic ceramide is known to be endogenously generated by aSMases (159, 160, 262). Before investigating the effect of ceramide-containing liposomes on J774 cells it was first necessary to examine whether free ceramide lipids displayed similar cytotoxicity against the J774 cells as was observed in the MDA435/LCC6 cells. The results in Figure 3.13 confirmed that the same trend of free ceramide cytotoxicity was observed, with the short-chain C6- and Cg-ceramides being the most active (IC50 values of 14.4 u M for both) and Ci6-ceramide showing no activity. Evaluation of the in vitro cytotoxicity of Ci6-ceramide liposomes in the J774 cell line demonstrated that Ci6-cer/CHEMS liposomes dramatically improved the cytotoxicity of Ci6-ceramide in these cells (Figure 3.14). Specifically, while the IC50 value of Ci6ceramide when exogenously applied to J774 cells in its free form was well in excess of 100 u M , its formulation into and delivery via C H E M S liposomes decreased the I C  50  to  36.1 u M , bringing it into the range of cytotoxicity observed with free C6-ceramide (14.4 uM). This cytotoxic effect was ceramide-specific and was not attributed to the C H E M S lipid, as control DPPC/CHEMS (50:50) liposomes were non-cytotoxic (Figure 3.14). Cellular uptake studies indicated that both the liposome and the ceramide components were internalized, as was evidenced by uptake of the [ H]CHE and [ C]Ci6-ceramide 3  labels, which both approached 50% after 24 hours (Figure 3.15).  14  This indicated that  under these conditions the Ci6-ceramide lipid was being delivered specifically by the liposomal carrier rather than by passive lipid exchange, as was previously the case for C^ceramide liposomes.  108  120  rrj 1E-8  1 —  |  1E-7  1 —  I 1E-6  1 —  I 1E-5  '—  1  I 1E-4  Ceramide Concentration (M)  Figure 3.13 Cytotoxicity of various acyl chain length free ceramide lipids on J774 murine macrophage cells. Cells were incubated with the indicated ceramide concentrations for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD.  109  120  1E-7  1E-6  1E-5  1E-4  Total Lipid/Ceramide Concentration (M)  Figure 3.14 Cytotoxicity of Ci -cer/CHEMS and DPPC/CHEMS liposomes on J774 cells. Cells were incubated with the indicated ceramide or total lipid concentrations for 72 hours and cell viability was measured using the M T T assay. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD. 6  110  Figure 3.15 Cellular uptake of Ci -cer/CHEMS liposomes by J774 cells. Uptake is expressed as a percentage of the total radioactivity added (corrected for binding with controls at 4°C) in order to allow for direct comparisons between liposomal and ceramide lipid to be made. Data are averaged means from two independent experiments conducted in triplicate; bars, SD. 6  Ill  3.5  DISCUSSION The field of sphingolipid biology is rapidly evolving, with great emphasis on  ceramide lipids in particular as mediators of diverse biological effects. involving the relationship between  ceramide lipids and apoptosis  Research  has received  considerable interest from both mechanistic (105, 106, 176, 177, 263) and therapeutic (58, 264) perspectives. Therapeutic approaches based on the modulation of intracellular ceramide levels in vitro is not a novel concept per se. The direct addition of exogenous, cell-permeable ceramides has been shown to induce apoptosis in many cell lines. Research based on this approach has largely focused on the C6-ceramide form due to its ease of solubility and therefore  delivery in aqueous media.  However, studies  investigating exogenous application of the more physiologically relevant long-chain ceramides such as Ci6-ceramide have been limited by difficulties with the poor solubility of natural ceramide in aqueous media and its reduced cell permeability characteristics. Therefore, the experimental objective of this chapter was to investigate the pro-apoptotic characteristics of free exogenous ceramides with respect to acyl chain length, and to then employ liposome-based delivery systems to enhance this activity through improved intracellular delivery. Initial studies examined the effect of different acyl chain length synthetic ceramides on in vitro cytotoxicity using the wild-type and mdr-1 gene transfected MDA435/LCC6 cell lines. A relationship between acyl chain length and cytotoxicity was observed, with short-chain C6-ceramide being most active (IC50 values in the 3-6 u M range) and the longer chain Ci6-ceramide being least active (IC50 values well in excess of 100 uM). The finding of potent activity of C6-ceramide is consistent with other research  112  in this field (265-267). As well, the minimal cytotoxicity observed with C2-ceramide is supported by work by Payne et al. (268) who found the cytotoxicity of C2-ceramide equivalent to control treatment in primary placental trophoblasts as measured by M T T O'Byrne et al. also failed to observe apoptosis in response to C2-ceramide  assays.  exposure (269). This is not always the case, however, as other research has shown potent cytotoxicity with C2-ceramide, suggesting that its pro-apoptotic effects may be cell-line specific (270). It is encouraging to note that there was very little difference in the sensitivity of wild-type versus resistant cell lines to the effects of ceramide.  This suggests that  ceramide-based therapies may be equally effective in the treatment of sensitive and M D R tumors that overexpress Pgp. The observed differences in cytotoxicity related to acyl chain length were correlated with the relative uptake of radioactive [ C]C - and [ C]Ci6-ceramide lipids. 14  14  6  These results demonstrated that short-chain ceramide uptake was more than 3-fold greater than that of long-chain ceramide (7 pmol cer/u.g protein versus 2 pmol cer/u.g protein),  implicating  ceramide cell-permeability characteristics  as  an  important  determinant of cytotoxicity. This highlights the importance of achieving intracellular delivery as a pre-requisite to ceramide-mediated apoptosis. The benefits of liposomebased delivery systems presented in Chapter 1, combined with the emerging role of ceramide as a bioactive lipid, led to the novel concept of incorporating ceramide lipids into liposome bilayers. The intrinsic activity of C6-ceramide identified it as the initial candidate for formulation into ceramide-based liposomes. Homogenous, primarily unilamellar vesicles  113  of approximately 100 nm average diameter containing up to 45 mole percent C6-ceramide in the liposome bilayer were successfully prepared.  These liposomes demonstrated  potent cytotoxic activity in both the wild-type and resistant MDA435/LCC6 cell lines, with IC50 values less than 20 u M whereas control (non-ceramide) liposomes were inactive.  However, cellular uptake studies revealed that the cytotoxicity was due to  exchange of the short-chain ceramide lipid from the liposome bilayer rather than through delivery by the liposome carrier. Thus, liposomal formulation of C6-ceramide offered no delivery advantage over free C6-ceramide. Because of this reduced C6-ceramide stability in the bilayer, the longer chain and more physiologically relevant Ci6-ceramide became an attractive alternative. Since the lack of activity of free Ci6-ceramide was attributed to poor intracellular delivery, it was speculated that its formulation into a liposomal delivery system may be sufficient to overcome this obstacle. The physico-chemical properties of Ci6-ceramide described in Chapter 1 presented significant formulation challenges and prevented proper hydration and dispersion of the lipid film when incorporated into DSPC/Chol-based liposomes above 15 mole percent.  This observation is consistent with those of Holopainen et al. who  observed extensive aggregation upon incorporation of high ceramide levels into M L V s designed to evaluate the biophysical effects of ceramide on lipid bilayers (271). Interestingly, despite the successful incorporation of 15 mole percent Ci6-ceramide into Ci6-cer/DSPC/Chol  (15:40:45) liposomes, this formulation showed no activity when  incubated with cells in vitro and the ceramide IC50 value remained greater than 100 u,M. This was attributed to a lack of liposome internalization by the target cells. Consistent with the postulation that C6-ceramide exchanged from the bilayer because of its short  114  acyl chain length, the longer chain (more hydrophobic) Ci6-ceramide did not appear to exchange from the liposome bilayer. However, since the liposomes were not internalized by the MDA435/LCC6 cells the ceramide component of the bilayer could not reach its intracellular target(s). Limited cellular uptake of liposomes is not uncommon for uncharged liposome formulations in the absence of specific targeting ligands. Therefore, the next step was to modify the liposome formulation in an attempt to increase association with the target cells.  Cholesteryl hemisuccinate was chosen for this purpose on the basis of three  specific attributes. combined  with  First, as discussed in Chapter 1, this lipid has been successfully the  non-bilayer  forming  (Hn  phase)  lipid  dioleoylphosphatidylethanolamine (DOPE) to stabilize its presence in liposome bilayers. Since Ci6-ceramide has similar shape-related properties to DOPE the objective was to extend the application of C H E M S to the stabilization of long-chain ceramide in bilayers. Second, the presence of electrostatic interactions due to the negatively charged C H E M S should cause the liposomes to interact more strongly with the cell surface and this may promote liposome internalization. Third, C H E M S is a pH sensitive lipid that, when protonated at pH -5.5, triggers liposome destabilization and should promote ceramide lipid release in endosomes, where pro-apoptotic ceramide is known to be produced via aSMase. Based on this rationale, Ci6-ceramide incorporation into C H E M S containing liposomes was investigated.  Indeed, Ci6-ceramide could be formulated into stable  liposomes at up to 50 mole percent (Ci6-cer/CHEMS, 50:50), which represented a substantial improvement over the non-CHEMS based formulations in which the maximum level of ceramide incorporation was 15 mole percent. Characterization of  115  these liposomes by quasi-elastic light scattering and cryo-TEM revealed uniform, primarily unilamellar, spherical vesicles of approximately 100 nm diameter.  Trapped  volume studies were consistent with those for conventional liposomes composed of phospholipids and cholesterol. Control liposomes composed of D P P C / C H E M S (50:50) were prepared in order to match the acyl chain length of the Ci6-ceramide component. Cytotoxicity studies showed a modest improvement in activity of Ci6-cer/CHEMS liposomes over Ci6-cer/DSPC/Chol formulations;  however, the IC50 value remained  above 100 u M . Cellular uptake studies revealed that the presence of C H E M S only enhanced liposome internalization by the MDA435/LCC6 cells to a small degree. Nevertheless, these results gave a preliminary indication that exogenous Ci6-ceramide may show activity i f the objective of efficient intracellular delivery could be met. Since modifications at the level of the liposome formulation were not sufficient to enhance liposome internalization, attention was turned to the target cell population. In order to evaluate the effectiveness of liposome-based delivery of Ci6-ceramide from a proof-of-principle perspective the J774 murine macrophage cell line, which has been previously demonstrated to internalize liposomes via endocytosis, was introduced. Cytotoxicity studies using free ceramide lipids confirmed that the J774 cells were affected by exogenous ceramides in a similar manner to the MDA435/LCC6 cells. Evaluation of the Ci6-cer/CHEMS liposomes in J774 cells showed enhanced cytotoxicity over both free Ci6-ceramide and Ci6-cer/DSPC/Chol formulations. Specifically, whereas the IC50 values for free Ci6-ceramide and Ci6-cer/DSPC/Chol liposomes were both greater than 100 u,M, Ci6-cer/CHEMS liposomes had an IC50 value of 36.1 u M . This approximates the cytotoxicity of free C6-ceramide in these cells (14.4 (J.M), and was  116  demonstrated to be a ceramide-specific effect since control DPPC/CHEMS liposomes were inactive.  Correlation of these results with cellular uptake studies confirmed  internalization of both the liposome and ceramide lipid components, both of which approached 50% after 24 hours. These results were very promising, as they demonstrated for the first time the successful formulation of a physiologically relevant, natural ceramide lipid into a liposome-based carrier system. Furthermore, whereas Ci6-ceramide was inactive when administered in its free form, this work demonstrated that its activity could be significantly enhanced by incorporation into a liposomal delivery vehicle.  Taken  together, these results provide an increased understanding of the basis for the differences in the cytotoxicity of exogenous short- and long-chain ceramide lipids, and demonstrate that successful intracellular delivery of exogenous, Ci6-ceramide can be used to trigger apoptosis.  This suggests the potential for developing therapeutic strategies based on  controlled ceramide delivery. The next step, then, was to develop these systems for in vivo applications and to evaluate their pro-apoptotic activity in an animal model. These goals formed the basis of Chapters 4 and 5 which follow.  117  CHAPTER 4 DEVELOPMENT OF AN IN VITRO EXCHANGE ASSAY TO A C C U R A T E L Y PREDICT T H E LIPID AND DRUG RETENTION PROPERTIES OF LIPOSOME-BASED DELIVERY SYSTEMS*  4.1  INTRODUCTION AND RATIONALE The results presented in Chapter 3 demonstrated the importance of intracellular  delivery for the development and application of exogenous therapeutically active inducers of apoptosis.  ceramide lipids as  Although cell-permeable C6-ceramide  showed activity when applied free in solution, the development of C6-ceramidecontaining liposomes for controlled ceramide delivery was limited by the apparent rapid ceramide exchange from the liposomal carrier into cellular lipid membranes. The ability of liposome-based systems to improve the therapeutic activity of bioactive lipids such as ceramide requires that the active lipid component be retained in the liposome bilayer throughout the delivery process. While the occurrence of rapid lipid exchange during cell culture experiments is certainly a frustration, this behaviour becomes even more problematic when such observations are made during the subsequent phase of in vivo characterizations in animal models, which are more costly and labor intensive than evaluations using cell culture systems. Both of the Ci6-ceramide containing formulations described in Chapter 3 appeared to retain the ceramide lipid for the duration of exposure to cells in vitro. However, our laboratory has observed that in vitro release of liposome contents is often not representative  of in vivo release characteristics (201, 272).  Consequently, before moving any ceramide liposomes forward for development and  *Adapted from: J A Shabbits, G N Chiu and LD Mayer (2002). Development of an in vitro exchange assay that accurately predicts in vivo drug retention for liposome-based delivery systems. Journal of Controlled Release, 84(3): 161-70.  118  evaluation in animal models it was necessary to develop an in vitro assay to predict the Ci6-ceramide retention properties that should be observed in vivo. The use of liposomes to deliver therapeutic lipids is a relatively recent application of liposome technology, and ceramide-based formulations designed for therapeutic applications have not been previously described in the literature.  However, there are  many well-characterized liposome formulations containing conventional anticancer agents that have already been developed for in vivo applications, and observations regarding the release of encapsulated drug contents from these systems can be applied to the development of the lipid exchange assay described here. Conventional  liposomal drug  formulations  undergo  extensive  in  vitro  optimization in order to achieve prolonged drug retention and stability before being moved forward to more advanced stages of testing. However, it is often the case that in vitro based assays do not accurately predict the liposomal drug retention properties actually observed in vivo.  In fact, it is not uncommon for formulations to exhibit  excellent drug retention properties in vitro, but to display almost complete drug release within minutes following systemic administration to animals (201).  For example,  cyclosporins incorporated into liposomes for the intended purpose of M D R modulation are rapidly released from the lipid carrier in the circulation, resulting in drug pharmacokinetic properties comparable to unencapsulated drug (273, 274). This calls into question the usefulness of current in vitro pre-clinical drug release assays, and highlights the need for more accurate predictors of true in vivo performance. Although several in vitro drug release assays have been developed (275-277), one of the most commonly used methods of measuring encapsulated drug release relies on  119  dialyzing the liposomal formulation against large volumes of buffer at physiological temperatures. The excess extravesicular buffer is intended to serve as a driving force to promote drug leakage from the liposome. Upon release from the liposomal carrier, free drug crosses the dialysis membrane and accumulates in the buffer system. Serum is frequently added to the dialysis buffer to more closely mimic the physiological environment (proteins and lipid constituents) that liposomes encounter in vivo. However, despite these efforts the drug leakage properties observed using dialysis-based systems often show a poor correlation with actual in vivo results.  It is believed that this  discrepancy is due to the inability of dialysis-based assays to mimic the large cellular membrane pool (lipid "sink") that exists in the physiological setting, comprising blood cells and tissues into which hydrophobic and amphipathic drugs can distribute after in vivo administration. Upon systemic administration, rapid drug leakage from liposomes may be observed as encapsulated hydrophobic or amphipathic drugs diffuse across the liposome bilayer and incorporate into these membranes.  A similar scenario may be  envisioned to occur for the exchange of lipid bilayer components such as ceramides. In order to study this latter scenario, an in vitro release assay was developed in which an excess of multilamellar vesicles were used as membrane "acceptors" for ceramide lipid exchange from "donor" liposomes. It is believed that the excess M L V s will mimic the physiological lipid membrane pool and, therefore, should more closely reflect the true in vivo retention properties of liposomes. This chapter describes the development and validation of the assay procedure and demonstrates its utility in predicting in vivo ceramide lipid retention properties. Results demonstrating the application of this assay to  120  the evaluation of drug release from conventional drug encapsulated liposomes are also presented.  4.2  HYPOTHESIS The hypothesis underlying the research presented in this chapter is that the  development of an in vitro drug release assay that uses large, multilamellar acceptor vesicles to simulate the physiological tissue and membrane lipid "sink" will allow for the lipid exchange characteristics of ceramide-based liposomes observed in vivo to be predicted in vitro, thereby facilitating the characterization and development of ceramidebased liposomes for in vivo applications.  4.3  MATERIALS AND METHODS  4.3.1  Materials A l l lipids were purchased from Avanti Polar Lipids (Alabaster, A L ) . Cholesterol  and Sephadex G-50 were obtained from the Sigma Chemical Company (St. Louis, MO). [ H]CHE was purchased from Perkin Elmer (Boston, M A ) , and [ C ] C H E and 3  14  [ H]verapamil were purchased from N E N (Boston, M A ) . [ C]C6- and [ C]Ci6-ceramide 3  14  14  were purchased from American Radiolabeled Chemicals Inc. (St. Louis, M O ) . [ C]doxorubicin-HCl was purchased from Amersham International (Buckinghamshire, 14  UK).  Non-radiolabeled doxorubicin-HCl and verapamil were obtained from Faulding  (Montreal, QC, Canada) and Sabex Inc. (Boucherville, QC, Canada), respectively. Spectra/Por dialysis tubing was purchased from Spectrum Laboratories (Rancho Dominguez, CA). Fetal bovine serum was obtained from Hyclone (Logan, UT). Pico-  121  fluor 40 scintillation cocktail was purchased from Packard Biosciences (Groningen, The Netherlands). Female SCJD/Rag-2 mice were bred in-house at the B C Cancer Agency animal facility (Vancouver, B C , Canada).  4.3.2  Preparation of Donor Large Unilamellar Vesicles (LUVs) Lipid films were prepared as previously described in Chapter 3, with radiolabels  incorporated as indicated.  A l l lipid ratios are specified on a mole:mole ratio unless  otherwise indicated. Lipid films containing ceramide or prepared for use as empty (nondrug encapsulated) liposomes were hydrated in 1 ml Hepes buffered saline (HBS; 20 m M Hepes/150 m M NaCl, pH 7.4) with heat and vortexing. Films to be used for conventional drug encapsulation were hydrated in 300 m M citrate buffer, pH 4.0 with heat and vortexing, followed by five freeze-thaw cycles (5 minutes liquid nitrogen freeze, 5 minutes 65°C water bath thaw).  Homogenously sized liposomes were then produced  following extrusion as previously described.  Liposome/ceramide lipid concentrations  were determined by liquid scintillation counting.  4.3.3  Preparation of Acceptor Multilamellar Vesicles (MLVs) Egg phosphatidylcholine and cholesterol lipids were weighed and combined at a  55:45 ratio as described above. [ H] or [ C]CHE was incorporated as indicated in order 3  14  to distinguish between the M L V and L U V populations. Lipids films were hydrated in 1 ml warm 300 m M sucrose with vortexing to yield M L V s , which were transferred to a 1.5 ml Eppendorf tube. The suspension was centrifuged at 4200 rpm for 10 minutes, after which the M L V s appeared as a supernatant layer with the sucrose buffer below. A n 18  122  guage needle syringe was passed through the M L V layer to withdraw the sucrose. The M L V s were resuspended in 0.5 ml HBS with vortexing and centrifuged again at 4200 rpm for 10 minutes, after which the M L V s appeared as a pellet. The HBS supernatant was  withdrawn and the  pellet was  washed  twice (two  cycles of 0.5 ml  HBS/vortex/centrifuge) before being resuspended in fresh HBS and stored at 4°C until needed.  4.3.4  Separation of L U V and M L V Populations Donor [ H]-LUVs (0.1 umoles) and 10 umoles of [ C]-acceptor M L V s were 3  14  combined in a 1.5 ml Eppendorf tube to yield a 100-fold molar excess of acceptor vesicles, and HBS was added to bring the final volume to 0.5 ml. A l l samples were run in triplicate, and control samples consisting of L U V or M L V populations alone were included. Samples were vortexed and centrifuged at 4200 rpm for 10 minutes to pellet the M L V s . The LUV-containing supernatant was collected into a scintillation vial. The MLV-containing pellet was then washed twice with 250 pi additions of fresh H B S followed by vortexing and centrifuging as above. Both washes were combined into a second scintillation vial. The pellet was then resuspended in 500 u.1 HBS and transferred to a third scintillation vial. The amount of radioactivity in each fraction was measured by liquid scintillation counting. Radioactivity associated with the supernatant and washes was combined to give an overall supernatant count, and the degree of cross-over contamination between populations was determined.  Radioactivity in the combined  washes was less than 10% of the supernatant-associated radioactivity.  123  4.3.5  Liposomal Encapsulation of Doxorubicin Liposomes radiolabeled with [ H]CHE were prepared in 300 m M citrate buffer, 3  pH 4.0. After extrusion the liposomes were passed down a Sephadex G-50 HBS (pH 7.4) column to exchange the external buffer and establish a pH gradient across the liposome membrane (inside acidic).  Four milligrams of doxorubicin in saline and 20 mg of  liposomes were aliquoted into separate glass test tubes and heated for 5 minutes in a 65°C waterbath.  [ C]doxorubicin was incorporated at 0.5 LiCi/mg to facilitate drug 14  quantitation. Doxorubicin was added to the liposomes while vortexing to achieve a final drug:lipid ratio of 0.2:1 (wt:wt). The liposomes were heated at 65°C with occasional vortexing for 10 minutes, cooled to room temperature and passed down a fresh Sephadex G-50 H B S column to remove any unencapsulated doxorubicin.  Doxorubicin and  liposome concentrations were determined by liquid scintillation counting.  4.3.6  Liposomal Encapsulation of Verapamil Sphingomyelin:cholesterol (55:45) liposomes labeled with  [ C ] C H E were 14  prepared in 300 m M citrate buffer, p H 4.0. One milligram of verapamil in saline was added to 10 mg of liposomes in a 5 mL glass vial to yield a 0.1:1 (wt:wt) drug to lipid ratio. [ H]verapamil was incorporated at 1 LiCi/mg to facilitate drug quantitation. The 3  vial was capped, inverted 5 times to mix and the external pH was adjusted to p H 7.4 by the addition of 1 ml of 0.5 M Na2HP04. The vial was inverted 5 times to mix and placed in a 56°C waterbath for 5 minutes, after which the vial was mixed again and heated for an additional 5 minutes. Liposomes were then passed down a fresh Sephadex G-50 H B S  124  column to remove unencapsulated verapamil. Verapamil and liposome concentrations were determined by liquid scintillation counting.  4.3.7  Ceramide Lipid/Drug Release from Liposomes Using Dialysis Assays A 750 p.1 aliquot of donor liposomes was placed into a 4 inch piece of Spectra/Por  dialysis tubing (molecular weight cut off of 12-14 kDa) sealed at both ends with clips. The dialysis tubing was placed into a beaker containing a 1000-fold excess of HBS with or without 30% FBS. Liposomes were incubated with stirring for 24 hours at 37°C. At various timepoints 50 pi aliquots were withdrawn from the tubing for drug analysis. Ceramide, doxorubicin and verapamil were quantitated by liquid scintillation counting.  4.3.8  Ceramide Lipid/Drug Release from Liposomes Using the MLV-Based Release Assay Various ceramide containing or drug-encapsulated donor L U V s and EPC/Chol  (55:45) acceptor M L V s were individually combined at a 1:100 mole ratio and incubated for the indicated times at 37°C in a revolving rack. The L U V and M L V populations were separated by centrifugation and washed as described above. The amount of ceramide lipid/drug in the pellet and supernatant fractions was then measured by liquid scintillation counting.  Controls consisting of L U V s or M L V s alone were also run.  The percent  ceramide lipid/drug retained was determined as: [(amt. ceramide or drug in supernatant)/(total amt. ceramide or drug at t=0)] x 100.  125  4.3.9  In Vivo Ceramide Lipid/Drug Release A l l animal studies were conducted according to procedures approved by the  University of British Columbia's Animal Care Committee and were performed in accordance with the current guidelines established by the Canadian Council of Animal Care.  Ceramide-containing or drug encapsulated  liposomes were diluted with  appropriate volumes of HBS so as to administer a dose of 100 mg/kg total lipid for ceramide-based liposomes, 20 mg/kg doxorubicin or 0.50 mg/kg verapamil by i.v. bolus (200 uL, tail vein) to female SCID/Rag2 mice (20 gram average weight; n=3/time point). At 1,4, and 24 hours post-administration the mice were sacrificed with C O 2 and blood was drawn by cardiac puncture. The blood was collected in microtainer tubes (EDTA anticoagulant) and centrifuged at 2200 rpm for 10 minutes to isolate the plasma, which was analyzed for drug and lipid content by scintillation counting.  4.4  RESULTS  4.4.1  Design of the MLV-Based Exchange Assay Procedure Figure 4.1 depicts a schematic of the overall ceramide lipid exchange assay  procedure.  Ceramide-containing donor L U V s of 100 nm average diameter were  incubated in Eppendorf tubes with a 100-fold molar excess of M L V s , which served as a lipid sink to mimic the physiological membrane pool. At time zero, all ceramide lipid is associated with the LUVs. Over the course of incubation, however, ceramide lipid can exchange from the L U V population and associate with the acceptor M L V membranes based on mass action equilibrium. These two populations can then be isolated by centrifugation and the amount of ceramide in each fraction is measured. The L U V and  126  M L V populations are not drawn to scale, and for illustrative purposes the ceramide is represented as a disproportionately large lipid.  Acceptor MLVs  Donor LUVs /*' Ceramide-containing LUVs combined imbined with acceptor MLVs MLVs at \?v 1:100 (mol:mol) L U V : M L V ratio  *-#V J : * ' -  *#\  I»vv-v  J®  Incubate at 37°C with constant rotation  1  Centrifuge at 2200 rpm for 10 minutes  1  J  r^N  5  L U V Supernatant M L V Pellet  Figure 4.1 A schematic illustration of the overall in vitro MLV-based assay. At time zero all ceramide lipid (represented as dark circles in the liposome bilayer) was associated with donor L U V s , which were combined with acceptor M L V s at a 1:100 (mole:mole) ratio and incubated at 37°C in a revolving rack. At various timepoints selected samples were centrifuged at 4200 rpm for 10 minutes. The M L V s formed a distinct pellet and the L U V s remained in the supernatant. Separation was >85% efficient. The two populations were assayed for ceramide lipid or drug content in order to measure the extent of donor to acceptor exchange.  127  4.4.2  Separation of Control and Ceramide-Containing Donor L U V and Acceptor M L V Populations In order to demonstrate the validity of this assay it was first necessary to verify  that various donor L U V formulations could be efficiently separated from acceptor M L V s by centrifugation. Centrifugation at 4200 rpm for 10 minutes resulted in the formation of a distinct M L V pellet while the L U V s remained in the supernatant. The composition of EPC/Chol (55:45) was chosen as the universal M L V acceptor since it represents an unsaturated and uncharged bilayer that is similar to many physiological membranes. A number of different donor L U V formulations were evaluated, however, in order to demonstrate that separation of the two vesicle populations by this technique was not highly dependent on lipid composition. Formulations that represent a broad range of membrane/bilayer properties were investigated: unsaturated (EPC/Chol, 55:45), saturated (DSPC/Chol,  55:45),  sterically  stabilized  (DSPC/Chol/PEG oo-DSPE, 20  50:45:5),  sphingolipid-based (SM/Chol, 55:45), cationic (DOTAP/DOPC/Chol, 10:45:45), anionic (DOPS/DSPC/Chol,  10:45:45), pH sensitive (DPPC/CHEMS,  50:50), short-chain  ceramide (C -cer/DSPC/Chol, 45:10:45), and long-chain ceramide (Ci -cer/CHEMS, 6  6  50:50). Regardless of the L U V composition, the efficiency of separation of L U V and M L V populations was consistently greater than 90%, with the exception of SM/Chol liposomes which was 89%. In all cases greater than 89% of the L U V s and less than 0.7% of the M L V s were recovered in the supernatant fraction, and less than 10% of the L U V s and greater than 96% of the M L V s were recovered in the pellet (Table 4.1).  This  indicated that the separation procedure was effective and could potentially be applied to a variety of liposome formulations.  128  Table 4.1 Separation of Empty [ H]-LUV Donor and [ C]-MLV Acceptor Vesicles By Centrifugation (mean ± SD) 3  14  a  LUV Formulation  % Recovery in Supernatant LUV MLV  EPC/Chol (55:45) DSPC/Chol (55:45) DSPC/Chol/PEG ooo-DSPE (55:45:5) SM/Chol (55:45) DOTAP/DOPC/Chol (10:45:45) DOPS/DSPC/Chol (10:45:45) DPPC/CHEMS (50:50) C -cer/DSPC/Chol (45:10:45) C, -cer/CHEMS (50:50)  93.56 ±4.72 97.08 ± 7 . 1 4 96.99 ± 0 . 5 0 89.90 ±0.42 96.08 ± 0.06 100 93.23 ± 1.07 96.01 ±4.11 96.88 ± 2.29  2  6  6  0.54 ± 0 . 1 6 0.50 ±0.01 0.30 + 0.01 0.47 ± 0 . 0 6 0.40 ± 0.00 0.41 ±0.03 0.62 ± 0 . 0 4 0.43 ±0.07 0.42 ± 0.07  % Recovery in Pellet LUV MLV 6.23 ± 0 . 9 0 7.58 ± 0 . 1 8 5.55 ± 4 . 5 0 9.84 ± 0 . 4 4 9.70 ± 3 . 0 7 0 6.08 ± 1.02 5.13 ± 3 . 2 7 3.87 ± 1.26  104.47 ± 1 . 0 8 103.89 ±2.41 105.18 ± 2 . 1 0 102.64 ± 0 . 9 0 97.88 ± 2 . 6 8 99.59 ± 0 . 0 3 96.87 ±0.27 106.87 ± 0 . 8 4 100.87 ±1.25  L U V and M L V populations were mixed at a 1:100 L U V : M L V (mole:mole) ratio and separated by centrifugation at 2200 rpm for 10 minutes. The M L V pellet was washed twice and combined with the supernatant fraction. The results represent the percent L U V s and M L V s in the supernatant and pellet after centrifugation as measured by liquid scintillation counting. Data represents the mean of two independent experiments conducted in triplicate. a  129  4.4.3  Evaluation of C6-Ceramide Retention Using Conventional In Vitro Dialysis Assays Figure  4.2  shows  the  profile  of  C6-ceramide  release  from  C6-  cer/DSPC/Chol/PEG oo-DSPE (45:10:40:5) L U V s following dialysis against a 1000-fold 20  excess of H B S (closed symbols) or H B S + 30% FBS (open symbols) at 37°C.  A  sterically stabilized liposome formulation containing PEG2000-DSPE was used in order to allow for direct comparisons between in vitro and later in vivo lipid exchange to be made. At 1, 4 and 24 hours three 50 uL aliquots were withdrawn from the dialysis bag and analyzed for [ C]C6-ceramide content. The results demonstrated extensive C6-ceramide 14  exchange from the L U V s over the dialysis period. The exchange followed a biphasic pattern with a rapid loss of greater than 50% of the ceramide lipid in the first 4 hours, followed by a more gradual exchange over the 4-24 hour time period. After 24 hours only 23.6%o and 21% of the ceramide lipid remained associated with the liposomes under the HBS and HBS + 30% FBS dialysis conditions, respectively.  4.4.4  Evaluation of C6-Ceramide Retention Following I.V. Bolus Administration The dialysis-based C6-ceramide exchange rates were correlated with actual in vivo  ceramide exchange following i.v. administration of the same liposomes to mice. Figure 4.3 shows the plasma C6-ceramide circulation profile for C6-cer/DSPC/Chol/PEG2oooDSPE (45:10:40:5) liposomes administered by i.v. bolus at a total lipid dose of 100 mg/kg.  Rapid ceramide exchange was observed, with less than 1% of the total  administered dose detectable in the plasma after 24 hours.  The percent C6-ceramide  remaining in circulation after 1, 4, and 24 hours was 7.46%, 1.67% and 0.56% respectively. In order to confirm that loss of plasma-associated ceramide lipid was not  130  -1—I—I—I—I—I—I—I—I—1—I—I—I—I—1—1—I—I—I—I—I—1—I—I—I— 0 2 4 6 8 10 12 14 16 18 20 22 24  Time (hours)  Figure 4.2 Profile of C -ceramide release from C -cer/DSPC/Chol/PEG2ooo-DSPE (15:10:40:5) donor L U V s following dialysis against HBS (open symbols) or HBS + 30% FBS (closed symbols) for 24 hours at 37°C. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD. 6  6  131  0  2  +  6  8  10  12  14  16  18  20  22  24  26  Time (hours)  Figure 4.3 Circulation profile of C6-ceramide (closed symbols) and bulk liposomal lipid (open symbols) following i.v. bolus administration (tail vein) of C6cer/DSPC/Chol/PEG oo-DSPE (15:10:40:5) liposomes to female Balb/c mice. A 100 mg/kg total lipid dose was administered by i.v. bolus in a 200 ul injection volume. At 1, 4 and 24 hours three mice were sacrificed with C 0 and blood was drawn by cardiac puncture. The blood was collected in microtainer tubes (EDTA anticoagulant) and centrifuged at 2200 rpm for 10 minutes to isolate the plasma, which was analyzed for [ C]C6-ceramide and [ H]CHE bulk liposomal lipid by scintillation counting. Each value represents the mean from two independent experiments; bars, SD. 20  2  14  3  132  attributed to overall elimination of the liposomes from the circulation, the bulk liposomal lipid marker [ H]CHE was also monitored. These results confirmed that the liposomes 3  remained in circulation over the 24 hour period while the ceramide lipid component did not (85.47%, 63.25% and 26.34% liposomal lipid remaining at 1, 4 and 24 hours, respectively). The in vivo ceramide exchange results demonstrated a faster and more extensive rate of ceramide exchange from the liposomes than was predicted by the dialysis assay system (r=0.776 and r=0.839 for HBS and HBS + 30% FBS, respectively). This confirmed the need for a better in vitro predictor of in vivo behaviour.  4.4.5. Evaluation of C6-Ceramide Retention Using the MLV-Based In Vitro Assay In order to test the in vivo predictive accuracy of the MLV-based assay, the C6cer/DSPC/Chol/PEG oo-DSPE (45:10:40:5) liposomes were used as donor L U V s and 20  incubated with acceptor M L V s according to the assay procedure described above. Figure 4.4 shows the profile of [ C]C6-ceramide release using this assay. 14  In contrast to the  release profiles predicted by dialysis-based assays, the drug release rates predicted by the MLV-based assay show a much better correlation with the behavior of these formulations in vivo (r=0.990). Specifically, 9.65%, 3.67% and 1.62% C6-ceramide was retained after 1, 4 and 24 hours respectively using the M L V exchange assay. These results suggest that the M L V acceptor population more accurately mimics the physiological environment that the liposomes encounter following systemic administration than dialysis against buffer or serum. Correlation coefficients for the comparisons made between the various assays are summarized in Table 4.2.  133  100  X3 CD  c  'CO CD  Cd CD "O  'E  CO 1  oCID O P  CD  -—i—<—i——r 2 4 6 8 1  1  1  10  i— —i— —i— —r —i— —i—>" 1  12  1  14  1  16  -1  18  1  20  22  24  26  Time (hours)  Figure 4.4 Profile of C -ceramide release from C -cer/DSPC/Chol/PEG oo-DSPE (15:10:40:5) donor L U V s using the MLV-based exchange assay. Ceramide-containing donor L U V s were incubated with acceptor M L V s for 24 hours at 37°C with constant mixing. At 1, 4 and 24 hours the samples were centrifuged for 10 minutes at 4200 rpm to separate the L U V and M L V populations. The M L V pellet was washed twice and combined with the supernatant fraction. The M L V and L U V fractions were analyzed for [ C]C6-ceramide content by liquid scintillation counting. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD. 6  6  20  14  134  Table 4.2 Correlation Coefficient (r) and Coefficient of Determination (r ) for Ceramide Lipid, Doxorubicin or Verapamil Release from Liposomes as Measured by the Dialysis and MLV-Based Assays Relative to Actual In Vivo Results 2  2  C6-ceramide  Dialysis with HBS r = 0.776 r - 0.602 r = 0.608 r = 0.370 r = 0.291 r = 0.085 2  DSPC/Chol/PEG o-DSPE/Dox 200  SM/Chol/Verapamil  2  2  Dialysis with FBS r = 0.839 r = 0.704 r = 0.673 r = 0.453 r = 0.361 r = 0.130 2  2  2  MLV-based Assay r = 0.990* r = 0.980* r = 1.000* r = 1.000* r = 0.864 r = 0.746 2  2  2  In vivo r = 1.000 r = 1.000 r = 1.000 r = 1.000 r = 1.000 r = 1.000 2  2  2  The correlation coefficient was calculated as: r = [n(Zxy) - (Zx)(Sy)]/sqrt{[(nEx )-(Ix) ][nEy )-(Sy) ]} Statistically significant from zero (p<0.05) a  2  2  2  2  135  4.4.6. Evaluation of Ci6-Ceramide Retention Using the MLV-Based In Vitro Assay Given the high correlation between in vivo and in vitro release for the C6ceramide formulation using the MLV-based exchange assay, the next step was to evaluate ceramide exchange from the Ci6-cer/CHEMS (50:50) formulation that showed promising in vitro cytotoxicity. Results presented in Figure 4.5 demonstrate that Ci6ceramide retention in these liposomes was very high, with 90.47%, 70.56% and 32.83% of the ceramide lipid remaining liposome-associated after 1, 4 and 24 hours, respectively. This is also in agreement with previously published results demonstrating that the rate of exchange of [ C]Ci6-ceramide between lipid vesicles was on the order of days (278). 14  These encouraging results suggested that these liposomes should retain the bioactive creamide lipid component in vivo and would be a suitable candidate to carry forward for in vivo evaluation.  4.4.7  Evaluation of the MLV-Based Assay as a Measure Encapsulated Conventional Drug Release  of  Liposomally  Additional experiments were completed to evaluate whether the utility of this new assay could be extended beyond the exchange of liposome-associated lipids to assess the release of conventional drugs encapsulated within the aqueous liposome core. Two conventional drugs were chosen for this evaluation: doxorubicin encapsulated into DSPC/CI10I/PEG2000-DSPE (55:40:5) liposomes, which represents a well-characterized formulation known to retain the encapsulated drug for extended periods (>24 hours) post i.v. administration, and verapamil encapsulated into SM/Chol (55:45) liposomes, which represents a liposome formulation that appears to retain the drug following in vitro dialysis but that releases >90% of the encapsulated contents within one hour post i.v.  136  Figure 4.5 Profile of Ci6-ceramide release from Ci6-cer/CHEMS (50:50) donor L U V s using the MLV-based exchange assay. Ceramide-containing donor L U V s were incubated with acceptor M L V s for 24 hours at 37°C with constant mixing. At the timepoints indicated samples were centrifuged for 10 minutes at 4200 rpm to separate the L U V and M L V populations. The M L V pellet was washed twice and combined with the supernatant fraction. The M L V and L U V fractions were analyzed for [ C]Ci6-ceramide content by liquid scintillation counting. Each value represents the mean from three independent experiments conducted in triplicate; bars, SD. 14  137  administration.  The results presented in Table 4.3 demonstrate that separation  efficiencies of more than 90% were achieved for the drug-loaded L U V and acceptor M L V populations. This indicated that the encapsulation of drug into L U V s did not adversely affect the ability to separate the donor and acceptor vesicle populations by centrifugation. The release of doxorubicin and verapamil from their respective liposome formulations was then measured as previously described by dialysis against HBS or HBS + 30%> FBS, using the MLV-based release assay, or by analysis of plasma following i.v. bolus administration. For the evaluation of doxorubicin release (Figure 4.6A), monophasic release kinetics were observed and greater than 85% of the encapsulated drug was retained by the liposomes over the 24 hour incubation period following dialysis against HBS or HBS + 30%) FBS. The MLV-based assay demonstrated biphasic doxorubicin release with 72% of the doxorubicin remaining liposome-associated after 4 hours and 58% remaining after 24 hours. In this case the MLV-based assay predicted more rapid doxorubicin release in the first 4 hours than was actually observed in vivo. Specifically, whereas the M L V based assay predicted 72% and 64% doxorubicin retention after 1 and 4 hours, respectively, the actual in vivo results predicted 98% and 75% retention at 1 and 4 hours. However, the final 24 hour doxorubicin retention result of 58% predicted by the M L V based assay corresponded well to the actual in vivo retention value of 55%, and the M L V based assay also demonstrated more accurate biphasic release characteristics than the monophasic dialysis-based assays. For the evaluation of verapamil release (Figure 4.6B), rapid triphasic drug release was observed following dialysis against HBS or HBS + 30% FBS, with approximately  138  35%, 18%) and 4% retained after 1, 4 and 24 hours, respectively. However, these results did not reflect the true extent of verapamil exchange observed in vivo. The MLV-based assay was more predictive, although the correlation did not reach statistical significance (p=0.33 for the MLV-based assay versus p=0.81 and p=0.76 for dialysis versus HBS and FBS, respectively). Both the MLV-based assay and the in vivo results showed extensive drug loss that displayed biphasic release characteristics. The in vivo retention results of 5%>, 4.5%) and 0% at 1, 4 and 24 hours, respectively, were closely predicted by the M L V based assay which demonstrated 11%, 6% and 3% retention at the same time points. The doxorubicin and verapamil release results, which are summarized in Figure 4.6, indicate that for both formulations the MLV-based assay showed a higher correlation with in vivo drug release than the dialysis-based systems, indicating again that it serves as a better predictor of true in vivo performance.  Table 4.2 provides a summary of the  correlation coefficients for each of the assay methods.  139  Table 4.3 Separation of Doxorubicin or Verapamil-Loaded [ H]-LUV Donor and [ C]-MLV Acceptor Populations by Centrifugation (mean ± SD) 3  14  a  L U V Formulation DSPC/Chol/PEG o-DSPE/Dox SM/Chol/Verapamil 200  % Recovery in Supernatant LUV MLV 90.58 ± 1.82 0.33 ±0.08 94.75 ±0.26 0.87 ±0.13  % Recovery in Pellet LUV MLV 7.98 ±0.35 106.17 ±0.83 3.29 ±0.44 101.36 ±0.90  doxorubicin was encapsulated at a 0.2:1 drug:lipid (wt:wt) ratio and verapamil at a 0.1:1 (wt:wt) ratio. L U V and M L V populations were mixed at a 1:100 L U V : M L V (mole:mole) ratio and separated by centrifugation at 2200 rpm for 10 minutes. The results represent the percent L U V s and M L V s found in the supernatant and pellet after centrifugation as measured by liquid scintillation counting.  140  o Q  - • - Dialysis vs HBS - • - Dialysis vs 30% FBS  20 -  — A — MLV-based Assay — • — In Vivo Results 0  B  0  2  4  6  8  10  12  14  16  18  20  22  24  26  Time (hours)  Figure 4.6 Release profiles of doxorubicin (A) and verapamil (B) from liposomes as measured by dialysis assays, the in vitro MLV-based assay, or from plasma collected following i.v. bolus liposome administration. Each value represents the mean from two independent experiments conducted in triplicate; bars, SD.  141  4.5  DISCUSSION Liposomes are well known as delivery vehicles for many conventional drugs such  as chemotherapeutic agents and antifungals (see references cited in Chapter 1). However, their utility as delivery systems for bioactive lipids incorporated into the liposome * membrane is just beginning to be realized. A n important step in the development of liposomes for either application is the evaluation of drug/lipid retention by the liposomal carrier.  This is essential for utilizing the liposome as a delivery vehicle and for  capitalizing on the inherent advantages of site-specific localization, mass-action delivery of contents and reduced drug toxicities described in Chapter 1. If the drug or bioactive lipid component is not adequately retained then the liposome can offer no delivery advantage, as was observed with the in vitro evaluation of the C6-ceramide formulation in Chapter 3.  Although the ultimate goal of developing liposomal formulations is to  optimize them for in vivo applications (ultimately in humans through clinical trials, but also in animal models for pre-clinical screening), it is first necessary to characterize their properties at the level of in vitro testing. As conventional drug encapsulated liposomal formulations were developed, so too were in vitro drug release assays based on methods such as dialysis (275-277). Although commonly used, this screening process is limited by its frequent inability to accurately predict actual in vivo liposome behaviour. This is likely due to its inability to mimic the extensive membrane lipid pool that exists in the physiological setting. The objective of this chapter, therefore, was to develop a more reliable in vitro system with which to evaluate the release characteristics of ceramidebased liposomes before moving them forward to more extensive in vivo evaluations. A large excess of M L V s was used as an acceptor lipid "sink" to promote exchange of  142  ceramide lipid from donor L U V s . The results presented in this chapter demonstrate that sucrose-containing M L V s can be separated from 100 nm L U V s with -90% efficiency, irrespective of L U V composition or the presence of encapsulated drug. Using the C6-cer/DSPC/Chol/PEG2ooo-DSPE liposomes from Chapter 3 as a model system, it was observed that the MLV-based assay demonstrated C6-ceramide exchange properties consistent with the C6-ceramide in vitro uptake results observed in Chapter 3 (loss of C6-ceramide from the liposomal carrier). Although the 24 hour C6ceramide exchange measured by the dialysis assay was consistent with the uptake results, the rate of release predicted by dialysis was much slower than that which was observed in vivo. This was resolved by the fact that the MLV-based assay showed a better correlation with in vivo C6-ceramide exchange than the dialysis systems (r=0.99 versus -0.80). The MLV-based assay was then used to evaluate Ci6-ceramide retention in the Ci6-cer/CHEMS (50:50) liposome bilayer. Since this formulation demonstrated superior cytotoxicity in the J774 cell line in vitro, it became the lead candidate for further evaluation.  Results from the MLV-based assay predicted that Ci6-ceramide should  remain associated with the membrane over 24 hours. This suggested that it was a suitable formulation for the next stage of in vivo analysis, which was the focus of Chapter 5. Given the apparent utility of this system for evaluating lipid exchange, its application to the release of encapsulated conventional drugs was also investigated. The results demonstrated that this MLV-based assay was a better predictor of both doxorubicin and verapamil release from liposomal carriers than dialysis-based systems, and showed a high correlation with the extent of observed in vivo release (r=1.0 and 0.86 for doxorubicin and verapamil, respectively). Therefore, not only is this system useful  143  for developing ceramide-based liposomes, it also has potential applications for the development of numerous liposomal systems based on other bioactive lipids or encapsulated agents.  144  CHAPTER 5 PHARMACOKINETIC EVALUATION AND ANTITUMOR ACTIVITY OF HIGH CERAMIDE CONTENT LIPOSOMES* 5.1  INTRODUCTION AND RATIONALE The  ability of liposomes to  overcome  the  difficulties associated  with  solubilization and intracellular delivery of long-chain (natural) Ci6-ceramide in vitro was demonstrated in Chapter 3 by the development of high content ceramide liposomes composed of Ci6-cer/CHEMS (50:50).  Whereas free Ci6-ceramide showed no  cytotoxicity when added to cells in culture, when delivered to cells in a liposomal formulation that was effectively internalized by macrophage-derived  J774 cells,  significant ceramide-specific cytotoxicity was observed. This implicated the utility of ceramide-based liposomes as a novel and biologically active ceramide delivery vehicle. However, in order to translate these in vitro observations into an in vivo model, it was first necessary to characterize the in vivo pharmacokinetic behavior of these formulations. Lessons learned from the development of therapeutically active liposomes with conventional drugs and small molecules encapsulated in the aqueous core have highlighted the importance of characterizing the drug retention properties of liposomebased formulations.  The same evaluation process is necessary for ceramide-based  liposomes in order to ensure that the biologically active lipid component remains liposome-associated for the duration of the delivery process.  The exchange assay  described in Chapter 4 provided an effective preliminary in vitro screening mechanism to predict how these formulations would behave in vivo.  Characterization of the Ci6-  cer/CHEMS (50:50) formulation using the MLV-based assay indicated that the liposomes  *Adapted from: J A Shabbits and LD Mayer. Antitumor activity of ceramide liposomes (in preparation).  145  should retain the bioactive lipid component over 24 hours following systemic administration.  This level of stability was sufficient to warrant more extensive  characterization in animal models. Therefore, the objectives of this thesis chapter were to characterize the in vivo behavior of these liposomes using pharmacokinetic analysis following i.v. bolus administration, and to evaluate the antitumor activity of this formulation in the J774 ascites tumor model.  5.2  HYPOTHESIS The hypothesis underlying the research presented in this chapter is that high  content ceramide liposomes with stability in vivo will exhibit antitumor activity against J774 cells growing as an ascites tumor in syngenic mice.  5.3  MATERIALS AND METHODS  5.3.1  Materials A l l lipids and cell culture materials were obtained as previously described in  Chapters 2 and 3. Female Balb/c mice were bred in-house at the B C Cancer Agency animal facility (Vancouver, B C , Canada).  5.3.2  Cell Line and Culture J774 murine macrophage cells were obtained and cultured as previously described  in Chapter 3.  146  5.3.3  Preparation of Liposomes Liposomes were prepared as previously described in Chapters 3 and 4.  5.3.4  Pharmacokinetic Analysis of Control and Ceramide Liposomes Pharmacokinetic studies were conducted in order to determine the in vivo  circulation longevity and ceramide retention properties of high Ci6-ceramide-content liposomes. A l l animal studies were conducted according to procedures approved by the University of British Columbia's Animal Care Committee and in accordance with the current guidelines established by the Canadian Council of Animal Care. (DPPC/CHEMS/PEG2000-DSPE,  47.5:47.5:5)  liposomes  containing  the  Control [ H]CHE 3  radiolabel, and ceramide-based (Ci6-cer/CHEMS/PEG ooo-DSPE, 47.5:47.5:5) liposomes 2  containing both [ H]CHE and [ C]Ci6-ceramide radiolabels were administered by i.v. 3  14  bolus (200 ul, tail vein) over approximately 5 seconds into female Balb/c mice (20 gram average weight) at a total lipid dose of 100 mg/kg. At 0.5, 1, 2, 4, 8, 12 and 24 hours three mice per time point were sacrificed with C O 2 and blood was drawn by cardiac puncture.  The blood was collected in microtainer tubes (EDTA anticoagulant) and  centrifuged at 2200 rpm for 10 minutes to isolate the plasma, which was analyzed for bulk liposomal lipid ([ H]CHE) and [ C]Ci6-ceramide lipid by liquid scintillation 3  14  counting. Prior to obtaining the plasma pharmacokinetic parameters, the log plasma lipid concentration versus time data were fitted to one- and two-compartment models using WinNonlin Version 1.1 (Pharsight Corp., Mountain View, CA). The appropriate model was selected on the basis of goodness of fit for each model tested using the Akaike  147  Information Criterion (AIC). The AIC values of 72.5, 72.3 and 53.5 obtained for the control liposomes, ceramide liposomes (total lipid) and ceramide liposomes (ceramide lipid), respectively, using a one-compartment model were not improved by using a twocompartment  model so the  one-compartment  model was used  for subsequent  pharmacokinetic analysis. The observed and predicted log plasma lipid concentration versus time graph shown in Figure 5.1 was also in agreement with the one-compartment model.  WinNonlin Version 1.1 software was then used to calculate the following  parameters: elimination half-life (t^) - In 2/k (where k=-2.303 x slope of the log cone, vs time graph) area under the curve (AUCo-><x) was calculated using the trapezoidal rule plasma clearance (CL ) = DoseiJAUCo^oo P  volume of distribution (Vd) = CLp/k  5.3.5  Establishment of the J774 Ascites Tumor Model Cell suspensions were prepared in Hank's Balanced Salt Solution at a  concentration of 2 x l 0 cells/ml and were innoculated i.p. into female Balb/c mice in an 6  injection volume of 0.5 ml. Animals were weighed daily and observed for morbidity and mortality. In particular, signs of ill health based on body weight gain due to ascitic tumor growth, altered gait and distension of the abdomen were monitored.  Animals were  terminated by C O 2 asphyxiation when significant tumor-related illness requiring euthanasia was observed.  148  c  o  10000  *4=>  D)  3  100 -I 0  1 5  1  1  1  10  15  20  25  Time (hours)  g  10000  100 -I  1  1  1  1  1  0  5  10  15  20  25  Time (hours)  Figure 5.1 Correlation plots comparing observed and predicted log plasma lipid concentration versus time graphs for control and ceramide liposomes modeled with WinNonlin 1.1 using a one-compartment i.v. bolus dosing model.  149  5.3.6  Evaluation of Antitumor Activity On day 0, l x l O cells/0.5 ml were injected i.p. into the mice. A total of four 6  mice/group were treated with saline, control liposomes or ceramide-based liposomes on day 1 (single-dose study) or days 1, 5, 9 (multi-dose studies). Animals were monitored as described above. Antitumor activity was measured as % Increase in Lifespan (%ILS) which was defined as: [(Median Survival ted)-(Median Survival ntroi)]/[MedianSurvivaI ntroi] x 100. trea  5.3.7  CO  CO  Statistical Analysis Statistical analysis was performed using one way analysis of variance ( A N O V A )  followed by Student-Newman-Keuls analysis with InStat Version 3.0 for Windows (GraphPad Software, Inc., San Diego, CA). Mean differences with ap value <0.05 were considered statistically significant.  5.4  RESULTS  5.4.1  Pharmacokinetic Analysis of Control and Ceramide Liposomes Following I.V. Bolus Administration Although an estimated prediction of the in vivo behaviour of Ci6-ceramide  liposomes with respect to ceramide lipid retention was obtained using the MLV-based assay described in Chapter 4, it was necessary to confirm these retention properties following systemic administration and to fully establish the pharmacokinetic parameters associated  with  these  liposomes  cer/CHEMS/PEG ooo-DSPE 2  in  vivo.  (47.5:47.5:5)  Formulations and  composed  of Ci6-  DPPC/CHEMS/PEG oo-DSPE 20  (47.5:47.5:5) were compared. Control and ceramide-based liposomes were administered  150  to mice by i.v. bolus and plasma collected at 0.5, 1, 2, 4, 8, 12 and 24 hours was analyzed by scintillation counting to determine the percent injected lipid dose remaining in the circulation. The pharmacokinetic behaviour of the control formulation was evaluated by following the radioactive [ H]CHE liposomal lipid marker. Results presented in Figure 3  5.2 demonstrated that the control liposomes were stable in the circulation over a 24 hour time period.  The liposomes were gradually eliminated from the plasma with an  elimination half-life of 10.7 hours, and 21.1% of the total lipid dose administered remained in the plasma after 24 hours. A plasma clearance value of 0.061 ml/hr and a plamsa A U C of 32.43 mg*hr/ml were obtained. A volume of distribution of 0.96 ml indicates that the liposomes are confined primarily to the blood/plasma, as the plasma volume of a mouse is approximately 1 ml.  These pharmacokinetic parameters are  summarized in Table 5.1. The pharmacokinetic behaviour of ceramide-based liposomes was evaluated from two perspectives. As with the control formulation, the behaviour of the liposome as a whole was monitored using the [ H]CHE lipid marker (Figure 5.2). 3  These results  demonstrated that the ceramide-containing liposomes were also stable in the circulation over 24 hours, although they displayed somewhat more rapid plasma elimination than control liposomes. This was evidenced by a shorter elimination half-life of 9.2 hours and a more rapid plasma clearance of 0.09 ml/hr. This was also confirmed by a smaller plasma A U C value of 22.28 mg*hr/ml. A volume of distribution of 1.2 ml is again consistent with the liposomes remaining in the blood/plasma (Table 5.1). Although the plasma elimination behaviour of the ceramide-containing formulations was more rapid  151  Figure 5.2 Plasma elimination profile of control (DPPC/CHEMS/PEG2000-DSPE, 47.5:47.5:5) and ceramide-based (Ci -cer/CHEMS/PEG oo-DSPE, 47.5:47.5:5) liposomes following i.v. bolus administration to female Balb/c mice. Bulk liposomal lipid was radiolabeled with [ H]CHE and ceramide lipid was radiolabeled with [ C ] C , ceramide. Mice were sacrificed at 0.5, 1, 2, 4, 8, 12 and 24 hours and plasma was analyzed for lipid as previously described. Data are means (n=3); bars, SD. 6  3  20  14  6  152  Table 5.1 Summary of Plasma Pharmacokinetic Parameters for Control and Ci6-Ceramide Containing Liposomes Following I.V. Bolus Administration at a Dose of 100 mg/kg Total Lipid ab  Control liposomes' [ H1CHE label Ceramide liposomes [ H]CHE label Ceramide liposomes' [ C]Cer label 3  ri  Dose (mg)  ty (hr)  (mg*hr/ml)  CL (ml/hr)  v (ml)  2.0  10.74 + 0.50  32.43 ± 1.12  0.061 ± 0.002  0.96 + 0.03  2.0  9.19 ±0.58  22.28 ± 0.99  0.090± 0.004  1.19 ±0.06  0.816  8.26 ±0.33  8.62 ±0.25  0.095 ± 0.003  1.13 ±0.04  2  AUCo->oo  P  d  .,_  3  1  14  elimination half-life (t ) = In 2/k, where k=-2.303 x slope of the log cone, vs time graph; area under the curve (AUCo->«>) was calculated using the trapezoidal rule; plasma clearance (CLp) = Dosei. /AUCo-**; volume of distribution (Vd) = CLp/k Data shown are mean ± S E M , n=3/group DPPC/CHEMS/PEG ooo-DSPE (47.5:47.5:5, mole:mole) Ci6-cer/CHEMS/PEG ooo-DSPE (47.5:47.5:5, mole:mole)  a  H  v  b  c  2  d  2  153  than control liposomes, 15.3% of the administered lipid dose remained in the plasma after 24 hours, indicating that the formulation was still stable in the circulation. In addition to monitoring the behaviour of the ceramide-containing liposomes as a whole, the behaviour of the ceramide lipid bilayer component itself was also measured by following the [ C]Ci6-ceramide radiolabel. A comparison of the [ H]CHE and [ C]Ci614  3  14  ceramide radiolabels associated with the ceramide containing liposomes revealed virtually identical plasma circulation profiles over the 24 hour period (Figure 5.2). This was an important observation because it confirmed that the bioactive Ci6-ceramide lipid remained associated with the liposome bilayer in the plasma.  This observation was  supported by a ceramide plasma elimination half-life of 8.26 hr and a plasma ceramide clearance of 0.95 ml/hr, both of which were similar to those obtained for the ceramidecontaining liposome as measured by [ H]CHE behaviour (Table 5.1). A plasma A U C 3  value of 8.62 mg*hr/ml was also obtained. This value cannot be directly compared with the A U C value obtained using the [ H]CHE data because the lipid doses for each are 3  necessarily different [0.82 mg Ci6-ceramide lipid versus 2.0 mg total lipid (ceramide + non-ceramide) in the formulation].  5.4.2  Evaluation of Antitumor Activity of Ceramide Liposomes in the J774 Ascites Tumor Model The efficacy of ceramide-based liposomes compared to controls was evaluated  following single and multiple dosing schedules in the J774 ascites tumor model. The first efficacy study employed a multiple dosing regimen (day 1, 5 and 9; cell innoculation day 0) for control or ceramide liposomes administered by i.v. bolus at a dose of 200 mg/kg total lipid.  Under these treatment conditions the saline and liposome control groups  154  displayed median survival times of 23 days, while the Ci6-ceramide containing liposome treatment group had a median survival time of 27 days. This corresponded to an ILS of 17.4% (Figure 5.3). A l l control animals were terminated on day 23 in accordance with the animal welfare guidelines. Since there was no standard deviation associated with the treatment group survival times it was not possible to calculate the statistical significance of the results. Given the importance of intracellular delivery of the ceramide component of the liposomes, subsequent studies were initiated to investigate  whether  direct i.p.  administration of the formulation to the site of the ascites tumor cells might improve the therapeutic response beyond that observed following i.v. bolus administration. This was done on the basis that maximizing the liposome contact with the target J774 cells would improve the likelihood of liposome endocytosis.  The first of these studies compared  control liposomes at 200 mg/kg total lipid with ceramide-containing liposomes at 100 mg/kg and 200 mg/kg total lipid doses administered i.p. one day after cell innoculation. The survival curves presented in Figure 5.4 indicate that groups treated with saline or control liposomes at 200 mg/kg total lipid had a median survival time of 24 days. The group treated i.p. with ceramide-liposomes at a dose of 100 mg/kg total lipid showed a median survival time of 26 days, which corresponded to an 8.3% ILS. Again, all treatment group animals were terminated on day 26 so the statistical significance of the 100 mg/kg treatment regimen could not be determined. Animals in the 200 mg/kg dosing group showed a median survival time of 27.5 days, which corresponded to a 14.6% ILS. Animals in this group showed survival to days 23, 25 and 30 (2 mice). Although these  155  iiitiiiiiiiiiiiiiiiiiiiMll• o CD  c  24 CO CD O  — • — Saline control —#— Control liposomes (200mg/kg) — A — Ceramide liposomes (200mg/kg) OH 2  I ' I  4 ,. 60  I  1  I  I  1  I  I  1  I  1  I  1  I  1  I  I  1  I  8 o Ji O i 12 14 16 18 20 22 24 26 28 30 32 34  Day  Figure 5.3 Evaluation of antitumor activity of Ci -cer/CHEMS/PEG ooo-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG2000-DSPE (47.5:47.5:5) liposomes in the J774 ascites tumor model. On day zero, l x l O cells were innoculated i.p. into female Balb/c mice (4 mice/group) and saline, control liposomes or ceramide liposomes were administered by i.v. bolus on days 1, 5, and 9 at the lipid concentrations indicated. Arrows indicate the days of treatment administration. Animals were weighed and monitored daily for survival. 6  2  6  156  44  CO •4—•  o c  E >  2  00 .y 1  OH  — • — Saline control — C o n t r o l liposomes (200mg/kg) —A—Ceramide liposomes (100mg/kg) —A—Ceramide liposomes (200mg/kg) I  Y  1  I  i ' i ' i ' i  4  6  8  1  i  1  i  1  i ' i ' i  1  i  1  i  1  i  1  i ' i ' i  10 12 14 16 18 20 22 24 26 28 30 32 34  Day  Figure 5.4 Evaluation of antitumor activity of Ci -cer/CHEMS/PEG oo-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG2000-DSPE (47.5:47.5:5) liposomes in the J774 ascites tumor model. On day zero l x l 0 cells were innoculated i.p. into female Balb/c mice (4 mice/group) and saline, control liposomes or ceramide liposomes were administered i.p. on day 1 at the lipid concentrations indicated. The arrow indicates the day of treatment administration. Animals were weighed and monitored daily for survival. 6  20  6  157  results did not reach statistical significance (p=0.190), the %ILS values suggest a trend toward an increased response at the higher dose. On the basis of these results a follow-up study using a multiple-dosing schedule with i.p. treatments at 200 mg/kg total lipid administered on days 1, 5 and 9 was then investigated.  Using this regimen the saline and 200 mg/kg control liposome groups  showed median survival times of 23 days, whereas the Ci6-ceramide containing liposome group at a dose of 200 mg/kg total lipid showed a median survival time of 33 days (Figure 5.5).  This corresponded to an ILS of 43.5%, which was determined to be  statistically significant (p=0.028).  mmmtum  4HJ  CO o CO  c i= > 2> I  Zi  CO H CD O  =8:  — • — S a l i n e control 0 _| — • — Control liposomes (200mg/kg) — A — Ceramide liposomes (200mg/kg) i ' i  4  t  6  t  1  i  1  8  i  1  i  i  1  1  i • i  1  i  1  i  1  i  1  i  1  i  1  i  1  i  10 12 14 16 18 20 22 24 26 28 30 32 34  Day  t  Figure 5.5 Evaluation of antitumor activity of Ci -cer/CHEMS/PEG2ooo-DSPE (47.5:47.5:5) versus DPPC/CHEMS/PEG2000-DSPE (47.5:47.5:5) liposomes in the J774 ascites tumor model. On day zero l x l 0 cells were innoculated i.p. into female Balb/c mice (4 mice/group) and saline, control liposomes or ceramide liposomes were administered i.p. on days 1, 5, and 9 at the lipid concentrations indicated. Arrows indicate the days of treatment administration. Animals were weighed and monitored daily for survival. 6  6  158  5.5  DISCUSSION The increasing attention being paid to the intracellular signaling and pro-apoptotic  properties of ceramide lipids has made them an exciting new target for therapeutic manipulation.  Dysregulation of ceramide production and/or metabolism has been  implicated in a number of disease states including atherosclerosis, insulin resistance, diabetes, cancer and multidrug resistance to chemotherapy (61, 279-284). Consequently, the ability to successfully modulate intracellular ceramide levels in a controlled and precise manner holds great therapeutic promise. Most of the efforts directed toward this goal thus far have utilized short-chain ceramide lipids. Two recent pre-clinical studies demonstrated the utility of synthetic, cell-permeable ceramides as potential therapeutic agents with anti-proliferative effects.  Charles et al. showed that C6-ceramide coated  balloon catheters prevent neointimal hyperplasia in rabbit carotid arteries (284), and Furuya et al. demonstrated that systemic administration of cell-permeable Cg-ceramide significantly reduced focal cerebral ischemia in spontaneously hypertensive rats (285). However, due to difficulties associated with effectively solubilizing and delivering the more physiologically relevant long-chain ceramides, approaches aimed at investigating the therapeutic effects of natural ceramide have primarily focused on indirect modulation of ceramide levels, either via enhanced ceramide production through increased metabolism of precursor sphingolipids (126, 127), or by using inhibitors of ceramide metabolism (132-134). Research presented in the preceeding chapters demonstrated that these solubility obstacles could be overcome by formulating Ci6-ceramide into liposomebased carrier systems. The encouraging cytotoxicity and cellular uptake data obtained following exposure of these liposomes to J774 cells in vitro led to the in vivo studies  159  presented in this chapter, which were aimed at translating the in vitro observations to an animal model. Initial studies examined the pharmacokinetic parameters of control and ceramidebased liposomes following i.v. bolus administration to Balb/c mice. This was done in order to confirm the stability of the liposomes and lipid components in the circulation. Control liposomes were labeled with the bulk lipid marker [ H]CHE, and the behavior of 3  ceramide-containing liposomes was monitored using both [ H]CHE and [ C ] C 3  14  16  ceramide radiolabels. This dual labeling approach allowed the behavior of the liposomes in general, and the ceramide lipid component in particular, to be simultaneously evaluated. Control and ceramide liposomes were administered at a total lipid dose of 100 mg/kg, which corresponded to a lipid dose of 2 mg total lipid per mouse for both formulations, and 0.816 mg Ci6-ceramide lipid for the ceramide-based formulation. Data were modeled in WinNonlin Version 1.1 using a one-compartment i.v. bolus dosing model. The pharmacokinetic parameters and liposome circulation longevity graph obtained from these studies revealed two key observations. First, although the ceramidebased liposomes showed more rapid plasma clearance than control liposomes (CLp=0.09 ml/hr ceramide versus 0.061 ml/hr control), the ceramide-based formulation was still stable in the circulation over the 24 hour period investigated.  Second, the plasma  clearance parameters for [ H]CHE and [ C]Ci6-ceramide were comparable (0.090 ml/hr 3  14  and 0.095 ml/hr, respectively). This was an important observation because it indicated that the Ci6-ceramide remained associated with the liposomal carrier, thereby confirming the ceramide retention properties predicted by the MLV-based assay in Chapter 4, and it  160  also confirmed that the liposomes were indeed acting as a carrier system for the ceramide lipid. In light of these encouraging stability results, the next studies were aimed at evaluating the efficacy of these formulations with respect to antitumor activity. The J774 cells that were used for in vitro studies were established as an ascites tumor model following i.p. innoculation.  Using a multi-dose treatment regimen, liposomes were  administered by i.v. bolus on days 1, 5 and 9 post cell innoculation. Mice treated with ceramide-based liposomes survived to day 27 whereas mice treated with control liposomes survived only until day 23. Although encouraging, this corresponded to a modest 17.4% ILS. It was then speculated that perhaps the liposomes would be more efficacious i f their interaction with the target tumor cells was maximized by administering the liposomes directly into the tumor site (i.p.). Both single-dose (day 1) and multi-dose (days 1, 5, 9) treatment regimens were evaluated. In the single-dose study there was trend toward increased response related to increased dose (8.3% ILS at 100 mg/kg and 14.6%o ILS at 200 mg/kg ceramide liposomes) compared to control liposomes. However, the effect of using the single dose i.p. approach was not superior to the multiple dose i.v. regimen.  Therefore, a multiple dosing regimen was investigated for i.p. treatment  administration. These results demonstrated statistically significant antitumor activity in mice treated with ceramide liposomes at 200 mg/kg compared to controls, and this corresponded to a 43.5% ILS (p=0.028). The results presented in this chapter demonstrate that endogenous ceramides can be formulated into liposomes that display pharmacokinetic parameters suitable for in vivo  161  applications.  Furthermore,  this work demonstrates that ceramide  delivered in  appropriately designed liposomal carriers can inhibit tumor growth in animal models. These results are very promising and provide proof-of-principle data that delivery of exogenous natural ceramide can provide therapeutic antitumor activity in vivo.  162  CHAPTER 6 SUMMARY OF RESULTS AND FUTURE DIRECTIONS The field of ceramide biology has received significant attention over the past decade as roles for this family of bioactive lipids in such diverse cellular responses as inflammation, proliferation, differentiation, senescence and apoptosis have emerged. Dysregulation of ceramide production and/or metabolism has been implicated in numerous disease states including diabetes, atherosclerosis, cancer and multidrug resistance to chemotherapy.  Although the mechanisms by which ceramide mediates  these effects are not fully understood, studies designed to understand the specific involvement of ceramides in these responses have begun to reveal many opportunities for exploiting these lipids in order to achieve specific therapeutic objectives. The work presented in this thesis comprised a series of experiments designed to test the hypothesis that modulating intracellular ceramide levels can be used as a therapeutic approach to achieve chemosensitization of multidrug resistant tumors and induce apoptosis.  Studies employing both indirect and direct approaches to ceramide  modulation yielded results that supported this hypothesis. Observations that cellular apoptosis is preceded by increases in intracellular ceramide, combined with evidence to suggest that multidrug resistance can arise due to overexpression of the ceramide metabolizing enzyme GCS, prompted studies to evaluate the effect of indirect ceramide modulation on the chemosensitivity of multidrug resistant tumor cells.  The results presented in Chapter 2 demonstrated that PDMP-induced  inhibition of pro-apoptotic ceramide metabolism to non-cytotoxic GlcCer resulted in significant chemosensitization of two M D R human breast cancer cell lines to Taxol® and  163  vincristine.  Interestingly, chemosensitization to the non-tubulin binding drugs  doxorubicin and cisplatin was not observed. Although this aspect of the results was not investigated further, it would be interesting to pursue the possible involvement of intracellular ceramide and ceramide metabolite transport via the vesicular trafficking pathway in the context of drug resistance and chemosensitization in light of these results. The successful chemosensitization results obtained with Taxol  and vincristine provided  preliminary evidence that therapeutic approaches aimed at increasing endogenous ceramide levels should enhance apoptosis. This conclusion, combined with research demonstrating that cell-permeable ceramides induce apoptosis when added to tumor cells in vitro, led to the experiments described in Chapter 3, which investigated the effect of direct ceramide modulation using exogenously applied synthetic ceramides. B y comparing the cytotoxicity and cellular uptake of synthetic ceramides with varying acyl chain lengths it was determined that the short-chain, cell-permeable C6-ceramide was internalized and cytotoxic to cells whereas the longer chain Ci6-ceramide was not internalized and therefore did not induce apoptosis. This difference established the importance of achieving intracellular ceramide delivery as a pre-requisite to apoptosis induction. The well established ability of liposomes to facilitate localized delivery of large amounts of carrier-associated agents to target cells prompted the subsequent formulation and development of a novel class of ceramide-based liposomes for this purpose. Although liposomes containing up to 45 mole percent C6-ceramide were successfully formulated into DSPC/Chol-based liposomes, these systems did not retain the ceramide lipid in the liposome bilayer when added to the breast cancer cells in culture. Rather, the  164  amphipathic nature of C6-ceramide allowed it to exchange from the liposome membrane into the cells where it exhibited cytotoxicity independent of the liposomal carrier, which was not internalized. Although the limited stability of these liposomes with respect to ceramide retention excluded them from further development in the context of this thesis, the successful incorporation of such a large amount of biologically active ceramide lipid into the liposome bilayer is in itself significant enough to warrant further investigation from a formulation perspective.  For example, it may be possible to improve the ceramide  retention properties by employing novel strategies to complex the ceramide lipids to other liposome bilayer components  or to metals present in the intra-liposomal buffer.  Successful formulation of stable C6-ceramide liposomes may provide yet another avenue from which ceramide delivery may be pursued. Returning to the research focus of this thesis, given the apparent rapid exchange of C6-ceramide from the bilayer, it was reasoned that this problem could potentially be avoided by focusing on the more hydrophobic Ci6-ceramide form. This approach offered the additional advantage of employing a more physiologically relevant and naturally occurring ceramide.  However, the unique physico-chemical properties of this longer-  chain ceramide limited its stable incorporation into DSPC/Chol-based liposomes to a maximum of 15 mole percent.  Consistent with the prediction of increased stability,  however, the liposome formulation containing Ci6-ceramide did not demonstrate appreciable ceramide exchange from the liposome bilayer upon addition to cells. However, because of limited internalization of liposomes by the breast cancer cells, the ceramide was not delivered intracellularly and minimal cytotoxicity was observed.  165  Although beyond the scope of this thesis, one potential solution to this problem may be to employ active liposome targeting techniques to facilitate liposome internalization. Liposome targeting using antibodies or ligands that bind to receptors known to be overexpressed and internalized by tumor cells could be investigated to promote endocytosis of ceramide-based liposomes. The approaches used in this thesis to address the problem of reduced liposome internalization involved making changes to the non-ceramide components and the target cell population.  liposome bilayer  Relatively recent developments in the  liposome field in the areas of triggered drug release, pH sensitive liposomes and fusogenic vesicles prompted an investigation of the cholesterol derivative C H E M S as an alternate non-ceramide lipid bilayer component.  Cholesteryl hemisuccinate  was  demonstrated to dramatically increase the limit of stable Ci6-ceramide incorporation from 15 to 50 mole percent, presumably by virtue of its lipid molecular shape. Cholesteryl hemisuccinate  should also provide the added benefit  of acid-induced liposome  destabilization upon intracellular delivery to endosomes, where natural ceramide is known to be endogenously generated. Although the negatively charged C H E M S lipid appeared to increase liposome association with the breast cancer cell lines somewhat, this alone was not sufficient to achieve adequate intracellular delivery. However, when the macrophage-derived J774 cells were used as target cells the Ci6-cer/CHEMS (50:50) liposomes were extensively endocytosed and the previously non-cytotoxic Ci6-ceramide became active.  These results collectively confirmed the importance of intracellular  delivery and demonstrated  that, once achieved, exogenously administered natural  ceramide could be used to induce apoptosis.  166  Although these studies moved away from chemosensitization strategies aimed at improving cellular response to conventional chemotherapy drugs, these results were significant because they demonstrated the utility of exogenous ceramide delivery as a therapeutic agent itself. Furthermore, the successful formulation of liposomes containing such a large amount of natural ceramide in the bilayer has not been previously described and presents a novel strategy for controlled ceramide delivery. The next step was to determine whether the encouraging in vitro activity could be translated into in vivo antitumor activity. It was apparent from the C6-ceramide exchange results that Ci6-ceramide must remain liposome-associated in vivo in order to be utilized for controlled ceramide delivery purposes. Although the in vitro studies suggested that the longer chain Ci6-ceramide should remain associated with its liposomal carrier, it is often the case that liposome behaviour observed in vitro is not predictive of in vivo performance. Therefore, before moving the Ci6-ceramide-based formulations forward to studies in animal models it was necessary to develop an in vitro release assay to accurately predict the in vivo ceramide retention properties of these novel liposomes. Previous studies using various methods to evaluate the release of conventional drugs encapsulated within the liposome core have demonstrated that the drug retention parameters obtained using in vitro assays do not accurately reflect in vivo retention properties.  This is likely attributed to the fact that current in vitro methods do not  adequately simulate the extensive lipid environment comprising cellular membranes into which drugs can distribute following systemic administration. Presumably the same scenario would apply to the exchange of ceramide lipids from liposome bilayers in vivo. Therefore, the work presented in Chapter 4 described the development and validation of a  167  novel in vitro exchange assay that employed an excess of acceptor M L V s to mimic the physiological lipid membrane pool.  This assay was used to evaluate the ceramide  retention properties of ceramide-based liposomes and confirmed the observations of extensive C6- and minimal Ci6-ceramide lipid exchange obtained in Chapter 3. This supported the decision to further investigate the behavior of Ci6-cer/CHEMS liposomes in an animal model. Although liposomes containing encapsulated chemotherapy drugs were not utilized for testing the hypothesis set out at the beginning of this thesis, the potential application of the MLV-based assay described in Chapter 4 to the improved evaluation of such conventional liposomal formulations was also demonstrated using doxorubicin and verapamil as model systems.  In addition, as a new generation of multifunctional  liposomes that incorporate features such as targeting ligands, exchangeable P E G lipids and other novel bioactive lipids are developed, it will become increasingly important to accurately measure the retention of these components in the liposome bilayer. It is anticipated that the in vitro assay described in Chapter 4 will be of value to the liposome community as a whole since it appears to be applicable to a diverse range of liposomal systems. On the basis of the encouraging in vitro results presented in Chapters 3 and 4, the objective of the research comprising Chapter 5 was to evaluate the pharmacokinetic behavior  and  antitumor  activity of the  Ci6-cer/CHEMS  formulation in vivo.  Pharmacokinetic studies demonstrated in vivo stability over 24 hours of both the ceramide-based liposomes in general and the ceramide-lipid component in particular. The antitumor activity of these systems was then evaluating using the J774 ascites tumor  168  model. Optimal antitumor activity was observed following a multi-dosing (days 1, 5, 9) regimen with i.p. liposome administration at 200 mg/kg.  This corresponded to a  statistically significant increase in animal survival (43.5% ILS) over non-ceramide based control formulations. In summary, this thesis demonstrates that modulation of intracellular ceramide can be used to chemosensitize resistant tumor cells and induce apoptosis in vitro, and provides proof-of-principle evidence that delivery of exogenous natural ceramide can provide therapeutic antitumor activity in vivo. Although this work is preliminary in nature, it provides evidence for the rational design of ceramide-based liposomes to enhance intracellular delivery of exogenous ceramide lipids, and demonstrates that this approach holds therapeutic promise as a novel strategy for cancer chemotherapy. It is anticipated that future developments, both in the field of ceramide-based therapeutics and targeted liposomal delivery systems, will facilitate the translation of these observations to even more effective systemic tumor treatments.  As well, the application of ceramide-  based systems may expand to include other therapeutic agents encapsulated within the aqueous core of ceramide-containing liposomes. 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