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Plasma lipoprotein distribution of amphotericin B formulations : potential role of phospholipid transfer… Patankar, Nilesh A. 2006

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PLASMA LIPOPROTEIN DISTRIBUTION OF AMPHOTERICIN B FORMULATIONS: POTENTIAL ROLE OF PHOSPHOLIPID TRANSFER PROTEIN by, N I L E S H A. P A T A N K A R B.Pharm., University of Mumbai, 1999 M.Pharm., University of Mumbai 2002 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T F O R T H E D E G R E E O F M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES ( P H A R M A C E U T I C A L SCIENCES) THE UNIVERSITY OF BRITISH C O L U M B I A August 2006 © Nilesh A. Patankar, 2006 A B S T R A C T The objectives of this study were to compare the plasma distribution profile of Amphotericin B (AmpB) following incubation of three commonly used formulations, Fungizone®, Abelcet® and Ambisome® and to determine i f phospholipid transfer protein (PLTP) facilitates the transfer of AmpB into high density lipoproteins (HDL) following incubation of AmpB-phospholipid complex within human plasma. Methods: Plasma lipoprotein distribution of AmpB was studied following incubation of Fungizone®, Abelcet® and Ambisome® at an AmpB concentration of 20 pg/mL within human plasma at 37°C for a range of incubation times. These plasmas were subsequently fractionated into their lipoprotein and lipoprotein deficient fractions by gradient density ultracentrifugation. Each fraction was analyzed for AmpB by high performance liquid chromatography (HPLC). Plasma phospholipid transfer protein (PLTP) assay was established and characterized in vitro using a commercially available kit. Plasma samples from six individual donors were analyzed for PLTP activity. PLTP activity observed from plasmas was then plotted against the percentage of AmpB recovered from the HDL fractions of the respective plasmas. H D L fractions of the same six plasmas were analyzed for the phospholipid content. HDL-phospholipid content was plotted against the percentage of AmpB recovered from the H D L fractions. In order to inhibit plasma PLTP activity, three different approaches were used. 1. Incubation of plasma with polyclonal antibody to PLTP. 2. Incubation of plasma with thimerosal. 3. Heating of plasma at 56°C for 1 hour. Results: Following incubation of Fungizone® within human plasma, the majority (~ 50-60%) of the incubated AmpB was recovered from the lipoprotein deficient fraction (LPDP). However, following incubation of lipid based formulations (i.e. Abelcet® and Ambisome®); i i the majority (~ 60-85%) of incubated AmpB was recovered from the H D L fraction. Plasmas containing elevated total cholesterol (TC) and triglycerides (TG) levels (plasmas with TC and TG >200 mg/dL) showed a four-fold higher AmpB-LDL association than plasmas with TC and TG levels <200 mg/dL following incubation of Fungizone® but did not show any difference in the distribution profile following incubation of Abelcet® and Ambisome®. A statistically significant positive correlation was observed between the plasma PLTP activity and the percentage of AmpB recovered from the HDL fraction following the incubation of Abelcet® but not following the incubation of Ambisome®. In addition, a positive correlation was observed between the percentage of AmpB recovered from H D L and HDL-phospholipids concentration. Among the various approaches used to inhibit PLTP activity, the only approach that did show significant inhibition (80%) in the PLTP activity was heat treatment of plasma. Conclusion: Incubation of phospholipid based formulations of AmpB within human plasma resulted in the majority of drug being recovered in the H D L fraction. Elevated plasma TC and TG levels altered the plasma distribution of AmpB following the incubation of Fungizone® but not following the incubation of Abelcet® and Ambisome®. Positive correlations reported between plasma PLTP activity and the percentage of AmpB recovered in the H D L fraction and between HDL-phospholipid concentration and the percentage of AmpB recovered in the H D L fraction together suggested indirect evidence that PLTP may co-transfer AmpB along with the phospholipids into H D L following incubation of Abelcet®. Among the different methods evaluated to inhibit plasma PLTP activity, heat treatment of plasma was the only approach that showed significant inhibition in the plasma ii i PLTP activity. However, due to limitations associated with this approach (i.e. non-specific denaturing of other plasma proteins and enzymes), it was not used in this set of studies. Future studies could employ following strategies, 1. Depletion of human plasma of PLTP by immunoprecipitation. This PLTP-depleted plasma will be used to study the distribution profile of AmpB following incubation of Abelcet®. 2. Development of PLTP antibody that will have PLTP neutralizing capacity. iv T A B L E O F C O N T E N T S ABSTRACT ii T A B L E OF CONTENTS v LIST OF T A B L E S ix LIST OF FIGURES x LIST OF ABBREVIATIONS xiii A C K N O W L E D G E M E N T xiv DEDICATION xv 1. I N T R O D U C T I O N 2 1.1 Amphotericin B 5 1.1.1 Description and physicochemical properties 5 1.1.2 Mechanism of action.. 6 1.1.3 Clinical uses of AmpB 9 1.1.4 Side effects/Toxicity 9 1.1.4.1 Acute Infusion Reactions 10 1.1.4.2 Nephrotoxicity , , 11 1.2 Formulations of Amphotericin B 13 1.2.1 Parenteral colloidal AmpB dispersion (Fungizone®) 13 1.2.2 Lipid-based AmpB formulations 15 1.2.2.1 AmpB-lipid Complex (ABLC/Abelcet®) 15 1.2.2.2 Liposomal Amphotericin B (Ambisome®) 18 1.2.2.3 Amphotericin B colloidal dispersion (ABCD/Amphotec®) 20 1.3 Lipoproteins 21 1.3.1 Structure of lipoproteins 22 1.3.2 Lipoproteins subclasses 23 1.3.2.1 Chylomicrons 24 1.3.2.2 Very Low Density Lipoproteins (VLDL) 24 1.3.2.3 Intermediate Density Lipoproteins (IDL) 24 1.3.2.4 Low Density Lipoproteins (LDL) 25 1.3.2.5 High Density Lipoproteins (HDL) 25 1.3.3 Lipoprotein Metabolism 27 1.3.3.1 Exogenous Pathway 28 1.3.3.2 Endogenous Pathway 29 v 1.4 Phospholipid transfer protein (PLTP) 29 1.4.1 Functions of PLTP 30 1.4.1.1 Phospholipid transfer activity 30 1.4.1.2 H D L conversion 31 1.5 Reverse cholesterol transport and PLTP '. 32 1.6 Role of Lipoproteins in the transport of hydrophobic drugs 34 1.6.1 Cyclosporine (CSA) 34 1.6.2 Halofantrine (Hf) 35 1.6.3 Eritoran (E5564) -. 35 1.6.4 AmpB ' 36 1.6.5 Compounds incorporated into lipid-based vesicles 37 1.7 Proposed mechanisms for improved therapeutic index of lipid based formulations ...38 1.8 Hypothesis 42 2. MATERIALS AND METHODS 45 2.1 Materials 45 2.1.1 Chemicals and Reagents 45 2.1.2 Human Plasma 45 2.1.3 PLTP Assay Kit 45 2.1.4 PLTP antibody 45 2.1.5 Phospholipid Assay Kit 46 2.2 Preparation of Analytes and Solutions 46 2.2.1 Fungizone® colloidal dispersion 46 2.2.2 Abelcet® suspension 46 2.2.3 Ambisome® liposomal suspension 46 2.3 Methods 47 2.3.1 Incubation of plasma with Amphotericin B-deoxycholate (Fungizone®) or Amphotericin B lipid complex (Abelcet®) or liposomal Amphotericin B (Ambisome®) 47 2.3.2 Separation of lipoprotein constituents 47 2.3.3 Non-lipoprotein control 48 2.3.4 Amphotericin B quantification 49 vi 2.3.4.1 Standard curve preparation 49 2.3.4.2 Determination of Amphotericin B content within the separated lipoprotein fractions (TRL, L D L , H D L and LPDP) 50 2.3.5 PLTP activity assay 51 2.3.5.1 Characterization of PLTP assay 52 2.3.6 Inhibition of plasma PLTP activity 53 2.3.6.1 Incubation of plasma with PLTP antibody 53 2.3.6.2 Incubation of plasma with Thimerosal 53 2.3.6.3 Heat treatment of plasma 54 2.3.6.4 Untreated plasma (Positive control) 54 2.3.6.5 Incubation of plasma with CETP antibody (Irrelevant control) 54 2.3.6.6 Evaluation of PLTP antibody for inherent PLTP activity 54 2.3.7 Measurement of phospholipid content from H D L 55 2.4 Statistical analysis 56 3. R E S U L T S 58 3.1 Part I: To assess the plasma distribution profile of Amphotericin B (AmpB) 58 3.1.1 Aim 1: To compare the distribution profile of AmpB following the incubation of Fungizone® (AmpB - sodium deoxycholate), Abelcet® (AmpB - phospholipid complex) and Ambisome® (liposomal AmpB) within plasmas from six different individuals 58 3.1.1.1 Experimental Design 58 3.1.1.2 Amphotericin B HPLC Quantification 59 3.1.1.3 Calculation of AmpB concentration in the plasma fractions 61 3.1.2 Aim 2: To assess the plasma distribution profile of AmpB following incubation within plasmas with Total Cholesterol (TC) and Triglyceride (TG) levels less than 200 mg/dL and those with TC and TG levels greater than 200mg/dL 67 3.2 Part II: To determine i f changes in PLTP activity alters the plasma distribution of AmpB, when incubated within human plasma as phospholipid based formulations. .71 3.2.1 Aim 1: To establish an in vitro PLTP activity assay within human plasma 71 3.2.1.1 Determination of acceptable plasma volume 71 3.2.1.2 Determination of acceptable incubation time 72 3.2.2 Aim 2: To determine i f a relationship exists between plasma PLTP activity and the percentage of AmpB recovered in the H D L fraction 73 3.2.2.1 Determination of PLTP activity from human plasmas used to study AmpB distribution 73 3.2.2.2 Determination of a relationship between plasma PLTP activity and the percentage of AmpB recovered in the H D L fraction 74 3.2.3 Aim 3: To determine i f a relationship exists between HDL-phospholipid concentration and the percentage of AmpB recovered in the H D L fraction 77 3.2.4 Aim 4: To develop and test different methods to inhibit plasma PLTP activity .79 vii 3.2.4.1 Incubation of plasma with PLTP antibody 79 3.2.4.2 Incubation of plasma with thimerosal..... 79 3.2.4.3 Heat treatment of plasma 79 3.2.5 Aim 5: To study the distribution profde of Abelcet® and Ambisome® in plasma heated at 56°C for 1 hour. . " 4. DISCUSSION 85 4.1 Incubation of AmpB formulations within human plasma 85 4.2 Self association of AmpB and the associated stability and toxicity of AmpB formulations 86 4.3 Physicochemical properties of AmpB formulations 87 4.4 Plasma distribution of AmpB 86 4.5 Effect of elevated TC and T G levels on plasma distribution of AmpB 92 4.6 Establishment and characterization of PLTP assay 94 4.7 Relationship between plasma PLTP activity and the percentage of AmpB recovered in the H D L fraction 95 4.8 Limitations of the study and future directions 99 4.9 Clinical significance 100 5. CONCLUSIONS 103 APPENDICES 105 APPENDIX A 106 APPENDIX B 107 APPENDIX C 108 R E F E R E N C E S 121 viii LIST O F T A B L E S T A B L E T i T i i? P A G E NO. T I T L E NO. 1 Physicochemical properties of Amphotericin B 5 2 Nature and configuration of currently available formulations of 2j Amphotericin B 3 Apolipoproteins of Human Plasma Lipoproteins 23 4 Major Classes of Human Plasma Lipoproteins: Some Properties 26 g Total cholesterol and triglyceride concentration of human plasmas ^ used in the of AmpB distribution study Representative linear calibration curves for Amphotericin B as 6 determined in the separated lipoprotein and non lipoprotein 59 fractions of human plasma for Fungizone® i x LIST O F FIGURES FIGURE ______ P A G E NO. T I T L E NO. 1 Molecular structure of Amphotericin B 5 Intercalation of AmpB with cell membrane sterols creates pores in 2 the membrane, causes leakage of electrolytes and lysis of cell ^ resulting in cell death. General structure of Lipoprotein 21 Lipoprotein metabolism and lipid transport 27 Schematic representation of reverse cholesterol transport process 32 Release of monomeric AmpB as a result of the rapid dissociation of Fungizone® and release of small amounts of intact monomeric 40 AmpB from lipid formulations to act on fungal cells Ultracentrifuge tube following ultracentrifugation in a Beckman SW41-Ti swinging bucket rotor at 40,000 rpm and 15°C for 18 ^ hours in a Beckman L8-80M ultracentrifuge. 8 Schematic representation of PLTP assay 52 Representative standard curves of Amphotericin B in (A) ^ triglyceride rich lipoprotein (TRL), (B) low density lipoproteins ^ (LDL), (C) high density lipoproteins (HDL) and (D) lipoprotein deficient plasma (LPDP) Representative chromatograms of Amphotericin B (lOpg/mL) in 10 (A) triglyceride rich lipoprotein (TRL), (B) low density 61 lipoprotein (LDL) Representative chromatograms of Amphotericin B (lOug/mL) in 11 (C) high density lipoprotein (HDL), (D) lipoprotein deficient 62 plasma (LPDP) x Effect of incubation time on the plasma distribution profile of 12 AmpB (20 pg/mL) after incubation of Fungizone® within human 64 plasmas at 37°C Effect of incubation time on the plasma distribution profile of 13 AmpB (20 pg/mL) after incubation of Abelcet® within human 65 plasmas at 37°C Effect of incubation time on the plasma distribution profile of 14 AmpB (20pg/mL) after incubation of Ambisome® within human 66 plasmas at 37°C Effect of elevated TC and TG on plasma distribution profile of 15 Amp B (20 pg/mL) after incubation of Fungizone® at 37°C for 68 60 min. *P<0.05 vs. Normolipidemic Effect of elevated TC and TG on plasma distribution profile of 16 AmpB (20 pg/mL) after incubation of Abelcet® at 37°C for 60 69 min. Effect of elevated TC and TG on plasma distribution profile of 17 AmpB (20 pg/mL) after incubation of Ambisome® at 37°C for 70 60 min. PLTP activity in 0, 3, 5, 10 and 20 pL of fresh human plasma 18 after incubating it with the assay components for 30 minutes at 72 37°C PLTP activity in 10 pL of fresh human plasma after incubating 19 it with the assay components for 0, 5, 10, 15, 20, 25, 30, 40, 50 73 and 60 minutes at 37°C PLTP activity from plasmas used (table 5) for the AmpB 20 distribution study after incubating 10 pL of these plasmas with 74 assay components for 30 minutes at 37°C Correlation between PLTP activity and % AmpB recovered in 2j the H D L fraction of plasma samples after incubation of ^ Fungizone® (20 pg/mL) within human plasma for 30 minutes at 37°C Correlation between PLTP activity and % AmpB recovered in 22 the H D L fraction of plasma samples after incubation of ^ Abelcet® (20 pg/mL) within human plasma for 30 minutes at 37°C xi Correlation between PLTP activity and % AmpB recovered in the H D L fraction of plasma samples after incubation of ^ Ambisome® (20 pg/mL) within human plasma for 30 minutes at. 37°C Correlation between phospholipid concentration in H D L and % 2^ AmpB recovered in the HDL fraction after incubation of ^ Abelcet® (20 pg/mL) within human plasma for 30 minutes at 37°C Correlation between phospholipid concentration in H D L and % 2^ AmpB recovered in the HDL fraction after incubation of ^ Ambisome® (20 ug/mL) with human plasma for 30 minutes at 37°C PLTP activity observed in an untreated plasma vs. heated plasma, plasma incubated with PLTP antibody, plasma 26 incubated with thimerosal and plasma incubated with CETP 80 antibody following incubation of lOpL plasma from each of the treatment groups with assay components for 30 min at 37°C Effect of heat treatment of plasma on the distribution profile of 27 AmpB (20 pg/mL) after incubation of Abelcet® within human 82 plasma at 37°C for 60 min. Effect of heat treatment of plasma on the distribution profile of 28 AmpB (20 ug/mL) after incubation of Ambisome® within 83 human plasma at 37°C for 60 min. 2£ Schematic representation of preferential distribution of ^ liposomal drug into HDL. xii LIST OF A B B R E V I A T I O N S A B L C Amphotericin B lipid complex AmpB Amphotericin B A N O V A Analysis of variance Apo A Apolipoprotein A A p o B Apolipoprotein B A p o C Apolipoprotein C Apo E Apolipoprotein E A U C Area under the curve CE Cholesteryl ester C E T P Cholesteryl ester transfer protein Cmax Concentration maximum CSA Cyclosporine A D M P C Dimyristoyl phosphatidylcholine D M P G Dimyristoyl phosphatidylglycerol DSPC Distearoyl phosphatidylcholine EDTA Ethylene diamine tetra acetic acid HDL High density lipoprotein HPLC High performance liquid chromatography HSPC Hydrogenated soy phosphatidylcholine IDL Intermediate density lipoproteins L D L Low density lipoproteins LPDP Lipoprotein deficient plasma ND Non detectable NaCl Sodium chloride PLTP Phospholipid transfer protein TC Total cholesterol TG Triglyceride TRL Triglyceride rich lipoprotein V L D L Very low density lipoproteins xiii A C K N O W L E D G E M E N T I would like to thank my research supervisor, Dr. Kishor M . Wasan for his guidance, encouragement and support throughout this project. I express my gratitude to him for his patience in allowing me to settle down in the early stages of graduate school and developing scientific creativity in me. I would also like to thank members of my research committee Dr. Helen Burt, Dr. Wayne Riggs, Dr. Marcel Bally, Dr. Kathleen MacLeod (chair) and Dr. John Hi l l (external examiner) for their support, insight and feedback into the development of this project. To my coworkers in the lab: A big thank you to Steve for spending those endless hours with me, going through and polishing my data and written material and also for teaching me gradient density ultracentrifugation technique. Thank you very much Carlos, for your constructive feedback and suggestions at various stages of my project. My special thanks to Verica for teaching me the handling and operation of HPLC instrument. Many thanks to Kristina, Olena, Mike and Sheila for their constant support and encouragement. I would also like to thank all the undergraduate students over the past two years with whom I had the pleasure of working with. I express my appreciation and gratitude to the Faculty of Pharmaceutical Sciences, UBC for providing me an opportunity to avail quality training in terms of research. I would like to thank my family and friends. It has always been through their love and support that I have accomplished all that I have. Last, but not least, Vinaya. Thank you for being there through my good times and bad. xiv D E D I C A T I O N This work is dedicated to my parents, Arun and Arundhati Patankar. Without your endless love and encouragement this would not have been possible. Thank you for everything Mom and Dad. xv Introduction i 1. INTRODUCTION Amphotericin B (AmpB) is a polyene macrolide antibiotic that was first introduced into clinical practice in 1958 and has remained the treatment of choice for most systemic fungal infections for over 45 years. However, the major problems with all polyenes are that they have limited oral bioavailability (due to water insolubility, thus requiring intravenous administration) and the toxicity associated with these compounds. Fungizone® (colloidal dispersion of AmpB with sodium deoxycholate) is the most widely used conventional formulation of AmpB. However, severe nephrotoxicity has been the greatest limitation associated with the clinical use of Fungizone®. Ameliorating this toxicity has been one of the major goals for the development of lipid based formulations of AmpB. Two commonly used lipid based formulations of AmpB are Abelcet® (AmpB-lipid complex) and Ambisome® (Liposomal AmpB). These formulations have been found to have an equivalent efficacy with lower toxicity compared to the conventional AmpB formulation (Fungizone®) (Arikan etal, 2001). Studies have shown that several compounds including AmpB (Wasan et al, 1993), cyclosporine (Wasan et al, 1997), nystatin (Wasan et al, 1997), annamycin (Wasan et al, 1994) associate with plasma lipoproteins. Lipoproteins are involved in the transport of a number of such compounds within the blood. A number of studies have reported that alteration in plasma lipoprotein distribution of AmpB may significantly alter the toxicity associated with AmpB (Wasan et al, 1998). 2 Incubation of Fungizone within human plasma, results in the preferential association of AmpB with high density lipoprotein (HDL) fraction, compared to other lipoprotein fractions (Wasan et al, 1998). However, AmpB also associates with the low density lipoprotein (LDL) fraction of plasma which is considered an important factor in the nephrotoxicity caused by AmpB. Studies using AmpB formulated into lipid excipients (Abelcet® and Ambisome®) have shown significantly greater HDL-association and lower LDL-association compared to the conventional formulation (i.e. Fungizone®) following incubation within human plasma. Several mechanisms have been proposed to explain the observed preferential distribution of AmpB into HDL. Those were based on the i) rapid uptake of lipid based formulations by tissues of reticuloendothelial system (RES) (Douglas et al, 1999), ii) relative affinity of AmpB for carrier lipids and for ergosterol and cholesterol (Juliano et al, 1987), iii) ability of formulation to release free and aggregated form of AmpB (Brajtburg et al, 1996), iv) role of phospholipases in liberating AmpB from lipid carriers (Swenson et al, 1998) and v) preferential distribution of AmpB into H D L when administered as a lipid based formulation (Wasan etal, 1996). PLTP is a specific phospholipid transfer protein that is observed in the plasma of human as well as other vertebrate species (Tall et al, 1983). It is mainly involved in the transfer of phospholipids in the plasma from triglyceride rich lipoproteins to HDL. Based on this activity of PLTP, we hypothesized that it may play an important role in co-transferring AmpB complexed with the phospholipids to H D L and therefore may promote its distribution into HDL. 3 The purpose of this work was to determine whether PLTP is involved in the transfer of AmpB complexed with the phospholipids into HDL. In order to test this hypothesis, the plasma distribution profiles of AmpB using three different formulations (Fungizone, Abelcet and Ambisome) were examined. An in vitro plasma PLTP activity assay method was established and characterized and used to measure the PLTP activity from human plasma. The experimental approaches used were to determine the relationship between plasma PLTP activity and the amount of AmpB distributed into H D L and to evaluate plasma lipoprotein distribution of AmpB in PLTP inhibited plasma. 4 1.1 Amphotericin B 1.1.1 Description and physicochemical properties Amphotericin B (AmpB) is a heptane polyene macrolide parenteral antifungal agent produced as fermentation by product of Streptomyces nodusus, an aerobic actinomycete (GilmanA. G.,2001). " . Macrolide ring C A R B O X Y L GROUP GROUP • ; ; : : v : : : : : '.v:: Figure 1: Molecular structure of Amphotericin B It is named for its amphoteric properties, which are acquired from a carboxyl group on the macrolide ring and a primary amino group on the mycosamine sugar (Figure 1). The physicochemical properties of solid AmpB are as follows: Table 1: Physicochemical properties of Amphotericin B Physical properties Deep yellow to golden-orange, granular powder, odourless. Chemical formula C 4 7 H 7 3 N 0 1 7 Molecular weight 924.084 LogP 1.29 (Aher etal., 1977) p K a Carboxyl pK a : 5.7 Amino pK a : 10.0 (Aher et a l , 1977) Solubility Insoluble in water, dissolves poorly in most pure solvents, exceptions being dimethyl sulfoxide (30 to 40 mg/mL) and dimethyl formamide (2 to 4 mg/mL). 5 1.1.2 Mechanism of action It has been noted (Hazan E. L. , 1951) that the effect of polyene macrolide antibiotics (including that of AmpB) is specific to fungi, but not to bacteria. It has been shown that the specificity of action of these antibiotics is due to their high affinity for the sterol components found in the biologic membranes of the fungi (Weber et al, 1965 ). The fungal cell membranes contain complex lipid particles, called sterols. The sterol found predominantly in the fungal cell membranes is 'ergosterol'. Although the human cell membranes contain sterols, the most critical advantageous property of AmpB is its significantly greater binding affinity for ergosterol (present only in fungal cells) over that for cholesterol (the primary sterol in the human cells). Thus, antifungal properties of AmpB have been attributed to its binding to ergosterol in the fungal cell membrane (Holz et al, 1970). Permeability studies in membrane systems have demonstrated that AmpB interacts directly with membrane sterols to produce pores in the membrane (Holz et al, 1970), disrupting membrane integrity and causing loss of potassium and other cellular contents, ultimately leading to the cell death. When the polyene antibiotic is added to the sterol-containing membrane system, it partitions into the lipid layer displacing sterol from its association with the membrane phospholipids to form a half pore. As shown in Figure 2, mycosamine sugar of polyene and the hydroxyl moiety of the sterol are aligned at the hydrophilic end of the polyene-sterol complex (at the membrane surface), while a single hydroxyl group will be found at the opposite hydrophobic end (Gale et al, 1984) of the half pore. It is suggested that two half pores come together, hydrogen bonds are formed between the single hydroxyl groups at the hydrophobic end of the polyene joining the ends of two half pores. This results in a full pore formation of a length sufficient to span 6 the normal biological membrane (Figure 2). Re-organization of the lipid then occurs, allowing sterol-polyene complex to form an aqueous pore consisting of an annulus of several AmpB molecules linked hydrophobically to the membrane sterols (Gilbert et al, 2002), (Wasan et al, 1993) (Figure 2). This configuration gives rise to a pore such that it has a hydrophobic exterior composed of sterol molecules and inner channel lined with the hydrophilic head groups of polyene ring (Figure 2), leading to altered permeability, leakage of vital cytoplasmic components and finally, death of the organism. 7 Figure 2: Intercalation of AmpB with cell membrane sterols creates pores in the membrane, causes leakage of electrolytes and lysis of cell resulting in cell death, (adapted from Gale, 1984) 8 Some researchers have proposed lipid peroxidation of cell membrane as another mechanism by which AmpB illicit its pharmacological effects (Brajtburg et al, 1985). According to this theory, AmpB has inhibitory effects on membrane enzymes: proton ATPase in fungal cells (Surarit et al, 1987) and N a + / K + ATPase in mammalian cells (Vertut-Doi et al, 1988). Inhibiting these enzymes would deplete the cellular energy reserves and reduce proliferative ability (Schindler et al, 1993). Lipid peroxidation induced by AmpB resulting in a corresponding increase in membrane fragility has been proposed as a possible mechanism by which membrane permeability changes occur as well (Brajtburg et al, 1985). 1.1.3 Clinical uses of AmpB AmpB has been the mainstay of the treatment of various fungal infections since 1960. Presently, it is available in two forms: oral and parenteral preparations. Oral preparations of AmpB are used rarely due to insufficient absorption from gastrointestinal tract and therefore poor bioavailability. The parenteral formulations are widely used in the treatment of fungal invasions such as systemic fungal infections (e.g. in immunocompromised patients), visceral leishmaniasis, aspergillosis, cryptococcus infections (e.g. meningitis), candidiasis, empirical treatment of febrile neutropenia in immunocompromised patients who do not respond to the broad-spectrum antibiotics. 1.1.4 Side effects/Toxicity Conventional intravenous (i.v.) AmpB (Fungizone®) is associated with a high incidence of adverse effects. The most frequent adverse reactions to conventional i.v. AmpB 9 include acute infusion reactions and nephrotoxicity. The less frequent adverse reactions reported are haematologic effects: Anaemia, agranulocytosis, coagulation disorders, altered prothrombin time, thrombocytopenia, leukopenia, eosinophilia, or leukocytosis. Cardiopulmonary effects: Hypotension, tachypnea, cardiac failure, cardiac arrest, cardiomyopathy, shock, pulmonary edema, hypersensitivity pneumonitis, arrhythmias (including ventricular fibrillation), dyspnea, and hypertension. Local reactions observed are erythema, pain, or inflammation at the injection site; phlebitis or thrombophlebitis. The two major problems with AmpB therapy are discussed below in detail: 1.1.4.1 Acute Infusion Reactions The infusion-related acute reactions most commonly observed with AmpB include fever, chills, hypotension, nausea, vomiting, headache, dyspnoea, anorexia and may occur 1 to 3 hours after initiation of i.v. infusions of conventional AmpB. Less common reactions include blurred vision, convulsions, numbness, tingling pain or weakness in hands or feet, shortness of breath, wheezing or tightness in chest, skin rash or itching and sore throat. Studies have suggested that AmpB can stimulate the release of prostaglandins (Gigliotti et al, 1987) as well as reactive cytokines, including Tumour Necrosis Factor- a (TNF- a) and Interleukin-1 from macrophages (Chia et al, 1990), (Louie et al, 1994), which are responsible for the infusion related toxicities such as fever, chills. Pre-medication to avoid these reactions is generally not recommended; however, it has been suggested that treatment with acetaminophen, meperidine, antihistamines (e.g., diphenhydramine), or corticosteroids can be administered promptly to treat a reaction if it occurs and then as pre-treatment prior to subsequent doses. In addition, AmpB therapy is 10 commonly initiated with low doses (test doses consist generally of 1 mg) followed by dose escalation as the anti-fungal treatment progresses (Gilman et al, 2001). 1.1.4.2 Nephrotoxicity Adverse renal effects in patients receiving conventional i.v. AmpB include decreased renal function and renal function abnormalities such as azotemia, hypokalemia, hyposthenuria, renal tubular acidosis, and nephrocalcinosis (Gilman et al, 2001). Increased B U N and serum creatinine concentrations and decreased creatinine clearance, glomerular filtration rate, and renal plasma flow occur in most patients receiving conventional i.v. AmpB. Nephrotoxicity is a major drawback and is observed in nearly half the patients with conventional i.v. AmpB therapy. The nephrotoxicity associated with AmpB is of more serious nature than the infusion-related adverse events and frequently necessitates interruption of therapy. In one report on 239 immuno-suppressed patients with invasive aspergillosis when treated with AmpB-deoxycholate, the incidence of nephrotoxicity (represented by two-fold increase in creatinine) was found in 53% of the cases. A significant number of patients (4.5%) had to go on renal dialysis and 60% of all patients died. (Wingard et al., 1999). Nephrotoxicity is ultimately the dose-limiting factor in many patients, particularly when the AmpB dose regimen is high, or AmpB is used in combination with other potentially nephrotoxic agents (aminoglycosides, cyclosporins, etc.), or in situations in which any renal damage is of extreme concern (e.g., in kidney transplant recipients) (Gilbert et al, 2002). 11 One of the possible reasons behind this nephrotoxicity was found to be the presence of high affinity low density lipoprotein (LDL) receptors expressed in the kidney which mediate the cellular uptake of the drug through the process of endocytosis (Ramaswamy et al, 2001). It has also been observed that elevation in L D L cholesterol levels in the plasma is associated with the higher AmpB-induced renal toxicity (Ramaswamy et al, 2001). Wasan et al have demonstrated the therapeutic importance of the influence of AmpB-lipoprotein association on the toxicity to L L C PK1 renal cells (a cell line derived from the proximal tubular cells of swine) (Wasan et al, 1994c) It was observed that AmpB was significantly less toxic to renal L L C PK1 cells when it was associated with high density lipoprotein (HDL) than when it was associated with L D L . However, L D L -associated AmpB was just as toxic to the cells as that of unbound AmpB. In addition, when high affinity surface receptors were decreased, LDL-associated AmpB was less toxic to L L C PK1 renal cells than was the unbound drug. These data suggested that an increased toxicity observed with the LDL-bound AmpB was as a result of its interaction with the high affinity L D L receptors located on the surface of the cell (Wasan et al, 1994c) Furthermore, an increase in the amount of AmpB-associated L D L would suggest an increase in the toxicity to the renal cells. In fact, studies in the transplant patients have demonstrated this link of AmpB-induced nephrotoxicity to L D L levels in the body (Wasan et al, 1998). Other mechanisms reported to be involved in the toxicity of AmpB suggest direct vasoconstrictive effects on renal arterioles and lytic action on tubular cell membrane, which may lead to vasocongestion and reduction in glomerular filtration rate (GFR) 12 ultimately resulting in tubular dysfunction. Studies on animals as well as on humans have shown that AmpB infusion in animal models decreased the renal flow within 45 minutes following infusion (Gilbert et al, 2002). Reduction in GFR was also accompanied with the loss of electrolytes and other cellular contents. Although generally reversible at lower doses, up to 10% of patients with significant kidney dysfunction on AmpB will require persistent dialysis after discontinuation of the antifungal (Groll et al, 1998). Vigorous hydration and sodium loading before and after AmpB infusions is probably the most accepted strategy for reducing nephrotoxicity but not all patients can tolerate extra fluids or sodium supplementation. Reducing the daily dosage of AmpB or intermittent dosing (e.g., every other day or three times per week) may slow the onset of renal dysfunction, but can potentially compromise the efficacy of antifungal therapy in patients with life-threatening infections. 1.2 Formulations of Amphotericin B AmpB has been the mainstay of antifungal therapy for more than 40 years. Several formulations of AmpB are available in the market, the primary ones of which are described below: 1.2.1 Parenteral colloidal AmpB dispersion (Fungizone®) The conventional AmpB formulation is a sterile, non-pyrogenic, lyophilised, colloidal dispersion of AmpB with sodium deoxycholate. Commercially, it is supplied in vials, each containing 50 mg AmpB and 41 mg sodium deoxycholate with 20.2 mg sodium phosphate as a buffer. Each ml of reconstituted dispersion contains 5 mg of AmpB. 13 Conventional AmpB is administered by slow intravenous infusion over a period of approximately 2 to 6 hours (depending on the dose). Since patient tolerance varies greatly, in most of the cases, the dosage of AmpB is individualized and adjusted according to the patient's clinical status (e. g., site and severity of infection, underlying causative agent, cardio-renal function, etc.). In patients with good cardio-renal function and a well tolerated test dose, therapy is usually initiated with a daily dose of 0.25 mg/kg of body weight. However, in those patients having severe and rapidly progressive fungal infection, therapy may be initiated with a daily dose of 0.3 mg/kg of body weight. In patients with impaired cardio-renal function or a severe reaction to the test dose, therapy is initiated with smaller daily doses (Meyer et al., 1992). Depending on the patient's cardio-renal status, doses are gradually increased by 5 to 10 mg per day to final daily dosage of 0.5 to 0.7 mg/kg. Total daily dosage ranges up to 1.0 mg/kg per day or up to 1.5 mg/kg when given on alternate days (Meyer et al, 1992). However, it is suggested that under no circumstances should a total daily dose of 1.5 mg/kg be exceeded. AmpB when administered i.v. as Fungizone® (1 mg/kg) follows three compartmental pharmacokinetic model (Gates et al, 1993). According to this model, AmpB first distributes into the intravascular space which is known as the central compartment. From there, it equilibrates into two peripheral compartments out of which one is slowly equilibrating (i.e. interstitial fluid of tissues such as skeletal muscle and skin) and the other is rapidly equilibrating (i.e. interstitial fluid of tissues such as liver, intestine and spleen). AmpB deoxycholate (Fungizone®) is highly protein bound and has high affinity to bind to the sterols. It has large volume of distribution (Vd) of 4 L/kg (Gates et al, 14 1993) and shows long terminal half life of 15 days and this is primarily due to slow redistribution from the peripheral tissues (Gates et al, 1993). The major adverse reactions to Fungizone® include nephrotoxicity and infusion-related toxicities that are discussed previously. Development of kidney toxicity which is manifested by renal vasoconstriction with a significant decrease in the glomerular filtration rate and renal plasma flow as well as renal electrolyte loss, often limits the use of this product. 1.2.2 Lipid-based AmpB formulations Despite its proven clinical efficacy, conventional AmpB therapy is associated with severe, life-threatening adverse reactions as mentioned above. Due to this limitation, a number of alternative formulations have been developed with the aim of improving the therapeutic index of AmpB by decreasing the toxicity and increasing the therapeutic efficacy. As a result, several lipid based formulations of AmpB were developed in the 1990s. These lipid based formulations were found to have equivalent therapeutic efficacy but with increased tolerance as compared to that of conventional intravenous AmpB. The commonly used lipid based formulations are described below: 1.2.2.1 AmpB-lipid Complex (ABLC/Abelcet®) AmpB-lipid complex is a sterile, non-pyrogenic suspension for intravenous infusion consisting of AmpB complexed with two phospholipids in a 1:1 drug-to-lipid molar ratio. The two phospholipids used for this purpose are L-dimyristoylphosphatidylcholine (DMPC) and L-dimyristoylphosphatidylglycerol (DMPG) (Hiemenz et al, 1996). 15 Commercially, Abelcet is provided as a sterile, opaque suspension in 20 mL glass, single-use vials, requiring dilution with 5% dextrose prior to administration. Each 20 mL vial contains 100 mg of AmpB, and each mL of Abelcet® contains: AmpB USP 5 mg, DMPC 3.4 mg, D M P G 1.5 mg, Sodium Chloride USP 9 mg and Water for Injection USP, quantity sufficient to make 1 mL. Prior to admixture, Abelcet® should be stored at 2°C to 8°C and protected from exposure to light. For preparation of the admixture for infusion, the appropriate dose of Abelcet® suspension is transferred to the i.v. infusion bag containing 5% Dextrose Injection USP under strictly aseptic conditions so that the final infusion concentration is 1 mg/mL. The diluted ready-for-use admixture is stable for up to 48 hours at 2°C to 8°C and an additional 6 hours at room temperature. The recommended daily dosage for adults and children is up to 5 mg/kg given as a single infusion. (Hiemenz et al., 1996). Abelcet® is administered by intravenous infusion at a rate of 2.5 mg/kg/hr. Renal toxicity of Abelcet®, as measured by serum creatinine levels, has been shown to be dose dependent. Physicochemical properties of lipids used as a carrier in case of lipid based products influence the distribution and pharmacokinetics of AmpB. AmpB administered as Abelcet® (5 mg/kg) show lower serum concentrations than that of Fungizone®, rapid clearance, larger volume of distribution and lower area under the curve (AUC). This may be due to rapid and extensive tissue distribution (Douglas S. et al., 1999). Toxicity and efficacy data in human clinical trials have shown that Abelcet® is equally or in some cases more effective and less nephrotoxic compared to conventional AmpB 16 formulation (Fungizone )(Arikan et al, 2001). Infusion-related reactions and other side effects were also found to be milder compared to Fungizone® and thus, did not require discontinuation of therapy (Arikan et al, 2001). While studying the structure of this formulation, Bailey et al (1990) have found that this formulation contained not only liposomal but also non liposomal structures. These non liposomal structures appeared as AmpB and lipids associated with each other to form ribbon-like structures. It was suggested that AmpB-lipid pairs are arranged in cylinders. The cylinders were aligned side by side and possessed two polar ends. They'also found that the percentage of AmpB incorporated into the lipid complex determined the structure and size of the vesicles and the amount of ribbon-like structures. It was observed that as the mole ratio of AmpB in the formulation was decreased, the overall toxicity was decreased due to reduction in the amount of free AmpB in the solution. At a 5% mole concentration of AmpB, the lipid vesicles formed were smaller than those formed in the absence of AmpB and it also contained some ribbon structures. However, when mole concentration of AmpB was increased to 25% and above, liposomal structures remained no longer and tightly packed ribbon structures were observed. Decreased toxicity observed with increased formation of ribbon structures may suggest that this decreased toxicity may be due to increased complex formation between AmpB and phospholipids and a corresponding decreased amount of free drug in the solution. Another mechanism behind decreased Abelcet® toxicity may be the association of AmpB with high density lipoproteins (HDL) in the plasma which will be discussed in detail in the later section. Along with reduced toxicity, another advantage of Abelcet® therapy is 17 that increasing the dose of Abelcet does not increase the concentration of the drug in kidneys significantly, hence it can be safely used at doses as high as 7 mg/kg (Hiemenz et al., 1996). 1.2.2.2 Liposomal Amphotericin B (Ambisome®) Liposomal AmpB is composed of unilamellar lipid vesicles with an average size of 60-70 nm. Phospholipids used in the formulation are hydrogenated soy phosphatidylcholine (HSPC) and distearoylphosphatidylglycerol (DSPG) along with cholesterol and AmpB in the ratio of 2:0.8:1:0.4 (Arikan et al., 2001). Cholesterol is incorporated to increase the packing of phospholipids and give more rigidity to liposomal vesicles. AmpB being hydrophobic drug is thought to be incorporated in the phospholipid bilayers of the liposomal structure. Ambisome® for injection is available as sterile, non pyrogenic lyophilized product for intravenous infusion. Each vial contains 50 mg of AmpB, USP entrapped within a liposomal membranes. The composition is as follows: Hydrogenated soy phosphatidylcholine 213 mg Cholesterol, NF 52 mg Distearoyl phosphatidylglycerol - 84 mg Alpha tocopherol, USP 0.64 mg Sucrose, NF 900 mg Disodium succinate hexahydrate 27 mg 18 Ambisome must be diluted with 5% Dextrose Injection to a final concentration of 1.0 to 2.0 mg/mL prior to administration. Ambisome® should be administered by intravenous infusion, using a controlled infusion device, over a period of approximately 120 minutes. The recommended daily dosage of Ambisome® for adult and children may range from 3 -6 mg/kg depending on the kind and severity of infection. The reconstituted product concentrate admixture is stable for up to 24 hours at 2°C to 8°C. Ambisome® (5 mg/kg) administered i.v. shows significantly higher C m a x , larger A U C , and smaller Vd compared to both Fungizone® and Abelcet®. This could be due to the prolonged circulation of these liposomes because of their smaller size (50-70 nm) and therefore lesser uptake by organs of reticuloendothelial system (RES). During the clinical trials, similar to Abelcet®; Ambisome® was found to be less toxic than conventional AmpB and severe toxic reactions were infrequent. The spectrum of activity of liposomal AmpB is found broad and similar to conventional AmpB. A specific advantage of Ambisome® over other formulations is that it remains in the circulation for a longer period of time. Pharmacokinetic studies with Ambisome® showed that although these vesicles are taken up by organs of reticuloendothelial systems similar to other lipid based formulations, the uptake of the drug by cells is slower (Lee et al, 1994),(Proffitt et al, 1991). Some of the factors responsible for the longer circulation of these liposomes may be their relatively small vesicle size (<100 nm) and relatively rigid lipid bilayer. Due to the longer circulation time, the formulation provides higher plasma drug levels and sustained delivery of drug to the target organs. 19 1.2.2.3 Amphotericin B colloidal dispersion (ABCD/Amphotec ) Amphotec® is a sterile, pyrogen-free, lyophilized powder for reconstitution and intravenous (/.v.) administration. It consists of a 1:1 (molar ratio) complex of AmpB and cholesteryl sulfate. Upon reconstitution, Amphotec® forms a highly organized disc shaped structure after two molecules of AmpB come together with two molecules of cholesteryl sulphate to form an amphiphilic tetramer. This formulation differs from Abelcet® and Ambisome® in that it contains a sterol based carrier whereas both Abelcet® and Ambisome® contain phospholipids as their carrier system. Amphotec® is reconstituted in Sterile Water for Injection and then administered (diluted in 5% Dextrose for Injection) by intravenous infusion at a rate of 1 mg/kg/hour. The recommended daily dosage of Amphotec® for adult and children is 3-4 mg/kg (Hiemenz etal, 1996). AmpB administered as an Amphotec® (5 mg/kg) shows higher C m a x , A U C and more rapid clearance than that of Fungizone®. Like other lipid based formulations, Amphotec® also showed improvement in the therapeutic index of AmpB as compared to conventional formulation i.e. Fungizone®. However, a drawback associated with the administration of Amphotec® that limits the clinical use of this product is its acute toxicity, as infusion related adverse events occur most frequently after its administration (White et al, 1998). Therefore, Abelcet® and Ambisome® are the two most commonly used lipid based AmpB-formulations in the market. Table 2 represents comparative chart of composition and structure of commercially available formulations of Amphotericin B. 20 Table 2: Nature and configuration of currently available formulations of Amphotericin B Commercial name Formulation Vehicle (wt/wt) Lipid configuration Fungizone® Colloidal dispersion Sodium deoxycholate (37%) -Abelcet® Lipid complex D M P C : D M P G (7:3) Ribbon-like Ambisome® Liposome HSPC, Cholesterol, DSPG (2:1:0.8) Unilamellar vesicle Amphotec Colloidal dispersion Cholesteryl sulphate (50%) Disk-like 1.3 Lipoproteins Lipoproteins are macromolecular complexes of lipids and proteins. Naturally-occurring lipids are insoluble in water and so need to be solubilised by association with specific carrier proteins, forming water soluble 'lipoproteins', in order to be transported around the body (Davis et al., 1996). The major sources of lipoproteins are the intestine and the liver. The primary carrier protein components of lipoproteins are apolipoproteins ("apo" refers to the protein in its lipid-free form) whereas the lipid components of lipoproteins are cholesterol esters, phospholipids, triglyceride and unesterified cholesterol (Davis et al, 1996). The lipid-protein complexes, in descending order of molecular mass, are: • chylomicrons • very low density lipoprotein (VLDL) • low density lipoprotein (LDL) • high density lipoprotein (HDL) 21 1.3.1 Structure of lipoproteins A l l lipoprotein particles are spherical in shape (Figure 3). Each lipoprotein complex consists of non polar lipid core made up of cholesterol esters and triglycerides and surface monolayer made up of amphipathic lipids (i.e. phospholipids and unesterified cholesterol) and specific carrier proteins called 'apolipoproteins' (Davis et al ,1996) Phospholipids-^ Figure 3 : General structure of Lipoprotein (adapted from http://www.peprotech.com/") A number of phospholipids are incorporated in the surface monolayer of lipoproteins; e.g. phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and sphingomyelin. Apolipoproteins are specialized proteins that form scaffolds in the surface monolayer of lipoproteins. Apolipoproteins help carrying lipids (triglycerides and cholesterol esters) between the liver and peripheral tissues. These are amphipathic in nature and contain polar and non-polar amino acid residues that help to solubilise the insoluble lipids incorporated within lipoproteins (Louie et al, 1994). Depending on their size, their Apoprotein T G and CE *— Cholesterol 22 reactions to specific antibodies and their characteristic distribution in the lipoprotein classes, at least nine different apolipoproteins are found in the lipoproteins of human plasma (Table 3). (Wasan et al, 1998) Table 3: Apolipoproteins of Human Plasma Lipoproteins (adapted from (Wasan et al. 1998)) Apolipoprotein Molecular weight Lipoprotein association Function (if known) Apo A-I 28,331 H D L Activates L C A T ; interacts with A B C transporter Apo A-II 17,380 HDL Apo A-IV 44,000 Chylomicrons, H D L ApoB-48 240,000 Chylomicrons ApoB-100 513,000 V L D L , L D L Binds to L D L receptor Apo-I 7,000 V L D L , L D L Apo-II 8,837 Chylomicrons, V L D L , H D L Activates lipoprotein lipase Apo C-III 8,751 Chylomicrons, V L D L , HDL Inhibits lipoprotein lipase A p o D 32,500 HDL Apo E 34,145 Chylomicrons, V L D L , HDL Triggers clearance of V L D L and chylomicron remnants With the exception of Apo B, all other apolipoproteins have appreciable water solubility (Wasan etal, 1998). 1.3.2 Lipoproteins subclasses Different combinations of lipids and apolipoproteins produce particles of different densities such that there is an inverse relationship between the lipid content and density; i.e. lipoproteins with larger amount of lipid relative to protein have lesser density than those containing lesser amount of lipids (Davis et al, 1996). Lipoproteins are classified according to their densities into five main classes: chylomicrons, very low density 23 lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). 1.3.2.1 Chylomicrons Chylomicrons are the largest lipoprotein particles (diameter of approximately 100-1000 nm) and are the least dense, containing a high proportion of triacylglycerols. Chylomicrons are synthesized in the intestine (Grundy, 1990). The apolipoproteins that predominate before the chylomicrons enter the circulation include ApoB-48 and Apo A-I, A-II and IV. ApoB-48 combines only with chylomicrons. 1.3.2.2 Very Low Density Lipoproteins (VLDL) Very low density lipoproteins are the next largest lipoproteins (diameter = 30-80 nm). These are synthesized in the liver. In addition to triacylglycerols, V L D L contains some cholesterol and cholesteryl esters. V L D L plays a major role in the transport of endogenously produced triacylglycerols from the liver to extrahepatic tissue (Salter et al., 1988). The apolipoproteins, found in V L D L are ApoB-100, Apo C-I, Apo C-II, Apo C-III and Apo E. 1.3.2.3 Intermediate Density Lipoproteins (IDL) Intermediate density lipoproteins are produced as a result of V L D L metabolism. IDL is rich in cholesterol ester and contain ApoB-100. 24 1.3.2.4 Low Density Lipoproteins (LDL) Low density lipoproteins are produced as a result of metabolism of V L D L as well as IDL. These are primary carriers of cholesterol in humans containing approximately 50% of the total cholesterol (Grundy S. M . , 1990). It contains ApoB-100 as its only apolipoprotein. 1.3.2.5 High Density Lipoproteins (HDL) High density lipoproteins are the smallest of the lipoproteins and also the densest because it contains the highest proportion of protein. HDL comprises several components derived from various sources-the liver, intestine, other lipoproteins, and other tissue (Grundy, 1990). HDL is responsible for removal of additional cholesterol from peripheral tissues and transports it to the liver for the excretion through the process known as reverse cholesterol transport (Fielding et al, 1995). The major apoproteins of H D L are Apo A-I and A-II. The surface coat of H D L also contains Apo C's and Apo E ' s . Table 4 represents the composition, size and density of the major lipoprotein complexes in brief. 25 Table 4: Major Classes of Human Plasma Lipoproteins: Some Properties [adapted from (Davis R. A. , 1996)] Characteristic Chylomicrons Very Low Density Lipoproteins Intermediate Density Lipoproteins Low Density Lipoproteins High Density Lipoproteins Abbreviation V L D L IDL L D L HDL Density (g/ml) <0.95 0.95-1.006 1.006-1.019 1.019-1.063 1.063-1.210 Diameter (nm) 75-120 30-80 25-35 18-25 5-12 Composition (% dry wt.) Protein 1-2 8 19 22 47 Triglycerides 86 55 23 6 4 Cholesterol 5 19 38 50 19 Phospholipid 7 18 20 22 30 Apolipoproteins Al, All B-48 CI, CII, CHI E B-100 ci, cii, cm E B-100 ci, cii, cm E B-100 Al, All CI, CII, cm E 26 Drugs like nystatin, ampB, cyclosporine are known to associate with lipoproteins present in the plasma. To understand the influence of this association on the pharmacological activity and toxicity of these compounds, it is important to understand the metabolism of plasma lipoproteins. 1.3.3 Lipoprotein Metabolism free fatty acids Figure 4: Lipoprotein metabolism and lipid transport [adapted from http://www.endotext.org/obesity/obesity 10/obesity 10.html L C A T : Lecithin cholesteryl acyl transferase, ABC1: ATP binding cassette protein 1. 27 1.3.3.1 Exogenous Pathway Following the intake of food, the dietary (exogenous) lipids are mobilized in the body by packaging into Chylomicrons (Figure 4). Chylomicrons are synthesized in the endoplasmic reticulum of the intestinal mucosal cells and consist largely of triglycerides along with small amounts of cholesterol esters and ApoB-48 as the major structural apolipoprotein. In addition to these, apoproteins Apo A-I, Apo A-II and Apo A-IV are also secreted with chylomicrons. They then move through the lymphatic system and enter the bloodstream via the thoracic duct (Grundy, 1990). After entering the systemic circulation, they acquire Apo E and Apo Cs from HDL. Apo C-II activates lipoprotein lipase in the endothelial cells of the capillaries of adipose, heart, skeletal muscle and lactating mammary tissues, allowing the hydrolysis of triglycerides and release of free fatty acids. Free fatty acids bind to circulating albumin and serve the following functions in these tissues (Grundy, 1990): a) They may be consumed as a source of energy by muscle; or b) They may be stored by adipose tissue following re-synthesis into triglycerides; or c) They may be consumed as source of fuel by liver or re-synthesized into triglycerides, used for the formation of hepatic triglyceride-rich lipoproteins. The remnants of chylomicrons, consisting mostly of the cholesterol esters in its core along with Apo E and Apo B-48, move through the bloodstream to the liver where, receptors in the liver bind to the Apo E in these remnants and mediate their uptake by endocytosis. In the liver, the remnants release their cholesterol and are degraded in lysosomes (Herz et al, 1988). 28 1.3.3.2 Endogenous Pathway When the diet contains more fatty acids than are needed immediately as an energy source, they are converted to triacylglycerols in the liver and packaged into very low density lipoproteins (VLDL) to transport endogenously derived triacylglycerols to extra-hepatic tissues. The nascent V L D L particles secreted into the circulation contain ApoB-100 as the major structural apolipoprotein and also contain Apo E. As nascent V L D L circulate, they are transformed into mature V L D L particles, which occurs by acquisition of cholesteryl esters, and Apo C-II and C-III. Mature V L D L particles, thus synthesized, then interact with lipoprotein lipase (activated by Apo C-II), resulting in the hydrolysis of triglycerides and release free fatty acids to the various tissues (primarily muscle and adipose tissue) (figure 4). V L D L remnants are removed from the circulation by hepatocytes through receptor-mediated endocytosis (Grundy, 1990). The loss of triacylglycerols converts some V L D L to V L D L remnants (also called IDL); further removal of triacylglycerols from V L D L produces L D L . Cholesterol-rich L D L carries cholesterol to extra hepatic tissues that have specific plasma membrane receptors which recognise Apo B-100. These receptors mediate the peripheral uptake of cholesterol and cholesteryl esters by the endocytosis process. 1.4 Phospholipid transfer protein (PLTP) In human plasma, the transfer of lipids between lipoprotein particles occurs via the action of two distinct lipid transfer proteins; cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP). CETP is a hydrophobic glycoprotein that is secreted primarily by the liver (Tall et al, 1993). The function of CETP is to facilitate the redistribution of cholesteryl esters from H D L to triglyceride-rich lipoproteins and of 29 triglycerides from triglyceride-rich lipoproteins to L D L and H D L . PLTP is mainly involved in the transfer of phospholipids from triglyceride-rich lipoproteins to HDL. It also plays an important role in the remodelling of H D L so that, together with CETP, it plays an important role in the pathway of reverse cholesterol transport by modifying the metabolic properties and size distribution of HDL (Huuskonen et al, 2000). CETP and PLTP belong to the same gene family; the lipid transfer/lipopolysaccharide binding protein family (Day et al, 1994). PLTP activity has been detected in all species studied thus far. However, in contrast, substantial CETP activity is found only in man, rabbit, chicken and trout (Guyard et al, 1998). A mature PLTP protein has a molecular weight of 81 kDa observed by sodium dodecylsulphate- polyacrylamide gel electrophoresis (SDS-PAGE) (Day et al, 1994). PLTP contains four cysteine residues with the potential to form two intra-chain disulfide bonds, whereby the bridge that is formed between cysteine residues 146 and 185 is found to be important for the structural integrity of the protein (Huuskonen et al, 1999). The N -terminal pocket of PLTP protein is critical for PLTP transfer activity and that the C-terminal pocket is required for H D L binding (Huuskonen et al, 1999). 1.4.1 Functions of P L T P 1.4.1.1 Phospholipid transfer activity PLTP transfers phospholipids from triglyceride-rich lipoproteins to H D L during lipolysis (Huuskonen et al, 2000). This function was discovered via an in vitro method in which radiolabeled phospholipid vesicles were incubated within H D L or plasma (Tall et al, 1981). By using this method it was discovered that the rate of transfer of radiolabeled 30 phosphatidylcholine from unilamellar egg phosphatidylcholine vesicles to HDL was sufficiently enhanced by the activity of PLTP (Huuskonen et al.,, 1999). These studies also demonstrated that PLTP is capable of transferring all common phospholipid classes non-specifically, except for phosphatidylethanolamine which is transferred more slowly. The physiological importance of PLTP in vivo is evident in PLTP-deficient mice as a study conducted using PLTP-deficient mice showed a total absence of transfer of phospholipids from V L D L to H D L in plasma, along with markedly decreased HDL levels (Jiang etal, 1999). 1.4.1.2 H D L conversion In addition to the phospholipid transfer, PLTP is also involved in the H D L conversion process by which the heterogeneity of HDL particles in the plasma is maintained. This process involves particle fusion rather than a net lipid transfer or particle aggregation. Phospholipid transfer process due to PLTP increases the surface pressure of HDL which releases Apo A-I from the surface (Lusa et al. 1996). In addition to human PLTP, mouse recombinant PLTP as well as pig PLTP also facilitate H D L conversion (Albers et al. 1995). Thus PLTP is involved in the conversion of HDL3 into a population of enlarged HDL2 particles together with a concomitant release of Apo-AI particles (Jauhiainen et al, 1993). 31 1.5 Reverse cholesterol transport and P L T P The transfer of cholesterol from the peripheral tissues to the liver for recycling or degradation into the bile is denoted as 'Reverse Cholesterol Transport' (RCT). This process begins with the formation of nascent HDL particles in the liver and small intestine, consisting of phospholipid bilayer along with lipid- free Apo A-I and A-II as major apolipoproteins. Cholesterol Bile salts Liver Apo-AI PL from TG-rich Jipoproteins PLTP Nascent discoidal H D L L D L Cholesterol A B C A l Nascent discoidal H D L + Peripheral cholesterol Peripheral Cell L C A T H D L 2 H D L 3 Figure 5: Schematic representation of reverse cholesterol transport process (adapted from Crestani M et al., 2004) PLTP: Phospholipid transfer protein, A B C A l : ATP binding cassette protein 1, SR-B1: scavenger protein B1 receptor, LDLr: Low density lipoprotein receptor. Nascent H D L particles are good acceptors of unesterified cholesterol (due to the presence of the apolipoproteins) (Fielding et al, 1995), which can come either from the peripheral cells or other lipoproteins. The efflux of cholesterol from the cells occurs via an interaction of the nascent HDL particles with transmembrane ATP-binding cassette A l 32 (ABCA1) (figure 5). This free cholesterol is then esterified to cholesteryl ester (CE) by the action of an enzyme called lecithin cholesterol acyl transferase (LCAT). As the cholesterol is esterified, it becomes hydrophobic and accumulates into the lipid core which then transforms the disc-shaped H D L particle into a sphere, now known as HDL-3 (Eisenberg et al, 1984), (Fielding et al, 1995) (figure 5). These HDL-3 particles are high in cholesterol esters and deficient in triglycerides (TG). Lipid transfer proteins present in the plasma assist in moving T G and CE into HDL-3. Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) are the two major lipid transfer proteins observed in the human plasma (Nunes et al, 2001). CETP help in the exchange of cholesteryl ester for triglycerides between H D L and T G rich lipoproteins like chylomicrons and V L D L . PLTP acquires phospholipids from Apo B-containing lipoproteins (Eisenberg et al, 1984) and transfer them to H D L particles. Subsequently mature HDL particles are formed which are known as HDL-2 consisting of CE and TGs as their lipid core. Cholesterol-enriched HDL may be taken up in the liver by receptor-mediated endocytosis or by partial and selective transfer of cholesterol and other lipids by binding to plasma receptor proteins called 'scavenger recepotor-B 1' (SR-B1) in the liver. Once cholesterol enters the liver, Apo A-I released is then utilized to generate new HDL particles. PLTP plays an important role during this process at two locations: 1. It acquires phospholipids from TG rich lipoproteins and transfer them to HDL particles. 2. It helps in the conversion of HDL-3 to HDL-2 by facilitating particle fusion as discussed earlier. 33 1.6 Role of Lipoproteins in the transport of hydrophobic drugs Previously it was assumed that protein binding of drugs is limited to aqueous proteins like albumin. However, studies have shown that compounds such as AmpB (Wasan et al., 1993), eritoran (Wasan et al, 2004), halofantrine (Cenni et al. 1995), cyclosporine (CSA) (Wasan et al, 1997), nystatin (Wasan et al, 1997), annamycin (Wasan et al, 1994) associate with the plasma lipoproteins. It has been observed that lipoproteins play a role not only in the transport of lipids in the blood but are also involved in the transport of a number of compounds. Drugs such as AmpB, CSA, eritoran, halofantrine, annamycin, nystatin have been shown to bind to plasma lipoproteins which results in the altered efficacy, pharmacokinetics, tissue distribution, toxicity and pharmacological action of these compounds after intravenous administration (Wasan et al, 1998b). 1.6.1 Cyclosporine (CSA) Cyclosporine (CSA) has been in clinical use as an immunosuppressant since 1983. A number of researchers have shown that CSA associates with plasma lipoproteins after incubation within human plasma resulting in the modification of its pharmacological activity (Wasan et al, 1998). Studies have shown increased antiproliferative effects when CSA is associated with the L D L and no change when it is associated with H D L or V L D L (Lemaire et al, 1988). CSA induced renal toxicity in kidney transplant patients with elevated cholesterol levels has been reported (Wasan et al, 1998). 34 1.6.2 Halofantrine (Hf) Halofantrine (Hf) is an effective antimalarial drug particularly against P. falciparum and other multi-drug resistant strains (Boudreau, 1988; Watkins, 1988 ). A number of clinical studies have shown decreased plasma HDL-cholesterol levels and increased plasma VLDL-triglyceride levels in malaria-infected versus non-infected patients ((Mohanty et al, 1992; Vernes, 1980)). A study by Cenni et al. has suggested that upon incubation within the human blood at 20°C, Hf interacts and binds mainly to L D L and HDL (Cenni et al, 1995). However, Charman et al. have shown that after i.v. administration of Hf (3 mg/kg) to pre- and postprandial beagles resulted in different in vivo Hf lipoprotein distribution profiles. In the fasted state, the distribution of Hf in plasma lipoproteins was as follows; 2% of the total Hf plasma concentration recovered in the V L D L fraction, 4.5% in the L D L fraction, 43% in the HDL and 50.5% in the LPDP (Humberstone et al.., 1995 ). However, in the fed state, Hf distribution showed 5-fold and 2-fold increases in the proportion of Hf present in the V L D L and L D L fractions at 0.5 and 2 h post dosing, relative to the fasted state (Humberstone et al, 1995). 1.6.3 Eritoran (E5564) Eritoran (E5564) is a structural analogue of the lipid portion of lipopolysaccharide and is an antagonist of lipopolysaccharide in animal and human endotoxemia models (Gotto et al, 1986 ). Recent studies by Rossignol et al. demonstrated that upon incubation of E5564 within human plasma, majority (~ 55%) of E5564 was recovered from HDL fraction. More importantly, this study indicated that H D L inactivates E5564, whereas long term antagonistic activity can be maintained when E5564 associates with L D L , triglyceride rich lipoproteins (TRL) and albumin (Rossignol et al. 2004) 35 1.6.4 AmpB A m p B associates extensively with lipoproteins after incubation within the human plasma. Brajtburg et al (1990) have shown that A m p B , when incubated within human serum for 60 minutes at 25°C, was equally associated with H D L and L D L . However, Wasan et A/.(1993) showed that A m p B , when incubated within the human serum at 37°C for 60 minutes, more than 50% of the drug was recovered from the H D L fraction. Since A m p B is very often administered to patients with abnormal serum cholesterol and triglyceride levels, its affinity for lipoprotein may have significant impact on the safety and efficacy of this drug (Lopez-Berestein et al, 1985). It has also been shown that L D L associated A m p B is more nephrotoxic than free A m p B or H D L associated A m p B . Patients with elevation in L D L cholesterol levels are more susceptible to A m p B induced renal toxicity (Oda et al, 2006). One of the possible reasons behind this toxicity was found to be the presence of high affinity L D L receptors expressed inside the kidney which mediate the cellular uptake of the drug through the process of endocytosis (Ramaswamy et al, 2001). Wasan et al. have demonstrated the therapeutic importance of the influence of A m p B -lipoprotein association on the toxicity to L L C -PK1 renal cells (a cell line derived from the proximal tubular cells of swine). It was observed that A m p B was significantly less toxic to renal L L C P K 1 cells when it was associated with H D L than when it was associated with L D L . However L D L associated A m p B was just as toxic to the cells as that of unbound A m p B . When high affinity surface receptor expression was decreased, L D L associated Amphotericin B was less toxic to L L C P K 1 renal cells than was the free, unbound drug. This data suggested that the increased toxicity observed with L D L bound A m p B may be a result of its interaction with the high affinity L D L receptors located on the surface of the cell (Wasan et al, 1994). 36 1.6.5 Compounds incorporated into lipid-based vesicles Number of previous studies as well as preliminary results of this project has demonstrated that incorporating compounds like nystatin (Cassidy et al, 1998), annamycin (Wasan et al, 1996), AmpB (Patankar et al, 2006) into lipid based vesicles significantly alters plasma lipoprotein distribution profile of these drugs. Annamycin is an anthracycline used in the treatment of human leukemia, lymphoma and breast cancer (Arcamone, 1985; Weiss et al 1986). A liposomal formulation of annamycin is composed of DMPC and DMPG. When liposomal annamycin at different concentrations was incubated within human plasma for 60 min at 37°C, more than 60% of the initial annamycin concentration was recovered in the H D L fraction (Wasan et al, 1996) . AmpB, when incubated within human plasma as Fungizone®, distributes preferentially into lipoprotein deficient fraction, whereas when incubated as a AmpB-phospholipid complex (i.e. Abelcet®) as well as a liposomal AmpB (Ambisome®), it mainly distributes into HDL (Wasan et al. 1993, Patankar et al. 2006). Nystatin is another polyene macrolide antibiotic and is used in the treatment of superficial fungal infections of the skin and mucous membranes (Medoff et al, 1983). Incorporation of nystatin into liposomal small unilamellar vesicles (SUV) provides a better alternative that can be administered safely at much higher concentrations than free nystatin (Wasan et al, 1997) .When liposomal nystatin was incubated within human plasma at 37°C for 5-120 min, the majority of nystatin was recovered in the HDL fraction (Wasan et al, 1997). These findings are similar to that observed with Abelcet® and liposomal annamycin. 37 Taken together, these findings may suggest that the preferential distribution of drug into H D L may not be a function of the drug itself but may be a function of the carrier lipids used in the formulations. A n interesting observation among the three formulations discussed here is that all these formulations contain D M P C and D M P G as their carrier phospholipids. Therefore, it can be interpreted that interaction of either D M P C or D M P G or together with H D L may facilitate the transfer of these drugs to H D L . Studies conducted in the past have investigated the interaction of liposomes and lipid-complexes with plasma lipoproteins. Scherphof (1983) demonstrated the transfer of phosphatidylcholine from small unilamellar vesicles to H D L (Scherphof et al. 1983). Surewicz et al. have reported the formation of thermally stable complexes between anionic phospholipids (i.e. D M P G ) and apolipoprotein A l (Surewicz et al. 1986) which is a principle constituent of H D L . However, P L T P as discussed earlier has an ability to transfer phospholipids in the plasma to H D L and therefore may have a significant potential to transfer drugs complexed with these phospholipids to H D L . 1.7 Proposed mechanisms for improved therapeutic index of lipid based formulations A s discussed in the earlier sections, l ipid based formulations of A m p B have been proved clinically less toxic than conventional A m p B formulation. The exact mechanism by which these formulations increase the therapeutic index of A m p B is unknown. However, based on the studies conducted in various labs, few mechanisms have been proposed: 38 Douglas (1999) suggested that as the lipid based AmpB enters the systemic circulation, it is rapidly taken up by macrophages by endocytosis and by tissues of RES. This phenomenon is commonly observed with Abelcet®. Subsequently drug is liberated from the lipid carrier and released slowly into the systemic circulation. Therefore, it is possible to have targeted and sustained delivery of the drug to infections in these RES organs and to other infection sites as macrophages are generally present at these sites (Mehta et al., 1994). Juliano et al. (1987) have shown selective transfer of drug from the donor lipid to the fungal cell membrane. This phenomenon is most likely to occur with lipid based formulations which are not readily removed from the blood circulation by RES. Studies conducted with liposomal AmpB have shown that AmpB encapsulated liposomal vesicles remain intact until they attach to the fungal cell wall (Adler-Moore, 1994). This theory is based on the relative affinity of the drug for carrier lipids and fungal ergosterol. According to it, AmpB has lower affinity for carrier lipids as compared to the ergosterol and has higher affinity to carrier lipids as compared to the cholesterol. Therefore liposomal AmpB remains intact until it comes in contact with the fungal cells, where the drug is released (Adler-Moore, 1994). In one of the models suggested (Brajtburg et al, 1996), only free AmpB can bind to the cell membranes. According to it, only self associated AmpB molecules can damage the mammalian cells whose membranes contain cholesterol whereas both self associated as 39 well as monomeric AmpB can bind and damage fungal and parasitic cells whose membrane contain ergosterol. Since, only free AmpB is active against cells, damaging activity of its formulations depends on their ability to dissociate AmpB from its carrier. In case of Fungizone®, bond between AmpB and deoxycholate is very weak and the drug is immediately dissociated from the carrier in the form of monomers as well as aggregates after the administration. Whereas, in case of lipid based formulations, affinity between AmpB and lipid is strong as compared to Fungizone® and amount of self associated AmpB molecules released is also very less (figure 6). Fungizone Self associated AmpB Monomer AmpB C ^ _ ^ ) 0 Fungal Cell Mammalian Cell \ t / Lipid associated AmpB Monomer AmpB Figure 6: Release of monomeric AmpB as a result of the rapid dissociation of Fungizone® and release of small amounts of intact monomeric AmpB from lipid formulations to act on fungal cells [adapted from (Brajtburg et al, 1996)]. 40 Swenson et al, (1998) studied the role of phospholipases in liberating AmpB from lipid carriers. It was proposed that phospholipases may play an important role in liberating AmpB from Abelcet® at the site of infection. So, drug lipid complex remains intact until it comes into contact with the phospholipases secreted by fungal cells [(Perkins et al, 1992), (Swenson et al, 1998)]. Many fungal and yeast cells produce extracellular phospholipases that can hydrolyze the major lipid component of AmpB lipid complex and cause release of drug in the presence of fungal phospholipases at the site of infection [(Perkins et al, 1992), (Swenson et al, 1998)]. It was suggested that host derived endogenous phospholipases along with the fungal phospholipases may play a role in the activity of Abelcet® in vivo. In order for AmpB to be selectively released at the site of infection, it is necessary for the drug lipid complex to remain intact till it reaches these sites. It is known that certain fungi are angiotropic and can disrupt the capillary lining sufficient for the drug lipid complex to escape from the circulation selectively at the site of infection where it would be broken down due to the action of fungal phospholipases and release the active drug. AmpB can also cause release of TNF-a and interleukin-1 which may lead to toxicities related to infusion. In vitro studies have shown that liposomal AmpB and lipid complex of AmpB do not cause release of these cytokines in contrast to conventional AmpB-deoxycholate (Chia et al, 1990). Continued efforts in K. Wasan's laboratory, in order to understand the reduced nephrotoxicity of lipid based formulations of AmpB have demonstrated that AmpB associated with H D L may be less nephrotoxic than L D L associated or free AmpB. One of 41 the possible reasons may be lack of expression of high affinity H D L receptors inside the kidney (Wasan et al, 1994). In addition, it has been observed that lipid associated AmpB is less likely to associate with L D L and more likely to associate with H D L (Wasan et al, 1996). Preliminary results observed in this project (results discussed in detail in the later sections of this dissertation) have demonstrated that AmpB, when formulated into phospholipids based formulation show significantly greater association with H D L following incubation within human plasma. There could be multiple factors responsible for the preferential distribution of AmpB into HDL. However, based on the phospholipid transfer activity of PLTP observed in the human plasma, we hypothesize that PLTP may co-transfer AmpB complexed with the phospholipids in the formulation into H D L and therefore may be responsible for the preferential distribution of AmpB into HDL. 1.8 Hypothesis Based on the background information and experimental evidence obtained earlier, we hypothesized that phospholipid transfer protein (PLTP) will be responsible for the transfer of AmpB into high density lipoprotein (HDL) when it is incubated within human plasma as a phospholipid complex. In order to test this hypothesis, the specific aims of my project were as follows; Part I: To study the plasma distribution profile of Amphotericin B (AmpB). Aim 1: To compare the plasma distribution profile of AmpB following the incubation of Fungizone® (AmpB - sodium deoxycholate), Abelcet® (AmpB - phospholipid 42 complex) and Ambisome (liposomal AmpB) within plasmas from six different individuals. Aim 2: To assess the plasma distribution profile of AmpB following incubation within plasmas with Total Cholesterol (TC) and Triglyceride (TG) levels less than 200 mg/dL and those with TC and TG levels greater than 200 mg/dL. Part II: To determine if changes in phospholipid transfer protein (PLTP) activity alters the plasma distribution of AmpB, when incubated within human plasma as phospholipid based formulations. Aim 1: To establish an in vitro PLTP activity assay within human plasma. Aim 2: To determine i f a relationship exists between plasma PLTP activity and the percentage of AmpB recovered in the high density lipoproteins (HDL) fraction. Aim 3: To determine i f a relationship exists between HDL-phospholipid concentration and the percentage of AmpB recovered in the H D L fraction. Aim 4: To develop and test different methods to inhibit plasma PLTP activity. 43 Materials and Methods 44 2. M A T E R I A L S AND M E T H O D S 2.1 Materials 2.1.1 Chemicals and Reagents Methanol, acetonitrile and dichloromethane were purchased from Fisher Scientific Canada (Toronto, ON). Thimerosal, sodium acetate and sodium bromide were purchased from Sigma (St. Louis. M O , USA). Fungizone®, Abelcet® and Ambisome® were purchased from Vancouver General Hospital pharmacy (Vancouver, BC). 2.1.2 Human Plasma Human plasma samples from individual donors having varying lipid profiles were purchased from Bioreclamation Inc. (Hicksville, NY) . These plasma samples were obtained from donations at FDA (US Food and Drug Administration) registered centres and pre-screened for Human T-cell Lymphotropic Viruses (HTLV), Hepatitis B surface antigen (HBsAG), Syphilis, Human Immunodeficiency virus (HIV) and Hepatitis C virus (HCV). 2.1.3 P L T P Assay Kit The PLTP activity assay kit was purchased from Cardiovascular Targets (NY, USA). 2.1.4 P L T P antibody The anti-PLTP antibody (rabbit polyclonal antisera with 0.02% sodium azide) was purchased from Novus Biologicals, USA. 45 2.1.5 Phospholipid Assay Kit Phospholipid assay kit was purchased from W A K O Chemicals (USA). 2.2 Preparation of Analytes and Solutions 2.2.1 Fungizone® colloidal dispersion Fungizone® supplied as lyophilized powder was reconstituted by adding 10 mL of sterile water for injection USP without a bacteriostatic agent to the vial. The vial was shaken continuously until the liquid was clear. Final concentration of AmpB in the reconstituted liquid was 5 mg/mL. 2.2.2 Abelcet® suspension Abelcet® was provided as a sterile, opaque suspension in a single use 20 mL glass vials. Each vial contained 100 mg of AmpB to give a final concentration of 5 mg/mL AmpB. 2.2.3 Ambisome® liposomal suspension Ambisome® was provided as a lyophilized product and was reconstituted prior to use by adding 12 mL of sterile water for injection USP to a vial containing 50 mg of AmpB to give a final concentration of 4 mg/mL AmpB. 46 2.3 Methods 2.3.1 Incubation of plasma with Amphotericin B-deoxycholate (Fungizone®) or Amphotericin B lipid complex (Abelcet®) or liposomal Amphotericin B (Ambisome®) To an ultraclear centrifuge tube (Beckman Instruments, Inc. Palo Alto, CA) , 3.0 mL of human plasma was added for incubation of AmpB formulation (n=6 for Fungizone®, n=6 for Abelcet® and n=6 for Ambisome®) and standard curve purposes. Contents of the tubes were pre-warmed to 37°C. The sample plasma was spiked with either 12 pL of Fungizone® (5 mg/mL) or 12 pL of Abelcet® (5 mg/mL) or 15 pL of Ambisome® (4 mg/mL). The final plasma concentration of AmpB for all three sample sets was 20 u.g/mL. Immediately after addition, the samples were vortexed and returned to 37°C and incubated for the required incubation time. After the incubation, these were cooled on ice for 30 minutes. Plasma used for the standard curve purpose was subjected to identical incubation and cooling times as the sample plasma. 2.3.2 Separation of lipoprotein constituents Sodium bromide solutions of different densities (8 = 1.006, 1.063, and 1.21 g/mL) were used for creating gradients on top of plasma samples. (Refer Appendix B for the composition of gradient density solutions). A l l density solutions were stored at 4°C prior to the layering of the gradient. To the previously cooled plasma sample, was added 1.02 g of sodium bromide (0.34 g sodium bromide per 1.0 mL plasma) in order to modify the density of the plasma to approximately 1.25 g/mL (Havel et al, 1955 Wasan et al, 1999). On the top of this plasma sample, 2.8 mL of sodium bromide solution (5=1.21 g/mL) was carefully layered. On top of this layer, 2.8 mL of sodium bromide solution (5 47 =1.063 g/mL) was layered, followed by a third layer of 2.8 mL of sodium bromide solution (8=1.006 g/mL). Ultracentrifuge tubes were balanced and placed into individual titanium buckets and capped. The buckets were placed into their respective positions on a SW 41 Ti swinging bucket rotor (Beckman Instruments, Palo Alto, CA) and centrifuged at 40,000 rpm for 18 hours at 15°C in a Beckman L8-80M ultracentrifuge (Beckman Instruments, Palo Alto, CA). Upon completion of the run, the ultracentrifuge tubes were carefully removed from the titanium buckets to prevent mixing of the layers. Each tube showed four visibly distinct regions represented by the TRL, L D L , H D L and LPDP fractions as shown in Figure 7. Subsequently, each fraction was removed by Pasteur pipette starting from the top and the volume of each fraction was measured. All sample and standard curve fractions were transferred to clean disposable test tubes, covered and stored at 4°C until further analysis. Very low density lipoproteins Low density lipoproteins High density lipoproteins Lipoprotein-deficient plasma Figure 7: Ultracentrifuge tube following ultracentrifugation in a Beckman SW41-Ti swinging bucket rotor at 40,000 rpm and 15°C for 18 hours in a Beckman L8-80M ultracentrifuge. 48 2.3.3 Non-lipoprotein control To ensure that the distribution of AmpB obtained in each of these fractions was a result of its association with each of the lipoprotein and lipoprotein deficient fraction and not a result of density of drug or formulation, previous studies in our lab (Kennedy et al., 1999) used LPDP as a control medium. Briefly, all the lipoprotein fractions from plasma were separated and LPDP was acquired by the technique of ultracentrifugation as described above and was dialyzed against a 0.9% sodium chloride solution for 24 hours at a temperature of 4°C. It was then used as a control medium for the incubation of AmpB formulations. Sample preparation was carried out by same procedure as described above and fractions separated were analyzed for the drug content. It was observed that the majority of the drug (>92%) was recovered in the density fraction from 1.21 to 1.25 g/ml, suggesting that the distribution of AmpB is not a function of drug or formulation density. 2.3.4 Amphotericin B quantification 2.3.4.1 Standard curve preparation Each plasma lipoprotein (TRL, L D L , HDL) and non-lipoprotein (LPDP) fractions were aliquoted (0.5 mL) into a series of tubes labelled: '0.625', '1.2', '2.5', and '5 pg/mL'. To the fifth tube labelled '10 pg/mL' was added 1 mL of the respective plasma lipoprotein or non-lipoprotein fraction. 2pL aliquot of either Fungizone® (5 mg/mL) or Abelcet® (5 mg/mL) or 2.5 pL of Ambisome® (4 mg/mL) was added to the tube labelled as '10 pg/mL' and vortexed for 10 seconds (s). From the '10 pg/mL' test tube, 0.5 mL of mixture was transferred to the '5 ug/mL' test tube. This mixture was vortexed for 10 s and then a 0.5 mL aliquot of this mixture was subsequently transferred to a '2.5 mL' test 49 tube. This procedure of serial dilution was repeated for the remaining test tubes. 0.5 mL of mixture from the '0.625 ug/mL' test tube was discarded to provide a final volume of 0.5 mL for all standard curve concentrations. 2.3.4.2 Determination of Amphotericin B content within the separated lipoprotein fractions (TRL, L D L , H D L and LPDP) The method used for the extraction and analysis of AmpB was based on the method developed by Wasan et al (1997). To an appropriately labelled test tube, 0.5 mL aliquot of plasma lipoprotein or non-lipoprotein fraction was added. To this sample tube and likewise the standard curve tubes, 3.0 mL of dichloromethane (DCM) was added. The mixture was vortexed for 1 minute and all samples were dried to completion under a stream of nitrogen at an ambient temperature. Once dried, the AmpB residue was extracted using series of methanol washes. Briefly, a 3.0 mL aliquot of methanol was added to the residue and vortexed for 1 minute. A l l test tubes were then centrifuged at 2000 rpm at 15°C for 2 minutes. The supernatant was transferred to a clean test tube. This procedure was repeated twice with 2.0 mL of methanol. The supernatant from each of the washes was pooled with the previous supernatants resulting in a final volume of 7 mL of methanol. This pooled methanol was then dried to completion under a steady stream of nitrogen at an ambient temperature. The residue was then reconstituted with 0.5 mL of methanol immediately prior to analysis, vortexed well for 30 s and centrifuged at 2500 rpm for 3 min at 15°C. Supernatant was collected and loaded into the HPLC autosampler; the injection volume was set at 20 pL. 50 H P L C apparatus The HPLC system consisted of a Waters 600 controller interfaced to a Waters 717 plus Autosampler and a Waters 486 Tunable Absorbance Detector. The detector was set to an U V absorption wavelength of 405 nm. A l l results were recorded on a Waters 746 Data Module integrator (Waters Corporation, Milford MA) . Attenuation was set to 32 and chart speed to 0.25 cm/min. Samples (100 pL) were injected onto a Waters SB-C18 column (4.6 x 150 mm, 5 pm particle size), pre fitted with a Zorbax SB-C18 guard column (4.6 x 12.5 mm, 5 pm particle size). Chromatographic separation was carried out using a mobile phase at an isocratic flow rate of 1.2 mL/min consisting of 10 m M sodium acetate: acetonitrile (70:30) mixture termed 'mobile phase A ' . 2.3.5 P L T P activity assay Buffer (10 m M tris, 150 m M NaCl, 2 m M EDTA; pH 7.4) and acceptor particles were chilled on ice prior to use. Donor and buffer were mixed (1:14 v/v ratio) to get a sufficient volume for the number of assays. 45 pL of this mixture was pipetted into each well of the microplate. To this mixture was added, 50 pL of acceptor. Finally, required volume (u.L) of PLTP source (plasma) was added and this mixture was immediately incubated for up to 30 minutes at 37°C. The fluorescence was read in the fluorescence spectrometer. The excitation filter used was 450 nm with a 50 nm window and the emission filter was 530 nm with a 25 nm window. Buffer blank: Buffer blank was prepared following the same procedure as described above but without the addition of PLTP source which was replaced by the same volume of assay buffer. 51 PLTP DONOR M O L E C U L E ACCEPTOR M O L E C U L E Measure Fluorescence Figure 8: Schematic representation of PLTP assay. 2.3.5.1 Characterization of PLTP assay Determination of acceptable plasma volume After addition of donor (premixed with buffer) and acceptor to the microplate well, increasing volumes (3 pL, 5 pL, 10 pL, 15 pL and 20 pL) of plasma as a PLTP source were added separately to this mixture, incubated for 30 min at 37°C and the fluorescence was measured. Determination of acceptable incubation time Using 10 pL plasma as a PLTP source, PLTP assay was carried out at different incubation times (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min and 60 min). 52 2.3.6 Inhibition of plasma P L T P activity Three different approaches were evaluated for this purpose; i) polyclonal antibody to PLTP that has previously been used to determine PLTP concentration (Nishida et al, 1997) was used to incubate with plasma, ii) thimerosal that is known to interfere with the structural integrity and therefore activity of PLTP (Huuskonen et al, 2000a) was incubated with plasma and, iii) heat treatment of plasma as shown by Desrumaux et al. (2004) was used to inhibit plasma PLTP activity. 2.3.6.1 Incubation of plasma with P L T P antibody Polyclonal antibody specific to PLTP was used for this purpose. Briefly, 1 uL of PLTP antibody was diluted with 40 pL of PLTP activity assay buffer (10 m M tris, 150 mM NaCl, 2 m M EDTA; pH 7.4). This antibody solution was incubated with 10 pL of human plasma at 4°C for 24 hours. 50 uL of this mixture was added to the microplate well containing 50 pL of acceptor and 3 uL of donor (hence, the final dilution of antibody in the assay mixture was 1:100), incubated at 37°C for 30 minutes and the increase in fluorescence was measured as described for the PLTP activity assay against blank. 2.3.6.2 Incubation of plasma with Thimerosal 10 uL of thimerosal solution (100 mM) (prepared in PLTP activity assay buffer) was incubated with 10 pL of human plasma in an eppendorf tube at 4°C for 24 hours. 20 pL of this mixture was added to the assay components comprising 30 uL buffer, 50 pL acceptor and 3 pL donor. Hence, the final concentration of thimerosal in the assay mixture was 10 mM. This assay mixture was incubated at 37°C for 30 minutes and the 53 increase in fluorescence was measured as described for the PLTP activity assay against blank. 2.3.6.3 Heat treatment of plasma Human plasma (quantity sufficient to carry out the PLTP assay) was heated in a temperature controlled water bath maintained at 56° for 1 hour. After 1 hour, plasma was removed from the water bath, brought back to the room temperature and PLTP activity was measured usinglO pL of this plasma against blank as described before. Controls used during PLTP inhibition experiments were as follows; 2.3.6.4 Untreated plasma (Positive control) Positive control was prepared by the same procedure that was followed for preparation of samples containing PLTP antibody (section 2.3.6.1) but without the addition of antibody which was replaced with 10 pL of assay buffer. 2.3.6.5 Incubation of plasma with C E T P antibody (Irrelevant control) Irrelevant control was prepared by the same procedure as that of section 2.3.6.1. However, the PLTP antibody was replaced with the CETP antibody. 2.3.6.6 Evaluation of P L T P antibody for inherent P L T P activity Since the PLTP antibody used in this experiment consisted of whole rabbit serum, it was tested for inherent PLTP activity. For this purpose, PLTP assay was carried out by the 54 same procedure as described before but using 10 uL of PLTP antibody instead of plasma as a PLTP source. 2.3.7 Measurement of phospholipid content from H D L A commercially available kit was used for this purpose. Briefly, 3.0 mL of colour reagent was pipetted accurately into the test tubes labelled 'standard', 'sample' and 'blank'. 20 pL of sample and standard were pipetted into appropriate test tubes. (Note: According to manufacturer's instructions, no difference in absorption is observed in the 'blank' by addition of 20 pL of water; therefore the addition of water is omitted.) The contents of the tube were mixed properly and all tubes were placed in a 37°C water bath for 10 minutes. 200 uL of contents from all tubes were pipetted into the microplate wells and the absorbance was measured using a spectrophotometer plate reader at 492 nm against 'blank' to determine the net absorbance. The phospholipid content of the HDL samples was calculated using the following equation: •^Sample X (-"Standard (mg/dL) C s a m ple (mg/dL) -^-Standard Where, A = Absorbance at 492 nm C = Phospholipids content (mg/dL) The HDL fractions obtained from plasmas that were used to study the AmpB distribution, were analyzed for the phospholipid content by the procedure described above. 55 2.4 Statistical analysis The differences in the distribution of AmpB for Fungizone®, Abelcet® and Ambisome® into the separated lipoprotein and lipoprotein-deficient fractions between plasma samples were analyzed by one way Analysis of variance (ANOVA) (InStat; GraphPad Software). Groups showing significant differences were identified using Student-Newman-Keuls post-hoc tests. Results were considered significant i f the probability of the result occurring by chance was less than 5% (P < 0.05). A l l data are expressed as mean ± SD of n=6 unless otherwise stated. 56 Results 57 3. R E S U L T S 3.1 Part I: To assess the plasma distribution profile of Amphotericin B (AmpB). 3.1.1 Aim 1: To compare the distribution profile of AmpB following the incubation of Fungizone® (AmpB - sodium deoxycholate), Abelcet® (AmpB - phospholipid complex) and Ambisome® (liposomal AmpB) within plasmas from six different individuals. 3.1.1.1 Experimental Design Fungizone®, Abelcet® and Ambisome® at an AmpB concentration of 20 pg/mL (n=6 for each formulation) were incubated within human plasmas from six different individuals (Table 1) for 5 to 120 minutes at 37°C. Following incubation, the plasmas were separated into their lipoprotein (consisting of triglyceride rich lipoproteins [TRL], low density lipoproteins [LDL] and high density lipoproteins [HDL]) and lipoprotein deficient (consisting of albumin and a-1 glycoprotein) plasma fractions by density gradient ultracentrifugation. A l l lipoprotein and lipoprotein deficient plasma fractions were assayed for AmpB content by high performance liquid chromatography and quantified using an external calibration curve (Wasan et. al. 1990). Table 5: Total cholesterol and triglyceride concentration of human plasmas used in the of AmpB distribution study. Plasma Total Cholesterol (TC) Triglycerides (TG) mg/dL mg/dL I 332 583 II 217 302 III 186 127 IV 178 147 V 176 122 VI 62 58 58 3.1.1.2 Amphotericin B H P L C Quantification In order to determine the concentration of AmpB from Fungizone®, Abelcet® or Ambisome® in each isolated plasma fraction, it was necessary to compare the area of the peak recovered from the HPLC chromatogram of the sample to a standard curve for the same fraction. External calibration curves were prepared for each of the separated fractions: TRL, L D L , H D L and LPDP for each assay. The standard curves for each of the plasma lipoprotein and non lipoprotein fraction were linear over a concentration range of 0.625 pg/mL tolO pg/mL. The coefficient of determination was greater than 0.99 for each of the regression line. Table 6: Representative linear calibration curves for Amphotericin B as determined in the separated lipoprotein and non lipoprotein fractions of human plasma for Fungizone®, Separated fraction Equation of the line Coefficient of determination (r2) Standard Curve Range (u.g/mL) T R L Area = 147744(conc.) - 55118 0.9994 0.625 to 10 L D L Area = 216945(conc.) + 9875.3 0.9942 0.625 to 10 H D L Area = 111297(conc.) - 23786 0.9991 0.625 to 10 LPDP Area = 144283(conc.) - 17054 0.9952 0.625 to 10 The retention time of the drug following extraction from lipoprotein and lipoprotein deficient fraction was approximately 8 to 10 minutes. Figures 10 and 11 show representative chromatograms of AmpB in TRL, L D L , H D L and LPDP.The area of each peak as recorded by a Waters 746 Data Module Integrator was used to generate a concentration point in the creation of a linear calibration curve for AmpB in each matrix (TRL, L D L , H D L and LPDP). Representative calibration curves for AmpB in each of the matrices are shown in Figure 9. The inter-assay variability of this assay as determined previously by calculating the coefficient of variation was less than 10 %. 59 A B y = 147744x - 55118 R2 = 0.9994 Concentration (ug/mL) y =216945x + 9875.3 R 2 = 0.9942 2500000 T 1 2000000 -£ 1500000 -< «1000000 -o. 500000 -o , , , , , 1 0 2 4 6 8 10 12 Concentration (ug/mL) D 1500000 51000000 y = 111297x- 23786 R2 =0.9991 4 6 8 10 concentration (fig/mL) 12 1500000 « 1000000 H I . £ 500000 y = 144283x- 17054 R2 = 0.9952 2 4 6 ! Concentration (ug/mL) 10 12 Figure 9: Representative standard curves of Amphotericin B in (A) triglyceride rich lipoprotein (TRL), (B) low density lipoproteins (LDL), (C) high density lipoproteins (HDL) and (D) lipoprotein deficient plasma (LPDP). Samples were prepared by adding Fungizone®/Abelcet®/Ambisome® to plasma fraction to obtain 10ug/mL of AmpB which was followed by serial dilution. 60 A B Figure 10: Representative chromatograms of Amphotericin B (lOug/mL) in (A) triglyceride rich lipoprotein (TRL), (B) low density lipoprotein (LDL) Scales represented are arbitrary 61 c 8 s s J3 D § a o Figure 11: Representative chromatograms of Amphotericin B (lOug/mL) in (C) high density lipoprotein (HDL), (D) lipoprotein deficient plasma (LPDP) Scales represented are arbitrary 62 3.1.1.3 Calculation of AmpB concentration in the plasma fractions The calibration curves for each of the fractions were utilized to determine the amount of drug (pg) present in each mL of the fraction. This concentration was then multiplied by the previously measured volume (mL) of the particular fraction obtained after step-gradient ultracentrifugation separation to give the total amount of AmpB recovered from the fraction. This procedure was applied to each of the plasma fractions. Total recovery as a percentage was calculated with the following formula. A recovery of > 80% was considered satisfactory considering loss of the drug during the repeated extraction steps and binding of the drug to the hydrophobic surface of the ultracentrifuge tube. An extraction efficiency of the assay was > 95 %. (Refer appendix A for representative calculations from each fraction.) fractions ^ (amount recovered in fraction), x 100 total amount incubated in the plasma Figure 12 shows plasma distribution profiles of AmpB following incubation of Fungizone® at an AmpB concentration of 20ug/mL within human plasma for 5, 30, 60 and 120 minutes at 37°C. It was observed that immediately after incubation (5 min.), majority of AmpB (64%) incubated initially was recovered from the LPDP fraction. 21% of AmpB was recovered from the HDL fraction. Whereas, L D L and TRL showed 12% and 2% of the incubated AmpB into them respectively. As the incubation time was increased, there was a slight variation in the percentage of AmpB recovered from each of the lipoprotein and non-lipoprotein fractions. However, the important observation was that overall distribution pattern remained the same 63 as even after 120 minutes of incubation, the order of percentage recovery of AmpB was LPDP>HDL>LDL>TRL. u > o u <u I -100 80 60 n g 40 20 0 30 minutes 60 minutes 120 minutes Incubation time (min) H TRL • L D L • H D L • LPDP Figure 12: Effect of incubation time on the plasma distribution profile of AmpB (20 ug/mL) after incubation of Fungizone® within human plasmas at 37°C. TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1 -glycoprotein) AmpB: Amphotericin B *P<0.05 vs. L D L (5min), #P<0.05 vs. H D L (5 min), +PO.05 vs. LPDP (5 min) using A N O V A Values are mean ± SD, n = 6 (six different plasmas) (Note: Refer Appendix C for the distribution profile in individual plasma) 64 Figure 13 shows plasma distribution profiles of AmpB following incubation of Abelcet* at an AmpB concentration of 20pg/mL within human plasma for 5, 30, 60 and 120 minutes at 37°C. As observed in the figure, majority of the AmpB (80%) incubated initially was recovered from the H D L fraction after 5 minutes of incubation. 20% AmpB was recovered from the LPDP fraction. However, L D L as well TRL fractions did not show any detectable amount of AmpB into them. As the incubation time was increased, there was no significant change observed in the distribution pattern of AmpB. T3 (U i -V > © i -a 100 80 60 40 20 0 ND ND ND ND ND ND ND ND H TRL • LDL • HDL • LPDP 5 minutes 30 minutes 60 minutes 120 minutes Incubation time (min) Figure 13: Effect of incubation time on the plasma distribution profile of AmpB (20 ug/mL) after incubation of Abelcet® within human plasmas at 37°C. TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) ND: Non detectable (below detection limit of the assay) AmpB: Amphotericin B *P<0.05 vs. H D L (5 min), #P<0.05 vs. LPDP (5 min) using A N O V A Values are mean ± SD, n = 6 (six different plasmas) (Note: Refer Appendix C for the distribution profile in individual plasma) 65 Figure 14 shows plasma distribution profiles of AmpB following incubation of Ambisome at an AmpB concentration of 20pg/mL within human plasma for 5, 30, 60 and 120 minutes at 37°C. As observed in the figure, greater percentage of AmpB (88%) incubated initially was recovered from the H D L fraction after 5 minutes of incubation compared to Fungizone® as well as Abelcet®. 10% AmpB was recovered from the LPDP fraction and about 2% was recovered from L D L fraction. TRL fraction did not show any detectable amount of AmpB into it. As the incubation time was increased, there was no significant change observed in the distribution pattern of AmpB. 8 > © u PS a = 100 80 60 40 20 ND ND ND ND 5 minutes 30 minutes 60 minutes 120 minutes Incubation time (min) Figure 14: Effect of incubation time on the plasma distribution profile of AmpB (20ug/mL) after incubation of Ambisome® within human plasmas at 37°C. TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) N D : Non detectable (below detection limit of the assay) AmpB: Amphotericin B. Values are mean ± SD, n = 6 (six different plasmas) (Note: Refer Appendix C for the distribution profile in individual plasma) 66 3.1.2 Aim 2: To assess the plasma distribution profile of AmpB following incubation within plasmas with Total Cholesterol (TC) and Triglyceride (TG) levels less than 200 mg/dL and those with TC and TG levels greater than 200mg/dL. The purpose of this data analysis was to re-analyze the results of A im 1 in a manner that will assist in differentiating the distribution of AmpB between plasmas with TC and TG levels < 200 mg/dL and those with TC and TG levels > 200 mg/dL. The data from the same set of plasmas used in Aim 1 were used in this analysis. The only exception was that of plasma VI, which was replaced with plasma containing TC=210 mg/dL and TG=237 mg/dL. Plasma distribution profiles of AmpB were compared following the incubation of Fungizone®, Abelcet® and Ambisome® within both the sets of plasmas for 60 minutes at 37°C. Figure 15 shows comparison of plasma distribution profile of AmpB (incubated as Fungizone®) between plasmas with TC and TG levels < 200 mg/dL and those with TC and TG levels > 200 mg/dL. As observed in the figure, plasmas with TC and TG levels greater than 200 mg/dL showed significantly greater percentage of AmpB recovered.from the L D L fraction which was accompanied with the proportionate decrease in the percentage of AmpB recovered from the LPDP fraction. No significant change in the percentage of AmpB recovered from the H D L and the TRL fractions was observed. 67 • T C and T G < 200 mg/dL • T C and T G > 200 mg/dL T R L LDL HDL L P D P Figure 15: Effect of elevated TC and TG on plasma distribution profile of Amp B (20 ug/mL) after incubation of Fungizone® at 37°C for 60 min. *P<0.05 vs. Normolipidemic (values are mean ± standard deviation, n=3) Normolipidemic: TC and TG < 200 mg/dL, Elevated TC and TG: TC and T G > 200 mg/dL (TC: Total cholesterol, TG: Triglycerides), TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) ND: Non detectable (below detection limit of the assay) *P<0.05 vs. L D L normolipidemic plasma, #P<0.05 vs. LPDP Normolipidemic plasma. 68 Figure 16 shows comparison of plasma distribution profile of AmpB (incubated as Abelcet ) between plasmas with TC and TG levels < 200 mg/dL and those with TC and TG levels > 200 mg/dL. No significant change in the distribution profile of AmpB was observed between the two sets of plasmas. <D 0 > O u m Q. E < 100 90 80 70 60 50 40 30 20 -10-1 ND ND 0 • TC and TG < 20o mg/dL • TC and TG > 200 mg/dL ND ND J l TRL LDL HDL L P D P Figure 16: Effect of elevated TC and TG on plasma distribution profile of AmpB (20 ug/mL) after incubation of Abelcet® at 37°C for 60 min. (values are mean ± standard deviation, n=3) Normolipidemic: TC and TG < 200 mg/dL, Elevated TC and TG: TC and T G > 200 mg/dL (TC: Total cholesterol, TG: Triglycerides), TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, HDL: High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and glycoprotein) N D : Non detectable (below detection limit of the assay) 69 Figure 17 shows comparison of plasma distribution profile of AmpB (incubated as Ambisome®) between plasmas with TC and TG levels < 200 mg/dL and those with TC and TG levels > 200 mg/dL. Similar to that of Abelcet®, no significant change in the distribution profile of AmpB was observed between the two sets of plasmas studied. T3 Qi >_ Qi > O O CQ a. E < 100 90 80 70 -j 60 50 40 30 20 10 0 • TC and TG < 200 mg/dL • TC and TG > 200 mg/dL ND TRL LDL HDL L P D P Figure 17: Effect of elevated TC and TG on plasma distribution profile of AmpB (20 ug/mL) after incubation of Ambisome® at 37°C for 60 min. (values are mean ± standard deviation, n=3) Normolipidemic: TC and TG < 200 mg/dL, Elevated TC and TG: TC and TG > 200 mg/dL (TC: Total cholesterol, TG: Triglycerides), TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and glycoprotein) ND: Non detectable (below detection limit of the assay) *P<0.05 vs. normolipidemic plasma (Values are mean ± SD, n=3) 70 3.2 Part II: To determine if changes in P L T P activity alters the plasma distribution of AmpB, when incubated within human plasma as phospholipid based formulations. 3.2.1 Aim 1: To establish an in vitro PLTP activity assay within human plasma. A commercially available kit was used for this purpose. General procedure for the assay was as described in the 'materials and methods' section. However, this assay method was characterized in terms of an acceptable plasma volume required as a PLTP source and an acceptable incubation time that will suit the set of experiments conducted in this project. 3.2.1.1 Determination of acceptable plasma volume PLTP assay was carried out in a normolipidemic plasma (TC= 167 mg/dL and TG=100 mg/dL) by a procedure described before using different plasma volumes (3 pL, 5 uL, 10 pL, 15 pL and 20 pL) as a PLTP source. Figure 18 represents PLTP activity observed using different plasma volumes. PLTP activity increased as plasma volume was increased. A plasma volume of 10 pL was chosen for the subsequent studies as it showed substantial PLTP activity and was also found easier to handle, with less pipetting errors compared to the lower volumes. 71 8000 -7000 -c 6000 --8 c >—i 5000 -<u u e 4000 -41 m 3000 -k. O 2000 • 1000 -10 15 —x— 20 25 Plasma Volume (uL) Figure 18: PLTP activity in 0, 3, 5, 10 and 20 uL of fresh human plasma after incubating it with the assay components for 30 minutes at 37°C. Data represented as mean ± SD, n = 6 3.2.1.2 Determination of acceptable incubation time Using 10 pL plasma as a PLTP source, PLTP assay was carried out using the same procedure as described before but at different incubation times (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, 50 min and 60 min). As observed in figure 19, PLTP activity was increased as the incubation time was increased. A n incubation time of 30 minutes was chosen for subsequent studies as substantial PLTP activity was observed at this time and was within the linear range of the activity curve. 72 9000 Blank 8000 -I * «— 10 uL plasma 1000 -I 0 0 10 20 30 40 50 60 70 Incubation Time (minutes) Figure 19: PLTP activity in 10 uL of fresh human plasma after incubating it with the assay components for 0, 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes at 37°C. Data represented as mean ± SD, n = 6 *P<0.05 vs. Blank. 3.2.2 Aim 2: To determine if a relationship exists between plasma P L T P activity and the percentage of AmpB recovered in the H D L fraction. 3.2.2.1 Determination of P L T P activity from human plasmas used to study AmpB distribution Using 10 uL plasma as a PLTP source and 30 min as an assay incubation time, PLTP activity was measured from six plasmas (refer table 5) that were used to study the plasma distribution profile of AmpB in Part I, Aim 1 of this project. As seen in figure 20, PLTP activity varied between different plasmas. 73 8000 -i >» 7000 to c <D 6000 •*-• "> c 5000 o 0) < o c 4000 D_ a> r -—I esc 3000 CL n 2000 jZ 1000 0 Blank II III IV Plasma Sample V VI Figure 20: PLTP activity from plasmas used (table 5) for the AmpB distribution study after incubating 10 uL of these plasmas with assay components for 30 minutes at 37°C. *P<0.05 vs. Blank. 3.2.2.2 Determination of a relationship between plasma P L T P activity and the percentage of AmpB recovered in the H D L fraction PLTP activity measured from six plasmas (table 5) was plotted against the percentage of AmpB recovered in the H D L fractions of the respective plasmas following incubation of Fungizone®, Abelcet® and Ambisome® at an AmpB concentration of 20 pg/mL for 30 min at 37°C. As observed in figure 21, following incubation of Fungizone®, there was no significant correlation observed between the two parameters studied. However, following incubation of Abelcet®, plasma with the highest PLTP activity among all showed highest percentage of AmpB recovered from the H D L fraction (figure 22) as opposed to plasma possessing the least PLTP activity which showed lowest percentage of AmpB recovered from the H D L fraction. Correlation analysis of this data showed that statistically significant positive correlation 74 existed between plasma PLTP activity and the percentage AmpB recovered in the HDL fraction. Similar analysis was conducted using the percentage AmpB recovered in the HDL fraction following incubation of Ambisome® and the plasma PLTP activity. However, no significant correlation was observed between the two parameters as observed in figure 23. _ 6000 >» •"S 5000 >. = ; | ~ 4000 I S 3000 h S 2000 0- a) o 1000 ± 0 R=0.41.NS 12 14 16 18 AmpB recovered in HDL (ug) 20 Figure 21: Correlation between PLTP activity and % AmpB recovered in the H D L fraction of plasma samples after incubation of Fungizone® (20 ug/mL) within human plasma for 30 minutes at 37°C. n = 6 NS: Statistically non significant. 75 8000 o. 5 7000 A 3? 6000 A I ~ 5000 A ? > 4000 A c w 3000 4. o> o (0 a> L _ o 3 U. 2000 -J 1000 -| 0 R=0.82 P<0.05 I 50 55 60 65 70 % AmpB Recovered in HDL 75 Figure 22: Correlation between PLTP activity and % AmpB recovered in the H D L fraction of plasma samples after incubation of Abelcet® (20 ug/mL) within human plasma for 30 minutes at 37°C. n = 6 8000 Q. j 7000 Q. J 6000 c ~ 5000 = > 4000 A a> o u ra c V u o 3 3000 2000 1000 A 0 R=0.40 NS 84 86 88 - i — 90 — i — 92 i 94 96 % AmpB Recovered in HDL Figure 23: Correlation between PLTP activity and % AmpB recovered in the H D L fraction of plasma samples after incubation of Ambisome® (20 ug/mL) within human plasma for 30 minutes at 37°C. n = 6, NS: Statistically non significant. 76 3.2.3 Aim 3: To determine i f a relationship exists between HDL-phospholipid concentration and the percentage of AmpB recovered in the H D L fraction. A commercially available kit (WAKO Chemicals, USA) was used for this purpose. The principle of this assay is as follows; Phospholipase D is allowed to react with the phospholipids in the sample, freeing choline. In the presence of choline oxidase and peroxidise, the free choline is then estimated by colorimetry, to determine phospholipid content. HDL fractions obtained following incubation of Abelcet® and Ambisome® within human plasmas for 30 minutes at 37°C during part I, Aim 1 of this project were analyzed for the phospholipid concentration by a procedure described before. Phospholipid concentration from these HDL fractions was plotted against the % AmpB recovered in the respective HDL fractions. Correlation analysis performed on this data showed that following incubation of Abelcet®, H D L fraction with the highest amount of AmpB in it showed highest amount of phospholipid content and a statistically significant positive correlation was observed between the HDL-phospholipid concentration and the % AmpB recovered in H D L (figure 24). However, no such correlation was observed between these two parameters following the incubation of Ambisome® (figure 25). 77 5 E O Q O I 2 c Q . ™ Q . (/) O 120 100 80 6 0 -40 • 20 • 0 50 R=0.92, P<0.05 i 5 55 60 65 70 % AmpB Recovered from HDL 75 Figure 24: Correlation between phospholipid concentration in H D L and % AmpB recovered in the H D L fraction after incubation of Abelcet (20 ug/mL) within human plasma for 30 minutes at 37°C. n = 6 _ 120 5 O) E h O X T5 C "5. ™ a. V) o SZ Q. 100 A 80 60 • 40 • 20 • 0 R=0.19, NS 84 i 86 - i — 88 — i — 90 92 i 94 96 % AmpB Recovered in HDL Figure 25: Correlation between phospholipid concentration in H D L and % AmpB recovered in the H D L fraction after incubation of Ambisome® (20 ug/mL) with human plasma for 30 minutes at 37°C, n = 6, NS: Statistically non significant. 78 3.2.4 Aim 4: To develop and test different methods to inhibit plasma P L T P activity. In order to inhibit the normal PLTP activity from the human plasma, three different approaches were evaluated. 3.2.4.1 Incubation of plasma with P L T P antibody Polyclonal antibody specific to PLTP was used for this purpose. Human plasma was incubated with the PLTP antibody for 24 hours at 4°C (as described in section 2.3.6.1) and PLTP activity was measured from 10 pL of this plasma. 3.2.4.2 Incubation of plasma with thimerosal Human plasma was incubated with thimerosal at 4°C for 24 hours (as described in section 2.3.6.2) and PLTP activity was measured from 10 pL of this plasma. 3.2.4.3 Heat treatment of plasma Required quantity of plasma sample was heated at 56°C for 1 hour, brought back to the room temperature and PLTP activity was measured from 10 pL of this plasma. PLTP activity observed from the three approaches was compared against that obtained from the positive control (i.e. untreated plasma) in order to measure the extent of inhibition. Figure 26 represents results obtained from this study. As observed in the figure, plasma heated at 56°C for 1 hour did show significantly less PLTP activity than that of the positive control. The extent of inhibition obtained was 80% of the positive control. However, plasma incubated with PLTP antibody as well as that incubated with the CETP antibody did not show significant inhibition in the PLTP activity when compared with the positive control. 79 Plasma incubated with thimerosal did not show significant inhibition in the PLTP activity. In addition, 10 uL of PLTP antibody was also tested to determine i f the antibody itself has any inherent PLTP activity and as observed in the figure 26, this antibody did not show any PLTP activity. These results together suggested that the only approach that was effective in inhibiting the plasma PLTP activity was heat treatment of plasma. 6000 5000 A Figure 26: PLTP activity observed in an untreated plasma vs. heated plasma, plasma incubated with PLTP antibody, plasma incubated with thimerosal and plasma incubated with CETP antibody following incubation of lOuL plasma from each of the treatment groups with assay components for 30 min at 37°C. Data represented as mean ± SD, n=6. *P<0.05 vs. untreated plasma (Blank). 80 3.2.5 Aim 5: To study the distribution profile of Abelcet® and Ambisome® in plasma heated at 56°C for 1 hour. 3.2.5.1 Experimental design Required volume of human plasma (TC=167 mg/dL, TG=100 mg/dL) was heated on a water bath at 56°C for 1 hour and cooled down to the room temperature. Abelcet® and Ambisome® at an AmpB concentration of 20 pg/mL were incubated within heat treated plasma and untreated plasma (control) for 60 minutes at 37°C. Following incubation, plasmas were separated into their lipoprotein and lipoprotein deficient fractions by density gradient ultracentrifugation and each fraction was analyzed for AmpB content by HPLC as described before. Figures 27 and 28 represent plasma distribution profiles of AmpB following incubation of Abelcet® and Ambisome® respectively in heat treated and untreated plasmas. As seen in these figures, heating of plasma did not have significant effect on the distribution profile of AmpB following incubation of Abelcet® as well as Ambisome®. 81 T3 2 > O U CQ a E < 100 90 80 70 60 50 40 30 20 10 0 ND ND ND ND i TRL LDL HDL I Control I Heated Plasma LPDP Figure 27: Effect of heat treatment of plasma on the distribution profile of AmpB (20 ug/mL) after incubation of Abelcet® within human plasma at 37°C for 60 min. TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) AmpB: Amphotericin B *P<0.05 vs. L D L (5min), #P<0.05 vs. H D L (5 min), +PO.05 vs. LPDP (5 min) using A N O V A Values are mean ± SD, n = 6 (six different plasmas) ND: Non detectable (below detection limit of the assay) 82 120 100 T3 2! > o o on m Q. E < 80 H 60 40 20 • Control O Heated Plasma TRL LDL LPDP Figure 28: Effect of heat treatment of plasma on the distribution profile of AmpB (20 ug/mL) after incubation of Ambisome® within human plasma at 37°C for 60 min. TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, HDL: High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1 -glycoprotein) AmpB: Amphotericin B *P<0.05 vs. L D L (5min), #P<0.05 vs. H D L (5 min), +PO.05 vs. LPDP (5 min) using A N O V A Values are mean ± SD, n = 6 (six different plasmas) N D : Non detectable (below detection limit of the assay) 83 Discussion 84 4. DISCUSSION The objectives of this research project were to compare the plasma distribution profile of AmpB following incubation of three commonly used formulations, Fungizone®, Abelcet® and Ambisome® and to determine i f phospholipid transfer protein (PLTP) facilitates the transfer of AmpB into high density lipoproteins (HDL) following incubation of AmpB-phospholipid complex within human plasma. 4.1 Incubation of AmpB formulations within human plasma To assess the plasma distribution of AmpB as Fungizone®, Abelcet® and Ambisome® in human plasma samples, a concentration of 20 pg of AmpB per millilitre of human plasma was chosen. In preliminary studies, while investigating plasma distribution profile of AmpB, Wasan et al. have demonstrated that AmpB's plasma distribution profile does not change significantly between AmpB concentrations of 5 to 50 pg/mL. Therefore, 20 pg/mL of AmpB concentration was chosen for the comparison purpose in this study. A l l AmpB formulations were incubated within human plasma at 37°C to simulate physiologic conditions. Following incubation, the samples were placed on ice to prevent redistribution of drug within plasma. At this temperature (0-4°C), the appearance of the lipoproteins change. It no longer exhibits a fluid like appearance, but rather is more solid (Cushley et al, 1987). Therefore, by rapidly cooling the lipoproteins from liquid to crystalline phase, AmpB becomes entrapped into its respective lipoprotein component and any transfer of drug between liporpoteins is effectively minimized. 85 4.2 Self association of AmpB and the associated stability and toxicity of AmpB formulations In aqueous suspensions of AmpB, three forms of AmpB coexist: monomers, water-soluble oligomers, and non-water-soluble aggregates (Legrand et al. 1992). Oligomers and aggregates are formed due to the ability of AmpB monomers to self associate when put into aqueous medium. When AmpB in suspension is below the threshold of AmpB self-association, toxicity against fungi is still observed but human toxic effects decline (Hartsel et al. 1996). In the current model of AmpB selective toxicity, soluble monomeric AmpB is very active toward ergosterol-containing membranes, but a self-associated oligomer damages sterol-free and cholesterol-containing membranes (Bolard et al. 1991). In addition, the oligomer seems to be more susceptible to auto-oxidation, which may enhance its toxicity (Lamy-Freund et al. 1993). After administration of Fungizone®, AmpB is released from the the carrier deoxycholate molecule and exist in the blood as monomers as well as in the self associated oligomers and aggregate form and the rate of aggregation increases strongly with dilution (Lamy-Freund et al. 1991). On the other hand, it has been shown that in vitro, the activity of liposomal or lipid complexed AmpB comes from the presence of free AmpB that is not bound to lipids (Jullien et al. 1990). It has been shown by number of studies that AmpB from liposomes as well as lipid complex is released much slower and in sustained manner (Brajtberg et al. 1996, Bhamra et al. 1997). Therefore, amount of free AmpB available after administration of Ambisome® and Abelcet® is much less compared to that of Fungizone®. In addition, due to 86 decreased availability of free AmpB from lipid based formulations there is less tendency of self association. Hence, lesser self association and therefore lesser aggregates formation could be one of the reasons behind the reduced human toxicity observed with lipid based formulations. 4.3 Physicochemical properties of AmpB formulations As discusses earlier in the introduction, AmpB is an amphoteric molecule with a carboxyl pKa of 5.5 and amino pKa of 10. Therefore ionization status of this molecule under physiological conditions may influence its plasma distribution profile. Product concentrates of Fungizone®, Abelcet® and Ambisome® have a pH of 5-7. As described earlier, after i.v. administration of Fungizone® free AmpB is released from its deoxycholate carrier. Free AmpB at a physiologic pH exist in an ionized form. Extent of ionization may influence its distribution and pharmacokinetics and may require further investigation. However, in case of lipid based formulations (Abelcet® and Ambisome®) AmpB is sequestered by lipid carriers and lesser free AmpB is available. Wasan et al. (2003) have earlier demonstrated that modifications in lipoprotein and liposomal charge may influence the distribution of drugs incorporated into liposomes. Since phospholipids specifically phosphatidyl glycerol used as carriers in Ambisome® and Abelcet® are negatively charged, it may be one of the factors responsible for the greater distribution of AmpB into HDL. 4.4 Plasma distribution of AmpB Wasan et al., have demonstrated that changes in temperature and liposomal lipid composition modify the distribution of AmpB in serum lipoproteins (Wasan et al, 1993). Due to the high affinity of AmpB for plasma lipoproteins, alterations in lipoprotein levels may have 87 significant impact on the pharmacokinetics and pharmacodynamics of this drug which could ultimately affect the pharmacological action. During the preliminary studies conducted in this project, plasma distribution profile of AmpB was examined following incubation of three different AmpB-formulations within human plasma. Results of this study and those from earlier studies conducted in our lab, have demonstrated that AmpB, when incubated within plasma as a colloidal dispersion with sodium deoxycholate (Fungizone®), distributes preferentially into the lipoprotein deficient plasma (LPDP) fraction followed by HDL, then L D L and TRL. These results are consistent with the results obtained by other researchers. Bhamra et al. (1997) have shown that following administration of Fungizone®, the majority of AmpB was associated with non-lipoprotein plasma proteins which are found in greater concentrations than lipoproteins in plasma. Brajtburg et al. (1984) demonstrated that plasma lipoproteins when separated from other proteins by sequential ultracentrifugation, only 25% of AmpB were found to be associated with plasma lipoproteins. However, when incubated as lipid based formulations, a significantly greater amount of AmpB was recovered from the HDL fraction. Following incubation of both Abelcet® and Ambisome®, the majority of AmpB was recovered in the H D L fraction followed by the LPDP and importantly, no detectable amounts of AmpB were recovered in the L D L or in the TRL fraction. Preferential distribution of AmpB into the LPDP fraction following incubation as Fungizone® may be a function of free drug available immediately after incubation (Bhamra et al, 1997). AmpB is known to be highly protein bound and more than 95% of AmpB administered intravenously as Fungizone® is known to bind with plasma proteins and lipoproteins (Bekersky et al, 2002). Previously, Bhamra et al.(1997) have shown in the in vitro studies that AmpB, when incubated within rat plasma as Fungizone®, released 88 immediately from its micellar carrier and became available as a free drug. However, sustained release of free AmpB took place from lipid based formulation (Abelcet®). Bekersky et al. (2002) have demonstrated that AmpB when administered i.v. as Fungizone®, is highly protein bound over a range of concentrations in human plasma, with human serum albumin (HAS) and alpha-1-glycoprotein being major contributors; however when administered as a small unilamellar liposomal vesicles, greater than 97% of AmpB remained associated with the liposomes after 4 hours of administration and much less free drug was available to bind with albumin and alpha-1-glycoprotein. Based on these findings, it can be speculated that preferential distribution of AmpB as Fungizone® into LPDP may be due to the greater availability of free AmpB and its. affinity for plasma proteins present in much greater numbers. However, when administered as a lipid based formulation, less free drug is available to bind to proteins. Another important observation in this study was that following incubation of lipid based formulations of AmpB within human plasma, distribution of AmpB into the HDL fraction increased significantly and the majority of the initially incubated AmpB was recovered from the HDL fraction. These results were consistent with some of the earlier findings. As discussed earlier, Wasan et al. (1990) first demonstrated that AmpB, when incorporated into multilamellar liposomes composed of DMPC: D M P G (7:3) and incubated within plasma, distributed preferentially into the H D L fraction. A liposomal formulation of annamycin (composed of D M P C and DMPG), when incubated within human plasma for 60. min at 37°C, more than 60% of the initial annamycin concentration was recovered in the HDL fraction (Wasan et al, 1996). Similarly, when liposomal nystatin was incubated within human plasma at 37°C for 5-120 min, the majority of nystatin was recovered in the H D L fraction (Cassidy et 89 al, 1997). An important observation in all three cases mentioned above was that, although the drug used in these studies was different, the type and composition of phospholipids used to encapsulate the drug were similar (i.e. DMPC/DMPG) (refer figure 29), which resulted in the hypothesis that preferential association of drug with H D L may not be a function of drug's interaction with H D L but may be a function of the phospholipids used as carriers. 1) Nystatin (1997) 2) Annamycin (1996) 3) Amphotericin B (2006) Figure 29: Schematic representation of preferential distribution of liposomal drug into H D L Wasan et al (1993) have shown that liposomal composition may affect the distribution of drug into different lipoprotein fractions. As a part of their study, AmpB incorporated into phospholipid vesicles (AmpB-phospholipid molar ratio 1:4) were incubated within human serum at 37°C for 1 h. Plasma was separated into H D L and L D L fractions and AmpB: phospholipid ratio in each fraction was determined. In the H D L fraction, 90% and 80% of the initial concentration of AmpB and phospholipids respectively, were found in a 1:3 molar ratio, while in the L D L fraction, AmpB and phospholipids were found in a 1:6 molar ratio. Since, AmpB : phospholipid molar ratio in HDL remained approximately the same as that 90 before the incubation, these results suggested that AmpB might have been co-transferred with the phospholipids used in the liposomes possibly as an intact complex. Several mechanisms have been proposed in order to explain the preferential distribution of drugs into the H D L fraction following administration/incubation within plasma as lipid based formulations. Cassidy et al. attributed their finding about preferential H D L distribution of nystatin with liposomal nystatin to the protein content (specifically Apo A) of the H D L particles as they observed decreased HDL-association of nystatin with the decreased HDL protein content. Kennedy (1999) studied the relationship between plasma distribution of AmpB as Abelcet® and coat lipid contents of the H D L particles; results of her studies suggested that the distribution of AmpB into H D L may be related to the HDL particle number, and more specifically, the H D L 3 (sub-population of HDL) particle number present in plasma. Another mechanism suggested was based on the ability of D M P G to form a thermally stable complex with Apo A l (Surewicz et al, 1986). These findings suggested that liposomes containing D M P G have an ability to target compounds to HDL. Therefore, i f the drug-DMPG complex is administered into the plasma as a liposome or as a drug-lipid complex, it may increase the distribution of the drug into the H D L fraction.. Damen et al. have done an extensive work investigating the association of phospholipids with HDL, specifically phosphatidylcholine with H D L subfractions {Damen et al, 1979, Damen et al, 1982). During their studies, it was determined that upon incubation of small unilamellar vesicle (SUV) liposomes containing D M P C within whole plasma, a rapid transfer of phospholipids occurred between SUV and HDL. This transfer of phospholipids was represented as an 91 exchange with the phospholipids of HDL rather than as a net transfer (Damen et al, 1982). Scherphof et al. have speculated that this exchange of phosphatidylcholine between SUV's and HDL may involve lipid transfer protein and the outer monolayer of the liposomal membranes. PLTP has an ability to transfer phospholipids in the plasma to H D L and therefore may have a significant potential to transfer drugs complexed with these carrier phospholipids to HDL. With the exception of HDL, all other lipoproteins are synthesized intracellularly. Chylomicrons, V L D L and L D L are synthesized within intestinal or hepatic cells and then secreted into the systemic circulation. HDL particles are synthesized extracellularly, which gives an opportunity for liposomal phosphatidylcholine to be incorporated into the membrane bilayer of the newly formed nascent H D L particles with the help of PLTP (Eisenberg, et al. 1984, Eisenberg et al, 1972,). So, i f the phosphatidylcholine is complexed with the drug molecule as in case of AmpB in Abelcet®, that molecule may also be incorporated into the HDL particle with the help of PLTP. Hence, we hypothesized that PLTP may play an important role in co-transferring AmpB complexed with the phospholipids to HDL and therefore, may promote its distribution into HDL. 4.5 Effect of elevated T C and T G levels on plasma distribution of AmpB Number of studies have shown that elevation in the L D L cholesterol levels of plasma is associated with the higher AmpB-induced nephrotoxicity. Koldin et al. (1985) have shown that LDL-associated AmpB, when administered to rabbits, increases the nephrotoxicity as compared to free AmpB. In another study, Barwicz et al. (1991) have shown decreased toxicity of AmpB in mice after inhibition of the AmpB-LDL interaction. Wasan et al. have 92 demonstrated the receptor mediated pathway for AmpB-induced nephrotoxicity as discussed earlier. As plasma samples used in this study were obtained from six different individuals varying in their lipid profile (total cholesterol and total triglyceride concentrations), these were divided into two sets, one containing TC and TG > 200 mg/dL and another containing TC and TG < 200 mg/dL. Distribution profile of AmpB was studied separately in the two sets of plasmas in order to determine the effect of variation in TC and T G levels. Among the three formulations tested (Fungizone®, Abelcet® and Ambisome®), Fungizone® was the only formulation that showed significant difference in the distribution profile of AmpB between two sets of plasmas. Plasma with elevated TC and T G levels showed 3-4 fold increase in the distribution of AmpB into L D L , which may increase the nephrotoxicity associated with Fungizone®, as more drug may be internalised into the cells via L D L receptors. However, AmpB's distribution profile remained the same following incubation of Abelcet® as well as Ambisome® irrespective of plasma TC and TG levels. Cholesteryl ester transfer protein (CETP) observed in human plasma is involved in the transfer of neutral lipids (cholesteryl ester and triacylglycerols) from H D L to L D L (Guyard et al, 1998). Wasan et al. (1994) have demonstrated that the presence of CETP in plasma facilitates the transfer of AmpB from H D L to L D L as it was observed that addition of CETP to plasma resulted in an increased distribution of AmpB into L D L following incubation of Fungizone®. Thus, the distribution of AmpB into L D L observed following incubation of Fungizone® may be related to CETP-facilitated distribution of cholesteryl ester (CE) between HDL and Apo B containing lipoproteins. In the same study, Wasan et al. further observed that addition of CETP did not facilitate the distribution of AmpB into L D L when AmpB was incorporated into negatively or positively charged liposomes composed of D M P C and D M P G 93 (7:3). Presence of these liposomes actually decreased CETP regulated transfer of CE from HDL to L D L . Desrumaux et al. (1999) have reported increase in mass and activity of CETP in case of hyperlipidemic patients (Desrumaux et al, 1999). Taken together, these findings suggest that the greater amount of AmpB recovered from the L D L fraction following incubation of Fungizone® within plasmas with higher TC and T G levels may be due to increased mass and activity of CETP in these plasmas. 4.6 Establishment and characterization of P L T P assay To analyze the PLTP activity in plasma, a commercially available kit was used. The assay method was based on the ability of PLTP to mediate the transfer of phospholipids from donor particles to acceptor particles. Donor molecules were made up of pyrene-labelled phospholipid vesicles where fluorescent molecule was in a self-quenched state and acceptor molecules were made up of HDL particles. Addition of plasma as a PLTP source to the mixture containing donor and acceptor resulted in the transfer of pyrene labelled phospholipids from donor to acceptor. Unquenching of fluorescent particles took place at the acceptor which was measured in terms of increase in the fluorescence intensity. Methods used in some of the previous studies used to involve donor containing radiolabeled phospholipids, and the appearance of radiolabel into an acceptor lipoprotein used to be measured (Damen et al, 1982). However, these methods require separation of donor and acceptor lipoproteins which can generally be achieved by precipitation assays (Damen et al, 1982) and are time consuming. In addition, alterations in lipoprotein composition can affect their behaviour in precipitation assays. The fluorescence based assay used in this project does not require separation of donor and acceptor and is significantly less time consuming. 94 General procedure for the PLTP assay, provided by manufacturer of the assay kit recommended a plasma volume of 3 pL as a PLTP source and incubation time of 8-20 minutes. However plasma volume of 3 uL was found inconvenient to handle with too many pipetting errors when the number of samples was large. In addition, considering subsequent set of studies in this project, PLTP activity beyond 20 minutes of incubation was required to be measured. Therefore, assay method was characterized in order to determine the acceptable plasma volume as a PLTP source and the acceptable incubation time for subsequent studies in this project. From the results obtained in characterization study, a plasma volume of 10 pL was chosen for subsequent studies as it showed substantial PLTP activity and was convenient in terms of handling and pipetting. Similarly, an incubation time of 30 minutes was chosen as substantial PLTP activity was observed at this time against that of blank sample which also lied on the linear part of the PLTP activity curve as observed in figure 17. In addition, this incubation time was found convenient when the relationship between plasma PLTP activity and AmpB distribution into the HDL fraction was determined in the subsequent studies. 4.7 Relationship between plasma P L T P activity and the percentage of AmpB recovered in the H D L fraction PLTP activity in plasmas that were used to study the AmpB distribution profile was measured by PLTP assay. As observed in figure 18, PLTP activity varied significantly between different plasmas. To evaluate whether PLTP is responsible for the transfer of AmpB into H D L when incubated within plasma as a lipid based formulation, relationship between the plasma PLTP activity and the percentage of AmpB recovered in the HDL fractions of the respective plasmas was studied. A significant positive correlation was observed between the plasma PLTP activity and the percentage of AmpB recovered from the 95 HDL fractions of Abelcet incubated plasmas but not in case of Ambisome incubated plasmas. This result was an indication of PLTP's role in the transfer of AmpB into HDL following incubation of Abelcet® within human plasma. In order to determine whether an ability of PLTP to transfer phospholipids to HDL is responsible for the transfer of drug complexed with the phospholipids, relationship between the phospholipid concentration in the HDL fractions and the percentage of AmpB recovered from the H D L fractions was also studied. Results showed a positive correlation between the HDL-phospholipid concentration and the percentage of AmpB recovered from the H D L fractions of Abelcet® incubated plasmas. No correlation was observed between these two parameters following incubation of Ambisome®. As described earlier, Abelcet® and Ambisome® are fundamentally two completely different formulations. In case of Abelcet, AmpB is complexed with the phospholipid carrier; therefore according to the hypothesis, PLTP may co-transfer AmpB-phospholipid complex into HDL which was reflected in the correlation analysis done. However, Ambisome® is a liposomal formulation and the orientation or arrangement of AmpB within phospholipid carriers may be completely different than that of Abelcet®. AmpB is incorporated within phospholipid bilayer and may not be complexed with these phospholipids as that of Abelcet®. Therefore, an ability of PLTP to co-transfer AmpB along with phospholipids may not be responsible for transfer of AmpB into H D L in this case. In addition, cholesterol present in Ambisome® formulation increases the packing density of phospholipid bilayers, increases their rigidity and reduces membrane permeability which makes these liposomes resistant to temperature and other proteins present in the plasma. This greater resistance of liposomes may also be preventing PLTP to bind to phospholipids in case of Ambsiome®. Therefore, greater H D L distribution of 96 AmpB observed following incubation of Ambisome® may not be a function of PLTP activity. Taken together, these results supported our hypothesis that PLTP may facilitate the transfer of AmpB along with the carrier phospholipids into HDL and therefore may be responsible for the preferential HDL-distribution of AmpB following incubation of Abelcet® within human plasma. However, recognizing the limitations of the correlation analysis, it was difficult to draw any concrete conclusion considering the limited sample size. Therefore, these results were considered only as indirect evidence of PLTP's role in the preferential distribution of AmpB into HDL. In order to obtain direct evidence, the strategy that we proposed was to inhibit the normal PLTP activity from human plasma and then study the distribution profile of AmpB following incubation of Abelcet® within PLTP-inhibited plasma. Three different approaches were evaluated to inhibit the PLTP activity from human plasma. In the first approach, polyclonal antibody specific to PLTP was used. This antibody was raised in rabbit plasma and was unpurified. It has previously been used in the studies to quantify PLTP protein concentration (Nishida et al, 1997). However, its ability to inhibit the PLTP activity was never tested. Due to commercial unavailability of the PLTP neutralizing antibody, this antibody was studied to observe i f it has any PLTP neutralizing capacity. Since the antibody was unpurified, different dilutions with the PLTP assay buffer were prepared and tested for the PLTP neutralizing capacity in the preliminary studies (data not shown). None of the dilutions showed statistically significant inhibition in the PLTP activity. However, incubation time between plasma and the antibody was very short (30 minutes) during these studies. Therefore, a 97 dilution of 1:100 was chosen and incubated within plasma for 24 hours in order to give sufficient time for the antibody to react with the PLTP. In the second approach that was evaluated, thimerosal (ethylmercurithiosalicylate) was used as a chemical inhibitor to inhibit the PLTP activity from human plasma. Some of the studies have previously shown that thimerosal may covalently modify either of the two free cysteine residues of PLTP which are responsible for the structural integrity and activity of PLTP (Huuskonen et al, 2000a). However, the concentration of thimerosal required to inhibit PLTP activity during these studies was very high (100 mM) which was not suitable for the set of studies conducted during this project. Therefore, a lower concentration of thimerosal (10 mM) was picked and incubated with human plasma to study if it has an ability to inhibit the PLTP activity. The third approach evaluated was heating of plasma at 56°C for 1 hour as studies have shown that heating plasma at these conditions leads to substantial loss of PLTP activity (Desrumaux et al, 2004). Among the different approaches evaluated, this was the only approach that did show significant inhibition in the plasma PLTP activity compared to the positive control. Distribution study carried out following incubation of Abelcet® and Ambisome® in heat treated plasma did not show significant difference in AmpB's distribution profile. However, it is difficult to derive any final conclusion based on this result as heating of plasma at 56 °C may not only inhibit the PLTP activity but it may also denature other proteins and lipoproteins present in the plasma. Therefore, this approach may not be appropriate for the set of studies conducted in this project. 98 Incubation of P L T P antibody as well as thimerosal within human plasma did not show any inhibition in the P L T P activity which indicated that P L T P antibody used may not have ability to inhibit phospholipid transfer due to P L T P and concentration of thimerosal used may not be sufficient to inhibit plasma P L T P activity. 4.8 Limitations of the study and future directions Plasma distribution study of A m p B carried out using Abelcet® and Ambisome® may have limited applicability due to the fact that Abelcet® and Ambisome® are utilized only as a second resort therapy due to the high cost associated with these formulations. Clinical trial data in which l ipid and lipoprotein profile of patients administered Abelcet® and Ambisome® were measured would be very informative in generating in vitro - in vivo correlation of the results obtained during this study. In addition, comprehensive economic analyses are required that w i l l look beyond drug acquisition expenses and include hospital costs and convenience because of the excess toxicity relating to the use of AmpB-deoxycholate. Correlation analysis between the P L T P activity and the percentage of A m p B recovered in the H D L fractions of Abelcet® incubated plasma did show a positive correlation between these two parameters. However, these results were considered only as indirect evidence of P L T P ' s role in the preferential distribution of A m p B into H D L because of the limited sample size available, as elimination of single sample from this data-analysis may significantly alter the outcomes. Having a larger sample size would significantly increase the importance of this data but may increase the cost of experiments. 99 Even though heating of plasma at 56°C for 1 hour did show inhibition in the plasma PLTP activity, it could not be used to study the drug distribution profile of AmpB due to limitations associated as discussed. Therefore, future studies in order to inhibit plasma PLTP activity may involve immunoprecipitation of PLTP from the plasma wherein an attempt will be made to deplete the plasma of PLTP by immunoprecipitation. This PLTP-depleted plasma will be used to study the distribution profile of AmpB following incubation of Abelcet®. If the results of this study show significantly less distribution of AmpB into H D L compared to the control (AmpB distribution in untreated plasma), then the next step will be to supplement PLTP-depleted plasma with purified PLTP and study the plasma distribution of AmpB again into it. Alternatively, PLTP antibody having an ability to inhibit PLTP activity may be developed in the lab. 4.9 Clinical significance The determination of pharmacokinetic and pharmacodynamics parameters is generally carried out in animal models or non diseased healthy human volunteers. This information is then extrapolated to calculate dose in the diseased population. However this information may not be accurate as any physiologic changes which may occur in diseased patients may not be present in healthy individuals or animals. For some drugs, dose that is reported to be non toxic in healthy volunteers or animals may be toxic in diseased patients (Roland M . , 1995). Disease states like cancer, AIDS, diabetes may alter plasma lipoprotein profile. Number of compounds like AmpB, nystatin, cyclosporine associate with lipoproteins as discussed earlier and are frequently administered to patients showing abnormal lipoprotein profiles (Roland M . , 1995) which may alter the toxicity and pharmacological activity of these drugs. Therefore, plasma distribution profile of AmpB studied in this project following incubation 100 of three different formulations within human plasmas having a wide range of TC and TG contents may provide useful information for a clinician in order to determine optimal dose of AmpB depending on the lipid profile of the patient. Results obtained from the distribution study may suggest that lipid-based formulations can be considered safer than conventional formulation (Fungizone®) in patients with elevated TC and T G levels (e.g. hyperlipidemic patients). Thus, introduction of lipid based formulations of AmpB with decreased toxicity and improved therapeutic index compared to Fungizone® has expanded the options available for clinicians and patients requiring treatment of invasive fungal infections. These preparations present an attractive option for patients requiring high or potentially nephrotoxic cumulative dose of AmpB. Results obtained from the PLTP activity measurement studies in part II of this project, provided indirect evidence of PLTP's involvement in the transfer of AmpB into HDL following incubation of Abelcet® within human plasma. Direct evidence that will support these results may provide a breakthrough in the area of research about understanding the reduced toxicity of lipid-based formulations of AmpB. 101 Conclusions 102 5. CONCLUSIONS Plasma lipoprotein distribution study demonstrated that A m p B associates with the lipoprotein as well as non-lipoprotein fractions of plasma. Association with individual lipoproteins varied depending on the type of formulation used and therefore efficacy and the toxicity potential of A m p B may depend on the type of formulation. Lipid-based formulations o f A m p B (Abelcet® and Ambisome®) showed significantly greater distribution of A m p B into H D L and reduced distribution into L D L compared to the conventional formulation (Fungizone®) which could be one of the reasons behind the reduced nephrotoxicity of l ipid based AmpB-formulations as discussed earlier. Elevation in the T C and T G levels of plasma altered the distribution profile of A m p B following incubation of Fungizone® but not following incubation of Abelcet® or Ambisome®. Hence, l ipid based formulations of A m p B may be preferred over Fungizone® in case of patients having an elevated l ipid profile (e.g. hyperlipidemic patients). A positive correlation was reported between plasma P L T P activity and the percentage of A m p B recovered in the H D L fraction which suggested indirect evidence of P L T P ' s role in the preferential distribution o f A m p B into H D L following incubation o f Abelcet®. In addition a positive correlation between phospholipid concentration in the H D L and the percentage of A m p B recovered in the H D L was observed. Taken together, these findings suggest that P L T P may co-transfer A m p B along with the phospholipids into the H D L when A m p B is incubated within human plasma as Abelcet®. 103 Among the different methods evaluated to inhibit plasma PLTP activity, heat treatment of plasma was the only approach that showed significant (80%) inhibition in the plasma PLTP activity. However, due to limitations associated with this approach (i.e. non-specific denaturing of other plasma proteins and enzymes), it was not suitable for the set of studies conducted in this project. 104 APPENDICES 105 APPENDIX A Representative table showing the method used to calculate the amount of AmpB recovered in each lipoprotein fraction, total AmpB recovered in the plasma and percentage recovery. Data represented is after incubation of Fungizone (20 pg/mL) with six different plasmas at 37°C/30 minutes. Plasma Sample Fraction Volume (mL) (A) Concentration fraction (ug/m external cal ( of AmpB in each L) obtained from ibration curve B) Total amount of AmpB in fraction (ng). (A x B) Total AmpB recovered (a+b+c+d) = e AmpB incubated (ng) % Recovery (e x 100)/60 TRL LDL HDL LPDP TRL LDL HDL LPDP TRL (a) LDL (b) HDL (c) LPD (d) Plasma I 1.4 2.5 4.9 3 1.0 1.1 5.0 8.6 1.4 2.7 24.5 25.9 54.5 60 90.9 Plasma II 1.5 2.5 4.9 3 1.0 1.0 5 8.8 1.5 2.6 24.2 26.3 54.6 60 90.9 Plasma III 1.4 2.3 5.1 2.9 1.4 0.4 5 7.3 2 1 25.5 20.8 49.3 60 82.1 Plasma IV 1.4 2.2 5.2 2.9 1.1 0.6 5.4 7.1 1.6 1.3 27.6 20.7 51.3 60 85.4 Plasma V 1.4 2.1 5.3 2.9 0.8 0.3 4.8 10.8 1.1 0.7 25.2 31.4 58.4 60 97.4 Plasma VI 1.4 2.6 5.3 2.9 0.8 0.3 4.9 9.9 1.2 0.6 25.6 29.1 56.4 60 94.0 Mean (n=6) 1.4 2.3 5.1 2.9 1.0 0.6 5.0 8.8 1.5 1.5 25.5 25.7 54.1 60 90.1 SD 0.1 0.2 0.2 0.1 0.2 0.3 0.2 1.4 0.3 0.9 1.2 4.3 3.3 0 5.6 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoprotein (comprising chylomicron and very low density lipoproteins), L D L : Low density lipoproteins, HDL: High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and glycoprotein). N D : Non detectable (below detection limit of the assay), SD: standard deviation. AmpB: Amphotericin B. 106 APPENDIX B Preparation and composition of density solutions employed for separation of plasma components into lipoprotein and lipoprotein-deficient fractions by gradient ultracentrifugation. Solution Density Composition Amount 1.006 g/mL Sodium chloride 11.4g 1M Sodium hydroxide 1 mL Water qslOOOmL 1.478 g/mL Sodium bromide 195.8 g 1.006 g/mL density solution 250 mL 1.063 g/mL 1.006 density solution 400 mL 1.478 density solution 55 mL 1.210 g/mL 1.006 density solution 200 mL 1.478 density solution 152.2 mL 107 APPENDIX C Distribution profile of AmpB using three different formulations with respect to time Fungizone®: Plasma distribution profile of Amphotericin B (20pg/mL) following incubation of Fungizone® in six human donor plasma samples for 5 minutes at 37°C Plasma Sample Amount of AmpB recovered (Mg) Amount Incubated (MS)' Total recovery (Mg) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) 1.8 10.8 9.4 34.9 60 56.8 94.7 Plasma II (TC=217 mg/dL, TG=302 mg/dL) 1.7 9.4 11.6 35.1 60 57.8 96.4 Plasma III (TC=186mg/dL, TG=127 mg/dL) 1.4 6 15.1 29.2 60 51.7 86.1 Plasma IV (TC=178mg/dL, TG=147 mg/dL) 1.4 5.5 14.2 29.6 60 50.8 84.6 Plasma V (TC=176mg/dL, TG=122 mg/dL) 0.9 4.9 13.9 48 60 67.8 112.9 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) 1.1 4.7 10.3 49.6 60 65.8 109.5 Mean (n=6) 1.4 6.9 12.4 37.8 60 58.4 97.4 SD 0.4 2.5 5.8 8.9 N A 7.1 11.7 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) N A : Not applicable. 108 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Fungizone in six human donor plasma samples for 30 minutes at 37°C Plasma Sample Amount of AmpB recovered (US) Amount Incubated (Hg) Total recovery (ug) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) 1.5 2.7 24.5 25.9 60 54.5 90.9 Plasma II (TC=217 mg/dL, TG=302 mg/dL) 1.4 2.6 24.2 26.3 60 54.5 90.9 Plasma III (TC=186mg/dL, TG=127 mg/dL) 2 1 25.5 20.8 60 49.3 82.1 Plasma IV (TC=178mg/dL, TG=147 mg/dL) 1.6 1.3 27.7 20.7 60 51.3 85.4 Plasma V (TC=176mg/dL, TG=122 mg/dL) 1.1 0.7 25.2 31.4 60 58.4 97.4 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) 1.2 0.6 25.6 29.1 60 56.4 94.0 Mean (n=6) 1.5 1.5 25.4 25.7 60 54.1 90.1 SD 0.3 0.9 1.2 4.3 N A 3.3 5.6 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) N A : Not applicable. 109 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Fungizone in six human donor plasma samples for 60 minutes at 37°C Plasma Sample Amount of AmpB recovered (ng) Amount Incubated (pg) Total recovery (ng) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) 3.8 5.0 13.1 18.2 60 40.0 66.7 Plasma II (TC=217 mg/dL, TG=302 mg/dL) 3.5 6.8 12.4 18.9 60 41.6 69.4 Plasma III (TC=186mg/dL, TG=127 mg/dL) 4.7 1.9 15.4 27.3 60 49.3 82.1 Plasma IV (TC=178mg/dL, TG=147 mg/dL) 6.5 1.7 19.5 28 60 55.7 92.9 Plasma V (TC=176mg/dL, TG=122 mg/dL) 4.8 1.6 17.9 28.8 60 53.2 88.7 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) 4.5 1.8 14.9 22.8 60 44.1 73.5 Mean (n=6) 4.7 3.4 15.6 24.2 60 48 79.9 SD 1.2 2.41 3.1 5.2 N A 6.9 11.6 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) N A : Not applicable. 110 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Fungizone in six human donor plasma samples for 120 minutes at 37°C Plasma Sample Amount of AmpB recovered (Mg) Amount Incubated (Mg) Total recovery (Mg) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) 3.6 6.5 16.5 21.7 60 48.2 80.3 Plasma II (TC=217 mg/dL, TG=302 mg/dL) 3.07 6.7 16.7 16 60 42.5 70.8 Plasma III (TC=186mg/dL, TG=127 mg/dL) 3.6 10.8 11.9 27:0 60 53.3 88.8 Plasma IV (TC=178mg/dL, TG=147 mg/dL) 3.5 8.9 11.9 23.4 60 47.7 79.5 Plasma V (TC=176mg/dL, TG=122 mg/dL) 3.9 5.2 26 21.3 60 56.4 94.1 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) 3.5 4.9 22.9 25.8 60 57.2 95.3 Mean (n=6) 3.5 7.2 17.7 22.5 60 50.9 84.8 SD 0.3 2.3 5.7 3.9 N A 5.7 9.6 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) N A : Not applicable. I l l Abelcet : Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Abelcet® in six human donor plasma samples for 5 minutes at 37°C Plasma Sample Amount of AmpB recovered (Mg) Amount Incubated (Mg) Total recovery (Mg) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) N D N D 52.8 8.7 60 61.6 102.6 Plasma II (TC=217 mg/dL, TG=302 mg/dL) N D N D 44.3 11.6 60 55.8 93.1 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D N D 46.1 15.03 60 61.1 101.9 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D N D 34.8 10.6 60 45.4 81.4 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D N D 47.0 13.5 60 60.5 100.9 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D N D 51.3 11.2 60 62.5 104.1 Mean (n=6) ND ND 46.0 11.8 60 57.8 97.3 SD N A N A 6.4 2.2 N A 5.2 8.7 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 112 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Abelcet in six human donor plasma samples for 30 minutes at 37°C Plasma Sample Amount of AmpB recovered (Mg) Amount Incubated (Mg) Total recovery (Mg) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) N D N D 32.1 14.5 60 46.7 77.8 Plasma II (TC=217 mg/dL, TG=302 mg/dL) N D N D 33 12.7 60 45.7 76.1 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D N D 28.9 20.7 60 49.6 82.7 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D N D 31.4 14.6 60 46.0 76.7 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D N D 24.8 22.7 60 47.5 79.1 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D N D 28.9 20.7 60 49.6 82.7 Mean (n=6) ND ND 29.2 18.6 60 47.9 79.2 SD N A N A 2.9 3.8 N A 1.7 2.9 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 113 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Abelcet in six human donor plasma samples for 60 minutes at 37°C Plasma Sample Amount of AmpB recovered (ng) Amount Incubated (ng) Total recovery (ng) % Recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) N D N D 34.1 14.5 60 48.6 81.0 Plasma II (TC=217 mg/dL, TG=302 mg/dL) N D N D 38.4 14.5 60 53 88.3 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D N D 32.2 8.5 60 40.7 67.8 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D N D 44.3 10 60 54.2 90.4 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D N D 43.9 11.4 60 55.3 92.2 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D N D 39.9 11.1 60 51.0 85.0 Mean (n=6) ND ND 38.8 11.7 60 50.5 84.1 SD N A N A 4.9 2.4 N A 5.3 8.9 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 114 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Abelcet in six human donor plasma samples for 120 minutes at 37°C Plasma Sample Amount of AmpB recovered (Hg) Amount Incubated (Hg) Total recovery (Hg) % Recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) N D N D 28.1 21.6 60 49.7 82.8 Plasma II (TC=217 mg/dL, TG=302 mg/dL) N D N D 31 17 60 48 79.9 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D N D 25.3 19.5 60 44.8 74.7 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D N D 29.7 22.8 60 52.5 87.5 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D N D 25.5 23.7 60 49.2 81.9 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D N D 27.9 19.9 60 47.8 79.7 Mean (n=6) ND ND 27.9 20.9 60 48.8 81.3 SD N A N A 2.5 2.7 N A 2.7 4.7 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoproteins (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha -1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 115 Ambisome : Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Ambisome in six human donor plasma samples for 5 minutes at 37°C Plasma Sample Amount of A ( mpB recovered tig) Amount Incubated (ng) Total recovery (ng) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) N D 1.5 42.7 4.9 60 49.2 81.9 Plasma II (TC=217 mg/dL, TG=302 mg/dL) N D 1.5 42.4 5.2 60 49.1 81.8 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D 0.5 40.3 5.0 60 45.8 76.3 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D 1 42.4 4.9 60 48.2 80.4 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D 0.9 45.3 3.4 60 49.6 82.7 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D 0.8 44.0 3.4 60 48.2 80.5 Mean (n=6) ND 1.0 42.9 4.5 60 48.4 80.6 SD N A 0.4 1.7 0.8 N A 1.4 2.3 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 116 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Ambisome in six human donor plasma samples for 30 minutes at 37°C Plasma Sample Amount of AmpB recovered (Hg) Amount Incubated (Hg) Total recovery (Hg) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) N D 2.6 38.5 4.4 60 45.5 75.9 Plasma II (TC=217 mg/dL, TG=302 mg/dL) N D 2.7 42.0 3.9 60 48.6 81.0 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D 1.5 39.3 2.6 60 43.4 72.4 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D 1.3 38.6 5.5 60 45.4 75.7 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D 1.2 45.3 2.6 60 49.1 81.9 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D N D 43.7 2.7 60 46.4 77.4 Mean (n=6) ND 1.6 41.2 3.6 60 46.4 77.4 SD N A 1.0 2.9 1.2 N A 2.1 3.6 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 117 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Ambisome in six human donor plasma samples for 60 minutes at 37°C Plasma Sample Amount of AmpB recovered (ns) Amount Incubated (ng) Total recovery (ng) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) 0.4 1.1 51.5 3.0 60 56.0 93.5 Plasma II (TC=217 mg/dL, TG=302 mg/dL) 0.4 1.2 49 3.1 60 53.7 89.6 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D 0.5 51.8 3.4 60 55.7 92.7 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D 0.9 50.2 3.9 60 55 91.6 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D 1.5 51.3 3.5 60 56.3 93.9 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D 1.1 49.5 5.9 60 56.6 94.3 Mean (n=6) 0.1 1.1 50.5 3.8 60 55.9 93.1 SD 0.2 0.2 1.2 0.9 N A 1.1 1.8 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 118 Plasma distribution profile of Amphotericin B (20pg/mL) after an incubation of Ambisome in six human donor plasma samples for 120 minutes at 37°C Plasma Sample Amount of AmpB recovered (Hg) Amount Incubated (Hg) Total recovery (Hg) % recovery T R L L D L H D L LPDP Plasma I (TC=332 mg/dL, TG=583 mg/dL) N D 2.2 52.4 5.2 60 59.8 99.6 Plasma II (TC=217 mg/dL, TG=302 mg/dL) N D 2.0 53.2 5.3 60 60.5 100.8 Plasma III (TC=186mg/dL, TG=127 mg/dL) N D 1.7 49.9 5.5 60 57.1 95.1 Plasma IV (TC=178mg/dL, TG=147 mg/dL) N D 1.3 51.4 4.5 60 57.2 95.4 Plasma V (TC=176mg/dL, TG=122 mg/dL) N D 1.3 55.6 4.8 60 61.8 102.9 Plasma VI (TC=62 mg/dL, TG=58 mg/dL) N D 1.8 51.2 4.6 60 57.6 96.0 Mean (n=6) ND 1.7 52.3 5 60 59 98.3 SD N A 0.3 2 0.4 N A 2.0 3.3 Data represented as n = 1 in each of the six different plasmas. TC: Total cholesterol, TG: Total triglycerides, TRL: Triglyceride rich lipoprotein (comprising chylomicrons and very low density lipoproteins), L D L : Low density lipoproteins, H D L : High density lipoproteins, LPDP: Lipoprotein deficient plasma (comprising plasma proteins mainly albumin and alpha-1-glycoprotein) ND: Non detectable (below detection limit of the assay) N A : Not applicable. 119 References 120 R E F E R E N C E S Adler-Moore, J. (1994). "Ambisome targeting to fungal infections." Bone Marrow Transplant 14 Suppl 5: S3-7. Adler-Moore, J. and R. T. Proffitt (2002). "AmBisome: liposomal formulation, structure, mechanism of action and pre-clinical experience." J Antimicrob Chemother 49 Suppl 1:21-30. Aher I. M . , S. G. (1977). Amphotericin B. Analytical Profiles of Drug Substances Florey K. New York, N Y , Academic Press. 6: 1-42. Albers, J. J., G. Wolfbauer, et al. (1995). "Functional expression of human and mouse plasma phospholipid transfer protein: effect of recombinant and plasma PLTP on H D L subspecies." Biochim Biophys Acta 1258(1): 27-34. Albers, J. J., J. H . Tollefson, et al. (1984). "Isolation and characterization of human plasma lipid transfer proteins." Arteriosclerosis 4(1): 49-58. Arikan, S. and J. H . Rex (2001). "Lipid-based antifungal agents: current status." Curr Pharm Des7(5): 393-415. Baas, B., K. Kindt, et al. (1999). "Activity and kinetics of dissociation and transfer of amphotericin B from a novel delivery form." A A P S PharmSci 1(3): E10. Babiak, J. and L. L. Rudel (1987). "Lipoproteins and atherosclerosis." Baillieres Clin Endocrinol Metab 1(3): 515-50. Bailey, E. M . , D. J. Krakovsky, et al. (1990). "The triazole antifungal agents: a review of itraconazole and fluconazole." Pharmacotherapy 10(2): 146-53. Barwicz, J., R. Gareau, et al. (1991). "Inhibition of the interaction between lipoproteins and amphotericin B by some delivery systems." Biochem Biophys Res Commun 181(2): 722-8. • Bekersky R. M . , F. D. E., Dressier J. W., Lee D. N . , Buell, WalshT. J., (2002). "Plasma protein binding of amphotericin B and pharmacokinetics of bound versus unbound amphotericin B after administration of intravenous liposomal amphotericin B (AmBisome) and amphotericin B deoxycholate." Antimicrob Agents Chemother 46: 834-40. Bennett, W. M . (1990). "Renal effects of cyclosporine." J A m Acad Dermatol 23(6 Pt 2): 1280-5; discussion 1285-7. Bhamra, R., A . Sa'ad, et al. (1997). "Behavior of amphotericin B lipid complex in plasma in vitro and in the circulation of rats." Antimicrob Agents Chemother 41(5): 886-92. 121 Bickel, M . H. (1975). "Binding of chlorpromazine and imipramine to red cells, albumin, lipoproteins and other blood components." J Pharm Pharmacol 27(10): 733-8. Bolard, J., P. Legrand, et al. (1991). "One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium." Biochemistry 30(23): 5707-15. Brajtburg, J. and J. Bolard (1996). "Carrier effects on biological activity of amphotericin B." Clin Microbiol Rev 9(4): 512-31. Brajtburg, J., S. Elberg, et al. (1985). "Involvement of oxidative damage in erythrocyte lysis induced by amphotericin B." Antimicrob Agents Chemother 27(2): 172-6. Brajtburg, J., W. G. Powderly, et al. (1990). "Amphotericin B : current understanding of mechanisms of action." Antimicrob Agents Chemother 34(2): 183-8. Cassidy, S. M . , F. W. Strobel, et al. (1998). "Plasma lipoprotein distribution of liposomal nystatin is influenced by protein content of high-density lipoproteins." Antimicrob Agents Chemother 42(8): 1878-88. Cenni, B., J. Meyer, et al. (1995). "The antimalarial drug halofantrine is bound mainly to low and high density lipoproteins in human serum." Br J Clin Pharmacol 39(5): 519-26. Chia, J. K. and E. J. McManus (1990). "In vitro tumor necrosis factor induction assay for analysis of febrile toxicity associated with amphotericin B preparations." Antimicrob Agents Chemother 34(5): 906-8. CrestaniM., D. F., Caruso D., Mitro N . , Gilardi F., Vigi l Chacon A . B . , Patelli R., Godio C , Galli G., (2004). " L X R (liver X receptor) and HNF-4 (hepatocyte nuclear factor-4): key regulators in reverse cholesterol transport." Biochem Soc Trans 32: 92-96. Cushley R. J., T. W. D., Parmar Y . I., Chana R. S., Fenske D. B., (1987). "Surface diffusion in human serum lipoproteins." Biochem Biophys Res Commun 146: 1139-45. Damen J., R. J., Scherphof G., (1982). "Transfer of [14C]phosphatidylcholine between liposomes and human plasma high density lipoprotein. Partial purification of a transfer-stimulating plasma factor using a rapid transfer assay." Biochim Biophys Acta 712: 444-52 DamenJ., W. M . , Scherphof G., (1979). "The in vitro transfer of [14C]dimyristoylphosphatidylcholine from liposomes to subfractions of human plasma high density lipoproteins as resolved by isoelectric focusing." FEBS Lett 105: 115-19. Danon, A . and Z. Chen (1979). "Binding of imipramine to plasma proteins: effect of hyperlipoproteinemia." Clin Pharmacol Ther 25(3): 316-21. 122 Davis R. A. , V . J. E. (1996). Structure, assembly and secretion of lipoproteins.. Biochemistry of lipids, lipoproteins and membranes. V . J. E. Vance D. E. New York, N Y , Elsevier, 473-493. Day J. R., A . J. J., Lofton C. E., Gilbert T. L., Ching A . F., Grant F. J., (1994). "Complete cDNA encoding human phospholipid transfer protein from human endothelial cells." J Biol Chem 269(12): 9388-91. Desrumaux C , A . A. , Bessede G., Verges B., Farnier M . , Persegol L. , Gambert P., Lagrost L. , (1999). "Mass Concentration of Plasma Phospholipid TransferProtein in Normolipidemic, Type Ha Hyperlipidemic, Type lib Hyperlipidemic, and Non-Insulin-Dependent Diabetic Subjects as Measured by a Specific ELISA." Arterioscler Thromb Vase Biol. 19: 266-75 Desrumaux C. M . , M . P. A . , Boisvert W. A. , Masson D., (2004). "Phospholipid transfer protein is present in human atherosclerotic lesions and is expressed by macrophages and foam cells.." J. Lip. Res. 44: 1453-61 Douglas S. (1999). "Lipid based Amphotericin B for the treatment of fungal infections. ." Pharmacotherapy 19(3): 306-323. Eisenberg, S. (1984). "High density lipoprotein metabolism." J Lipid Res 25(10): 1017-58. Eisenberg S., B. D. W., Levy R. I. , (1972). "The metabolism of very low density lipoprotein proteins. II. Studies on the transfer of apoproteins between plasma lipoproteins.." Biochim Biophys Acta 280: 94-104 Fielding, C. J. and P. E. Fielding (1995). "Molecular physiology of reverse cholesterol transport." J Lipid Res 36(2): 211-28. Gale, E. F. (1984). Mode of action and resistance mechanisms of polyene macrolides. . Macrolide Antibiotics: Chemistry, Biology, and Practice. S. Omura. London, Academic Press Inc.: 425-55. Gardier, A . M . , D. Mathe, et al. (1993). "Effects of plasma lipid levels on blood distribution and pharmacokinetics of cyclosporin A." Ther Drug Monit 15(4): 274-80. Gates, C. and R. J. Pinney (1993). "Amphotericin B and its delivery by liposomal and lipid formulations." J Clin Pharm Ther 18(3): 147-53. Gigliotti, F., J. L. Shenep, et al. (1987). "Induction of prostaglandin synthesis as the mechanism responsible for the chills and fever produced by infusing amphotericin B." J Infect Dis 156(5): 784-89. Gilbert D. (2002). "Amphotericin B nephrotoxicity." J Antimicrob. Chemother. 49(S1): 37-41. 123 Gilbert, N . , S. Corden, et al. (2002). "Comparison of commercial assays for the quantification of H B V D N A load in health care workers: calibration differences." J Virol Methods 100(1-2): 37-47. Gilman A . G. (2001). The Pharmacological Basis of Therapeutics. New York, McGraw-Hill, * Medical Publishing Division. Groll, A . H. , S. C. Piscitelli, et al. (1998). "Clinical pharmacology of systemic antifungal agents: a comprehensive review of agents in clinical use, current investigational compounds, and putative targets for antifungal drug development." Adv Pharmacol 44: 343-500. Grundy S. M . (1990). Cholesterol and Atherosclerosis / Diagnosis and Treatment. Philadelphia, Lippincott. Guyard D. V. , D. C , Gambert P., Lallemant C , Lagrost L . (1998). "Phospholipid and cholesteryl estr transfer activities in plasma from 14 vertebrate species. Relation to atherogenesis susceptibility. ." Comp Biochem Physiol B Biochem Mol Biol 120(3): 517-25. Hamilton-Miller, J. M . (1973). "Chemistry and biology of the polyene macrolide antibiotics." Bacteriol Rev 37(3): 166-96. Hartsel, S. and J. Bolard (1996). "Amphotericin B: new life for an old drug." Trends Pharmacol Sci 17(12): 445-9. Havel R. J., E. H. A . , Bragdon J. H. , (1955). "The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. .". J Clin Invest 34: 1345-53 Hazan E. L. , B. R. (1951). "Fungicidin, an antibiotic produced by a soil actinomycete. ." Proc. Soc. Exp. Biol. Med 76: 93-97. Herz J., H . U . , Rogne S., Myklebost O., Gauspohl H. , Stanley K . K. , (1988). "Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor." Embo J7(13): 4119-27. Hiemenz, J. W. and T. J. Walsh (1996). "Lipid formulations of amphotericin B: recent progress and future directions." Clin Infect Dis 22 Suppl 2: SI33-44. Holz, R. and A . Finkelstein (1970). "The water and nonelectrolyte permeability induced in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B." J Gen Physiol 56(1): 125-45. Huuskonen J., E. C. (2000). "Phoshpolipid transfer protein in lipid metabolism." Curr Opin Lipidol 11(3): 285-289. 124 Huuskonen, J., V . M . Olkkonen, et al. (2000a). "Phospholipid transfer is a prerequisite for PLTP-mediated H D L conversion." Biochemistry 39(51): 16092-8. Huuskonen J., W. G., Jauhiainen M . , Ehnholm C , Teleman O., Olkkonen V. M . , (1999). "Structure and phospholipid transfer activity of human PLTP: analysis by molecular modelling and site directed mutagenesis." J Lip Res 40(6): 1123-30. Jauhiainen, M . , J. Metso, et al. (1993). "Human plasma phospholipid transfer protein causes high density lipoprotein conversion." J Biol Chem 268(6): 4032-6. Jiang, X . C , C. Bruce, et al. (1999). "Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels." J Clin Invest 103(6): 907-14. Juliano, R. L., C. W. Grant, et al. (1987). "Mechanism of the selective toxicity of amphotericin B incorporated into liposomes." Mol Pharmacol 31(1): 1-11. Jullien, S., J. Brajtburg, et al. (1990). "Affinity of amphotericin B for phosphatidylcholine vesicles as a determinant of the in vitro cellular toxicity of liposomal preparations." Biochim Biophys Acta 1021(1): 39-45. Kennedy, A . L . and K. M . Wasan (1999). "Preferential distribution of amphotericin B lipid complex into human HDL3 is a consequence of high density lipoprotein coat lipid content." J Pharm Sci 88(11): 1149-55. Koldin, M . H. , G. S. Kobayashi, et al. (1985). "Effects of elevation of serum cholesterol and administration of amphotericin B complexed to lipoproteins on amphotericin B-induced toxicity in rabbits." Antimicrob Agents Chemother 28(1): 144-5. Lamy-Freund, M . T., S. Schreier, et al. (1991). "Characterization and time dependence of amphotericin B: deoxycholate aggregation by quasielastic light scattering." J Pharm Sd 80(3): 262-6. Lamy-Freund, M . T., V . F. Ferreira, et al. (1993). "Effect of aggregation on the kinetics of autoxidation of the polyene antibiotic amphotericin B." J Pharm Sci 82(2): 162-6. Larabi, M . , N . Pages, et al. (2004). "Study of the toxicity of a new lipid complex formulation of amphotericin B." J Antimicrob Chemother 53(1): 81-8. Lee, J. W., M . A . Amantea, et al. (1994). "Pharmacokinetics and safety of a unilamellar liposomal formulation of amphotericin B (AmBisome) in rabbits." Antimicrob Agents Chemother 38(4): 713-18. Legrand, P., E. A . Romero, et al. (1992). "Effects of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes." Antimicrob Agents Chemother 36(11): 2518-22. 125 Lemaire, M . , W. M . Pardridge, et al. (1988). "Influence of blood components on the tissue uptake indices of cyclosporin in rats." J Pharmacol Exp Ther 244(2): 740-3. Lopez-Berestein, G., V . Fainstein, et al. (1985). "Liposomal amphotericin B for the treatment of systemic fungal infections in patients with cancer: a preliminary study." J Infect Djs 151(4): 704-10. Louie, A. , A . L. Baltch, et al. (1994). "Comparative capacity of four antifungal agents to stimulate murine macrophages to produce tumour necrosis factor alpha: an effect that is attenuated by pentoxifylline, liposomal vesicles, and dexamethasone." J Antimicrob Chemother 34(6): 975-87. Lusa, S., M . JaUhiainen, et al. (1996). "The mechanism of human plasma phospholipid transfer protein-induced enlargement of high-density lipoprotein particles: evidence for particle fusion." Biochem J 313 ( Pt 1): 275-82. MBewu A D . and Durrington P. N . (1990). "Lipoprotein (a): structure, properties and possible involvement in thrombogenesis and atherogenesis." Atherosclerosis 85(1): 1-14. Massey, J. B., D. Hickson-Bick, et al. (1985). "Fluorescence assay of the specificity of human plasma and bovine liver phospholipid transfer proteins." Biochim Biophys Acta 835(1): 124-31. Mehta, R. T., T. J. McQueen, et al. (1994). "Phagocyte transport as mechanism for enhanced therapeutic activity of liposomal amphotericin B." Chemotherapy 40(4): 256-64. Meunier, F. (1994). "Alternative modalities of administering amphotericin B: current issues." J Infect 28 Suppl 1: 51-6. Meyer R. D. (1992). "Current Role of Therapy with Amphotericin B." Clin Infect Dis 14(1): S154-S160. Morton, R. E. (1990). "Interaction of lipid transfer protein with plasma lipoproteins and cell membranes." Experientia 46(6): 552-60. Murdoch, S. J., G. Wolfbauer, et al. (2002). "Differences in reactivity of antibodies to active versus inactive PLTP significantly impacts PLTP measurement." J Lipid Res 43(2): 281-89. Nishida, H. I. and T. Nishida (1997). "Phospholipid transfer protein mediates transfer of not only phosphatidylcholine but also cholesterol from phosphatidylcholine-cholesterol vesicles to high density lipoproteins." J Biol Chem 272(11): 6959-64. Nunes, V . S., E. C. Quintao, et al. (2001). "Plasma lipases and lipid transfer proteins increase phospholipid but not free cholesterol transfer from lipid emulsion to high density lipoproteins." B M C Biochem 2: 1. 126 Oda M . N . , H . P. L. , Beckstead J. A . , Redmond K. A. , Antwerpen R. V. , Ryan R. O., (2006). "Reconstituted high density lipoprotein anriched with the polyene antibiotic Amphotericin B." J Lip Res 47: 260-267. Oram, J. F., G. Wolfbauer, et al. (2003). "Phospholipid transfer protein interacts with and stabilizes ATP-binding cassette transporter A l and enhances cholesterol efflux from cells." J Biol Chem 278(52): 52379-85. Perkins, W. R., S. R. Minchey, et al. (1992). "Amphotericin B-phospholipid interactions responsible for reduced mammalian cell toxicity." Biochim Biophys Acta 1107(2): 271-82. Petit, C., M . Cheron, et al. (1998). "In-vivo therapeutic efficacy in experimental murine mycoses of a new formulation of deoxycholate-amphotericin B obtained by mild heating." J Antimicrob Chemother 42(6): 779-85. Proffitt, R. T., A . Satorius, et al. (1991). "Pharmacology and toxicology of a liposomal formulation of amphotericin B (AmBisome) in rodents." J Antimicrob Chemother 28 SupplB: 49-61. Ramaswamy, M . , K . D. Peteherych, et al. (2001). "Amphotericin B lipid complex or amphotericin B multiple-dose administration to rabbits with elevated plasma cholesterol levels: pharmacokinetics in plasma and blood, plasma lipoprotein levels, distribution in tissues, and renal toxicities." Antimicrob Agents Chemother 45(4): 1184-91. Rodicio, J. L. (2000). "Calcium antagonists and renal protection from cyclosporine nephrotoxicity: long-term trial in renal transplantation patients." J Cardiovasc Pharmacol 35(3 Suppl 1): S7-11. Roland M . , T. T. (1995). Clinical Pharmacokinetics: Concepts and Applications. Baltimore, M D , Williams and Wilkins, 248-266. Sabra, R. and R. A . Branch (1991). "Mechanisms of amphotericin B-induced decrease in glomerular filtration rate in rats." Antimicrob Agents Chemother 35(12): 2509-14. Salter, A . M . and D. N . Brindley (1988). "The biochemistry of lipoproteins." J Inherit Metab DJs 11 Suppl 1:4-17. Schindler, J. J., R. P. Warren, et al. (1993). "Immunological effects of amphotericin B and liposomal amphotericin B on splenocytes from immune-normal and immune-compromised mice." Antimicrob Agents Chemother 37(12): 2716-21. Schneider W. J. (1989). "The low density lipoprotein receptor." Biochim. Biophys. Acta 988: 303-317. Surarit, R. and M . G. Shepherd (1987). "The effects of azole and polyene antifungals on the plasma membrane enzymes of Candida albicans." J Med Vet Mycol 25(6): 403-13. 127 Surewicz, W. K. , R. M . Epand, et al. (1986). "Human apolipoprotein A-I forms thermally stable complexes with anionic but not with zwitterionic phospholipids." J Biol Chem 261(34): 16191-97. Swenson, C. E., W. R. Perkins, et al. (1998). "In vitro and in vivo antifungal activity of amphotericin B lipid complex: are phospholipases important?" Antimicrob Agents Chemother 42(4): 767-71. Tall A . R. (1981). "Incorporation of phosphatidylcholine into spherical and discoidal lipoproteins during incubation of egg phosphatidylcholine vesicles with isolated high density liporproteins or with plasma." J Biol Chem 256(4): 2035-44. Tall A . R. (1993). "Plasma cholesteryl ester transfer protein." J Lipid res 34(8): 1255-74. Tall, A . R., L. R. Forester, et al. (1983). "Facilitation of phosphatidylcholine transfer into high density lipoproteins by an apolipoprotein in the density 1.20-1.26 g/ml fraction of plasma." J Lipid Res 24(3): 277-89. Tomii, Y . (2002). "Lipid formulation as a drug carrier for drug delivery." Curr Pharm Pes 8(6): 467-74. Vertut-Doi, A. , P. Hannaert, et al. (1988). "The polyene antibiotic amphotericin B inhibits the Na+/K+ pump of human erythrocytes." Biochem Biophys Res Commun 157(2): 692-7. Wasan K. M . (1996). "Modifications in plasma lipoprotein concentration and lipid composition regulate the biological activity of hydrophobic drugs." J Pharmacol Toxicol Methods 36: 1-11 Wasan K. M . , C. S. M . , Ramaswamy M . , Kennedy A. , Strobel F. W., Ng S. P., Lee T. Y. , (1999). "A comparison of step-gradient and sequential density ultracentrifugation and the use of lipoprotein deficient plasma controls in determining the plasma lipoprotein distribution of lipid-associated nystatin and cyclosporine.." Pharm Res 16: 165-169. Wasan K. M . , P. P. H. , Ramaswamy M . , Wong W., Donnachie E. M . , and Brunner L. J. (1997). "Differences in lipoprotein lipid concentration and composition modify the plasma distribution of cyclosporine." Pharm Res 14: 1613-20 Wasan, K. M . (1996b). "Modifications in plasma lipoprotein concentration and lipid composition regulate the biological activity of hydrophobic drugs." J Pharmacol Toxicol Methods 36(1): 1-11. Wasan, K. M . , G. A . Brazeau, et al. (1993). "Roles of liposome composition and temperature in distribution of amphotericin B in serum lipoproteins." Antimicrob Agents Chemother 37(2): 246-50. Wasan, K. M . and S. M . Cassidy (1998). "Role of plasma lipoproteins in modifying the biological activity of hydrophobic drugs." J Pharm Sci 87(4): 411-24. 128 Wasan, K. M . , S. M . Cassidy, et al. (1999b). "A comparison of step-gradient and sequential density ultracentrifugation and the use of lipoprotein deficient plasma controls in determining the plasma lipoprotein distribution of lipid-associated nystatin and cyclosporine." Pharm Res 16(1): 165-9. Wasan, K. M . and J. S. Conklin (1997b). "Enhanced amphotericin B nephrotoxicity in intensive care patients with elevated levels of low-density lipoprotein cholesterol." Clin Infect Dis 24(1): 78-80. Wasan, K. M . , A . L. Kennedy, et al. (1998b). "Pharmacokinetics, distribution in serum lipoproteins and tissues, and renal toxicities of amphotericin B and amphotericin B lipid complex in a hypercholesterolemic rabbit model: single-dose studies." Antimicrob Agents Chemother 42(12): 3146-52. Wasan, K. M . and G. Lopez-Berestein (1994). "Modification of amphotericin B's therapeutic index by increasing its association with serum high-density lipoproteins." Ann N Y Acad Sci 730: 93-106. Wasan, K. M . and G. Lopez-Berestein (1996c). "Characteristics of lipid-based formulations that influence their biological behavior in the plasma of patients." Clin Infect Dis 23(5): 1126-38. Wasan, K. M . and G. Lopez-Berestein (1997c). "Diversity of lipid-based polyene formulations and their behavior in biological systems." Eur J Clin Microbiol Infect Dis 16(1): 81-92. Wasan, K. M . and R. E. Morton (1996d). "Differences in lipoprotein concentration and composition modify the plasma distribution of free and liposomal annamycin." Pharm Res 13(3): 462-8. Wasan, K. M . , R. E. Morton, et al. (1994b). "Decreased toxicity of liposomal amphotericin B due to association of amphotericin B with high-density lipoproteins: role of lipid transfer protein." J Pharm Sci 83(7): 1006-10. Wasan, K. M . and R. Perez-Soler (1995). "Distribution of free and liposomal annamycin within human plasma is regulated by plasma triglyceride concentrations but not by lipid transfer protein." J Pharm Sci 84(9): 1094-100. Wasan, K. M . , P. H . Pritchard, et al. (1997d). "Differences in lipoprotein lipid concentration and composition modify the plasma distribution of cyclosporine." Pharm Res 14(11): 1613-20. Wasan, K. M . , M . Ramaswamy, et al. (1997e). "Physical characteristics and lipoprotein distribution of liposomal nystatin in human plasma." Antimicrob Agents Chemother • 41(9): 1871-5. 129 Wasan, K. M . , M . G. Rosenblum, et al. (1994c). "Influence of lipoproteins on renal cytotoxicity and antifungal activity of amphotericin B." Antimicrob Agents Chemother 38(2): 223-7. Wasan, K. M . and O. Sivak (2003). "Modifications in lipoprotein surface charge alter cyclosporine A association with low-density lipoproteins." Pharm Res 20(1): 126-9. Wasan, K. M . , K. Vadiei, et al. (1990). "Pharmacokinetics, tissue distribution, and toxicity of free and liposomal amphotericin B in diabetic rats." J Infect Dis 161(3): 562-6. Weber, M . M . and S. C. Kinsky (1965). "Effect Of Cholesterol On The Sensitivity Of Mycoplasma Laidlawii To The'Polyene Antibiotic Filipin." J Bacteriol 89: 306-12. White, M . H. , R. A . Bowden, E. S. Sandler, M . L . Graham, G. A . Noskin, J. R. Wingard, M . Goldman, J. A . van Burik, A . McCabe, C. B. Miller, M . Gurwith, B. Carole., (1998). "Randomized, double-blind clinical trial of amphotericin B colloidal dispersion vs. amphotericin B in the empirical treatment of fever and neutropenia." Clin Infect Dis 27: 296-302. Wingard J. R., K . P., Lee L. , (1999). "Clinical significance of nephrotoxicity in patients treated with amphotericin B for suspected or proven aspergillosis." Clin Infect Dis 29: 1402-07. 130 

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