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The plasma lipoprotein distribution of amphotericin b lipid complex is influenced by the coat lipid content… Kennedy, Allison Louise 1999

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THE PLASMA LIPOPROTEIN DISTRIBUTION OF AMPHOTERICIN B LIPID COMPLEX IS INFLUENCED BY THE COAT LIPID CONTENT OF HIGH DENSITY LIPOPROTEINS by ALLISON LOUISE KENNEDY B.Sc, The University of Lethbridge, 1996 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) (Division of Pharmaceutics and Biopharmaceutics) We accept this thesis as conforming-tp the .required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1999 © Allison Louise Kennedy, 1999 in presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P h a r m a r ^ u 4 i m 1 c % \ l < = V ^ > ^ The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The purpose of this study was to compare the human plasma lipoprotein distribution of amphotericin B (AmpB; Fungizone®) and amphotericin B lipid complex (ABLC; Abelcef®) in different human plasma samples, and subsequently determine the relationship of the drug distribution to lipid and protein composition and concentration of these separated fractions. Independent of plasma lipoprotein lipid and protein concentration, the majority of AmpB was recovered in the lipoprotein-deficient (LPD) plasma fraction following the incubation of free AmpB, while the majority of AmpB was recovered in the high-density lipoprotein (HDL) fraction following the incubation of ABLC. It was also observed that increases in HDL coat lipid content (which contains free cholesterol and phospholipid) resulted in less AmpB recovered in this fraction following the incubation of ABLC. However, increases in the total triglyceride to total protein ratio within HDL resulted in more AmpB recovered in this fraction following the incubation of free AmpB. It was further observed that when HDL coat lipid content (fC + PL) was artificially elevated by dithionitrobenzoate (DTNB), the percentage of AmpB recovered in this fraction was significantly decreased compared to controls following the incubation of ABLC. In addition, it was further observed that the majority of the AmpB recovered in the HDL fraction following the incubation of ABLC was found in the HDL3 fraction. Taken together, these findings suggest that the HDL coat lipid content (more specifically HDL3) may be an important factor in determining which lipoprotein amphotericin B iii associates with following the incubation of ABLC. These results may be an important consideration when evaluating the pharmacokinetics, toxicity and activity of these compounds following administration to patients with altered high-density lipoprotein profiles, patients such as those with cancer, liver and kidney disease, and patients with HIV/AIDS. TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES xi LIST OF ABBREVIATIONS xiii ACKNOWLEDGMENTS xiv DEDICATION xv CHAPTER ONE: Introduction 1 1.1 Lipoproteins 4 1.1.1 Apolipoproteins 7 1.1.1.1 Apolipoprotein A 8 1.1.2 Triglyceride-rich lipoproteins - chylomicrons and very low density lipoproteins 9 1.1.3 Low-density lipoproteins 10 1.1.4 High-density lipoproteins 12 1.2 Liposomes 14 1.2.1 Biological fate of liposomes 19 1.2.2 Use of liposomes in therapy 21 1.3 Amphotericin B 22 1.3.1 The physical characteristics of amphotericin B 23 1.3.2. Amphotericin B lipid complex ....26 1.3.3 Mechanism of action 29 V 1.3.4 Pharmacokinetics 32 1.4 Lipoproteins as Transporters of Hydrophobic Drugs 34 1.4.1 Antidepressants 34 1.4.2 Vitamin E and Antiarrhythmic Agents 35 1.4.3 Cyclosporine 36 1.4.4 Amphotericin B 36 1.5 Significance of Proposed Study 38 Hypothesis 39 CHAPTER TWO: Materials and Methods 40 2.1 Chemicals and Reagents 41 2.2 Preparation of Analytes and Solutions 41 2.2.1 Amphotericin B solution 41 2.2.2 Amphotericin B lipid complex 42 2.2.3. DTNB solution 42 2.3 Heterogeneity of Human Plasma Lipoprotein Profiles 43 2.3.1 Pre-screening of human plasma 43 2.4 Lipoprotein Separation Techniques 43 2.4.1 Step-gradient ultracentrifugation 43 2.4.1.1 Treatment of plasma with AmpB or ABLC 43 2.4.1.2 Separation of plasma lipoprotein constituents 44 2.4.1.3 Controls 45 2.4.2 Step-gradient ultracentrifugation of HDL2 / HDL3 47 2.4.2.1 Treatment of plasma with AmpB or ABLC 47 vi 2.4.2.2 Separation of plasma lipoprotein constituents 47 2.4.2.3 Controls 48 2.4.3 Gradient gel electrophoresis 49 2.4.3.1 Electrophoresis buffer 49 2.4.3.2 Gradient gel formation 49 2.4.3.3 Sample preparation and electrophoresis 50 2.4.3.4 Gel staining and destaining 51 2.5 Amphotericin B Quantification 51 2.5.1 Standard curve preparation 51 2.5.2 Determination of amphotericin B content within the separated lipoprotein fractions 52 2.5.2.1 VLDL and LDL 52 2.5.2.2 HDL 52 2.5.2.3 LPD 53 2.5.3 HPLC Apparatus 53 2.6 Lipid and Protein Content Analysis of Lipoprotein and Lipoprotein-deficient plasma 54 2.6.1 UV spectrophotometry apparatus 54 2.6.2 Determination of total cholesterol concentration 54 2.6.3 Determination of free cholesterol concentration 56 2.6.4 Determination of cholesteryl ester concentration 57 2.6.5 Determination of total triglyceride concentration 57 2.6.6 Determination of phospholipid concentration 59 2.6.7 Determination of total protein concentration 61 Statistical Analysis 63 CHAPTER THREE: Results 64 3.1 Amphotericin B HPLC Quantification 65 3.2 Lipid and Protein Analysis 66 3.3 Establishment of Experimental Design 72 3.3.1 Experimental design 72 3.4 Experimentation Utilizing Step Gradient Ultracentrifugation 73 3.4.1 Lipid and protein composition of separated plasma fractions 73 3.4.2 AmpB distribution within separated plasma fractions 87 3.4.3 Effect of lipoprotein composition on AmpB and ABLC distribution. ...91 3.5 HDL2 / HDL3 Step-Gradient Ultracentrifugation 101 3.5.1 Lipid and protein composition of separated fractions 101 3.5.2 AmpB distribution within HDL2 and HDL3 107 3.6 Gradient Gel Electrophoresis 109 3.7 Experimentation Utilizing DTNB-Modification of Plasma Lipoprotein Profiles 109 CHAPTER FOUR: Discussion 115 4.1 Lipoprotein Distribution of Amphotericin B 116 4.1.1 Amphotericin B 116 4.1.2Amphotericin B lipid complex 119 4.2 Limitations of the Study 123 4.3 Clinical Ramifications 124 4.4 Future Studies 129 4.5 Conclusion 132 viii REFERENCES 134 IX LIST OF TABLES Table 1. The Apolipoproteins 7 Table 2. Pharmacokinetic parameters and tissue distribution of drug after a single intravenous dose (lmg/kg) to a normolipidemic rabbit model 33 Table 3. Representative linear calibration curves for amphotericin B as determined in the separated lipoprotein and lipoprotein-deficient plasma fractions for plasma sample 3 65 Table 4. Total and lipoprotein plasma cholesterol concentrations from seven different human plasma samples 74 Table 5. Total and lipoprotein plasma cholesteryl ester concentrations from seven different human plasma samples 76 Table 6. Total and lipoprotein plasma free cholesterol concentrations from seven different human plasma samples 77 Table 7. Total and lipoprotein plasma triglyceride concentrations from seven different human plasma samples 78 Table 8. Total and lipoprotein plasma phospholipid concentrations from seven different human plasma samples 80 Table 9. Total lipoprotein and lipoprotein-deficient human plasma protein concentrations from seven different human plasma samples 81 Table 10. Total and lipoprotein cholesteryl ester and triglyceride concentrations (lipoprotein core lipid content) from seven different human plasma samples.82 Table 11. Total and lipoprotein free cholesterol and phospholipid concentrations (lipoprotein coat lipid content) from seven different human plasma samples.83 Table 12. Lipoprotein total cholesterol to total protein ratios (TC:TP) from seven different human plasma samples 85 Table 13. Lipoprotein total triglyceride to total protein ratios (TG:TP) from seven different human plasma samples 86 Table 14. Lipoprotein total cholesterol to total triglyceride ratios (TC:TG) from seven different human plasma samples 88 X Table 15. Lipoprotein phospholipid to total protein ratios (PL:TP) from seven different human plasma samples 89 Table 16. The lipoprotein and lipoprotein-deficient human plasma distribution of AmpB (20 pg/mL of plasma) incubated at 37°C for 60 minutes in seven different human plasma samples 90 Table 17. The lipoprotein and lipoprotein-deficient human plasma distribution of ABLC (20 pg/mL of plasma) incubated at 37°C for 60 minutes in seven different human plasma samples 91 Table 18. Correlation analysis comparing amount of AmpB recovered to lipid and protein content and composition of the separated lipoprotein and lipoprotein-deficient plasma fractions following the incubation of AmpB (20 pg/mL) for 60 minutes at 37°C in plasma from seven different human subjects 98 Table 19. Correlation analysis comparing amount of ABLC recovered to lipid and protein content and composition of the separated lipoprotein and lipoprotein-deficient plasma fractions following the incubation of ABLC (20 pg/mL) for 60 minutes at 37°C in plasma from seven different human subjects 100 Table 20. HDL2, H D L 3 and LPD plasma lipid and protein concentrations 106 Table 21. Total lipid and protein concentration in control and DTNB-treated lipoprotein and lipoprotein-deficient plasma Ill Table 22. Important trends observed in the comparison of AmpB and ABLC distribution within plasma lipoproteins and the content and composition of these lipoprotein fractions ; 117 Table 23. Molar concentration of lipoproteins in normal fasting patients 118 XI LIST OF FIGURES Figure 1. The structure of a lipoprotein particle 5 Figure 2. The LDL Receptor Pathway 11 Figure 3. Reverse Cholesterol Transport 13 Figure 4. Schematic representation of a liposome 15 Figure 5. The bilayer structure of large multilamellar (MLV) and small unilamellar vesicles (SUV) 17 Figure 6. The structure of multilamellar and large unilamellar vesicles 18 Figure 7. Structure of Amphotericin B 24 Figure 8. Structure of Amphotericin B Lipid Complex 28 Figure 9. The interaction of amphotericin B with sterol-containing membranes 31 Figure 10. A representation of the separation of plasma lipoprotein fractions by density gradient ultracentrifugation 46 Figure 11. Representative chromatograms for (A) 0 pg/mL and (B) 20 pg/mL concentration time points in the standard curve utilized for the determination of AmpB distribution in plasma following the incubation of 20 pg of AmpB per milliliter of plasma for 60 minutes at 37°C 67 Figure 12. Representative chromatogram for 20 pg/mL concentration point in the standard curve utilized for the determination of AmpB distribution in the HDL fraction of plasma following the incubation of 20 pg of AmpB per milliliter of plasma for 60 minutes at 37°C 68 Figure 13. Representative cholesterol assay standard curve 69 Figure 14. Representative triglyceride assay standard curve 70 Figure 15. Representative protein assay standard curve 71 Figure 16. Distribution of AmpB and ABLC (20 pg/mL) in human plasma following incubation for 60 minutes at 37°C for (A) patient sample 1 and (B) patient sample 2 92 X l l Figure 17. Distribution of AmpB and ABLC (20 pg/mL) in human plasma following incubation for 60 minutes at 37°C for (A) patient sample 3 and (B) patient sample 4 93 Figure 18. Distribution of AmpB and ABLC (20 pg/mL) in human plasma following incubation for 60 minutes at 37°C for (A) patient sample 5 and (B) patient sample 6 94 Figure 19. Distribution of AmpB and ABLC (20 pg/mL) in human plasma following incubation for 60 minutes at 37°C for (A) patient sample 7 95 Figure 20. The amount of amphotericin B (AmpB) recovered within the high-density lipoprotein fraction (HDL) versus the total triglyceride to total cholesterol ratio (TG:TC) after incubation of 20 pg/mL for 60 minutes at 37°C 99 Figure 21. The amount of amphotericin B (AmpB) recovered within the high-density lipoprotein fraction (HDL) versus the coat lipid content (fC + PL) after incubation of 20 pg/mL for 60 minutes at 37°C 102 Figure 22. The amount of amphotericin B (AmpB) recovered within the triglyceride-rich lipoprotein fraction (TRL) versus the total triglyceride to total protein ratio (TG:TP) after incubation of 20 pg/mL for 60 minutes at 37°C 103 Figure 23. The amount of amphotericin B (AmpB) recovered within the triglyceride rich lipoprotein fraction (TRL) versus the total triglyceride to total cholesterol ratio (TG:TC) after incubation of 20 pg/mL for 60 minutes at 37°C 104 Figure 24. Distribution of AmpB and ABLC (20 pg/mL) in human plasma following incubation for 60 minutes at 37°C 108 Figure 25. Figure 26. Human plasma samples run on the HDL gradient gel format 110 Distribution of ABLC (20 pg/mL) in control and DTNB-treated human plasma following incubation for 60 minutes at 37°C 114 xiii LIST OF ABBREVIATIONS ABLC amphotericin B lipid complex ACAT acyl-CoA:cholesterol transferase AmpB amphotericin B Apo apolipoprotein AUC area under the curve CE cholesteryl ester CETP cholesteryl ester transfer protein Cmax concentration maximum DCM dichloromethane DMF dimethylformamide DMPC dimyristoylphosphatidylcholine DMPG dimyristoylphosphatidylglycerol DTNB dithionitrobenzoate EDTA ethylenediaminetetraacetic acid fC free cholesterol HDL high-density lipoprotein HPLC high performance liquid chromatography HTGL hepatic triglyceride lipase IDL intermediate-density lipoproteins LCAT lecithimcholesterol acyltransferase LDL low-density lipoprotein LPD lipoprotein-deficient LPL lipoprotein lipase LRP lipoprotein receptor protein LUV large unilamellar vesicles MeOH methanol MLV multilamellar vesicles MPS mononuclear phagocyte system MWCO molecular weight cutoff Nys nystatin PL phospholipid PLTP phospholipid transfer protein QES quasi-electric light scattering tl/2 half-life TBE tris - boric acid - EDTA TC total cholesterol TEMED N,N,N',N' - tetramethylethylenediamine TG total triglyceride TP total protein TRL triglyceride-rich lipoprotein Vd volume of distribution VLDL very low-density lipoprotein xiv ACKNOWLEDGMENTS I would like to thank my supervisor, Dr. Kishor Wasan, for his guidance and patience while providing me the opportunity to further my studies. But most of all thank you for believing in me and taking a chance sight unseen. I would also like to thank the members of my research committee: Dr. Kathleen MacLeod (Chair), Dr. Helen Burt, Dr. John Hill (External), Dr. Haydn Pritchard, and Dr. Wayne Riggs for their support and feedback into this project To my co-workers in the lab: Manisha, the best rabbit wrangler I know, thank you for everything. I aim to be half as organized as you when I grow up. Kathy, thank you for all of your support and letting me "act like a grad student". Wes, I thank you for the many lunch hours we spent together talking about everything and nothing. I would also like to thank all of the undergraduate students over the past two years with whom I have had the pleasure of working with. To my girls, Lisa, Kara and Heather. It has always been through your love and support that I have accomplished all that I have. I love you and miss you always. This one's for the Band. And last, but not least, Randy. Thank you for being there through my good times and bad. "it's a pleasure that I have known, and it's a treasure that I have gained" V N. Finn XV DEDICATION This work is dedicated to my parents, Colin and Audrey Kennedy. The greatest gift you ever gave was the belief in the power of education. Everyday, and in every way, you instilled in me a dream. Without your endless encouragement and support (and $$$), this would not have been possible. Thank you for everything Mom and Dad. 1 Chapter 1 Introduction 2 The pharmacokinetics and toxic effects of a number of drugs differ when administered to diseased patients as compared to healthy controls [Gibaldi and Perrier 1982 and Rowland and Tozier 1989]. This often makes it difficult to determine the most effective dose of these drugs for administration to diseased patients. This is due to the fact that the maximum tolerated dose and pharmacokinetics of a drug are determined in healthy animals and human volunteers [Gibaldi and Perrier 1982 and Rowland and Tozier 1989]. These data are subsequently extrapolated and used for the diseased patient population. In the case of some water insoluble compounds and compounds incorporated into lipid-based vesicles, the dose that is deemed non-toxic in healthy animals and humans is ineffective and/or toxic when administered to the diseased patient [Sgoutas et al., 1986, Kasiske et al., 1988, Wassef et al, 1986, Rodl and Khoshsorur 1990 and Andrade et al., 1993]. Disturbances in lipid metabolism (e.g., hypertriglyceridemia and hypocholesterolemia) commonly occur during infection [Wasan and Cassidy 1998]. Early studies have indicated a number of mechanisms by which infection can cause the increase of triglycerides (TG): (1) increased hepatic de novo synthesis of fatty acids leading to increased secretion of chylomicrons and very-low density lipoproteins (VLDL) (2) increased adipose tissue lipolysis with the mobilized fatty acid being re-esterified into triglycerides in the liver and then re-secreted as triglyceride rich lipoproteins rather than being oxidized, and (3) decreased levels of lipoprotein lipase (LPL) leading to decreased clearance of triglyceride-rich lipoproteins [Vitols et al., 1985, Grunfeld et al., 1992, Feingold et al., 1993, and Umeki 1993]. Furthermore, cancer cells presumably require additional cholesterol for the formation of new-membrane material and metabolism requirements, as evidenced by the development of hypocholesterolemia and a decrease in plasma LDL cholesterol concentrations in leukemia patients [Vitols et al., 1985 and Umeki 1993]. Since many hydrophobic compounds predominantly distribute into plasma lipoproteins upon incubation in plasma, changes in plasma triglyceride and cholesterol concentrations could affect not only the plasma distribution of these compounds, but may also have a bearing on their pharmacokinetics and pharmacodynamics [Wasan and Cassidy 1998]. For a water insoluble compound such as the antifungal agent amphotericin B (AmpB), several lines of evidence indicate that increases in serum low-density lipoprotein cholesterol (LDL-C) concentrations are associated with increases in amphotericin B-induced kidney toxicity [Wasan and Conklin 1997]. However, when amphotericin B was incorporated into non-toxic phospholipids to form an amphotericin B-lipid complex (ABLC), amphotericin B-induced kidney toxicity was substantially reduced [Gates and Pinney 1993, Hay 1994, Meuneir 1994, De Marie et al., 1994, Fromtling 1995 and Bolard and Andremont 1995,]. Furthermore, it has been demonstrated that the majority of amphotericin B binds to high-density lipoproteins (HDL) when ABLC is incubated in human serum [Wasan et al., 1993] and that amphotericin B was less toxic to kidney endothelial cells when bound to HDL than when bound to LDL [Wasan et al., 1993]. 4 Based on these preliminary findings the main objective of this project will be to determine if changes in lipoprotein composition and concentration affect the distribution of amphotericin B and amphotericin B lipid-complex (ABLC) in plasma lipoproteins. 1.1 Lipoproteins Plasma lipoproteins are a heterogeneous population of soluble, macromolecular aggregates of lipid and protein that serve to transport water insoluble nutrients (phospholipid, cholesteryl ester (CE) and triacylglycerol (TG)) through the vascular and extravascular body fluids to cells that require them for anabolic and energy purposes [Davis 1996, Harmony et al, 1996]. Lipoproteins transport these cellular nutrients from the liver and intestine to other tissues in the body for storage or catabolism in the production of energy. Lipoproteins are also known to be involved in other biological processes including coagulation and tissue repair as well as immune reactions [Mbewu and Durrington 1990 and Durrington 1996]. Lipoproteins are roughly spherical particles consisting of a non-polar lipid core (TG and CE) surrounded by a surface monolayer of amphipathic lipids (phospholipid and unesterified cholesterol) and specific proteins called apolipoproteins [Wasan and Cassidy 1998] (Figure 1). The types of phospholipids incorporated into the coat of the lipoprotein include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingomyelin. Since lipids, in general, have lower buoyant densities than proteins, lipoproteins with a larger amount of lipid relative to protein will have a lower density than lipoproteins with a smaller s 6 lipid-to-protein ratio [Wasan and Cassidy 1998]. Lipoproteins are traditionally classified according to their density and are divided into five main categories: chylomicrons, very-low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). These are broad classifications and within each group there is heterogeneity in terms of particle size and composition. Briefly, chylomicrons are characterized by a diameter of approximately 100 - 1000 nm, thus making them the largest of the lipoproteins. They are synthesized by the intestine and contain a core, which is rich in triglyceride derived from dietary fat. Slightly smaller than chylomicrons are very-low density lipoproteins (VLDL) which have a diameter between 30 -80 nm and are also triglyceride rich. The liver synthesizes VLDL. Intermediate-density lipoproteins (IDL) have a lipid core, which is comprised mainly of cholesteryl esters (CE) with some triglyceride (TG). These lipoproteins are the product of VLDL metabolism by lipoprotein lipase (LPL). Low-density lipoproteins (LDL) in turn, are the product of IDL metabolism in which almost all of the remaining TG have been hydrolyzed to produce a lipoprotein core comprised almost entirely of CE. LDL is the major cholesterol-carrying lipoprotein in humans and has a diameter averaging 18 - 25 nm. HDL is the smallest and most dense lipoprotein with an average diameter of only 7-12 nm. HDL is a diverse population in both structure and function, and contains a lipid core that contains both CE and TG of varying ratios. 7 1.1.1 Apolipoproteins All lipoproteins have specialized proteins, known as apolipoproteins, embedded within the surface monolayer. These apolipoproteins are amphipathic in nature and contain polar and non-polar amino acid residues that help to solubilize and stabilize the insoluble lipids incorporated within the lipoprotein [Davis and Vance 1996 and Harmony et al., 1996]. There are numerous classes and sub-classes of apolipoproteins, all of which serve a variety of functions, one of which being to act as ligands for cell-surface receptors (Table 1). There are four major types of apolipoproteins, as well as numerous minor apolipoproteins, each with its own-subclass; apolipoprotein B (apo B), apolipoprotein A (apo A), apolipoprotein C (apo C), and apolipoprotein E (apo E). Table 1. The Apolipoproteins Apolipoprotein Main Functions Apo A-I Structural for HDL. Ligand for HDL binding. LCAT cofactor Apo A-II Structural for HDL. Ligand for HDL binding. LCAT cofactor Modulator of LPL and HTGL activity (possible) Apo A-IV Ligand for HDL binding. LCAT activator Apo (a) Structural for LP(a). Structural analogy with plasminogen Apo B-48 Structural for chylomicrons Apo B-100 Structural for VLDL, IDL and LDL. LDL receptor ligand Apo C-I LCAT and LPL inhibitor Apo C-II LCAT and LPL inhibitor Apo C-III LPL inhibitor. Modulator of uptake of triglyceride-rich lipoproteins by LRP. ApoD Unknown Apo J Membrane protection (possible) 8 One common characteristic of apolipoproteins is the presence of amphipathic helical structures, which are believed to be responsible for the binding of the apolipoprotein to the surface coat lipid of the lipoprotein [Segrest et al., 1974 and Gotto et al., 1986]. Each a-helix is comprised of two sections. A polar, hydrophilic section is surrounded by the plasma (aqueous) compartment, while the non-polar hydrophobic section is found embedded within the lipoprotein surface coat [Segrest et al., 1974 and Gotto et al, 1986]. All of the apolipoproteins have been shown to be water-soluble. An exception to this rule is apolipoprotein B, which has been found to be insoluble in water. 1.1.1.1 Apolipoprotein A Of particular interest to the proposed project is the class of lipoproteins termed apolipoprotein A. In man the two major A apolipoproteins are AI and All, with apo AI being most abundant [Cheung and Albers 1982]. These apolipoproteins are synthesized in the liver and intestine from where they are secreted as phospholipid rich discs called nascent HDL. Most of the apo AI is present in plasma and extravascular tissue fluid in HDL. There is however a small percentage of apo AI which is not present in lipoproteins, and is therefore generally referred to as pre-beta HDL. Apo AI is necessary for HDL structural integrity while both apolipoproteins act as ligands for HDL binding, as well as a cofactor for lecithin:cholesterol acyltransferase (LCAT). It has been suggested that apo AH has the ability to act as a modulator of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activity [Bhatnagar and Durrington 1997]. This however, has yet to be clearly established. 9 1.1.2 Triglyceride-rich lipoproteins - chylomicrons and very-low-density lipoproteins The process of chylomicron formation is quite complex, beginning with the hydrolysis of triacylglycerols into free fatty acids and monoacylglycerols in the intestinal lumen. Cholesterol esters are hydrolyzed by cholesterol esterase to release free cholesterol and fatty acids. The long-chain fatty acids and cholesterol are then re-synthesized in the intestinal epithelial cells. These are re-assembled within cells into TG and then, on the backbone of apo B-48, a variant of apolipoprotein B, into TG-rich particles, chylomicrons. As the chylomicron particles enter the systemic circulation, they acquire apolipoprotein E and apolipoprotein C (which are required for their metabolism) from HDL. Enterocytes secrete chylomicrons into the thoracic duct from where chylomicrons enter plasma. Within minutes the chylomicron is rapidly metabolized, with the triacylglycerols being hydrolyzed by lipoprotein lipase (LPL). LPL is located on the surface of capillary endothelial cells and is activated by the apolipoprotein CII on the chylomicron particle. The residual lipoprotein, termed a chylomicron remnant, is removed from the circulation by the liver. VLDL is synthesized and secreted by the liver and is involved in the transport of endogenously synthesized triacylglycerols from the liver to peripheral tissue. The VLDL particle that is initially secreted by the liver is in a nascent form and acquires cholesteryl ester, apolipoprotein CII, CHI and E from HDL. Like chylomicrons, VLDL-TG is hydrolyzed by LPL, leaving a VLDL remnant, which is also known as IDL. This IDL particle has a CE rich core and is subject to two possible fates. The liver can take up IDL via the LDL receptor mediated pathway, or it can be converted to LDL by interaction with hepatic lipase. The interaction with hepatic lipase further hydrolyzes the remaining TG and 10 the apolipoprotein C's and apolipoprotein E are also lost. The remaining particle, still bearing the apolipoprotein B is now a LDL particle. 1.1.3 Low-density lipoproteins In humans, LDL is composed of approximately 50% cholesterol, and is the major cholesterol carrying lipoprotein with apo B-100 as its only apolipoprotein. It has been found that in other animal species, such as ruminants and some rodents, that HDL is the major cholesterol carrying lipoprotein [Davis 1991]. As mentioned previously, LDL is the product of the interaction of IDL with the enzyme hepatic lipase. It has however been suggested that LDL can also be synthesized and secreted directly by the liver [Myant 1990]. LDL is removed from the circulation primarily via the LDL-receptor pathway (Figure 2). Discovered in the early 1970's, the LDL receptor is one of the best-understood receptor models. The receptor is initially synthesized on the rough endoplasmic reticulum (RER), after which it undergoes post-translational modification. The mature receptor is translocated to the cell surface where it is localized into specialized regions known as coated pits [Harrison and Kirchhausen 1983]. LDL particles which bind to the LDL-receptors in the coated pits are internalized to form coated vesicles known as endosomes [Goldstein et al., 1985]. Once within the endosome, ATP-driven proton pumps decrease the pH within the endosome, thus resulting in the dissociation of the LDL particle from the LDL receptor [Stone et al., 1983]. 12 The release of cholesterol has three major regulatory effects on the cell [Goldstein et al., 1985 andMyant 1990]: i) the down regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase), the rate limiting enzyme involved in de novo cholesterol synthesis ii) activation of acyl-coenzyme A cholesterol acyl transferase (ACAT), the enzyme responsible for the esterification of cholesterol to cholesteryl ester iii) the down regulation of the LDL-receptor synthesis These combined effects allow the cell to selectively utilize either dietary cholesterol or synthesized cholesterol. 1.1.4 High-density lipoproteins High-density lipoproteins are comprised of a heterogeneous population that is subdivided into different classes based on apolipoprotein and phospholipid content. The primary function of HDL is the transport of cholesterol from the peripheral tissues to the liver in a process known as reverse cholesterol transport (Figure 3). The liver and intestine secrete nascent, discoid HDL containing apo AI, All and apo E along with phospholipid and free cholesterol [Dominiczak 1997]- Some apo-C-containing discoid forms are generated from VLDL and chylomicrons during TG hydrolysis [Dominiczak 1997]. The free cholesterol in the nascent HDL is converted into cholesteryl ester by lecithimcholesterol acyltransferase (LCAT). LCAT transfers a fatty acid moiety from phosphatidylcholine (lecithin) to the cholesterol, producing a cholesteryl ester, which is more hydrophobic than free cholesterol and is located in the core of the HDL particle. This increases the size of the particles and 13 14 changes the disk-shaped particles into spheres known as H D L 3 . The core of the HDL3 particle is rich in CE but deficient in TG. Cholesteryl esters carried by HDL are either transported to the liver or transferred to triglyceride rich lipoproteins (TRL) - VLDL and chylomicrons. This is facilitated by cholesterol ester transfer protein (CETP), in exchange for TG, phospholipids, apo A-I, apo E and apo C [Dominiczak 1997]. Transfer of TG to HDL further increases its size. The resulting mature HDL particle, called HDL2 contains a core comprised of mainly TG, and is subjected to the action of hepatic lipase (HTGL). The interaction of the HDL2 particle with hepatic lipase results in the hydrolysis of the triacylglycerol, thus regenerating the H D L 3 particle. This HDL3 particle can acquire further cholesterol from peripheral tissue and interact with TG-rich lipoproteins again, thus the cycle of cholesterol removal continues. 1.2 Liposomes Many drugs, either in clinical use or in development, have properties that are far from ideal. They may have poor solubility, rapid metabolism, instability under physiological conditions or unfavorable biodistribution leading to toxicity. One attempt at achieving a solution to these problems has been to associate the drugs with a variety of drug carriers. Liposomes, (i.e., phospholipid membrane vesicles) are the most advanced of the drug carriers with several products either approved for clinical use or in advanced trials (Figure 4) [Allen 1996]. 15 16 Liposomes are bilayer structures comprised of natural or synthetic phospholipids, fatty acids, proteins, and sterol [Singh and Perdue 1998]. Bangham and co-workers initially observed that when dried phospholipids were introduced to an aqueous solution, either sterile water or normal saline, the phospholipids tended to form structures similar in appearance to mammalian membranes [Bangham and Home 1964, Bangham et al., 1965 and Bangham et al., 1974]. Phospholipids contain a hydrophilic head attached to a hydrophobic tail. When hydrated, bilayered membranes form with the hydrophilic heads facing out, protecting the hydrophobic tail from the aqueous environment. Liposomes have been classified into three main categories largely based on their appearance and physical characteristics such as homogeneity and size. The most common liposome is the multilamellar vesicle (MLV) which is sometimes thought to be visually comparable to an onion. Multilamellar vesicles are composed of concentrically smaller lipid bilayers surrounding an aqueous compartment (Figure 5). The size of these vesicles is heterogeneous in nature and ranges between 100 nm and approximately 2 pm in diameter [Yatvin and Lelkes 1982, Ostro 1987]. Upon sonication, MLVs reduce in size to between 50 and 100 nm in diameter. As well, the structure changes to a smaller, single bilayered vesicle called a small unilamellar vesicle (SUV). The last type of liposome commonly found is the large unilamellar vesicle (LUV) which is comprised of a single large aqueous compartment surround by a single phospholipid bilayer. The size of a LUV is generally around 1 pm (Figure 6). 18 0 o "55 E 03 .2 o i/> o > "35 E 03 £3 «J cn O 00 O GO cd O i-i -»—> 00 cn o o "S o > ^ .—< -(-> 'o & S-i B o 00 s —' (U > § cd • -3 _o ^ * § .2 o> o a GO d O P E cd I O cn O <D a" cd tu GO u* cd £ ° s> cd cn > H 00 Jd ^ O « u u u 1 II T—i cd cn 2 cd ^ g -O j d o rt 2 cn C <L> T3 5 cd cd cn 'rt 3 cd H ^ T3 C o oo S-H c^d X> a • i - H ' G O G cn cd 00 }^ GO <D (S/) ^ oo 5 ' ^ CD 00 o o> 19 Liposomes are versatile drug carriers, which can be used to solve problems of drug solubility, instability and rapid degradation. Both hydrophilic and hydrophobic drugs can be associated with liposomes, and special techniques have been developed for the efficient loading of weak acids and weak bases into liposomes [Allen 1996]. The polarity of a drug determines where it partitions in a liposome [Storm and Crommelin 1997]. Lipophilic drugs partition into the liposomal membrane, and water-soluble drugs concentrate in the internal aqueous phase [Storm and Crommelin 1997]. Liposomes can target a drug to the intended site of action in the body, thus enhancing its therapeutic efficacy (drug targeting, site-specific delivery). Liposomes may also direct a drug away from those body sites that are particularly sensitive to the toxic action of it (site-avoidance delivery) [Storm and Crommelin 1997]. Liposomes can also act as a depot from which the entrapped compound is slowly released over time. Such a sustained release process can be exploited to maintain therapeutic (but non-toxic) drug levels in the bloodstream or at the local administration site for prolonged periods of time [Storm and Crommelin 1997]. As well, drugs incorporated in liposomes, in particular those entrapped in the aqueous interior, are protected against the action of detrimental forces such as degradative enzymes present within the host [Storm and Crommelin 1997]. Conversely, the patient can be protected against detrimental toxic effects of drugs. 1.2.1 Biological fate of liposomes The biological fate of liposomes is dependent upon a variety of factors. Liposome size, type, bilayer rigidity, transition temperature and liposome surface charge are all factors that play 20 an important role in determining the ultimate fate of a liposome. For example, multilamellar vesicles have been reported to be rapidly cleared from the blood while negatively charged liposomes have a shorter blood residence time than similar positively charged and neutral liposomes [Schneider 1985 and Lopez-Berestein 1989]. Once in the body, parenterally administered liposomes are readily taken up by the mononuclear phagocyte system (MPS) [Raz et al., 1981, Kasi et al., 1984, Perez-Soler et al., 1985, Senior 1987 and Gregoriadis 1988,]. The cells of the MPS are found in all tissues and organs where they help to maintain homeostasis and immune regulation. However, these cells are mainly found circulating in the bloodstream or residing in the spleen, liver, lung and bone marrow. Liposomes may also interact with plasma lipoproteins. It has been suggested that lipid-drug complexes may associate with specific lipoprotein fractions, which may in turn act as a transport system complementary to the MPS [Damen et al., 1979 and Scherphof etal., 1983]. Upon reaching the area in which the liposomes are to act, the contents of liposomes can enter a cell in a number of ways. The contents may first be released into the extracellular fluid before then crossing the cell membrane. This release of the liposomal contents can either occur close to the cell or immediately upon entering the circulation. Cells of the MPS may also internalize intact liposomes. Liposomes are internalized, and the resulting endosome fuses with lysosomes. The lysosomal enzymes degrade the liposomal structure, thus releasing the free drug into the cell. Another possible method involves the exchange of lipids between the liposomes and the cell membranes. If the liposome contains phospholipids 21 found in the cellular membrane, an exchange of phospholipid may occur between the two "membranes" thereby facilitating the transport of drug into the cell [Scherphof et al, 1983]. Another proposed mechanism for internalization of liposome contents involves the incorporation of the liposome into the cell membrane. The outer layer of the liposome may fuse directly with the cell membrane and lead to the release of the inner contents of the liposome into the cytoplasm of the cell. 1.2.2 Use of liposomes in therapy For several years prior to the clinical approval of liposomal products, a great deal of effort was spent in basic research. Research into liposome composition, size, stability, drug loading, pharmacokinetics and pharmacodynamics were all necessary in order to understand how this type of delivery system could be used to achieve improved drug efficacy and/or decreased drug toxicity. Recently, liposomal drug delivery systems have come of age, with three antifungal and two anticancer preparations having received final approval for clinical use in a number of countries around the world [Allen 1998]. A number of other liposomal formulations of drugs, including anticancer, antibacterial and anti-inflammatory drugs, are in early-to-late clinical trials [Allen 1998]. Ligand- and antibody-targeted applications, designed to further increase site-specific delivery of liposomal drugs, are currently being explored in a number of laboratories. Liposomal drug delivery systems have been extensively researched for use in treating infections, particularly fungal infections [Lopez-Berestein et al, 1985, Wasan and Lopez-Berestein 1995, and Allen 1998]. The first liposomal drug delivery system to be marketed 22 was a formulation of amphotericin B in late 1990. AmBisome® was first approved for sale in the Republic of Ireland, followed by the approval, again in Europe, of an amphotericin B colloidal dispersion (Amphotec® [Amphocil®]) and an amphotericin B lipid complex (Abelcet®) early in 1995. The first approval in the US for marketing the lipid formulation of amphotericin B came in late 1995 for Abelcet®. All of these formulations have been shown to be effective at reducing the severe kidney toxicity of free amphotericin B in patients with systemic fungal infections. These formulations have also been instrumental in clearing the infection from many patients who could no longer tolerate the dose intensity of the free drug that was necessary to control their infection [Lopez-Berestein et al., 1985]. 1.3 Amphotericin B The incidence of invasive and opportunistic fungal infections is rising in Canada. This is due to increasing use of myelosuppressive chemotherapy, the spread of AIDS, and increasing use of invasive procedures for monitoring patients [Singh and Perdue 1998]. Invasive fungal infections increase mortality in both severely ill and immunocompromised patients [Singh and Perdue 1998]. Unlike bacterial infections, for which there are a number of systemic agents directed toward at least nine separate targets, there are only six systemic antifungals directed at three distinct targets [Rapp et al., 1997]. The primary reason for this is that, unlike their bacterial counterparts, fungi are eukaryotes. Consequently they are similar to mammalian cells, and thus the two have many potential drug targets in common [Rapp et al., 23 1997]. It is difficult to inhibit a specific target in the fungal cell without producing similar toxicity in the mammalian host. To date, the drug of choice for these infections has been amphotericin B because of its fungicidal effect at therapeutic levels. Invasive aspergillosis and candidiasis are often seen in immunocompromised patients, particularly those with hematologic malignancies or in recipients of bone marrow or organ transplants who are receiving immunosuppressant treatment [Singh and Perdue 1998]. Also at risk of invasive fungal infections are older patients, those with diabetes mellitus, those receiving inadequate nutrition and those with indwelling catheters that have undergone multiple surgery [Singh and Perdue 1998]. Unfortunately, these infections can require high-dose amphotericin B therapy for long periods, precipitating nephrotoxicity and electrolyte abnormalities. 1.3.1 The physical characteristics amphotericin B Amphotericin B is a heptaene macrolide antifungal derived from a strain of Streptomyces nodosus. Amphotericin B has a molecular weight of 923.62, and is designated chemically as [1R-(1R*, 3S*, 5R*, 6R*, 9R*, 11R*, 15S*, 16R*, 17R*, 18S*, 19E, 21E, 23E, 25E, 27E, 29E, 3IE, 33R*, 35S*, 36R*, 37S*)]-33-[(3-Amino-3, 6-dideoxy-p-D-mannopyranosyl)-oxy]-l, 3, 5, 6, 9, 11, 17, 37-octahydroxy-15, 16, 18-trimethyl-13-oxo-14, 39-dioxabicyclo[33.3.2] nonatriaconta-19, 21, 23, 25, 27, 29, 31-heptaene-36-carboxylic acid (Figure 7). 24 25 In order to isolate amphotericin B from Streptomyces nodosus, whole broth is mixed with isopropanol (1:1) and adjusted to pH 10.5 [Asher et al., 1977]. The filtrate is neutralized, the alcohol evaporated and the resulting powder (40 - 70% pure) washed with water and acetone, and vacuum dried [Asher et al., 1977]. Slurrying with a 2% CaCh methanol solution separates amphotericin A (filtrate) and amphotericin B (precipitate). The B fraction is then slurried with acidic DMF, followed by dilution of the filtrate in methanol and precipitation with water while maintaining pH 5. The precipitate (75-80% pure) is again dissolved in acidic DMF, diluted with pure methanol, and precipitated with water. Amphotericin A is several times less active than amphotericin B, and is usually encountered as a contaminant of B [Asher et al., 1977]. Amphotericin B is an amphoteric molecule with a carboxyl pKa of 5.7 and an amino pKa of 10.0 [Asher et al., 1977]. Amphotericin B is practically insoluble in water, alcohol, and in ether; slightly soluble in dimethylformamide and methyl alcohol; soluble in dimethyl sulphoxide and propylene glycol [USP 1995]. Due to the fact that amphotericin B contains an internal hemi-ketal ring, it has been suggested that the ketal-form may be in equilibrium with an open keto-form in solution [Asher et al., 1977]. However, 13C-NMR studies have provided no evidence for a keto-form in DMSO solution [Asher et al., 1977]. Dry amphotericin B powder appears stable for long periods of time at room temperature [Asher et al., 1977]. It is however suggested that amphotericin B powder should be stored at 2-8°C. Reconstituted colloidal dispersions should be protected from light and are stable for 24 hours at room temperature or 1 week when refrigerated. 26 Because of its poor water solubility, the clinically utilized form of amphotericin B (Fungizone®) consists of micelles of amphotericin B complexed with the bile salt deoxycholate as a solubilizing agent. Each vial of amphotericin B (Fungizone®) contains a sterile, nonpyrogenic, lyophilized cake providing 50 mg amphotericin B and 41 mg sodium deoxycholate with 20.2 mg sodium phosphates as a buffer [Squibb 1998]. Prior to injection, each of the lyophilized formulations is prepared by reconstitution with sterile water for injection without preservatives, then dilution with glucose injection 5% with a pH above 4.2 to the desired final concentration [USP 1995]. 1.3.2 Amphotericin B lipid complex Amphotericin B has been the mainstay of invasive antifungal therapy for nearly 40 years. Although the conventional form of injectable amphotericin B has offered the advantage of a fungicidal effect, many of its adverse effects - such as electrolyte imbalances, nephrotoxicity, and chills and fever during administration - have prevented widespread use of the agent [Wasan et al., 1993]. Due to these adverse effects, a number of alternative formulations have been developed with the goal of increasing the tolerance of amphotericin B without compromising the antifungal effects of the drug. As a result, amphotericin B is now commercially available as amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, and liposomal amphotericin B [AHFS 1998]. Amphotericin B lipid complex (ABLC) is not considered a true liposome, but a lipid-complex. The first lipid formulation to be studied in humans was the Juliano/Lopez-Berestein formulation (ABLC). It was composed of large multilayered liposomes of 27 heterogeneous size, or multilamellar vesicles (MLVs). The MLV was comprised of a mixture of two phospholipids, dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) in a 7:3 ratio (mol/mol), and contained 5 mol% of amphotericin B [Lopez-Berestein et al., 1985]. A reduction in cellular toxicity in humans was observed [Lopez-Berestein et al., 1985]. Over time it was eventually determined that the 5 mol% Juliano/Lopez-Berestein formulation was a heterogeneous mixture of both liposomal and ribbon-like, non-liposomal structures. Amphotericin B associated with the ribbon-like non-liposomal structures was complexed, and formed non-liposomal lipid-stabilized aggregates of amphotericin B with a diameter of about 2-1 lpm [Storm and Crommelin 1997]. Amphotericin B and lipid are arranged in a phase-separated 1:1 interdigitated complex [Lopez-Berestein et al., 1985]. It is suggested that amphotericin B-lipid pairs are arranged in cylinders. The hydrophobic polyene region of amphotericin B is aligned with the lipid hydrocarbon chains, and the polar hydroxyl groups face towards the center of the cylinder pores [Storm and Crommelin 1997]. The cylinders are aligned side by side and possess two polar ends (Figure 8) [Storm and Crommelin 1997]. Amphotericin B lipid complex (ABLC; Abelcet®) consists of a 1:1 molar ratio of amphotericin B complexed to a phospholipid vehicle composed of a 7:3 molar ratio of L-cc-dimyristoylphosphatidylcholine (DMPC) to L-a-dimyristolylphosphatidylglycerol (DMPG). Each milliliter of commercially available amphotericin B lipid complex suspension contains 5 mg of amphotericin B, 3.4 mg of DMPC, 1.5 mg of DMPG, and 9 mg of sodium chloride. The suspension occurs as a yellow, opaque liquid with a pH of 5.7 [AHFS 1998]. 28 29 1.3.3 Mechanism of action As a polyene (heptaene) antibiotic, amphotericin B, like the similar polyene macrolide, nystatin, has been shown to have antifungal and antiviral properties resulting from its high affinity for sterols found in biologic membranes. Amphotericin B usually is fungistatic in action at concentrations obtained clinically, but may be fungicidal in high concentrations or against very susceptible organisms [AHFS 1998]. Early on, researchers found that the effects of polyene antibiotics were specific to fungi, but not to bacteria [Hazan and Brown 1951]. Researchers later discovered that polyenes had the ability to alter membrane permeability in certain fungi, thus causing a release of cellular components. Over time it was shown that this specificity of action of the polyene antibiotics was due to a requirement of sterol being present in the membrane; more specifically the presence of 3p-hydroxy sterol in the plasma membrane [Weber and Kinsky 1965 and Lampen 1966]. It was also shown that in order for a biological membrane to be sensitive to polyenes it must contain a planar ring system, a hydrophilic side chain at the c-l7 position, as well as the afore mentioned 3p-hydroxy sterol [DeKruijff et al., 1974]. Although both fungal and mammalian membranes contain P-hydroxy sterols, both amphotericin B and nystatin appear to show selectivity for the ergosterol-containing membranes of fungi over those membranes comprised of cholesterol (i.e., mammals) [Norman et al., 1972 and DeKruijff et al., 1974]. The non-specific binding of amphotericin B to sterols; binding both ergosterol and cholesterol, however, has resulted in amphotericin B having a narrow therapeutic index [Storm and Crommelin 1997]. 30 Spectroscopic and permeability studies in membrane systems have suggested that amphotericin B interacts directly with membrane sterol to produce pores in the membrane which leads to an increase in membrane permeability and the eventual leakage of cellular components [Holz and Finkelstein 1970]. Cell death occurs in part as a result of permeability changes. Molecular models show that sterol (S) can be present on both sides of the double-bond region of the polyene molecule (P), effectively forming a "polymer-like" structure. This polymer is of the type (-P-S-)n and can form a closed cylinder that will have a hydrophobic exterior composed of sterol molecules and an inner channel lined with the hydrophilic head groups of the polyene ring. The hydroxyl moiety of the sterol and the mycosamine sugar of the polyene are situated at the hydrophilic end of the complex (at the membrane surface), while at the opposite, hydrophobic end of the complex a single hydroxyl group will be found (Figure 9). Upon addition of a polyene to a sterol-containing membrane system, the polyene would be drawn into the lipid layer, displacing the sterol from its association with the membrane phospholipids and eventually forming a sterol-polyene complex. Subsequent organization of the lipid would then occur allowing the individual sterol-polyene complexes to form channels. It is believed that two half pores come together, with hydrogen bonds occurring between the single hydroxyls at the hydrophobic ends of the polyene joining the ends of the two half pores. This results in a full pore of a length sufficient enough to span the normal biological membrane (Figure 9). 31 rr O CL UJ rr O CL oo O o - I 9: < _ l m D O OO 0 0 0 0 0 0 0 n o n n o o n CD C) O O O O O 0 0 O 0 O O OO"' Q O O O n n, n n n r> n nr> ^ U J V r -_i o O LJJ CL CO g CO t-o \ Q CL _1 o X CL 00 o X CL O rr H I t-00 g CO T^OOO O O O _i >- CO X CL 0 3 rr O o rr >- O x z o rr O i CL < 2 +-> co ' C x ! o £ co r v CO C C X G O H OJ • « .3 co ^4 o CJ -5 O* W >^ C D bJO rt O o DM « a .S s ^ 0 tJ) 1 1 — 1 o ^ • o C D C D -I—> co co CO C D £ C D O QQ C D C D -t-> c On DH « D O w co c « .2 £ C D X I -i-> C C D — X C D + - » t—1 o ** 5 a -o 1=3 ^ cd co S 2 CO rt o co C C D C ° x! C D rt X o co C D t-i O OH rt CO C D 32 1.3.4 Pharmacokinetics The pharmacokinetic behavior of amphotericin B is relatively complicated and has been best described by a three-compartment model [Gates and Pinney 1993]. According to this model, amphotericin B first distributes into a central compartment (e.g., the intravascular space). From this central compartment, amphotericin B equilibrates with two peripheral compartments. One of the compartments is a slowly equilibrating compartment with a larger volume of distribution. This compartment most likely represents distribution to the interstitial fluid of tissues such as skeletal muscle and skin. The other compartment is a rapidly equilibrating compartment with a small volume of distribution. This compartment most likely represents distribution to the interstitial fluid of tissues such as liver, spleen, and intestine. Amphotericin B deoxycholate is highly protein bound (90-95%), and its affinity to bind to cholesterol in cell membranes contributes to its large apparent volume of distribution of 4 L/kg [Wasan et al., 1990, Gates and Pinney 1993 and Chavanet et al., 1994]. The terminal half-life (ti/2) is approximately 15 days and this is primarily due to the slowly equilibrating compartment [Gates and Pinney 1993]. When amphotericin B is associated with a lipid molecule, the physicochemical properties of the lipid molecules influence the distribution of amphotericin B in the body, however, this influence lasts only as long as amphotericin B is complexed to the phospholipids. The pharmacokinetic profile of amphotericin B and ABLC, in a rabbit model, following the administration of a single dose can be seen in Table 2 [Wasan et al., 1998]. This study 33 showed that ABLC serum concentrations were lower than those for amphotericin B deoxycholate were. Table 2. Pharmacokinetic parameters and tissue distribution of drug after a single intravenous dose (1 mg/kg) to a normolipidemic rabbit model. Pharmacokinetic parameters Tissue distribution (ug of Amp B/g of tissue) AUC t 1 / 2a tiaP v v ss CL Kidney Liver Lung Spleen Heart (ug«h/mL) (h) (h) (mL/kg) (mL/kg) Fungizone® 11.3 ±2.5 0.2 ± 10.8 1252 ± 88.8 ± 1.07 ± 2.54 ± 0.88 + 3.50 ± 0.07 + 0.1 ±3.5 187 19.4 0.17 0.54 0.10 1.65 0.06 ABLC 2.1 ± 0.3 0.08 0.7 ± 5542 ± 471.6 ± 0.87 ± 4.81 ± 2.72 ± 6.85 ± 0.05 ± ±0.04 1.5 706 58.6 0.14 0.86 1.19 1.73 0.06 Data are expressed as means ± standard deviation. AUC - area under the concentration time curve, V s s - volume of distribution at steady state, CL - clearance, t 1 / 2a - distribution half-life and t1/2(3 - terminal half-life It is implied that this lower serum concentration may represent a rapid clearance of ABLC from the serum and subsequent sequestration by the MPS, as would be suggested by an increased clearance and larger steady-state volume of distribution for ABLC [Gates and Pinney 1993]. This theory is supported by the work of Wasan et al., (1998) presented in Table 2, which shows that in a single-dose pharmacokinetic study involving a rabbit model, the clearance (CL) and volume of distribution at steady state (Vss) of ABLC are increased over amphotericin B. As well, the tissue concentrations of ABLC are increased over those of amphotericin B. Specifically, the concentration of ABLC found within the liver, lung and spleen are significantly higher than amphotericin B levels in those same organs. Although the pharmacokinetic behavior of ABLC has not been fully described, some conclusions can be made [Rapp et al., 1997]: 34 1. ABLC has increased tissue distribution compared with amphotericin B deoxycholate 2. The enhanced tissue penetration should produce higher intracellular concentrations of amphotericin B 3. This should translate into a clinical benefit in the treatment of invasive mycosis by allowing more drug to reach the infected tissue 1.4 Lipoproteins as Transporters of Hydrophobic Drugs It has recently emerged that lipoproteins have a larger biological significance than simply that of lipid transport. They are also involved in the transport of a number of hydrophobic compounds. Drugs such as amphotericin B (AmpB), cyclosporine (CSA) and halofantrine (HF) have been shown to bind with plasma lipoproteins, resulting in the modification of the efficacy, tissue distribution, toxicity and pharmacological activity of these compounds [Wasan and Cassidy 1998]. 1.4.1 Antidepressants It has been suggested that the in vivo variability in response to a number of antidepressants may be due in part to the varying plasma lipid levels within patients [Bickel 1975, Danon and Chen 1979, Brinkschulte and Breyer-Pfaff 1980, Pike et al., 1982 and Shireman et al., 1983]. Bickel showed than when imipramine, a tricyclic antidepressant, was incubated in human blood that the drug was bound to three major blood components, membranes of red blood cells, albumin and lipoproteins, with equal affinity and capacity for both albumin and 35 lipoproteins [Bickel 1975]. Danon and Chen reported that the binding of imipramine to plasma lipoproteins was higher in hyperlipoproteinemic patients than in normal subjects and correlated well with both plasma cholesterol and triglyceride levels [Danon and Chen 1979]. As well, Brinkschulte and Breyer-Pfaff investigated the contribution of lipoproteins to the plasma binding of two other antidepressants, amitriptyline and nortriptyline [Brinkschulte and Breyer-Pfaff 1980]. It was reported that lipoproteins contributed 40 - 52% to total binding of these compounds. Taken together, these studies provide evidence that hydrophobic antidepressants interact with lipoproteins and that this interaction is influence by the variation in plasma lipid levels. 1.4.2 Vitamin E and Antiarrhythmic Agents A number of studies have reported that lipoproteins are the major transporters of vitamin E in the circulation, particularly apolipoprotein B containing (TRL, LDL) lipoproteins and nascent lipoproteins and chylomicrons [Rajaram et al., 1974, Vuilleumier et al., 1983, Gurusinghe et al., 1988, Traber et al., 1990, Traber and Packer 1994, Aten et al., 1994 and Traber 1994]. Furthermore, numerous studies have demonstrated that antiarrhythmic agents associate with lipoproteins [Ho and Sirois 1984, Ho and Sirois 1985, Urien et al., 1985, Northcote et al., 1986, Linden et al., 1990, Oravcova et al., 1994 and Seifert et al., 1997]. Ho and Sirois have demonstrated competitive binding of quinidine, propanolol and 4-OH-propanolol to human plasma TRL and LDL and that the nature and concentration of some ions may affect the in vitro binding of these compound to lipoproteins present in human serum [Ho and Sirois 1984, Ho and Sirois 1985]. Urien and co-workers reported that nicardipine was mainly bound to lipoproteins, orosomucoid, albumin and erythrocytes in 36 human blood [Urien et al., 1985]. They found that the determinants of nicardipine binding to lipoproteins were triglyceride, phospholipids and cholesterol ester. These findings suggest that not only the structure of surface apolipoproteins but also the plasma level of each lipoprotein fraction and drug lipophilicity are factors which may determine the interaction of these compounds with plasma lipoproteins. 1.4.3 Cyclosporine Cyclosporine has also been shown to bind to lipoproteins upon incubation in human plasma, resulting in a modification of its pharmacological effect [Awni et al., 1989, Awni and Sawchuk 1986 and Wasan et al., 1997]. Several investigators have reported decreases in cyclosporine activity in those patients that have hypertriglyceridemia and increases in cyclosporine toxicity in those with hypolipidemia [Nemunaitis et al., 1986, de Groen et al., 1987 and De Kippel et al., 1992]. In addition, Lemaire and co-workers have suggested that the drug's availability to tissue and thus its activity and toxicity may depend on the specific class of lipoprotein to which the drug is bound [Lemaire and Tillement 1982]. 1.4.4 Amphotericin B Amphotericin B is another example of a drug that is insoluble in water and binds to lipoproteins both in vivo and in vitro [Wasan 1996 and Wasan and Lopez-Berestein 1997]. Initially, Brajtburg and co-workers examined the interactions of amphotericin B with human serum lipoproteins [Brajtburg et al., 1984]. Their studies showed amphotericin B to be equally associated with HDL and LDL after 60 minutes of incubation at 25°C. However, Wasan and associates found that when amphotericin B was incubated in human serum for 60 37 minutes at 37°C, over 75% of the initial amphotericin B concentration incubated was recovered within the serum HDL fraction [Wasan et al., 1993]. It is suggested that the binding of amphotericin B to lipoproteins may have a major impact on the safety of this drug since amphotericin B is often administered to patients with abnormal serum cholesterol and triglyceride metabolism [Lopez-Berestein 1988, Pontain et al., 1989, Chabot et al., 1989 and Wasan et al., 1990]. There is growing evidence that suggests that increases in cholesterol concentrations increase the renal toxicity of amphotericin B, while an elevation in serum triglyceride levels decrease amphotericin B induced renal toxicity. Preliminary findings suggest that patients with higher serum LDL-cholesterol levels, and in turn a greater binding of amphotericin B with serum LDL are more susceptible to amphotericin B induced kidney toxicity [Wasan and Conklin 1997]. Koldin and co-workers demonstrated elevated amphotericin B induced nephrotoxicity when LDL-associated amphotericin B was administered to hypercholesterolemic rabbits [Koldin et al., 1985]. Barwicz and co-workers have demonstrated decreased toxicity in mice in conjunction with the inhibition of the amphotericin B-LDL interaction [Barwicz et al., 1991]. Furthermore, Chavanet and co-workers have observed that an increase in serum triglyceride concentration led to a reduction in amphotericin B toxicity [Chavanet et al., 1994]. These human and animal studies provide preliminary evidence that serum LDL cholesterol and serum triglyceride levels have a major impact on the renal toxicity of amphotericin B. 38 1.5 Significance of Proposed Research Understanding how variations in plasma lipid concentrations affect hydrophobic drug interactions with lipoproteins could help explain the changes in the pharmacokinetics and pharmacodynamics of amphotericin B when administered to patients that exhibit modifications in their lipoproteins and lipid metabolism. These findings may also be extrapolated to other compounds incorporated into phospholipid complexes of similar composition. 3 9 HYPOTHESIS The human plasma lipoprotein distribution of amphotericin B (AmpB) and amphotericin B lipid complex (ABLC) is dependent on the lipid composition - specifically, the total cholesterol, esterified cholesterol, free cholesterol, total triglyceride, and phospholipid content - of four characteristically distinct groups within human plasma: i) triglyceride rich lipoproteins (consisting of very low density lipoproteins and chylomicrons) (TRL) ii) low-density lipoproteins (LDL) iii) high-density lipoproteins (HDL) iv) lipoprotein-deficient plasma (LPD). Furthermore, the incorporation of amphotericin B in lipid complexes consisting of DMPG and DMPC alters the plasma lipoprotein distribution of the drug when compared to the lipoprotein distribution of the free, non lipid complex formulation of amphotericin B. Chapter 2 Material and Methods 41 2.1 Chemicals and Reagents Pooled human plasma was obtained from the Vancouver Red Cross (Vancouver BC, Canada). Methanol (MeOH), dichloromethane (DCM), acetonitrile and other organic solvents were purchased from Fisher Scientific Canada (Toronto ONT, Canada). Sodium bromide and sodium acetate was purchased from Sigma Chemical Co. (St. Louis MO, USA). Cholesterol Reagent, Cholesterol Calibrators, Triglyceride (INT) Reagent, Triglyceride Calibrators, and Protein Assay kits were purchased from Sigma Diagnostics (St. Louis MO, USA). Free Cholesterol analysis kits and Phospholipid analysis kits were purchased from Boehringer Mannheim (Laval QUE, Canada). Acrylamide, bis-Acrylamide, and other electrophoresis reagents were purchased from Biorad Laboratories (Canada) Ltd (Mississaugua ONT, Canada). 2.2 Preparation of Analytes and Solutions 2.2.1 Amphotericin B solution Amphotericin B (5 mg/mL) was reconstituted by the addition of 10 mL sterile water for injection directly into the sterile vial. The vial was shaken immediately until the colloidal solution was clear. The solution was protected from light and stored at 4°C for the duration of the experiment. 42 2.2.2 Amphotericin B lipid complex The method of lipid-complex formation containing AmpB has been previously described [Mehta et al., 1987 and Lopez-Berestein et al., 1985]. Briefly, AmpB dissolved in methanol is mixed with chloroform solutions of dimyristoyl phosphatidylcholine [DMPG] and dimyristoyl phosphatidylglycerol [DMPG] [Mehta et al., 1987 and Lopez-Berestein et al., 1985]. Organic solvents are removed by evaporation under vacuum, with the resultant powder containing a 7:3 (wt/wt) ratio of DMPC:DMPG and a total lipid to drug ratio of 1:1 (wt/wt). The mean diameter of these lipid-based vesicles are less than 5 um as determined by quasi-electric light scattering (QES) using a Nicomp submicron particle sizer, Model 270 (Pacific Scientific, Santa Barbara, CA; USA) [Wasan and Conklin 1997 and Wasan and Cassidy 1998]. 2.2.3 DTNB solution Preparation of a DTNB 15 mM solution was accomplished by dissolving 60 mg of DTNB powder in 10 mL of phosphate buffer solution (pH 7.4). The phosphate buffer was made by preparing a 0.1 M solution of K2HPO4 and a 0.1 M solution of KH2PO4; the pH of one solution was brought to pH 7.4 by the dropwise addition of the other solution [Wasan et al., 1997]. 43 2.3 Heterogeneity of Human Plasma Lipoprotein Profiles 2.3.1 Pre-screening of human plasma Randomly selected human plasma, obtained from the Red Cross (Vancouver BC, Canada), was provided freshly drawn (i.e. removed from the donor within the previous 48 hours) and frozen. 2.4 Lipoprotein Separation Techniques 2.4.1 Step-gradient ultracentrifugation For the separation of lipoprotein plasma components by step-gradient ultracentrifugation, sodium bromide density solutions were carefully layered on top of the plasma sample in order of highest to lowest density. The density of the plasma was initially altered such that it had the greatest density of all layers in the gradient. Samples were ultracentrifuged overnight and separation of the lipoprotein fractions was accomplished in a single spin. Each distinct layer was removed and separated for further analysis. 2.4.1.1 Treatment of plasma with free or liposomal amphotericin B To an Ultraclear centrifuge tube (Beckman Instruments, Inc.; Palo Alto CA, USA), 2.88 mL and 3.0 mL of human plasma was added for sample (n=3 for AmpB and n=3 for ABLC) and standard curve (n=6) purposes. The contents of all tubes were pre-warmed to 37°C. To the samples tubes was added either 12 uL of an AmpB 5 mg/mL solution or 12 uL of an ABLC 5 mg/mL suspension. The final concentration for both AmpB and ABLC formulations was 44 20 pg/mL of amphotericin B in human plasma. Immediately after addition, the samples were returned to 37°C and incubated for 60 minutes whereupon they were removed and cooled on ice for 30 minutes. A concentration of 20 pg/mL was chosen as it is the peak concentration observed in the plasma of mice after the administration of an I.V. bolus dose. The incubation period was 60 minutes for these studies due to the fact that research by our lab has shown that the distribution phase of ABLC after administration lasts 60 minutes. Finally, an incubation temperature of 37°C was chosen to simulate body temperature. Thus taken together, these three conditions are thought to mimic physiological conditions. Plasma employed for standard curve purposes was subjected to identical incubation and cooling times as the sample plasma. 2.4.1.2 Separation of lipoprotein constituents Briefly, all density solutions, 8 = 1.006, 1.063, and 1.21 g/mL, were stored at 4°C prior to the layering of the gradient. To the previously cooled standard curve and sample plasma was added 1.02 g of accurately weighed 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, Hatch and Lees 1968 and Wasan et al., 1997]. Once the sodium bromide had dissolved into the plasma, 2.8 mL of the highest density sodium bromide solution (8 = 1.21 g/mL) was carefully layered on top of the plasma. Using the same volume of 2.8 mL, the next highest sodium bromide solution (8 = 1.063 g/mL) was layered on top of the sample, followed by 2.8 mL of the lowest density solution (8 = 1.006 g/mL). 45 The ultracentrifuge tubes were balanced, 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, Inc.; Palo Alto Ca, USA) and centrifuged at 40,000 rpm for 18 hours at a temperature of 15°C in a Beckman L8-80M Ultracentrifuge (Beckman Instruments, Inc.; Palo Alto CA, USA). Upon completion of the run, the ultracentrifuge tubes were carefully removed from the titanium buckets. Each tube showed four visibly distinct regions represented by the TRL, LDL, HDL, and LPD fractions (Figure 10). Subsequently, each of the layers was removed by Pasteur pipette from top to bottom layer respectively and the volumes of each of the fractions were measured. Fractions removed for standard curve purposes were pooled together. All sample and pooled standard curve fractions were transferred to clean test tubes, covered, and stored at 4°C until further analysis. 2.4.1.3 Controls A proper set of experimental controls was required in order to determine that the lipoprotein distribution of amphotericin B was a result of the association of amphotericin B with the plasma lipoproteins and not some other function. Lipoprotein-deficient plasma was utilized as the control medium [Wasan et al., 1997]. The lipoprotein-deficient fraction was acquired employing the technique of step-gradient ultracentrifugation as described above. The lipoprotein-deficient plasma was dialyzed (MWCO = 1000) against a 0.9% sodium chloride solution for 24 hours at a temperature of 4°C and then used as the control medium for incubation of AmpB or ABLC. Preparation of the samples with AmpB or ABLC as well as separation of constituents was carried out utilizing identical techniques as those described above for step-gradient ultracentrifugation. 46 Triglyceride rich lipoproteins Low density lipoproteins High density lipoproteins Lipoprotein-deficient plasma Figure 10: A representation of the separation of plasma lipoprotein fractions in an Ultraclear® 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. Separation is based on the density of the plasma components; therefore the constituents of low density will rise to the top and those of heavier density will remain at the bottom. 47 After ultracentrifugation, the removal of each "lipoprotein" fraction was estimated by comparison to previously separated lipoprotein fractions. That is, the layer removed where TRL would normally reside was based on previous separations of actual plasma in which the layers were visible. In this way, removal of the non-lipoprotein fractions in the control medium was estimated as close to the placement of their separated lipoprotein counterparts in plasma. 2.4.2 Step-Gradient ultracentrifugation of HDL2/HDL3 2.4.2.1 Treatment of plasma with free or liposomal amphotericin B To an Ultraclear centrifuge tube (Beckman Instruments, Inc.; Palo Alto CA, USA), 1.64 mL and 1.7 mL of human plasma was added for sample (n=3 for AmpB and n=3 for ABLC) and standard curve (n=6) purposes. The contents of all tubes were pre-warmed to 37°C. To the samples tubes was added either 6.4 pL of an AmpB 5 mg/mL solution or 6.4 pL of an ABLC 5 mg/mL suspension. The final concentration for both AmpB and ABLC formulations was 20 pg/mL of amphotericin B in human plasma. Immediately after addition, the samples were returned to 37°C and incubated for 60 minutes whereupon they were removed and cooled on ice for 30 minutes. Plasma employed for standard curve purposes was subjected to identical incubation and cooling times as the sample plasma. 2.4.2.2 Separation of lipoprotein constituents Briefly, solutions of all densities (density [8] = 1.00, 1.19, and 1.25 g/ml) were stored at 4°C prior to the layering of the gradient. To the previously cooled plasma used for standard curves and the plasma samples was added 0.936 g of accurately weighed sodium bromide in 48 order to modify the density of the plasma to approximately 1.40 g/ml [Groot et al., 1982]. Once the sodium bromide had dissolved into the plasma, 1.70 ml of the highest-density sodium bromide (8=1.25 g/ml) was carefully layered on top of the plasma. By using the volume of 6.45 ml, the next highest sodium bromide solution (8 = 1.19 g/ml) was layered on top of the sample, followed by 1.70ml of the lowest-density solution (8 = 1.00 g/ml). The ultracentrifuge tubes were balanced, 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, Inc.) and centrifuged at 40,000 rpm for 21 h at a temperature of 15°C in a Beckman L8-80M Ultracentrifuge (Beckman Instruments, Inc.). Upon completion of the run, the ultracentrifuge tubes were carefully removed from the titanium buckets. Each of the layers was removed from the top to the bottom layer with the following volumes; TRL/LDL - 2.0 mL, HDL2 - 2.4 ml, HDL3 - 4.8 ml, and LPD - bottom 4.2 ml. Fractions removed for standard curve purposes were pooled together. All sample and pooled standard curve fractions were transferred to clean test tubes, covered, and stored at 4°C until further analysis. 2.4.2.2 Controls LPD was used as the control medium. The LPD was acquired by the technique of step-gradient ultracentrifugation as described above for HDL2/HDL3. The LPD was dialyzed as previously described and then used as the control medium for the incubation of AmpB or ABLC. Preparation of the samples with AmpB or ABLC, as well as separation of the 49 constituents was carried out by techniques identical to those described above for HDL2/HDL3 step-gradient ultracentrifugation. 2.4.3 Gradient gel electrophoresis 2.4.3.1 Electrophoresis buffer The gradient gel electrophoresis buffer consisted of Tris base (90 mM), boric acid (80 mM), EDTA (2 mM); pH 8.35 [Rainwater et al., 1997]. The procedure for the formation of the TBE buffer was as follows: 10.90 g Tris, 4.95 g H3BO4 and 1.12 g Na2EDTA were dissolved in distilled water and the solution was made up to 1 L. 2.4.3.2 Gradient gel formation Each of the two acrylamide stock solutions used to construct the gradient was made up in TBE buffer, which was previously described. The two stock solutions were made as follows: a. 292.95 g/L Acrylamide and 17.05 g/L fe-acrylamide (31% total, 5.5% crosslinker). b. 28.8 g/L Acrylamide and 1.2 g/L te-acrylamide (3.0% total, 4% crosslinker). Both solutions were subsequently filtered immediately following preparation. Working solutions were made just before gel casting: c. 31%: To each 30 mL of stock solution a, 1.5 mL freshly prepared ammonium persulfate (1 g/lmL) and 20 pL TEMED were added. d. 3%: To each 30 mL of stock solution b, 1.5 mL freshly prepared ammonium persulfate (1 g/mL) and 20 pL of TEMED were added. 50 The gradients were formed using a Biorad Model 385 Gradient Gel Former. To the reservoir chamber of the gradient former, 19.2 mL of 3% to-acrylamide was added, followed by 19.2 mL of 31% 6/5-acrylamide to the mixing chamber of the gradient former. Into the mixing chamber was placed a magnetic stir bar. Gels were poured within 10 minutes of the addition of the polymerization agent, TEMED, and allowed to polymerize for a minimum of 1 hour before subsequent use. 2.4.3.2 Sample preparation and electrophoresis In a 0.6 mL eppendorff tube, 40 pL of ultracentrifugal < 1.20 fraction and 10 pL of a solution consisting of: sucrose (40%) and bromophenyl blue (0.05%) were vortexed. To the gels, 20 pL of the above mixture was added. For each gel, a spacer solution was prepared. Four parts of a solution having a salt concentration equivalent to background salt concentration of the lipoprotein sample were mixed with one part of a solution consisting of: sucrose (40%) and bromophenyl blue (0.05%). To each of the unused sample lanes in the gel, 20 pL of spacer solution was applied. The high molecular weight standards calibration kit (Amersham Pharmacia Biotech, Baie d'Urfe QUE, Canada), a mixture containing thyroglobulin (17 nm diameter), ferritin (12.2 nm) lactate dehydrogenase (8.16 nm), and albumin (7.1 nm) was used as calibrators for the HDL subclasses. To each gel that was run, 20 pL of standard was added. 51 Samples were electrophoresed in a PROTEAN® II xi Cell, utilizing a Power Pac 1000 at 10°C in the following sequence: 15 min at 15 V, 20 min at 70 V, 24 hr at 125 V (constant voltage; 3000 volt-hours). 2.4.3.3 Gel staining and destaining Gels were immediately exposed after electrophoresis to sulfosalicylic acid (10%) for one hour. After the fixation step, gels were stained for one hour by exposing the gels to a solution containing Coomassie G-250 (0.04%) in methanol and acetic acid. Gels were destained in acetic acid (10%) and methanol (30%) until the background was clear. 2.5 Amphotericin B Quantification 2.5.2 Standard curve preparation To a series of test tubes labeled: 0, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5, and 10 pg/mL was added 0.5 mL of pooled standard curve fraction and to a 20 pg/mL test tube was added 0.96 mL of the same pooled fraction. A 4 u L aliquot of either AmpB 5 mg/mL solution or ABLC 5 mg/mL suspension was then added to the 20 pg/mL test tube, and vortexed for 10 s. From the 20 pg/mL test tube, 0.5 mL was transferred to the 10 pg/mL test tube. This mixture was vortexed for 10 s whereupon a 0.5 mL aliquot of this mixture was subsequently transferred to the 5 pg/mL test tube. This procedure of serial dilution was carried out for the remaining standard curve tubes (not including the 0 pg/mL test tube). Note, 0.5 mL from the 0.039 pg/mL test tube was discarded to provide a final volume of 0.5 mL for all standard curve concentrations. 52 2.5.2 Determination of amphotericin B content within the separated lipoprotein fraction 2.5.2.1 V L D L and L D L To an appropriately labeled test tube, a 0.5 mL aliquot of sample was added. To these sample tubes, as well as the standard tubes, was added 3.0 mL of dichloromethane (DCM). The mixture was vortexed for 10 s and all samples were dried to completion under a steady stream of nitrogen at ambient temperature. Once dried, amphotericin B was extracted from the residue using a methanol wash. Extraction efficiency was determined to be > 90% [Wasan et al., 1997]. Next, a 3.0 mL aliquot of methanol was added to the residue and vortexed for 20 s. The mixture was allowed to stand for 5 minutes and was vortexed again for 20 s. This methanol was then dried to completion under a steady stream of nitrogen at ambient temperature. Immediately prior to analysis, the residue was reconstituted with 0.5 mL of methanol and injected onto the column. 2.5.2.2 H D L To an appropriately labeled test tube, a 1.0 mL aliquot of sample was added. To these sample tubes as well as the standard curve tubes was added 3.0 mL of dichloromethane (DCM). The mixture was vortexed for 10 s and all samples were dried to completion under a steady stream of nitrogen at ambient temperature. Once dried, amphotericin B was extracted from the residue using a single methanol wash. A 3.0 mL aliquot of methanol was next added to the residue and vortexed for 20 s. The mixture was allowed to stand for 5 minutes and was vortexed again for 20 s. This methanol was then dried to completion under a steady stream of nitrogen at ambient temperature. Immediately prior to analysis, the residue was reconstituted with 0.250 mL of methanol and injected onto the column. 53 2.5.2.3 LPD To an appropriately labeled test tube, a 0.5 mL aliquot of sample was added. To these sample tubes as well as the standard curve tubes was added 3.0 mL of dichloromethane (DCM). The mixture was vortexed for 10 s and all samples were dried to completion under a steady stream of nitrogen at ambient temperature. Once dried, amphotericin B was extracted from the residue using a series of methanol "washes". Briefly, a 3.0 mL aliquot of methanol was added to the residue and vortexed for 20 s. All test tubes were then centrifuged at 2000 rpm for 2 minutes at 15°C. The supernatant was transferred to a clean test tube and the procedure repeated an additional two times with 2.0 mL methanol. The supernatant from each of the three extraction steps was pooled with the previous supernatant to provide a final volume of approximately 7 mL of methanol. This pooled methanol was then dried to completion under a steady stream of nitrogen at ambient temperature. Immediately prior to analysis, the residue was reconstituted with 0.5 mL of methanol and injected onto the column. 2.5.3 HPLC apparatus The HPLC system consisted of a Waters 600 Controller interfaced to a Waters 717P|US Autosampler and a Waters 486 Tunable Absorbance Detector. The detector was set at an UV absorbance wavelength of 405 nm and an absorbance sensitivity of 0.05 absorbance units -full scale. All results were recorded on a Waters 746 Data Module integrator (Waters Corporation; Milford MA, USA). Attenuation was set to 64 and chart speed to 0.25 cm/min. Samples (100 pL volume) were injected onto a Zorbax SB-C18 column (4.6 x 150 mm; 5 pm particle size), pre-fitted with a Zorbax Reliance SB-C18 guard column (4.6 x 12.5 mm; 5 pm 54 particle size) (Rockland Technologies Inc., Napean ONT, Canada). Chromatographic separation was carried out at ambient temperature. The mobile phase employed an isocratic flow and consisted of a 10 mM sodium acetate: acetonitrile [70:30 (wt/wt)] mixture termed "mobile phase A". Flow was at a rate of 1.5 ml/min. 2.6 L i p i d & Protein Content Analysis of Lipoprotein and Lipoprotein-Deficient Plasma The lipid and protein concentration of each lipoprotein and lipoprotein-deficient fraction was analyzed by enzymatic assay utilizing colorimetric reactions. Standard curves were prepared for each analysis and all solutions employed were prepared immediately prior to analysis. 2.6.1 U V spectrophotometry apparatus All spectrophotometric measurements were performed using a Hewlett Packard 8452A Diode Array Spectrophotometer interfaced with a Hewlett Packard Vectra N2 Personal Computer utilizing HP89532A UV-Vis software. UV absorbance readings for total cholesterol, free cholesterol, total triglyceride, phospholipid, and total protein concentrations were measured at a wavelength of 506, 500, 500, 500, and 750 nm respectively. Lipid and protein content were determined from their respective standard curves or calculated using the equations provided in the following sections. 2.6.2 Determination of total cholesterol concentration Total cholesterol determination was performed utilizing an enzymatic procedure based on a modification of the method of Allain et al., (1974). This method measures both free 55 cholesterol and cholesteryl ester concentration by first hydrolyzing the cholesteryl esters to free cholesterol and then reacting all of the free cholesterol in the sample with the chromagen in the presence of cholesterol oxidase and peroxidase enzymes. The enzymatic reactions are as follows: cholesteryl esters + H 20 c h o l e s t e r o 1 e s t e r a s e cholesterol + fatty acids cholesterol + 0 2 c h o'* s t e r o 1 o x i d a s e cholest-4-en-3-one + H 20 2 2 H 2 0 2 + 4-aminoantipyrine + phenol p e r p x i d a s e quinoneimine + 4 H 20 Initially, cholesteryl esters are hydrolyzed by cholesterol esterase to produce cholesterol and fatty acids. Cholesterol, both that produced by hydrolysis as well as any free cholesterol already present in the sample, is oxidized in the presence of cholesterol oxidase to cholest-4-en-3-one and hydrogen peroxide (H202). The H 20 2 is then coupled with 4-aminoantipyrine and p-hydroxybenzene-sulfonate by the catalyst, peroxidase, to yield a highly coloured quinoneimine dye, which has an absorbance maximum of 505 nm. The Cholesterol Reagent® (Sigma Diagnostics; St. Louis MO, USA) was reconstituted with 100 mL of distilled water. After addition of water, inversion and gentle swirling facilitated dissolution and mixed the contents of the vial. Any unused reconstituted Cholesterol Reagent® was stored at 4°C for up to 60 days. To a series of test tubes labeled 0, 12.5, 25, 50, 100, and 200 mg/dL was added a 10 pL aliquot of stock solutions corresponding to their respective concentrations; these were employed for the standard curve. Similarly, a 10 pL aliquot from each separated fraction - TRL, LDL, HDL, and LPD - was also measured. To each of the standard curve and sample aliquots was added 1 mL of Cholesterol Reagent®. 56 The resulting mixture was vortexed for 10 s and incubated at 37°C for 5 minutes. Using an UV spectrophotometer, absorbance readings of all solutions were measured at 506 nm in a cuvette of 1 cm pathlength. Total cholesterol concentrations of each sample were subsequently determined directly from the standard curve. 2.6.3 Determination of free cholesterol concentration Unesterified cholesterol (free cholesterol) concentration determination was accomplished by the use of a series of enzymatic steps similar to those employed in the determination of total cholesterol. Free, unesterified cholesterol is oxidized in the presence of cholesterol oxidase to produce A4-cholestenone and H2O2. The H2O2 is then reacted with 4-aminophenazone and phenol in the presence of peroxidase to yield the chromagen, 4-(p-benzo-quinone-monoimino)-phenazone and water [Stahler et al., 1977 and Trinder 1969]. The chromagen has an absorbance maximum of 505 nm. The enzymatic reactions are as follows: cholesterol + 0 2 c h 9 ' e s t e r o l o x i d a s e A4-cholestenone + H 20 2 2 H2O2 + 4-aminophenazone + phenol p e r o x i d a s e 4-(p-benzo-quinone-monoimino)-phenazone + 4H 2 0 Free cholesterol reagent was prepared in the following manner. To the contents of a bottle containing 45 mL of 4-aminophenazone 2 mmol/L solution was added 0.5 mL of a solution containing cholesterol oxidase > 12 U/mL and peroxidase > 8 U/mL. To this mixture was added 45 mL of a phenol 20 mmol/L solution. The resultant free cholesterol reagent was then mixed by gentle swirling and inversion and was stable for 2 weeks when stored at 4°C. 5 7 To an appropriately labeled test tube was added a 10 pL aliquot from each separated fraction - TRL, LDL, HDL, and LPD; a reagent blank test tube containing 10 pL of distilled water was also prepared. A 1 mL aliquot of the prepared free cholesterol reagent was subsequently added to the sample and reagent blank test tubes. The contents of each test tube were mixed and incubated for 10 minutes at 37°C. The absorbance of the sample solution was then read against that of the reagent blank (AA) in a cuvette of 1 cm pathlength at a wavelength of 500 nm. The concentration of free cholesterol in each sample was determined by the equation: Equation 1. C = 585 x AA [mg/dL] 2.6.4 Determination of cholesteryl ester concentration Cholesteryl ester concentration of each separated lipoprotein fraction was determined by subtraction of the free, unesterified cholesterol concentration, from the total cholesterol concentration of the same sample. Equation 2. Cholesteryl ester = total cholesterol - free cholesterol 2.6.5 Determination of total triglyceride concentration The method for determination of total triglyceride was based on a modification of the method of Bucolo and David [Bucolo and David 1973]. Triglycerides are hydrolyzed by lipase to glycerol and free fatty acids. The resulting glycerol produced is then measured by coupled enzyme reactions catalyzed by glycerol kinase (GK), glycerol-1-phosphate dehydrogenase, and diaphorase. The enzymatic reactions involved in this process are as follows: 58 triglyceride l i p o p r o t e i n l i p a s e glycerol + fatty acids glycerol + ATP s ' v c e r o l k i n a s e glycerol-1-phosphate + ADP glycerol-l-phosphate + NAD g l v c e r o M ' p h o s p h a t e D A P + NADH NADH + INT d j a p J j e m s e formazan + NAD Initially triglycerides are hydrolyzed by lipoprotein lipase to produce glycerol and free fatty acids. The glycerol is subsequently phosphorylated by adenosine-5-triphosphate (ATP), in the presence of glycerol kinase (GK), to produce glycerol-l-phosphate (G-l-P) and adenosine-5-diphosphate (ADP). The enzyme glycerol-l-phosphate dehydrogenase (G-l-PDH) then catalyses the oxidation of G-l-P to dihydroxyacetone (DAP) with the concomitant reduction of nicotinamide adenine dinucleotide (NAD) to NADH. The NADH is oxidized with the simultaneous reduction of 2-[p-iodophenyl]-3-p-nitrophenyl-5-phenyl-tetrazolium chloride (INT) to produce INTH (formazan) and NAD in the presence of the catalyst, diaphorase. The product formazan is highly coloured with an absorbance maximum wavelength at 500nm. The Triglyceride (INT) Reagent® (Sigma Diagnostics; St. Louis MO, USA) was reconstituted with 20 mL of distilled water. After addition of water, contents of the vial were mixed by inversion and gentle swirling to facilitate dissolution. Any unused reconstituted Triglyceride (INT) Reagent® was stored at 4°C for up to 5 days. To a series of test tubes labeled 0, 15.625, 31.25, 62.5, 125, and 250 mg/dL was added a 10 pL aliquot of stock solution corresponding to their respective concentrations; these were employed for the standard curve. Similarly, a 10 pL aliquot from each separated fraction - TRL, LDL, HDL, 59 and LPD - was also measured. To each of the standard curve and sample aliquots was added 1 mL of Triglyceride (INT) Reagent®. The resulting mixture was vortexed for 10 s and incubated at 37°C for 5 minutes. Using an UV spectrophotometer, absorbance readings of all solutions were measured at 500 nm in a cuvette of 1 cm pathlength. Total triglyceride concentrations of each sample were subsequently determined directly from the standard curve. 2.6.6 Determination of phospholipid concentration Phospholipid concentration measurements (specifically phosphatidylcholine) were performed based on the following test principle. Phospholipids are catalyzed by the enzyme, phospholipase D to produce choline and phosphatidic acids. The choline is subsequently oxidized in the presence of choline oxidase to yield betaine and hydrogen peroxide ( H 2 O 2 ) . The last step of this series of reactions involves a similar reaction previously described for total and free cholesterol determination. Hydrogen peroxide combines with 4-aminophenazone and phenol in the presence of peroxidase to produce 4-(j?-benzo-quinone-monoimino)-phenazone (the chromagen) and water [Trinder 1969, Takayama et al., 1977]. The chromagen is a highly coloured dye, which has an absorbance maximum at 500 nm. The series of reactions are as follows: 60 phospholipids + H 20 p h o g p h o l i p a s e D choline + phosphatidic acids choline + 2 0 2 + H 20 choline oxidase betaine + 2 H 20 2 2 H 20 2 + 4-aminophenazone + phenol peroxidase 4-(p-benzoquinone-monoimino)phenazone + 4H 20 Phospholipid reagent was prepared by dissolving the contents of one enzyme reagent bottle in 40 mL of provided buffer solution. This working reagent solution contained phenol 20 mmol/L, 4-aminophenazone 8 mmol/L, phospholipase D > 1000 U/L, choline oxidase > 1400 U/L, and peroxidase > 800 U/L and was stable for up to two weeks when stored at 2 to 8°C. To an appropriately labeled test tube was added a 10 pL aliquot from each separated fraction - TRL, LDL, HDL, and LPD. A standard test tube containing 10 pL of a 54.1 mg/dL choline chloride solution (equivalent to 300 mg phospholipids per dL) was also prepared. The reagent blank test tube contained a similar volume of distilled water. To the sample, standard, and reagent blank test tubes was added a 1.5 mL aliquot of the previously prepared phospholipid reagent solution. The contents of each tube were mixed and incubated at 37°C for 10 minutes whereupon the absorbance of the samples ( A A ) and standard ( A A s t d ) were read against the reagent blank within 2 hours. Concentration of phospholipid in each sample was calculated based on the following equation: Equation 3. C = 300 x A A [mg/dL] A A s t d 61 2.6.7 Determination of total protein concentration The procedure utilized for determination of total protein is based on Peterson's modification of the micro-Lowry method [Peterson 1977]. This method employs sodium dodecylsulfate, found in the Lowry Reagent, to facilitate the dissolution of relatively insoluble proteins. The Lowry Reagent, an alkaline cupric tartrate reagent, then complexes with the peptide bonds and forms a blue-purple colour when the phenol reagent, Folin and Ciocalteu's Phenol Reagent is added [Lowry et al., 1951]. Absorbance is read at a suitable wavelength between 500 and 800 nm and the sample protein concentrations are determined from a calibration curve. Lowry Regent solution was prepared by reconstituting a bottle of Lowry Reagent Modified, with 40 mL of distilled water. The solution was mixed by inversion and gentle swirling to aid in dissolution. Folin and Ciocalteu's Phenol Reagent Working Solution was prepared by mixing 5 mL of 2N Folin and Ciocalteu's Phenol Reagent with 25 mL of distilled water (i.e. a 1 part to 5 part mixture). Unused Lowry Reagent solution and Folin and Ciocalteu's Phenol Reagent Working Solution were stored at room temperature for a maximum of two weeks. Protein Standard Solution, equivalent to bovine serum albumin 400 pg/mL, was prepared by adding to the vial 5 mL of distilled water. Contents of the vial were then mixed by inversion and gentle swirling. Any unused Protein Standard Solution was stored at 4°C and was stable for up to 3 months. A protein standard curve was prepared by diluting the Protein Standard Solution in distilled water to a volume of 1 mL in a series of test tubes as follows: 62 Protein Standard Solution Water (mL) Protein Concentration (mL) (pg/mL) 0 1.00 0.875 0.750 0.500 0.250 0 0 0.125 0.250 0.500 0.750 1.00 50 100 200 300 400 For all separated TRL fractions, a 300 pL aliquot of sample was added to an appropriately labeled test tube and diluted with 0.70 mL of distilled water for a final volume of 1.0 mL. Similarly, for all LDL and HDL fraction samples, a 100 pL aliquot of sample was diluted to 1.0 mL with 0.900 mL distilled water. For the LPD total protein measurements, 10 pL of sample was diluted to 1 mL with 0.990 mL distilled water. To both standard curve and sample test tubes was added 1.0 mL of Lowry Reagent Solution; the mixture was vortexed for 10 s and allowed to stand at ambient temperature for 20 minutes. Immediately following, 0.5 mL of Folin and Ciocalteu's Phenol Reagent Working Solution was added to each test tube and vortexed for 10 s. The resulting colour was subsequently allowed to develop over the next 30 minutes whereupon the absorbance of each standard and sample solution was measured at a wavelength of 750 nm within 30 minutes. Protein concentrations of the diluted samples were determined directly from the standard curve. Final protein concentrations of the separated fractions were calculated based on their initial dilution factor. 63 Statistical Analysis Differences in the lipid and protein content of the separated lipoprotein fractions between the different plasma samples were determined by one-way ANOVA (InStat; GraphPad Software). Similarly, differences in the distribution of AmpB or ABLC within the separated lipoprotein and lipoprotein-deficient fractions between plasma samples were also determined by one-way ANOVA. Post tests were performed using the Tukey-Kramer test. Differences between the distribution of AmpB and ABLC into the separated lipoprotein fractions for each patient plasma sample were determined by student t-test. Correlation coefficients between the amount of AmpB recovered within the TRL, LDL, and HDL plasma fractions and the amount of lipid within these fractions was determined using Pearson's Test. Differences were considered significant if p was < 0.05. All data are expressed as mean ± standard deviation. Chapter 3 Results 65 3.1 Amphotericin B HPLC Quantification In order to determine the concentration of AmpB (Fungizone®) or AmpB (ABLC) within each separated lipoprotein and lipoprotein-deficient fraction it was necessary to compare the area of the peak recovered from the HPLC chromatogram of the sample to a standard curve for that same fraction. External calibration curves were prepared for each and every separated fraction from each of the seven patient plasma samples. Put simply, a separate standard curve was prepared for each of the four separated fractions: TRL, LDL, HDL, and LPD; and these four standard curves were repeated for each of the seven different plasma samples. The standard curves for each plasma lipoprotein and lipoprotein-deficient fraction were linear for AmpB over a range of 0.039 to 20 pg/mL; the correlation coefficient was greater than 0.994 for each regression line (Table 3). Table 3. Representative linear calibration curves for amphotericin B as determined in the separated lipoprotein and lipoprotein-deficient plasma fractions for plasma sample 3 Separated fraction in Equation Coefficient of Standard Curve which calibration determination Range curve was determined (r2) (pg/mL) Amphotericin B TRL LDL HDL LPD cone. - (area+65812)/539086 cone. = (area+104768)/469604 cone. = (area+352280)/lE+06 cone. = (area+95843)/355930 0.9989 0.9964 0.9981 0.9948 0.039 to 20 0.039 to 20 0.039 to 20 0.039 to 20 The retention time of the drug following extraction from the lipoprotein and lipoprotein-deficient fractions was approximately 12.34 minutes; no detectable peaks were observed at 66 this retention time in the fraction blank (0 pg/mL of AmpB) of the standard curve (Figure 11). The area of each peak as recorded by the Waters 746 Data Module Integrator was used to generate a concentration point in the creation of the lipoprotein and lipoprotein-deficient fraction linear calibration curve for amphotericin B. This calibration curve (Table 3) was then utilized in determining the concentration of amphotericin B in the appropriate plasma sample. A representative chromatograph for amphotericin B in HDL is shown in Figure 12. The intra-assay variability for all of the calibration curves was between 2 and 7%. 3.2 Lipid and Protein Analysis For every separated lipoprotein and lipoprotein-deficient fraction of each plasma sample, lipid and protein values were calculated. All lipid and protein concentrations were determined by colorimetric enzyme analysis kits and were measured using an U V spectrophotometer. For the determination of lipoprotein and lipoprotein-deficient plasma total cholesterol, total triglyceride, and total protein, standard curves were used. These standard curves were prepared along with the lipoprotein and lipoprotein-deficient fractions for each of the seven patients. Figure 13-15 display representative standard curves for total cholesterol, triglyceride and protein respectively. The total cholesterol and triglyceride standard curves were linear over a range of 12.5 to 200 mg/dL and 15.625 to 250 mg/dL respectively. The intra-assay variability for both cholesterol 67 CD -G 85' S I 0 II 82" e6 ' s 9Kfr 19" \ W F T T I l II 00 -<—> g ' S O H CD bJO C I cd ^ . g oo " O H c OJQ o cN c o • i—i • f-H V-i H—> oo *3 u o m •(-> cd oo CD -t-< C T3 C cd toX) l-H CD -<—> o 43 O H >-H S cd o -(—> cd B o t-H CD CD > *H—> cd H—> e CD oo CD O H CD «*"•< »»1 ^ » a B: cd <+-< O c 'H—> cd o cd 6 00 cd O H C^-I O S-H CD § 6 CD -t-> CD T3 CD 43 CD N CD £ CD TD (-1 cd T3 C cd 00 >-l CD O H CQ < O DTj zL o CN C+H O c o • ^ H 4-> cd =3 a _g CD 43 68 69 70 71 72 and triglyceride analysis was found to be less than 1%. The standard curve for total protein utilized a concentration range of 50 to 400 pg/ml for total protein determination. Intra-assay variability for this assay was determined to be approximately 1%. The unknown sample concentrations of each of the separated fractions were measured directly from their respective standard curves. For the other lipid components of the lipoprotein and lipoprotein-deficient plasma fractions (i.e., free cholesterol, cholesteryl ester, and phospholipid) measurement of their concentrations was determined directly by equation as previously described. 3.3 Establishment of Experimental Design 3.3.1 Experimental design AmpB (Fungizone®) and ABLC (Abelcet®) (20 pg/mL) were incubated in vitro in human plasma from seven separate subjects for 60 minutes at 37°C. Following incubation, the plasma was partitioned into its lipoprotein and lipoprotein-deficient plasma fractions: TRL -consisting of chylomicrons and very-low-density lipoproteins, LDL - consisting of intermediate and low-density lipoproteins, HDL - consisting of all subclasses of HDL, and LPD - consisting of all non-lipoprotein constituents of plasma. Separation of the plasma components was accomplished by step-gradient ultracentrifugation. Each lipoprotein fraction was analyzed for cholesterol (total, esterified, and unesterified), triglyceride, phospholipid, and protein concentration, as well as AmpB and ABLC distribution. A comparison of AmpB and ABLC distribution to the lipoprotein lipid profile was then performed in order to determine which, if any, relationship may exist in determining the plasma lipoprotein distribution of AmpB and ABLC. 73 3.4 Experimentation Utilizing Step-Gradient Ultracentrifugation Human plasma comprised of varying lipid content and composition was obtained from seven different subjects. AmpB (Fungizone®) or AmpB (ABLC) (20 pg/mL) was incubated in human plasma for 60 minutes at 37°C. Following incubation, the samples were separated into their four different lipoprotein and lipoprotein-deficient fractions by step-gradient ultracentrifugation. The lipid and protein content of each fraction was measured, as was the distribution of AmpB or ABLC in each fraction. 3.4.1 Lipid and protein composition of separated plasma components When human plasma was partitioned into its lipoprotein fractions by step-gradient ultracentrifugation, differences were observed in the total cholesterol concentrations of the seven plasma samples. Total cholesterol concentrations in the seven plasmas ranged from a low of 77.57 ± 2.95 to a high of 219.61 ± 16.21 mg/dl. TRL cholesterol concentrations ranged from 10.59 ± 0.09 to 70.02 ± 3.31 mg/dl; LDL cholesterol concentrations ranged from 36.82 ± 1.13 to 102.22 ± 5.42 mg/dl; and HDL cholesterol concentrations were between 23.28 ± 0.65 and 65.76 ± 4.45 mg/dl (Table 4). The following differences in the esterified cholesterol concentration were observed. The cholesteryl ester concentration in the TRL fractions varied between 6.95 ± 0.52 and 38.87 + 3.89 mg/dl, while the concentration in the LDL fractions ranged from 25.78 ± 0.79 to 75.60 ± 2.60 mg/dl. Total HDL cholesteryl ester concentration was found to range from 13.77 ± 0.56 CO rt a CO "S, c CD > C« s <g CA o CD o c o o s u (/I o *o o 6 g '53 -4-> s o rt -4-» O H Tf Cjt JQ rt H rt o H £ S <u o JS O I -TS S -a S © .ST -S 2 "S H a. E rt rt & C« « CO S Cw i a~53o o g 1 s s J2 m O N CN +1 r--m N O O +1 00 CN ro CN CO +1 CN 00 N O ro =tfc "p. CO rt B CN N O +1 N O O N C N O T-H © +1 O N m CN i n +1 CN CN CN O CN =tfc CD CO rt s CO r^t O N +1 N O oo I T ) CN O N >o in +1 un C N in cn +1 T-H CN cn u "p-rt s ca rt CD 1—H CO rt s CO rt in =tfc u CO rt s ca rt N O in +1 T J -I T ) 00 00 O N N O o O N cn O N O i > CN +1 +1 +1 +1 IT) r-. N O r -O N CN CN oo in in CN m N O un N O CD t CO rt c a rt N O O N N O +1 m r-N O o CN r--© +1 r-cn N O in CN un O N cn N O m N O O N CN cn cn +1 +1 +1 +1 +1 m oo 00 cn N O i n N O r - cn cn *—H N O •>* O N 00 N O O N 00 N O O N T—H 00 O N cn cn O in 00 in N O cn O cn o T—H CN* cn +1 +1 +1 +1 +1 +1 +1 O N ,—1 cn N O oo cn CN in <—1 CN O N O N O © 00 00 cn o <—• -3- m T—( cn r -"0*H CO rt s ca rt 75 to 50.84 ± 7.60 mg/dl and the total cholesteryl ester concentration in the seven plasma samples ranged from 53.84 ± 2.36 to 161.95 ± 18.29 mg/dl (Table 5). After separation of the seven human plasma samples into their lipoprotein and lipoprotein-deficient fractions by step-gradient ultracentrifugation, the following differences in the plasma samples were observed. Within the TRL fraction, the free cholesterol concentration of the seven plasma samples varied between 3.64 ± 0.46 and 31.79 ± 3.22 mg/dl. The free cholesterol concentration in the LDL fraction was found to range from 11.05 ± 0.34 to 36.65 ± 2.62 mg/dl. Free cholesterol concentrations in the HDL fraction from the seven plasma samples ranged from 6.99 ± 0.19 to 26.30 ± 4.76 mg/dl while total plasma free cholesterol concentration ranged from a low of 23.74 ± 0.74 to a high of 119.86 ± 1.63 mg/dl (Table 6). When the seven patient plasma samples were separated into their lipoprotein and lipoprotein-deficient components, the following differences in triglyceride concentration were observed. Total triglyceride concentrations in the seven plasma samples ranged from 83.05 + 2.38 to 258.20 ± 20 mg/dl. TRL triglyceride concentrations ranged from 31.34 ± 1.00 to 175.10 ± 11.92 mg/dl; LDL triglyceride concentrations ranged from 14.33 ± 2.14 to 102.27 ± 3.02 mg/dl; and HDL triglyceride concentrations were found to be between 20.11 ± 0.36 and 63.84 ± 2.40 (Table 7). Each of the seven plasma samples utilized in this study was found to have different total and individual lipoprotein fraction phospholipid values. Total phospholipid concentrations in the seven plasma samples ranged from 86.28 ± 4.10 to 294.83 ± 3.67 mg/dl. TRL phospholipid "E, I 1 l - H « •3 c > s e g C/3 « O a u o c o o 2 u -4—» CA *o o U •c cu -*-» co <D rt J "E, c "53 -*-> 8 CL, O (X rt -*-» o H 3 rt H rt o H s 2 J o -o J2 cn a T3 a & rt C O rt s CO OS VO CN OS uo r o 00 00 •<* CN i-H 1-H CN +1 +1 +1 +1 •* u-> U"> 00 OS uo vq r o 00 uo VO r-- OS i-H CN o +1 u-> Os vd 4t "E, 00 rt s CO rt os +1 o o OS C N f N o +1 00 C N r o vo u~> d +l r o os OS o +1 o OS 00 rO *E< U0 J U "EH O r--VO i-H +1 u-> CN S O ju "E-C N +1 O VO uo o vo o OS C N OS VO uo 1—< o VO d d 00 o o r o v d +1 +1 +1 +1 +1 +1 +1 o U1 r-- VO U0 VO uo oo r > VO t - - CO vd d r o vd OS os uo r o CO r o OS o OS uo r - - VO C O O vo C O d C N 1-H d ^r' C N vd +1 +1 +1 +1 +1 +1 +1 o o o co vo uo OS uo r - v q os o C O C O 00 U O uo C N os OS C O C N C N uo OS os C N vq o o C O C O +1 +1 +1 C N C N C N 00 d 00 C N C N r o 00 rt d 00 rt 00 rt 00 rt 00 rt 00 rt Plasn Plasn Plasn Plasn Plasn Plasn co rt a CO J2 "EH a C5 2 CH > CD co a co C o CD O e o o 2 CD -*-» CO CD "o O T3 CD «3 •c CD H-» CO CD rt s J "EH G '53 2 DH o QH rt +^ o H N© <u 3 H , 0 ) O B la S,~5JD ja o a Of O H W la 3 CQ H W O H "Sio S _ .a a •*=> H a © o -a » S O H W s.l >H * * 2 o TJ eu Q . ~O3D .Sf> J 1-CU O H s « C/i « a — ! m O m © CN l - H +1 +1 + 1 +1 CN O N O N co N O r-; CO O N oo CN >n O N T — | • — 1 N O O co r - H +1 +1 +1 O N co 00 O N N O N O od O N N O © +1 N O co O N i n +1 00 i n T—I CN O +1 O N 00 1—H CN CO N O +1 N O 00 O N m r-+1 o O N 00 CN CN CN +1 00 o i n N O CN oo +1 o N O O N i n CN CN CN T—1 i n +1 +1 -H +1 CO o CN T - H O N CO i n O N CO N O i n 00 CN 1 — 1 1 r - CO CO m CO CO N O i n r—' i n O N © ' CN © ' CO CN T - H i n +1 +1 +1 +1 +1 +1 +1 m CO CN i n O m o N O N O O N oo O N r -T — ' i"-" N O i n , — H O N N O CN 1 — 1 CN CO CN CN T—H r—I CN co CN CN CO CN CO +1 +1 +1 r - CN O N r-; N O CN O N T—H T - H T - H CO T - H CN co in N O t -% % % -tfc JD CD 3H T S JD JD JD "EH *E. "EH "EH "EH "EH "EH on rt oo rt d oo 00 rt 00 rt 00 rt 00 rt Plasn Plasn Plasn Plasn Plasn Plasn Plasn CO a S J "E, B 3 si c <u > CO B co (3 O '-a -4—» e (L> O a o o <u •c u o >> •c rt a S3 • i—t <D +-» 2 o-o .& -*-» o H JU 05 H o H CA O a -ge a w I K 3 CA a Q B CA a 2 -O TS ~O3D o ._ p -a a oi o g 2 CU s u !*1 Q. a B « CO OS E CA C J o -a P S I 00 CO CN +1 UO o CO oo UO CO CN +1 o CN co CO rt co rt OS CN +1 O CN 00 UO CN CN 00 CN +1 o co T t T t CO rt B CO rt Os r -+1 CN UO © uo CN VO OS r-CO uo © d i — i i—< +1 +1 +1 OS >—1 oo 00 d co 00 CN T t CN UO CO +1 T t CO T t oo T - H Os +1 vo oo uo oo +1 CN CO T t o 00 00 CN J Os vq uo oo CN CO - H 1-H +1 +1 +1 +1 +1 co VO vo vo OS CO oo uo uo T t vd vd oo d >—' T t CN CN CO rt B CO rt B J CM CO rt B co rt OS o d +1 T t CO CO CN CN O CO +1 <N CN O 1.92 T t vq O O T t O i - H CO +i +1 +1 +1 o os vo T t CO uo T t uo r--d CN CO co uo CO rt co rt c--uo oo +1 T t T t CN CN CO o vo . - H T t t - ; CO CN UO +1 +1 +1 OS r - T t Os oo UO T t CO T t CO VO CN oo uo +1 uo OS CO CO CN CO +1 o 00 uo .—4 CN CO T t uo VO =tfc JU J U JU J U JU J U JU "&> "E, "E, "E, "Ev CO rt B CO rt 79 values ranged from 10.65 ± 1.23 to 81.13 ± 2.55 mg/dl; LDL phospholipid values were found to be between 25.87 ± 2.16 and 69.92 ± 2.45 mg/dl; while HDL phospholipid values ranged from 31.45 ± 3.25 to 100.94 ± 2.33 mg/dl (Table 8). The following differences in total protein concentration were observed when the seven plasma samples were separated into their lipoprotein and lipoprotein-deficient fractions. Total protein concentration values ranged from 4406.47 ± 262.52 to 4750.01 ± 506.68 mg/dl. TRL protein concentration values ranged from 7.04 ± 0.88 to 74.56 ± 8.85 mg/dl; LDL protein concentration values ranged from 19.15 + 0.32 to 98.05 ± 3.07 mg/dl; while HDL protein concentration values were found to be between 128.89 ± 15.86 and 318.91 ± 18.02 mg/dl (Table 9). The following differences in the lipoprotein core lipid concentration (cholesteryl ester plus total triglyceride) were observed between the seven plasma samples. Total core lipid concentration values ranged from a low of 112.32 ± 4.11 to a high of 376.73 ± 21.57. TRL core lipid concentration values ranged from 30.96 ± 1.49 for sample 4 to 216.41 ± 12.06 for sample 3. LDL core lipid concentration values ranged from a low of 40.44 ± 1.02 for sample 1 to a high of 122.16 ± 2.63 for sample 2. HDL core lipid concentration values ranged from 34.02 ± 2.38 for sample 1 to 107.97 ± 11.68 for sample 4 (Table 10). Table 11 shows the lipoprotein coat lipid concentrations (free cholesterol plus phospholipid) of the seven patient plasma samples. Total lipoprotein coat lipid concentration ranged from fi OJ CO fi O is fi <u o fi o o 12 PH CO O . f i O H rt B co "E, .c '53 2 O H o Cu -*-> o H 00 rt H rt O H T S "SJD a cu O T S •a o g gf Q H W K 3 o> p Q H, it ° a 2.1 •a « CU Q , OX •Sf'hJ cu ft a rt C O rt a CO +i oo tN vd oo ju *E, oo rt CO jrt EC co +1 uo oo 1-H o CN C N J U 00 rt co rt T t C N CN +1 VD C O uo ON CN d T — I + 1 o vo l > uo ,_, UO uo vq O N CN C O +1 +1 +1 vo C O r o .vo CN 00 vd T t T t 00 O N C N r _ 1 C N UO C N oo i-H O N r -oo C O C O r o T t C N r o ^ H ' C N d d +1 +1 +1 +1 +1 +1 +l uo T t ON T t CN ON uo T t ON CN o uo 1-H r o C N T t C O ON r -d o vo oo vo o r o o uo oo uo VO r o r o C N UO T t CN u-i C N CN CN C N C N +1 +1 +1 +1 +1 +1 +1 r -oq VO r o C N t--C N oo oo ON UO C N ON u-i CN v d uo ON C N 00 C O VO od uo ON VO r o CN r o oo C N UO 00 1-H i-H 00 i-H VO UO uo CN co i-H 1—H C N C N +1 +1 +1 +1 +1 +1 +1 uo VO T t UO ON i-H ON T t vo r-- UO r o r o © t > r o uo uo C O C N r o r o 00 r o J U 'E, oo rt a co jrt K T t =tfc J U "E. uo J U "EH VO J U "E. 00 00 00 rt rt rt G co fi CO fi CO rt rt rt ju "EH 00 rt co rt PH 81 rt B 3 O -fi <u ,<u c <u > <u cn s cS fi O "3 B c <u o fi o o .fi '53 -*-> o V H O H B J c <u 'o I B <u "? fi '53 o tn O H O O H c .fi '53 H-» s O H O .& *rt -*-» o H H co rt o H 2 a O H ,.2-Q M XI S rt E CO rt -J •d oi, E <U O Q *• J S O .Sf> .S-Si CU P tt ° C. Q H X) ~Dh a XJ ~53D a CU ."2 CU u co a '3 O w o r s. WD * O a o a - J xi DSD a — a a rt in rt a CO rt CN T t VO 00 oo UO T t UO T t vq uo VO ON UO CN T t 1—H vd T t ,—J VO VO T t i — i o VO 1 - H CN 1—1 co CO uo 1 - H CO +1 +1 +1 +1 +1 + 1 +1 r - CO 1 - H T t 1—1 ON .—1 T J - CN CN O o UO uo vd ON T t od d vd CO o T t VO CN uo VO T t r - uo f- vo ON T t T t T t T t T t T t T t CO T t VO CN ,_, vo OO VO o CN CN oo o T t d vd r-^  ,—< uo d T t uo uo CO UO CO CN 1 - H CO CO uo i — i co +1 +1 +1 +1 +1 +1 +1 OO CO VO CO o ,_< _ CO t - ; CN 1 - H uo vo T t 1—H CO r> T t od CN d uo T t 00 uo oo ON o CN T t 1—1 T t T t VO T t T t T t T t T t T t T t vo oo T t ON ON CN 00 CN UO r - - r » O CO uo ,—I d od T t od V/"*\ I-H i - H VO CO CN 1—H +1 +1 +1 +1 +1 + 1 +1 Os T - H ON ON r-~ 1—H o O S 00 i - H VO vq ON UN oo UO UO ON uo 00 UO CN o CO i—t CO CN CN CO C N CN uo OO OO o co r - oo CO uo 1 - H p d vd CO 1 - H uo' CN CO +1 +1 +1 +1 +1 +1 +1 uo CN . VO r - OO r - H uo 1— 1 oo o 1—; T t CO p ON uo T t oo u-i ON od uo CO CN CO r - ON 00 uo CN uo T t o 00 oo vq ^ CN vq UO d 00 CN r - H CO - H +1 +1 +1 +1 +1 +1 +1 VO ON o VO CO O uo o C l p oo r- T t d uo ON I r - CO 1 - H CN T t J U "On 00 rt CO rt CN =tfc JU 00 rt B CO rt co J U tn, oo rt B J cu T t <U 00 rt B uo cu 00 rt B CO Jrt CU vo J U " O H 00 rt s CO rt <u r—I O H 00 rt B CO rt vo C o o rt cfi C u o I B <u C '53 H—» 2 o. o O H : i - § T3 O S .& I S CO CO cu rt c fi co <u rt co - r H u 1? <H co 5J co £; SH CO OH «a « 13 CD > c3 ^ 8 o rt H CD •s o o "2 'ex <u o o g '53 2 ex o ex CO (3 O <u o e o o CD 'C CD o 'B +-* o +H T3 co UJ CD H—> CO "o o rt B «> S CD rt co g « ex g o 3 eu O TJ d o g u a w In 3 52 « J a « H , cu O TS i 1 2 •S -3 H el 2 > to co s s eu o o H on s £.1 eu 2.1 •rt « H . 3 O XJ fc.fc.~-. eu Q , DD « O g JU a. & « « S 1 I1 si + I +1 CN cn CN' C N O +1 o un CN +1 cn vd r-cn cn N O C N +1 N O T - H CN CN C N J D N O un +1 C N cn ON ON CN oo un" +1 r -ON ON ON un C N +1 •*t ON un oo cn cn un un +1 +1 +1 o T—1 un © ON un N O ON cn J D J D U~> =tfc J D "H. o un un +1 cn oo cn ^ f cn N O N O NO =tfc J D ON un +1 o cn NO cn cn 00 cn oo N O o ON oo N O o •<+ CN T f CN CN 00 +1 +1 +i +1 +1 +1 +1 CN O r - -o oo un r -ON N O T f 61.807 -<t' cn o o T - H CN o T f un T f o 61.807 C N un r - oo T f © 2 « 1-4 °°. C—' CN N O cn o ON CN cn ON i > T—1 CN CN 00 O +1 +1 +1 +1 +1 +1 +1 N O ON N O r-» oo 00 T f ON un c-- N O T f © ,—1 r-' N O cn un cn un ON T—1 CN T - H J D oo a 00 ccj 00 00 rt 00 rt 00 rt 00 rt Plasn Plasn Plasn Plasn Plasn Plasn Plasn £3 <D "S O o *2 'cx "5 o o g '53 -*-» o I-I C M O CX co £3 O c u o o o "2 'jjx "3 J=l ex co o X o « CO u *o xi o <u <u g CO £X a a 2 & •a o ox o, V o t * H i cd co I § c J '53 ° -O H g 0 a 1 I o -3 H S3 2 > 5U co s a 05 O H * fc, H * H g o "d i " ~53D S cu 2 S S o T J fc.fc.~Sy CU Q , SL © S IS J fc. cu a « « a -2 §' as + o o CN • ' T - H +1 O N N O CN T i - CN O N N O T—< oo ro ro IT) l - H O N N O i — ! N O +1 + 1 +1 +1 +1 +1 O N co , — I oo O N T — I T - H O N ro CN O N T - H © 00 © , — < N O N O CN O N O 00 CN CN CN ro 1 ro CN T -H © T f N O © ro N O T f in r-" +1 +1 +1 +1 N O CN IT) O N N O l-H ro © T f ' l-H N O ro in 00 </-> O N © ro N O +1 N O CN r-" T l " mple #1 mple #2 mple #3 CO cd cd GO cd Plasn Plasn Plasn 0) co cd co cd CO cd a M © © +1 CN O N 00 N O m CN i n T f C O N O N O T f 00 i > © O N CN oo C O CN T f T-I +1 +1 +1 +1 +1 + 1 T f C O T - H © C O — H i—< l-H N O T f in C O ro O N od CN N O CN in N O CN T f C O co cd a CO cd m co +1 T f i n © © N O oo C O C N m N O © © q O N 00 in N O C O in C O T f C N +1 +1 +1 +1 +1 +1 +1 T f © C O r- C N m m oo o C O r- T f © T f T f C O T f r-" N O T f T f 00 T f N O O N m O N N O q CN +1 ON CN CN m N O % "tfc % J J J J J J BH BH B-co cd CO cd P M 84 161.28 ± 1.32 to 386.99 ± 6.59. TRL coat lipid concentration values ranged from 22.40 ± 2.74 for sample 4 to 112.29 ± 2.06 for sample 7. LDL coat lipid concentration values ranged from a low of 43.33 ± 3.08 for sample 3 to a high of 97.42 ± 3.02 for sample 5. HDL coat lipid concentration values ranged from 54.66 ± 6.62 for sample 1 to 147.26 ± 6.30 for sample 5. The lipoprotein plasma total cholesterol to total protein ratio (TC:TP) from the seven different human plasma samples showed the following differences. TRL total cholesterol:total protein values ranged from 0.64 ± 0.10 for sample 2 to 3.66 ± 0.53 for sample 5. LDL TC:TP values ranged from a low of 0.70 ± 0.03 for sample 7 to a high of 2.72 ± 0.44 for sample 5. HDL TC:TP values ranged from 0.17 ± 0.03 for sample 6 to 0.66 ± 0.01 for sample 3 (Table 12). Table 13 shows the differences that were observed in the total triglyceride to total protein (TG:TP) ratio for the seven plasma samples. TRL total triglyceride:total protein values ranged from 2.52 ±0.13 for sample 5 to 6.41 ± 1.32 for sample 2. LDL TG:TP values ranged from a low of 0.35 ± 0.04 for sample 7 to a high of 0.79 ±0.13 for sample 2. HDL TG:TP values ranged from 0.10 ± 0.01 for sample 3 to 0.67 ± 0.90 for sample 1. The following differences in the total cholesterol to total triglyceride (TC:TG) ratio within the separated lipoprotein fractions for the seven human plasma samples were observed. TRL total cholesterol:total triglyceride values ranged from 0.80 ± 0.01 for sample 5 to 3.20 ± 0.20 for sample 3. LDL TC:TG values ranged from a low of 0.22 ± 0.01 for sample 5 to a high of & g CA a a -a , ox S P i a ob o g' a w CA a cu Q CA a o -a 0 , 0 * o & 12.1 , CU CU ^2 \mm H W M 1.5 O Xt CU W O •Sf 3 OX CU a , S ICO rt s CA « |5 CN T t ,—1 CN ro wo O 0 0 O O O O © d d d d d d +1 +1 +1 +1 +1 +1 +1 00 T i - VO ro ,—, t - - vo I—* ro vo CN ro CN d d d d d d d ro UO vo T t T t ro O i - H 0 T t 1 - H O d d d O d d d +1 +1 +1 +1 +1 +1 +1 CN T t 0 P 4 CN CN 0 ON 00 T t T t r» r-I - H 1—H CN CN I - H d 0 O vo VO ro uo ro CN I - H 0 uo l - H O d d d d d d d +1 +1 +1 +1 +1 +1 +1 CN T t ON 0 VO 0 ro UO VO ON ro vq T t i - H d d ro ro H r ^ cd a co CN <U 1^  00 rt s J r o J U 00 rt B CA rt ju ta, 00 rt a CA rt uo VO r -J U <u <u 00 rt CA rt 00 rt CA rt 00 rt a CA rt CO CO s S c CO O l-i ,g -*-» 2 a, "ed o o •*H ii T3 'C •u o >, "oo •fi o -4—» #g '53 •*-» o PH o P H cn JU 3 H CO a CU ;Q OD £ eu © TJ Q ft M .-is*.! co a hH O TJ a cu 2 I S O TJ CU u >>> OH ft "a* o B S os « & Cfl — o u n c n c n c n O N © o © © © © © ' © © o ©' © © +1 + 1 +1 +1 +1 +1 +1 r - u n © C N T - H N O C N T - H C N T - H T - H T - H © ' © ' © ' © © © ' © c n c n © c n T f T - H T - H T - H © © T - H © © © ' © © o © ' © +1 +1 +1 +1 +1 +1 +1 u n ON N O T f N O ON u n r - r - N O u n u n c n © ' © o © © ' © ' © 00 C N T—H ON c n c n u n C N c n C N q T - H T f © © ' T - H ©' T - H © © © ' +1 +1 +1 +1 +1 +1 +1 r-» T - H C N o C N T f r - . u n T f r- u n ON C N T f N O T f c n " C N C N c n -Jfc " p H oo cd B CO CN T ^ H oo cd s cn -tfc J J " p . oo cd a co cd T f "P, 00 cd co cd u n -tfc J J 00 cd s co j d N O =tfc " p -00 cd B CO cd J H If oo cd B CO cd 87 0.87 ± 0.04 for sample 6. HDL TC:TG values ranged from 0.54 ± 0.09 for sample 5 to 1.26 ± 0.07 for sample 3 (Table 14). Table 15 shows the phospholipid to total protein (PL:TP) ratio of each of the separated lipoprotein fractions from the seven human plasma samples. TRL total phospholipid:total protein values ranged from 0.51 ± 0.09 for sample 2 to 2.69 ± 0.26 for sample 5. LDL PL:TP values ranged from a low of 0.71 ± 0.01 for sample 7 to a high of 1.78 ± 0.28 for sample 5. HDL PL:TP values ranged from 0.12 ± 0.01 for sample 3 to 1.07 ± 1.47 for sample 1. 3.4.2 AmpB distribution within separated plasma component fractions Tables 16 and 17, as well as Figures 16-19 describe the human plasma lipoprotein and lipoprotein-deficient plasma distribution of AmpB and ABLC in seven different plasma samples as well as compare the distribution of AmpB versus ABLC within the same plasma sample. The lipoprotein and lipoprotein-deficient plasma distribution of AmpB and ABLC incubated at 37°C for 60 minutes in seven different human plasma samples showed the following differences. For plasma incubated with AmpB, a range of total drug recovered based on initial drug incubated (% recovery) was found in each fraction. Plasma sample 1 showed the greatest amount of drug recovered in the TRL fraction with approximately 12% of initial drug incubated being recovered in the TRL fraction of plasma sample 1. For plasma sample 5, approximately 1% of total drug incubated was recovered in the TRL fraction. S +J cu o IO u °-. I o .2* .2-IS -ro 00 ON vo o o ro o o © d d d d d d +1 +1 +1 +1 +1 +1 +1 UO vo ON T t r» uo 00 CN ON U0 oo d d d d d d CA CU C *-cu o IQ a > o ,5 .&! 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Plasma was separated into triglyceride-rich lipoprotein (TRL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein-deficient plasma (LPDP) fractions. (A) patient sample 1 (B) patient sample 2. Data is represented as mean ± standard deviation; *p < 0.05 vs. AmpB 93 I 100 80 60 40 20 0 > © 2-PQ a S < TRL LDL HDL LPDP • AmpB • A B L C B T3 TRL LDL HDL LPDP • AmpB • ABLC Figure 17: Distribution of AmpB and ABLC (20 |ug/mL) in human plasma following incubation for 60 minutes at 37°C. Plasma was separated into triglyceride-rich lipoprotein (TRL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein-deficient plasma (LPDP) fractions. (A) patient sample 3 (B) patient sample 4. Data is represented as mean ± standard deviation; *p < 0.05 vs. AmpB 94 8 CU > © cu ca < 100 80 60 40 20 0 TRL L D L HDL LPDP • AmpB • A B L C B 1 100 cu > o a. 0 s 80 60 40 20 0 A i l TRL L D L HDL LPDP • AmpB • A B L C Figure 18: Distribution of AmpB and ABLC (20 |ig/mL) in human plasma following incubation for 60 minutes at 37°C. Plasma was separated into triglyceride-rich lipoprotein (TRL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein-deficient plasma (LPDP) fractions. (A) patient sample 5 (B) patient sample 6. Data is represented as mean ± standard deviation; *p < 0.05 vs. AmpB 95 73 U © a 20 80 60 40 0 TRL L D L HDL LPDP • AmpB • A B L C Figure 19: Distribution of AmpB and ABLC (20 fig/mL) in human plasma following incubation for 60 minutes at 37°C. Plasma was separated into triglyceride-rich lipoprotein (TRL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein-deficient plasma (LPDP) fractions. (A) patient sample 7. Data is represented as mean ± standard deviation; *p < 0.05 vs. AmpB 96 When the LDL fraction was examined, the range of drug recovered in this fraction was less than when compared to TRL. The highest percent recovery was found in plasma sample 1, with around 6% of initial drug incubated being recovered in this fraction, while the least amount of drug based on initial drug incubated was found in plasma sample seven, with approximately 1.3% initial drug incubated being recovered. For the separated HDL fractions, the highest percent recovery was seen with plasma sample 6, at 12%, while the lowest amount of drug recovered was found in plasma sample 2, with only 1.5% of the initial drug incubated being recovered from the HDL fraction. As can be seen from Table 16, the majority of the drug initially incubated in each of the seven plasma samples was recovered from the lipoprotein-deficient plasma fraction. Within the seven plasma samples, the amount of drug recovered in the LPD fraction ranged from 67% to 89%. Within the seven plasma samples, total percent recovery was greater than 83%> in all cases, with the majority of plasma samples showing greater than 90% recovery. For plasma incubated with ABLC, all samples showed less than 1% recovery of ABLC within the TRL fraction. In the LDL fraction, all seven plasma samples showed less than a 2% recovery of initial ABLC incubated. As can be seen from Table 17, the majority of initial ABLC incubated in each of the seven plasma samples was recovered in the HDL fraction. All seven plasma samples showed greater than 50% ABLC recovery in the HDL fraction, with the majority of the plasma samples having greater than 70% ABLC recovering in the HDL fraction. Within the seven plasma samples, total percent recovery was greater 97 than 87%, with six of the seven plasma samples showing greater than 90% recovery of initial drug incubated. As can be seen from Tables 16 and 17, when AmpB was incubated in seven different human plasma samples, the majority of drug recovered was found within the lipoprotein-deficient fraction. In contrast, when AmpB was incubated in seven different human plasma samples as ABLC, the majority of drug recovered was clearly found within the high-density lipoprotein (HDL) fraction. 3.4.3 Effect of lipoprotein composition on AmpB and ABLC distribution In order to determine the effect of lipoprotein content and composition on the distribution of AmpB and ABLC within each separated lipoprotein fraction, comparisons of the amounts of AmpB or ABLC recovered and the lipoprotein content and composition of each separated fraction were performed. The values obtained for each of the seven patient plasma samples were compared. The following relationships were calculated: AmpB or ABLC to (i) total cholesterol, (ii) cholesteryl ester, (iii) free cholesterol, (iv) triglyceride, (v) phospholipid, (vi) total protein, (vii) core lipid, and (viii) coat lipid concentrations. Tables 18 and 19 present the correlation coefficients for the analyses performed within each of the four separated fractions for both the AmpB and ABLC formulation respectively. Within the HDL fraction of the seven plasma samples, a positive correlation exists between AmpB and the total triglyceride to total cholesterol ratio (TG:TC). In assessing the linear relationship between AmpB and the TG:TC ratio of HDL, a correlation coefficient of 0.80 (p 98 < 0.05) was calculated (Table 18, Figure 20). Therefore, as the TG:TC ratio increased within the HDL fraction, there was a proportional increase in the amount of AmpB recovered from that fraction as well. Table 18. Correlation analysis comparing amount of AmpB recovered to lipid and protein content and composition of the separated lipoprotein and lipoprotein-deficient plasma fractions following the incubation of AmpB (20 pg/ml) for 60 minutes at 37° C in plasmas from seven different human subjects3. r value Component or ratio TRL-AmpB LDL-AmpB HDL-AmpB LPD-AmpB TC -0.44 -0.32 -0.59 NA CE NA fC -0.10 -0.06 0.47 NA TG 0.11 0.20 0.22 NA PL -0.34 -0.71 -0.20 NA TP -0.32 -0.63 0.58 Core lipid content 0.10 -0.72 -0.50 NA (CE + TG) Coat lipid content -0.30 -0.50 0.02 NA (fC + PL) TC:TP ratio -0.49 0.39 -0.30 NA TG:TP ratio 0.70 0.31 0.01 NA PL:TP ratio -0.56 0.47 -0.10 NA TG:TC ratio 0.60 0.11 0.80c NA "Calculations are based on the Pearson correlation coefficient values with significance. Abbreviations: AmpB, amphotericin B; TRL, triglyceride-rich lipoproteins (which includes very low-density lipoproteins and chylomicrons); LDL, low-density lipoproteins; HDL, high-density lipoproteins; LPD, lipoprotein-deficient plasma (which includes oc-1 glycoprotein and albumin); TC, total cholesterol; CE, cholesteryl ester; fC, free cholesterol; TG, total triglycerides; PL, phospholipid; TP, total protein. ""NA, not applicable; analysis was not performed due to a lack of sufficient data. CP < 0.05. Few strong linear relationships existed between AmpB and the individual lipid components of TRL; only that relationship involving the amount of AmpB recovered to the total triglyceride to total protein ratio (TG:TP) (r = 0.70) was apparent (Table 18). A 99 r m O 00 I 1 ~1 1 I 1 1 Tj- <N O 00 O T f <S i - H l - H i—I 1CIH ui pajaAODaj gduiy jo o/ G in m c o s-Q H a U H a H o .in o 2 I o cd cd 1 / 3 in cu <t; cd I £ b .S o 2 S ca to > <u 65 s 3 C H O S ft < B PQ i s .5 u CD of) O »-( c CS • $ H a ® CD V CD OH GO * C 2 CD O O "In O c o cd cd o C O 00 CD CD a CD CJ c o C H ^ 1 - 1 cu cd p., cd cu U o CD t~~- C/D m CD S-H C H CD VH C O 6 cd CD f-a C U D C/5 Ui CD > Q o • l-H -t-> o cd cd C/> CD • —i cd VH C -c S H - l f j h-l CD VH ,o orj n o CN 100 positive relationship between total triglyceride to total cholesterol ratio (TG:TC) (r = 0.64) does exist; however that association is not as strong as the TG/TP ratio. There were no apparent linear relationships between AmpB and any of the lipid or protein components of LDL. A negative relationship between phospholipid and the amount of AmpB recovered in the LDL fraction does exist (r=-0.71); however, the association is not as strong as that seen with TG:TC/HDL (Table 18). Table 19. Correlation analysis comparing amount of AmpB recovered to lipid and protein content and composition of the separated lipoprotein and lipoprotein-deficient plasma fractions following the incubation of ABLC (20 pg/ml) for 60 minutes at 37° C in plasmas from seven different human subjects3. r value Component or ratio TRL-AmpB* LDL-AmpB** HDL-AmpB LPD-AmpB TC -0.26 -0.33 -0.37 NA CE NA fC 0.02 0.25 -0.66 NA TG 0.33 0.57 -0.16 NA PL -0.15 -0.17 -0.66 NA TP 0.01 -0.15 0.04 Core lipid content 0.21 -0.14 0.27 NA (CE + TG) Coat lipid content -0.12 -0.01 -0.76c NA (fC + PL) TC:TP ratio -0.62 -0.21 -0.42 NA TG:TP ratio 0.71 0.21 0.23 NA PL:TP ratio -0.60 0.03 0.03 NA TG:TC ratio 0.75 0.33 -0.04 NA "Calculations are based on the Pearson correlation coefficient values with significance. Abbreviations: ABLC, amphotericin B lipid complex; TRL, triglyceride-rich lipoproteins (which includes very low-density lipoproteins and chylomicrons); LDL, low-density lipoproteins; HDL, high-density lipoproteins; LPD, lipoprotein-deficient plasma (which includes a-1 glycoprotein and albumin); TC, total cholesterol; CE, cholesteryl ester; fC, free cholesterol; TG, total triglycerides; PL, phospholipid; TP, total protein. ''NA, not applicable; analysis was not performed due to a lack of sufficient data. CP<0.05. *n=4 patients; 3 patients had non detectable AmpB concentrations **n=5 patients; 2 patients had non detectable AmpB concentrations 101 There was a strong negative correlation between the amount of ABLC recovered and the coat lipid content (fC + PL) of the HDL fraction (r = -0.76, p < 0.05) (Table 19, Figure 21). Therefore, as the concentration of coat lipid decreased within the HDL fraction, there was a proportional increase in the amount of AmpB recovered from that fraction. As well, a negative correlation between the concentration of phospholipid or free cholesterol present in HDL, and the amount of drug recovered in that fraction exists. These correlation's however, r = -0.66 and r = -0.66 are not as strong as the HDL/coat lipid content correlation (Table 19). Few strong linear relationships existed between ABLC and the individual lipid components of LDL. Within the TRL fraction of the seven plasma samples, a strong positive correlation exists between ABLC and the total triglyceride to total protein ratio (r = 0.71), as well as the total triglyceride to total cholesterol ratio (r = 0.75) (Table 19, Figures 22 - 23). Therefore, as the ratio of total triglyceride to total protein and the ratio of total triglyceride to total cholesterol present in the TRL fraction increased, there was a proportional increase in the amount of ABLC recovered from that fraction as well. 3.5 HDL2/HDL3 Step-Gradient Ultracentrifugation 3.5.1 Lipid and protein composition of separated fractions When human plasma was partitioned into the HDL subfractions, HDL2 and H D L 3 by step-gradient ultracentrifugation, the following differences in the total cholesterol concentrations between the two fractions was seen. Within the HDL2 fraction a total cholesterol 102 HM O a a <u fi o U .9* J 03 O U o o o o o o o o o o 1QH HI D i a V JO o/ 0 c o •53 ^ O . r—J £ a co CD - r t * il fi fi 'fi ° r f i o 03 CD S-H co cd T3 <D > O OH CD »- + ^ y CQ H-> < fi w CD 00 c - ° C o CD PH -(—> o r f i GO ' f in HH-> c C D * £ > ^ ° V co ^ C § IS CD O CD co fi O cd cd O CD C O cd • -cd g | O o fi o 2 c «4H « w -4-> fi fi o S cd CD .fi H <N co fi co i-H CD > h-l Q c o co H—> fi co co 5H « CD PH ^ CD Cd r f i cd Q CO C CD co y g PH CD cd o m H-> cd co C D cd .a 103 r l > h <N o — r 00 o r O Pi H H a. H a CN —I fN O a « V cd CD CD H £d 13) H ~ O CD c .S <D cd 2- -»-* CD O > O o CD VH c V > C H CD oo * * IS CD 00 o o o c o CD 1 tD h - l CD PQ cd 3 t * C -t—> CJ CD g oo O CD po VH V- 1 o . cd CD « -^H cd -<-> CD oo CD VH C H •S3 O +-> H C o c S3 C oo S 13 o _ THX U I P9J3A0D9J 319V jo % § b Cd § CD H .2 o fc C H i-S3 u o cn p VH cd S-H oo CD cd 3 6 C oo | JES C C H O ^sj VH « .O VH ^ c S g m CN cd M - H o oo cd o ^ •a c cd CD X ) *-> o cG o •S '-3 o 104 H a U H O H «3 *-i "C ^ i cd CD S-H o S3 V > cx CD C O * 4 3 .2 •ti o CD CD 4 3 co S3 o cd o O c o I s •t i co o CD <-> > $ 0 O CD -•-> -H O OQ S S & CQ '£ .S 13 VH •+-> CD 2 4 3 +-> 1 g § 2 o H s w cd C O CD \n 4 3 CD cd 4^  3$ cd o o co H—> c CD co S3 o CD co i—I *—' o cd CD CD VH PH 4 3 cd d CO S3 CD U- CO CD O -H r- ex cd t-n co . «s cu cd p" s S3 co •a _3 O 4 3 i-. « h-l too 11 =L S3 C_? CO tN cd C O cd ^ 53 <N ' C D -4—> «^  o ^ 2 K r Pi ^ ^3 S3 O ^ '+3 S3 cd CD O -H o <+H o •S -3 o 105 concentration of 12.63 mg/dL was found, as compared to 45.77 mg/dL total cholesterol for HDL3 (Table 20). The following differences in the free cholesterol concentration between HDL2 and H D L 3 were observed. A total of 12.16 mg/dL free cholesterol was measured in the HDL2 fraction. This level was much lower than the recorded values for H D L 3 , which were measured at 37.43 mg/dL free cholesterol (Table 20). After separation of human plasma into the HDL subfractions HDL2 and HDL3, the following differences in the cholesteryl ester concentration of the two fractions were observed. Approximately 4%, or 0.46 mg/dL of the total cholesterol concentration found within the HDL2 fraction was determined to be cholesteryl ester. This compared to 18%, or 8.34 mg/dL cholesteryl ester for HDL3 (Table 20). When HDL was separated into two of its subfractions, HDL2 and HDL3, the following differences in triglyceride concentration were observed. Within the HDL2 subtraction of HDL, 40.27 mg/dL was measured as the triglyceride level within that fraction. This was less than the 78.31 mg/dL that was measured for the HDL3 fraction (Table 20). When HDL was separated into HDL2 and HDL3, the following differences in phospholipid concentration were observed. It was found that HDL2 has higher phospholipid levels, at 40.16 mg/dL, than HDL3 levels, which were measured at around 20.78 mg/dL (Table 20). 106 OT EH O o c o u a '53 s P H -a "2 co " ? P '53 -*-» 2 P . o p. h-l Q © C N J O 2 rt H CJ T . C ^ © CO g .s-e w -J taw ^ o NJ B | a ta w N O N O un ro ro CN d CN 00 < < +1 +1 +1 161.28 33.98 5439.25 d +1 CN CN N O C O d +1 C O N O CN X u o 53 >*-» OT JO "o XI o "rt -*-> O H O N ro +1 ro T f r-^  ro O +1 N O CN s co -*-> OT JO "o U CO co i i fa CN ro +1 T i -ro 06 NO CN d +1 N O T f 0 OT w CO -*-» OT co o u N O 0 N O 1/1 CN N O ro d CN +l +1 +1 _ i T f ro .—1 00 d ro r- CN CN 00 O 00 r- O N N O wi CN +1 +1 +1 00 U l O CN T f d d T f T f T f N O CN o H co •a •c co o B "rt -*-» O H w "2 'E, O H OT o XI PH PH H g '53 - 4 - » o PH "rt O H N O II c o • i-H > CO OT l+l CO s CO ^ rt ^ I'd 5 OT W OT CO co 5 5 l-H X PH O -4—» CO o CO P 5 II t d < IQ £ 107 The following differences in total protein concentration were observed when HDL was separated into two of its subfractions, H D L 2 and HDL3. The HDL3 subtraction of HDL was found to have a total protein level of 823 mg/dL. This was higher that those levels measured for HDL2. HDL2 levels were measured at 264.41 mg/dL total protein (Table 20). 3.5.2 AmpB distribution within separated H D L 2 and H D L 3 plasma components Figure 24 shows the human plasma HDL2 and HDL3, as well as lipoprotein-deficient fraction distribution of AmpB and ABLC in plasma, as well as compare the distribution of AmpB versus ABLC within the HDL2 and HDL3. The distribution of AmpB and ABLC in the HDL2 and H D L 3 and LPD fractions of plasma incubated at 37°C for 60 min showed the following differences. For plasma incubated with AmpB and separated into HDL2 and HDL3 lipoprotein fractions, only 6% of the total drug incubated was recovered from the HDL3 fraction, with the remaining drug being found within the LPD fraction (Figure 24). For plasma incubated with ABLC and separated into HDL2 and HDL3 lipoprotein fractions, >95% of the initial concentration of the drug incubated was recovered from the H D L 3 fraction, with the remaining drug located within the LPD fraction (Figure 24). 108 o <N O o o 00 o v© o <N s u o i p e j j uid|Ojdodi| u i p 9 J 9 \ o o a . i gdmy % c 3 .2 B -t-> CO 'fa 'H. CD c $3 •*-H CD bfj 'o a « * —H cd O co o cd OH PH cd B a cd Q tn S Q 1 1 o CN .S r-H t d PQ ' cd T3 co El co cd eg QQ £ & cd 6 B < % O PH c r < 2 U •r-i H_, Cd co 2! at co CD H-> c o co c CD cd M^H CQ OH B < C O > O O V P H * cd > CD T3 cd -a C cd CO +1 C cd CD co cd T3 CD •4—< c CD co CD i-t PH CD PH .2 Q cd *j t d 109 3.6 Gradient Gel Electrophoresis The method for separating HDL into two of its subclasses, HDL2 and HDL3 was modified from a method published by Groot et al., (1982). This method utilized a different rotor system for ultracentrifugation than conventionally used in my laboratory setting. Thus it was necessary to make modifications to this method for my purpose. It was however confirmed, through the use of gradient gel electrophoresis, that the method for separating HDL into its subfractions, HDL2 and HDL3 was indeed separating as expected (Figure 25). 3.7 Experimentation Utilizing DTNB-Modification of Plasma Lipoprotein Profiles Preliminary investigations to artificially modify the human plasma lipoprotein profile were previously conducted within the lab [Wasan et al., 1997]. Human plasma pre-treated for 18 h with DTNB was separated into the four lipoprotein and lipoprotein-deficient fractions - TRL, LDL, HDL, and LPD. The total cholesterol, cholesteryl ester, free cholesterol, total triglyceride, total phospholipid, and total protein concentrations of each of the four fractions were measured and the values compared to those of the control plasma. Table 21 illustrates the total lipid and protein concentrations of the four lipoprotein and lipoprotein-deficient fractions for control and DTNB treatment groups. When compared to control plasma, a decrease in the LDL and HDL total cholesterol concentration for DTNB-treated plasma was observed. A slight increase in the TRL total cholesterol concentration for LANE 1 LANE 2 LANE 3 LANE 4 ^ 2b l a lb -Figure 25: Human plasma samples run on the HDL gel format. Lane 1 is HDL3 separated by ultracentrifugation; la-HDL3a, lb-HDL3b, c is a dye remnant. Lane 2 is HDL2 separated by ultracentrifugation; 2a-HDL2b, 2b-HDL2a. Lane 3 is total HDL (including both HDL3 and HDL2) separated by ultracentrifugation. Lane 4 contains the standard; T-thyroglobulin; F-ferritin; C-catalase; L-lactate dehydrogenase; A-bovine serum albumin; c is a dye remnant Ill —, cd 2 B o a H ,53 . . O H 03 a a •B M s s & h O cu Q £ 8 3 2 CU O fi >-- °-43. O •Hf .2-X J CA cU S 1 fi D . i t ° b 3 TS ~0JD s g •a & CU TS ce S CU u j i t H 8 O P ^ o g a, w CU a S « « B cn « CM V O V O CN CO CN © +1 < < © +1 +1 f -O N CO CO u o CN ON CO CO v q V O CN 1-H 1—* H^ © c o +1 +1 +1 +1 +1 o o CN O N o o CN t-^  T t CO u o r-1-H V O CN CO o d CN l > CN t - -o o © T f CN O U0 00 o o T t V O T-H CO uo CO i > +1 +1 +1 +1 +1 CO O N V O o 00 1-H u o v q ON uo OO V O CN CO O N CO 1—1 CN o o T t CN VO o VO o o c o 00 VO 00 +1 © +1 1-H +1 © +1 1-H +1 o T t v o o o © o u o o o T-H CN 00 c o t > T t CO ON l > VO +1 T t o T t T t VO v q o d +1 u o CN ON CO ON +1 CN v o T t CN +1 c o ON -_l ,_, r - < ^ g o 3 1 $ « o w 43 o 43 CO •e CO o CO " 5 8 Z rt CO o gel H P H U "2 5=1 "o 8 PH _ o 4 3 O P H H I H o r~-<-H CN CN --H . . © CN +1 < < +1 +1 O ON ^ CO VO ^ H H vo VO ON 00 i-H CN ON CO CN CO v o ON v o © T t ON 1—H CN ©• © T-H +1 +1 +1 +1 +1 ON uo © CO u o o o Os r -T t © uo v d CO T t CN CN VO © v q r-CN T t CO © CN CN VO -H © H © © +1 +1 +1 +1 +1 CO o o uo © ON r— T t o o VO CN CO u-i v d © 00 ON uo ON r -© CN ON CO CN CN © CN O CN +1 +1 +1 +1 +1 © o CN © o o ON T t UO UO ON v d CN CN CO v d c o 1-H uo uo T t T t +1 © v q 00 © T t OO c-CN +1 CN OO ON VO T t CN © +1 o o u o r--T t © +1 u o ON l - l l ' l 1-H T—I i—  1 CO O •*-» . z i <H co C B W g -a a O 43 „ . O ps w ^ V j CO a co ^ O 2 43 O r-H U H 2 8 43 PL, PH o ^ O * J 43 O P H H vo S3 O '"I '> CO ha •a CO I+l fi rt s CO « rt _o co J 5 tH 42 O - O X * -m o co fi H II 112 DTNB-treated plasma was observed, with no change in LPD total cholesterol concentration observed as compared to control plasma. Within the TRL and HDL lipoprotein fractions, an increase in free cholesterol concentration was observed in DTNB-treated plasma as compared to control. Again, a decrease in LDL free cholesterol concentration was seen in DTNB-treated plasma as compared to control plasma (Table 21). The total cholesteryl ester concentration for both LDL and HDL was seen to decrease in DTNB-treated plasma as compared to control plasma. Overall, there was seen an increase in free cholesterol concentration in DTNB-treated plasma (65%) as compared to control plasma (52%). The overall cholesteryl ester concentration in DTNB-treated plasma was shown to decrease (34%) as compared to control plasma (47%). Total phospholipid values were lower in the DTNB-treated lipoprotein fractions LDL and HDL as compared to control plasma. No change was observed when the TRL DTNB-treated fraction was compared to control plasma (Table 21). Triglyceride levels did not seem to be affected by pre-treatment of plasma with DTNB (Table 21). Total protein levels within the four fractions of the DTNB-treated plasma showed the following differences. There was a decrease in total protein levels of the DTNB-treated LDL and HDL fractions. Other fractions did not seem to be affected by plasma pre-treatment with DTNB. 113 When ABLC was incubated in plasma pretreated with DTNB for 18 h, the percentage of AmpB recovered in the HDL fraction was significantly decreased and the percentage recovered in the LPD fraction was significantly increased compared to controls. (Table 21, Figure 26). 1 1 4 T3 a u i o s-a © • o o o o o o o o suoiptuj iiiajojdodji ui P9J9A039J 3iavj° % cd 43 s . a OO VH _H VH c tu cd o 6 ^ S3 bO ^ "C -i-> <u o cd cj tu -S3 VH H cl OH CU OO S3 cd Q CU H—> o VH C H O C H T3 Cl „ cd cd 00 O VH -*-> VH o o c/i <u bO cd s oo cd bfj CO o * <N oo w tu Q H - l CH • l-H tU o VH H-H VH o o oo > o o V O H * > cd u H4 PQ o VH £2 O o « • l -H 5 "cd oo -O 3 I V © CN bp c * i .2f3C3 0 > VH tU O H T3 S ^ cd PH • tn PH OO , _ J c S. *0 2 VH 1 a I S oo 52 o 42 a OH oo G cd H—> oo +1 2 <-> •c^- tu T3 tU VH VH • i-H • ^H tU tu VH C H CU ^ _ VH O O oo VH VH - -H C H C H cd o ° •§ a a ^ 3 3 Q Chapter 4 Discussion 116 4.1 Lipoprotein Distribution of Amphotericin B The objective of this research was to determine the plasma lipoprotein distribution of amphotericin B (Fungizone®) and amphotericin B lipid complex (ABLC) in different human plasma samples, and subsequently determine the relationship of the drug distribution to lipid and protein composition and concentration of these separated fractions. Independent of plasma lipoprotein lipid and protein concentration, the majority of AmpB was recovered in the LPD fraction following the incubation of free AmpB, while the majority of AmpB was recovered in the HDL fraction following the incubation of ABLC. It was also observed that increases in HDL coat lipid content (which contains free cholesterol and phospholipid) resulted in less AmpB recovered in this fraction following the incubation of ABLC. However, increases in the TG:TP ratio within HDL resulted in more AmpB recovered in this fraction following the incubation of free AmpB. It was further observed that when HDL coat lipid content (fC + PL) was artificially elevated by DTNB, the percentage of AmpB recovered in this fraction was significantly decreased compared to controls following the incubation of ABLC. In addition, it was further observed that the majority of the AmpB recovered in the HDL fraction following the incubation of ABLC was found in the H D L 3 fraction. 4.1.1 Amphotericin B When amphotericin B (Fungizone®) was incubated in human plasma, the majority of drug was recovered in the lipoprotein-deficient fraction (Table 16, Figures 16-19). These results 117 are consistent with the results of other researchers. Bhamra and associates have shown, through the use of a rat model, that the majority of amphotericin B is found to bind the nonlipoprotein plasma proteins that are found in greater concentrations than the lipoproteins in plasma [Bhamra et al., 1997]. Brajtburg and associates reported in 1984 that only 25% of amphotericin B is bound to lipoproteins when separated from the other plasma proteins by sequential ultracentrifugation. Within the lipoprotein fractions of the plasma samples themselves however, the majority of amphotericin B was found to be associated with high-density lipoproteins (Table 16, Figures 16-19). In an attempt to determine the relationship between drug/lipoprotein association and lipoprotein composition and concentration, all components of the lipoproteins were considered. Trends in the relationship of drug distribution to lipid or protein content and concentration of the lipoprotein fractions are summarized in Table 22. Within the high-density lipoprotein fraction, it was observed that as the total triglyceride to total cholesterol ratio (TG:TC) increases, the amount of amphotericin B recovered within that fraction also increases (r = 0.80, p < 0.05). Table 22: Important trends observed in the comparison of AmpB and ABLC distribution within plasma lipoproteins and the content and composition of these lipoprotein fractions. Lipoprotein Lipoprotein Amphotericin B Correlation Fraction Profile Recovery Amphotericin B t TG:TP t AmpB TRL 0.70 LDL tPL •I AmpB -0.71 HDL t TG:TC t AmpB 0.80 ABLC TRL t TG:TC t ABLC 0.75 TRL T TG:TP t ABLC 0.71 HDL t fC + PL i ABLC -0.76 118 These findings suggest that amphotericin B lipoprotein distribution following the incubation of Fungizone® is regulated by different high-density lipoprotein components, specifically triglyceride and cholesterol. This may indicate that the association of amphotericin B with high-density lipoproteins is a function of the particle number. The molar concentration of HDL, i.e. the number of HDL particles that circulate in the bloodstream, exceed that of any other lipoprotein (Table 23) [Patsch et al., 1981]. Simply put, amphotericin B may associate with HDL mainly because it is the most abundant lipoprotein particle within the bloodstream. One must, however, take into account the fact that a relatively small amount of amphotericin B is actually recovered within the high-density lipoprotein fraction (Table 16). As previously mentioned, most of the amphotericin B incubated in human plasma does not associate with lipoproteins at all, but with the lipoprotein-deficient fraction, which contains other plasma proteins such as albumin and a-1-glycoprotein. Amphotericin B is over 95% protein bound, which suggests that within the lipoprotein-deficient fraction, the majority of amphotericin B is bound to nonlipoprotein plasma proteins. Table 23: Molar concentrations of lipoproteins in normal fasting patients Lipoprotein pm Mol % No of particles X10'12/mL VLDL 0.1 0.7 7 IDL 0.04 0.3 3 LDL 1.6 10 100 HDL2 1.5 9 90 HDL3 12/7 80 800 Although the amount of amphotericin B recovered from the low-density lipoprotein fraction is very small compared to the lipoprotein-deficient fraction, it is important to note that within LDL, as the concentration of phospholipid increases, the amount of amphotericin B 119 recovered in the LDL fraction proportionally decreases (r = -0.71, p < 0.05). This relationship is supported by the work of others. A known modifier of polyene binding action is the cholesterol-to-phospholipid ratio in the plasma environment. Specifically, phospholipid can decrease polyene-sterol interactions [Brajtburg et al., 1984]. Thus, it has been shown that as you increase the amount of phospholipid present, you can decrease amphotericin B binding. 4.1.2 Amphotericin B lipid complex The studies presented within show that the majority of ABLC is seen to associate with high-density lipoproteins. The incorporation of amphotericin B into lipid complexes composed of DMPC and DMPG, results in an overall increase in the amount of drug recovered in the lipoprotein fractions, and specifically the high-density lipoprotein fraction as compared to the traditional amphotericin B colloidal dispersion. Following the incubation of ABLC in seven different human plasma samples, and the subsequent separation of the lipoprotein and lipoprotein-deficient plasma fractions, certain relationships were observed. Within all human plasma samples the majority of ABLC was recovered in the high-density lipoprotein fraction (Table 17, Figures 16-19). Within HDL, a relationship between the percentage of ABLC recovered from HDL and the coat lipid content of the HDL fraction appears evident (Table 22). As the HDL coat lipid content (i.e. free cholesterol and phospholipid) increases, the amount of ABLC recovered in that fraction decreases (r = -0.76, p < 0.05). That is, the higher the coat lipid content of HDL, the less ABLC tends to associate with that fraction. 120 This relationship is supported by work done with DTNB. As discussed previously, DTNB is a sulfhydryl inhibitor, which prevents LCAT from esterifying free cholesterol to cholesteryl ester. My hypothesis was that through the use of DTNB, I would be able to inhibit the enzymatic activity of LCAT. This would lead to an increase in the free cholesterol content of the lipoproteins, thereby increasing the coat lipid content of HDL. Through this modification, one would thereby expect to see less ABLC associated with high-density lipoproteins. As shown in Table 21 and Figure 26, when the coat lipid content of HDL was increased a significant decrease in the amount of ABLC recovered within HDL was seen with a subsequent increase in the amount of ABLC recovered in the lipoprotein-deficient fraction. This suggests that the coat lipid content of HDL does indeed play a role in dictating the preferential distribution of ABLC into HDL. Within HDL specifically, the majority of ABLC was found within the HDL3 fraction (Figure 24). HDL2 contains a higher proportion of all lipids, while H D L 3 has a higher protein content (Table 20). This is due to the fact that H D L 3 contains both apolipoprotein AI and apolipoprotein All, while HDL2 is comprised of only apolipoprotein AI particles. Work concurrent with the research reported here, which was carried out by another member of my lab, showed a similar ABLC distribution pattern. The majority of ABLC was recovered within HDL3 (data not shown). The results obtained showed slightly different distribution patterns than those reported here. However, this is in line with research 121 presented here as different human plasma samples were utilized. This shows that even within HDL subclasses, ABLC distribution changes with changes in HDL content and concentration. Further DTNB experiments carried out within the lab showed that when ABLC was incubated in human plasma pretreated with DTNB, and the plasma subsequently separated into HDL2 and HDL3, the amount of ABLC recovered with HDL3 significantly decreased (data not shown). This data provides further evidence that suggests that as the HDL, and more specifically the HDL3 coat lipid content increases, the amount of ABLC recovered within this fraction decreases. As with amphotericin B, it is my feeling that the preferential association of amphotericin B lipid complex with high-density lipoproteins is due, in part, to particle number. Within normolipidemic human plasma, it has been reported that the HDL2-to-HDL3 particle ratio, based on cholesterol concentration, is around 0.45 [Anchisi et al., 1995]. This ratio suggests that there are approximately twice as many HDL3 particles present in human plasma as HDL2 particles. Other researchers have reported an HDL2-to-HDL3 particle ratio, based on cholesterol concentration, as low as 0.11, suggesting almost eight times as many HDL3 particles as HDL2 particles (Table 23) [Patsch et al., 1981]. This theory is supported by preliminary research carried out within the laboratory. Observations showed that decreases in the HDL2-to-HDL3 particle ratio in plasma from different subjects results in a greater percentage of amphotericin B recovered in the HDL fraction (r = -0.70, p < 0.05) following the incubation of ABLC [Kennedy and Wasan 1999]. That is, as the number of HDL3 122 particles increased in human plasma, more ABLC was recovered from the high-density lipoprotein fraction. It has been suggested that the distribution of ABLC may be governed by lipoprotein lipid concentration. If the preferential distribution of amphotericin B lipid complex with high-density lipoproteins was a function of total lipid mass, instead of particle number, one would expect to see the majority of ABLC recovered from the HDL2 subclass. As previously reported, HDL2 has a greater amount of all of the lipids found within lipoproteins. This however is not the case. This is also supported by the fact that between all lipoprotein subclasses, triglyceride-rich lipoproteins and low-density lipoproteins contain a greater percentage by weight of all lipids. If the distribution of ABLC was simply due to lipid mass, one would expect to see the majority of drug associating with either of these two classes. If the plasma lipoprotein distribution of ABLC was dependent on the triglyceride or cholesterol (total, esterified, unesterified) concentration within each lipoprotein fraction, a greater percentage of the drug should be recovered in the TRL or LDL fractions respectively. Again, this is simply not what is observed. It has also been suggested that the preferential distribution of amphotericin B lipid complex with HDL may be due to the formation of apolipoprotein Al-phospholipid complexes [Surewicz et al., 1986, Gazzara et al., 1997 and Lambert et al., 1998]. Surewicz and associates showed that the association of Apo AI with multilamellar liposomes comprised of acidic phospholipids, such as DMPG, rapidly formed thermally stable complexes over a wide temperature range. As previously mentioned, HDL2 particles are comprised of 123 apolipoprotein AI alone, while HDL3 is comprised of both apolipoprotein AI and All. It has been reported that apolipoprotein AI makes up between 60 - 70% of the protein content of HDL [Ritter et al., 1977 and Patsch et al., 1981]. In a normolipidemic situation, the amount of apolipoprotein AI is equally distributed between HDL2 and HDL3, with apolipoprotein All being found only within HDL3 [Koga et al., 1983]. HDL2 is comprised of 2 mol% apolipoproteins, while H D L 3 is comprised of 5 mol% [Patsch et al., 1981]. If the distribution pattern of ABLC was due solely to a complex between DMPG and apolipoprotein AI, it is my feeling that ABLC would be more equally distributed between HDL2 and HDL3, as there are roughly equal amounts of apolipoprotein AI found within these HDL subclasses. However, the research presented within showed no relationship between HDL total protein content and ABLC distribution within this fraction. Coupled with the findings of other investigators, the results of these studies suggest that the distribution of ABLC into high-density lipoproteins appears to be related to the HDL particle number, and more specifically, the H D L 3 particle number within plasma. 4.2 Limitations of the Study The major limitation of this work is the lack of a broader spectrum of subject plasmas. Only seven human plasma samples were utilized in these experiments. By incorporating more plasma samples with a broader range of lipoprotein profiles into this study, a better understanding of the relationships between drug distribution and lipid or protein content of the lipoproteins could have been established. As well, the application of the modified 124 HDL2/HDL3 ultracentrifugation technique to a larger number of plasma samples would have provided a clearer picture into what drives the preferential distribution of ABLC into high density lipoproteins. Furthermore, it is my feeling that it would also have been beneficial to determine HDL2 and HDL3 particle numbers based on an actual count of the number of apolipoproteins present. 4.3 Clinical Ramifications As discussed in the introduction, the pharmacokinetics and toxic effects of a number of drugs differ when administered to diseased patients as compared to healthy controls [Gibaldi and Perrier 1982 and Rowland and Tozier 1989]. This often makes it difficult to determine the most effective dose of these drugs for administration to diseased patients. In the case of some water insoluble compounds, and compounds incorporated into lipid-based vesicles, the dose that is deemed non-toxic in healthy animals and humans is ineffective and/or toxic when administered to the diseased patient [Sgoutas et al., 1986, Wassef et al., 1986, Kasiske et al., 1988, Rodl et al., 1990 and Andrade et al., 1993]. During the past 15 years, there has been a dramatic increase in the prevalence of fungal infections around the world. This is due to the improvement in the recognition and diagnosis of fungal infections, as well as the prolonged survival of patients who are immunocompromised. These are patients such as those with cancer, liver disorders, patients with HIV/AIDS, kidney disease or diabetes. 125 Research into the lipoprotein profile of patients with cancer indicates that those suffering from cancer are also characterized by low HDL-cholesterol levels [Anchisi et al., 1995]. Chao and associates (1975) report that HDL-cholesterol levels are significantly reduced in 14 types of cancer. In the serum of a healthy population, HDL3 is the main HDL subtraction. Anchisi and associates report that the normal HDL2/HDL3 ratio is 0.45 or approximately twice as many H D L 3 particles as H D L 2 particles. In the serum of a patient with cancer, one sees an inversion of the normal profile caused by a sharp reduction in the number of HDL3 particles coupled with an increase in the percentage of HDL2 particles present. This leads to an increase in the HDL2/HDL3 ratio. Anchisi et al., reports an HDL2/HDL3 ratio of 2.00 ± 0.30 (p < 0.01 vs. normal subjects) in those patients possessing solid tumors. This indicates that in cancer patients, there are twice as many HDL2 particles than HDL3 particles. Although the free cholesterol and phospholipid levels are not reported for this study, one would expect to see a decrease in the amount of ABLC associated with high-density lipoproteins in this case due to a decrease in HDL3 particle number. With a decrease in ABLC associating with HDL, one would see an increase in association with other lipoproteins, such as TRL and LDL. If this is the case, this suggests that perhaps ABLC may be more nephrotoxic in cancer patients. As with cancer sufferers, it has been reported that patients suffering from liver disease also display abnormal lipoprotein profiles. In general, with liver disease one sees an overall decrease in high-density lipoprotein levels. HDL3-cholesterol levels are decreased with no change seen in HDL2-cholesterol levels. The lipoprotein profile of patients with chronic active hepatitis show a decrease in HDL3 levels, with no apparent change in H D L 2 [Koga et 126 al., 1983]. With hepatitis, HDL3-cholesterol levels are reduced by half when compared to controls [Okazaki et al., 1981]. Koga and associates also report that in a number of patients with liver cirrhosis, little to no H D L 3 particles are found. In those cases where H D L 3 levels are present, the HDL3-cholesterol levels are one third of control levels [Koga et al., 1983]. In specific cases of liver disease, such as patients with hepatocellular dysfunction and cholestasis, the apolipoprotein AI/AII ratio is reported to be higher in HDL2 than in HDL3 [Koga et al., 1983]. This suggests that in these cases, there are more apolipoprotein AI particles in HDL2. This is in opposition to what is found normally, that being equal levels of apolipoprotein AI in both HDL2 and HDL3. It has also been observed that in those patients with severe hepatocellular dysfunction and cholestasis, an increase in the apolipoprotein AI/AII ratio corresponds to a decrease in HDL3 levels [Koga et al., 1983]. Due to the fact that LCAT activity is also decreased in patients with liver disease, HDL3 levels may be decreased due to insufficient synthesis of necessary precursor lipoproteins (nascent HDL) and concurrently with impairment of the maturing of nascent HDL by inactive LCAT [Koga et al., 1983]. The conversion of HDL2 to HDL3 might also be impaired in liver disease in which one sees the decreased activity of hepatic triglyceride lipase (HTGL). Taken together, the two points can lead to decreased serum levels of HDL3 in patients with liver damage. Kajiyama and associates report that both HDL-cholesterol and HDL-triglyceride levels are decreased in cases of acute hepatitis, chronic aggressive hepatitis, liver cirrhosis, fatty liver, hepatoma and cholelithiasis. In each case HDL2 levels are low but within normal reported values, while HDL3 levels are significantly decreased [Kajiyama et al., 1983]. In all cases as 127 well, LCAT activity was found to be decreased. In agreement with Koga et al., Kajiyama and associates feel that liver parenchymal damages may decrease the synthesis of nascent HDL, which leads to a decrease in mature HDL. The increased serum bile acids and inhibited LCAT observed in many hepatobiliary diseases inhibit the conversion of nascent HDL into mature HDL [Hamilton et al., 1974]. It has also been suggested that the lowered HDL3-cholesterol levels and HDL3 particle numbers result from hepatocellular dysfunction causing the inhibition of protein and lipid synthesis [Kajiyama et al., 1983]. In all cases of liver disease, H D L 3 particle numbers seem to be decreased due to the decreased activity of enzymes such as LCAT and HTGL. It would be expected then that if these patients were to be administered ABLC for the treatment of systemic fungal infections often seen in those patients with liver disease, an alteration in the lipoprotein distribution of ABLC would be observed. Specifically, due to the decreased levels of H D L 3 , and HDL in general, less ABLC would associate with these particles. As in the case of patients with cancer, this suggests that perhaps ABLC nephrotoxicity may increase in those patients with liver disease. Amphotericin B is also often administered to patients with HIV/AIDS. It has been reported that in those patients with AIDS, one sees a decrease in serum cholesterol levels, with a doubling in serum triglyceride levels [Grunfeld et al., 1992]. As well, a 37% decrease in both HDL3 and HDL2 levels is seen. This decrease in HDL levels in most likely due to the fact that a significant decrease in apolipoprotein AI levels, from 124 mg/dL to 86 mg/dL, is observed [Grunfeld et al., 1992 and Feingold et al., 1993]. The HDL that is present is t 128 characterized as being triglyceride rich and deficient in cholesterol. This indicates a decrease in HTGL activity, commonly seen in those patients with liver disease. Similar to cancer and liver disease cases, patients with various degrees of kidney disease have also been reported to have lipid and lipoprotein abnormalities. In those patients with nephrotic syndrome, decreased LCAT enzyme activities and low levels of HDL and apolipoproteins AI and AH have been reported [Feltz et al., 1974, Saku et al., 1988, Appel et al., 1991 and Kamanna et al., 1998]. In those patients with end stage renal disease, decreased LCAT enzyme activities as well as decreased synthetic rates of HDL and apolipoprotein AI are commonly seen [Fu et al., 1990 and Appel et al., 1991]. In those patients with end stage renal failure on continuous ambulatory peritoneal dialysis is it common to see decreased HDL-cholesterol levels [Serdyuk et al., 1997]. It is fair to suggest that in these patients the toxicity of both amphotericin B (Fungizone®) and ABLC may be altered due to the alterations in the lipid and lipoprotein profile of the patient. Finally, diabetic patients whom are not insulin dependent are characterized by low levels of HDL-cholesterol [Albers et al., 1999]. In those patients whom are insulin dependent, higher levels of HDL-cholesterol as compared to controls are seen. As well, higher levels of apolipoprotein AI and consequently a higher apolipoprotein AI/AII ratio have been reported [Albers et al., 1999]. This suggests a higher HDL2/HDL3 particle ratio. In this case as well, ABLC would likely have an altered lipoprotein distribution pattern due to the increases in HDL2, and/or decreases in HDL3 particles. 129 As discussed above, amphotericin B (Fungizone®) and ABLC are commonly administered to patients with cancer, liver and kidney disease, diabetes and HIV/AIDS. The lipid and lipoprotein profile of these patients is characterized by a decrease in HDL-cholesterol levels, and more specifically, H D L 3 particle numbers. Through my research I have shown that ABLC preferentially associates with high-density lipoproteins due to the coat lipid content of HDL, and possibly the fact that these are the most abundant particles in serum. Decreases in HDL particle number seen in many immunocompromised patients may effect the lipoprotein distribution of ABLC. Specifically, changes in the coat lipid content of HDL, or more specifically H D L 3 , may lead to a decrease in the amount of ABLC that will associate with that fraction. This will lead to more ABLC being recovered within other lipoprotein fractions such as LDL, or with the lipoprotein-deficient fraction. This may lead to an increase in the toxic side effects of ABLC upon administration. 4.4 Future Studies The results of these experiments have shown that the concentration of high-density lipoprotein found in plasma plays an important role in determining the distribution of ABLC. More specifically, it may be a function of the HDL2-to-HDL3 particle ratio that determines the distribution of ABLC. While previous research has shown the HDL2/HDL3 ratio is altered in patients commonly administered amphotericin B, the lipoprotein distribution of this drug in these patients has not be elucidated. Observing the distribution pattern in these patients would offer up a world of information into what effect dyslipidemia has on the efficacy and toxicity of hydrophobic compounds. 130 One drawback to this research is the fact that ABLC is utilized only as last resort therapy due to the cost associated with administering this compound. It would be beneficial however to undertake a clinical study in which the lipid and lipoprotein profile of patients administered ABLC could be examined along with the lipoprotein distribution of the drug. This would aid in providing information into exactly how the coat lipid content of HDL dictates the distribution of ABLC into this lipoprotein fraction. Correlating toxic side effects of amphotericin B to the HDL2/HDL3 ratio of immunocompromised patients may help in predicting whether or not patients will suffer toxicity related side effects when administered amphotericin B. Studies have shown that decreased HDL3 levels are due to a decrease in the activity of LCAT, the enzyme responsible for the esterification of free cholesterol to form cholesteryl ester [Feltz et al., 1974, Saku et al., 1988, Appel et al., 1991and Kamanna et al., 1998]. It is my belief that future studies may be necessary to elucidate what role, if any, LCAT plays in the lipoprotein distribution of amphotericin B. It would also be relevant to investigate what role, if any, HTGL plays in affecting the lipoprotein distribution of amphotericin B and other similar hydrophobic drugs. Grundy and associates have reported that there is an inverse relationship between HTGL activity and HDL-cholesterol levels. More specifically, an increase in HTGL activity reduces HDL particle numbers in the plasma [Grundy et al., 1999]. It may also be beneficial to investigate the roles of other enzymes such as cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) in lipoprotein distribution of hydrophobic drugs. Research suggests that changes in HDL size and 131 composition can lead to altered interactions between HDL, LCAT and CETP [Sparks et al., 1989 and Sparks et al., 1992]. In hypertriglyceridemic situations, the transfer of CETP to H D L 3 is significantly reduced, resulting in an increase in transfer of cholesteryl ester to HDL2 particles [Sparks et al., 1995]. HIV/AIDS patients have been shown to have triglyceride levels double those of normal controls. Thus, CETP may play an important role in HDL lipid composition, and as a result, amphotericin B distribution. PLTP has been shown to promote the mass transfer of phospholipids between lipoproteins and mediate the conversion of HDL into larger (HDL2) and smaller (HDL3) particles [Albers et al., 1999 and Cheung et al., 1999]. Increases in PLTP have been shown to increase apolipoprotein AI, and consequently, HDL2 particle numbers [Cheung et al., 1999]. These changes may effect the lipoprotein distribution of amphotericin B if they occur in those patients administered this compound. While the research presented within did not show a correlation between the amount of apolipoprotein present and the distribution of ABLC, I feel that this is an important area that should not be overlooked. One very important difference between HDL2 and HDL3 is the fact that HDL3 contains both apolipoproteins AI and AH, while HDL2 is comprised of only AI. This is a very important difference one in which a variety of experiments should be designed in order to conclusively rule out either of these apolipoproteins playing a role in the distribution of hydrophobic compounds. With the recent advances in transgenics, these models should be utilized in determining the role of a variety of enzymes and apolipoproteins in drug distribution [Rodriqueza et al., 1998 and Semple et al., 1998]. Studies in which the lipoprotein distribution of amphotericin B is investigated in HDL deficient, or apolipoprotein 132 AI or All knockout mice may be beneficial. This may provide a wealth of information on the effects of HDL alteration on the pharmacology of Fungizone® and ABLC. 4.5 Conclusions In conclusion, these studies have demonstrated that the plasma lipoprotein distribution of amphotericin B is altered when incorporated into a lipid complex composed of DMPC and DMPG. Specifically, the majority of conventional amphotericin B (Fungizone) associates with the lipoprotein-deficient plasma fraction (LPD), while amphotericin B lipid complex (ABLC) is found to associate predominantly with high-density lipoproteins (HDL). The results suggest that the preferential association of ABLC with HDL is a consequence of HDL coat lipid content (i.e. phospholipid and free cholesterol). As the HDL coat lipid content increases, the amount of ABLC recovered within this fraction decreases. Furthermore, it has been demonstrated through two different methods that it is changes in the coat lipid content of the HDL subclass, H D L 3 , which defines the preferential distribution of ABLC into HDL. Since H D L 3 has a lower percentage by weight of coat lipids than does HDI2, the lower percentage of ABLC recovered in the HDL fraction as the HDL coat lipid content increases may be a function of the number of H D L 3 particles found within the HDL fraction. 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