INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms Internationa! A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 1352310 Disposition kinetics of amphotericin B in rats Wu, Yuhong, M.S. The University of Arizona, 1993 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 DISPOSITION KINETICS OF AMPHOTERICIN B IN RATS by YUHONG WU A Thesis Submitted to the Faculty of the DEPARTMENT OF PHARMACEUTICAL SCIENCES In Partial Fulfillment of the Requirement For the Degree of MASTER OF SCIENCE In the Graduate College THE UNIVERSITY OF ARIZONA 19 9 3 2 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED:. APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown below: 3-U-fS Sherry Chow, Ph.D. Assistant Professor of Pharmaceutical Sciences Date 3 TO MY DEAR HUSBAND ZHENGWEI ZHAO 4 ACKNOWLEDGEMENTS The author wishes to express her sincere gratitude to her advisor, Dr. M. Mayersohn, for providing guidance, inspiration, and continued support and encouragement during the course of this research. His honest devotion, constructive criticism, effective discussions and suggestions greatly helped the author to accomplish this project. The author expresses her sincere gratitude for the advice, help, encouragement, and friendship of Dr. H. Chow. Sincere appreciation is also due to Dr. S. H. Yalkowsky for serving as a member of committee. In addition, the author wish to sincerely thank all the individuals in the pharmacokinetic group. The author takes this chance to thank her parents for their love, support and guidance though these years. Also, this work would not have been possible without the encouragement, understanding and overwhelming support from her dear husband. 5 TABLE OF CONTENTS page LIST OF ILLUSTRATIONS 8 LIST OF TABLE 10 ABSTRACT 11 CHAPTER 1. 2. STATEMENT OF PROBLEM 12 1.1 Statement of Problem 13 1.2 Objectives 17 1.3 Organization 17 LITERATURE REVIEW 19 2.1 Chemistry 19 2.2 Stability 21 2.3 Pharmacology 22 2.3.1 Mechanisms of Action 22 2.3.2 Oxidation-Dependent Mechanism 23 2.4 2.5 Toxicity 24 2.4.1 Nephrotoxicity 25 2.4.2 Prevention of Toxicity 28 2.4.3 Liposomes 29 Pharmacokinetics 31 2.5.1 Human Studies 31 2.5.2 Animal Studies 35 6 TABLE OF CONTENTS continued page 2.6 3. 35 EXPERIMENTAL SECTION 39 3.1 Materials and Instruments 39 3.2 Pharmacokinetic Studies 41 3.2.1 Catheter 41 3.2.2 Animal Surgery 42 3.2.3 Pharmacokinetic Studies 43 3.3 Sampling 44 3.4 Preparation of Intravenous Solution 45 3.5 Sample Preparation 46 3.6 AmB Assay by HPLC 46 3.7 Calculations 47 3.8 Creatinine 50 3.8.1 Creatinine Assay 50 3.8.2 Creatinine Clearance 51 3.9 4. Analysis Methods Data Analysis 52 3.9.1 Model-Independent Method 52 3.9.2 Model-Dependent Method 57 3.9.3 Statistical Analysis 58 RESULTS 61 4.1 Analytical Method 61 4.2 Stability of AmB in Urine 62 7 TABLE OF CONTENTS.....continued page 4.3 Pharmacokinetic Data 5. DISCUSSION 65 92 5.1 Discussion 92 5.2 Conclusions 98 5.3 Suggestions for Further Work 98 APPENDIX A APPENDIX B APPENDIX C REFERENCE Serum and Urine AmB Concentration versus Time 100 Plot of Serum AmB Concentration versus Time 108 Fitted Curves Generated by ADAPT II 116 124 8 LIST OF ILLUSTRATIONS page Figure 1. Chemical Structure of AmB, Cholesterol and Ergosterol 20 Figure 2. Mechanism of Nephrotoxicity 27 Figure 3. Three Compartment Model 33 Figure 4. UV Absorption Spectrum of AmB 48 Figure 5. Plot of Standard Curve of AmB in Serum 49 Figure 6. The Plot of Relationship between Urine Excretion Rate and Time 54 Figure 7. Two Compartment Model Describing AmB 60 Figure 8. Representative Chromatograms of AmB and Internal Standard in Serum 63 Plot of Mean AmB Serum Concentration versus Time for High Dose 76 Figure 10. Plot of Mean AmB Serum Concentration versus Time for Medium Dose 77 Figure 11. Plot of Mean AmB Serum Concentration versus Time for Low dose 78 Figure 12. Plot of Mean Steady-State Concentration versus Dose 79 Figure 13. Plot of Half-life versus Dose 80 Figure 9. Figure 14. Plot of Volume of Distribution versus Dose .. 81 Figure 15. Plot of Systemic Clearance versus Dose 82 Figure 16. Plot of Renal Clearance versus Dose 83 Figure 17. Plot of Renal Clearance versus AmB Serum Concentration 84 9 LIST OF ILLUSTRATIONS continued page Figure 18. Plot of Renal Clearance versus AmB Steady-state Concentration 85 Figure 19. Plot of AmB Renal Clearance versus Time in the Low, Medium, and High Dose Groups 86 Figure 20. Plot of Fraction of the Dose of AmB Excreted into the Urine versus Dose 87 Figure 21. Plot of the Individual Creatinine Clearance before and after AmB Treatment in the Low, Medium and High Dose Groups 88 Figure 22. Plot of Comparison of Creatinine Clearance before and after AmB dosing 89 Figure 23. Plot of Renal Clearance versus Creatinine Clearance 90 Figure 24. Plot of Renal Clearance versus Urine Flow ... 91 10 LIST OF TABLES page TABLE 1. TABLE 2. Factors Responsible for the Increased Prevalence of Fungal Infections 15 The Major Invasive Deep Fungal Infections in Different Immunocompromised Patients 16 TABLE 3. Pharmacokinetic Parameters of AmB in Humans .. TABLE 4. HPLC Methods Used to Analyze AmB 38 TABLE 5. Intra-Day Assay Results of AmB 64 TABLE 6. Inter-Day Assay Results of AmB 64 TABLE 7. Stability of AmB in Urine 66 TABLE 8. Summary of Pharmacokinetic Parameters 72 TABLE 9. Creatinine Clearance 74 TABLE 10. Summary of Pharmacokinetic Parameters (ModelDependent Method) 34 75 11 ABSTRACT Amphotericin B (AmB) is widely used for the treatment of systemic infections. The current therapeutic regimens for this drug are complex because of limited pharmacokinetic information. Male Sprague-Dawley rats were randomly divided into three different dose groups, receiving 0.332, 0.838, 1.323 mg/kg of AmB. A two-step infusion procedure was employed to rapidly achieve steady-state conditions. Serum samples and urine samples were collected and assayed by HPLC. Halflife, volume of distribution (Varea) and systemic clearance (CLs) are (± SD): 953.2 ± 300.6 min, 5.14 ± 1.53 L/kg and 3.72 ± 0.8 ml/min*kg, respectively. There are no significant differences for any parameter as a function of dose. However, renal clearance decreases when the total infused dose increases. We conclude that volume of distribution and systemic clearance are linear over the dose range studied. The dose-dependent renal clearance may be due to either the nephrotoxicity associated with AmB or saturable secretion. 12 CHAPTER 1 STATEMENT OF PROBLEM Amphotericin B (AmB), a polyene antibiotic, is a natural fermention product produced by Streptomyces nodous ( Trejo et al., 1963) which was originally isolated from soil from the Orinoco River region in Venezuela in 1953. Streptomyces nodous co-produces two polyene antibiotics, the tetraene amphotericin A (AmA) and the hapatene amphotericin B. Both antibiotics were found to possess a broad spectrum of antifungal action (Steinberg et al., 1956), but AmA was not further developed. AmB remains the drug of choice for most forms of deep-seated fungal infections, despite its serious side effects and the introduction of newer anti-fungal compounds. Diseases caused by fungi are called mycoses or fungal infections, which range from very common mild chronic infections affecting the skin, via deep cutaneous or subcutaneous infections, to acute or chronic systemic infections. Systemic fungal 13 infections may occur in normal hosts, however, in immunocompromised or physiologically compromised patients, these infections are a major cause of morbidity and mortality (Gold et al., 1984; Bodey et al., 1984). For example, aspergillosis and candidiasis are among the leading causes of deaths from infections in patients with leukemia. A high incidence of fungal infections also occurs in people who receive intensive chemotherapy, broad-spectrum antibiotics and have potential defects in neutrophil functions (Table 1 and Table 2). 1.1 Statement of the Problem The incidence of fungal infections has increased dramatically during the last decade. Deep-seated mycoses are beginning to create serious problems for clinicians working with certain populations of patients, which include patients that have cancer or are immunocompromised or physiologically compromised. There have been a number of recent surveys which provide an impression of the importance of fungal infections in severely ill patients. For example, disseminated candidiasis is rapidly becoming a major threat to cancer patients, especially those with hematologic malignancies (Bodey et al., 1984). Recent data have indicated that over a 4-year period, 11.9% of patients with acute leukemia suffered from fungal infections caused by Candida sp. and torulopsis glabrata (Maksymiuk et al., 1984). The need for effective antifungal therapies has been felt more and more acutely with the emergence of the acquired immune deficiency syndrome (AIDS) and AIDS- 14 related complex (ARC), which are often associated with systemic fungal infections. Recent data from the Centers for Disease Control reveal that between 100,000 and 200,000 Americans meet the criteria for AIDS and ARC, and that between 1 and 2 million healthy Americans have already been infected. Of patients showing antibodies to the human immune deficiency virus, according to estimates of the Centers for Disease Control, between 5% and 25% will progress to AIDS and 25% will develop one of the ARC diseases. As expected, a significant number of these cases will be associated with various fungal infections. One yeast-like fungus, Cryptococcus neoformans is reported to be the fourth most common cause of lifethreatening infections in AIDS patients (Kovacs et al., 1985). Data from the Centers for Disease Control for 3170 AIDS patients registered at the Centers between May 1983 and June 1984 indicate that a total of 58% of the reported cases of AIDS have been associated with various mycoses (Chandler et al., 1985). The main antifungal drugs available now belong to two families, the polyene and azole groups. Although there are a number of antifungal drugs available, which are used for the treatment of serious fungal infections, most of them have drawbacks such as efficacy, adverse effects, and the development of resistance. AmB remains the drug of choice for the treatment of systemic fungal infections (Graybill et al. 1983). Usage of this drug has become more prevalent during the past decade as a result of a dramatic increase in the number of patients having defective immune response. The agent has been in clinical use for over 30 years, yet the 15 Table 1 Factors Responsible for the Increased Prevalence of Fungal Infections AIDS and longer surviving AIDS patients More aggressive cancer chemotherapy Increase in transplantation operations More aggressive intensive care medicine New and use of more prosthetic devices Broader spectrum antibiotic usage Greater travel and exposure to foci of endemic mycoses Increase in intravenous drug abuse More parenteral nutrition and use of lipid suspension Table 2 The Major Invasive Deep Fungal Infections in Different Immunocompromised Patients Candida Aspergillus neutropenia neutropenia AIDS AIDS lymphoma lymphoma infants burns transplantation transplantation corticos neonates burns corticosteroids teroids diabetes corticos sarcoidosis corticos teroids teroids chronic granu drug abuse lomatous disease TPN a: a total parenteral nutrition Cryptococcus Histoplasma 17 of AmB remain poorly understood. The current therapeutic dosage regimens of AmB are based on years of empirical evidence and anecdotal experience. Furthermore, the use of AmB, is often limited by its acute and chronic toxic effects such as renal impairment, hypokalemia and anemia (Butler et al.,1966). Because of these adverse effects, different clinicians attest to a variety of administration methods and accompanying premedication in order to minimize or prevent the acute reactions to AmB. Understanding the disposition kinetics and predicting toxicity as a result of dosing becomes a particularly important issue in optimizing the therapeutic use of this antifungal drug. 1.2 Objectives of Research The major objectives of this thesis research are to: (1) develop analytical methods in order to quantitate AmB in different biological fluid samples. (2) characterize the disposition kinetics of AmB and examine its association with nephrotoxicity after administration of different doses of AmB to rats. 1.3 Organization of Thesis This thesis is presented in five chapters. The first chapter is a brief introduction to this study. The second chapter gives a background knowledge and a brief review of the literature of AmB stability, mechanisms of action, toxicity and 18 pharmacokinetics. Chapter three describes the experiments which were conducted. Chapter four contains the results. Chapter five provides the discussion, conclusions and recommendations for further research. 19 CHAPTER 2 LITERATURE REVIEW 2.1 Chemistry The chemical structure of AmB was elucidated in 1970 and is shown in Figure 1 (Mechlinski et al.,1970). The molecule consists of a large macrolide lactone ring with 37 carbon atoms. One side of the macrolide ring is composed of a rigid lipophilic chain of seven conjugated double bonds and on the opposite side of the ring there is a similar number of hydroxyl groups. The molecule is amphipathic and this feature of its structure is believed to be important in its biological mechanisms of action. The macrolide ring also contains a six-membered ketalic ring and the amino sugar mycosamine is bonded to the ring through an a -glycosidic linkage. Two functional groups, carboxylic acid and amino, form the basis for chemical AMPHOTERICIN B OH OH O COOH NH2 CHOLESTEROL ERGOSTEROL Figure 1. Chemical structure of amphotericin B, cholesterol and ergosterol. 21 modification of AmB. Crystalline AmB is insoluble in water and most solvents except dimethylsulfoxide (DMSO). The commercial product of AmB, Fungizone, is a mixture of AmB, deoxycholate and sodium phosphate buffer. The detergent deoxycholate produces an aqueous micellar suspension rather than a solution of the drug for parenteral administration. When diluted in a 5% glucose injection, Fungizone forms a colloidal dispersion suitable for intravenous injection. However, the presence of salts such as sodium or potassium chloride or acidic pH leads to aggregation of the colloid (Barrriere, 1990). 2.2 Stability AmB has an extended unsaturated chromophore and the presence of a variety of functional groups which make it sensitive to diverse chemical and physical attack. When exposed to extremes of pH, light and heat, AmB exhibits poor stability and reduction of bioactivity (Waksman et al., 1965). Products resulting from degradation are usually biologically inactive. AmB was very stable ( < 5% loss) in human serum at -20°C for up to 14 days, with only a 10% loss of drug after 6 months. But loss of AmB exceeded 60% after six months in DMSO solution (Edmonds et al, 1989). AmB is administered intravenously to patients due to its poor gastrointestinal absorption. Kintzel studied the stability of AmB and found that in 5% dextrose 22 solution it was stable at concentrations of 0.92,1.2 and 1.4 mg/ml when stored at 6°C and 25°C for up to 36 hours (Kintzel et al., 1991). No precipitation, turbidity, gas formation or color change was observed. 2.3 Pharmacology AmB shows a high order of in vitro activity against many species of fungi. Histoplasma capsulatum, Coccidiodes immitis, Candida species, Blastomycse dermatiaidis, Cryptococcus neoformans, Aspergillus fumigatus have been shown to be inhibited in vitro by concentrations of AmB ranging from 0.03 to 1.0 mcg/ml ( Shadomy et al. 1978). AmB has no effect on bacteria and viruses. 2.3.1 Mechanisms of Action The major mechanism of action of AmB is attributed to its ability to bind to the fungal cells. AmB can bind more strongly to ergosterol, the main component of the fungal cell membrane, than to cholesterol, the main component of the mammalian cell membrane. This binding results in an increase in membrane permeability that produces loss of protons and cations from the cell. The clinical usefulness of AmB is due to its binding to ergosterol. However, its toxicity is associated with its binding to cholesterol. The binding mechanism is based on the AmB structure. There are two types of interactions between AmB and ergosterol or cholesterol, specific binding and 23 nonspecific binding (Herve et al., 1989). The hydrogen bonds, specific binding, are formed between hydroxyl groups of the sterol and the carboxyl groups of the AmB molecule. This binding is strengthened by participation of the amino group of the amino sugar side chain of the AmB molecule (see Figure 1) (Brajtburge et al., 1990). The second type of interaction, nonspecific binding, involves the rigid chain of the seven conjugated double bonds of AmB and the whole sterol molecule (see Figure 2) (Herve et al., 1989). Herve and coworkers also conclude that the flat shape of most ergosterol conformers having the double bond located at C-22 of the alkyl side chain was responsible for the greater sensitivity to AmB of ergosterol- containing membranes compared with the sensitivity to AmB of cholesterol-containing membranes. 2.3.2 Oxidation-Dependent Mechanism The action of AmB on fungal cells is believed to involve more than one mechanism (Brajtburg et al, 1990). It has been reported that the lethal effects of higher concentrations of AmB on Candida albicans are not a simple consequence of its membrane-permeabilizing effects, but involves oxidative damage to the cell (Sokol-Anderson et al., 1986 a,b). AmB induces a cascade of oxidative reactions and causes the formation of free radicals during its auto-oxidation process (Lamy-Freund et al., 1985). These radicals may have several different effects on cells, such as stimulating the immune system. 24 AmB markedly augmented the polymorphonuclear leukocyte immunoglobulin Gmediated ingestion of opsonized sheep erythrocytes; this effect was inhibited by superoxide dismutase (Gresham et al., 1988). The experiments done by Brajtburg indicate that hemolysis of erythrocytes and cell death may be inhibited by extracellular catalase and potentiated in the presence of ascorbic acid and other prooxidants (Brajtburg et al., 1989). In contrast, extracellular scavengers and prooxidants did not affect prelytic or prelethel AmB- induced K+ leakage from cells. This type of damage by AmB is not linked to cell membrane permeability and is not dependent on the kind of sterol. 2.4 Toxicity Despite the development of alternative antifungal agents, AmB remains the primary drug for the treatment of life-threatening fungal infections. However, the usage of AmB is limited by its side effects, acute and chronic toxicities. Infusionrelated acute toxicities have included fever, chills, nausea, vomiting, anorexia, headache, bronchospasm, hypotension and anaphylaxis. Chronic toxicities are nephrotoxicity, bone marrow suppression and renal losses of potassium and magnesium. Clements investigated 50 patients receiving I.V. infusion of AmB with a mean cumulative dose of 582 mg. Acute toxicities accompanying infusion occurred in 66% of all patients. Nephrotoxicity occurred in 60% of patients and baseline serum creatinine values in these patients rose by a mean of 1.55 mg/dL, with 25 increases in creatinine ranging from 0.2 to 5.2 mg/dL (Clements et al., 1990). In an attempt to reduce the incidence of toxicity and to increase patient acceptance of therapy, special protocols for infusion of AmB and management of toxicities have evolved. Premedication before infusion is one of the methods used for controlling acute toxicities. Diphenhydramine, acetaminophen, hydrocortisone, and meperidine are suggested to be potentially useful premedications, but there are few reports documenting the efficacy of these agents (Winn et al., 1963). In addition to the use of premedication, standard guidelines for the administration of AmB have been developed and incorporated into standard clinical practice. AmB is usually administered a test dose of 1 mg diluted in 100-250 ml of 5 % dextrose in water and infused over 1-2 hours ( Graybill et al., 1983 ). If infusion-related toxicities occur, infusion rates are often slowed further or therapy may be interrupted temporarily (Bannett et al., 1985). However, toxicities continue to occur with usage of AmB despite current methods of infusion and premedication. 2.4.1 Nephrotoxicity Nephrotoxicity has been regarded as one of the most serious adverse effects to AmB therapy. Early reports indicated that as many as 80 to 90% of patients who received the drug developed some degree of renal dysfunction (Butler et al., 1964). The clinical presentation of nephrotoxicity includes azotemia, renal tubular acidosis, impaired concentrating ability, and renal sodium, potassium and magnesium wasting, 26 with consequent dehydration, hypokalemia and hypomagnesemia. The mechanism of amphotericin B associated nephrotoxicity is poorly understood. Amphotericin B causes renal damage by several postulated mechanisms (Said et al., 1980). Figure 2 illustrates the potential pathways by which AmB induces nephrotoxicity. The main mechanism may involve effects on membrane permeability, an extension of the mechanism of antifungal activity on the cells of the host. As stated in the previous section, AmB damages cell walls of fungal and cell membranes of the host by binding to a sterol, ergosterol of the fungal cell and cholesterol of mammalian cells. This binding action creates pores, increasing the permeability of the membrane which results in leakage of cellular constituents and electrolytes followed by cell lysis and death (Sabra et al., 1990). In addition to the alterations in the permeability of the luminal membranes of the tubular epithelium (Andreoli et al., 1973), modifying renal blood flow (RBF) and glomerular filtration rate (GFR) acutely by increased solute delivery to the macula densa and activating the tubuloglomerular feedback (TGF) mechanism is also a possible explanation (Cheng et al., 1982; Gerkins et al., 1980). Tubuloglomerular feedback, a regulatory mechanism coordinating and regulating the function of both proximal and distal tubules, is manifested as an immediate constriction of afferent arterioles evoked by increased amounts of monovalent ions at the macula densa region of the distal renal tubules (Wright et al., 1977; Schnermann et al., 1975). The end result is ischemic damage to the kidney. Impaired urinary concentrating ability and distal renal tubular acidosis with secondary AMPHOTERICIN B SOLUTE PERMEABILITY N«CI UPTAKE MACULA DENSA ACTIVATION TUBULO-GLOMERULAR FEEDBACK A |GLOMERULAR T FILTRATION RATE 1 I RENAL BLOOD FLOW -• iRENAL VASCULAR T RESISTANCE L RENAL BLOOD FLOW RENAL VASCULAR RESISTANCE OXYGEN DELIVERY m.T.A.L. AMPHOTERICIN B HYPOXIC INJURY m.T.A.L. CELL PERMEABILITY OXYGEN DEMAND I ACTIVE TRANSPORT I ELECTROLYTES Figure 2. Potential pathways by which amphotericin B causes alterations in renal cell permeability; initial alterations in renal hemodynamics (A) and renal tubular cell injury (B) in the medullary limb of the thick ascending limb of the loop of Henle. 28 potassium wasting are associated with these effects (Bhathena et al., 1978). The responsiveness of TGF seems to be influenced by sodium ion concentration. Salt loading can block TGF and reduce the nephrotoxicity. Stein reported that administration of sodium, 90 meq/day, during each day of amphotericin B therapy is effective in decreasing nephrotoxicity associated with amphotericin B (Stein et al., 1989). Mannitol, an osmotic diuretic, prevents amphotericin B nephrotoxicity by causing vasodilation in the renal tubules, maintaining GFR, and promoting flushing of the tubules. Other drugs such as furosemide and aminophylline are reported to reduce the toxicity (Gerkens et al., 1980). 2.4.2 Prevention of Toxicity Several approaches have been developed over the past few years in an effort to overcome the toxicities associated with the clinical use of AmB and these include: (1) alternative antifungal agents have been developed; (2) derivatives of AmB have been synthesized and studied; (3) the combination of AmB with other drugs has been used to treat the fungal infections; and (4) AmB was incorporated into lipid vesicles in an effort to increase antifungal activity and decrease toxicity. The main antifungal drugs belong to two families: polyene and azole (Medoff et al.,1983). The polyene family include three main products: nystatin, amphotericin B and natamycin. Of these, only amphotericin B can be used parenterally and in the treatment of systemic fungal infections (Bindschadler et al., 1969). The development 29 of the azole antifungal class has meant that there are alterative drugs for the treatment of systemic fungal infections (Fromtling et al., 1988). Among the large number of azole antifungal drugs , four drugs - miconazole, ketoconazole, fluconazole and itraconazole - are currently available for the treatment of systemic fungal infections. However, most have drawbacks in terms of efficacy, adverse effects and the development of resistance. Derivatives of AmB have been synthesized to study the antifungal activity and toxicity. The free carboxylic acid group of AmB has formed the basis for the preparation of ester derivatives and the free amino group has been used to prepare N-acyl, N-methylated, N-glycosylated and N-guanidine derivatives of amphotericin B (Schaffner et al, 1987). Ester derivatives of AmB were active in vitro and in vivo. Also, they were less nephrotoxic and better tolerated than amphotericin B. However, clinical testing was discontinued following reports of progressive neurological dysfunction and diffuse white matter degeneration in patients receiving prolonged high-dose treatment with the methyl ester (Ellis et al., 1982). Most other derivatives were both less toxic and active than amphotericin B (Schaffner et al., 1987). None has achieved clinical importance. 2.4.3 Liposomes Another alternative approach is to use liposomes as a carrier of amphotericin B. Liposomes are microscopic vesicles consisting of concentrically arranged 30 phospholipid bilayers (Bangham et al., 1974; Szoka et al., 1980). These multilamellar vesicles display a prominent lipid compartment and a minor aqueous compartment formed by the intralayer spaces. Since AmB is highly lipophilic with a general structure consisting of a mycosamine group and a series of seven double bonds, which makes AmB compatible with incorporation into a phospholipid bilayer. The rationale for the work on AmB liposomes was that the targets of liposomes include organs such as liver and spleen that are rich in macrophage and which are also frequent targets of systemic fungal infections. This consideration made liposome AmB development attractive. Besides liposomes, other drug-lipid formulations such as colloidal sulfate also have been developed to reduce the toxicity of amphotericin B since 1982. In 1983, Lopez-Berestein et al. first used multilamellar amphotericin B liposomes composed of dimyristoylphosphatidylcholine, dimyristoyl- phosphatidylglycerol and amphotericin B (molar ratio 7 : 3 :1) to treat mice infected with a lethal dose of Candida albicans. All animals given one dose of AmB- containing liposomes (4 mg/kg) survived for longer than 42 days while all animals in the untreated control group died within eight days after infection (Lopez-Berestein et al., 1983). In addition, liposomes have been used as a vehicle for AmB in the treatment of cancer patients with fungal infections (Lopez-Berestein et al., 1987). Patients tolerated liposome AmB much better than AmB in the form of Fungizone. Increased doses could be administered, and a significant clinical improvement was observed in some patients. The mechanism of action of liposome AmB in improving 31 the therapeutic index of AmB has not been completely elucidated. 2.5 Pharmacokinetics After nearly 30 years of use, AmB remains the mainstay of therapy for systemic fungal infections. The pharmacokinetics of AmB are not well known. The available information on pharmacokinetics and metabolism is too inadequate to provide a rational basis for designing less toxic but efficacious dosage regimens. Dosage regimens are often dictated by toxicity rather than by the findings of controlled clinical studies. Decisions on selecting daily dosage, total dosage, duration of therapy, and whether or not to use AmB are often determined by the clinical experience of the physician in treating fungal infections. Adult patients exhibit a large volume of distribution, 3 to 4 L/kg, and a long terminal elimination-phase half-life, which ranges from 11 to 16 days (Atkinson et al., 1978). No metabolites of AmB have yet been identified in in vivo and in vitro studies (Christiansen et al., 1985). Renal and biliary clearances were found to account for 10-15% of the systemic clearance depending on the species used in the investigation. 2.5.1 Human Studies The pharmacokinetics of AmB have been described in two patients (Atkinson et al., 1978) (Table 3). In these patients, the distribution kinetics of AmB were described by a three-compartment model, a central compartment and quickly and 32 slowly equilibrating compartments illustrated in Figure 3. The elimination phase half-life of AmB is approximately 15 days due to the slow return of AmB from the slowly equilibrating peripheral compartment. The volume of this peripheral compartment accounts for 80% of the total distribution volume of 4 L/kg, yet its intercompartment clearance, 9 ml/min, is only 10% that of the rapidly equilibrating peripheral compartment. AmB concentration in the slowly equilibrating peripheral compartment is higher than serum concentration when therapy is stopped. Renal excretion is a relatively minor pathway for the elimination of AmB, accounting for only 3% of total elimination. Studies in children demonstrated a higher clearance and shorter half-life (Benson et al., 1989). The total clearance, apparent volume of distribution, and elimination half-life were 0.46 ml/min* kg, 0.76 L/kg, 18.1 h, respectively. In addition, they found a strong inverse correlation between patient's age and clearance of AmB. Two implications of this finding are possible. First, younger children with high clearance perhaps receive inadequate dosages of AmB for the treatment of fungal infection. Second, older children are being exposed to excessive doses of AmB. However, no correlations exist between changes in serum creatinine and patient age, clearance, total dosage, or duration of therapy. The distribution of AmB in tissues was obtained at autopsy from eight patients (Christiansen et al., 1985). The highest concentrations of AmB were found in the liver; in one patient the amount of AmB in the liver was 41% of the total dose Renal Elimination Non-Renai Elimination Figure 3. Pharmacokinetic model of amphotericin B distribution and elimination. After intravenous injection into the central compartment (Vc), amphotericin B distributes into fast-(Vf) and slow-(Vs) equilibrating peripheral compartments. The rate of this distribution is governed by the intercompartmental clearances Qf and Qs ( from Atkinson and Bennett, 1978 ). Table 3 Ref. Pharmacokinetic Parameters of AmB in Humans No.patients CL (ml/min*kg) Atkinson adult et al. 2 Chabot et al.b Stark et al. Koren et al. Benson et al adult Varea T1/2 (L/kg) (h) a 0.43 ± 0.08 3.99 ± 0.40 0.17 3.8 17 3.72 ± 3.91 3.10 ± 2.32 17.7 ± 17.6 0.43 ± 0.08 0.38 ± 0.02 9.93 ± 1.50 0.46 ± 0.20 0.76 ± 0.52 18.1 ± 6.65 14 children 9 children 13 children 9 T1/2 not reported, terminal T1/2 was 15 days standard deviations not reported 35 administered. High concentrations were also found in the spleen, kidney and lung. In total, these tissues account for 20-50% of the total amount recovered. The heart contains measurable concentration, as do fat and muscle, but amounts are much lower. 2.5.2 Animal Studies Craven et al. (1979) studied the kinetics of AmB in dogs. They used the dog as an animal model with bile duct cannulation and found that excretion of AmB in the bile accounted for 3% of the dose within 10 days, while excretion in the urine was prolonged and greater (21%). The half-life of AmB in dogs is 4 days, the volume of distribution is 6.7 L/kg and systemic clarence is about 0.73 ml/min»kg. The distribution of AmB in tissues was determined by Niki et al. (1990) in rats that were treated with daily intraperitoneal doses for 7 days. Rats were sacrificed 24 h after the last dose. The highest concentrations of AmB were found in the spleen and liver. Low concentrations were detected in serum. 2.6 Analytical Methods A variety of microbiological assays have been available to measure the activity of AmB. Most bioassays involve either serial tube dilution or agar diffusion with a variety of indicator organisms, including Paecilomyces varioti, Candida tropicalis and Saccharomyces Cerevisiae (Bannatyne et al., 1977; Bindschadler et al., 1969). 36 Bioassay requires 16-36 hour of incubation. In addition, natural antifungal activity in the blood of normal individuals and other antifungal reagents may interfere with the assay. Wide variations exist in the accuracy, precision, sensitivity and specificity of the bioassay which make it difficult to compare data from different publications. Radiometric bioassay for AmB (Drazin et al., 1976; Hopfer et al., 1977) was relatively sensitive, accurate and precise. This method measured AmB based on the estimation of potassium or rubidium ions. The addition of amphotericin B to a cell suspension leads to the leakage of ions from the cells, in proportion to the concentration of drug present. However, this method requires expensive equipment or facilities for handing radioactive compounds. Furthermore, the reagents are costly and have a relatively short shelf-life (Drazin et al., 1976), and the test results are affected by other antifungal agents (Hopfer et al., 1977). A reverse-phase high performance liquid chromatographic (HPLC) method was first applied by Mechlinski (Mechlinski et al., 1974) for quality control analysis of the components of different classes of polyene macrolides. Subsequently, NillsonEhle et al. (1977) used HPLC to measure AmB in biological fluids. The chromatographic separation was performed using a reverse phase C18 column and eluent monitored at 405 nm. The limit of detection of this method was 0.02 ug/ml compared with 0.05-0.1 ug/ml in the microbiological method (Green et al., 1965; Bindschadler et al., 1969). HPLC methods have been widely used and Table 4 summarizes the available HPLC methods. HPLC has proven to offer faster, more 37 accurate and more reproducible results compared pharmacokinetic studies and routine clinical use. to bioassay for both In addition, HPLC offers improved sensitivity and specificity and is easier to standardize than bioassay. Table 4 HPLC Methods Used to Assay AmB Ref. Kobayashi et al. Column I.S.a C18 Mobile Phase acetate buffer, MeOH, acetonitrile, THF Mayhew et al. C18 p-nitrophenol b Granich et al. C18 ANN Bach et al. C18 diperse yellow acetonitrile, EDTA acetonitrile, EDTA acetonitrile, MeOH 42 dye a : I.S. represent internal standard b : ANN represent l-amino-4-nitronaphthalene EDTA 39 CHAPTER 3 EXPERIMENTAL SECTION 3.1 Materials and Instruments The materials used in the study were: (1) Amphotericin B: Fungizone ( each vial contains 50 mg AmB, 41 mg deoxycholate and 20.2 mg sodium phosphate, Lot # C0217A/43730, E.R. Squibb & Sons, Princeton, NJ) was obtained commercially. Pure AmB powder was supplied by E.R. Squibb & Sons (Batch # 20-914-59718) A stock solution of AmB (550 ug/ml) was prepared by dissolving AmB in methanol and DMSO (1:1, v/v) in a volumetric bottle covered with aluminum foil and stored at -20°C. Sonication was used to assist the dissolution of AmB in the solvent. 40 (2) Internal standard: l-Amino-4-nitronaphthaIene (ANN) was purchased from Aldrich Chemical Co. (Milwaukee, Wisconsin) and was used as the internal standard (I.S.) in the HPLC assay. A stock solution of internal standard (170 ug/ml) was prepared by dissolving ANN in methanol and stored at -4°C. (3) Creatinine assay kit: Creatinine colorimetric assay kits were purchased from Sigma Chemical Co. (St. Louis, MO) and used for measurement of creatinine concentrations in serum and urine samples. (4) Reagents used for animal study: Sterile 0.9% sodium chloride solution (Kendall McGaw Laboratories, Inc.), ethyl ether (Fisher Scientific) and 5% dextrose injection (Baxter Healthcare Corporation) were used in the surgery and for intravenous infusion. (5) Other chemical reagents: Dimethyl sulfoxide (DMSO) was used for preparing the AmB stock solution. KH2P04 (Aldrich Chemical Co.), Na2HP04 (Fisher Scientific Inc.) were used for preparing buffer solutions. Methanol (Fisher Scientific Inc.) and acetonitrile (Fisher Scientific Inc.) are HPLC grade and used in the mobile phase. The instruments used in the study were: (1) HPLC system: A high pressure liquid chromatograph system (a single piston pump, Model 110 B Beckman Associates) was used with a reversed-phase Nova Pak® C18 (3.9 x 150 mm, particle size 5/*m, Waters, Diversion of Millipore) stainless steel column. The effluent was monitored at 405 nm using a fixed wavelength UV-visible 41 absorbance detector (Model 160, Beckman Associater). The output was connected to both a 10-mV potentiometric integrator (Model 3390 A, Hewlett Packard) and recorder (Model 585, Linear Instruments Co.) (2) UV spectrophotometer: UV/VIS spectrophotometer (Model Lambda 3A, Perkin-Elmer) was used to measure the creatinine concentration in serum and urine samples. Disposable semi-micro cuvettes (VWR Scientific Inc.) were used to hold the test solution. (3) Materials used for surgery: Silastic (Dow Corning Corporation), PE 50 (Clay Adams Inc.) and 22 gauge needles (Becton Dickinson and Company) were used for making catheters. Metabolic cages (Nalge Company), infusion pumps (syringe infusion pump 22, Harvard Apparatus), 10 ml glass syringes (Propper & Sons. Inc.) and 1 ml sterile plastic syringes (Becton Dickinson and company) were used in the study. (4) Centrifuge: A Model TJ-6 centrifuge (Beckman) was used for separating serum. 3.2 Pharmacokinetic Studies 3.2.1 Catheter The catheter is made from PE 50, silastic and a metal portion. The metal portion is simply a segment (3-4 mm) of 22 a gauge needle. The needle is placed 1/3 - 1/2 of the way into the PE 50, and the silastic is forced over the PE 50, then both pieces of tubing are tied to the metal piece. This particular cannula is bent to 42 accommodate the jugular vein. The cannulas are bent by wrapping around glass tubing and immersing into hot water. 3.2.2 Animal Surgery Male Sprague-Dawley rats (220 g - 320 g) supplied by Harlan Sprague Dawley Inc. (Indianapolis, IN ) were used in the study. The rats were allowed to acclimate to the facility for one week prior to the experiment. Forty-eight hours before the experiment, two cannulas were implanted (both right and left jugular vein), one catheter for long term infusion and another catheter for sampling during the infusion (a preliminary study has shown that AmB is not adsorbed onto the catheter). The surgical procedures were modifications of Weeks (Weeks et al., 1964). Briefly, rats were placed under light ether anesthesia and the dorsal-neck-shoulder blade area shaved for exteriorizing the catheters. The right and left ventral neck were shaved for jugular vein cannulation. One small incision was made in the skin between the shoulder blades and other incisions were made over the area on the ventral neck (either right or left) where the jugular vein can be seen to pulsate below the skin surface. The jugular vein was isolated and ligated. A probe was pushed subcutaneously from the dorsal neck opening through the ventral neck opening and the PE 50 end of the cannula was pulled through the probe to the ventral side. The cannula was inserted into the jugular vein and tied. The cannula was fixed to the neck muscle by a suture. Both incisions were sutured. About 2-3 cm of the cannula 43 filled with saline protuded from the shoulder blade area and the cannula was plugged with a pin. 3.2.3 Pharmacokinetic Studies According to the preliminary study, it appears that a minimum of 40 hours (four half-lives) is required to reach 94% of the steady-state plasma concentration. Therefore, a two-step infusion method proposed by Wagner (Wagner et al., 1974) was used in the study. Simulations were performed by substituting the measured pharmacokinetic parameters of AmB ( Chow et al. 1992) into the equations derived by Wagner to estimate the plasma concentrations at different times as well as the times required to reach the steady-state condition based on the infusion rates chosen. Simulation results suggest that by employing this two-step infusion method we can achieve the desired steady state concentration within eight hours. Based on the simulation, a protocol using a forty min fast infusion rate followed by a 520 min slow infusion rate was chosen. The infusion rate used for the fast infusion was 0.025 ml/min and the infusion rate used for the slow infusion was 0.01 ml/min. Male Spraque-Dawley rats were used as the animal model. Twelve rats were randomly selected and divided into three groups to receive different doses (low, medium and high dose). The long term intravenous infusion was given through a left jugular vein catheter which was surgically implanted as described previously. During the infusion, rats were housed separately in metabolic cages and had access to water 44 and food. Blood samples were collected at appropriate times via a right jugular vein catheter (implanted two days prior to the experiment) during infusion and after infusion. Urine samples were collected at different time intervals during and after drug administration. Rats were weighed before drug infusion. Intravenous infusion solutions were prepared immediately before the study. Infusion syringes were covered with aluminum foil to protect from light. At the end of the fast infusion, (40 mins, 0.025 ml/min infusion rate), the infusion line (with one end connected to the implanted catheter and another end connected to the infusion syringe) was switched to another infusion syringe which contained the slow infusion solution. The second infusion rate was 0.01 ml/min during an infusion time of 520 mins. 3.3 Sampling The blood samples were collected prior to drug administration and at appropriate times up to 90 hours after the end of the infusion. Blood samples (0.35 ml) were obtained at the desired times from the implanted cannula. One tenth ml of blood (which was subsequently re-injected and followed by 0.4 ml of saline) was withdrawn prior to sampling in order to avoid an artifact due to sample contamination by saline or blood trapped in the cannula. Time points for taking blood samples were 0, 40, 370, 460, 560, 840, 1420, 1930, 2300, 3035, 3640, 4486, 5115, and 6165 min. Once blood samples were collected, they were centrifuged at 45 3000 rpm for 3 mins. Serum was collected and placed into a brown glass vial and stored at -20°C until analysis. During and after AmB infusion, urine cups were covered with aluminum foil and put in an ice bath to protect the sample from light to avoid degradation. The urine samples were collected at different time intervals up to 130 hours after the end of infusion. The volume of urine was measured and the samples were stored at 20°C before analysis. 3.4 Preparation of Intravenous Solution I.V. infusion solutions were prepared immediately before the experiment. AmB, in the original Fungizone bottle (containing 50 mg AmB), was diluted with 10 ml sterile water to form a 5 mg /ml suspension. Five percent sterile dextrose injection was used for further dilution to the desired concentration. The resulting infusion solution is clear without presence of precipitation. The concentrations of the infusion solutions used for the fast infusion in the three different dose groups were 199.2, 796.4, and 1990.8 g/ml. The concentrations of the three slow infusion solutions were 21, 80, and 200 g/ml. The infusion solutions were protected from light by aluminum foil. Intravenous solution stability (in 5% dextrose ) was examined by Kintzel (Kintzel, 1991). The intravenous solutions at concentrations of 920,1200,1400 ug/ml were stable at room temperature for up to 36 hours. No precipitation, turbidity, gas 46 formation or color change was observed. 3.5 Sample Preparation Serum samples were treated with methanol before analysis by HPLC. Three hundred 1 methanol (containing a certain amount of internal standard) was added to 100 1 serum sample or standard serum sample. The mixture was vortexed and centrifuged at 3000 rpm for 10 mins. The supernatant was concentrated under nitrogen at room temperature, then reconstituted with 150 1 mobile phase and injected onto the HPLC system. Urine samples were diluted with 1 to 4 volumes of mobile phase according to the AmB concentration. The diluted urine samples were vortexed for 1 min and centrifuged at 3000 rpm for 3 mins. One hundred 1 supernatant of the diluted urine sample was added to 100 1 mobile phase containing a certain amount of internal standard. The mixture was vortexed and injected onto the HPLC column. 3.6 AmB Assay by HPLC AmB concentrations were determined using the modification of the HPLC method described in the literature (Granich et al., 1986). HPLC methods have proven to offer a faster, more accurate and more reproducible alternative to bioassay for pharmacokinetic study. A reversed phase Nova-Pak C18 (Waters) column was employed in the assay. Analysis was performed with a Beckman Model 110B liquid 47 chromatograph system with a fixed wavelength detector. The mobile phase consisted of Na2HP04-KH2P04 buffer (4 mM, pH 7.0) : acetonitrile = 69 : 31 (v/v). The mobile phase was filtered though a 0.45 m Nylon 66 filter membrane and degassed by sonication under vacuum. A flow rate of 1.2 ml/min was used. The sample injection size was 100 1. The ultraviolet spectrum of AmB in DMSO/methanol is shown in Figure 4 indicating maximum absorption at 405 nm and 382 nm. Since the maximum absorption of the internal standard is about 405 nm, the chosen wavelength of 405 nm was near the maximum for both compounds. AmB and internal standard had retention times of 3.5 min and 5.5 min, respectively. 3.7 Calculations Standard curves for the assays were constructed and least-squares linear regression analysis was performed. Standard curves were linear from 0.015 g/ml to 1.75 g/ml. An example of a standard curve is shown in Figure 5. The lowest correlation coefficient among the standard curves for any assay was 0.97. The intraday and inter-day reproducibilities of the method were examined by HPLC injection during one day and on each of three different days, respectively. The intra-day coefficients of variation of the assay in three different days ranged from 1.89 % to 9.09 % at 0.05 ug/ml and ranged from 1.96 % to 4.12 % at 0.5 ug/ml. For the interday assay, the coefficient of variation was 8.33 % at 0.05 ug/ml and 3.33 % at 0.5 ug/ml. Unknown concentrations of AmB were calculated by comparing the observed 48 1 1 1 • 1 1 1 ' 406 t 1 38? 1 AMPHOTERICIN B (DMSO/CH3OH) LU U z < CD cc 3f»3 o on 1 CD < > Z3 345 t 250 > 300 1 350 1 1 400 V « 1 450 . 1 500 WAVELENGTH (nmj Figure 4. Ultraviolet absorption spectrum of amphotericin B in DMSO/CH,OH solution (concentration 5.45 /ig/ml). :.477X —0.0247 = 0.999 3- 0.000 0.500 1.000 1.500 2.000 AmB serum concentration Figure 5. Plot of standard curve of amphotericin B in serum. Peak area ratio versus AmB serum concentration. 50 peak height ratio of AmB to internal standard as a function of AmB concentration on the standard curves. 3.8 Creatinine 3.8.1 Creatinine Assay Creatinine clearance is a useful index for evaluating glomerular filtration rate and for assessing renal function. Low creatinine clearances occur in patients with nephritis and renal obstruction. It is necessary, therefore, to develop methods to measure creatinine concentration. The most commonly used method for measuring creatinine concentration is the Jaffe alkaline picrate method described by Jaffe 100 years ago. However, the method is subject to interference from endogenous constituents (glucose, acetoacetic acid, acetone, etc.) of plasma and urine, and it is influenced by analytical condition. The assay always gives an overestimated value of the creatinine concentration. Numerous modifications have been made to the Jaffe method in order to improve the specificity of the assay. The method used in this experiment was developed by Slot (Slot et al., 1965). The basic principle is that under acid conditions the creatinine-picrate color fades faster than the interfering chromogens. The diagnostic creatinine kit is based on this principle and it is widely used in hospital and clinical laboratories due to its simplicity, although it is a non-specific method. 51 The procedure for this method is as follows: 0.1 ml water, 0.1 ml of standard creatinine solution (concentration of 0.3 mg/dl of creatinine) and 0.1 ml test serum or diluted urine sample were each separately added to three different cuvettes. Onetenth ml alkaline picrate solution was added to all three cuvettes, mixed well and maintained at room temperature for 8 mins. The initial absorbance of the standard and test (versus blank as reference) was determined at 500 nm. Then, 0.1 ml acid reagent was added to the cuvette, mixed immediately and thoroughly. After reacting for exactly 5 min, the final absorbance was determined. Creatinine concentration was obtained by using the following equation: initial A. - final A, me mQ Creatinine P^] = x 3^ dl initial As - final As dl (1) where initial At and finial At are initial and final absorbances of the test sample and initial \ and final As are initial and final absorbances of the standard creatinine sample. 3.8.2 Creatinine Clearance Creatinine concentrations in serum and urine samples were measured before and after drug administration in order to compare differences in creatinine clearance (CLcr) values. Creatinine clearance can be calculated by the ratio of creatinine excretion rate in urine determined over selected time intervals and the serum concentration. The 52 equation is as follows: CLcr = bXuJAt » ~C~ (2) where Ccr is the creatinine concentration in serum and A Xucr/A t is the excretion rate of creatinine in urine over the time interval T. Excretion rate is given by: AXuJAt = Cur'cr V°lur T (3) where Cur cr is the creatinine concentration in urine and Volur is the volume of urine over the time interval T. 3.9 Data Analysis The pharmacokinetic data were analyzed by both the model-independent and model-dependent procedures. 3.9.1 Model-Independent Method In the model-independent procedure, AmB serum concentrations were plotted versus time on semi-logarithmic graph papers. Data in the post-distribution phase of AmB concentration versus time profile were analyzed by log linear regression to obtain the slope which presents the terminal disposition rate constant, 6. The disposition half-life, TH, was calculated as follows: 53 where fi is the terminal phase rate constant. T1/2 was also estimated based on urine data. The relationship between In excretion rate (In A Xu/A t) versus time at the midpoint of the urine collection interval should yield a straight line with a slope of fi, as illustrated in Figure 6. The fi value can be obtained by using linear regression analysis. Harmonic mean half-lives (Hn/2) and variance were estimated by using a jacknife technique (Lam et al., 1985) in order to present the population mean halflives. A harmonic mean half-life can be determined from the following equation: Ht, = n n ' <— + —+••• + —) *1/2, 1 *1/2, 2 *1/2, n (5) where n is the number of subjects. The standard deviation of the harmonic mean half-life is given by: E (//,. \ (ti-1) i=l SD = (6) - Hf where Hj and H are given by: "> - ~—i + *1/2,1 *1/2,2 + ... + r1—i + *1/2,1-1 1 " H = - Y,HI n i-1 *l/2,i+l + ... + r <7> *l/2^i (8) InAXu/At Tmid Figure 6. A plot of the relationship between in urine excretion rate (In AXu/At) and time at the midpoint of urine collection. The slope is -p. 55 The AmB area under the serum concentration versus time curve (AUC) was calculated using the trapezoidal rule. The equation is as follows: auc; = icA + Iekc, • cwx<,., - <,)] • £ 2 2<=i (»> p where Cj is the concentration at time t;. AUCn°° can be calculated by the term, Cn/B, in equation 9, where Cn is the last measured concentration. The first moment of the AmB concentration-time profile is the total area under the curve (AUMC) resulting from a plot of the product of AmB concentration and time versus time. AUMC is given by: aumc; = lc,tf * Iekca + 2 2j=i - oi+ ,Jr- * p (10) pz By using serum data together with urine data, other pharmacokinetic parameters can be calculated. The systemic clearance CLS was obtained using the following equation: CL = Y,dose S AUCQ (11) where S dose is the total dose infused (including the fast infusion dose and the slow infusion dose), AUC0°° is the area under the concentration-time curve from time 0 to infinity. Also, CL was calculated from: 56 a, = where Kq is the slow infusion rate, K0 / ss (12) is the AmB serum concentration at steady- state condition. Renal clearance, CLr, was estimated by the following relationship. CL, = Xu',j '•!AUCtJ (13) where Xutl12 is the amount of AmB excreted into urine during the time interval tl to t2, AUCtlt2 is the area under the curve during the corresponding time intervals. Renal clearance was also obtained by employing urinary excretion rate versus plasma concentration at the midpoint of the collection interval. This relationship can be described by: CLr = AX" 1 Af (14) ^'mid CLr can be obtained from the slow of the above relationship by performing a linear regression analysis of plots of A Xu/A t versus Cmid. Renal clearance at steady-state condition was the ratio of urinary excretion rate at the steady-state condition and the serum steady-state concentration. The apparent volume of distribution, usually termed Varca, is given by: 57 r, E dose area = ^ AUCq • p (15) where 2 dose is the total dose infused. The volume at the steady-state, Vss, is given by ( Perrier et al., 1982): v " = Dose uose AUC AUMC-T.AUC. T-AUC, 1 1 _ —2 -]-AT AUC. ^ 2AUC1 2AUC AUC (16) where Tj is the duration of the first infusion, T2 is the duration of the second infusion, A T is the time interval between these two infusions, AUCj and AUC2 are the areas under the AmB serum concentration versus time profiles for infusion one and infusion two, respectively. Fexc, the fraction of the AmB dose excreted into urine is determined experimentally and can be calculated by: Clr F = " CLS (17) 3.9.2 Model-Dependent Method The AmB plasma concentration - time curve can be described by a multicompartment model according to literature reports and on the basis of data obtained from this laboratory. AmB data is best described by a two compartment model based on our previous experiments. 58 In this two compartment model, AmB is assumed to distribute instantaneously into the central compartment which represents tissues that are highly perfused by blood. The drug is simultaneously but more slowly distributed into a peripheral or tissue compartment which is poorly perfused by blood. The situation is illustrated in Fig. 7. Model-dependent analysis was conducted by using the computer program ADAPT II (D'Argenio et al., 1992). We have fit the data using the following sequence: first, setting up the model file with the differential equations describing the two compartment model; second, entering the experimental data; and, last, using weighted least squares regression to estimate the pharmacokinetic parameters. Initial estimated values are obtained from the analysis of preliminary results of experiments conducted in this laboratory. Micro-rate constants K12, K21, K10 and volume Vc were estimated by ADAPT n. 3.9.3 Statistical Analysis CLS, CLr, V, Fex, Tlj2 and other pharmacokinetic parameters as a function of dose were examined by linear regression analysis. The slope of the regression line was tested for statistical significance. One-way analysis of variance was used to examine the variance among three groups. Creatinine clearances (before and after drug administration) were examined by a paired Students t test. 59 A p value < 0.05 was considered to be significant. All statistical analyses were performed with use of the MINITAB statistical package (Minitab Inc, state college, PA). XI Central Comoartment 21 12 Denpherai X2 Comoartment 10 Figure 7. Pharmacokinetic model for AmB distribution and elimination. X, is the amount of drug in the central compartment. X2 is the amount of drug in the peripheral compartment. K12 and K21 are the rate constants between the two compartments. K10 is the elimination rate constant from the central compartment. 61 CHAPTER 4 RESULTS 4.1 Analytical Method AmB concentrations in serum and urine samples were determined by using the HPLC assay described earlier. Briefly, the mobile phase (4mM NajHPO,, KH2P04 buffer, PH=7.0, and acetonitrile, 69:31 v/v) was pumped at a flow rate 1.2 ml/min through a C18 Nova-Pak column. The column effluent is monitored at 405 nm using a UV absorbance detector. nitronaphthalene) were separated AmB and internal standard (l-amino-4from other interfering compounds. Chromatograms of blank serum and spiked AmB serum sample are shown in Figure 8A and 8B. In the chromatogram, AmB and internal standard were completely separated, with retention times of 3.59 min and 4.96 min, respectively. The urine sample was diluted 1 to 4 times according to the AmB concentration. The mobile phase was used to dilute the urine due to low solubility 62 of AmB in water. In addition, the mobile phase can improve the resolution of the peak. Standard curves for AmB were established by linear least-square regression of the area ratio of amphotericin B/internal standard versus the AmB concentration (r2 > 0.99). The standard curve was shown in Figure 5. Unknown concentrations of AmB in the authentic samples were determined by comparison with the AmB standard curve. The linear range of the standard curve was from 0.015 ug/ml to 1.75 ug/ml. The within-day precision was assessed by analyzing triple samples for each concentration in one day for three days, while the between-day variation was examined by analyzing the results of three days. Two different AmB concentrations, 0.05 ug/ml and 0.5 ug/ml, were examined. The results are shown in Table 5. The within-day % CV ranged from 1.89% to 9.09% for two different concentrations. The between-day % CV was 9.12% and 3.33% for the concentrations of 0.05 ug/ml and 0.5 ug/ml, respectively. The detection limit of the method for AmB in biological fluids was 15 ng/ml at a sensitivity setting of 0.008 a.u.f.. 4.2 Stability of AmB in Urine According to the literature (Shadomy et al., 1973), light, temperature and pH of the medium are thought to affect the stability of AmB. Wiest et al.(1991) found (A) (B) r© Figure 8. (A) Blank serum. (B) Spiked serum sample with AmB concentration of 0.5 jug/ml. 3.59 min and 4.96 min are retention times of AmB and internal standard, respectively. Table 5. Day Intra-day Reproducibility of the AmB Assay in Spiked Serum Samples (n=3) Spiked conc. (Mg/ml) Observed conc. (Mg/ml) SD CV% 1 0.050 0.500 0.048 0.510 0.003 0.010 6.25 1.96 2 0.050 0.500 0.053 0.510 0.001 0.021 1.89 4.12 3 0.050 0.500 0.044 0.530 0.004 0.011 9.09 2.08 Table 6. Inter-day Reproducibility of the AmB Assay in Spiked Serum Samples (n=9) Spiked con. (fxg/ml) 0.050 0.500 Observed con. (jug/ml) 0.048 0.515 SD CV% 0.004 0.017 9.12 3.33 65 a peak for a degradation product of AmB in the chromatogram when AmB was heated, and in contact with acid or base. In this study, the stability of AmB in rat urine was examined. During 24 hours, the initial concentration declined by up to 43% at room temperature, without protection from the light. However, no peak was found for a degradation product. Therefore, precautions were taken in handing urine samples to minimize the degradation. Aluminum foil was used to cover the urine cup to protect from light and an ice bath was used to keep urine at a low temperature during urine collection. With such precautions, AmB concentrations in urine remained above 95% of the initial concentration at all times up to 24 hours (see Table 6). The results suggest that amphotericin B in urine was stable under these condition. 4.3 Pharmacokinetic Data Male Sprague-Dawley rats were randomly divided into three different dose groups. The experiment was designed to administer the drug with a two-step infusion procedure to rapidly achieve a steady-state condition. The high dose group was given a total dose of 4210.0 ug/kg, including 3069.8 ug/kg for the fast infusion rate and 1900 ug/kg for the slow infusion rate. The medium dose group was given a 1323.1 ug/kg total dose, with 837.8 ug/kg for the fast infusion rate and 331.5 ug/kg for the slow infusion rate. In the low dose group, a total dose of 380.3 ug/kg was given, with 284.1 ug/kg and 96.3 ug/kg from the fast and slow infusions, respectively. Table 7. Stability of AmB in Urine with Protection from Light and Kept at Low Temperature in Glass Tubes (n=2). Time (hour) 0 4 16 24 % Initial Concentration Remaining 102.8 102.1 103.1 96.0 Stability of AmB in Urine in Plastic Tubes with the Same Precautions as Above (n=l) Time (hour) 0 4 16 24 % Initial Concentration Rmaining 112.3 102.8 101.9 109.5 67 The serum concentration versus time profiles for the three different dose groups are shown in Figures 9-11. The individual plasma and urine data for all animals are summarized in Appendix A and B. AmB concentrations in blood can reach steady-state conditions in about 300 mins after the initiation of the slow infusion. The mean AmB steady-state concentrations for the low, medium and high dose groups were 0.066 ± 0.017 ug/ml, 0.143 ± 0.023 ug/ml and 0.551 ± 0.12 ug/ml, respectively. Linear regression analysis of the mean AmB serum concentration at steady-state as a function of dose indicates that the mean steady-state concentration increased linearly when the dose increased (Figure 12). The terminal disposition rate constant, 6, was calculated by linear regression of the last several points in the serum concentration - time profile. Linear regression of the rate constants as a function of dose indicates that the parameter is doseindependent. One-way analysis of variance indicates that there were no significant differences in rate constant among the three groups. The half-life can be estimated from the rate constant obtained from the plasma data or from linear regression of urinary excretion rate versus the midpoint of the urine collection interval (Table 8). The half-lives (± SD) were 917 ± 252.6 min, 1043.3 ± 139.0 min and 909.5 ± 443.7 min in the high, medium and low dose groups, respectively. Linear regression of half-life versus dose indicates that half-life was dose-independent, as illustrated in Figure 13. One-way analysis of variance ( ANOVA) indicates that there were no significant differences in half-life among those three groups. 68 The harmonic mean half-life is more meaningful than the arithmetic mean half-life because the harmonic mean half-life represents the population mean of the half-life under investigation. The harmonic mean half-life can be obtained based on the method described in Chapter Three. The harmonic mean half-lives (± SD) were 866.24 ± 239.25 min, 1030.48 ± 124.69 min and 767.78 ± 1303.68 min for the high, medium, and low dose groups, respectively. The volume of distribution (Varea) in the high, medium and low dose groups were 5.29 ± 1.50 L/kg, 5.89 ± 1.34 L/kg and 4.43 ± 1.65 L/kg, respectively, and these results are shown in Table 8. Linear regression of volume of distribution versus dose indicates that volume of distribution is dose-independent (Figure 14). One-way analysis of variance indicates that there were no significant differences in this volume among those three groups. The systemic clearances were determined to be 4.03 ± 0.67 ml/min*kg, 3.92 ± 0.81 ml/min»kg and 3.52 ± 0.72 ml/min* kg for the high, medium and low dose groups, respectively. Systemic clearance values calculated from the steady-state condition ( i.e., K^/C^ ) were very similar to those determined from area analysis (Table 8). Linear regression of systemic clearance versus dose indicates that systemic clearance is dose-independent, as illustrated in Figure 15. ANOVA indicated there were no significant differences in clearance among the three groups. However, renal clearances were 0.37 ± 0.04 ml/min*kg, 0.86 ± 0.24 ml/mimkg and 0.76 ± 0.33 ml/min*kg for the high, medium and low dose groups, respectively. The 69 renal clearances calculated from the steady-state condition were very similar to those above cited (Table 8). The linear regression of renal clearance versus dose indicates that renal clearance decreases when the total infused dose increases, suggesting dosedependent characteristics (Figure 16). One-way analysis of variance suggests that renal clearance of the high dose group was significantly different from both the low and medium dose groups. However, there is no significant difference in renal clearance between the medium and low dose groups, suggesting that the alteration of renal clearance is not proportional to the total infused dose. Figures 17 and 18 illustrate the profiles of renal clearance ( estimated from the rate method ) versus serum concentration at corresponding time and the renal clearance versus the serum concentration at the steady-state condition. Both graphs indicate that renal clearance is inversely related to AmB serum concentration. In the graph of renal clearance versus time in the high dose group (Figure 19), it seems that renal clearance increased over time. However, with the high variation, it is difficult to conclude that renal clearance increased with time. There were no alterations in renal clearance as a function of time in the medium and low dose groups (Figure 19). Urinary excretion accounted for 9.4 ± 1.2%, 22.0 ± 5.0% and 23.1 ± 13.0% of total elimination in the high, medium, and low dose groups, respectively. The linear regression of the fraction of the dose excreted in the urine versus dose suggested that this fraction decreased when the infused dose increased, which is in agreement with the change in renal clearance (Figure 20). 70 Renal clearance only accounted for 10-20% of total body clearance. The mode of non-renal AmB elimination remains to be determined. Serum and urine creatinine concentrations were determined using the picric acid assay. Creatinine clearance was estimated using the method described in Chapter Three and these results are shown in Table 8. Comparison of creatinine clearance before and after AmB treatment in the three different dose groups is shown in Figure 21. High AmB dose treatment resulted in significantly decreased creatinine clearance (Figure 22), which suggests there is a change in glomerular filtration rate. This finding agrees with the alteration in AmB renal clearance. There were no significant decreases in creatinine clearances after AmB treatment in the medium and low dose groups. The relationship between renal clearance and creatinine clearance is shown in Figure 23. Both renal clearance and creatinine clearance were found to decrease in the high dose group. Model-dependent methods were also used to analyze the data. Serum data were analyzed by the ADAPT n computer program developed by D'Argenio et al. (1992) for nonlinear regression. Both two compartment and three compartment models were designed and used to fit the experimental data. Based on the results of the F test ( F value can be calculated based on weighted sum of squares and degree of freedom) ( Boxenhaum et al., 1974), the two compartment model was chosen as the best model to describe the pharmacokinetic characteristics of AmB. Micro constant, K12, K21, K10, and central volume of distribution, Vc, were estimated 71 by running the ADAPT II program. The non-linear regression fitting curves are provided in Appendix C. The computer iteratively adjusted the values of all the parameters of the model to minimize the differences between the theoretical curves describing the serum concentration and observed data points. Parameters estimated from the computer program depend on the initial estimates given. Sometimes, giving different initial estimates results in different computer-estimated parameters. However, the initial estimates used in the fitting were obtained from the results of previous work done in this laboratory ( Cai, Masters Thesis, 1991). This initial estimation problem is responsible for the high variation of parameter valves among the individual animals. 72 Table 8. Dose Mg/kq Summary of Pharmacokinetic Parameters of AmB Based Upon Model-Independent Analysis of the Data. AUC 'rain/ml 0 min" T1/2a min T1/2b mm Varea L/kq Vss L/kq 3782.6 4145.9 4989.5 3921.9 AVG SD 810.7 1161.1 1489.6 861.7 0.00065 0.00094 0.00058 0.00103 0.00080 0.00022 1066.2 737.2 1194.8 672.8 917.8 252.6 845.1 1019.1 805.8 1004.3 918.4 109.1 7.17 3.80 5.78 4.42 5.29 1.50 2.94 2.16 4.01 2.12 2.81 0.89 1220.0 1500.0 1672.2 900.2 AVG SD 300.6 300.0 492.2 280.1 0.00056 0.00074 0.00066 0.00073 0.00067 0.00008 1237.5 936.5 1050.0 949.3 1043.3 139.0 949.3 1260.0 924.0 911.8 1011.3 166.6 7.25 6.76 5.14 4.40 5.89 1.34 2.75 2.92 2.53 1.33 2.38 0.72 323.7 351.8 439.5 395.3 391.2 AVG SD 106.6 117.8 99.6 114.4 116.8 0.00045 0.00057 0.00108 0.00120 0.00117 0.00089 0.00036 1540.0 1215.8 641.7 557.5 592.3 909.5 443.7 529.0 1050.0 558.9 962.5 679.4 756.0 237.3 6.76 4.82 4.40 2.88 2.86 4.43 1.65 1.78 1.40 1.44 1.45 1.07 1.43 0.25 73 Table 8 (continued) Summary of pharmacokinetic parameters Clearance, ml/min kg CLsd CLre CLpf 4.66 3.57 3.35 4.55 4.03 0.67 3.69 4.70 2.70 4.18 3.82 0.85 0.42 0.38 0.33 0.36 0.37* 0.04 0.41 0.39 0.37 0.40 0.39* 0.02 0.37 0.28 0.33 0.30 0.32* 0.04 4.24 3.19 3.02 4.19 3.66 0.64 0.090 0.106 0.099 0.079 0.094* 0.012 0.078 0.077 0.098 0.067 0.080* 0.013 4.06 5.00 3.39 3.21 3.92 0.81 3.94 5.03 3.17 6.56 4.68 1.47 0.77 1.15 0.59 0.92 0.86 0.24 0.72 1.12 0.62 0.82 0.82 0.22 0.45 0.76 0.49 0.58 0.57 0.14 3.29 3.85 2.80 2.29 3.06 0.67 0.190 0.230 0.174 0.287 0.220 0.050 0.111 0.084 0.143 0.182 0.130 0.042 3.04 2.99 4.75 3.46 3.35 3.52 0.72 3.55 4.98 2.75 2.44 2.34 3.21 1.10 0.76 1.34 0.59 0.53 0.60 0.76 0.33 0.63 1.14 0.80 0.62 0.74 0.79 0.21 0.60 0.95 0.44 0.56 0.58 0.62 0.19 2.28 1.65 4.16 2.93 2.75 2.75 0.93 0.250 0.448 0.124 0.153 0.179 0.231 0.130 0.197 0.317 0.100 0.162 0.172 0.190 0.080 CLSC CLr8 cC Fexc ' i *Fexc Plasma data Urine data From dose/AUC From K q /CSS From excretion rate vs plasma concentration From excretion rate vs plasma concentration at steady-state From amount excreted vs AUC Non-renal clearance from, Clsc - Clre Fraction of dose excreted unchanged in urine (Clre/Clsc) Fraction of dose excreted unchanged in urine determined experimentally Significant difference 74 Table 9 Sununary of Creatinine Clearance Before and After Dosing Dose Before dosing ug/kg csea AXucr/Atb 323.7 351.8 439.5 395.3 391.2 AVG SD 1.84 1.90 1.84 1.30 1.50 1.68 0.26 8.84 9.82 9.60 10.81 8.92 9.60 0.80 1220.0 1500.0 1672.2 900.2 AVG SD 2.90 1.43 1.89 2.00 1.82 0.84 3782.6 4145.9 4989.5 3921.9 AVG SD 1.10 1.21 2.21 1.86 1.60 0.53 after dosinq CLcrC cse AXucr/At CLcr 17.27 17.95 17.28 27.53 19.70 19.95 4.35 1.81 2.10 2.89 2.13 1.22 2.03 0.60 8.23 9.95 8.78 10.34 9.46 9.35 0.86 16.36 16.45 10.06 16.07 25.68 16.92 5.59 7.34 8.93 11.86 6.88 8.75 2.25 11.30 25.39 19.07 11.62 16.85 6.73 2.02 2.54 2.06 2.19 2.20 0.24 7.66 8.14 10.33 8.08 8.55 1.20 16.93 13.03 15.24 12.46 14.42 2.06 6.04 9.62 9.46 8.73 8.46 1.66 19.89 28.81 14.86 16.18 19.94 6.29 3.10 2.50 3.03 2.98 2.90 0.27 7.78 7.50 6.90 7.67 7.46 0.39 9.09 10.87 7.91 8.88 8.99 0.89 ®: Cse is the concentration of creatinine in serum, unit is ng/ml. : excretion rate of creatinine in urine, jig/min. creatinine clearance, ml/min«kg. Table 10. Summary of Pharmacokinetic Parameters of AnB Obtained by Model-Dependent Fitting Method. V= CLS Dose *12 K21 *10 Aig/kg 1/min 1/min 1/min L/kg 3782.6 4145.9 4989.5 3921.9 AVG SD 0.0025 0.0090 0.0024 0.0041 0.0045 ±0.0031 0.0012 0.0020 0.0010 0.0026 0.0017 ±0.0008 0.0027 0.0068 0.0040 0.0027 0.0040 ±0.0019 1.865 0.712 0.979 1.869 1.356 ±0.600 5.01 4.82 3.90 4.99 4.68 ±0.53 1220.0 1500.0 1672.2 900.2 AVG SD 0.0035 0.0226 0.0045 0.1984 0.0572 ±0.0945 0.0009 0.0078 0.0013 0.0140 0.0060 ±0.0062 0.0019 0.0023 0.0041 0.0059 0.0036 ±0.0018 1.981 2.260 0.958 0.491 1.423 ±0.836 3.79 5.11 3.93 2.92 3.94 ±0.90 323.7 351.8 439.5 395.3 391.2 AVG SD 0.0025 0.1582 0.0043 0.0219 0.0014 0.0377 ±0.0679 0.0017 0.0192 0.0060 0.0336 0.0027 0.0132 ±0.0148 0.0012 0.0030 0.0020 0.0023 0.0021 0.0021 ±0.0007 2.320 0.691 2.439 1.626 1.689 1.753 ±0.696 2.86 2.10 4.81 3.82 3.51 3.42 ±1.02 ml/min kg 1.000 Dose= 380.3 ug/kg 0.100-r 11 i 0.010-- 0.001 0 500 1000 1500 2000 2500 Time (min) Figure 9. Plot of mean AmB serum concentration versus time in the low dose group. Average infused dose was 380.3 jug/kg. Each point is the mean of five rats. The cross-hatched vertical bars represent the standard deviation. The solid line connects the points. 77 Dose= 1323.1 ug/kg 1.000-- 0.100c <L> <J o o 0.010 + 0.005 1000 2000 3000 Time (min) Figure 10. Plot of mean AmB serum concentration versus time in the medium dose group. Average infused dose was 1323.1 /ig/kg. Each point is the mean of four rats. The cross-hatched vertical bars represent the standard deviation. The solid line connects the points. 78 10.000 Dose =4210.0 ug/kg| cn 3 c o 1.000- 0.100c (D O c o CJ 0.01040.005 1000 2000 3000 4000 Time (min) Figure 11. Plot of mean AmB serum concentration versus time in the high dose group. Average infused dose was 4210.0 /ng/kg. Each point is the mean of four rats. The cross-hatched vertical bars represent the standard deviation. The solid line connects the points. 0 1 2 3 4 Thousands Dose (ugfcg) Figure 12. Plot of mean AmB serum concentration at steady-state as a function of dose. Each point is the mean of four rats. The solid line represents the linear regression analysis of the data forced through the origin ( Y = 0.000129 X; r=0.99 ). 5 80 !500 o high dose • medium dose A low dose 2000 1500--. A A 0 0 O • • • 1000- 500- • O o° £ « ' \ 1000 2000 3000 1. 4000 » 5000 6000 Dose( / u,g/kg) Figure 13. Plot function of dose. of AmB half-life as a 81 O high dose • medium dose A low dose A O • O O A A O •• • A 0 - " " i i i 1000 2000 3000 4000 5000 6000 Dose (ju,g/kg) Figure 14. Plot of volume of distribution of AmB as a function of dose. 82 O High dose • Medium dose A Low dose 8 - A 4-1- 0 qd • H 1000 O 1 2000 DOSE 1 3000 1 4000 O h 5000 6000 G^g/kg) Figure 15. Plot of AmB systemic clearance as a function of dose. 83 2.000 O high dose • medium dose A low dose .500c E E 1.000- o 0.500- 0.000 0 1000 2000 3000 4000 5000 6000 Dose (Aig/kg) Figure 16. Plot of AmB renal clearance as a function of dose. Renal clearance determined from excretion and plasma concentration. The solid line is a linear regression fit of the data ( Y=-0.00011X+0.883; r=0.40; p>0.05 ). 84 2.000 cn c O high dose • medium dose A low dose 1.500O E 1.000 + A * • t: o °^ o °' 500 '^c 0.000 0.000 o o 0.500 1.000 AmB serum concentration (ug/ml) Figure 17. Plot of AmB renal clearance versus AmB serum concentration. Renal clearance determined from excretion rate and plasma concentration. 85 1.500 O high dose • medium dose A low dose A • J? 1.000 c E A® A • AA • _i 0.500 o <8 0.000 0.000 o I 1 U 0.200 0.400 0.600 I 0.800 1.000 AmB serum concentration at steady-state condition (ug/ml) Figure 18. Plot of AmB renal clearance versus AmB serum concentration at steady-state. Renal clearance determined from excretion rate and plasma concentration. 0.400 - • 0.200 0.000 1500 2000 0.400 -- 0.200-- 0.000 -j 0 1 1 1 1 500 1000 1500 2000 2500 0.400-- 0.000 -I 0 1 1 1 1 500 1000 1500 2000 2500 Time (mln) Figure 19. Plot of AmB renal clearance as a function of time in the low (A), medium (B), and high (C) dose groups. Renal clearance determined from excretion rate and plasma concentration. Each point is the mean of four rats. The crosshatched vertical bars represent the standard deviation. 87 0.600 O high dose • medium dose A low dose A 0.400 0.300 + • 0.200 v - * . . A 0.100 0.000 0 i i 1 t 1 1000 2000 3000 4000 5000 6000 Dose (fj,g/kg) Figure 20. Plot of the fraction of the dose of AmB excreted into the urine as a function of dose. This fraction calculated from, CLr/CLs. The solid line represents the linear regression fit of the data (Y=-0.000037X + 0.254; r2=0.40; p>0.05). Figure 21. Plot of the individual creatinine clearance before and after AmB treatment in the low (A), medium (B) and high (C) dose groups. The circles with vertical cross-hatched bars represent the mean and standard deviation. after dosing before dosing 30- 20- low dose medium dose high dose * significant difference Figure 22. Bar graphs illustrating the mean creatinine clearance values prior to and after AmB treatment as a function of dose. The cross-hatched vertical bars represent the standard deviations. 90 2.000 O high dose • medium dose A low dose ^ 1.500-cn c E \ E 1.000- L- o 0.500- 0.000 0 10 20 CLcr (ml/min kg) Figure 23. Plot of the relationship between the AmB renal clearance and creatinine clearance in three different groups. The solid line is a regression fit of the data (Y=0.0575X-0.0564; r=0.375; p<0.05). Value in parentheses not used in analysis. 30 91 2.000 O High dose • Medium dose A Low dose 1.500 O _c 1.000 J, u d 6 A O •& 0.500 8 0.000 0.000 0.020 0.040 0.060 0.080 0.100 Urine Flow (ml/min) Figure 24. Plot of the renal clearance versus urine flow. 92 CHAPTER 5 DISCUSSION AND CONCLUSIONS 5.1 Discussion Obtaining an accurate concentration measurement in biological samples is the foundation for studying the pharmacokinetic characteristics of AmB. The HPLC method that we modified offers a sensitive, precise, specific and reproducible measurement of the plasma and urine concentrations of AmB. The final chromatographic conditions adopted represented a compromise between analysis time, peak shape symmetry, and resolution from other interfering substances. Our method for extraction of serum samples uses methanol as a 93 deproteinizing agent with a 1 : 3 volume ratio of sample : organic solvent. Deproteinization is relatively complete and the supernatant may be quickly evaporated in only a few minutes under a steam of nitrogen. Precautions were taken during urine collection. These precautions have proven to efficiently avoid degradation of AmB during the urine collection (i.e., protection from light and cooling on ice). For nearly 30 years AmB has been the principal chemotherapy agent used to treat systemic mycoses. However, the pharmacokinetic information about this drug remains limited. The results of this study provide information on half-life, volume of distribution, and clearance of AmB in an animal model. The linearity of the disposition kinetics of AmB has also been determined in the study. Based upon results from preliminary studies (Chow et al., 1992), it will take at least 40 hours to reach 94% of AmB steady-state plasma concentration via a constant rate infusion. The traditional approach to attain and maintain an immediately desired steady-state drug plasma concentration is achieved by administering a simultaneous intravenous bolus and infusion. However, this regimen may lead to potential toxicity to AmB whose kinetic behavior was described by a multicompartment model. In addition, an intravenous bolus of AmB causes precipitation of the drug due to its low water solubility ( based on a preliminary study). Therefore, a two-step infusion method proposed by Wagner (Wagner et al., 1974) was used to rapidly achieve AmB steady-state plasma concentrations. An 94 initial simulation was performed by substituting the pharmacokinetic parameters into equations defined by Wagner. Based on the computer simulation and a preliminary study, steady-state conditions are achieved within eight hours. The AmB steady-state plasma concentration is related to the dose infused (Figure 12). At the steady-state, the systemic clearance estimated based on steady state concentration and infusion rate should have minimal error if a real steady-state has been attained. Also, the renal clearance under this condition should also be accurate. Atkinson and Bennett (1978) reported the pharmacokinetic results in humans. They found that the behavior of AmB is best described by a three compartment model. The terminal half-life of 15 days is due to the slow return of AmB from the slowly equilibrating peripheral compartment. The volume of the peripheral compartment accounts for an average 80% of the total distribution volume of 4 Liters/kg. The half-life reported by Atkinson was calculated by measuring AmB concentrations over several days ( from the fortieth day in one patient and from the sixth day in another patient) after cessation of therapy. In our study, a two compartment model was chosen to fit the plasma data based on the results of a statistical test. The half-life of AmB is approximately 15 hours. Although there are some variations in the half-lives among three groups, the linear regression indicates that half-life is dose-independent. The central volume of distribution is about 1.2 L/kg and accounts for 23% of the total volume of distribution. The differences in half-life between literature reports and our results are due to the different species 95 studied, although other factors such as analytical methods used to determine AmB concentrations may have played a role. In addition, Atkinson and Bennett (1978) reported that the rate of transport from the peripheral tissues to the central compartment is relatively slow when compared to the rate of transport from the central compartment to the peripheral compartment. Similarly, we observed the same phenomena in our study as well as in a preliminary study in this laboratory. The rate constant of the drug moving from the peripheral compartment to the central compartment is one-fourth of the rate constant from the central compartment to the peripheral compartment. This information, along with the analysis provided by Atkinson and Bennett (1978), supports the hypothesis of prolonged AmB storage in tissues. The large volume of distribution observed coincides with this hypothesis. Renal clearance was reported to account for the elimination of less than 10 % of the total dose (Bindschadler et al., 1969). In our study, renal excretion accounted for 10 -20 % of the total dose. This difference may be attributed to species difference. In rhesus monkeys, high concentrations of AmB have been found in bile following intravenous administration (Lawrence et al., 1980). In dogs, serum AmB concentrations were about 19 % higher during periods of biliary obstruction and biliary elimination was estimated to be 19 % of the dose (Craven, et al., 1979). Therefore, biliary elimination and urinary elimination may account for a certain fraction of the dose. The fate of the remaining 60 % - 70 % of the dose of AmB is 96 unknown. A possible reason for failure to find most of the administered dose might be metabolic conversion of AmB. However, no metabolites of AmB have been identified both in human and animals. Another possible reason is due to the limitation of assay methods, such that very low concentrations of AmB excreted over a prolonged period may be undetectable. This undetectable drug in biological samples is likely to be the portion of the missing dose. The hypothesis of prolonged tissue storage of AmB agrees with this observation. The renal clearance has been found to account for 5 % of creatinine clearance, similar to the result reported by Aktinson. Systemic clearance of AmB was about 3 ml/min*kg, and was found to be dose-independent over the dose range studied. This is consistent with the preliminary results from this lab and results reported by Chabot (1989). However, renal clearance was found to decrease significantly from 0.80 ml/min* kg to 0.4 ml/min* kg as dose increased. The renal excretion of drugs usually involves three processes: glomerular filtration, renal tubular secretion, and passive or active reabsorption from the renal tubular lumen. Glomerular filtration is a passive process and may be assumed to be a function of the free concentration of drug in plasma, if the glomeruli are intact. Renal tubular secretion is an active and saturable process because it needs carriers and energy to accomplish transport. Renal tubular reabsorption of most drugs involves passive diffusion of unionized molecules from the renal tubular lumen. Therefore, the rate of reabsorption is proportional to the concentration gradient of diffusible drug across 97 the renal tubular boundary, and may be affected by urine flow rate and by the urine pH. Renal clearance from our study was found to be similar or even lower than glomerular filtration ( product of unbound fraction of drug obtained from the literature and GFR ) depending on the unbound fraction used to calculate glomerular filtration. This comparison dose not provide a clear indication of the mechanisms responsible for renal excretion of AmB. The alteration of renal clearance can result from a number of underlying mechanisms. First, one may consider alteration in the glomerular filtration process. Glomerular filtration may be affected by both the integrity of the nephron and plasma protein binding. Impairment of renal function after high dose AmB treatment is a possible explanation for the observed dose-dependent renal clearance. Signs of renal impairment noted in our study, by measuring creatinine clearance, tends to support the above hypothesis. Alterations in plasma protein binding are known to affect glomerular filtration and renal clearance. Renal clearance usually increases when plasma protein binding decreases. For example, progressive saturation of protein binding at high drug concentration can increase the unbound fraction and consequently, increase renal clearance. However, the direction of alteration in renal clearance does not agree with the observed results, suggesting a minimal contribution from this process. A second consideration is the existence of active renal tubular secretion. The dose-dependent renal clearance can also be explained by the saturation of the active secretion process at high AmB 98 concentrations. A plot of renal clearance versus plasma amphotericin B concentration results in a profile characteristic of a saturable active secretion process (Figure 20). Finally, there is the possible existence of tubular passive reabsorption. There are no known alterations in urine flow rate and urine pH as the AmB doses increased (Figure 24). Thus, alterations in the reabsorption process are least likely to be responsible for the dose-dependent renal clearance. Because renal clearance is a small part (10-20 %) of systemic clearance, the change in renal clearance will not dramatically affect total body clearance. 5.2 Conclusion The most important conclusion of this research is the observation that elimination half-life, volume of distribution, and systemic clearance of amphotericin B are dose-independent in the rat. However, the dose-dependent renal clearance may be due to either the nephrotoxicity associated with AmB or saturable active secretion. 5.3 Suggestions for Further Work Further work may emphasize (1) characterizing the distribution, metabolism and elimination of amphotericin B (2) understanding factors affecting the disposition kinetics of Amphotericin B (3) examining amphotericin B-associated nephrotoxicity 99 as a function of dose, dose rate and disease states. 100 APPENDIX A Serum and urine AmB concentration versus time data after administration of AmB. Table A.l AmB Concentration (jLtg/ml) vs Time ( Low Dose ) Dose la Dose 2b Dose 3° Dose 4d Dose 5e 40 0.091 0.077 0.128 0.139 0.165 370 0.049 0.041 0.070 0.069 0.095 460 0.055 0.046 0.049 0.102 0.072 560 0.049 0.045 0.069 0.095 0.081 840 0.029 0.049 0.039 0.051 0.040 1420 0.026 0.039 0.023 0.017 0.027 1930 0.017 0.026 0.012 0.014 0.011 Time (min) 323.7 : 351.8 439.5 : 395.3 e: 391.2 Mg/kg Mg/kg /ig/kg Mg/kg Mg/kg Table A.2 Time (min) Dose 1 1220.0 M9/kg AmB Concentration ( n q / m l ) vs Time ( Medium Dose ) Dose 2 1500.0 Mg/kg Dose 3 1672.2 jLtg/kg Dose 4 900.2 (ig/kg 40 0.411 0.340 1.160 0.198 370 0.154 0.119 0.222 0.081 460 0.129 0.134 0.208 0.098 560 0.111 0.122 0.180 0.098 840 0.051 0.098 0.101 0.097 1420 0.071 0.072 0.066 0.075 1930 0.030 0.073 0.043 0.067 2300 0.051 0.038 0.047 0.059 3035 0.024 0.030 0.022 0.031 Table A.3 Time (min) Dose 1 3782.6 /ig/kg AmB Concentration (|zg/ml) vs Time (High Dose) Dose 2 4145.9 /xg/kg Dose 3 4989.5 /xg/kg Dose 4 3921.9 Mg/kg 40 1.410 3.020 3.360 1.350 370 0.532 0.464 0.907 0.505 460 0.448 0.567 0.882 0.499 560 0.323 0.475 0.595 0.415 840 0.219 0.229 0.271 0.263 1420 0.105 0.141 0.184 0.153 1930 0.109 0.126 0.117 0.130 2300 0.072 0.121 0.088 0.122 3035 0.054 0.027 0.047 0.033 3640 0.032 0.033 0.065 0.026 104 Table A.4 DOSE (ug/kg) 3782.6 Urine Amphotericin B Concentration versus Tims Data TIME (min) 0-363 363-547 547-798 798-1359 1359-2018 2018-2279 2279-3006 3006-3614 3614-4655 4655-5175 5175-6131 6131-8048 VOLUME (ml) EXC. RATE (ug/min) 10 12.5 12 13 6 5 8 8 12 5 8 11 0.037 0.056 0.043 0.027 0.014 0.016 0.006 0.006 0.003 0.002 0.0008 0.0004 0-362 362-549 549-801 801-1363 1363-2023 2023-2281 2281-3008 3008-3616 3616-4655 4655-5177 5177-6133 6133-8053 8 8 12 16 8 4 10 8 12 2 10 13 0.038 0.054 0.047 0.022 0.018 0.018 0.011 0.009 0.004 0.003 0.002 0.0005 0-348 348-546 546-798 798-1360 1360-2020 2020-2277 2277-3013 3013-3610 3610-4650 4650-5169 5169-6127 6127-8046 7.5 8 16 19 12 5 10 6 14 8 12 22 0.043 0.097 0.085 0.038 0.031 0.028 0.012 0.012 0.008 0.008 0.004 0.002 4145.9 4989.5 3921.9 0-346 346-600 600-801 801-1364 1364-2020 2020-2279 2279-3002 3002-3608 3608-4651 4651-5166 5166-6126 6126-8042 7.5 12 5 9 7.5 3 11 5.5 8.5 1.5 5 10 0.042 0.059 0.030 0.014 0.017 0.013 0.009 0.005 0.002 0.001 0.001 0.0003 0-395 395-573 573-880 880-1448 1448-2273 2273-3069 3069-3690 3690-4529 4529-5731 8 4 4 6 8 10 8 10 12 0.021 0.023 0.011 0.006 0.006 0.003 0.002 0.001 0.0005 0-390 390-605 605-899 899-1447 1447-2272 2272-3067 3067-3689 3689-4530 4530-5730 10 9.5 9 10 13 12 9 12 20 0.017 0.036 0.026 0.011 0.011 0.007 0.005 0.001 0.001 0-406 406-598 598-812 812-1475 1475-2138 2138-2519 2519-3315 3315-3884 3884-4877 4877-6771 19 7 4 8 3.8 3 4.5 3 8.2 12 0.049 0.044 0.033 0.018 0.016 0.013 0.009 0.007 0.005 0.002 1220.0 1550.0 106 900.2 0-404 404-597 597-809 809-1475 1475-1890 1890-2275 2275-3067 3067-3635 3635-4630 4630-6525 10 3.5 3 13 6 4 10.5 4.5 7 26 0.026 0.024 0.017 0.014 0.010 0.008 0.006 0.004 0.001 0.001 0-407 407-591 591-814 814-1474 1474-1891 1891-2273 2273-3072 3072-3640 10 3 3.8 11.5 3.5 4.8 20 11 0.014 0.009 0.013 0.005 0.002 0.002 0.002 0.001 0-407 407-594 594-813 813-1475 1475-1890 1890-2273 2273-3070 3070-3635 11 4.5 3 9 3 4 7.5 5.2 0.016 0.015 0.013 0.011 0.006 0.004 0.001 0.0007 0-360 360-610 610-809 809-1471 1471-1986 1986-2276 6 4.7 2 6 3.5 2.5 0.009 0.015 0.007 0.003 0.0006 0.0004 0-363 363-612 612-810 810-1472 1472-1986 1986-2281 2281-3014 7 6.5 3.5 9 3.5 2 7.5 0.009 0.013 0.015 0.006 0.002 0.002 0.0004 351.8 107 391.2 0-365 365-613 613-811 811-1474 1474-1987 1987-2282 2282-3015 6 4 4 10 4 4 10 0.014 0.017 0.007 0.008 0.003 0.002 0.0005 108 APPENDIX B Serum AmB concentration versus time plots in rats after administration of - .52 5 / 0.100 •• • c o CJ CD E < 0 010 500 1500 1000 2000 2500 Dose=351 8 £ig/kg 0.100 -- •• 0.010 500 1000 1500 2000 2500 Time (min) O VO 1 000 l)ose = 395 -5 /jg/kg 0 100 0.010 - 0 001 500 1000 2000 2500 1.000 Dose=391.2 ng/kg 0 100 0.010 I— 0.001 500 1000 Time (min 2000 2500 AmB Concentration III (/ig/ m l ) 1 000 Uose= 1220 /zg/kg ~ 0 100 —I. 0010 500 1000 1— 1500 2000 -I 2500 I 1 3000 3500 4000 1.000 Pose= 1500 0 //g/t-g - o 0 100 0.010 500 1000 1500 2000 lime (min) 1 1 1 2500 5000 3500 4000 AmB Concentrotion (/xg/ml) £11 AmB Concentrotion (/xg/ml) 10 000 f>ose = 3782 6 /jg/kg 1.000 0.100 - - —I 0.010 - 500 1000 1500 2000 f 3000 3500 4000 10.000 Dose-4145 9 itq/Uq 1.000 -- ••• 0.100 -- 0.010 500 1000 1500 2000 Time (niin) 3000 3500 4000 10.000 Dose=4989 5 ng/kg 1.000 •* 0.100 -- 0.010 500 -I (- 1000 1500 2000 2500 3000 3500 4000 10.000 Dose=3921.9 /aq/kg 1.000 0.100 0.010 500 1000 1500 2000 Time (min) 2500 3000 3500 4000 116 APPENDIX C Serum AmB concentration versus time fitted curves generated by program ADAPT n. 10.0-q Dose=323.7 ug/kg 1.0- 3 C o •H •P J" o. i - •P C <D o c 8 o.oi- ffl e < 1. O E - 3 11 || | l I I I I I | I I I I I I I I I |I I I I I I I I I| I I I I I I I I I | I I I I I I I I I | 0.50k 1.00k l.SOk 2.00k 2.SOk Time (min) 10.0-q 3 l. o-d C o •P 00 o.i- Dose=351.8 ug/kg •H •p c a) o c o O o.oi - CQ I 1•OE-3 "I I I I I I I I I | I I I I I 1 I I I | I I I I I I I I I | I I b • i i i i i | • i i (• i i J I | 0.50k 1.00k 1.50k 2.00k Time (min) Note: symbols represent the observed data, line represents the fitted curve generated by ADAPT II. 2.60k AmB concentration (ug/ml) AmB concentration (ug/ml) O O H ""i O H H O H O O \ K) cno AmB concentration (ug/ml) J ' ' ' "ill i • • i mil | I l i l i mil c 8 120 10.0-^ Dose=1220.0 ug/kg 1.0- 0 .1- 0 . 01 - . 0E-3 11111111111111 11 11|I IItII I I 11lll11 I I I 11I Il11 II I I11 I I I 11 I I I 11 II I I i I l I | 0.50k 1.00k 1.50k 2.00k 2.50k 3.00k 3.50k Time (min) 10 0 —i Dose=1500.0 ug/kg 1.0- o. i- 1111111111 0 . 50k 1111111 n 111H1111111 u 111111 u 11} 111111111111111111 * I 1.00k 1.50k 2.00k Tim© (min) 2.50k 3.00k 3.50k 10. 0 -a Dose=1672.2 ug/kg 1 . 0-= 0.1- 1 'I I I I I I I 1 I | I I I I I I I I I| I I I I I I I I I I I I 0.50k 1.00k i M I |) M I 1 I I I I | 1.50k 3.00k 3.50k Time (min) 10.0-q Dose=900.2 ug/kg 1.0- 0 .1- 0 . 01 - l.OE-3 0. 50k 1.00k 1. 50k 2.00k Time (min) 2.50k 3.00k 3.50k AmB concentration (ug/ml) AmB concentration (ug/ml) O K K O M O O CO AmB concentration (ug/ml) AmB concentration (ug/ml) K O O i i i 1111ii H TO i i -/> ' i i M i iiI X 3 (D D N O 0 W (0 II w vj 00 3 H3 CO • cn 0 % CO o *• o JP $ O- J—* 124 REFERENCE: Andreoli, T.E., "On the Anatomy of Amphotericin B Cholesterol Pores in Lipid Bilayer Membranes." Kidney Int., Vol.4, pp.337-345, 1973. Atkinson A.J., J.E. Bennett, "Amphotericin B Pharmacokinetics in Humans". Antimicrob. Agents Chemother., Vol. 13, pp 271-276, 1978. Bangham, A.D., M.W. Hill and N.G.A. Miller, " Preparation and Use of Liposomes as Models of Biological Membranes". Methods in Membrane Biology. E.D. Korn, Ed. pp. 1-68, PLenum. New York, NY, 1974 Bannatyne, R.M., and R. Cheung. "Discrepant Results of Amphotericin B Assays on Fresh versus Frozen Serum Samples." Antimicrob. Agents Chemother., Vol. 12, pp. 550, 1977. Barriere, S.L. "Pharmacology and Pharmacokinetics of Traditional Systemic Antifungal Agents". Pharmacotherapy, Supplement Vol. 10, No. 6, pp. 134s-140s, 1990. Bennett JE: Antifungal agents. In: Mandell GL, Douglas RG, Bennett JE, edds. Principles and practices of infectious diseases, 2nd ed. New York: John Wiley, 1985:263-270. Benson, J.M. and M.C. Nahata, "Pharmacokinetics of Amphotericin B in Children." Antimicrobial Agents and Chemotherapy, Vol. 33, No. 11, pp. 1989-1993, 1989. Bhathena, D.B., W.F. Bullock, C.E. Nuttall, R.G. Luke, " The Effects of Amphotericin B Therapy on the Intrarenal Vasculature and Renal Tubules in Man". Clin. Nephrol., Vol. 9, pp. 103-110, 1978. Bindschadler, D. D., J.E. Bennett, "Pharmacological Guide to the Clinical Use of Amphotericin B". Journal of Infectious Diseases, Vol. 120, pp. 427-36, 1969. Bodey, G.P., "Candiasis in Cancer Patients". Am. J. Med., Vol. 77, pp. 13-19, 1984. Boxenhaum, H.G., S. Riegelman, and R.M. Elashoff, " Statistical Estimations in Pharmacokinetics ". Journal of Pharmacokinetics and Biopharmaceutics, Vol. 2, pp. 123-148, 1974. 125 Brajtburg, J., G.S. Kobayashi, G.S. and G. Medoff, "Effects of Ascorbic Acid on the Antifungal Action of Amphotericin B." J. Antimicrob. Chemother;, Vol. 24, pp. 333337, 1989. Brajtburg, J., W.G. Powerly and G.S. Kobayashi, "Amphotericin B: Delivery Systems." Antimicrob. Agents Chemother, Vol.34, pp. 381-384, 1990. Butler, W.T., J.E. Bennet, D.W. Ailing and P.T. Wertlake, "Nephrotoxicity of Amphotericin B . Early and Late Effects in 81 Patients". Ann. Intern. Med., Vol.61, pp.175-187, 1964. Cai, Y." Disposition Kinetics of Amphotericin B in Rats ". Master's Thesis, 1991. Chabot, G.G., R. Pazdur, F. Valeriote and L. Baker, "Pharmacokinetics and Toxicity of Continuous Infusion Amphotericin B in Cancer Patients." Journal of Pharmaceutical Sciences, Vol.78, No.4, pp. 307-310, 1989. Chandler, F. W., "Pathology of the Mycoses in Patients with Acquired Immunodeficiency Syndrome." Current Topics in Medical Mycology. M.R. McGinnis, Ed.: pp. 1-23, Springer-Verlag. New York, NY, 1985. Cheng, J.T., R.T. Witty, R.R. Robinson and W.E. Yarger, "Amphotericin B Nephrotoxicity: Increase Renal Resistance and Tubule Permeability." Kidney Int., Vol.22, pp.626-633, 1982. Chow, H., Y. Cai and M. Mayersohn, "Disposition Kinetics of Amphotericin B in Rats, The Influence of Dose." Drug Metabolism and Disposition, Vol. 20, pp. 432-435, 1992. Christiansen, E. Bernard, J. Gold and D. Armstrong, "Distribution and Activity of Amphotericin B in Humans."/. Infec. Diseases, Vol. 152, pp.1037, 1985. Clements, J.S., J.E. Peacock. "Amphotericin B Revisited: Reassessment of Toxicity." The American Journal of Medicine, Vol. 88, pp. 5-22N - 5-27N, 1990. Cosgrove, R.F. and J.E. Fairbrother, "A Bioassay Method for Polyene Antibiotics Based on the Measurement of Rubidium Efflux from Rubidium-Loaded Yeast Cells". Antimicrobial Agents and Chemotherapy, Vol. 11, pp. 31-33, 1977. Craven, P., T. Ludden, D. Drutz, W. Rogers, K. Haegele and H. Skrdlant, "Excretion Pathways of Amphotericin B." J. Infec. Disease, Vol. 140, pp. 329, 1979. 126 D'Argenio, D.Z. and A. Schumitzky, "ADAPT: Mathematical Software for Pharmacokinetic/Pharmacodynamic System Analysis." Developed and Supported by the Biomedical Simulations Resource, University of Southern California, Los Angeles, CA 90089-1451, 1992. Drazin, R.E. and R.I. Lehrer, "Rubidium Release: A Rapid and Sensitivity Assay for Amphotericin B." J. Infect. Dis., Vol. 134, 238-244, 1976. Edmonds, L.C., L. Davidson and J.S. Bertino, "Solubility and Stability of Amphotericin B in Human Serum". Therapeutic Drug Monitoring, Vol. 11, pp. 323-326, 1989. Ellis, W.G., R.A. Sobel and S.L. Nielsen, "Leukoencephalopathy in Patients Treated with Amphotericin B Methyl Ester". Journal of Infectious Diseases, Vol. 146, pp. 12537, 1982. Fromtling, R.A., "Overview of Medically Important Antifungal Azole Derivatives". Clinical Microbiology Reviews,Vol. 1, pp. 11-5, 1988. Gale, E.F., "The Release of Potassium Ions from Candida albicans in the Presence of Polyene Antibiotics." Journal of General Microbiology, Vol. 80, pp.451-65, 1974. Gerkens, J.F. and R.A. Branch. "The Influence of Sodium Status and Furosemide on Canine Acute Amphotericin B Nephrotoxicity." J. Pharmacol. Exp. Ther., Vol. 172, pp. 85-9, 1980. Gold, J. W. M., " Opportunistic Fungal Infections in Patients with Neoplastic Disease". Am. J. Med., Vol. 76, pp. 458-463, 1984. Goodman, L.S. and A. Gilman. "The Pharmacological Basis of Therapeutics". Macmillan: New York, pp. 1219-1221, 1985. Granich, G., G. Kobayashi and D. Krogstad. "Sensitive High-Pressure Liquid Chromatographic Assay for Amphotericin B Which Incorporates an Internal Standard." Antimicrob. Agents Chemother., Vol. 29, pp. 584, 1986. Graybill, J. and P. Craven, "Antifungal Agents Used in Systemic Mycoses". Drug, Vol. 25, pp.41-62, 1983. Green, W.R., J.E. Bennett and R.D. Goos, "Ocular Penetration of Amphotericin B." Archives of Ophthalmology, Vol. 73, pp. 769-775, 1965. 127 Gresham.H.D., J.A. McGarr, P.G. Shackelford and E.J. Brown, "Studies on the Molecular Mechanisms of Human Fc Receptor-Mediated Phagocytosis: Amplification of Ingestion Is Dependent on the Generation of Reactive Oxygen Metabolites and Is Deficient in Polymorphonuclear Leukocytes from Patients with Chronic Granulomatous Disease." J. Clin. Invest., Vol. 82, pp. 1192-1201, 1988. Herve, M., J.C. Dubouzy and E. Borowski, "The Role of the Carboxyl and Amino Groups of Polyene Macrolides in Their Interactions with Sterols and Their Selective Toxicity. A 31 P-NMR Study." Biochem. Biophys. Acta, Vol 980, pp.261-272, 1989. Hopfer, R.L. and D. Groschel, "Radiometric Determination of the Concentration of Amphotericin B in Body Fluids." Antimicrob. Agents Chemother., Vol. 12, pp. 733735, 1977. Kintzel, P.E. and P. E. Kennedy, "Stability of Amphotericin B in 5% Dextrose Injection at Concentrations Used for Administration through a Central Venous Line". Am. J. Hosp. Pharm., Vol. 48, pp. 283-285, 1991. Koren, G., A. Lau, J. Klein, C. Golas, M. Bologa-Campeanu, S. Soldin, "Pharmacokinetics and Adverse Effects of Amphotericin B in Infants and Children." J. Pediatr, Vol.113, pp. 559-563, 1988. Kovacs, J.A., A.A. Kovacs, M. Polis, W.C. Wright, V.J. Gill, C.U. Tuazon, E.P. Gehmann, H.C. Lane, et al., "Cryptococcosis in the Acquired Immunodeficiency Syndrome." Ann. Intern. Med., Vol.103, pp. 533-538, 1985. Lam, F., C. Hung, D. Perrier, "Estimation of Variance for Harmonic Mean HalfLives." J. Pharm. Sci., Vol. 74, pp. 229, 1985. Lamy-Freund, M.T., V.F.N. Ferreira, and S. Schreier, "Mechanism of Inactivation of the Polyene Antibiotics Amphotericin B: Evidence for Radical Formation in the Process of Autoxidation." J.Antibio., Vol. 38, pp. 753-757, 1985. Lawrence, R.M., P.D.Hoeprich, A.A. Jagdis, N. Monji, A.C. Huson," Distribution of Doubly Radiolablled Amphotericin Methyl Ester and Amphotericin B in the Nonhuman Primate, Macaca Mulatta." J. Antimicrob. Chemother., Vol. 25, pp. 45-8,1980. Lopez-Berestein, G., R. Mehta, R.L. Hopfer, K. Mills, L. Kasi, K. Mehta and V. Fainsterin. "Treatment and Prophylaxis of Disseminated Infection Due to Candida Albicans in Mice with Liposome-encapsulated Amphotericin B." J. Inf. Dis., Vol. 147, pp. 939, 1983. 128 Lopez-Berestein, G., G.P. Bodey, L.S. Frankel, "Treatment of Hepatosplenic Candidisis with Liposomal-Amphotericin B." Journal of Clinical Oncology, Vol. 5, pp. 310-7, 1987. Maksymiuk, A.W., S. Thongpraset, R. Hopfer, M. Luna, V. Fainstain and P. Bodey, "Systemic Candidiasis in Cancer Patients." Am. J. Med., Vol.77(4D), pp. 20-27,1984. Mechlinski, W., C. Schaffner and P. Ganis, "Structure and Absolute Configuration of the Polyene Macrolide Antibiotic Amphotericin B". Tetrahedrom Lett., Vol. 44, pp. 3873-3876, 1970. Mechlinski, W. and C.P. Schaffner, "Separation of Polyene Antifungal Antibiotics by High-Speed Liquid Chromatography." J. Chromatogr., Vol. 99, pp. 619-633, 1974. Modeff, G., J. Brajtburg, G.S. Kobayashi and J. Bolard," Antifungal Agents Useful in Therapy of Systemic Fungal Infections". Annual Review of Pharmacology and Toxicology, Vol. 23, pp. 303-30, 1983. Niki, Y., E.M. BernardH. Schmitt and W.P. Tong, "Pharmacokinetics of Aerosol Amphotericin B in Rats." Antimicrobial Agents and Chemotherapy, Vol. 34, No. 1, pp. 29-32, 1990. Nilsson-Ehle, I., T.T. Yoshikawa, M.C. Schotz and L.B. Guze, "Quantitation of Amphotericin B with Use of High-Pressure Liquid Chromatography." J. Infect. Dis., Vol. 135, pp. 414-422, 1977. Perrier, D. and M. Mayersohn," Noncompartment Determination of the Steady-State Volume of Distribution for Any Mode Administration ". J. Pharm. Sci, Vol. 71, pp. 372-374, 1982. Rocci Jr, M.L., W.J. Jusko, "Lagran Program for Area and Moments in Pharmacokinetic Analysis". Computer Program in Biomedicine, Vol. 16, pp 203-216, 1983. Sabra, R and R. Branch," AmB Nephrotoxicity", Drug Saf, Vol. 5, pp. 94, 1990. Said, R., P. Marin, H. Anicama, A. Quintanilla, " Effect of Mannitol on Acute Amphotericin B Nephrotoxicity". Res. Exp. Med., Vol. 177, pp. 185-90, 1980. Schaffner, C.P., " Amphotericin B derivatives ". Recent Trends in the Discovery, Development and Evaluation of Antifungal Agents, pp.595-618, Prous Science 129 Publishers, Barcelona, 1987. Schnermann, J., "Regulation of Single Nephron Filtration Rate by Feedback: Facts and Theories", Clin. Nephrol., Vol. 3, pp. 75, 1975. Shadomy, S., D.L. Brummer and A.V. Ingroff, "Light Sensitivity of Prepared Solutions of Amphotericin B." Amer. Rev. Resp. Dis., Vol. 107, pp. 303-304, 1973. Slot, C., " Plasma Creatinine Determination - A New and Specific Jaffe Reaction Method". Scand. J. Clin. Lab. Invest., Vol. 17, pp. 381, 1965. Sokol-Anderson, M. L., J. Brajtburg and G. Medoff, "Amphotericin B-induced Oxidative Damage and Killing of Candida Albicans". Journal of Infectious Diseases, Vol. 154, pp. 76-83, 1986a. Sokol-Anderson, M. L., J. Brajtburg and G. Medoff, "Sensitivity of Candida Albicans to Amphotericin B Administered as Single or Fractionated Doses". Antimicrobial Agents and Chemotherapy, Vol. 29, pp.701-702, 1986b. Sokol-Anderson, M. L., J.E. Sligh, S. Elberg, J. Brajtberg, G.S. Kobayashi and G. Medoff, "Role of Cell Defense against Oxidative Damage in the Resistance of Candida Albicans to the Killing Effect of Amphotericin B". Antimicrobial Agents and Chemotherapy, Vol. 32, pp. 702-705, 1988. Starke, J.R., E.O. Mason, W.G. Kramer and S.L. Kaplan, "Pharmacokinetics of Amphotericin B in Infants and Children." J. Infect. Dis., Vol. 155, pp. 766-774, 1987. Stein, R.S. and J.A. Alexander. "Sodium Protects against Nephrotoxicity in Patients Receiving Amphotericin B"Am. J. Med. Sc., Vol. 298, pp. 299, 1989. Steinberg, B. A., W.P. Jambor and L.O. Suydam, "Amphotericin A and B: Two New Antifungal Antibiotics Processing High Activity against Deep-Seated and superficial Mycoses". Antibiotics Annual 1955 - 1956, pp. 574-578. Szoka, F. and D. Papahadjopoulos, "Comparative Properties and Methods of Preparation of Lipid Vesicles (liposomes)". Annu. Rev. Biophys. Bioeng. Vol. 6, pp. 465-506, 1980. Trejo, W. H., and R.E. Bennett, "The Amphotericin Producing Organism". Journal of Bacteriology, Vol. 85, pp. 436-439, 1963. 130 Wagner, J.," A Safe Method for Rapidly Achieving Plasma Concentration Plateaus". Clin. Pharmacol. Titer., Vol. 16, pp 691, 1974. Waksman, S.A., H.A. Lechevalier and C.P. Schaffner, "Candicidin and Other Polyenic Antifungal Antibiotics". Bull. Wld. Hlth. Org., Vol.33, pp. 219-226, 1965. Weeks, J.R., J.D. Davis, "Chronic Intravenous Cannulas for Rats". J. Appl. Physiol., Vol. 19, pp. 540-1, 1964. Wiest, D.B., W.A. Maish, S.S. Garner, " Stability of Amphotericin B in Four Concentrations of Dextrose Injection." Am. J. Hos. Pharm., Vol. 48, pp. 2430-3,1991. Winn W.A., "Coccidioidomycosis and AmB", Med. Clin. North. Am., Vol. 47, pp. 1131-1148, 1963. Wright, F.S. and J.P. Briggs, "Feedback Regulation of Glomerular Filtration Rate." Am. J. Physiol. Vol. 233, pp. Fl, 1977.
* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project