ANTIMICRoBIAL AGENTS AND CHzMOTHzRAPY, Nov. 1977, p. 582-590 Copyright 0 1977 American Society for Microbiology

Vol. 12, No. 5 Printed in U.S.A.

Comparative Pharmacology of Amphotericin B and Amphotericin B Methyl Ester in the Non-Human Primate, Macaca mulatta F. A. JAGDIS,t P. D. HOEPRICH,l* R. M. LAWRENCE,1 AND C. P. SCHAFFNER2 Department of Internal Medicine, School of Medicine and the California Primate Research Center, University of California, Davis, California 95616,1 and the Waksman Institute ofMicrobiology, Rutgers University, Rutgers, New Jersey 089032

Received for publication 31 May 1977

The phannacokinetics of amphotericin B methyl ester hydrochloride (AME) and commercial deoxycholate-stabilized amphotericin B (AMB) were compared after single doses of 5 mg and 1 mg/kg of body weight, respectively, given intravenously in a period of 3 h to adult female rhesus monkeys (Macaca mulatta). By bioassay, the concentrations of AME were 12.2 to 7.2 times higher in the serum and 7.8 to 2.5 times higher in the urine during the 8 h after infusion. The decline in concentrations of the drugs in sera was consistent with a three-compartment, open pharmacokinetic model; rate constants of transfer of the drugs between the compartments and volumes of distribution were calculated. The overall rate of elimination from the central compartment (the bloodvascular space) was about four times greater for AME than for AMB. Serum urea nitrogen and creatinine concentrations were mildly and transiently increased after infusion of AME, whereas the more severe azotemia that followed infusion of AMB persisted for 5 days. AME was less toxic and achieved a greater urinary outfall than AMB. As the antifungal activity of AME is comparable to that of AMB by testing in vitro, further study is warranted.

Amphotericin B (AMB) has been the drug of choice for treatment of systemic fungal infections since 1955 (17, 18, 37). However, not all isolates of pathogenic fungi are susceptible to it (19). Moreover, AMB virtually always causes adverse reactions (18, 37); permanent kidney damage is the most important injury and is often the factor that limits parenteral therapy. It was speculated that adverse reactions engendered by injection of the AMB suspension might be mitigated by use of a water-soluble derivative. To this end, the N-acyl derivative was synthesized (28, 33, 41); it was soluble in water (e.g., as the sodium salt) but was greatly reduced in antifungal activity. Aided by the determination of the absolute chemical structure of AMB (34), the obverse semisynthesisconversion of AMB from amphotericity to cationicity-was accomplished through esterification of the carboxyl (6, 33). Acid salts of the esters retained antifungal activity. Of a series of esters, the methyl ester has attracted most attention because it: (i) attains a molecular or near molecular dispersion in water (42); (ii) is significantly less toxic than AMB (2, 5-8, 14, t Present address: #402-1571 Mortimer St., Victoria, British Columbia, Canada V8P 3AK.

15, 22, 23, 27); and (iii) retains antifungal activity (5, 6, 15, 19, 33). We studied the pharmacological effects and pharmacokinetics after administration of single intravenous (i.v.) doses of amphotericin B (deoxycholate-stabilized suspension) and amphotericin B methyl ester hydrochloride (AME) to non-human primates, Macaca mulatta. MATERIALS AND METHODS Drugs. AMB was purchased as the deoxycholatestabilized phosphate-buffered, commercial formulation for i.v. administration (Fungizone, E. R. Squibb & Sons, New Brunswick, N.J.). It was prepared for infusion with sterile distilled water according to the manufacturer's instructions. AME was synthesized at the Waksman Institute of Microbiology, Rutgers University, New Brunswick, N.J. (33). The batch used in this work was

free of AMB by physicochemical analyses and was 75% antifungally active drug. The AME was dissolved in sufficient sterile distilled water to yield a final concentration of 0.2% (wt/vol); it was then passed through a 0.45-,um-pore size membrane filter. Neither storage of AME as a dry powder at -20°C (periods up to 6 months), solution in distilled water and membrane filtration, nor dilution for i.v. injection in 5% glucose solution (v.i.) was associated with detectable loss of antifungal activity.

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AMPHOTERICIN B METHYL ESTER IN RHESUS MONKEYS

Primates. Seven adult female rhesus monkeys (M. mulatta) weighing between 5.2 and 8.3 kg were used. They were housed individually in indoor cages at the California Primate Research Center, Davis, and were fed Purina Monkey Chow (Ralston Purina Co., St. Louis, Mo.). Two monkeys were used twice, with recovery intervals of 43 and 55 days between experiments, respectively. Experimental protocol. The monkeys were anesthetized by intramuscular injection of ketamine hydrochloride (Ketaset, Bristol Laboratories, Veterinary Products, Syracuse, N.Y.) in doses of 100 to 200 mg initially followed by 20 to 50 mg at intervals of 1.0 to 1.5 h as needed. The amphotericins were injected i.v. in doses calculated as milligrams per kilogram of body weight. Each dose was made up to a final volume of 100 ml in sterile 5% glucose solution and was administered over a 3-h periodto mimic a minimal period for infusion of AMB as it is used in the treatment of humans (18, 37). Specimens of serum (from venous blood clotted in glass tubes) and urine (catheter indwelling in urinary bladder through 4 h postinfusion; in and out catherization thereafter) were collected before infusion and at the following times (hours) after infusion: 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 120, and 168 h. The specimens were stored frozen at -20°C in snap-top polycarbonate tubes until bioassays were performed. Hemoglobin and hematocrit determinations and urinalyses were carried out by standard laboratory methods. Serum urea nitrogen and creatinine were measured by an ABA Biochromatic Analyzer (Abbott; Pasadena, Calif.). Bioassay. Large agar plates (30 by 30 cm) were prepared, using a culture medium of the following composition (per liter): D-glucose, 10 g; peptone, 9.4 g; yeast extract, 4.7 g; beef extract, 2.4 g; NaCl, 10 g; agar, 23.5 g; final pH 6.0 to 6.2 (formula kindly provided by D.M. Isaacson, Squibb Institute for Medical Research, Princeton, N.J.). The agar (1 mm deep) was seeded with a test strain of Paecilomyces variotii (NIH 5605) to produce a final concentration of 2 x 104 spores per ml. For assay of specimens containing AMB, wells (5 mm in diameter) were cut into the agar; triplicate 0.2-ml portions of each specimen were placed in each well. For assay of specimens containing AME, stainless-steel well plates (six equidistant dimples of 0.5-ml capacity with 0.5-mm-diameter orifices) were placed on the agar surface. Again, triplicate 0.2-ml portions of specimens were placed in each well. Triplicates of standards containing known amounts of each drug diluted in serum (for assay of sera) or phosphate-buffered saline (for assay of urines: Dulbecco formula, purchased from GIBCO, Grand Island, N.Y.) were applied to the same plates. Diameters of the zones of inhibition were measured after 48 h of incubation at 35°C. The concentrations were determined by comparison of average zone diameters of the specimens with corresponding standards, according to the method of Cooper (9). The minimal assayable concentrations varied with specimen and drug-for AMB: serum, 0.20 ,ug/ml; urine, 0.90 Ag/ ml; and for AME: serum, 0.22 mg/ml; urine, 0.50 ;Lg/ml. All of the specimens from a given animal

583

were assayed simultaneously.

Pharmacokinetic calculations. The decline in concentration of each drug in the serum of each animal was plotted as a function of time (semilog), and each curve was individually analyzed. Such analysis was done to avoid curve averaging, which could lead to bias problems (46). The initial equations were derived by the "feathering" or back projection technique (29). The parameters of each equation were then refined by a digital computer, nonlinear least-squares regression program (40). The newly derived intercepts on the y-axis were next corrected for time of infusion because the drugs were not injected as a bolus (31). Pharmacokinetic rate constants were then detennined by the formulae deduced by Rescigno and Segre (38). Volumes of distribution were calculated by the formula given in Table 4; the values of peak concentrations in serum used in the calculations were derived by applying the Loo-Riegelman correction (31).

RESULTS Serum concentrations. Concentrations in the serum during the entire study period of 7 days are shown in Table 1. Postdose specimens were first obtained 30 min after infusion; for all doses, the concentrations in sera were highest 30 min postdose. The mean (30 min) peak concentration after infusion of AMB in a dose of 1 mg of drug per kg of body weight was 1.25 ,ug/ml, whereas injection of 5 mg of AME per kg yielded a mean peak concentration of 14.98 ,ug/ml (11.98 times higher). Higher doses of AME also gave concentrations in the serum that exceeded those of AMB (factors of 8.64 and 9.60 for doses of 15 and 10 mg/kg); however, these doses of AME caused intravascular hemolysis (both doses) and death (15 mg/kg). Decline pattems of mean concentrations for each drug (AMB, 1 mg/kg; AME, 5 mg/kg) were similar except for higher concentrations obtained with AME (Fig. 1). At 4 h after infusion, the concentration of AMB had declined by 20%, whereas AME had fallen off by 40% of the peak concentration (ratio of AME/ AMB, 7.72). At 48 h postinfusion, the concentration of AMB had declined by 78% and AME by 93% (ratio of AME/AMB, 3.61). Both drugs were detectable in serum obtained 7 days post-

infusion. A linear-decline pattern resulted when the serum concentrations of each drug were plotted against time on log-log scales (Fig. 2A). This kind of fall-off has been described for AMB (4). The slope of the curve for AME was greater than that for AMB; i.e., AME disappeared from the blood more rapidly than AMB. Urine concentrations. Although concentrations of drugs in urine varied much more than in serum, the concentrations of AME always

584

JAGDIS ET AL.

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exceeded those of AMB (ratios of AME/AMB, 7.76 at 30 min and 2.52 at 8 h postinfusion). At each time, concentrations in urine were approximately twice the corresponding concentrations in serum. When concentrations in urine were plotted against time on log-log scales, lineardecline patterns were obtained (Fig. 2B). As in serum, the slope of the curve for AME was greater than that for AMB, indicating a more rapid urinary excretion of AME. Data on the quantity of each drug excreted in urine were obtained only for the period of 4 h postinfusion (catheter indwelling in bladder). Thereafter, collections were incomplete as urine was obtained by catheterization at the time of venipuncture. The quantity of AME put out in urine in the 4 h immediately after i.v. infusion was almost 10 times greater than the urinary outfall of AMB, when expressed as percentage of dose injected (Table 2). Pharmacokinetics. Feathering of each serum decline curve on semilogarithmic plots in-

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585

AMPHOTERICIN B METHYL ESTER IN RHESUS MONKEYS

VOL. 12, 1977

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i.v. infusions of the drugs into non-human primates (M. mulatta) over periods of 3 h are plotted against time. The insert depicts the curves during the first 8 h on expanded scales. Semilogarithmic plot.

FIG. 2. Mean concentrations of AMB (0,1 mg/kg) and AME (0, 5 mg/kg) in specimens of serum (A) and urine (B) obtained after i.v. infusions of the drugs into non-human primates (M. mulatta) over periods of 3 h are plotted against time. Log-log plots.

2. Urinary excretion of AMB and AME by non-human primates (M. mulatta) during the 4 h immediately after i.v. injection of 1 mg and 5 mg ofdrug per kg ofbody weight, respectively

TABLE

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a Run 2.

Drug/dose

(mg)

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AME/35.5

AME/41.5

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Dose

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140 177

8,058

9,065

2.7 2.8 22.7 21.8

586

ANTIMICROB. AGENTS CHEMOTHER.

JAGDIS ET AL.

postinfusion day 7. As a result, the decline in concentrations was best described by a triexponential equation: (1) S = Ae-a- + Be-b' + Ce-ct

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where S is the serum concentration at time t. The intercepts on the y-axis of each phase are A, B, and C; a, b, anO " are obtained from the slopes of the three linear phases. The parameters of equation 1 for each animal are shown in Table 3 after processing by nonlinear, least-square regression. Because there are three linear phases, each phase has a half-life. The biological half-life of a drug is determined from the terminal linear phase (29). Thus with both drugs: (2) 4 = ln 2/c where c is the slope of the terminal phase from equation 1. Thus, the biological half-life for AMB is 275 h, and for AME it is 97 h. The pharmacokinetic model assigned was a three-compartment open model (Fig. 3). The drug is introduced into the central compartment and enters and exchanges with two peripheral, communicating compartments. Removal of drug from the system is assumed to occur only from the central compartment. The pharmacokinetic constants for this model are shown in Table 4. Peripheral compartment 1 has relatively fast (k12 and k2l) and peripheral compartment 2 relatively slow (kl3 and k3j) exchange rates. The overall elimination constant of AME from the central compartment (kio) is about four times that of AMB (respective mean values of 0.1221 and 0.0290). The calculated volumes of distribution were not significantly different. Toxicity. Hemolysis, indicated by hemoglobinemia and hemoglobinuria, was evident 2 to 3 h after infusion of 15 and 10 mg of AME per kg of body weight. Of the two animals given 15 mg of AME per kg, animal 7182 died 48 h postinfusion, and 7184 was sacrificed due to apparent fatal illness. One animal (7186) given 10 mg of AME per kg also had detectable hemoglobinemia and hemoglobinuria, but the process was not severe, for the animal recovered without any complications, suffering only a decrease in hemoglobin concentration from 15.9 g/100 ml preinfusion to 11.9 g/100 ml on postinfusion day 7. No evidence of hemolysis was detectable with the 5-mg/kg dose of AME or the 1-mg/kg dose of AMB: the hemoglobin concentrations remained constant. The mean concentrations of serum urea nitrogen and creatinine measured during the entire study period are in Fig. 4. Twenty-four hours after the infusion of 5 mg of AME per kg

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VOL. 12, 1977

AMPHOTERICIN B METHYL ESTER IN RHESUS MONKEYS

of body weight, maximal concentrations (35 and 1.5 mg/100 ml for urea and creatinine, respectively) were observed; normal values were re-established within 24 h. In contrast, greater elevation in concentrations of urea nitrogen and creatinine occurred after infusion of 1 mg ofAMB per kg ofbody weight. Maximal concentrations persisted for 1 to 2 days after the infusion (44 to 46 mg and 1.7 mg/100 ml of urea nitrogen and creatinine, respectively). There was a leisurely decline to normal values over the subsequent 48 h; e.g., normalcy was attained by the postinfusion day 5. DISCUSSION The maximum serum concentration observed after a dose of 1 mg of AMB per kg of body weight in humans is in the range of 0.4 to 2.5 Ag/ml (3, 4, 12, 13, 30, 32, 37, 43-45). Elimination of AMB is slow in humans. Thus, at 1 week after infusion, concentrations in the serum ranged from 0.1 to 0.3 ,ug/ml (4, 13). NonI.V DOSE PERIPHERAL

COMPARTMENT 2

CENTRAL

human primates appear to behave similarly: 30 min after single i.v. doses of 1 mg of AMB per kg, the concentrations in serum were 1.2 and 1.3 ,ug/ml; 7 days postinfusion, AMB was 60

AMPHOTERICIN B METHYL ESTER 5 mg/ kg

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FIG. 4. Mean concentrations of urea nitrogen and creatinine in sera obtained after i.v. infusions of AMB and AME into non-human primates (M. mulatta) over periods of 3 h are plotted against time.

TABLE 4. Pharmacokinetic rate constants (three-compartment open model) of the disappearance of AMB and AME from serum of non-humran primates (M. mulatta) after i.v. infusion (calculated volumes of distribution of each drug are listed) Rate constants (h-9) Vda Drug (mg/kg) Monkey (dose in jAg) k2, k3l k13 klo k12 0.6533 0.0616 0.0149 0.0269 7f89 0.8780 1,855 AMB, 1 (5,200) 0.0607 0.0066 0.0310 7193 0.7083 0.4896 2,121 (6,400) 0.0612 0.0108 0.0290 0.7932 0.5715 Mean 1,988 133 0.0005 0.0042 0.0021 0.0849 0.0819 SEMb

AME, 5

6896 (i) (35,000) 7186 (40,000) 6896 (ii) (35,500) 3200 (41,500) Mean

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0.4306

0.1861

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0.0093

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SEMb

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588

JAGDIS ET AL.

still detectable in serum at concentrations of

Comparative pharmacology of amphotericin B and amphotericin B methyl ester in the non-human primate, Macacca mulatta.

ANTIMICRoBIAL AGENTS AND CHzMOTHzRAPY, Nov. 1977, p. 582-590 Copyright 0 1977 American Society for Microbiology Vol. 12, No. 5 Printed in U.S.A. Com...
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