INTRODUCTION

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Concurrent with the increasing number of patients with impaired host defenses, invasive fungal infections have emerged as important causes of morbidity and mortality in hospitals (1, 12). Therapeutic options have been expanded by the introduction of antifungal triazoles (15) and lipid formulations of amphotericin B (13), but each of the individual compounds has its specific limitations of safety, spectrum, and efficacy (11, 23). With the emergence of unusual fungal pathogens and drug resistance, there is an indisputable need for new, well-tolerated antifungal agents with novel modes of action and improved efficacy in immunocompromised patient populations.

The pradimicins are a new class of fermentation-derived antifungal antibiotics structurally characterized by a dihydrobenzonaphthacene quinone skeleton substituted with a D amino acid and a disaccharide side chain (16). The proposed mechanism of action of these agents consists of a calcium-dependent complexing with the saccharide portion of fungal cell wall mannoproteins, which leads to perturbation of the integrity and function of the fungal cell membrane and, ultimately, cell death (21). The pradimicins have non-cross-resistant, broad-spectrum fungicidal activity in vitro, being effective against Candida spp., Aspergillus spp., Cryptococcus neoformans, and other fungal pathogens (7, 14, 17, 18). Preclinical in vivo studies have demonstrated promising safety and efficacy in experimental models of invasive fungal infections in both normal and immunocompromised animals (9, 10, 14, 17-20).

Little is known, however, about the specific disposition of this new class of compounds in plasma and tissues. The purpose of this study was therefore to investigate the plasma pharmacokinetics and tissue distribution of BMS 181184, a water-soluble pradimicin FA-2 derivative with potent in vitro and in vivo antifungal activity.

	MATERIALS AND METHODS

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Study drug. BMS 181184 (250-mg vials; Bristol-Myers Squibb, Princeton, N.J.) was provided as a lyophilized powder, maintained at 4°C, and freshly reconstituted in sterile normal saline-sterile 5% dextrose in water (1:1, vol/vol) to a 50-mg/ml solution prior to dosing of animals.

Animals. Healthy female New Zealand White rabbits (Hazleton, Denver, Pa.) weighing 2.5 to 3.5 kg were used in all experiments. They were individually housed and maintained with water and standard rabbit feed ad libitum according to National Institutes of Health guidelines for laboratory animal care (4) and in fulfillment of American Association for Accreditation of Laboratory Animal Care criteria. Vascular access was established in each rabbit by the surgical placement of a subcutaneous silastic central venous catheter as previously described (22).

Single-dose studies. Four groups of three rabbits each were studied. Animals received BMS 181184 at 10, 25, 50, or 150 mg/kg of body weight as a single steady intravenous (i.v.) bolus (2 mg/s). Plasma samples were drawn immediately before administration of the compound and then at 0.16, 0.5, 1, 2, 3, 5, 6, 7, and 24 h after its administration.

Multiple-dose studies. Four groups of three rabbits each were studied. After the single-dose pharmacokinetics study was completed, the animals continued to receive BMS 181184 at 10, 25, 50, or 150 mg/kg of body weight as a single i.v. bolus once a day for a total of 16 days. On day 15, plasma samples were drawn immediately before dosing and then at 0.16, 0.5, 1, 2, 3, 5, 6, 7, and 24 h postdosing. On day 16, rabbits were euthanized by i.v. administration of pentobarbital 1 h after administration of the compound and samples from the brain, cerebrospinal fluid (CSF), vitreous fluid, lung, liver, spleen, and kidney were obtained for determination of their drug levels at the time of near-peak levels in the plasma.

Processing of blood and tissue specimens. Blood samples were collected in heparinized syringes. Plasma was separated by centrifugation and stored at 80°C until assayed. Tissue samples were carefully dissected and stored at 80°C. Before the assay, the tissue specimens were thawed, and three portions of approximately 1 g each were weighed for each sample (balance model AE 163; Mettler Instrument Corp., Hightstown, N.J.). The specimens were thoroughly rinsed with phosphate-buffered saline, pH 7.4 (Quality Biological, Inc., Gaithersburg, Md.). Buffer solution remaining on the tissue surface was removed by blotting with Micro Wipes (Scott Paper Company, Philadelphia, Pa.). The specimens were then reweighed and homogenized with phosphate-buffered saline, pH 7.4 (1:3 [wt/wt]). Tissue specimens were homogenized four times, for 30 s each, in a Tissumizer (Tekmar, Cincinnati, Ohio) equipped with a 10N head. To avoid tissue heating, homogenization was performed with the sample being placed in an ice bucket. A 100-µl sample of the resulting homogenate was subjected to assay, and the BMS 181184 concentration per gram of tissue was calculated. Standards and quality control samples were similarly prepared by homogenizing normal tissues in phosphate-buffered saline, pH 7.4 (1:3 [wt/wt]), and adding known amounts of the compound. Blank samples of these homogenates, containing no BMS 181184, were also extracted to ensure the absence of interfering peaks.

Analytical method. Levels of BMS 181184 in plasma and tissue homogenates were determined by an accurate, sensitive, reproducible, and specific high-performance liquid chromatographic (HPLC) method developed and fully validated at the Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, N.J. The reference standard of the compound (Bristol-Myers Squibb, Princeton, N.J.) was 94% pure. The method involved precipitation of proteins by addition of 1.2 ml of methanol to each standard, control, or unknown rabbit plasma specimen, or tissue homogenate, followed by centrifugation and removal of the methanolic layer. The methanolic supernatant was then evaporated to dryness at 40°C under a stream of nitrogen, and the sample was reconstituted in 250 µl of the mobile phase (50 mM sterile potassium phosphate buffer-acetonitrile, 80/20 [vol/vol]; Fisher Scientific, Fair Lawn, N.J.) for injection onto the HPLC column. The injection volume was 180 µl, and the flow rate was 1.3 ml/min. BMS 181184 eluted at approximately 3.5 to 4.5 min from a C₁₈ analytical column maintained at 35°C (Ultrasphere; Beckman Instruments, Fullerton, Calif.) and was detected by its UV absorbance at 510 nM. Quantification was based on the peak area-concentration response of the calibration standard. Ten-point standard curves were linear from 0 to 200 µg/ml, with r² values of greater than 0.97. The lower limit of quantification was 0.2 µg/ml. Accuracies were within 15%, and intra- and interday variability (precision) was <10%.

Pharmacokinetic analysis. Pharmacokinetic parameters for BMS 181184 were determined by performing both compartmental and noncompartmental analyses. Concentrations of BMS 181184 were fit to a two-compartment open model, with i.v. bolus input and elimination from the central compartment, by iterative weighted nonlinear least-squares regression with the ADAPT II computer program (5). Weighting was achieved by using the inverse of the square root of the observation variance. Model selection was guided by Akaike's information criterion (24). The model fit the data well, with a mean coefficient of determination (r²) of 0.992 (r² = 0.969 to 1.00), and the regression lines through the plot of observed versus fitted concentrations did not differ from the line of identity. Fitted parameters were total clearance (CL_T), distributional clearance (CL_D), volume of distribution (V), volume of distribution of the central (V₁) and the peripheral (V_p) compartments, distributional half-life (t_1/2), and elimination half-life (t_1/2). Model-independent parameters of maximum (C_max) and minimum (C_min) concentrations were determined directly from concentration-time profiles. The area under the plasma concentration-time curve from 0 to 24 h (AUC_0-24) was determined by the linear trapezoidal rule (8).

Statistical analysis. Differences between the means of pharmacokinetic parameters across the four dosage levels were evaluated by the Kruskal-Wallis nonparametric analysis of variance (ANOVA) test. For comparison of two dosage levels or dosage schedules, Student's t test or Welch's t test was used, as appropriate. Simple linear regression and one-way ANOVA were utilized to assess dose linearity. A two-tailed P value of <0.05 was considered statistically significant.

	RESULTS

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Single-dose studies. The concentration-versus-time curves of BMS 181184 in plasma following single-dose administration are shown in Fig. 1a, and calculated pharmacokinetic parameters for the four dosage levels are listed in Table 1.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Single-dose plasma pharmacokinetics of BMS 181184. (a) Concentration-versus-time curves after administration of 10, 25, 50, and 150 mg of the compound/kg. Each point plots the mean level for three rabbits each at that time. (b) Plot of the AUC0-24/dose ratios for each rabbit and the mean ± the standard error of the mean (SEM) for each dosage group. For a drug that follows linear kinetics, such a plot would have a slope of 0. For BMS 181184, however, the significantly negative slope of the means (P < 0.001; ANOVA) is indicative of the compound's nonlinear kinetics.

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Multiple-dose studies. The concentration-versus-time profiles of BMS 181184 in plasma following once-daily administration of 10, 25, 50, or 150 mg/kg over 15 days are shown in Fig. 2a, and the corresponding pharmacokinetic parameters are listed in Table 2.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Multiple-dose plasma pharmacokinetics of BMS 181184. (a) Concentration-versus-time curves after administration of 10, 25, 50, and 150 mg of the compound/kg over 15 days. Each point plots the mean level for three rabbits each at that time. (b) Plot of the ratios of AUC0-24/dose for each rabbit and the mean ± the standard error of the mean (SEM) for each dosage group. Similar to the single-dose data, the significantly negative slope of the means (P < 0.005; ANOVA) demonstrates the nonlinear kinetics of BMS 181184 after multiple dosing.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   AUC0-24 values at the four investigated dosage levels after single-dose and multiple-dose administration of BMS 181184 (mean values for three rabbits each ± standard errors of the means [SEM]). Note the decrease in the AUC0-24 after multiple once-daily dosing at 150 mg/kg compared to the next-lower dosage level (*, P < 0.01; Student's t test) and compared to the AUC0-24 obtained after one single dose of 150 mg/kg (*, P < 0.05; Student's t test).

Distribution in tissues. Mean levels of BMS 181184 in tissues at near-peak plasma concentrations after multiple dosing over 16 days are shown in Table 3. The highest drug levels were detected in the kidney, where the compound accumulated progressively with increasing dosages up to levels exceeding concurrent plasma concentrations by a factor of 1.6; substantial, apparently AUC-proportional drug disposition occurred in the lung, liver, and spleen, with concentrations well above the MICs for most pathogenic fungi (Fig. 4). Drug levels in brain tissue, CSF, and vitreous fluid were lower than those measured in other tissues but were detectable at concentrations exceeding 1 µg/g or 1 µg/ml.

View larger version (11K): [in this window] [in a new window]   FIG. 4.   Concentrations of BMS 181184 in tissues after multiple once-daily dosing over 16 days (mean values for three rabbits each ± standard errors of the means [SEM]; 50- and 150-mg/kg dosage levels only). Note the apparent accumulation of drug within the kidney (*, P < 0.001 versus 50 mg/kg; Student's t test).

Toxicity. There were no statistically significant differences in the mean plasma creatinine, BUN, bilirubin, and hepatic transaminase levels when values determined after 15 days of treatment were compared to those obtained at baseline. One rabbit at the 150-mg/kg dosage level had elevated plasma creatinine and BUN values at the end of treatment (1.5 and 33 mg/dl, respectively). Throughout administration of the compound, no apparent clinical abnormalities were observed in BMS 181184-treated rabbits and no abnormal weight changes were noted. However, the body fluids and skin of BMS 181184-treated rabbits showed a red discoloration, and, similarly, most of their tissues exhibited red discoloration at autopsy.

	DISCUSSION

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BMS 181184 was the first member of the pradimicin family of antifungal antibiotics to be selected for clinical development. It exemplifies the potent and broad-spectrum activity of this novel class of antifungal compounds against clinically relevant fungi both in vitro and in vivo. Considering also their unique fungicidal mechanism of action and their lack of cross-resistance with amphotericin B and antifungal triazoles, the pradimicins, as a class, clearly warrant further investigation for their use in treatment of invasive fungal infections. The present study was performed in order to characterize the compartmental pharmacokinetics of BMS 181184 and to quantify the concentrations of the compound achievable at clinically relevant peripheral sites.

Independent of the duration of administration, BMS 181184 demonstrated nonlinear, dose-dependent kinetics with significant differences in dose-normalized C_max and AUC_0-24 values across the dosage range assessed in this study. Plasma data fit well to a two-compartment open model, with a rapid initial distribution followed by a slower elimination from the central compartment. Consistent with a nonlinear disposition, there was an increase in clearance and a reciprocal shortening of the drug's t_1/2 with increasing dosage. The V was approximately equivalent to that of total body water but increased in a nonlinear fashion at the highest dosage level.

Multiple once-daily dosing over 15 days did not result in drug accumulation in the plasma. Indeed, there was a significant decrease in the AUC_0-24 at the 150-mg/kg dosage level compared to the corresponding value after administration of one single dose and the AUC obtained after multiple dosing at 50 mg/kg. This coincided with a significant increase in CL_T and a decrease in the t_1/2 but also an expansion of the V.

Bearing in mind the limitations of tissue concentration ratios taken from a single time point of the dosing interval (6), there was substantial uptake of the drug in the lung, liver, and spleen after multiple dosing. Most notably, however, BMS 181184 appeared to accumulate within the kidney in an almost dose-proportional manner, with concentrations six to eight times higher than simultaneously measured concentrations in liver and lung tissues, respectively, and exceeding corresponding plasma drug concentrations by a factor of 1.6 after multiple once-daily dosing at 150 mg/kg.

Although the metabolic pathways of BMS 181184 have not yet been elucidated, renal mechanisms appear to play an important role in the disposition of this pradimicin derivative. On-file data from unpublished studies by the manufacturer, using rats and BMS 181184 dosages of 12, 60, and 300 mg/kg, showed that up to 58% of a single dose could be detected in the urine within the first 24 h after administration. Along with nonlinear increases in the AUC, the CL_T, renal clearance, and urinary recovery as a percentage of the dose, as well as the compound's V, increased with the dose. Values for renal clearance were lower than the estimated value for the glomerular filtration rate in the rat. After multiple once-daily dosing at 300 mg/kg, urinary recovery was greater on day 24 than on day 1, whereas there were no differences at the 10- and 60-mg/kg dose levels (2).

Our findings are well in agreement with those obtained in the rat studies. Taken together, they suggest saturable renal tubular reabsorption of the compound, resulting in enhanced renal excretion and/or accumulation within the kidney with increasing dosage and time. However, the observed two-step increase in CL_T (i.e., an initial change from 10 to 25 and 50 mg/kg and a second major change at the highest dosage level) makes it also likely that BMS 181184 undergoes significant protein binding and that protein binding becomes saturated at higher concentrations and/or prolonged exposure, leading to renal excretion of free drug via glomerular filtration and accumulation in the kidney.

Good penetration into CSF and/or brain tissue is essential for any drug targeted for the treatment of invasive fungal infections. Although the levels of penetration of BMS 181184 into CSF and brain tissue were relatively low, the measured concentrations reflect only a static assessment of a dynamic process, with the possibility of a different equilibrium at later time points and completely different states of tissue inflammation and/or necrosis.

Since the assessment of toxicity was not the primary goal of our study, no tissue from the liver or kidney was sampled for histological examination. Nevertheless, with the exception of a variable red discoloration of body fluids and tissues, BMS 181184 appeared to be well tolerated over a typical treatment duration, with no evidence of clinical or laboratory abnormalities or abnormal weight changes. However, the first phase I multiple-dose study in normal human volunteers revealed increases in hepatic transaminase levels which resulted in the discontinuation of clinical development of the compound. In that study, transient, apparently dose-related increases in ALT (less than or equal to World Health Organization [WHO] grade 2) and, to a lesser extent, AST and gamma-glutamyltranspeptidase were observed. The two dose levels studied were 0.75 and 1.5 mg/kg, each given every 12 h over 7 days. Overall, 9 of 12 subjects developed abnormal ALT values. At the 0.75-mg/kg dose level, four of six subjects exhibited a WHO grade I ALT value (1.25 to 2.5 times the upper normal limit); at the 1.5-mg/kg dose level, five of six subjects had an elevated ALT level, two of them being in the WHO grade 2 range (2.6 to 5 times the upper normal limit) (3). In preclinical toxicity studies performed by the manufacturer, monkeys, mice, and rats did not display evidence of hepatic toxicity. Only when dogs received multiple doses of greater than 60 times (i.e., 90 mg/kg/day) the dosage administered to human volunteers were mild elevations of ALT levels and subacute perivascular inflammation observed (2). Thus, there was no animal model which reliably predicted hepatic toxicity of BMS 181184 in humans.

In summary, BMS 181184, the first derivative of a new and unique class of antifungal antibiotics selected for further development, demonstrated nonlinear plasma pharmacokinetics, with dose- and time-dependent increases in clearance at high dosages and evidence of dose-dependent accumulation within the kidney and substantial penetration into most peripheral sites of clinical relevance. Increased elimination at dosages potentially required for therapeutic efficacy might be overcome by dividing the administration of the drug. High-level efficacy in vivo and suitable pharmacokinetic properties would favor the further development of congeners of this promising class of compounds.