In vitro and in vivo studies to characterize the clearance mechanism and potential cytochrome P450 interactions of anidulafungin

Abstract

Anidulafungin is a novel semisynthetic echinocandin with potent activity against Candida (including azole-resistant isolates) and Aspergillus spp. and is used for serious systemic fungal infections. The purpose of these studies was to characterize the clearance mechanism and potential for drug interactions of anidulafungin. Experiments included in vitro degradation of anidulafungin in buffer and human plasma, a bioassay for antifungal activity, in vitro human cytochrome P450 inhibition studies, in vitro incubation with rat and human hepatocytes, and mass balance studies in rats and humans. Clearance of anidulafungin appeared to be primarily due to slow chemical degradation, with no evidence of hepatic-mediated metabolism (phase 1 or 2). Under physiological conditions, further degradation of the primary degradant appears to take place. The primary degradation product does not retain antifungal activity. Anidulafungin was not an inhibitor of cytochrome P450 enzymes commonly involved in drug metabolism. Mass balance studies showed that anidulafungin was eliminated in the feces predominantly as degradation products, with only a small fraction (10%) eliminated as unchanged drug; fecal elimination likely occurred via biliary excretion. Only negligible renal involvement in the drug's elimination was observed. In conclusion, the primary biotransformation of anidulafungin is mediated by slow chemical degradation, with no evidence for hepatic enzymatic metabolism or renal elimination.

Figures

FIG. 1.

Chemical structure of anidulafungin. *, position of the 14C radiolabel.

FIG. 2.

Changes in HPLC-MS/MS peak areas for anidulafungin and its primary degradation product over time in pH 8.0 phosphate-buffered saline at room temperature. Each data point is the mean of three determinations. Linear regression lines are shown for each curve.

FIG. 3.

In vitro concentration-time profiles of anidulafungin and its primary degradation product following incubation of 8 μg/ml anidulafungin in pH 7.4 phosphate-buffered saline (A) and human plasma (B), both at 37°C. Each data point is the mean of three determinations. The concentrations indicated for the degradation product were estimated from chromatographic peak areas, relative to anidulafungin peak areas, by applying a mass spectrometer sensitivity factor of 4 derived from the results presented in Fig. 2.

FIG. 4.

Lack of hepatic metabolism of anidulafungin in rat and human hepatocytes as shown by the percentage of 10 μM anidulafungin remaining over time. Values are the means of three determinations. Error bars have been omitted for clarity, but the coefficient of variation ranged from 1.7 to 3.1% for rat hepatocytes and 1.3 to 9.4% for human hepatocytes.

FIG. 5.

Loss of anidulafungin activity over time, as determined by bioassay, following incubation of 8 μg/ml of anidulafungin in phosphate-buffered saline, pH 7.4, at 37°C. Each point represents a single determination. The percentage of biological activity at each time point was expressed as 100 times the concentration at that time point divided by the starting concentration (8.7 μg/ml).

FIG. 6.

Concentration-time profile for drug-derived radioactivity in plasma and blood following i.v. administration of [14C]anidulafungin (5 mg/kg) to male Sprague-Dawley rats (n = 3 per time point). Standard deviation bars have been omitted from the blood values for clarity. The coefficient of variation ranged from 4 to 17% for plasma and from 1 to 8% for blood.

FIG. 7.

Mean (and standard deviation) plasma concentration-time profiles of anidulafungin (i.e., parent drug) and drug-derived radioactivity following a single i.v. infusion of [14C]anidulafungin to adult healthy male subjects in a phase 1 clinical study (n = 9). Standard deviation bars have been omitted for clarity. The coefficient of variation ranged from 11 to 32% for radioactivity and from 14 to 39% for anidulafungin.