Short Communication Metabolites of Caspofungin Acetate, a Potent Antifungal Agent, in Human Plasma and Urine

Caspofungin acetate (MK-0991) is a semisynthetic pneumocandin derivative being developed as a parenteral antifungal agent with broad-spectrum activity against systemic infections such as those caused by Candida and Aspergillus species. Following a 1-h i.v. infusion of 70 mg of [H]MK-0991 to healthy subjects, excretion of drug-related material was very slow, such that 41 and 35% of the dosed radioactivity was recovered in urine and feces, respectively, over 27 days. Plasma and urine samples collected around 24 h postdose contained predominantly unchanged MK-0991, together with trace amounts of a peptide hydrolysis product, M0, a linear peptide. However, at later sampling times, M0 proved to be the major circulating component, whereas corresponding urine specimens contained mainly the hydrolytic metabolites M1 and M2, together with M0 and unchanged MK-0991, whose cumulative urinary excretion over the first 16 days postdose represented 13, 71, 1, and 9%, respectively, of the urinary radioactivity. The major metabolite, M2, was highly polar and extremely unstable under acidic conditions when it was converted to a less polar product identified as N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine g-lactone. Derivatization of M2 in aqueous media led to its identification as the corresponding g-hydroxy acid, N-acetyl-4(S)hydroxy-4-(4-hydroxyphenyl)-L-threonine. Metabolite M1, which was extremely polar, eluting from HPLC column just after the void volume, was identified by chemical derivatization as des-acetylM2. Thus, the major urinary and plasma metabolites of MK-0991 resulted from peptide hydrolysis and/or N-acetylation. Systemic fungal infection often can be lethal in vulnerable patient populations, such as those immunocompromised with AIDS, or receiving cancer chemotherapy or immunosuppressive therapy following organ transplants. For such patients, new therapies are needed because of the ineffectiveness, resistance to, or toxicity of existing treatments (Horsburgh and Kirkpatrick, 1983; Rex et al., 1994, 1995). Newer echinocandins, such as LY303366 (Petraitis et al., 1998) and pneumocandins, e.g., caspofungin (MK-0991; Fig. 1), exhibit excellent activity against yeast and Pneumocystis carinii infection in animals and show potential as clinical antifungal candidates. Caspofungin (Adefarati et al., 1992; Graybill et al., 1997a,b; Powles et al., 1998) is a semisynthetic, water soluble, broad spectrum lipopeptide with potent fungicidal activity, e.g., against serious Candida and Aspergillus infections. MK-0991 is a complex, cyclic hexapeptide with 16 asymmetric centers, a molecular weight of 1093 Da and low lipophilicity (log P 21.2) (Qin et al., 1999). MK-0991 is a potent inhibitor of (1,3)-b-D-glucan synthesis, an essential cell wall homopolysaccharide found in many pathogenic fungi (Geogopapadakou and Tkacz, 1995). As a result of this functional selectivity toward the fungal cell wall enzyme, the side effects due to MK-0991 in humans are expected to be low. In addition, MK-0991 has shown synergistic effects in the in vitro activities of amphotericin B and fluconazole against Cryptococcus neoformans, an infection often incurable in patients with AIDS (Franzot and Casadevall, 1997). This report describes the metabolism of MK-0991 in humans after i.v. administration to healthy volunteers. Experimental Procedures Materials. Specimens of plasma and urine were obtained from healthy adult volunteers given a single 70-mg [H]MK-0991 i.v. dose (1-h infusion) (specific activity 3.0 mCi/mg), with sample collection over 27 days. Urine samples were treated with albumin to a final concentration of approximately 0.5% to prevent binding of drug to containers. An authentic sample of metabolite M0 was prepared at Merck Research Laboratories, and its structure confirmed by MS and NMR analysis. All other chemicals were of analytical grade and solvents of HPLC grade. Samples were stored at 270°C until analysis. Radioanalysis. Aliquots of HPLC fractions from plasma and urine samples were mixed with liquid scintillation cocktail and analyzed by scintillation spectrophotometry. Samples of individual or pooled (across time or subjects) plasma from humans (24 and 30 h; days 5/6, 11/12, and 19/20 pools) were deproteinized by the addition of a mixture of acetonitrile (ACN) and methanol containing 0.1% trifluoroacetic acid (TFA), followed by vortex mixing and centrifugation. The resulting supernatants then were evaporated on a SpeedVac system, and the residues reconstituted in 0.1% TFA-H2O. Aliquots of these samples were analyzed by HPLC using a DuPont Zorbax RX C8 column (4.6 3 250 mm, 5 mm) eluted isocratically at 1 ml/min with 5 mM pentanesulfonate (S5) in 0.5% acetic acid-water for 10 min, followed by a 30-min linear gradient to 80% ACN. The effluent was mixed with Packard Ultima Flow scintillation cocktail flowing at 1.4 ml/min before being analyzed by a Packard 500TR radiochemical detector (system A). A second system used 1 Abbreviations used are: MK-0991: L-743872, Caspofungin acetate, CANCIDAS, 1-[(4R,5S)-5-[(2-aminoethyl)amino)-N-(10,12-dimethyl-1-oxotetradecyl)-4hydroxy-L-ornithine]-5-[(3R)-3-hydroxy-L-ornithine] pneumocandin B0 diacetate (salt), CAS registry no. 179463-17-3; MS, mass spectrometry; LC, liquid chromatography; ACN, acetonitrile; TFA, trifluoroacetic acid. Send reprint requests to: S. K. Balani, DuPont Pharmaceuticals Co., StineHaskell Research Center, Bldg. 110, 1090 Elkton Rd., Newark, DE. E-mail: [email protected] 0090-9556/00/2811-1274–1278$03.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 11 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 76/859391 DMD 28:1274–1278, 2000 Printed in U.S.A. 1274 at A PE T Jornals on July 6, 2017 dm d.aspurnals.org D ow nladed from