Identification of [C]Fluasterone Metabolites in Urine and Feces Collected from Dogs after Subcutaneous and Oral Administration of [C]Fluasterone

The objective of this research was the identification of the metabolic profile of fluasterone, a synthetic derivative of dehydroepiandrosterone, in dogs treated orally or subcutaneously with [4-C]fluasterone. Separation and characterization techniques used to identify the principal metabolites of fluasterone in urine and feces included high-performance liquid chromatography (HPLC), liquid scintillation spectrometry, HPLC/tandem mass spectrometry, and NMR. In urine, the majority of the radioactivity was present as two components that had apparent molecular weights consistent with their tentative identification as monoglucuronide conjugates of 4 -hydroxy-16 -fluoro-5-androsten17 -ol and X( or )-4 -dihydroxy-16 -fluoro-5-androsten-17 -ol. The identification of the monoglucuronide conjugate of 4 -hydroxy-16 -fluoro-5-androsten-17 -ol was also supported by NMR data. In support of this identification, these metabolites were cleaved with glucuronidase enzyme treatment, which gave rise to components with molecular weights again consistent with the aglycones of a monohydroxylated, 17-keto reduced (dihydroxy) fluasterone metabolite and a dihydroxylated, 17-keto reduced (trihydroxy) fluasterone metabolite. In feces, nonconjugated material predominated. The primary metabolites eliminated in feces were the two hydroxy fluasterone metabolites arising from 17-reduction (16 -fluoro-5-androsten-17 -ol and 16 -fluoro-5-androsten-17 -ol) and 4 -hydroxy-16 -fluoro-5-androsten-17 -ol that was present in urine in glucuronide form. Fluasterone (Fig. 1) is a synthetic derivative of dehydroepiandrosterone (DHEA), an important intermediate in both testosterone and estrogen biosynthesis. In addition to its role as an intermediate in steroid hormone synthesis, DHEA and its sulfate conjugate (DHEAS) have been shown to have numerous direct physiological activities, including immunomodulatory and antiglucocorticoid effects, and are believed to be important for the development and function of the central nervous system. In humans, DHEA is synthesized by the adrenal cortex, gonads, brain, and gastrointestinal tract. At certain times, DHEA and DHEAS constitute the most abundant steroid hormones in the circulation. Levels of DHEAS decrease rapidly after birth, increase to a peak at approximately 20 to 30 years of age, and then decrease again gradually over time (Rainey et al., 2002). Because of this, supplemental intake of DHEA and DHEAS is popular as an “aging remedy.” In addition, DHEA and DHEAS are often used by athletes to improve performance and are also said to increase longevity and improve mood, cognition, and sexuality (Allolio and Arlt, 2002). Most interestingly, DHEA is a an inhibitor of cancer induction in a wide range of in vivo experimental models for human cancer, including rat mammary gland (Li et al., 1994; McCormick et al., 1996), mouse mammary gland (Schwartz, 1979), mouse skin (Pashko et al., 1984), mouse colon (Nyce et al., 1984; Osawa et al., 2002), mouse lung (Schwartz and Tannen, 1981), mouse lymphatic system This work was supported by the National Institutes of Health Division of Cancer Prevention and Control [Contract N01-CN-43306]. Parts of this work were previously presented as a poster as follows: Zhan Q, Hill JM, Chao A, Green JS, Luybimov A, Kapetanovic IM, and Thomas BF (2007) Characterization of the metabolites of [C]fluasterone excreted in dog urine and feces using LC/MS/MS. American Association of Pharmaceutical Scientists 2007 Annual Meeting; 2007 Nov 10–16; San Diego, CA. American Association of Pharmaceutical Scientists, Arlington, VA. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.023614. ABBREVIATIONS: DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; LC/MS/MS, liquid chromatography/tandem mass spectrometry; LC/MS, liquid chromatography/mass spectrometry; 17 -OH fluasterone, 16 -fluoro-5-androsten-17 -ol; 17 -OH fluasterone, 16 -fluoro-5-androsten-17 -ol; HPLC/UV/LSS, high-performance liquid chromatography/UV/liquid scintillation spectrometry; LSS, liquid scintillation spectrometry; HPLC, high-performance liquid chromatography; HPLC/LSS, high-performance liquid chromatography/liquid scintillation spectrometry; HPLC/MS/MS, high-performance liquid chromatography/tandem mass spectrometry; 4 -17 -diOH fluasterone, 4 -hydroxy-16 fluoro-5-androsten-17 -ol; X( or )-4 -17 -triOH fluasterone, X( or )-4 -dihydroxy-16 -fluoro-5-androsten-17 -ol; APCI, atmospheric pressure chemical ionization; G6PDH, glucose-6-phosphate dehydrogenase. 0090-9556/09/3705-1089–1097 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 5 U.S. Government work not protected by U.S. copyright 23614/3457526 DMD 37:1089–1097, 2009 Printed in U.S.A. 1089 at A PE T Jornals on N ovem er 6, 2017 dm d.aspurnals.org D ow nladed from (Perkins et al., 1997), rat liver (Moore et al., 1986), rat thyroid (Moore et al., 1986), and rat prostate (McCormick et al., 2007). Depending on the hormonal milieu, DHEA has either estrogen-like or androgen-like effects (Ebeling and Koivisto, 1994). For example, in premenopausal women DHEA is either an estrogen antagonist, possibly through the competitive binding of its metabolite 5-androstene3 , 17-diol, and estradiol to the estrogen receptor, or an androgen through its metabolism to androstenedione and testosterone. These estrogenic and androgenic properties can produce some significant adverse effects. Indeed, it has been shown that postmenopausal women with elevated serum androgens (including high DHEAS concentrations) are at an increased risk of breast cancer (Dorgan et al., 1996, 1997; Hankinson et al., 1998). In men with normal testosterone levels, DHEA produces predominantly estrogenic effects. Thus, treatment of men with DHEA can lead to gynecomastia and other unwanted side effects. These sex hormone-related side effects limit the therapeutic utility of DHEA in humans. The adverse effects seen with DHEA prompted the development of fluasterone, which in certain experimental paradigms seems to be devoid of the estrogenic and androgenic activity but retains many of DHEA’s therapeutic properties (Schwartz and Pashko, 1995). Fluasterone is currently of sufficient interest that the National Cancer Institute has sponsored its preclinical development. Biotransformation pathways for DHEA and steroid-based therapeutics can be extensive, involving both Phase I and Phase II processes, and can produce additional compounds that may retain the pharmacological or toxicological properties of the parent or possess new and unanticipated biological activities. Because the metabolism and disposition of fluasterone have not been previously thoroughly investigated, this investigation, of importance for any pharmaceutical, is particularly important for this close structural analog of such an important endogenous compound as DHEA. Indeed, the Food and Drug Administration mandates that such studies be performed and that reliable and accurate qualitative and quantitative methods for drugs and their metabolites in biological fluids be used to adequately assess the pharmacokinetic and pharmacodynamic processes of candidate therapeutics. Therefore, the main objectives of these studies were to 1) develop methods that allow for the separation of fluasterone and its metabolites in a variety of biological matrices; 2) obtain radiochromatographic profiles of biological fluids and matrices from dogs dosed with [C]fluasterone; 3) acquire spectrometric information on isolated metabolites using liquid chromatography/(tandem) mass spectrometry (LC/MS/MS or LC/MS, respectively) and NMR spectroscopy; and 4) elucidate the structure of urinary and fecal metabolites of [C]fluasterone in dogs. The metabolism and elimination profiles determined in dogs receiving [C]fluasterone orally and subcutaneously are reported in this article. The pharmacokinetic and tissue distribution profiles of [C]fluasterone are to be presented elsewhere. Materials and Methods Chemicals. Radiolabeled [4-C]fluasterone (lot 9676-29-07 from Midwest Research Institute, Kansas, MO) had a radiochemical purity of approximately 97% and specific activity of 58.3 mCi/mmol. Fluasterone (lot 99973-1/91) was supplied by Proquina (Orizaba, Mexico). Standards of 16 -fluoro-5-androsten17 -ol (17 -OH fluasterone) and 16 -fluoro-5-androsten-17 -ol (17 -OH fluasterone) were supplied by Dr. Marvin L. Lewbart (Department of Medicine, Jefferson Medical College, Philadelphia, PA). Sulfatase-free -glucuronidase, bacterial from Escherichia coli, was supplied with phosphate buffer Sigma G8396 (Sigma-Aldrich, St. Louis, MO). -Glucuronidase-free sulfatase, type VI, from Aerobacter aerogenes was supplied with 0.01 M Tris, pH 7.5, Sigma S1629 (Sigma-Aldrich). Soluene-350 tissue solubilizer and Ultima Gold scintillation mixture were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Dosing and Collection of Biological Samples. Groups of male beagle dogs received either a single subcutaneous administration of [C]fluasterone in 30% hydroxypropyl-cyclodextrin at 1 mg/kg (n 4) or a single oral administration of [C]fluasterone in corn oil at 15 mg/kg (n 4) in a study performed by the Toxicology Research Laboratory at the University of Illinois at Chicago. Dosage formulations were analyzed by high-performance liquid chromatography/UV/liquid scintillation spectrometry (HPLC/UV/LSS) and proved to be within 10% of target before dosing. Blood samples were collected in lithium heparin tubes 0.5 and 2 h after subcutaneous administration and 1 and 4 h after oral administration of [C]fluasterone. Blood was centrifuged at 3700 rpm for 15 min to separate red blood cells and plasma. Urine was collected into containers placed on dry ice through 48 h after dosing. Feces collected for metabolic profiling were removed from the cages at intervals of 0 to 8, 8 to 24, and 24 to 48 h postdosing. All of the collected samples were stored at approximately 80°C. The collected urine and feces and aliquots of plasma and red blood cells were shipped frozen by the Toxicology Research Laboratory at the University of Illinois at Chicago and stored at RTI International (Research Triangle Park, NC) at approximately 80°C until analyzed. Determination of Radioactivity in Samples. Weighed aliquots of thawed urine and plasma were added directly to vials containing scintillation mixture (Ultima Gold). Each feces collection was thawed and homogenized with an approximately equal weight of deionized, distilled water. Weighed aliquots of feces homogenates and red blood cells (0.1–0.3 g) were digested in Soluene350 (2 ml) and then neutralized and bleached by the addition of approximately 125 l of 70% HClO4 followed by the addition of approximately 0.3 ml of 30% H2O2. After the samples were decolorized, scintillation mixture was added to the samples. Samples prepared as described above were analyzed for radioactivity content by liquid scintillation spectrometry (LSS) using a Packard Tri-Carb 2100TR Liquid Scintillation Spectrometer (PerkinElmer Life and Analytical Sciences). Eluent fractions (1 ml) from high-performance liquid chromatography (HPLC) analyses were collected directly into vials containing scintillation mixture and also analyzed by LSS. Treatment of Urine and Plasma with Glucuronidase or Sulfatase Enzymes. Varying amounts of glucuronidase enzyme were reconstituted in urine and heated at 37°C for approximately 22 h. Likewise, aliquots of urine and plasma were treated with varying amounts of sulfatase and allowed to hydrolyze at 37°C for up to approximately 94 h. Analysis by HPLC. Analysis by HPLC was accomplished using a Phenomenex (Torrance, CA) Luna 5m C18(2) column (150 4.6 mm) and two gradient systems, each varying the amount of acetonitrile and water in the mobile phase over the time course of linear gradient changes at a flow rate of 1.5 ml/min. For HPLC Method 1, the initial proportions of acetonitrile/water, 5:95 (v/v), were maintained for 5 min after sample injection; changed over 5 min to 10:90 and held constant for 6 min; changed over 6 min to 40:60 and held for 8 min; then changed over 5 min to 70:30 and held for 5 min; and finally changed over 2 min to 95:5 and held for 8 min. For HPLC Method 2, used to provide increased chromatographic resolution for the separation of fluasterone, 17 -OH fluasterone, and 17 -OH fluasterone, the initial proportions of acetonitrile/water, 70:30 (v/v), were maintained for 15 min after sample injection, changed to 100% acetonitrile over 2 min, and held for 7 min. Metabolite Profiles. Aliquots of untreated urine and feces (after extraction with methanol) from all the collection samples that afforded analysis were FIG. 1. The structure of fluasterone with atom numbering. 1090 BURGESS ET AL. at A PE T Jornals on N ovem er 6, 2017 dm d.aspurnals.org D ow nladed from profiled for fluasterone and its metabolites by high-performance liquid chromatography/liquid scintillation spectrometry (HPLC/LSS) (eluent collected at 1-min intervals) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) using an on-line radioactivity detector ( -RAM; IN/US Systems, Tampa, FL) using HPLC Methods 1 and 2. In addition, enzyme-treated aliquots of selected urine collections were profiled using the same procedures as those for untreated urine. Methanol extracts of plasma were also profiled by HPLC/LSS. Unless otherwise stated, results reported below are from analysis using HPLC Method 1. For preliminary HPLC/MS/MS analyses, the mass spectrometer was operated in positive ionization mode using the atmospheric chemical ionization probe in series after the UV and -RAM radioactivity detector (IN/US Systems). The time difference between a peak detected at each detector was approximately 0.15 min. All the data were recorded using Analyst software (MDS Sciex, Concord, ON, Canada). When glucuronides were suspected, a TurboIon Spray ion source (Applied Biosystems, Foster City, CA) was operated in negative ion mode with the following operating conditions: curtain gas 40, nebulizer gas 75, vaporizer temperature 600°C, needle voltage 4500 V, collisionally activated dissociation 4, and collision energy 30 V. Information-dependent analysis was used during the data acquisition. An MS survey scan was conducted, followed by an information-dependent analysis experiment to select the two most intense ions for MS/MS analysis. The fragmentation patterns of these ions were collected in the MS/MS experiments and analyzed for qualitative identification using the Metabolite ID software (Thermo Fisher Scientific Inc., Waltham, MA). Urinary metabolites were isolated by combining and concentrating fractions collected from multiple HPLC injections of dog urine. Proton NMR spectra of standards and the isolated urinary metabolites were obtained on a Bruker (Billerica, MA) AVANCE 300 NMR spectrometer in d6-dimethyl sulfoxide at ambient temperature using a 5-mm QNP probe. Proton spectra, proton-proton correlation spectra, and proton-carbon correlation spectra of isolated urinary metabolites in D2O were obtained on a Varian, Inc. (Palo Alto, CA) INOVA 500 NMR spectrometer at ambient temperature using a 5-mm broadband probe. Chemical shift calculations were performed using ACD Proton Predictor version 10.02 (Advanced Chemistry Development, Inc., Toronto, ON,