The participation of rat hepatic P-450 in the conversion of 17 beta-estradiol to catechol estrogens was examined by means of enzyme reconstitution and immunoinhibition studies. It was thus demonstrated that three rat liver microsomal cytochrome P-450 forms, designated P-450UT-A, P-450PCN-E, and P-450ISF-G, each contribute to the 2- and 4-hydroxylation of 17 beta-estradiol catalyzed by hepatic microsomal preparations. Two of these enzymes, P-450UT-A and P-450PCN-E, are expressed constitutively, are male-specific, and are regulated by testosterone as well as influenced by the administration of various chemicals. Consistent with these observations, 17 beta-estradiol 2- and 4-hydroxylation activities both increased rapidly during puberty in male rats and were induced by treatment of rats with phenobarbital or pregnenolone 16 alpha-carbonitrile. Castration of male rats at birth or at 5 weeks of age suppressed the levels of 17 beta-estradiol 2- and 4-hydroxylase activities measured at 10 weeks of age. This suppression of activity was reversed upon administration of testosterone during the neonatal period (days 1 and 3 of life) or by capsule implantation at 5 weeks of age. These patterns of 17 beta-estradiol 2- and 4-hydroxylation are discussed in terms of the previously characterized response of the multiple rat hepatic P-450 forms to ontogenic, hormonal, and xenobiotic factors.
Cytochrome P450 17A1 (CYP17A1; also P450c17and P450sccII) is a critically important enzyme in humans that catalyzes the formation of all endogenous androgens. It is an atypical cytochrome P450 enzyme in that it catalyzes two distinct types of substrate oxidation. Through its hydroxylase activity, it catalyzes the 17α-hydroxylation of pregnenolone to 17α-OH pregnenolone. Subsequently, through its C17,20lyase activity, it can further convert 17α-OH pregnenolone to the androgen dehydroepiandrosterone, which is a precursor to androstenedione, testosterone, and dihydrotestosterone. The importance of androgens in diseases such as prostate cancer has been appreciated for decades and the discovery of extra-testicular formation of androgens has helped clarify the pathology of the disease, especially the castrate- resistant disease. Therefore, specific inhibition of CYP17A1 by therapeutic intervention has been an area of considerable effort in several research laboratories. This basic research has led to the discovery of several promising drug candidates followed by the conduct of several clinical trials. Recently, all these efforts have culminated in the first approval by FDA of an inhibitor of CYP17A1 for the treatment of castrate-resistant prostate cancer. Ongoing clinical trials are now evaluating the agent in earlier stages of prostate cancer and even rare forms of androgen-dependent breast cancer. Accordingly, this review focuses on the biochemistry, chemistry, and clinical inhibitors of CYP17A1.
The thiol-containing androgens 17 beta-hydroxy-10 beta-mercaptoestr-4-en-3-one and 19-mercaptoandrost-4-ene-3,17-dione were synthesized and tested in human placental microsomes for their ability to suicide inhibit aromatase. Both compounds showed time-dependent, pseudo-first-order rates of inactivation of aromatase with Ki's of 106 and 34 nM and kcat's of 3.2 X 10(-3) and 1.2 X 10(-3) s-1 respectively for 1 and 2 at 30 degrees C. Diffusion dialysis failed to reactivate aromatase previously inactivated by either compound, and both compounds required that NADPH and O2 be present for the time-dependent inactivation of the enzyme. The presence of the substrate, androst-4-ene-3,17-dione (5.0 microM), protected the enzyme from inactivation while cysteine (1.0 mM) failed to protect aromatase from inactivation by either compound. The above evidence demonstrates that both compounds are potent suicide inhibitors of aromatase.
An automated gas chromatographic/mass spectrometric assay is described for the antiepileptic drug valproic acid (VPA) and 14 of its metabolites in plasma or urine. Quantitative analysis of the parent drug and its biotransformation products was carried out with the aid of trimethylsilyl derivatives, and was performed by selected ion monitoring gas chromatography/mass spectrometry (normally of [M-CH3]+ species) using an HP 5790 mass selective detector (MSD) quadrupole mass spectrometer. The analysis was fully automated, in that simple injection, data acquisition, integration, quantification and report functions were carried out during unattended operation by an HP 59970C ChemStation computer system. The method exhibits good accuracy and high precision, with correlation coefficients greater than 0.990 for all standard curves. Replicate analyses of pooled plasma samples over a 4 month period exhibited an inter-day variation of less than 15% for the parent drug and ten of its metabolites. Moreover, the high dynamic range of the MSD instrument permitted quantification of VPA and minor metabolites thereof (e.g. the hepatotoxic terminal olefin, delta 4-VPA) at levels as disparate as 260 micrograms ml-1 (VPA) and 14 ng ml-1 (delta 4-VPA) in a single analysis. The high stability and sensitivity of the assay, combined with the fully automated features of the instrumentation, make the method ideally suited to expanded clinical studies and for the routine monitoring of potentially high-risk patients on VPA therapy.
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