A series of 17 alpha-acetylenic steroids was examined with regard to ability to inactivate human liver microsomal cytochrome P-450 (P-450) IIA4, an enzyme involved in the oxidation of a number of drugs, carcinogens, and steroids, including estrogens and progestogens. Of the eight compounds tested, gestodene was found to be particularly active as a mechanism-based inactivator of P-450 IIIA4. Inhibition of both microsomal nifedipine oxidation and 17 alpha-ethynylestradiol (EE) 2-hydroxylation was dependent upon NADPH and gestodene concentration. Rates of inactivation were pseudo first order-values of kinactivation = 0.4 min-1 and Ki = 46 microM and a partition ratio of 9 were calculated. The kinactivation is approximately 50-fold greater than estimated for EE and is one of the highest reported for P-450 mechanism-based inactivators. Spectrally detectable P-450 was also destroyed in microsomes, but several experiments indicate that little covalent binding to amino acid residues of P-450 IIIA4 occurs. Microsomal inactivation of P-450 could be blocked by the presence of other P-450 IIIA4 substrates, and several activities catalyzed by other P-450s were not inhibited under conditions in which greater than 90% of P-450 IIIA4 was inactivated. Consideration of structure/activity relationships among the 17 alpha-acetylenic steroids examined indicates that the delta 15 double bond is critical but is not in itself sufficient for the inactivation process, which is postulated to result from attack of P-450 on the substituted acetylenic carbon and lead to porphyrin N-alkylation. The effectiveness of this mechanism-based inactivator may account for reports of increased estrogen and steroid levels in some women using gestodene in oral contraceptives.
PIP: A toxicologist at Vanderbilt University in Nashville, Tennessee conducted various in vitro studies of several 17 alpha-acetylenic steroids to determine their ability to inactivate human liver microsomal cytochrome P-450 (P-450) IIIA4. (This enzyme oxidizes several drugs, carcinogens, and estrogens and progestogens.) These 17 alpha-acetylenic steroids included ethinyl estradiol (EE), desogestrel, gestodene, 3 varieties of norethisterone, levonorgestrel, and 3-ketodesogestrel. Gestodene was the most active mechanism based inactivator of P-450 IIIA4. Further the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) and gestodene concentration contributed significantly to inhibition of both microsomal nifedipine oxidation and 17 alpha ethinyl estradiol 2-hydroxylation. The k inactivation (0.4 min 1 tenth) was about 50 times greater than calculated for EE and one of the highest reported for P-450 mechanism based inactivators. Even though the researcher did not find spectrally detectable P-450 in microsomes because they had been destroyed, several experiments found that little covalent binding to amino acid residues of P-450 IIIA4 indeed occurs. Further he learned that the existence of other P-450 IIIA4 substrates could block microsomal inactivation of P-450. Moreover various activities catalyzed by other P-450s were not inhibited when 90% of P-450 IIIA4 had been inactivated. Even though the research found the delta 15 double bond critical, it was not sufficient for the inactivation process. He theorized that the inactivation process begins when P-450 attacks the substituted acetylenic carbon which results in porphyrin N-alkylation. The effectiveness of this mechanism based inactivator may explain increased estrogen and steroid levels in various women using oral contraceptives with gestodene.