Identification of CYP3A4 as the principal enzyme catalyzing mifepristone (RU 486) oxidation in human liver microsomes.

Various complementary approaches were used to elucidate the major cytochrome P450 (CYP) enzyme responsible for mifepristone (RU 486) demethylation and hydroxylation in human liver microsomes: chemical and immunoinhibition of specific CYPs; correlation analyses between initial rates of mifepristone metabolism and relative immunodetectable CYP levels and rates of CYP marker substrate metabolism; and evaluation of metabolism by cDNA-expressed CYP3A4. Human liver microsomes catalyzed the demethylation of mifepristone with mean (+/-SD) apparent K(m) and Vmax values of 10.6 +/- 3.8 microM and 4920 +/- 1340 pmol/min/mg protein, respectively; the corresponding values for hydroxylation of the compound were 9.9 +/- 3.5 microM and 610 +/- 260 pmol/min/mg protein. Progesterone and midazolam (CYP3A4 substrates) inhibited metabolite formation by up to 77%. The CYP3A inhibitors gestodene, triacetyloleandomycin, and 17 alpha-ethynylestradiol inhibited mifepristone demethylation and hydroxylation by 70-80%; antibodies to CYP3A4 inhibited these reactions by approximately 82 and 65%, respectively. In a bank of human liver microsomes from 14 donors, rates of mifepristone metabolism correlated significantly with relative immunodetectable CYP3A levels, rates of midazolam 1'-and 4-hydroxylation and rates of erythromycin N-demethylation, marker CYP3A catalytic activities (all r2 > or = 0.85 and P < 0.001). No significant correlations were observed for analyses with relative immunoreactive levels or marker catalytic activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. Recombinant CYP3A4 catalyzed mifepristone demethylation and hydroxylation with apparent K(m) values 7.4 and 4.1 microM, respectively. Collectively, these data clearly support CYP3A4 as the enzyme primarily responsible for mifepristone demethylation and hydroxylation in human liver microsomes.

Antiprogestin pharmacodynamics, pharmacokinetics, and metabolism: implications for their long-term use.

Antiprogestins represent a relatively new and promising class of therapeutic agents that could have significant impact on human health and reproduction. In the present work, the pharmacodynamics, pharmacokinetics, and metabolism of mifepristone (MIF), lilopristone (LIL), and onapristone (ONA) in humans are reviewed, and characteristics bearing important clinical implications are discussed. Although MIF has gained notoriety as an "abortion pill," antiprogestins may more importantly prove effective in the treatment of endometriosis, uterine leiomyoma, meningioma, cancers of the breast and prostate, and as contraceptive agents. MIF pharmacokinetics display nonlinearities associated with saturable plasma protein (alpha 1-acid glycoprotein, AAG) binding and characterized by lack of dose dependency for parent drug plasma concentrations (for doses greater than 100 mg) and a zero-order phase of elimination. LIL and ONA pharmacokinetics are less well characterized but ONA does not appear to bind AAG and displays a much shorter t1/2 than the other agents. The three antiprogestins are substrates of cytochrome P450 (CYP) 3A4, an enzyme exceedingly important in human xenobiotic metabolism. Even more implicative of likely drug-drug interactions subsequent to their long-term administration are recent data from our laboratory indicating that they inactivate CYP3A4 in a cofactor- and time-dependent manner, suggesting that complexation and induction of the enzyme may occur in vivo via protein stabilization. Moreover, it has been demonstrated that MIF increases CYP3A4 mRNA levels in human hepatocytes in primary culture, indicative of message stabilization and/or transcriptional activation of CYP3A4 expression. Finally, MIF has also been shown to inhibit P-glycoprotein function. Whether LIL and ONA share these latter two characteristics with MIF has not yet been determined but they illustrate properties that, in addition to diminished antiglucocorticoid activities and altered pharmacokinetic characteristics, warrant consideration during the development of these and never antiprogestational agents.

Antiprogestin-mediated inactivation of cytochrome P450 3A4.

Based on previous observations of very short periods of linearity for antiprogestin metabolite formation and the presence of a common tertiary amine moiety in each compound as the principal site of their metabolism, we hypothesized that mifepristone, lilopristone and onapristone are oxidized by cytochrome P450 (CYP) 3A4 to reactive nitroso species that complex the heme of the enzyme, thereby inactivating it. Upon preincubation with human liver microsomes in the presence (but not the absence) of NADPH, mifepristone inhibited midazolam 1'-hydroxylation, a marker of CYP3A4 catalytic activity, very potently (IC50 approximately 3.5 mumol/l) and extensively (by approximately 87%). Lilopristone and onapristone also displayed NADPH and time-dependent inactivation of CYP3A4 with characteristics very similar to mifepristone. These data support antiprogestin-mediated inactivation of CYP3A4 and suggest the potential for drug-drug interactions and time-dependent nonlinearities in pharmacokinetics upon their long-term administration.

Cytochrome P4503A4-mediated N-demethylation of the antiprogestins lilopristone and onapristone.

The metabolism of two newer antiprogestational agents, lilopristone and onapristone, was investigated using human liver microsomes, and evidence was obtained supporting a principal role of cytochrome P450 (CYP) 3A4 in their N-demethylations. Kinetic studies with microsomes from three organ donors indicated lack of biphasic kinetics at substrate concentrations up to 200 microM, consistent with a single enzyme mediating the oxidations. Selective chemical inhibitors of CYP1A2 (furafylline), CYP2C9 (sulfaphenazole), CYP2D6 (quinidine), and CYP2A6/2E1 (diethyldithiocarbamic acid) did not affect initial rates of metabolism of either steroid. Gestodene and triacetyloleandomycin (selective for CYP3A enzymes) inhibited the demethylations of both antiprogestins by up to 77%. Rabbit polyclonal antibodies to CYP3A4 decreased initial rates of N-demethylation of the antihormones by up to 82%, whereas antibodies to CYP2C9 were not inhibitory. Collectively, these data thus suggest potential drug-drug interactions of these promising new therapeutic agents with concomitantly administered CYP3A4 substrates.

Pharmacokinetics and its role in small molecule drug discovery research.

Pharmacokinetics (PK), which describes the disposition of a drug in the body, should be a primary consideration in the selection of a drug candidate, ultimately contributing to its eventual clinical success or failure. Accordingly, a sound understanding of PK concepts and an appreciation of the judicious use of PK and related (e.g., metabolism, transporter) data in drug discovery can be beneficial to those involved in the process. This review defines important PK parameters (e.g., clearance, volume of distribution, half-life), describes methods of PK data analysis (noncompartmental vs. compartmental) and provides an overview of additional concepts such as allometric scaling, PK/pharmacodynamic modeling, and nonlinear PK. Furthermore, the role and strategic use of PK screens in drug discovery are discussed.