[In vitro metabolic interaction between diphenytriazol and steroid hormone drugs].

AIM: To observe the metabolic interaction between diphenytriazol and steroid hormone drugs, and provide some useful information for clinical medication. METHODS: The steroid hormone drugs which may be co-administrated with diphenytriazol were selected, such as mifepriston, estradiol, medroxyprogesterone acetate, progesterone, norethisterone and so on. Diphenytriazol was incubated with each drug in rat liver microsome. The residual concentration of diphenytriazol or steroid hormone drugs in the microsomal incubates was determined by reversed-phase high-performance liquid chromatography, separately. The inhibition constants (K(i)) for each of them were calculated. RESULTS: The inhibition constant K(is) of diphenytriazol for the metabolism of mifepristone, estradiol, medroxyprogesterone acetate, progesterone and norethisterone were (201.3 +/- 1.0), (94 +/- 4), (128.7 +/- 2.2), (64 +/- 5) and (80 +/- 4) micromol x L(-1), respectively. The inhibition constants K(i) of steroid hormone drugs for the metabolism of diphenytriazol was (66.9 +/- 2.2) micromol x L(-1) for estradiol, (60.0 +/- 2.3) micromol x L(-1) for medroxyprogesterone acetate, (163 +/- 10) micromol x L(-1) for progesterone and (88 +/- 5) micromol x L(-1) for norethisterone, respectively. CONCLUSION: Diphenytriazol shows metabolism interaction with steroid hormone drugs such as estradiol, medroxyprogesterone acetate, progesterone and norethisterone.

Mechanism-based inactivation of cytochrome P-450-3A4 by mifepristone (RU486).

Mifepristone (RU486), an 11beta-substituted nor-steroid containing a 17alpha-1-propynyl group used clinically as an antiprogestin agent for medical abortions, was demonstrated to be a selective mechanism-based inactivator of human cytochrome P-450-3A4 (CYP-3A4). The loss of testosterone 6beta-hydroxylation activity was time- and concentration-dependent as well as requiring metabolism of mifepristone in a purified CYP-3A4 reconstituted system. The inactivation exhibited pseudofirst-order kinetics. The values for KI and kinactivation were 4.7 microM and 0.089 min-1, respectively. The reduced-CO spectrum of CYP-3A4 was decreased by 76%, whereas approximately 81% of the activity was lost following incubation with mifepristone in the reconstituted system in the presence of NADPH. However, the Soret peak of the inactivated CYP-3A4 was slightly increased. High-performance liquid chromatography analysis of the incubation mixture showed that the peak containing the heme dissociated from the inactivated CYP3A4 was almost identical with that seen for the -NADPH control. Covalent binding of [3H]mifepristone to apoCYP3A4 was demonstrated by SDS-PAGE and high-pressure liquid chromatography analyses of the reconstituted system containing CYP-3A4, NADPH-CYP reductase, cytochrome b5 and lipids in the presence of NADPH. The stoichiometry was determined to be approximately 1 mol of mifepristone bound per 1 mol of CYP-3A4 inactivated. Therefore, the mechanism of inactivation of CYP-3A4 by mifepristone involves irreversible modification of the apoprotein at the enzyme active site instead of being the result of heme adduct formation or heme fragmentation. Mifepristone exhibits selectivity for CYP-3A4 as evidenced by the fact that it did not show mechanism-based inactivation of CYPs 1A, 2B, 2D6, and 2E1, although a competitive inhibition of CYP 2B1 and 2D6 was observed.

PIP: This study demonstrates mifepristone (RU-486) as a potent and selective mechanism-based inactivator of cytochrome P-450-3A4 (CYP-3A4) via irreversible modification of the apoprotein. The results of this clinical research indicate that loss of testosterone 6-beta-hydroxylation activity was time- and concentration-dependent, as well as requiring metabolism of mifepristone in purified CYP-3A4 reconstituted system. Inactivation using several different concentrations of mifepristone exhibited pseudo-first-order kinetics. Reduced-CO difference spectrum of CYP-3A4 decreased by 76%, whereas approximately 81% of CYP-3A4 activity was lost following incubation with mifepristone, indicating the occurrence of N-heme adduct formation detected by HPLC and UV-visible spectroscopy. The peak containing the heme dissociated from the mifepristone-inactivated CYP-3A4 was almost identical with that of the -NADPH control when the incubation mixtures were analyzed by HPLC. 3H-mifepristone proved to be covalently bound to the apoCYP-3A4 by HPLC and SDC-PAGE. The stoichiometry for the binding of the mifepristone was determined to be 1.02 +or- 0.15, approximately 1 mol of mifepristone bound per mole of inactivated CYP-3A4. In summary, mifepristone has been shown to be a potent mechanism-based inactivator of human CYP-3A4. The mechanism of the inactivation showed to involve irreversible modification of the apoprotein at the enzyme active site instead of heme adduct formation or heme fragmentation.

Medical abortion.

PIP: In 1996, an application was submitted to the US Food and Drug Administration for the use of mifepristone (RU-486) plus the prostaglandin misoprostol in medical abortion. Over 100,000 women in more than 20 countries have received this regimen, which results in pregnancy termination in 92.7-99.0% of treated women. This article presents state-of-the-art information on medical abortion. Reviewed are its pharmacokinetics and metabolism, mechanism of action, and history of use. The article outlines a standard protocol that includes RU-486 administration at the first visit (day 1), misoprostol administration at the second visit (day 3), and post-treatment examination at the third visit (days 14-20) and suggests counseling guidelines. It discusses the contraindications and potential complications of abortifacient agents. Finally, the article compares the experience in the US and Europe of medical versus surgical abortion in terms of effectiveness, complications, and acceptability.^ieng

Antiprogestins: mechanism of action and contraceptive potential.

Antiprogestins are characterized by substitutions at the 11 beta and 17 alpha positions of the steroid ring system and bind strongly to both progesterone and glucocorticoid receptors. Although they function predominantly as antiprogestins and antiglucocorticoids, on occasion they display progestin agonistic and even antiestrogenic properties. The most common clinical use of the antiprogestin mifepristone is to induce a medical abortion in the early stages of pregnancy. Progesterone maintains the endometrium, transforming it from a proliferative to a secretory state. It also facilitates the luteinizing hormone surge, which initiates ovulation. As a consequence, antiprogestins may also have contraceptive potential. Although antiprogestins do delay ovulation, this effect is inconsistent unless high doses are given, and under these circumstances, the antiprogestin effect is associated with unopposed estrogen action on the endometrium. Very low doses of antiprogestins do not affect hormonal secretion or ovulation or alter bleeding patterns, but they do have contraceptive potential by inducing profound alterations in endometrial morphology. Mifepristone is also a very effective and safe postcoital agent. This new class of pharmacological agents has numerous other gynecological and obstetrical indications, such as endometriosis, uterine myoma, and expulsion of the fetus in the case of fetal death in utero. Antiprogestins may also be used in the treatment of steroid-dependent tumors. There are also therapeutic implications consequent to their antiglucocorticoid properties.

PIP: Antiprogestins have a 17-alpha substitution, which promotes higher binding affinity to both progesterone and glucocorticoid receptors, and an 11-beta substitution, which appears to be responsible for the antagonistic action. Antiprogestins also exhibit progestin agonistic and even antiestrogenic properties. Induction of a medical abortion in very early pregnancy is the most frequent clinical use of the antiprogestin mifepristone (RU-486). Antiprogestins may someday be used as a contraceptive since very low doses of antiprogestins cause considerable changes in endometrial morphology. At these low doses, they do not affect hormonal secretion or ovulation or bleeding patterns. At high doses, they delay ovulation but are associated with unopposed estrogen action on the endometrium. At low doses, antiprogestins do not always delay ovulation. RU-486 is a very effective and safe postcoital contraceptive. Antiprogestins may be beneficial in treating endometriosis, uterine myoma, and steroid-dependent tumors (e.g., breast cancer). They may be used to induce labor in cases of intrauterine fetal death. Antiprogestins may prove useful in treating Cushing's syndrome due to ectopic adrenocorticotropic hormone secretion, in lowering intraocular pressure in glaucoma, and in preventing the progression of viral diseases in humans. They also appear to have even more potential benefits, such as treatment of burns.

Progesterone receptor antagonists and prostaglandins in human fertility regulation: a clinical review.

Progesterone receptor antagonists have been developed by substitutions at the 11-beta and 17 side-chain positions of the progestagen norethisterone. The most studied progesterone receptor antagonists are mifepristone (Mifegyne; Roussel-UCLAF; RU486) and ZK98734 and ZK98299 (Schering AG). These compounds bind avidly to the progesterone receptor and glucocorticoid receptor but have essentially no binding to the mineralocortocoid, oestrogen or androgen receptors. Mifepristone also binds avidly to albumin, resulting in a half-life of approximately 24 h after oral administration. Progesterone receptor antagonists can induce menstruation by a direct action upon the endometrium. They have also been shown to exert weak progesterone agonist actions in certain circumstances and to modulate pituitary hormone secretion by antagonizing the feedback actions of progesterone. Moreover, they release prostaglandin F2 alpha and E2 from human endometrium or early pregnancy decidua and reduce the metabolism of these eicosanoids. Clinically, progesterone receptor antagonists have been used in trials of menstrual regulation, abortion and induction of labour, and during treatment of breast or ovarian cancer, some forms of hypertension and meningioma. Progesterone receptor antagonists have been administered to approximately 70,000 women in 18 countries as medical abortifacients. They have been proven, especially when combined with prostaglandin analogues, to be as effective as surgical methods of termination of pregnancy. Progesterone receptor antagonists have focussed international attention on menstrual regulation, abortion and the rights of women to regulate their fertility.

Levels of the antiprogestin RU 486 and its metabolites in human blood and follicular fluid following oral administration of a single dose.

Using high-pressure liquid chromatography (HPLC) the antiprogestin RU 486 and two of its metabolites (N-monodemethyl RU 486 and propargyl RU 486) were measured in plasma and follicular fluid of 21 women requesting laparoscopic sterilization. Pretreatment of the women involved ovulation induction with clomiphene and HCG. RU 486 (100 mg) was administered orally and 1 h later blood samples were withdrawn. Thirty-four hours later, at laparoscopy, samples of both blood and follicular fluid were collected. During the 34-h period the average plasma level of RU 486 decreased from 1.93 mumol/l to 0.91 mumol/l, i.e. by -50%. The latter concentration of RU 486 was not significantly different from that found in follicular fluid (0.79 mumol/l). The monodemethyl metabolite exhibited significantly higher plasma levels (3.09 mumol/l) than RU 486 1 h after administration. Thirty-four hours later these levels had decreased to 0.92 mumol/l, i.e. by 70%. In follicular fluid, the levels of the monodemethyl metabolite (1.76 mumol/l) were significantly higher than those of RU 486 (0.79 mumol/l). Because of background noise, only approximate values were established for the propargyl metabolite. These were 0.67 and 0.40 mumol/l, respectively, in plasma and 0.42 mumol/l in follicular fluid. The results indicate that RU 486 and two of its major metabolites can readily cross the blood-follicle barrier of human pre-ovulatory follicles.

A novel approach to human fertility control: contragestion by the anti-progesterone RU 486.

PIP: The introductory presentation for a session on antiprogestins summarizes the action of RU 486 relating to its use as a contragestive agent. RU 486, or mifepristone (Roussel-Uclaf), is a progestin analogue resembling norethindrone with a longer 17-alpha side chain and a 11-beta phenyl group that confers the progesterone antagonist activity. It binds the progesterone receptor with a half-life of about 20 hours. Since progesterone receptors are only located in a few tissues, notably the decidualized endometrium and the corpus luteum, its action can be targeted. RU 486 interrupts the cycle regardless of whether fertilization occurred. A single 600 mg dose arrests over 80% of pregnancies up to 41 days, then a decreasing proportion, down to 60% at 10 weeks, due to a greater chance of incomplete evacuation. There have been no recorded side effects in 4000 trails. The effect of prostaglandins is magnified when progesterone action is suppressed. 400 trials have confirmed that over 98% successful termination of pregnancy can be obtained if 600 mg RU 486 is followed by 1 mg synthetic prostaglandin 36 hours later. RU 486 has also tested successfully for delivery of dead fetuses, cervical dilation, a postcoital contraceptive, and as an antiglucocorticoid agent. It has been submitted to the French Ministry of Health, and will probably be approved for marketing in 1988. Clinical trials are underway in 15 countries. This drug is a useful contragestive agent, active in the beginning of the continuum of pregnancy, and may defuse the abortion issue.^ieng

3H-labelled RU 38486: characterization of binding sites in human uterine cytosol.

The behaviour of the antifertilizing synthetic steroid RU 38486 towards human uterine progestin receptor was investigated. RU 38486 competed in the same order of magnitude as progesterone for the [3H]R 5020 binding site of progestin receptor, whereas R 5020 was unable to compete against [3H]RU 38486. This apparent contradiction could be explained by means of HPLC-chromatography. HPLC-chromatography with an anion exchange column (MonoQ, Pharmacia, Uppsala, Sweden) showed that [3H]RU 38486 forms at least two stable complexes with uterine cytosol, on one hand with serum albumin, which presents almost 90% of bound radioactivity, and on the other hand with the two native progestin receptor forms, corresponding to 4 S and 8 S receptor forms in sucrose density gradient analysis. Whether reduced binding of salt-activated RU 38486 receptor complexes to DNA-cellulose is due to reduced activation is still uncertain and remains to be further investigated.

Comparative bioavailability of a lipophilic steroid.

17beta-Acetoxy-2alpha-chloro-3-(p-nitrophenoxy)imino-5alpha-androstane (I) is a lipophilic steroid with postimplantive antifertility activity in laboratory animals. The bioavailability of micronized I from solutions and suspensions was compared in four groups of adult female Wistar rats. Each group received varying concentrations of micronized 3H-I (specific activity of 0.38--8.94 muCi/mg) in sesame oil by oral gavage. Samples of whole blood and urine collected following drug administration were assayed for radioactive content. Calculation of the mean area under the blood radioactivity versus time curve, when corrected for the quantity of drug administered, indicated that a substantially larger fraction of the dose was absorbed in the two instances where I was present only in solution. A linear relationship between the amount of I absorbed based on whole blood radioactivity and urinary excretion and the administered dose was found primarily for groups receiving the drug in solution. Preliminary results in humans indicate that 3H-I was absorbed to a much greater extent following oral administration of the drug in sesame oil than when admixed with lactose.

Disposition of ORF 9326, a novel contragestational steroid, in animals.

The disposition of ORF 9326 [17BETA-acetoxy-2alpha-chloro-3(p-nitrophenoxy) imino-5-androstane], an O-aryl oxime of 2beta-chlorodihydrotestosterone acetate, was studied in rats, dogs, monkeys and rabbits. Intravenous administration of 3H-ORF 9326 dissolved in PEG-400 to rats, dogs and monkeys resulted in a rapid decline of radioactivity in blood followed by a terminal slope suggesting long retention of radioactivity. Apparent half lives of radioactivity in blood were calculated to be from 50--95 hours for the three species, which peak levels of radioactivity in whole blood occurring within 4--7 hours after administration of the compound. Tissue distribution studies in the rat and dog indicate that body fat is one of the major depot areas for the drug and/or its metabolites. The major route of excretion for ORF 9326 and/or its metabolites in dog and rat is biliary whereas in monkey and rabbit it appears to be renal. Greater than 90% of the radioactive compounds excreted in the urine of dogs and monkeys following intravenous administration of 3H-ORF 9326 appear to be in the form of conjugates.