Oxidation of lynestrenol by the fungus Cunninghamella elegans.

Transformation of lynestrenol (19-nor-17alpha-pregn-4-en-20-yn-17beta-ol) (1) was carried out by incubation with Cunninghamella elegans to obtain 19-nor-17alpha-pregn-4-en-20-yn-3-one-10beta,17beta-diol (2), 19-nor-17alpha-pregn-4-en-20-yn-3-one-6beta,17beta-diol (3), and 19-nor-17alpha-pregn-4-en-20-yn-3beta,6beta,17beta-triol (4). Metabolite 4 was identified as a new compound. These metabolites were structurally characterised on the basis of spectroscopic techniques.

Identification of the human cytochrome P450 enzymes involved in the in vitro biotransformation of lynestrenol and norethindrone.

This study examined the cytochrome P450 (CYP) enzyme selectivity of in vitro bioactivation of lynestrenol to norethindrone and the further metabolism of norethindrone. Screening with well-established chemical inhibitors showed that the formation of norethindrone was potently inhibited by CYP3A4 inhibitor ketoconazole (IC(50)=0.02 microM) and with CYP2C9 inhibitor sulphaphenazole (IC(50)=2.13 microM); the further biotransformation of norethindrone was strongly inhibited by ketoconazole (IC(50)=0.09 microM). Fluconazole modestly inhibited both lynestrenol bioactivation and norethindrone biotransformation. Lynestrenol bioactivation was mainly catalysed by recombinant human CYP2C9, CYP2C19 and CYP3A4; rCYP3A4 was responsible for the hydroxylation of norethindrone. A significant correlation was observed between norethindrone formation and tolbutamide hydroxylation, a CYP2C9-selective activity (r=0.63; p=0.01). Norethindrone hydroxylation correlated significantly with model reactions of CYP2C19 and CYP3A4. The greatest immunoinhibition of lynestrenol bioactivation was seen in incubations with CYP2C-Ab. The CYP3A4-Ab reduced norethindrone hydroxylation by 96%. Both lynestrenol and norethindrone were weak inhibitors of CYP2C9 (IC(50) of 32 microM and 46 microM for tolbutamide hydroxylation, respectively). In conclusion, CYP2C9, CYP2C19 and CYP3A4 are the primary cytochromes in the bioactivation of lynestrenol in vitro, while CYP3A4 catalyses the further metabolism of norethindrone.

Monoamineoxidase activity in certain brain regions associated with xanthurenic acid excretion in rats on oral contraceptive steroids.

Monoamineoxidase (MAO) activity in some regions of brain and urinary xanthurenic acid were investigated in female rats administered with oral contraceptive (OC) steroids-ethynyl estradiol (Ees), lynestrenol (Ly) and both in combination (Cm) for 75 days. The MAO activity was reduced significantly in most regions specially in cortex-the extent of reduction being 65 per cent with Ees, 51 per cent with Ly and 69 per cent with Cm treatments. In the hypothalamus, the activity was decreased by 50 per cent with Ees, 38 per cent with Ly and by 40 per cent with Cm treatments. In the corpus striatum the activity was reduced by 14 per cent with Ly treatment, 25 per cent with Cm treatment and in the midbrain by 59 per cent with Ees treatment only. The concentration of xanthurenic acid in urine was higher by 55 per cent with Ees, 109 per cent with Ly and by 120 per cent with Cm treatments. These changes in MAO activity and level of xanthurenic acid excretion indicate the possible alteration in the metabolism of neurotransmitter, associated with prolonged use of OC.

Metabolism of lynestrenol: characterization of 3-hydroxylation using rabbit liver microsomes in vitro.

In spite of the absence of oxygen at C-3, lynestrenol (17 alpha-ethynyl-4-estren-17 beta-ol) has a marked progestational activity. It is known to be metabolized to norethindrone (17 alpha-ethynyl-4-estren-17 beta-ol-3-one) by 3 beta-hydroxylation and dehydrogenation. In the present study this conversion of lynesterol to norethindrone, via the formation of 3 alpha-hydroxylynestrenol, was investigated using rabbit liver microsomes in vitro. Two hydroxylated metabolites, 3 alpha-hydroxy-lynestrenol and 3 beta-hydroxy-lynestrenol were separated and identified by GLC and GC-MS analyses. In the course of incubation, the concentration of 3 alpha-hydroxy-lynestrenol was much higher than that of the 3 beta-hydroxy isomer suggesting that the metabolic pathway in the conversion to norethindrone proceeds predominantly via 3 alpha-hydroxylation of lynestrenol.

Sulphation of contraceptive steroids.

The sulphation of a number of contraceptive steroids by rabbit tissue in vitro was investigated. With liver tissue the three synthetic gestagens (norethisterone, norgesterel and lynestrenol) were sulphated at different rates and none was sulphated as rapidly as dehydroepiandrosterone; sulphation occurred at the tertiary 17 beta-hydroxyl group. The synthetic oestrogen ethynyloestradiol was sulphated more rapidly than dehydroepiandrosterone, both mono and disulphates being formed. Of the other tissues studied, sulphation occurred with stomach and lung but not with heart, spleen, muscle, kidney or adipose tissue. These in vitro studies provide confirmation of in vivo findings regarding sulphate conjugates of the synthetic steroids.

Serum levels and pharmacokinetics of norethisterone after ingestion of lynestrenol: its relation to dose and stage of the menstrual cycle.

The peak concentration, peak time, the area under the serum concentration time curve (AUC) and half-life of serum norethisterone (NET) after a single application of lynestrenol (LYN) to female volunteers demonstrated that 0.7 mg NET is bioequivalent to 1 mg LYN which is rapidly converted to NET. There was a decrease of the peak values and an increase of half-life of NET during the periovulatory and luteal phase which was, however, not significant due to the great individual differences. The shift of the peak time to longer intervals and the increase of half-life of NET after ingestion of higher LYN doses indicate a certain limitation of the metabolic capacity of the liver. One of the volunteers who complained of nausea and vertigo after the administration of 5 mg LYN, showed the highest serum values of NET. The large interindividual variations of the serum levels of synthetic steroids demonstrate a possible risk of contraceptive safety in women with low steroid levels and possibly a coherence between extremely high serum levels of synthetic steroids and side effects.

[Pharmacokinetics of norethindrone and lynestrenol studied by HPLC (author's transl)].

The concentration of norethindrone in plasma samples from subjects receiving norethindrone (norethisterone) and lynestrenol orally was measured by high pressure liquid chromatography (HPLC). Norethindrone is a synthetic gestagen widely used in contraceptive formulations, and lynestrenol is also a synthetic gestagen which is metabolized to norethindrone in humans. But the evaluation of plasma norethindrone levels in subjects receiving lynestrenol has not yet been reported. After the administration of norethindrone, the peak level of norethindrone in the plasma was obtained within 2h, and the peak concentration in the plasma was about 3.5 ng/ml/mg norethindrone. During a period of 2-6hs after the administration of norethindrone, the half life of norethindrone in the plasma was approximately 1.8h, and during the period of 6-24hs, half life was variable. On the other hand, after the administration of lynestrenol, the peak level of norethindrone in the plasma was obtained within 4h, and the peak concentration of norethindrone was about 1.9 ng/ml/mg lynestrenol. During a period of 4-12hs, the half life of norethindrone was about 2.5h. The peak of the norethindrone level after the administration of lynestrenol was lower and appeared later than that after the administration of the same dose of norethindrone. Norethindrone in plasma in subjects receiving lynestrenol could be measured for a longer period than in those receiving the same dose of norethindrone. These results suggest that lynestrenol is stored in fat tissue and is slowly metabolized to norethindrone.

PIP: The concentration of norethindrone in plasma samples from subjects receiving norethindrone (norethisterone) and lynestrenol orally was measured by high pressure liquid chromatography (HPLC). Norethindrone is a synthetic gestagen widely used in contraceptive formulations, and lynestrenol is also a synthetic gestagen which is metabolized to norethindrone in humans. But the evaluation of plasma norethindrone levels in subjects receiving lynestrenol has not yet been reported. After the administration of norethindrone, the peak level of norethindrone in the plasma was obtained within 2 hours, and the peak concentration in the plasma was about 3.5 ng/ml/mg norethindrone. During a period of 2-6 hours after the administration of norethindrone, the half-life of norethindrone in the plasma was approximately 1.8 hours, and during the period of 6-24 hours, half-life was variable. On the other hand, after lynestrenol administration, the peak level of norethindrone in the plasma was obtained within 4 hours, and the peak concentration of norethindrone was about 1.9 ng/ml/mg lynestrenol. During a period of 4-12 hours, the half-life of norethindrone was about 2.5 hours. The peak of norethindrone level after lynestrenol administration was lower and appeared later than that after administration of the same dose of norethindrone. Norethindrone in plasma in subjects receiving lynestrenol could be measured for a longer period than in those receiving the same dose of norethindrone. These results suggest that lynestrenol is stored in fat tissue and is slowly metabolized to norethindrone. (author's)

Tissue distribution of 14C-lynestrenol in pregnant rats.

The tissue distribution of 4-14C-lynestrenol (17 alpha-ethynyl-oestr-4-en-17 beta-ol) following oral administration to pregnant rats was studied by whole body autoradiography and liquid scintillation counting. Pregnant females were sacrificed on days 10, 12, 14 and 19 of gestation, in each case 5 hours after oral administration of 43 microCi 4-14C-lynestrenol per animal. The isotopelabelled compound was distributed throughout most tissues, including the fetuses. The highest concentrations were found in the liver, while there was lower activity in the fatty tissues and the activity in the fetuses was comparable with that in the brain. The placental transfer was verified by the results of liquid scintillation counting. The concentration of labelled substance in the fetuses increased with the duration of pregnancy.

Bioavailability of Lynestrenol.

The bioavailability of lynestrenol and ethynyloestradiol from a capsule formulation was superior to a normal tablet formulation. Bioavailability, as measured by the area under the plasma concentration curves, showed, particularly in the case of the capsule formulation, that all of the administered lynestrenol was converted to norethisterone.

PIP: An in vivo research study demonstrated that the bioavailability of lynestrenol and ethynyloestradiol administrered in capsule form was equal to or slightly greater than when it was administered in normal tablet form. Lynestrenol was metabolized to norethisterone both in vivo and in vitro in previous investigations. 11 normal males were administer either 1 capsule containing 2.5 mg lynestrenol and 0.05 mg ethynyloestradiol or tablets containing the same dose of steroids. Blood samples were taken just before and 1,2,4,8, and 24 hours after the drugs were administered. 8 days later the subjects were given the opposite formulations and blood samples were again obtained at the same time intervals. Radioimmunoassay was used to assess levels of free and conjugated ethynyloestradiol and norethisterone in the serum samples. Biovailability assessments were made by calculating the areas under the time-concentration curves. Results demonstrated that the conversion of lynestrenol to norethisterone in vivo was complete. Unconjugated ethynyloestradiol mean values, 2 and 4 hours after administration of the drugs, were higher for capsules than for tablets. For blood samples taken at other time periods the mean values of unconjugated ethynyloestradiol were similar for both capsules and tablets. For conjugated ethynyloestradiol the mean values and the areas under the curve, for blood samples obtained at each time interval, showed no significant difference for tablets and capsules.

Comparative metabolism of 17alpha-ethynyl steroids used in oral contraceptives.

The metabolism of mestranol, ethynylestradiol, norethynodrel, norethindrone, ethynodiol diacetate, lynestrenol, and norgestrel is reviewed. The estrogenic components of the oral contraceptives, mestranol or ethynylestradiol, have nearly identical metabolic pathways since mestranol is rapidly and almost completely converted to ethynylestradiol The major fraction of the drugs plus metabolites is excreted in the urine as conjugated materials. All of the 17beta-ethynyl progestins reviewed follow similar metabolic paths. For three of these, norethynodrel, ethynodiol diacetate and lynestrenol, a principal metabolite is norethindrone. Biotransformation to more polar metabolites and conjugation proceed rapidly for these three precursor drugs and norethindrone. Norgestrel follows metabolic paths similar to those of norethindrone. However, the ethyl moiety at the C-13 position appears to slow the metabolism of this steroid so that biotransformation to more polar metabolites and the conjugation of these steroids does not proceed as rapidly as that of the other progestins. The high progestational potency of norgestrel may be attributed to this slow rate of biotransformation. Some of the pharmacokinetic parameters derived from the research reports reviewed here are summarized. The compounds appear to be readily absorbed, and they and their metabolites are excreted to a greater extent in the urine than in the feces.

[Postpartum contraception: effect of lynestrenol during the lactation period (author's transl)]. / Kontrazeption post partum: Beeinflussung der Stillperiode durch Lynestrenol.

167 women were treated with 0.5 mg lynestrenol/day for a period of 8-32 weeks immediately postpartum. The lactation periods of 67 of the women were observed during the stay in hospital (maximum 7 days) in comparison with a control group (n = 20) of untreated women in the puerperium. There was no quantitative difference in milk production between the lynestrenol-treated women and the control group. In order to exclude a possible thromboembolic effect the lynestrenol the thrombocyte aggregation was determined in all women at 4-weekly intervals as a measure of a thrombotic tendency. During the entire period of the investigation the mean PAT rating remained between I and II. Among the clinical parameters a raised amenorrhea rate of 21% and a larger proportion (39%) of breakthrough bleedings were notable. The responded easily to treatment.

Metabolites of lynestrenol acetate in the bile of rats after intravenous administration; a comparison with lynestrenol.

Some aspects of the metabolism of lynestrenol acetate, an orally active contraceptive compound, were studied in female rats. Lynestrenol acetate is stable in gastric and intestinal juice in vitro. After intravenous administration of lynestrenol acetate and lynestrenol with a -14C label in the nucleus approximately 40% of the administered radioactivity was excreted in the bile within 90 min. After administration of lynestrenol acetate labelled in the ester group, 6% of the radioactivity was found in the bile. This means that the greater part of the lynestrenol acetate had lost its acetate group during the process of metabolism. There was an important difference between the autoradiograms of the thin layer patterns of post-hydrolysis extracts after administration of [4-14C]lynestrenol acetate and those after administration of [1'-14C]lynestrenol acetate and [4-14C]lynestrenol: the major metabolite of [4-14C]lynestrenol acetate did not appear on the autoradiograms of [1'--14C]lynestrenol acetate and [4-14C]lynestrenol. This indicates that lynestrenol acetate was altered in the nucleus in the presence of the acetate group. The acetate group itself was removed, either when the alterations took place, or after it had been completed. The results of IR, NMR and mass spectrometry analysis indicate the introduction of a 15alpha hydroxyl group. Results of gas-liquid chromatography and thin layer chromatography indicate that a second important metabolic is 19-nor-17alpha-pregn-20-yne-3alpha, 17beta-diol. The main conclusions are: 1. A part of the lynestrenol acetate is metabolized and excreted in the bile, the acetate group still being present. 2. Lynestrenol acetate is to some extent metabolized via another pathway than lynestrenol. This indicates that esterification of a steroid can lead to deviation from the metabolic pathway of the free original steroid.

PIP: The metabolism of lynestrenol acetate (LA), an oral contraceptive, w as studied in female rats. LA is stable in gastric and intestinal juice in vitro, and is partly absorbed unchanged after oral administration. 40% of the radioactivity is excreted in the bile within 90 minutes, when labelled in the nucleus. Administration of LA labelled in the acetate group resulted in only 6% of the radioactivity in the bile, indicating that most of LA lost the acetate group during metabolism. Complex formation with silver shows that more than 80% of the metabolites of LA still contained the 17 alpha-ethinyl group. Thin layer analysis revealed that the major metabolite of (4 carbon-14)LA did not appear in the autoradiograms of (1" carbon-14)LA or (4 carbon-14)L. LA seems to be altered in the nucleus in the presence of the acetate group. IR, NMR and mas spectrophotometric analysis indicate the introduction of a 15 alpha hydroxyl group. A second major metabolite of LA and L seems to be 19-nor-17 alpha-pregn-20-yne-3 alpha, 17 beta-diol. These results indicate that esterification of a steroid can lead to alteration in the metabolic pathway of the free steroid.

Interaction of contraceptive progestins and related compounds with the oestrogen receptor. Part I: Effect on (3H)oestradiol distribution pattern in the ovariectomized rat.

The distribution pattern of oestradiol in ovariectomized rats as a function of time has been studied following intravenous adminstration of the tritiated hormone. Oestrogen specific binding with limited capacity was observed in the uterus, vagina, anterior pituitary, adrenals, preoptic area, hypothalamus, amygdala, septum and tractus diagonalis. Maximal uptake of oestradiol in the pituitary occurred within 5 min, in the uterus 60 min after injection, and remained almost unchanged at this level for more than two hours. The binding capacity per mg tissue decreased in the order pituitary, uterus, vagina, preoptic area, adrenals, hypothalamus, amygdala, spetum and tractus diagonalis. The hormone concentration in these tissues one hour after (3H)oestradiol injection was lowered by previous administration of ethinodiol, norethinodrel, lynestrenol and norethindrone, whereas medroxyprogesterone, chlormadinone, megestrol and methyllynestrenol had no effect. The same results were obtained, when instead of the steroid alcohols the corresponding acetate esters were administered. For norgestrel, oestrenol and nortestosterone the effect in the dose range studied was limited to the pituitary and preoptic area. For lynestrenol the inhibition of oestradiol binding in the target tissues was almost the same when the progestin was given 60 and 5 min before oestradiol, whereas in the case of administration 30 min after oestradiol no inhibition was observed. The reduction of oestrogen binding appeared to be dose-dependent, but the dose required to obtain a certain effect for the uterus was four times as high as for the pituitary. Discrepancies between previous studies and the implications of the present findings for the mechanism of action of ovulation inhibition by these progestins are discussed.

Interaction of contraceptive progestins and related compounds with the oestrogen receptor. Part II: Effect on (3H)oestradiol binding to the rat uterine receptor in vitro.

Incubation of the 105 000 times g supernatant of rat uterus homogenate with (3H)oestradiol resulted in an oestrogen specific binding of limited capacity to a macromolecule sedimenting in the 8-9S region after density gradient centrifugation. The contraceptive progestins of the 19-nortestosterone series were able to interfere with oestradiol binding in contrast to the hydroxyprogesterone derivatives chlormadinone, medroxyprogesterone and megestrol. The interaction appeared to be competitive. The strongest inhibition of oestradiol binding was observed in the presence of ethinodiol, followed by northinodrel, lynestrenol and norethindrone respectively. Norgestrel was almost inactive. Of the related structures tested oestrenol displayed more activity than norethindrone, nortestosterone and ethisterone were less active and 6alpha-methyllynestrenol showed only border line activity. In comparison with norethinodrel and norethindrone, lynestrenol and oestrenol appeared in vitro to be stronger competitors for oestradiol than in vivo (Part I; Van Kordelaar et al. 1975). This may be due to the great difference in lipophilic character, which is reflected in the RM values of these compounds. From the results obtained it may be concluded, that the presence of a 17alpha-ethynyl substituent promotes receptor binding, whereas the introduction of methyl substituents in the position 6, 10 and 18 causes the opposite effect. The relationship between the various ring A structures and the affinity to the receptor is discussed.

[Competition between estradiol and various progestogens at the level of the rat hypothalamus]. / Compétition entre l'oestradiol et différents progestagènes au niveau de l'hypothalamus du rat

PIP: Short-term cultures (32 hours) of rat hypothalamus were incubated with tritiated estradiol and either medroxyprogesterone, Ro 4-8347, norgestrel, or lynestrenol to find out whether the progestagens would compete for estrogen receptors. Each culture was preincubated for 8 hours in medium 199, washed, incubated for 16 hours with progestagen (preincubation experiment), and incubated a further 8 hours with labeled estrogen (control or preincubation experiment) or estrogen and the progestagen (simultaneous experiment). Ro 4-8347 (1,6-dehydro-6-chlororetroprogesterone) .6 ng/ml inhibited estradiol uptake when it was added before the estrogen. Lynestrenol and norgestrel displaced estradiol in simultaneous cultures only. Medroxyprogesterone reduced estradiol uptake when it was added both before and during the estradiol incubation.^ieng

Metabolism of 4-14C lynestrenol in Thai women.

PIP: The metabolism of 4-14-carbon lynestrenol was studied in Thai women, and the results were compared with a similar study of European women. The half-lives of radioactivity in plasma and urine, and the levels of urinary radioactivity found in free, sulphate, and acid-hydrolyzed fractions were similar to those reported in the European study. The levels of radioactivity observed in enzyme-hydrolyzed urinary extracts were significantly lower among Thai women (p=.01), though the difference was probably due to the use of a different enzyme prepararation. The chromatographic mobilities of free and enzyme-hydrolyzed urinary extracts were similar for both groups, though the levels for the sulphate fractions were so variable in the Thai group that they defied comparison. The results indicate that the metabolism of lynestrenol in European and Thai women is similar, and that the observed differences are unlikely to have biological significance.^ieng