Differential off-target glucocorticoid activity of progestins used in endocrine therapy.

The glucocorticoid receptor (GR) regulates transcription of genes involved in multiple processes. Medroxyprogesterone acetate (MPA), widely used in the injectable contraceptive Depo-MPA (DMPA), has off-target effects via the GR, which may result in side-effects in endocrine therapy. However, very little is known about the GR activity of other progestins used in endocrine therapy. This study compared GR activities for several progestins, using whole cell binding, dose-response, and GR phosphorylation assays, in both a cell line model and peripheral blood mononuclear cells (PBMCs). MPA, etonogestrel (ETG) and nestorone (NES) exhibit greater relative binding affinities for the GR than levonorgestrel (LNG) and norethisterone/norethindrone (NET) and are partial GR agonists for transactivation but agonists for transrepression on synthetic promoters in COS-1 cells. MPA is a potent agonist for endogenous GR-regulated GILZ and IL6 genes in PBMCs. While ETG and NES also display agonist activity on IL6, they have little effect on GILZ. In contrast, LNG and NET exhibit little to no activity in transactivation models, while both exhibit some transrepressive activity but are generally less potent and/or efficacious than MPA. Antagonist and phosphorylation assays confirmed that MPA and NES act via the GR on endogenous genes in PBMCs. Our results suggest GR-mediated dose-dependent and gene-specific transcriptional side-effects are likely to occur at physiologically relevant concentrations in vivo for MPA, may possibly occur selectively for ETG and NES, but are unlikely to occur for LNG and NET. This suggests that these progestins will exhibit differential side-effects in endocrine therapy via the GR.

Norethindrone alters growth, sex differentiation and gene expression in marine medaka (Oryzias melastigma).

Norethindrone (NET) is a widely used synthetic progestin, which appears in water environments and threatens aquatic organisms. In this study, marine medaka (Oryzias melastigma) larvae were exposed to 7.6 and 80.1 ng/L NET for 190 days. The effects of NET on growth, sex differentiation, gonad histology and transcriptional expression profiles of hypothalamic-pituitary-gonadal (HPG) axis-related genes were determined. The results showed that exposure to 80.1 ng/L NET caused an all-male marine medaka population and significantly decreased the growth of males. Exposure to 7.6 ng/L NET increased the ratio of males/females in the marine medaka population, decreased the growth of males and delayed the ovary maturation in females. However, the sperm maturation was accelerated by 7.6 or 80.1 ng/L NET. In females, the transcription levels of cytochrome P450 aromatase (cyp19a1a) and progesterone receptor (pgr) in ovaries, glucocorticoid receptor (gr) and vitellogenin (vtg) in livers were suppressed after exposure to 7.6 ng/L NET, which may cause delayed ovary maturation. In males, NET significantly decreased the transcription levels of follicle stimulating hormone ß (fshß) and Luteinizing hormone ß (lhß)in the brain, Estrogen receptor ß (erß)，gr and pgr in the liver, and vitellogenin receptor (vtgr) in the testes, while NET of 80.1 ng/L led to a significant up-regulation of steroidogenic acute regulatory protein (star) in the testes of males. These results showed that NET could influence growth, sex differentiation and gonadal maturation and significantly alter the transcriptional expression levels of HPG axis-related genes.

Differential metabolism of clinically-relevant progestogens in cell lines and tissue: Implications for biological mechanisms.

Steroid hormones regulate a variety of physiological processes, including reproductive function, and are widely used in hormonal therapy. Synthetic progestogens, or progestins, were designed to mimic progesterone (P4) for use in contraception and hormonal replacement therapy in women. Medroxyprogesterone acetate (MPA) and norethisterone (NET) are the most widely used injectable contraceptives in the developing world, while other progestins such as levonorgestrel (LNG), etonogestrel (ETG) and nestorone (NES) are used in or being developed for other forms of contraception. As concerns remain about the most appropriate choice of progestin and dosage, and the associated side-effects, the mechanisms and biological effects of progestins are frequently investigated in various in vitro mammalian cell line and tissue models. However, whether progestogens are differentially metabolised in different cell types in vivo or in vitro is unknown. For nine mammalian cell lines commonly used to investigate progestogen mechanisms of action, we developed and validated an ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) protocol for simultaneously quantifying the metabolism of the above-mentioned steroids. We show for the first time that, while 50-100% of P4 was metabolised within 24 h in all cell lines, the metabolism of the progestins is progestin- and cell line-specific. We also show that MPA and NET are significantly metabolised in human cervical tissue, but to a lesser extent than P4. Taken together, our findings suggest that differential progestogen metabolism may play a role in cell-specific therapeutic and side-effects. Relative affinities for binding to steroid receptors as well as potencies, efficacies and biocharacters for transcriptional activity of progestins, relative to P4, are most frequently determined using some of the cell lines investigated. Our results, however, suggest that differential metabolism of progestins and P4 may confound these results. In particular, metabolism may under-estimate the receptor-mediated intrinsic in vitro binding and dose-response values and predicted endogenous physiological effects of P4.

Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants.

M01A82W, M11A82W and M01A82WS72I are three cytochrome P450 BM3 (CYP102A1) variants. They can catalyze the hydroxylation of testosterone (TES) and norethisterone at different positions, thereby making them promising biocatalysts for steroid hydroxylation. With the aim of obtaining more hydroxylated steroid precursors it is necessary to probe the steroidal substrate diversity of these BM3 variants. Here, three purified BM3 variants were first incubated with eight steroids, including testosterone (TES), methyltestosterone (MT), cholesterol, ß-sitosterol, dehydroepiandrosterone (DHEA), diosgenin, pregnenolone and ergosterol. The results indicated that the two 3-keto-Δ4-steroids TES and MT can be hydroxylated at various positions by the three BM3 mutants, respectively. On the contrary, the three enzymes displayed no any activity toward the remaining six 3-hydroxy-Δ5-steroids. This result indicates that the BM3 mutants prefer 3-keto-Δ4-steroids as hydroxylation substrates. To further verify this notion, five other substrates, including two 3-hydroxy-Δ5-steroids and three 3-keto-Δ4-steroids, were carefully selected to incubate with the three BM3 variants. The results indicated the three 3-keto-Δ4-steroids can be metabolized to form hydroxysteroids by the three BM3 variants. On the other hand, the two 3-hydroxy-Δ5-steroids cannot be hydroxylated at any position by the BM3 mutants. These results further support the above conclusion, therefore demonstrating the 3-keto-Δ4-steroid substrate preference of BM3 mutants, and laying a foundation for microbial production of more hydroxylated steroid intermediates using BM3 variants.

Probing the binding of two 19-nortestosterone derivatives to human serum albumin: insights into the interactions of steroid hormone drugs with functional biomacromolecule.

Norethindrone acetate (NETA) is a fatty acid ester of norethindrone (NET) that can convert to its more active parent compound NET when orally administered. To study the interactions of NETA and NET with human serum albumin (HSA), we applied fluorescence spectroscopy, circular dichroism (CD), and molecular docking. The effects of metal ions on the HSA-NETA/NET system were also explored. Fluorescence data showed that the quenching mechanism of HSA by NETA and NET was consistent with a static model and that the binding constant of NETA was higher than that of NET. Thermodynamic parameters indicated that hydrogen bonds and van der Waals forces were the main forces maintaining the stability of the HSA-NETA/NET complex. Molecular modeling studies revealed that NETA and NET were bound within subdomain IIA of HSA, in accordance with the site probe results. Synchronous fluorescence spectroscopy, CD, and three-dimensional fluorescence spectroscopy further confirmed that the binding of NETA/NET to HSA changed the secondary structure of the protein. All other metal ions, except for Ca(2+) , decreased the K value of the HSA-NETA/NET system with enhancement of the maximum effectiveness of NETA/NET. Three commercially available steroid hormone drugs influenced the binding ability of NETA on HSA to different extents. This study provides novel insights into the interactions between HSA and NETA/NET, as well as a solid foundation for future research on drug pharmacokinetics and pharmacodynamics. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of estradiol and progestogens on human breast cells: regulation of sex steroid receptors.

OBJECTIVES: To examine the effects of 17ß-estradiol (E2) and progestogens, used in hormone therapy, on estrogen receptors (ER), progesterone receptors (PR), and human breast tumor cell growth. MATERIALS AND METHODS: MCF-7 cells were incubated in pure E2 (1 nM and 10 nM) as well as in E2 in conjunction with 10 nM progestogens, including progesterone (P4), medroxyprogesterone acetate (MPA), norethisterone acetate (NET), and cyproterone acetate (CPA). Cell proliferation, apoptosis, expression of caspase-3, and both ER and PR isoforms were evaluated. RESULTS: Caspase-3 was significantly diminished in cultures with only E2, whereas ERα significantly increased. A significant increase of caspase-3 in addition to the entire abolishment of E2-induced augmentation of ERα was observed in 1 nM E2 plus MPA and 10 nM E2 plus NET, whereas PR isoform B (PRB) was significantly increased. The ratios of apoptosis: proliferation significantly increased in 1 nM E2 plus progestogens (except P4) and 10 nM E2 plus NET. The changes of the PRA/PRB ratio were inversely related to the changes of the apoptosis to proliferation ratio. Significant increase of ERß and PRB was noted in the E2 plus MPA or NET, in addition to a significant increase of ERα and decrease of PRA in the E2 plus CPA, as well as an increase of ERα and decrease of PRA and PRB in the E2 plus P4. CONCLUSIONS: The combination of E2 and various progestogens resulted in diverging effects on ERs and PRs expressions, which induced different effects on MCF-7 cell growth. Compared with P4, aberrant hormone and biological activity of synthetic progestin, by way of altered receptor expression, may be an important factor in affecting breast cell growth.

Metabolism of norethisterone in the greyhound.

RATIONALE: Norethisterone has been used as a successful oral contraceptive in humans for many years. It was recently permitted for use as an oestrus suppressant in racing greyhounds. To monitor the use of norethisterone as part of a routine drug surveillance programme, knowledge of its metabolism was required to enable detection. METHODS: Gas chromatography/mass spectrometry and selective derivatisation techniques have been used to identify urinary metabolites of norethisterone following oral administration to the greyhound. Metabolites were extracted using solid-phase and liquid-liquid extraction techniques. RESULTS: Several metabolites were identified, including reduced, mono-, di- and trihydroxylated steroids. The major metabolites observed were 17α-ethynyl-5ß-estrane-3α,17ß-diol, 17α-ethynyl-5α-estrane-3ß,17ß-diol, three 17α-ethynylestranetriol stereoisomers and two 17α-ethynylestranetetrol stereoisomers. The major metabolites were predominantly excreted as glucuronic acid conjugates and detection of the administration of norethisterone was possible for up to 8 days post-dose using the methods described. The nandrolone metabolites, 19-norepiandrosterone, estranediol and 19-noretiocholanolone, were also identified in the post-administration samples collected up to 8 h after dosing the treated animals. CONCLUSIONS: The urinary metabolites identified in this study have further increased the knowledge of steroid metabolism in the greyhound, providing information to support routine drug testing programmes for greyhound racing.

Comparison of serum and cervical mucus hormone levels during hormone-free interval of 24/4 vs. 21/7 combined oral contraceptives.

BACKGROUND: This study analyzes levels of progesterone, estradiol, norethindrone (NET) and ethinyl estradiol (EE) in serum and levels of NET in cervical mucus on the last day of the hormone-free interval (HFI) in users of 24/4 [norethindrone acetate (NETA)/EE-24] vs. 21/7 (NETA/EE-21) regimens. STUDY DESIGN: This was a randomized controlled, crossover, equivalency trial. Subjects were randomized to receive NETA/EE-24 or NETA/EE-21 for 2 months and then switched between study drugs. Blood and cervical mucus samples were obtained on Days 12-16 and on the last day of the HFI. RESULTS: From April 2010 to November 2011, 32 subjects were enrolled with 18 subjects completing all study visits. There were no statistically significant differences in either day 12-16 (p=.54) or last hormone-free day (p=.33) cervical mucus NET concentrations between the regimens. On the last day of the HFI, median serum progesterone levels did not differ significantly; however, users of NETA/EE-24 had higher levels of serum NET (p<.001) and users of NETA/EE-21 had higher levels of serum estradiol (p=.01). CONCLUSION: This data supports the fact that inhibition of the pituitary-ovarian axis occurs during oral contraceptive use and during the HFI. We demonstrated that a reduced HFI of 4 days resulted in better suppression of the ovarian hormone production, thereby reducing the risk of ovulation and potential contraceptive failure.

Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies.

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16ß. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16ß-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16ß-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2ß.

The influence of gill and liver metabolism on the predicted bioconcentration of three pharmaceuticals in fish.

The potential for xenobiotic compounds to bioconcentrate is typically expressed through the bioconcentration factor (BCF), which has gained increased regulatory significance over the past decade. Due to the expense of in vivo bioconcentration studies and the growing regulatory need to assess bioconcentration potential, BCF is often calculated via single-compartment models, using K(OW) as the primary input. Recent efforts to refine BCF models have focused on physiological factors, including the ability of the organism to eliminate the compound through metabolic transformation. This study looks at the ability of in vitro biotransformation assays using S9 fractions to provide an indication of metabolic potential. Given the importance of the fish gill and liver in metabolic transformation, the metabolic loss of ibuprofen, norethindrone and propranolol was measured using rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) gill and liver S9 fractions. Metabolic transformation rates (k(M)) were calculated and integrated into a refined BCF model. A significant difference was noted between BCF solely based on K(OW) and BCF including k(M). These studies indicate that the inclusion of k(M) in BCF models can bring predicted bioconcentration estimates closer to in vivo values.

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR.

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)alpha and hERbeta integrating target-ligand interactions and liquid chromatography-high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-(1)H, 2D-COSY, 2D-NOESY, and (1)H-(13)C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.

Doping in sport: 3. Metabolic conversion of oral norethisterone to urinary 19-norandrosterone.

The detection of 19 norandrosterone (19-NA) in a competitor's urine sample is taken as prima facie evidence of administration of nandrolone or other 19-norsteroid but a potential problem is that administration of norethisterone, a progestogen used for menstrual disorders and for hormonal contraception, also results in the excretion of 19-NA that can exceed the laboratory reporting threshold of 2ng/mL. The contribution of norethisterone to urinary 19-NA with and without 19-norandrostenedione, a known norethisterone tablet impurity, requires evaluation. Preparations containing, either <2ng or 1microg 19-norandrostenedione impurity per 5mg of norethisterone, administered to female volunteers (n=10) in doses comparable to those used for menstrual disorders (5mg three times daily for 10 days), resulted in maximal 19-NA concentrations of 51 and 63ng/mL, respectively. The maximal concentration of 19-NA, 2h post-administration of a single 1microg dose of 19-norandrostenedione, was 2.4ng/mL. These results prove unequivocally that norethisterone is metabolized to 19-NA and that there is only a minor contribution from the impurity 19-norandrostenedione. Administration to women (n=30) of a single contraceptive tablet containing norethisterone (1mg) with one of the highest proportions of the impurity 19-norandrostenedione ( approximately 0.5microg, 0.05%, w/w) resulted in a urinary 19-NA concentration of 9.1ng/mL, with a maximum concentration ratio of 19-NA to the norethisterone metabolite 3alpha,5beta-tetrahydronorethisterone of 0.36. We provide data that should remove the need for time-consuming follow-up investigations to consider whether doping with 19-norandrogens has occurred.

Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay.

The recent identification of tetrahydrogestrinone (THG), a non-marketed designer androgen used for sports doping but previously undetectable by established mass spectrometry-based urine drug screens, and its production by a facile chemical modification of gestrinone has raised concerns about the risks of developing designer androgens from numerous marketed progestins. We therefore have used yeast-based in vitro androgen and progesterone bioassays to conduct a structure-activity study assessing the intrinsic androgenic potential of commercially available progestins and their derivatives, to identify those compounds or structures with the highest risk of forming a basis for such misapplication. Progestins had a wide range of androgenic bioactivity that was not reliably predicted for individual steroids by their progestin bioactivity. 17alpha-Hydroxyprogesterone and 19-norprogesterone derivatives with their bulky 17beta-substituents were strong progestins but generally weak androgens. 17alpha-Ethynylated derivatives of testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone such as gestrinone, ethisterone, norethisterone and norgestrel had the most significant intrinsic androgenicity of all the commercially marketed progestins. Facile chemical modification of the 17alpha-ethynyl group of each of these progestins produces 17alpha-methyl, ethyl and allyl derivatives, including THG and norbolethone, which further enhanced androgenic bioactivity. Thus by using the rapid and sensitive yeast bioassay we have screened a comprehensive set of progestins and associated structures and identified the ethynylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives as possessing the highest risk for abuse and potential for conversion to still more potent androgens. By contrast, modern progestins such as progesterone, 17alpha-hydroxyprogesterone and 19-norprogesterone derivatives had minimal androgenic bioactivity and pose low risk.

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.

Can 19-nortestosterone derivatives be aromatized in the liver of adult humans? Are there clinical implications?

CONTEXT: Previous studies in postmenopausal women have demonstrated that, after oral administration of norethisterone, a small proportion of the compound is rapidly converted into ethinylestradiol. The shape of the concentration - time curve suggested that this occurred in the liver. The results were confirmed by in vitro investigations with adult human liver tissue. In 2002, it was shown that, after oral treatment of women with tibolone, aromatization of the compound occurred, resulting in the formation of a potent estrogen, 7 alpha-methyl-ethinylestradiol. The result has been called into question, because the adult human liver does not express cytochrome P450 aromatase, which is encoded by the CYP 19 gene. Moreover, it has been claimed that the serum level of 7 alpha-methyl-ethinylestradiol measured by gas chromatography/mass spectrometry was an artifact. REPLY: Aromatization of steroids is a complex process of consecutive oxidation reactions which are catalyzed by cytochrome P450 enzymes. The conversion of the natural C19 steroids, testosterone and androstenedione, into estradiol-17beta and estrone is dependent on the oxidative elimination of the angular C19-methyl group. This complex key reaction is catalyzed by the cytochrome P450 aromatase, which is expressed in many tissues of the adult human (e.g. ovary, fat tissue), but not in the liver. However, 19-nortestosterone derivatives are characterized by the lack of the C19-methyl group. Therefore, for the aromatization of these synthetic steroids, the action of the cytochrome P450 aromatase is not necessary and the oxidative introduction of double bonds into the A-ring can be catalyzed by other hepatic cytochrome P450 enzymes. The final key process in the formation of a phenolic A-ring, both in natural androgens and 19-nortestosterone derivatives, is the enolization of a 3-keto group to the C2-C3-enol or the C3-C4-enol moiety, which occurs without the action of enzymes. CONCLUSION: 19-nortestosterone derivatives (norethisterone, norethynodrel, tibolone) can readily be aromatized in the adult human liver. This leads to the formation of the potent estrogens ethinylestradiol from norethisterone or norethynodrel and 7 alpha-methyl-ethinylestradiol from tibolone. This may have clinical consequences, e.g. the elevated risk of venous thromboembolic disease in premenopausal women treated with high doses of norethisterone for bleeding disorders, or the elevated risk of stroke or endometrial disease in postmenopausal women treated with tibolone.

The effects of synthetic 19-norprogestins on osteoblastic cell function are mediated by their non-phenolic reduced metabolites.

The key role of estrogens on osteoblastic cell function is well documented; however, the role of progesterone (P) and synthetic progestins remains controversial. While several reports indicate that P has no significant effects on bone cells, a number of clinical studies have shown that 19-norprogestins restore postmenopausal bone loss. The mechanisms by which 19-norprogestins induce estrogen-like effects on bone cells are not fully understood. To assess whether the actions of 19-norprogestins on osteoblasts are mediated by their non-phenolic metabolites, we studied the effects of norethisterone (NET), levonorgestrel (LNG), and two of their A-ring reduced derivatives upon cell proliferation and differentiation in neonatal rat osteoblasts. Osteoblast function was assessed by determining cell DNA, cell-associated osteocalcin and calcium content, alkaline phosphatase activity, and mineral deposition. P failed to induce changes on osteoblasts, while NET and LNG exerted a number of actions. The most striking finding was that the 3beta,5alpha- and 3alpha,5alpha-tetrahydro derivatives of NET and LNG induced osteoblast proliferation and differentiation with higher potency than those exerted by their parent compounds, mimicking the effects of estradiol. Interestingly, osteoblast differentiation and mineral deposition induced by NET and LNG were abolished by finasteride, a 5alpha-reductases inhibitor, while the potent effect on osteoblast proliferation induced by progestin derivatives was abolished by a steroidal antiestrogen. Results demonstrate that A-ring reduced derivatives of NET and LNG exhibit intrinsic estrogen-like potency on rat osteoblasts, offering a plausible explanation for the mechanism of action of 19-norprogestins in bone restoration in postmenopausal women and providing new insights for hormone replacement therapy research.

Lack of aromatisation of the 3-keto-4-ene metabolite of tibolone to an estrogenic derivative.

Tibolone is used for the treatment of climacteric symptoms in postmenopausal women. It is metabolised in a tissue-specific manner so that while some metabolites exert estrogenic effects on bone and the CNS, others are thought to protect the breast and endometrium from estrogenic stimulation. Tibolone is a 7alpha-methyl derivative of 19-norethynodrel. Since the introduction of synthetic progestagens for therapeutic use there has been considerable controversy as to whether they can undergo aromatisation to give rise to the potent estrogen, ethinylestradiol. In this study, we examined whether the delta-4-ene (7alpha-methyl norethisterone) metabolite of tibolone, which has a similar delta-4-ene A-ring structure to that of the estrone precursor, androstenedione, could undergo aromatisation to the potent estrogen, 7alpha-methyl ethinylestradiol. For these studies, JEG-3 choriocarcinoma cells were employed as they have a very high level of aromatase activity. TLC and HPLC procedures were developed to separate phenolic from non-phenolic compounds and were initially used to confirm that JEG-3 cells readily aromatised androstenedione to estrogens (up to 74%). The aromatisation of androstenedione to estrogens by these cells could be completely blocked with the potent aromatase inhibitor letrozole. When [(3)H] 7alpha-methyl norethisterone was incubated with JEG-3 cells no evidence for its conversion to [(3)H] 7alpha-ethinylestradiol was obtained. Radioactivity detected on the TLC plate or HPLC fractions where standard 7alpha-methyl ethinylestradiol was located, revealed that similar levels were present when 7alpha-methyl norethisterone was incubated with culture medium alone or with JEG-3 cells in the absence or presence of letrozole. From these investigations, it is concluded that 7alpha-methyl norethisterone does not undergo aromatisation to an estrogenic derivative.

Synthetic progestins used in HRT have different glucocorticoid agonist properties.

The synthetic progestins, medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-EN or NET-A), are widely used as female contraceptive agents and in hormone replacement therapy (HRT). Competitive binding revealed that MPA displays a higher relative binding affinity than NET-A and progesterone (prog) for the human GR (Kd of 4.2 nM for dexamethasone (dex) and Ki's of 10.8, 270 and 215 nM for MPA, NET-A and prog, respectively). Furthermore, MPA displays much greater glucocorticoid (GC) transactivation agonist potency than NET-A or prog (EC50s of 1.1, 7.2, >1000 and 280 nM for dex, MPA, NET-A and prog, respectively) and much greater GC agonist potency for transrepression than NET-A or prog (EC50s of 0.21, 2.7, >100 and 26 nM for dex, MPA, NET-A and prog, respectively). In addition, MPA induces phosphorylation of the GR at Ser 211 to a much greater extent than NET-A or prog and protects the GR from partial trypsin digestion in vitro to a much greater extent than NET-A or prog at saturating concentrations. Together these results suggest that the differences in biological activity of the progestins are not merely due to differences in their affinity for the GR but also due to the induction of different conformational changes in the liganded-GR. MPA and NET-A therefore display very different GC-like properties compared to each other and to prog, and are likely to exhibit different side effects via the GR.

The interaction between St John's wort and an oral contraceptive.

OBJECTIVES: The popular herbal remedy St John's wort is an inducer of cytochrome P450 (CYP) 3A enzymes and may reduce the efficacy of oral contraceptives. Therefore we evaluated the effect of St John's wort on the disposition and efficacy of Ortho-Novum 1/35 (Ortho-McNeil Pharmaceutical, Inc, Raritan, NJ), a popular combination oral contraceptive pill containing ethinyl estradiol (INN, ethinylestradiol) and norethindrone (INN, norethisterone). METHODS: Twelve healthy premenopausal women who were using oral contraception (>3 months) received a combination oral contraceptive pill (Ortho-Novum 1/35) for 3 consecutive 28-day menstrual cycles. During the second and third cycles, the participants received 300 mg St John's wort 3 times a day. The serum concentrations of ethinyl estradiol (day 7), norethindrone (day 7), follicle-stimulating hormone (days 12-16), luteinizing hormone (days 12-16), progesterone (day 21), and intravenous and oral midazolam (days 22 and 23) were determined in serial blood samples. The incidence of breakthrough bleeding was quantified during the first and third cycles. RESULTS: Concomitant use of St John's wort was associated with a significant (P <.05) increase in the oral clearance of norethindrone (8.2 +/- 2.7 L/h to 9.5 +/- 3.4 L/h, P =.042) and a significant reduction in the half-life of ethinyl estradiol (23.4 +/- 19.5 hours to 12.2 +/- 7.1 hours, P =.023). The oral clearance of midazolam was significantly increased (109.2 +/- 47.9 L/h to 166.7 +/- 81.3 L/h, P =.007) during St John's wort administration, but the systemic clearance of midazolam was unchanged (37.7 +/- 11.3 L/h to 39.0 +/- 10.3 L/h, P =.567). Serum concentrations of follicle-stimulating hormone, luteinizing hormone, and progesterone were not significantly affected by St John's wort dosing (P >.05). Breakthrough bleeding occurred in 2 of 12 women in the control phase compared with 7 of 12 women in the St John's wort phase. The oral clearance of midazolam after St John's wort dosing was greater in women who had breakthrough bleeding (215.9 +/- 66.5 L/h) than in those who did not (97.5 +/- 37.2 L/h) (P =.005). CONCLUSION: St John's wort causes an induction of ethinyl estradiol-norethindrone metabolism consistent with increased CYP3A activity. Women taking oral contraceptive pills should be counseled to expect breakthrough bleeding and should consider adding a barrier method of contraception when consuming St Johns wort.

Vasodilating effect of norethisterone and its 5 alpha metabolites: a novel nongenomic action.

Estrogens are generally administered in hormone replacement therapy in combination with synthetic progestins. Studies of cardiovascular risk factors in postmenopausal women have shown a variety of responses according to the molecular structure of the progestin used in hormone replacement therapy schemes. The present study sets out to determine the vasoactive effects of norethisterone and its 5alpha-dihydro (5alpha-norethisterone) and -tetrahydro (3alpha,5alpha-norethisterone and 3beta,5alpha-norethisterone) metabolites in isolated precontracted rat thoracic aorta. The addition of norethisterone and 3alpha,5alpha-norethisterone in rat aorta exhibited a potent, concentration-response inhibition of noradrenaline-induced contraction, while 5alpha- and 3beta,5alpha-norethisterone had very little, if any, vasorelaxing effect. Relaxation to norethisterone and 3alpha,5alpha-norethisterone had very rapid time-courses and it was neither affected by the absence of endothelium nor by the inhibitor of nitric oxide synthase, Nomega-nitro-L-arginine methyl ester (L-NAME). The addition of specific anti-androgen, anti-progestin and anti-estrogen compounds and protein synthesis inhibitors did not preclude the vasorelaxing effect of norethisterone and its 3alpha,5alpha-reduced metabolite. The results strongly suggest that these effects are not mediated by nuclear sex steroid hormone receptors. The overall data document a novel nongenomic endothelium-independent vasorelaxing action of a 19-nor synthetic progestin and one of its A-ring-reduced derivatives.