In vitro exposure to environmentally relevant concentrations of norgestrel affects sperm physiology and reproductive success of the Pacific oyster Crassostrea gigas.

Progestins in aquatic environments are of increasing concern, as shown by the results of toxicological studies on adult invertebrates with external fertilization. However, their potential effects on the gametes and reproductive success of such animals remain largely unknown. Thus, the current study assessed the effect of in vitro exposure of environmentally relevant concentrations (10 ng/L and 1000 ng/L) of norgestrel (NGT) on the sperm of Pacific oyster Crassostrea gigas, analyzing sperm motility, ultrastructure, mitochondrial function, ATP status, characteristic enzyme activities, and DNA integrity underlying fertilization and hatching success. The results showed that NGT increased the percentage of motile sperm by elevating intracellular Ca2+ levels, Ca2+-ATPase activity, creatine kinase activity, and ATP content. Although superoxide dismutase activity was enhanced to eliminate reactive oxygen species generated by NGT, oxidative stress occurred, as indicated by the increase in malonaldehyde content and damage to plasma membranes and DNA. As a consequence, fertilization rates decreased. However, hatching rates did not alter significantly, possibly as a result of DNA repair processes. This study demonstrates oyster sperm as a useful, sensitive tool for toxicological research of progestins and provides ecologically relevant information on reproductive disturbance in oysters resulting from exposure to NGT.

Norgestrel causes digestive gland injury in the clam Mactra veneriformis: An integrated histological, transcriptomics, and metabolomics study.

The potential adverse effects of progestins on aquatic organisms, especially non-target species, are of increasing concern worldwide. However, the effect and mechanism of progestin toxicity on aquatic invertebrates remain largely unexplored. In the present study, clams Mactra veneriformis were exposed to norgestrel (NGT, 0, 10, and 1000 ng/L), the dominant progestin detected in the aquatic environment, for 21 days. NGT accumulation, histology, transcriptome, and metabolome were assessed in the digestive gland. The bioconcentration factor (BCF) was 386 and 268 in the 10 ng/L NGT group and 1000 ng/L NGT group, respectively, indicating efficient accumulation of NGT in the clams. Histological analysis showed that NGT led to the swelling of epithelial cells and blurring of the basement membrane in the digestive gland. Differentially-expressed genes and KEGG pathway enrichment analysis using a transcriptomic approach suggested that NGT primarily disturbed the detoxification system, antioxidant defense, carbohydrate and amino acid metabolism, and steroid hormone metabolism, which was consistent with the metabolites analyzed using a metabolomic approach. Furthermore, we speculated that the oxidative stress caused by NGT resulted in histological damage to the digestive gland. This study showed that NGT caused adverse effects in the clams and sheds light on the mechanisms of progestin interference in aquatic invertebrates.

Assessment of porphyrogenicity of drugs and chemicals in selected hepatic cell culture models through a fluorescence-based screening assay.

Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence-based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase-1 [ALAS-1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS-1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, ß-estradiol, hydroxychloroquine sulfate, alpha-methyldopa, D (-) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi-well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

Molecular insights into how SHBG dimerization exerts changes on ligand molecular recognition.

Sex hormone binding globulin (SHBG) is a homodimeric glycoprotein and is the major carrier protein for sex steroids in plasma, regulating sex hormone availability in most vertebrate groups. Although it was initially thought that human dimeric SHBG bound a single ligand at the homodimer interface, studies demonstrated that dimeric SHBG binds a ligand to each subunit with similar affinity. In fact, the findings from recent experimental studies suggest that ligand binding to the SHBG dimer involves a complex allosteric mechanism involving conformational changes that limit observations of the presence of allosteric regulation. Therefore, we combined structural data with molecular dynamics simulations using Molecular Mechanics Generalized-Born Surface Area (MMGBSA) to dissect the structural and energetic basis for molecular recognition between five ligands whose affinities and binding positions on SHBG are known, i.e., 3ß,17α-diol; 3ß,17ß-diol; DHT; norgestrel (NOG); and estradiol (E2), and monomeric and dimeric SHBG. Protein-ligand complexes, involving dimeric SHBG saturated with two ligands on each subunit, reproduce the experimental affinity tendency and allow the observation that dimerization exerts disparate effects on binding affinity, characteristic of negative cooperativity for E2, DHT, and NOG, whereas 3ß-17α-diol and 3ß-17ß-diol lack allostery.

Reproductive effects of synthetic progestin norgestrel in zebrafish (Danio rerio).

The aim of this study was to assess the adverse effects of synthetic progestin norgestrel (NGT) on the reproduction of zebrafish by measuring the egg production, histology and transcriptional expression profiles along the hypothalamic-pituitary-gonadal (HPG) axis in adult zebrafish. After a pre-exposure period of 7 days, adult zebrafish were exposed to 6, 29 and 69 ng L-1 NGT for 21 days. The results showed that exposure to 69 ng L-1 NGT led to a significant up-regulation of follicle stimulating hormone, beta polypeptide (fshb), luteinizing hormone, beta polypeptide (lhb), progesterone receptor (pgr), estrogen receptor 1 (esr1) and androgen receptor (ar) genes in the brains, as well as significant up-regulation of hydroxysteroid 20-beta dehydrogenase (hsd20b) and hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2) genes and down-regulation of 11-beta-hydroxylase (cyp11b) gene in the ovaries of females. In the testes of males, an overall down-regulation of steroidogenic acute regulatory protein (star), cytochrome P450-mediated side-chain cleavage enzyme (cyp11a1), cyp11b, hsd20b, hydroxysteroid 17-beta dehydrogenase type 3 (hsd17b3), hsd11b2 and ar genes were observed following exposure to different treatments of NGT. These transcriptional alterations imply that NGT could exhibit the potent progestogenic and androgenic activities in zebrafish. Egg production as well as histology in the ovaries and testes was not affected by NGT. Taken together, the overall results demonstrated that NGT could significantly affect transcriptional expression levels of genes related to HPG axis in zebrafish, and whether that change translates to additional physiological effects is needed further research.

Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling.

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue 'Norgestrel' is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel's neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.

Biotransformation of progesterone and norgestrel by two freshwater microalgae (Scenedesmus obliquus and Chlorella pyrenoidosa): transformation kinetics and products identification.

Natural and synthetic steroid hormones such as progesterone and norgestrel in the aquatic environment may cause adverse effects on aquatic organisms. This study investigated the biotransformation of progesterone and norgestrel in aqueous solutions by two freshwater microalgae Scenedesmus obliquus and Chlorella pyrenoidosa and elucidated their transformation mechanisms. More than 95% of progesterone was transformed by the two microalgae within 5d. For norgestrel, almost complete transformation by S. obliquus was observed after 5 d, but nearly 40% was remained when incubated with C. pyrenoidosa. The results also showed that these two compounds were not accumulated in the algal cells. Biotransformation was found to be the main mechanism for their loss in the aqueous solutions, and it followed the first-order kinetic model. For progesterone, three main transformation products, i.e. 3ß-hydroxy-5α-pregnan-20-one, 3,20-allopregnanedione and 1,4-pregnadiene-3,20-dione, and six minor androgens were identified. For norgestrel, only two transformation products, 4,5-dihydronorgestrel and 6,7-dehydronorgestrel, were identified for the first time. Hydroxylation, reduction and oxidation are proposed to be the main transformation pathways. Among the two microalgae species, S. obliquus was found more efficient in the transformation of the two target compounds than C. pyrenoidosa. The results clearly demonstrated the capability of the two microalgae to transform the two progestogens. The biotransformation and products could have significant environmental implications in the fate and effects of the two steroids.

Degradation of norgestrel by bacteria from activated sludge: comparison to progesterone.

Natural and synthetic progestagens in the environment have become a concern due to their adverse effects on aquatic organisms. Laboratory studies were performed to investigate aerobic biodegradation of norgestrel by bacteria from activated sludge in comparison with progesterone, and to identify their degradation products and biotransformation pathways. The degradation of norgestrel followed first order reaction kinetics (T1/2 = 12.5 d), while progesterone followed zero order reaction kinetics (T1/2 = 4.3 h). Four and eight degradation products were identified for norgestrel and progesterone, respectively. Six norgestrel-degrading bacterial strains (Enterobacter ludwigii, Aeromonas hydrophila subsp. dhakensis, Pseudomonas monteilii, Comamonas testosteroni, Exiguobacterium acetylicum, and Chryseobacterium indologenes) and one progesterone-degrading bacterial strain (Comamonas testosteroni) were successfully isolated from the enrichment culture inoculated with aerobic activated sludge. To our best knowledge, this is the first report on the biodegradation products and degrading bacteria for norgestrel under aerobic conditions.

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.

Delivery of ethinylestradiol from film forming polymeric solutions across human epidermis in vitro and in vivo in pigs.

Film forming polymeric solutions may present an alternative to the common transdermal dosage forms such as patches or gels. To evaluate the potential of these systems for transdermal drug delivery the permeation of ethinylestradiol from four formulations with different polymers was tested across heat separated human epidermis. The formulation with the best results was then modified by incorporating chemical enhancers to further increase the efficiency of the delivery system. Finally, drug delivery from the developed film forming systems was compared to a commercially available transdermal patch in vitro as well as in vivo in pigs. Among the tested preparations the formulation with polyurethane-14-AMP-acrylates copolymer (DynamX) showed the highest ethinylestradiol permeation. The drug transport was further increased with the incorporation of oleic acid as penetration enhancer, especially when used in combination with propylene glycol. The enhancing effect of oleic acid/propylene glycol was concentration-dependent and increased disproportionately with rising enhancer content. The film forming solution showed a higher ethinylestradiol permeation through heat separated human epidermis than the commercial EVRA patch in vitro and achieved measurable plasma concentrations of ethinylestradiol in vivo in pigs. These promising results encourage the further development of film forming polymeric solutions as novel transdermal dosage form.

Comparative progestational activity of norgestimate, levonorgestrel-oxime and levonorgestrel in the rat and binding of these compounds to the progesterone receptor.

The progestational activity of norgestimate (NORG), levonorgestrel-oxime (LNG-oxime) and levonorgestrel (LNG) were compared in a pregnancy maintenance study in rats. The compounds were administered subcutaneously to pregnant rats at several doses, blood samples were collected repeatedly, and the concentration of LNG was measured in these samples. It could be demonstrated that following the administration of NORG and LNG-oxime, LNG was a major metabolite present in the serum. The pharmacological response in rats treated with NORG and LNG-oxime could be related to the systemic exposure of these animals to metabolically derived LNG. Thus, both NORG and LNG-oxime can be regarded as pro-drugs of LNG, the latter being almost exclusively responsible for the pharmacological activity of both pro-drugs. This notion was further supported by studies on the comparative binding affinity of these compounds to rabbit and human progesterone receptor (PR). LNG exhibited the highest binding affinity of the compounds studied. Relative binding affinity (RBA) values of LNG using progesterone as reference (100%) were found to be 125% for rabbit PR (rPR), 143% for human uterine PR (hPR) and 125% for recombinant hPR, respectively. In contrast to LNG, NORG exhibited only a low affinity to the PR, which is documented by RBA values of 1.2% for rPR, 3.2% for uterine hPR and 9% for recombinant hPR. The corresponding values of LNG-oxime were 30% (rPR), 20% (uterine hPR) and 18% (recombinant hPR), respectively. Thus, the combined experimental evidence of the present study does not support the view of NORG being a progestogen on its own as has been suggested by others.

Metabolism of the oral contraceptive steroids ethynylestradiol, norgestimate and 3-ketodesogestrel by a human endometrial cancer cell line (HEC-1A) and endometrial tissue in vitro.

Human endometrial cancer cells and human endometrial tissue have been extensively used to study steroid hormone action and metabolism. The natural estrogens estradial (E2) and estrone (E1) are known to be metabolized by both cells and tissue with the interconversion of the two steroids and the formation of sulphate conjugates. The aim of the present work was to see if the commonly used oral contraceptive steroids ethynylestradiol (EE2), norgestimate (Ngmate) and 3-ketodesogestrel (3-KDG) were metabolized by a human endometrial cancer cell line (HEC-1A) and human endometrial tissue in vitro. Metabolites were analysed by on-line radiometric HPLC. Endometrial tissue was obtained from women undergoing dilation and curettage or hysterectomy operations. In preliminary studies with endogenous estrogens, HEC-1A cells were able to interconvert E1 and E2; the equilibrium favouring the formation of E2. Normal endometrial tissue extensively converted E2 to E1, tumour tissue appeared to catalyse this reaction much less avidly. In addition sulphate conjugates were formed by normal tissue from some patients. Cell line and endometrial tissue was able to hydrolyse estrone 3-sulphate. With EE2 as substrate there was no evidence of phase I metabolism by cell line or tissue. However, conversion to the presumed 3-sulphate conjugate was observed following incubation with normal tissue from some women. Deacetylation of the progestogen Ngmate to norgestrel oxime (NgOx) was complete within 24 h. There was also some loss of the oxime moiety to give norgestrel (Ng) following incubation with HEC-1A cells. Metabolism of Ngmate was also complete within 24 h following incubation with endometrial tissue. There were both qualitative and quantitative differences in metabolite formation between tissue obtained from different women. In contrast, 3-KDG was relatively resistant to metabolism by cell line and tissue. The major metabolite formed by HEC-1A cells accounted for only 3.3 +/- 0.4% of total added radiolabelled steroid and co-chromatographed with 3 alpha-hydroxydesogestrel. Smaller amounts of other radiometabolites were formed. No phase I metabolites of 3-KDG were formed by normal endometrial tissue, however small amounts of radiometabolites appeared to be formed by malignant tissue. These studies have provided evidence to suggest that the oral contraceptives EE2, Ngmate and 3-KDG are metabolized in the human endometrium. Knowledge of the metabolism of these in target tissues such as the endometrium may be pertinent considering the possibility that metabolites may exert specific effects.

PIP: In England, pharmacologists and a biochemist at the University of Liverpool used an established human endometrial cancer cell line (HEC-1A) and human endometrial tissue in vitro to confirm that HEC-1A and tissue metabolize oral contraceptive (OC) steroids. They used on-line radiometric high-performance liquid chromatography to analyze metabolic activity. Surgeons obtained the endometrial tissue from women undergoing dilatation and curettage or hysterectomy at the Royal Liverpool University Hospital. Earlier research showed that HEC-1A cells interconvert estrone (E1) and 17 beta-estradiol (E2), with E2 predominating in the equilibrium. In this in vitro study, healthy endometrial tissue extensively changed E2 to E1, while malignant tissue caused this conversion to a much lesser extent. The healthy endometrial tissue of some patients formed sulphate conjugates. Both HEC-1A and endometrial tissue hydrolyzed E1 3-sulphate. They did not bring about phase I metabolism when ethinyl estradiol (EE2) was the substrate. Yet, incubation with healthy tissue from some women did lead to conversion of the presumed 3-sulphate conjugate. Incubation with HEC-1A cells completely removed the acetyl group from norgestimate, resulting in mainly norgestrel oxime (55.1% of metabolites) within 24 hours. It also resulted in some norgestrel (16.3%). Incubation with endometrial tissue also brought about complete metabolism of norgestimate within 24 hours. The tissue from different women brought about qualitative and quantitative differences. HEC-1A and endometrial tissue did not metabolize much of 3-ketodesogestrel (3-KDG). In fact, the major metabolite formed by HEC-1A was 3 alpha-hydroxydesogestrel, which made up 3.3% of total added radiolabeled steroid. Healthy endometrial tissue did not produce any phase I metabolites of 3-ketodesogestrel, while tumor tissue may have produced a small amount of radiometabolites. These findings indicate that the endometrium does metabolize the OC EE2, 3-KDG, and norgestimate.

Receptor binding of norgestimate--a new orally active synthetic progestational compound.

Binding of the new progestagen, norgestimate (D-(-)-13 beta-ethyl-17 beta-acetoxy-17-ethinyl-4-gonen-3-one-oxime), and its metabolites (levonorgestrel-3-oxime, levonorgestrel-17-acetate and levonorgestrel) to the progesterone receptor was investigated by competition experiments using cytosol from human myometrial tissue. Compared to R5020, a highly potent synthetic ligand for progesterone receptor analysis, the L-isomer of norgestimate shows only a weak specific behaviour with regard to binding to the progesterone receptor from uterine cytosol with an RBA value of 0.8%, whereas the D-isomer of this compound is characterized by a lack of binding activity to the progesterone receptor. Levonorgestrel-3-oxime, one of the possible metabolites of norgestimate, binds to the progesterone receptor with an RBA value of 8%, whereas levonorgestrel-17-acetate, the other possible metabolite of norgestimate, binds with a binding affinity of 110% which is in the same order of magnitude as levonorgestrel itself. The competition experiments suggest that norgestimate is a prodrug and that the metabolites, levonorgestrel and levonorgestrel-17-acetate, which actively bind to the progesterone receptor, must first be formed from the parent drug via metabolic processes in vivo. These are the actual biologically active compounds which are responsible for the gestagenic potency.

Preclinical evaluation of norgestimate, a progestin with minimal androgenic activity.

Norgestimate is a novel progestin with highly selective progestational activity and minimal androgenicity. In rabbits, norgestimate binds to uterine progestin receptors, stimulates the endometrium, and inhibits ovulation. Norgestimate acts directly on target organs, stimulating rabbit endometrium when injected into the uterine cavity and inhibiting luteinizing hormone-releasing hormone-stimulated luteinizing hormone release in dispersed rat pituitary cells in culture. Norgestimate has no estrogenic activity, and like other progestins, it suppresses the action of estrogen. Unlike some other progestins, it is relatively free of androgenic activity. Norgestimate and its 17-deacetylated metabolite demonstrate very poor affinity for androgen receptors compared with levonorgestrel and gestodene and do not exhibit androgenic activity when measured as the stimulation of prostatic growth in immature rats. Norgestimate's lack of affinity for human sex hormone-binding globulin is further evidence of its minimal androgenicity.

PIP: The androgenic activity of progestins used in oral contraceptives (OCs) may be responsible for the adverse changes in lipid and lipoprotein metabolism, so researchers are continuing to work on developing progestins that reduce androgenicity. They have developed norgestimate (NGM) which is a progestin with improved selectivity. i.e., extent of maximization of progestational potency and minimization of androgenic potency. The receptor binding affinities of ngm and its major metabolite, 17-deacetylated norgestimate for the progestin receptor are on par with progesterone. Their binding affinities are only 0.003 and 0.013 times that of dihydrotestosterone, respectively, while the affinities for levonorgestrel and gestodene are 0.220 and 0.154 times that of dihydrotestosterone, respectively. They have almost no affinity for human sex hormone binding globulin (SHBG) in vitro. clinical research of 40 women using combined OCs with NGM and ethinyl estradiol over 4 cycles shows that NGM does not prevent estrogen-induced rises in SHBG levels indicating its minimal androgenicity. In addition, NGM successfully suppresses ovulation in rabbits, rats, hamsters, and mice by preventing the preovulatory rise of luteinizing hormone and, in rats, by targeting the hypothalamic/pituitary axis. NGM also allows rats and rabbits to effectively maintain pregnancy by stimulating the endometrium. It also has no estrogenic activity in vivo and does not bind to estrogen receptors in vitro. Specifically NGM prevents estrogen-induced vaginal cornification. These findings from various preclinical research and 1 clinical study demonstrate NGM to be a selective progestin which reduces androgenicity.

Mechanisms of hormonal and antihormonal action of contraceptive progestins at the molecular level.

19-Nor synthetic progestins undergo extensive metabolism at the target cells. The resulting metabolic conversion products interact with putative steroid receptors within the cells, and through those interactions, they may exert either agonistic, synergistic and antagonistic hormonal effects. Studies conducted in our laboratories have disclosed that norethisterone (NET) and D-(1) norgestrel (LNG), two widely used contraceptive progestins, are biotransformed to several A-ring reduced (dihydro and tetrahydro) derivatives. The resulting metabolites 5 alpha-dihydro NET (5 alpha-NET) and 5 alpha-dihydro LNG bind with relative high affinity to the progesterone and androgen receptors. To gain insight into the underlying molecular events mediating the mode of action of NET and its neutral metabolites, we have examined the expression of their biological effects at target organs by using the rabbit uteroglobin gene model and the beta-glucuronidase activity of the mouse kidney. The results of a series of experiments seem to indicate that the enzyme-mediated formation of the 5 alpha (trans A/B ring junction) NET derivative results in a significant diminution of its progestational and androgenic potencies. Furthermore, 5 alpha-NET acquire a potent anti-progestational/contragestational effect as assessed in the female rabbit. These results demonstrated that 5 alpha-reduction of 19-nor progestins exerts a paradoxical effect, at least in terms of their hormone-like effects. The overall data are in line with the concept that metabolism of synthetic progestins at hormone-sensitive organs modulates their mechanisms of action.

Metabolism of the oral contraceptive steroids ethynylestradiol and norgestimate by normal (Huma 7) and malignant (MCF-7 and ZR-75-1) human breast cells in culture.

Human breast cancer cells are used extensively for the study of steroid hormone action. It is known that in both receptor positive and receptor negative cell lines there is considerable metabolism of the natural estrogens, estradiol (E2) and estrone (E1) with interconversion of the two steroids and formation of sulphate and glucuronide conjugates. The aim of the present work was to see if the commonly used oral contraceptive steroids (OCS) ethynylestradiol (EE2) and norgestimate (Ngmate) were metabolized in human breast cancer cell lines (MCF-7 and ZR-75-1) and a normal breast cell line (Huma 7). MCF-7, ZR-75-1 and Huma 7 cells were maintained in Dulbeccos Modified Eagles Medium (DMEM) containing foetal calf serum (FCS) insulin and hydrocortisone. In addition, ZR-75-1 cells required epidermal growth factor (EGF) and E2 while MCF-7 cells required only EGF. On reaching confluence cells were transferred to DMEM containing charcoal-stripped FCS, insulin and hydrocortisone. 48 h later this medium was renewed, radiolabelled steroid ([3H]E1; [3H]E2; [3H]EE2, [3H]Ngmate; [3H]E1-SO4; 1 nM; 0.2 microCi) was added and incubation was for 24 or 48 h. Following incubation, the medium was removed and radioactive steroid extracted with ether. Metabolites were analysed by on-line radiometric HPLC. All the cell lines were able to interconvert E1 and E2; the equilibrium favouring the formation of E2 in MCF-7 and ZR-75-1 and E1 in Huma 7 cells. E1 and E2 also underwent phase II metabolism to form their respective estrogen sulphates, this activity being most marked in the Huma 7 cell line. In addition to sulphotransferase activity, the study with E1 sulphate demonstrated sulphatase activity in both normal and cancer cells. There appeared to be no difference in extent of hydrolysis, with both E1 and E2 formed. With EE2 as substrate there was no evidence of phase I metabolism in any of the cell lines but there was conversion to the presumed 3-sulphate conjugate. The percentage formation of this metabolite was very much greater in Human 7 cells (64.1 +/- 9.6% after 24 h) than in MCF-7 and ZR-75-1 cells (7.4 +/- 5.3% and 10.6 +/- 4.1%, respectively after 24 h). In all the cell lines deacetylation of the progestogen Ngmate to norgestrel oxime was complete within 24 h. In addition there was evidence of loss of the oxime moiety to give norgestrel.(ABSTRACT TRUNCATED AT 400 WORDS)

Progestin-rhenium complexes: metal-labeled steroids with high receptor binding affinity, potential receptor-directed agents for diagnostic imaging or therapy.

In order to investigate the possibility of developing diagnostic imaging agents for steroid-positive tumors that are labeled with the readily available radionuclide technetium-99m, we prepared four conjugate systems in which a progestin is linked to a metal chelate system. Three of these are bis-amino bis-thiol (BAT or N2S2) systems and are linked through carbon-21 of progesterone or the 17 alpha- or 11 beta-position of a nortestosterone type progestin. The fourth, an amino-amido-thiol-alcohol chelate (N2OS) system, is linked at the 16 alpha,17 alpha-positions of a dihydroprogesterone. As a model for technetium-labeled complexes, all four chelate systems were converted to their oxo-rhenium complexes. Of the four possible diastereomers in the 16 alpha,17 alpha-system, only one was isolated, while of the four possible diastereomers in the other systems, a syn pair and an anti pair (linker methylene vs rhenium-oxo, relative to the N2S2 plane) were separated in the 17 alpha-substituted series, a syn pair was isolated in the 21-substituted series, and a syn pair and the two individual anti diastereomers were separated in the 11 beta-substituted series. In competitive radiometric receptor binding assays, the 21-, 17 alpha-, and 16 alpha,17 alpha-linked systems had low affinity for the progesterone receptor (less than 0.3% that of promegestone (R5020) or 2% that of progesterone). By contrast, the two anti diastereomers of the 11 beta-linked system had affinities that were 10% and 44% that of R5020 (or 64% and 283% that of progesterone) and the syn pair had an affinity 25% that of R5020 (or 161% that of progesterone). The latter finding indicates that it is possible to prepare metal-labeled steroids that retain high affinity for steroid receptors. These and related systems, when complexed with radioactive metals, may be useful in vivo as receptor-directed agents for diagnostic imaging or therapy of steroid receptor-positive tumors.

Metabolism of norgestimate by human gastrointestinal mucosa and liver microsomes in vitro.

The metabolism of the progestogen oral contraceptive norgestimate has been studied in vitro using human intestinal mucosa and human liver microsomes. Metabolites have been separated using radiometric high-performance liquid chromatography (HPLC) and identified by co-chromatography with authentic standards and by mass spectrometry. Histologically normal colon was obtained from 6 patients undergoing various resections and the mucosa mounted between 2 perspex (Ussing) chambers. 2 h after addition of [3H]norgestimate to the mucosal chamber, more than 95% of the radioactivity was present in that chamber. Metabolite analysis showed 38.1 +/- 11.6% (mean +/- SD; n = 8) of drug present was norgestimate, 49.2 +/- 14.5% as 17-deacetyl norgestimate and 8.1 +/- 4.5% as conjugated metabolites. Small amounts of 3-keto norgestimate, norgestrel and uncharacterized metabolites were found. Norgestimate was also metabolized by stomach tissue with 17-deacetyl norgestimate again being the main metabolite found. Microsomes were prepared from 6 human livers. Metabolism was studied over a 5 h time-course in the absence and presence of NADPH. Deacetylation to 17-deacetyl norgestimate took place in the absence of the cofactor. In the presence of NADPH, after 5 h incubation only 30.5 +/- 14.6% (mean +/- SD) of steroid present was norgestimate. The major metabolite formed was 17-deacetyl norgestimate which accounted for 39.3 +/- 20.5%. Less than 2% was present as 3-keto norgestimate but 10.0 +/- 2.3% was identified as norgestrel and 15.5 +/- 8.9% as uncharacterized metabolites. We also examined the microsomal breakdown of [3H]17-deacetyl norgestimate. This was NADPH and oxygen dependent. Norgestrel and other metabolites were formed. This study has demonstrated that norgestimate is rapidly deacetylated by both gut wall and liver. The deacetylated metabolite can then be further metabolized.

Gastrointestinal metabolism of contraceptive steroids.

A number of oral contraceptive steroids undergo first-pass metabolism in the gastrointestinal mucosa. Ethinyl estradiol (mean systemic bioavailability 40% to 50%) is extensively metabolized, principally to a sulfate conjugate. In vivo studies that use portal vein catheterization and the administration of radiolabeled ethinyl estradiol have shown that the fraction of steroid metabolized in the gut wall is 0.44. In vitro studies with jejunal biopsy samples or larger pieces of jejunum or terminal ileum mounted in Ussing chambers have indicated that more than 30% of added ethinyl estradiol is sulfated. The progestogen desogestrel is a prodrug that is converted to the active metabolite 3-ketodesogestrel. Substantial first-pass metabolism of desogestrel occurs in the gut mucosa, with evidence from Ussing chamber studies for the formation of the active metabolite. Another progestogen, norgestimate, is also metabolized by the gut wall in vitro of which the principal metabolite is the deacetylated product, norgestrel oxime. It seems very likely that this will also occur in vivo. Drug interactions occurring in the gut wall have been reported with ascorbic acid (vitamin C) and paracetamol.