Heterocyclic androstane and estrane d-ring modified steroids: Microwave-assisted synthesis, steroid-converting enzyme inhibition, apoptosis induction, and effects on genes encoding estrogen inactivating enzymes.

d-ring-fused and d-homo lactone compounds in estratriene and androstane series were synthesized using microwave-assisted reaction conditions. Microwave-irradiated synthesis methods were convenient and effective, and provided high yields with short reaction times. Their inhibition of C17,20-lyase and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities were studied in in vitro enzyme assays. d-ring-fused triazolyl estrone analog 24 showed potent inhibition of NADH-complexed 17ß-HSD1, with a binding affinity similar to that of the substrate estrone; its inhibition against NADPH-complexed 17ß-HSD1 was markedly weaker. Compound 24 also significantly and selectively reduced proliferation of cancer cell lines of gynecological origin. This estrane triazole changed the cell cycle and induced apoptosis of HeLa, SiHa, and MDA-MB-231 cancer cells, measured by both increased subG1 fraction of cells and activation of caspase-independent signaling pathways. A third mode of anti-estrogenic action of 24 saw increased mRNA expression of the SULT1E1 gene in HeLa cells; in contrast, its 3-benzyloxy analog 23 increased mRNA expression of the HSD17B2 gene, thus showing pronounced pro-drug anti-estrogenic activity. Estradiol-derived d-ring triazole compound 24 thus acts at the enzyme, gene expression and cellular levels to decrease the production of active estrogen hormones, demonstrating its pharmacological potential.

Minor chemical modifications of the aminosteroid derivative RM-581 lead to major impact on its anticancer activity, metabolic stability and aqueous solubility.

The aminosteroid (AM) RM-581 is built around a mestranol backbone and has recently emerged as this family's lead candidate, showing in vitro and in vivo potency over different types of cancer, including high fatality pancreatic cancer. To extend the structure-activity relationships (SAR) to other estrane analogs, we synthesized a focused series of RM-581 derivatives at position C3 or C2 of its steroidal core. These new AM derivatives were first tested on a large selection of prostate, breast, pancreatic and ovarian cancer cell lines. The impact of these modifications on metabolic stability (human liver microsomes) was also measured. A SAR study revealed a fine regulation of anticancer activity related to the nature of the substituent. Indeed, the addition of potential prodrug groups like acetate, sulfamate or phosphate (compounds 8, 9 and 10) at C3 of the phenolic counterpart provided better antiproliferative activities than RM-581 in breast and pancreatic cancer cell types while maintaining activity in other cancer cell lines. Also, the phosphate group was highly beneficial on water solubility. However, the bulkier carbamate prodrugs 6 (N,N-dimethyl) and 7 (N,N-diethyl) were less active. Otherwise, carbon homologation (CH2) at C2 (compound 33) was beneficial to metabolic stability and, in the meantime, this AM conserved the same anticancer activity as RM-581. However, the replacement of the hydroxy or methoxy at C3 by a hydrogen or an acetyl (compound 17 or 21b) was detrimental for anticancer activity, pointing to a crucial molecular interaction of the aromatic oxygen atom at this position. Overall, this work provided a better knowledge of the structural requirements to maintain RM-581's anticancer activity, and also identified minor structural modifications to increase both metabolic stability and water solubility, three important parameters of pharmacological development.

Women's fertility cues affect cooperative behavior: Evidence for the role of the human putative chemosignal estratetraenol.

Previous studies demonstrating that women's body odor during ovulation is perceived as more attractive suggest that exposure to women's chemosignals of high fertility increases mating motivation. Building on previous evidence showing that cooperative behaviors are perceived as attractive, in the current study we investigated whether chemosignals of women's fertility affect men's tendency to behave cooperatively. In the first experiment we found that in the presence of women's body odor during ovulation, men increase their tendency to apply a cooperative strategy, while their tendency to apply an individualistic strategy decreases. To examine the mechanism underlying this effect, we tested a different sample of men exposed to the putative human pheromone estratetraenol (estra-1,3,5(10),16-tetraen-3-ol) or to a control solution. Exposure toestratetraenol compared with control yielded strikingly similar effects of increased cooperation. The results indicate that women's chemosignals of high fertility increase mating motivation among man, encouraging them to act in a cooperative manner toward others, a response that may highlight their attractive qualities and thus attract mates. We further conclude that estratetraenol may serve as one of the biological agents that mediate the behavioral effects of women's chemosignals of fertility on social behavior.

Chemical synthesis of C3-oxiranyl/oxiranylmethyl-estrane derivatives targeted by molecular modeling and tested as potential inhibitors of 17ß-hydroxysteroid dehydrogenase type 1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a promising therapeutic target known to play a pivotal role in the progression of estrogen-dependent diseases such as breast cancer, and endometriosis. This enzyme is responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2) and its inhibition would prevent the growth of estrogen-sensitive tumors. Based on molecular modeling with docking experiments, we identified two promising C3-oxiranyl/oxiranylmethyl-estrane derivatives that would bind competitively and irreversibly in the catalytic site of 17ß-HSD1. They have been synthesized in a short and efficient route and their inhibitory activities over 17ß-HSD1 have been assessed by an enzymatic assay. Compound 15, with an oxiranylmethyl group at position C3, was more likely to bind the catalytic site and showed an interesting, but weak, inhibitory activity with an IC50 value of 1.3 µM (for the reduction of estrone into E2 in T-47D cells). Compound 11, with an oxiranyl at position C3, produced a lower inhibition rate, and the IC50 value cannot be determined. When tested in estrogen-sensitive T-47D cells, both compounds were also slightly estrogenic, although much less than the estrogenic hormone E2.

Case Study: Atypical δ13 C values of urinary norandrosterone.

Isotope ratio mass spectrometry (IRMS) has been established in doping control analysis to identify the endogenous or exogenous origin of a variety of steroidal analytes including the 19-norsteroid metabolite norandrosterone (NorA). NorA can be found naturally in human urine in trace amounts due to endogenous demethylation or in situ microbial degradation. The administration of nortestosterone (nandrolone) or different prohormones results in the excretion of urinary NorA. Usually, this can be detected by IRMS due to differing δ13 C values of synthetic 19-norsteroids compared to endogenous reference compounds. The consumption of uncastrated pig edible parts like offal or even meat may also lead to a urinary excretion of NorA. In order to determine the δ13 C values of such a scenario, urine samples collected after consumption of a wild-boar-testicle meal were analyzed. IRMS revealed highly enriched δ13 C values for urinary NorA, which could be related to the completely corn-based nutrition of the animal. Isotopic analysis of the boar's bristles demonstrated a dietary change from C3 -based forage, probably in winter and spring, to a C4 -based diet in the last weeks to months prior to death. These results supported the interpretation of an atypical test result of a Central European athlete's doping control sample with δ13 C values for NorA of -18 ‰, most probably caused by the consumption of a wild boar ragout. As stated before, athletes should be fully aware of the risk that consumption of wild boar may result in atypical or even adverse analytical findings in sports drug testing.

Targeting Cytochrome P450 (CYP) 1B1 Enzyme with Four Series of A-Ring Substituted Estrane Derivatives: Design, Synthesis, Inhibitory Activity, and Selectivity.

Cytochrome P450 (CYP) 1B1 is involved in the bioactivation of procarcinogens and drug resistance. To obtain selective CYP1B1 inhibitors over CYP1A1, we synthesized four series of estrane derivatives: (1) 12 estrone (E1)- and 17ß-estradiol (E2)-derivatives bearing a 3- or a 4-pyridinyl core at C2, C3, or C4, (2) eight estrane derivatives with different sulfur groups at C3, (3) 19 E1- and E2-derivatives bearing distinct aryls at C2, and (4) five D-ring derivatives. E2-derivatives were more active than oxidized E1-analogues, thus highlighting the key role of 17ß-OH for interaction with CYP1B1. 2-(4-Fluorophenyl)-E2 was the best CYP1B1 inhibitor (IC50 = 0.24 µM), with a selectivity index (SI) of 20 over CYP1A1. Furthermore, the addition of a C17α-ethynyl group as D-ring modification improved the selectivity index to 25 with only a slight loss of activity (IC50 = 0.37 µM). Our docking results showed that these compounds fit better into the CYP1B1 binding site than that of CYP1A1.

In vitro evaluation of the effects of anti-fungals, benzodiazepines and non-steroidal anti-inflammatory drugs on the glucuronidation of 19-norandrosterone: implications on doping control analysis.

We have studied whether the phase II metabolism of 19-norandrosterone, the most representative metabolite of 19-nortestosterone (nandrolone), can be altered in the presence of other drugs that are not presently included on the Prohibited List of the World Anti-Doping Agency. In detail, we have evaluated the effect of non-prohibited drugs belonging to the classes of anti-fungals, benzodiazepines, and non-steroidal anti-inflammatory drugs on the glucuronidation of 19-norandrosterone. In vitro assays based on the use of either pooled human liver microsomes or specific recombinant isoforms of uridine diphosphoglucuronosyl-transferase were designed and performed to monitor the formation of 19-norandrosterone glucuronide from 19-norandrosterone. Determination of 19-norandrosterone (free and conjugated fraction) was performed by gas chromatography - mass spectrometry after sample pretreatment consisting of an enzymatic hydrolysis (performed only for the conjugated fraction), liquid/liquid extraction with tert-butylmethyl ether, and derivatization to form the trimethylsilyl derivative. In parallel, a method based on reversed-phase liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization with acquisition in selected reaction monitoring mode was also developed to identify the non-prohibited drugs considered in this study. Incubation experiments have preliminarily shown that the glucuronidation of 19-norandrosterone is principally carried out by UGT2B7 (39%) and UGT2B17 (31%). Inhibition studies have shown that the yield of the glucuronidation reaction is reduced in the presence of the anti-fungals itraconazole, ketoconazole, and miconazole, of the benzodiazepine triazolam and of the non-steroidal anti-inflammatory drugs diclofenac and ibuprofen, while no alteration was recorded in the presence of all other compounds considered in this study. Copyright © 2015 John Wiley & Sons, Ltd.

Revisiting the metabolism of 19-nortestosterone using isotope ratio and high resolution/high accuracy mass spectrometry.

The synthetic anabolic androgenic steroid 19-nortestosterone is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) due to its performance-enhancing effects. Today, doping controls focus predominantly on one main urinary metabolite, 19-norandrosterone glucuronide, which offers the required detection windows for an appropriate retrospectivity of sports drug testing programs. As 19-norandrosterone can also be found in urine at low concentrations originating from in situ demethylation of other abundant steroids or from endogenous production, the exogenous source of 19-norandrosterone needs to be verified, which is commonly accomplished by carbon isotope ratio analyses. The aim of this study was to re-investigate the metabolism of 19-nortestosterone in order to probe for additional diagnostic long-term metabolites, which might support the unambiguous attribution of an endo- or exogenous source of detected 19-nortestosterone metabolites. Employing a recently introduced strategy for metabolite identification, threefold deuterated 19-nortestosterone (16,16,17-(2)H3-NT) was administered to one healthy male volunteer and urine samples were collected for 20 days. Samples were prepared with established methods separating unconjugated, glucuronidated and sulfated steroids, and analytes were further purified by means of high-performance liquid chromatography before trimethylsilylation. Deuterated metabolites were identified using gas chromatograph/thermal conversion/isotope ratio mass spectrometer comprising an additional single quadrupole mass spectrometer. Additional structural information was obtained by gas chromatography/time-of-flight mass spectrometry and liquid chromatography/high resolution mass spectrometry. In general, sulfo-conjugated metabolites were excreted for a longer time period than the corresponding glucuronides. Several unexpected losses of the arguably stable isotope labels were observed and characterized, attributed to metabolic reactions and sample preparation procedures. The detection window of one of the newly detected metabolites was higher than currently used metabolites. The suitability of this metabolite to differentiate between endo- or exogenous sources could however not be verified conclusively.

[Effect of the steroid molecule structure on the direction of its hydroxylation by the fungus Curvularia lunata].

The main and side products of hydroxylation by the C. lunata VKPM F-981 mycelium of fourteen delta(4)-3-ketosteroids of the estrane, androstane, and pregnane series and six of their delta(5)-3beta-hydroxy analogues were identified by H1 PMR spectroscopy and comparison with standard samples. The obtained experimental data are considered in terms of the triangular model of the enzyme-substrate interaction. The dependence of the direction of hydroxylation of steroid molecules and the orientation of hydroxy groups on the structure of the initial substrate was revealed.

Determination of nandrolone metabolites in human urine: comparison between liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry.

Nandrolone (19-nortestosterone) is an androgenic anabolic steroid illegally used as a growth-promoting agent in animal breeding and as a performance enhancer in athletics. Therefore, its use was officially banned in 1974 by the Medical Commission of the International Olympic Committee (IOC). Following nandrolone administration, the main metabolites in humans are 19-norandrosterone, 19-norethiocolanolone and 19-norepiandrosterone, and their presence in urine is the basis of detecting its abuse. The present work was undertaken to determine, in human urine, nandrolone metabolites (phase I and phase II) by developing and comparing multiresidue liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) methods. A double extraction by solid-phase extraction (SPE) was necessary for the complete elimination of the interfering compounds. The proposed methods were also tested on a real positive sample, and they allow us to determine the conjugated/free fractions ratio reducing the risk of false positive or misleading results and they should allow laboratories involved in doping control analysis to monitor the illegal use of steroids. The advantages of LC/MS/MS over GC/MS (which is the technique mainly used) include the elimination of the hydrolysis and derivatization steps: it is known that during enzymatic hydrolysis several steroids can be converted into related compounds and deconjugation is not always 100% effective. The validation parameters for the two methods were similar (limit of quantification (LOQ) <1 ng/mL and percentage coefficient of variance (CV%) <16.4), and both were able to confirm unambiguously all the analytes, thus confirming the validity of both techniques.

Urinary nandrolone metabolite detection after ingestion of a nandrolone precursor.

INTRODUCTION: Quantities of various anabolic/androgenic steroids have been found in dietary supplements without their presence being disclosed on the label. The aim of this study was to quantify the excretion patterns of the diagnostic metabolites, 19-norandrosterone (19-NA), and 19-noretiocholanolone (19-NE) after ingestion of small doses of 19-nor-4-androstene-3,17-dione (19-norandrostenedione). METHODS: Eleven males and nine females entered the laboratory in the morning after an overnight fast. An initial urine sample was collected, and volunteers then ingested 500 mL of water containing 5 g of creatine monohydrate and 1.0, 2.5, or 5.0 microg of 19-norandrostenedione. The volume of each urine void was measured, and an aliquot was taken. Samples were analyzed for the metabolites 19-NA and 19-NE by GCMS. RESULTS: Baseline urinary 19-NA concentrations were 0.19 +/- 0.14 ng x mL. Ingestion of the supplement resulted in peak mean urinary 19-NA concentrations of 0.68 +/- 0.36, 1.56 +/- 0.86, and 3.89 +/- 3.11 ng.mL in the 1.0-, 2.5-, or 5.0-microg trials, respectively. Under current WADA regulations, ingestion of the 1.0-microg dose produced 0 positive doping tests, 5 subjects (20%) tested positive in the 2.5-microg trial, and 15 subjects (75%) had urinary 19-NA concentrations exceeding 2 ng x mL after ingesting creatine containing 5.0 microg of the steroid. The recovery of the ingested dose was highly variable between individuals, with values ranging from 11% to 84% (mean +/- SD = 47% +/- 18%). CONCLUSIONS: Ingestion of trace amounts of 19-norandrostenedione can result in transient elevations of urinary 19-NA and 19-NE concentrations. The addition of as little as 2.5 microg of 19-norandrostenedione to a supplement (0.00005% contamination) appears sufficient to result in a doping violation in some individuals.

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.

Regioselective oxyfunctionalization of unactivated carbons in steroids by a model of cytochrome P-450: osmiumporphyrin complex/tert-butyl hydroperoxide system.

tert-Butyl hydroperoxide catalyzed by (5,10,15,20-tetramesitylporphyrinate) osmium(II) carbonyl [Os(TMP)CO] complex was found to be a highly efficient versatile oxidant for C-H carbons in steroid substrates. When reacted with representative steroids with an estrane, pregnane, 5beta-cholane, or 5alpha-cholestane structure, regioselective oxyfunctionalization and/or oxidative degradation occurred to give a variety of novel and uncommon derivatives in one step.

Formation of 19-norsteroids by in situ demethylation of endogenous steroids in stored urine samples.

The formation of 19-norsteroids by demethylation of endogenous steroids in stored urine samples was observed. Suspicious urine samples (i.e. containing trace amounts of 19-norandrosterone and 19-noretiocholanolone) were selected and spiked with deuterated analogues of androsterone and etiocholanolone at concentrations corresponding to high endogenous levels (4 microg/mL). After incubation, respective 19-norsteroids (19-norandrosterone-d4 and 19-noretiocholanolone-d5) were identified in these samples by high-resolution mass spectrometry. The transformation of the 5 beta-isomer (etiocholanolone) yields about three-fold higher concentrations, compared to the 5 alpha-isomer. A significant temperature dependence was observed by comparison of reaction kinetics at room temperature (23+/-2 degrees C) and 37 degrees C. Concentrations of 19-norandrosterone-d4 and 19-noretiocholanolone-d5, respectively, were 2.7 and 3.6 times higher at elevated temperature. The conversion of androsterone-d4 to 19-norandrosterone-d4 did not exceed a relative amount of 0.1%. Incubation of the urine samples with androsterone-d4-glucuronide led to the production of 19-norandrosterone-d4-glucuronoide. A partial stabilization was observed after addition of metabolic inhibitors (e.g. EDTA). The application of the incubation experiments described may contribute to the clarification of adverse analytical findings regarding low levels of 19-norsteroid metabolites.

Endogenous nandrolone metabolites in human urine: preliminary results to discriminate between endogenous and exogenous origin.

When administered to human subjects, nandrolone is metabolized into two main products, 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Recent studies demonstrated the endogenous production of these compounds in man at concentrations very close to the threshold of the International Olympic Committee (IOC), i.e. 2 ng/ml. Because the possibility of reaching or exceeding this fateful limit is difficult to exclude, a complementary biochemical parameter is necessary for the differentiation of endogenous 19-NA and 19-NE production from residues resulting from nandrolone consumption. We measured the endogenous concentrations of 19-NA and 19-NE in 385 urine samples from professional football players, and we studied the phase II metabolite composition in individuals excreting the highest concentrations. The results showed that around 30% of endogenous 19-norandrosterone was sulfo-conjugated, whereas 100% of 19-norandrosterone was excreted conjugated to a glucuronic acid when nandrolone was administered. This significant qualitative difference appears to be a promising complementary criterion to more definitively conclude about an athlete's culpability, especially when nandrolone metabolites are found in the low ng/ml range.

Ultraviolet spectroscopic evidence for decreased motion of the active site tyrosine residue of delta 5-3-ketosteroid isomerase by steroid binding.

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the highly efficient conversion of delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and intramolecular transfer of the 4 beta-proton to the 6 beta-position. Tyr-14 is the critical general acid and Asp-38 is the general base involved in catalysis. The UV absorption bandwidths of Tyr-14 were much narrower than those of the other two tyrosines (Tyr-55 and Tyr-88) of isomerase or of the N-acetyltyrosine ethyl ester in aqueous solution, suggesting that Tyr-14 is restricted in its mobility. Further immobilization of this residue occurs upon steroid binding. Thus, 5 alpha-estrane-3,17-dione, an A-ring saturated steroid, induces significant narrowing of the tyrosine absorption bands (pi-->pi*) of the main peak (279.5 nm) and the shoulder (285.5 nm) of Tyr-14, with no significant changes in lambda max. No effects of steroid binding were found on the absorption bandwidths of Tyr-55, Tyr-88, or the phenylalanine residues. The ratio of the absorbance (Amax) at the absorption maximum (lambda max) to that at lambda max plus 4 nm (Amax +4) was used as a measure of peak sharpness. Specifically, the ratios of A279.5/A283.5 (main peak) and A285.5/A289.5 (shoulder) of Tyr-14 of the free enzyme at 25 degrees C were 1.25 and 1.89, respectively, and they increased to 1.41 and 2.70, respectively, in the complex. A more precise measurement of the band narrowing from 4.2 to 3.1 nm between the inflection points was obtained from the derivative spectra. The absorption bands of free and steroid-bound isomerase were narrowed significantly by lowering the temperature and were broadened by denaturation, suggesting that the unusual peak-sharpening effects induced by steroid binding arise from the restricted motion of Tyr-14, as well as from more directional hydrogen bonding resulting from the displacement of water molecules from the active site and decreased flexibility of the protein. Larger enthalpy of the sharpening effects was observed for the steroid-bound enzyme (-0.527 +/- 0.016 kcal/mol) than for the free enzyme (-0.250 +/- 0.018 kcal/mol) by lowering the temperature, indicating that interactions of Tyr-14 with its environment, which restrain its motion, are stronger in the steroid-bound enzyme than in the free enzyme. Hydrogen-bonding modes of Tyr-14, mobility of the active site, and protein flexibility are the environmental factors determining the absorption bandwidths of the critical Tyr-14 residue.

Clinical experience with the recently developed progestogens.

The third generation of combination estrogen/progestogen oral contraceptives (OCs) first became available in the early 1980s. The gonanes (e.g., norgestimate, desogestrel, and gestodene) are alternatives to the long-standing progestogens, norethindrone and norgestrel/levonorgestrel. While the newer compounds are related to levonorgestrel, their biochemical structure differs. All, however, are strongly progestogenic with respect to ovulation inhibition and are very selective in their affinity for endometrial progesterone receptors. At present, experience with the third-generation combined OCs is relatively limited, and extensive comparative data have yet to be accrued. Nevertheless, these ultra-low-dose compounds appear to be as efficacious as the traditional OCs, while their cycle control may be slightly superior. In addition, the incidence of minor side effects, such as nausea, weight gain, and mastalgia, compares favorably with that of the earlier OCs. In combination with estrogen, these new progestogens have revealed a neutral or possibly beneficial effect on lipid/lipoprotein metabolism. Thus, the newer progestogens do not appear to have adverse effects on the cardiovascular system and offer a range of noncontraceptive health benefits.

PIP: A study using data from the United Kingdom (UK), Denmark, and Sweden and an ongoing study in the UK found a significant association between estrogen content of high dose combined oral contraceptives (OCs) and all thromboses except venous thrombosis of lower limb. In fact, the ongoing study and another study both found a distinct association between progestogen dose and arterial disease. These association prompted formulators to decrease OC estrogen and progestogen (norethindrone and levonorgestrel) content. These new formulations entered the market after 1975. The early 1980s witnessed the introduction of yet another generation of genane progestogens which were developed in hoped of reducing the incidence of thrombosis. Clinical trials found their contraceptive effectiveness to be comparable to those of the older demonstrated that their effect on associated incidence of bleeding is similar to the older OCs and falls over time. Moreover, even though the 3rd generation OCs have brought on the same side effects (weight gain, mastalgia, and nausea) as the others, the frequency has been at least, and often less, than the others. So far data have not implicated the 3rd generation OCs in impairing glucose tolerance. In addition, research has demonstrated that 3rd generation formulations cause a small increase in high density lipoprotein and either did not change or reduced low density lipoprotein and total cholesterol levels. Thus the new combined OCs do not promote atherosclerotic changes in lipid metabolism. Even though more research is needed, the data have indicated that physicians should continue to prescribe the 3rd generation OCs.

Steroids in equine testes: the identification of endogenous 19-hydroxy and 19-nor neutral steroids by gas chromatography--mass spectrometry.

After homogenization of testicular tissue from stallions aged 1, 2 and 5 years, the unconjugated and conjugated steroids were isolated by a combined solvent-solid extraction procedure. The conjugates were further separated into glucuronides and sulphates by chromatography using Sephadex LH-20. After enzyme hydrolysis and solvolysis of the respective conjugate classes, the three extracts, unconjugated steroids, aglycones and solvolysed sulphates, were purified by chromatography using Kieselgel 60H columns. Five fractions were resolved from each extract; an aliquot of each fraction was derivatized to form the methoxime-trimethylsilyl ethers and the steroids were identified by combined gas chromatography-mass spectrometry. The results have shown that in stallion testes (1) steroidogenesis proceeds by both the 4-ene and the 5-ene pathways, (2) age-linked changes occur in both unconjugated and sulphoconjugated steroid fractions and (3) 19-hydroxy androgens and the 19-nor (C18) neutral steroids (19-norandrostenedione and 19-nortestosterone) are detected only in the unconjugated fraction whereas oestrene, the isomers of oestradiol and of 5(10)-oestrene-3,17-diol are the only steroids detected in the sulphoconjugate fraction. It is suggested that the unconjugated 19-oxygenated androgens present in stallion testes are converted to 19-nor neutral steroids by a reverse aldol reaction and a mechanism showing the putative intermediates in their formation is illustrated.

Interaction of A-nor, A, 19-dinor, and A-homo-5 alpha-androstane derivatives with the androgen receptor and the epididymal androgen-binding protein.

The equilibrium affinity constant for rat prostate androgen receptor and epididymal androgen binding protein (ABP) has been determined for thirty-four potential progestogens. Three A-nor-, four A,19-dinor-, and one A-homo-5 alpha-androstane derivative bind to the androgen receptor (KD less than 0.5 muM). Five of these compounds also bind to ABP with an affinity of the same order of magnitude. "Anordrin" (compound 24) and "Dinordrins" (compounds 10, 14, 15, 16, 17), which are potential female contraceptives, do not bind with high affinity to the androgen receptor or to ABP. The following modifications in A-nor derivatives favour binding to the receptor as compared to ABP: 19-nor substitution (compound 1), C-18 methyl homologation (compound 5), 2 alpha-ethinylation (compound 22). One 2 alpha-allenyl A-nor derivative (compound 25) and one A-homo derivative (compound 34) bind almost exclusively to ABP. The interaction with either binding protein is decreased by oxidation or esterification of the hydroxyl group at C-17, and by addition of a 17 alpha-ethinyl group. The latter modifications are likely to increase the specificity of androstane derivatives for receptors other than androgen binding proteins, such as the progesterone receptor.