Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Identification of early response genes and pathway activated by androgens in the initial segment and caput regions of the regressed rat epididymis.

To identify the initial response to androgens and estrogens in the orchidectomized, regressed epididymis, we determined the gene expression changes triggered by the administration of either of two metabolites of testosterone, 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2), in the regressed rat epididymis. Adult rats were orchidectomized and 8 d later implanted with either empty implants (control), DHT-filled-, or E2-filled-polydioxanone implants. Rats were euthanized 12 h, 1 d, and 7 d later, and RNA was extracted and probed on Rat230-2.0 Affymetrix arrays. Probe sets that respond to DHT or E2 were identified at early time points; although the expression of some was repressed, the expression of many others was either transiently or chronically elevated. Nerve growth factor receptor (Ngfr) and S100 calcium binding protein G (S100g) were two E2 up-regulated genes detected at 12 h. Among the genes that showed a dramatic early response to DHT were endothelin 1 (Edn1), bone morphogenetic protein 4 (Bmp4), and IGF binding protein 3 (Igfbp3), which were suppressed, and IGF-I (Igf1), which was induced. Genes that were up- or down-regulated by DHT were classified based on biological function. Using PathwayStudio 4.0, we identified genes that were linked and directly influenced either the expression or regulation of one another. Epidermal growth factor and IGF-I play an important role in the pathway due to their function in regulation and expression of many other genes. These results provide novel insights into the impact of androgen action on the expression of genes that are important for epididymal function.

New steroidal aromatase inhibitors: suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death.

BACKGROUND: Aromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells. RESULTS: The new steroids inhibit hormone-dependent proliferation of MCF-7aro cells in a time and dose-dependent manner, causing cell cycle arrest in G0/G1 phase and inducing cell death with features of apoptosis and autophagic cell death. CONCLUSION: Our in vitro studies showed that the two steroidal AIs, 3a and 4a, are potent inhibitors of breast cancer cell proliferation. Moreover, it was also shown that the antiproliferative effects of these two steroids on MCF-7aro cells are mediated by disrupting cell cycle progression, through cell cycle arrest in G0/G1 phase and induction of cell death, being the dominant mechanism autophagic cell death. Our results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer.

Biologic effects of equilin sulfate in postmenopausal women.

In order to determine the relative potency of equilin sulfate (EqS), a major constituent of conjugated equine estrogens, 15 women received oral doses of EqS (0.15, 0.31, and 0.625 mg) for 25 days. Doses of 0.31 and 0.625 mg significantly stimulated hepatic globulins. This stimulatory effect ranged from being 1.5 to 8 times greater than the effects of comparable doses of estrone sulfate and conjugated equine estrogens. A significant stimulation in high-density lipoprotein-cholesterol occurred with as little as 0.15 mg of EqS. Elevations in the high-density lipoprotein/low-density lipoprotein-cholesterol ratio occurred with EqS, which resulted in an approximately 4-fold greater response than that achieved with comparable doses of conjugated equine estrogens. The fasting urinary calcium/creatinine ratio was only significantly lowered with 0.625 mg of EqS and was less potent than conjugated equine estrogens in this regard. It is concluded that EqS is a potent estrogen that contributes significantly to the hepatic stimulatory effects of conjugated equine estrogens. These data also provide support for the suggestion that there may be a dissociation in potency between estrogenic effects on liver and bone.

Comparative electrophysiological effects of Org 6001, a new orally active antidysrhythmic agent, and lignocaine on human ventricular muscle.

1 The electrophysiological effects of Org 6001, a new orally active antidysrhythmic agent, have been compared with those of lignocaine on the human ventricular action potential in vitro.2 Org 6001 (4 to 16 mg/l) greatly reduced the maximum rate of depolarization (MRD) of the human ventricular action potential but had no effect on resting membrane potential or action potential amplitude.3 The action potential duration at the 50% repolarization level, but not at the 90% repolarization level, was significantly reduced by Org 6001. The absolute refractory period was unchanged.4 Lignocaine, at a concentration (4 mg/l) within the therapeutic range, had no significant effect on any measured parameter, either in muscle exposed to a normal (4.0 mM) or high (5.4 mM) extracellular potassium concentration ([K(+)](o)).5 Higher concentrations of lignocaine (8 to 16 mg/l) did, however, reduce MRD at both [K(+)](o) without changing resting membrane potential or action potential amplitude. The action potential duration was decreased slightly by these higher concentrations of lignocaine whilst the absolute refractory period was lengthened.6 Org 6001 was found to be more potent than lignocaine in reducing MRD but, unlike lignocaine, the absolute refractory period was not prolonged. These compounds, therefore, differed in their electrophysiological effects on human ventricular muscle although both are characterized as being class 1 antidysrhythmic drugs.

The effect of aminosteroid, ORG 6001, on hypothermia induced ventricular fibrillation in the cat.

1 The effect of the antidysrhythmic aminosteroid, ORG 6001, on hypothermia-induced ventricular fibrillation was investigated in cats anaesthetized with pentobarbitone. 2 ORG 6001 (total dose, 10 mg/kg, by intravenous injection) reduced both the incidence of fibrillation and the temperature at which it occurred. The number of animals that survived to 16 degrees C was increased. 3 This protective effect of ORG 6001 could not be explained by changes in respiratory acidosis, plasma concentrations of sodium and potassium, or by changes in the action potential of excised hypothermic ventricular muscle. The hypothermia-induced elevation of blood lactate was less in cats treated with the aminosteroid. 4 Over a limited temperature range, ORG 6001 prolonged the P wave and QRS duration and shortened the QTc interval. ST segment elevation was slightly reduced in the drug-treated group. J deflections were observed but were not correlated with the development of fibrillation. 5 The onset of fibrillation was not considered to be due to temperature differences between the myocardium and arterial blood or between localized areas of the left ventricular wall.

The effects of the hypocholesteremic compound 3 beta-(beta-dimethylaminoethoxy)-androst-5-en-17-one on the sterol and steryl ester composition of Saccharomyces cerevisiae.

When yeast was grown in the presence of 10(-4) M 3 beta-(beta-dimethylaminoethoxy)-androst-5-en-17-one (DMAE-DHA), the compound 2,3;22,23-dioxidosqualene (DOS) accumulated. Total free sterol was reduced by about 30%, whereas almost no steryl esters were found. The same drug at lower concentration (3 x 10(-6) M) caused a slight increase in steryl ester production, and a 24% reduction in free sterol content. The marked accumulation of ergostra-5,7,22,24(28)-tetraen-3 beta-ol with 3 x 10(-6) M DMAE-DHA indicated that the C24-28 reductase is especially sensitive to the action of the drug.

Control of obesity in Avy/a mice by 5alpha-androstan-17-one.

Treating VY/WfL-Avy/a mice with 5 alpha-androston-17-one, a mammalian glucose-6-phosphate dehydrogenase inhibitor, prevented the mice from becoming obese. The weight difference between treated and control Avy/a mice was mainly due to a decreased accumulation of triacylglycerol. The compound did not suppress appetite, had no detectable toxicity and did not affect the lipogenesis rates in the liver and carcass. The weight-controlling effect of 5alpha-androstan-17-one in Avy/a mice was reversible upon withdrawal of treatment.

Effect of somatostatin analogues and 17-alpha-dihydroequilin on rat brain opiate receptors.

Two somatostatin analogues (SA) and 17-alpha-dihydroequilin (E) inhibited the stereospecific binding of 3H-naloxone in vitro to rat brain opiate receptors in the absence of sodium. The addition of sodium indicated the SA to be greater or similar in potency to somatostatin as opiate agonists and E to be an opiate antagonist. The results indicate that SA and certain steroids may affect endorphin containing neurons.

Effects of an aminosteroid, ORG 6001, on various membrane phenomena of non-myelinated nerve fibres of the rat.

The aminosteroid ORG 6001 (3 alpha-amino-5 alpha-androstan-2 beta-ol-17-one hydrochloride) was shown to be a compound with a rather strong local anaesthetic action. Compound action potentials evoked in non-myelinated nerve fibres of the desheathed rat vagus with the single sucrose gap method showed, in presence of this drug, a decrease in amplitude and increase in conduction time comparable with the effect of lidocaine. Since the total membrane resistance represented by the amplitude of the electrotonic potential was not affected by ORG 6001, it is likely that ORG 6001 reduces the transient increase in sodium conductance during the action potential, a mechanism also postulated for lidocaine. This is confirmed by the decrease of the post-tetanic hyperpolarization in the presence of both ORG 6001 and lidocaine. Since neither sodium pump activity nor the Na/K coupling ratio was affected by ORG 6001, the increase of the potassium-activated response was explained by an increased sodium permeability during rest. The local anaesthetic action of these drugs was even seen with a concentration (20--40 micron) sufficient to produce antiarrhythmic effects in vivo in man if the nerve was stimulated frequently (1--4 c.p.s.).

Antiarrhythmic, metabolic and hemodynamic effects of Org 6001 (3alpha-amino-5alpha-androstan-2beta-ol-17-one-hydrochloride) after coronary flow reduction in pigs.

The antiarrhythmic activity of the aminosteroid Org 6001 was investigated in young pigs (20-28 kg). Ventricular arrhythmias were induced by restriction of the flow in the left anterior descending coronary artery (LAD) to 25% of its control value during a period of 30 minutes. Nine out of 30 control animals died in this period due to ventricular fibrillation. None of the 19 animals treated with Org 6001 (5-10 mg/kg) or the 12 animals treated with lidocaine (2.75-3.50 mg/kg) fibrillated. Moreover, the number of premature ventricular beats was greatly reduced in pretreated groups compared with the untreated group (P less than .001). The first derivative of left ventricular pressure decreased with 25% (P less than 0.001) after administration of Org 6001. However, during 30 minutes of LAD flow reduction to 25% of control, the adverse effects of Org 6001 were less than those of lidocaine. Myocardial lactate production indicated some delay in onset of ischemia. However, there was no indication that this beneficial effect was long-lasting. When after 30 minutes of LAD flow reduction to 25% of control, the LAD was completely occluded between its second and third branch, all untreated animals fibrillated within 120 minutes, whereas 4 of the 19 animals treated with Org 6001 and 3 of the 12 treated with lidocaine survived. It is concluded that Org 6001 has antiarrhythmic properties in the ischemic pig heart which compare favorably with those of lidocaine.

The antidysrhythmic aminosteroid, ORG 6001, reduces the ST-segment elevation produced by coronary occlusion in the dog.

ST-segment elevation following temporary coronary artery occlusion was measured from nine epicardial leads in open-chest anaesthetized dogs. This was greatly reduced by the prior administration of the anti-dysrhythmic aminosteroid, ORG 6001. It is suggested that this effect is related either to a reduction in the extent and degree of myocardial ischaemia or to prevention of K+ egress from ischaemic cells.

Investigations to characterize a new anti-arrhythmic drug, ORG 6001 including a simple test for calcium antagonism.

1 The compound Org 6001 (3alpha-amino-2beta-hydroxy-5alpha-androstan-17-one hydrochloride) was found in recent experiments to exhibit anti-arrhythmic activity. Evidence is presented in this paper concerning its mode of action. 2 Org 6001 was 1.8 times more potent than procaine as a local anaesthetic on desheathed frog nerve. 3 Org 6001 had no effect on the resting potential of isolated cardiac muscle of rabbit, but greatly reduced the maximum rate of depolarization tion (MRD). The action potential duration TAPD) WAS MARGINALLY PROLONGED IN ATRIAL AND VENTRICULAR MUSCLE. 4 Org 6001 preferentially shortened APD in that part of the Purkinje system in which APD is normally longer than elsewhere, so that APD

Antiarrhythmic and hemodynamic effects of an aminosteroid (Org 3001) in the digitalized dog.

Org 6001 (3alpha-amino-5alpha-androstan-2beta-ol-17-one-hydrochloride) is an orally non-hormonal aminosteroid possessing antiarrhythmic activity. In 13 dogs the efficacy of the drug against ouabain-induced ventricular tachycardia (VT) was studied. VT was produced by a mean dose of 67.5 +/- 18.7 mug/kg ouabain, administered by continuous infusion of 25 mug/min. 20 min after the onset of VT an 0.125 mg/kg/min infusion of Org 6001 was initiated, doubling the dose every 10 min. In all dogs VT was reverted into normal sinus rhythm (NSR) by a dose of 9.72 +/- 7.07 mg/kg (0.87-20.75 mg/kg) Org 6001. The duration of VT ranged from 27-61 min (mean 47.1 +/- 11.4 min), including the 20 min waiting period. NSR persisted in 8 dogs until the experiment was terminated (90 min after onset of VT), while in 5 dogs VT returned after 3-23 min sufficient to revert VT into NSR. A bolus injection of Org 6001 (10 mg/kg) gave an immediate return to NSR in 3 dogs, in which VT was provoked again by administration of a second dose of ouabain after the 90 min period had elapsed. Though the interaction of ouabain makes a quantitative analysis of the negative inotropic effects difficult, it appeared that there uas no major hemodynamic deterioration during and after treatment with Org 6001. During digitalization there was a significant increase in the first derivative of left ventricular systolic pressure (peak LVdP/dt) from 2340 +/- 600 to 3650 +/- 1070 mm Hg/sec and in peripheral resistance, while heart rate decreased. During VT, left ventricular systolic pressure (LVSP) and mean aortic pressure (MAP) dropped by approximately 20 mm Hg, while heart rate increased significantly. After treatment with Org 6001, LVSP and MAP further decreased to 128 +/- 30 mm Hg (p less than 0.05) areased to 128 +/- 30 mm Hg (p less than 0.05) and 112 +/- 20 mm Hg respectively. Peak LVdP/dt fell from 3650 +/- 1390 to 2780 +/- 970 mm Hg/sec (p less than 0.05). Heart rate had dropped to 126 +/- 22 beats/min (p less than 0.05). During the first 30 min after Org 6001 infusion was stopped none of the parameters showed significant changes, although peak LVdP/dt rose slightly. It is shown in the present investigation that Org 6001 has effective antiarrhythmic properties in controlling ouabain-induced VT with acceptable cardiodepressant actions.

Cardiovascular effects of an antiarrhythmic aminosteroid ORG 6001 in the anesthetized pig.

The hemodynamic effects of 3alpha-amino-5alpha-androstan-2beta-ol-17-one-hydrochloride (ORG 6001), an aminosteroid possessing antiarrhythmic properties, were studied in 14 anesthetized pigs. ORG 6001 was administered i.v. cumulatively until a toxic level was reached. Doses less than 20mg/kg had, in spite of the negative inotropic properties of ORG 6001, no effect on the pump function of the heart, as the afterload was reduced and the filling pressure was not significantly altered. After administration of 40 mg/kg A-V conduction was depressed and second degree A-V block appeared. This was associated with deterioration of the pump function and for 6 of the 14 animals this was fatal. In the survivors, the alterations were reversible after cessation of drug administration. Since ORG 6001 has been shown to be effective against arrhythmias at doses less than 20 mg/kg, though in a different species (dog), it seems likely that it can be used safely for antiarrythmic treatment.

Interaction of steroids with the transport system of glucose in human erythrocytes.

Steroids inhibit the exchange transport of glucose in human erythrocytes. The extent of inhibition is roughly correlated to the affinity of the steroids to the membrane lipids. All C-21-steroids tested show a competitive inhibition while the C-19-steriods show different types of inhibition. 5Beta-androstane-3,17-dione acts as a competitive inhibitor. The inhibition by testosterone is of mixed type, while with androst-4-ene-3,17-dione and 5alpha-androstane-3,17-dione a non-competitive inhibition is observed. In this case two inhibitor molecules can be bound per transport molecule. The "non-competitive" inhibitors compete also to some extent with the glucose binding. This effect, however, is at high inhibitor concentrations masked by the more powerful non-competitive inhibition. Competitive and non-competitive inhibitors compete with each other. The structural requirements for the different types of inhibition are discussed.

Antiarrhythmic, haemodynamic and metabolic effects of 3alpha-amino-5alpha-androstan-2beta-ol-17-one hydrochloride in greyhounds following acute coronary artery ligation.

1 The antiarrhythmic, haemodynamic and metabolic effects of a new amino steroid, ORG6001, have been investigated in experimental acute myocardial infarction in anaesthetized greyhounds. 2 ORG6001 administered either intravenously (2-10 mg/kg) or orally (50 mg/kg) significantly reduced the incidence of ventricular ectopic beats in the first 30 min after ligation of the left anterior descending coronary artery. 3 In dogs pretreated with ORG6001, metabolic changes indicative of myocardial ischaemia (lactate production and potassium efflux) were less marked than those occurring in control animals. 4 Antiarrhythmic doses of ORG6001 caused only minimal transient haemodynamic effects. 5 These results suggest that ORG6001 may possess distinct advantages over presently-used antiarrhythmic drugs in the prevention and treatment of the early arrhythmias which occur after myocardial infarction.