Excretion and metabolism of desogestrel in healthy postmenopausal women

The metabolism of desogestrel (13-ethyl-11-methylene-18,19-dinor-17-pregn-4-en-20-yn-17-ol), a progestagen used in oral contraceptives and hormone replacement therapy, was studied in vivo after a single oral administration of 150 μg [¹⁴C]-labeled desogestrel and 30 μg ethinylestradiol under steady state conditions to healthy postmenopausal women. After this oral administration, desogestrel was extensively metabolized. The dosed radioactivity was predominantly (60%) excreted via urine, while about 35% was excreted via the feces. Desogestrel was metabolized mainly at the C3-, C5-, C6- and C13-CH₂CH₃ positions. At the C3-position, the 3-keto moiety was found and in addition, 3β-hydroxy and 3-hydroxy groups were observed in combination with a reduced Δ⁴-double bond (5-H). Hydroxy groups were introduced at the C6- (6β-OH), the C13-ethyl (C13-CH₂CH₂OH) and possibly the C15- (15-OH) position of desogestrel. Conjugation of the 3-hydroxy moiety with sulfonic acid and conjugation with glucuronic acid were also major metabolic routes found for desogestrel in postmenopausal women. The 3-keto metabolite of desogestrel (the biologically active metabolite) was the major compound present in plasma at least up to 24 h after administration of the radioactive dose. Species comparison of the metabolic routes of desogestrel after oral administration indicates that in rats and dogs desogestrel is also mainly metabolized at the C3-position, similar to what is now found for postmenopausal women. Most other metabolic routes of desogestrel were found to differ between species. Finally, major metabolic routes found in the present study in postmenopausal women are in line with outcome of previous in vitro metabolism studies with human liver tissue (microsomes and postmitochondrial liver fractions) and intestinal mucosa.     

In vitro evaluation of estrogenic, estrogen antagonistic and progestagenic effects of a steroidal drug (Org OD-14) and its metabolites on human endometrium

The human endometrial model for in vitro evaluation of estrogenic, estrogen antagonistic, and progestagenic effects of endogenous steroids, natural products or synthetic drugs was applied to the study of Org OD-14, an analog of norethynodrel developed by Organon International, Oss, The Netherlands, and some of its metabolites. Estrogen antagonistic actions of Org OD-14 and its 4-ene isomer were evident from their ability to suppress the enhancement of PGF2 alpha output elicited by estradiol on fragments of secretory endometrium and to decrease the rate of output of the prostaglandin by proliferative tissue, already stimulated by endogenous estrogens. These inhibitory effects were similar to those obtained with progesterone and do not appear to involve competition for the estrogen receptor since the antiestrogen 4-hydroxyamoxifen was not active in parallel incubations of proliferative endometrium. The progestagenic effects of Org OD-14 and its 4-ene isomer were also evident from their capability to enhance estradiol 17 beta-dehydrogenase activity and glycogen accumulation in specimens of proliferative endometrium. Estrogenic effects of the 3 alpha- and 3 beta-hydroxy metabolites of Org OD-14 were demonstrated by their stimulatory actions on PGF2 alpha output during incubations of secretory endometrium. The estrogenic and progestagenic actions of these compounds are in general agreement with their relative affinity for binding to the estradiol and progesterone receptors, although their actions may be influenced by intracellular metabolism in the endometrial tissue. For instance, the similarity in progestagenic activity of Org OD-14 and the 4-ene isomer, contrasting with their different affinities for the progesterone receptor, may result from in situ isomerization of Org OD-14 to the 4-ene metabolite.     

Regulation of the pituitary 5 alpha-reductase activity by gonadotropin releasing hormone and testosterone in the adult male rat

Intact or castrated adult male rats were treated for nine days with GnRH (10 micrograms/day), the synthetic GnRH goserelin (100 micrograms/day) or the GnRH-antagonist Org 30276 (250 or 500 micrograms/day). In some series, 1 mg testosterone propionate was administered alone, or in combination with goserelin or Org 30276. The in vitro metabolism of [1 alpha,2 alpha-3H]testosterone by pituitary and hypothalamic homogenates was investigated in combination with the estimation of plasma concentrations of testosterone and gonadotropins. No qualitative or quantitative differences were observed in hypothalamic testosterone metabolism or in the pituitary 17 beta-hydroxysteroid dehydrogenase activity. Testosterone administration to intact male rats decreased the pituitary 5 alpha-reductase activity and LH, while administered to castrated rats, it was able to suppress totally the castration-induced increase of the 5 alpha-reductase activity and of the gonadotropin secretion. The drastic decrease of the plasma levels of testosterone, observed after a prolonged treatment with GnRH, goserelin or Org 30276 was not accompanied by an increased pituitary 5 alpha-reductase activity. Injected to castrated rats, it was observed that the castration-induced increase of the pituitary 5 alpha-reductase was further stimulated by GnRH, totally suppressed by goserelin and partially suppressed by Org 30276. Concomitant administration of goserelin or Org 30276 and testosterone propionate to castrated rats resulted in a further decrease of the pituitary 5 alpha-reductase activity, compared to the castrated, GnRH-analogue treated rats. These data indicate that the pituitary 5 alpha-reductase enzyme system is controlled by both direct steroidal and indirect GnRH-mediated mechanisms.     

Metabolism of 4-hydroxyandrostenedione and 4-hydroxytestosterone: Mass spectrometric identification of urinary metabolites

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography-mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3beta-hydroxy-5alpha-androstane-4,17-dione (2) and 3alpha-hydroxy-5beta-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3xi,4xi-dihydroxy-5xi-androstan-17-one (4, 6-11) except 3alpha,4alpha-dihydroxy-5beta-androstan-17-one (5). Out of the 17beta-hydroxylated analogs 4-hydroxytestosterone (18), 3beta,17beta-dihydroxy-5alpha-androstan-4-one (19), 3alpha,17beta-dihydroxy-5beta-androstan-4-one (20), 5alpha-androstane-3beta,4beta,17beta-triol (21), 5alpha-androstane-3alpha,4beta,17beta-triol (26) and 5alpha-androstane-3alpha,4alpha,17beta-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation.     

Androgen metabolism by rat epididymis. 3. Effect of castration and anti-androgens.

The in vivo and in vitro metabolism of 3H-testosterone by rat epididymis and the changes in epididymal weight have been studied after castration and treatment with anti-androgens. The utilization of 3H-testosterone was greatly reduced after castration as was the formation of 5alpha-reduced 17 beta-hydroxy metabolites. The formation of the 17 -keto metabolites was unaffected. Castration had no effect on the ratio between water and ether soluble radioactivity. Administration of testosterone propionate, necessary for giving normal stimulated prostate weight (150 mug/day), restored the metabolism of testosterone to approximately normal values. Estradiol benzoate and progesterone inhibited metabolism of testosterone in vitro and greatly reduced the formation of DHT (17 beta-hydroxy-5alpha-androstan-3-one) and 3 alpha-diol(5 alpha-androstane-3 alpha-17 beta-diol) by experiments both in vivo and in vitro. No effect of cyproterone acetate could be demonstrated on either the in vitro or in vivo metabolism of testosterone. Castration for 14 days reduced the epididymal weight to about 30% of that found in intact animals. Administration of testosterone propionate restored the epididymal weight to about 80% of normal. Estradiol benzoate and cyproterone acetate given to intact rats led to a decrease in the epididymal weight. Progesterone had no such effect. In 14 days castrated rats receiving testosterone propionate all three anti-androgens reduced the weight of the epididymis. In conclusion, our results show that the metabolic conversion of testosterone in epididymis to DHT and 3 alpha-diol is dramatically dependent on the hormonal status of the animal; castration or treatment with anti-androgens causes a reduced formation of the "active" androgens whilst testosterone replacement treatment restores the metabolism of testosterone to normal.     

Age and sex differences in the metabolism of C19-delta5-steroids in human amniotic epithelium

In vitro transformation of [4-(14)C]-dehydroepiandrosterone and [4-(14)C]-5-androstenediol in human amniotic epithelium from the first and the second trimester was studied. Total conversion of both substrates used and formation of their "more polar" metabolites increased with advancing gestational age. The reduction of 17-oxo or the oxidation of 17 beta-hydroxy groups reached a maximum between weeks 17 and 20. A significant decrease in the conversion of delta 5-3 beta-hydroxy group from week 11 was evident. No significant sex differences were observed.     

Kinetic control of steroidogenesis by steroid concentration in guinea pig adrenal microsomes

For clarification of the effects of steroid concentration on steroidogenesis of adrenal microsomes, the kinetic parameters, Km and kcat, were determined in the steady-state for progesterone and 17 alpha-hydroxyprogesterone metabolism catalyzed by P-450C21 and P-450(17 alpha lyase) in guinea pig adrenal microsomes. At a high concentration of progesterone, it was equally metabolized by P-450C21 and P-450(17 alpha lyase), while at a low concentration, it was hydroxylated at 17 alpha-position with twice higher rate than at 21-position. 17 alpha-Hydroxyprogesterone is apparently metabolized preferentially by P-450C21 at any concentration. Although the productions of deoxycortisol and androstenedione from 17 alpha-hydroxyprogesterone were strongly inhibited by progesterone, androstenedione formation from progesterone was not inhibited by a high concentration of progesterone. The addition of liposomal P-450C21 to the reaction medium containing adrenal microsomes caused a decrease in the concentration of 17 alpha-hydroxyprogesterone released into the medium in the steady state reaction, but this had no effect on the activity of androstenedione formation from high concentrations of progesterone. It thus follows that androstenedione is produced by successive monooxygenase reactions without the release of 17 alpha-hydroxyprogesterone from P-450(17 alpha lyase) at a high concentration of progesterone, which is the condition of the adrenal microsomes in vivo.     

Studies on anabolic steroids--6. Identification of urinary metabolites of stenbolone acetate (17 beta-acetoxy-2-methyl-5 alpha-androst-1-en-3-one) in human by gas chromatography/mass spectrometry

The metabolism of stenbolone acetate (17 beta-acetoxy-2-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. Nine metabolites were detected in urine either as glucuronic or sulfuric acid aglycones after oral administration of a single 50 mg dose to a male volunteer. Stenbolone, the parent compound, was detected for more than 120 h after administration and its cumulative excretion accounted for 6.6% of the ingested dose. Most of the stenbolone acetate metabolites were isolated from the glucuronic acid fraction, namely: stenbolone, 3 alpha-hydroxy-2-methyl-5 alpha-androst-1-en- 17-one, 3 alpha-hydroxy-2 xi-methyl-5 alpha-androst-17-one; 3 isomers of 3 xi, 16 xi-dihydroxy-2-methyl-5 alpha-androst-1-en-17-one; 16 alpha and 16 beta-hydroxy-2-methyl-5 alpha-androst-1-ene-3, 17-dione; and 16 xi, 17 beta-dihydroxy-2-methyl-5 alpha-androst-1-en-3-one. Only isomeric metabolites bearing a 16 alpha or a 16 beta-hydroxyl group were detected in the sulfate fraction. Interestingly, no metabolite was detected in the unconjugated steroid fraction. The steroids identities were assigned on the basis of their TMS ether, TMS enol-TMS ether, MO-TMS and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. Data indicated that stenbolone acetate was metabolized into several compounds resulting from oxidation of the 17 beta-hydroxyl group and/or reduction of A-ring delta-1 and/or 3-keto functions with or without hydroxylation at the C16 position. Finally, comparison of stenbolone acetate urinary metabolites with that of methenolone acetate shows similar biotransformation pathways for both delta-1-3-keto anabolic steroids. This indicates that the position of the methyl group at the C1 or C2 position in these steroids has little effect on their major biotransformation routes in human, to the exception that stenbolone cannot give rise to metabolites bearing a 2-methylene group since its 2-methyl group cannot isomerize into a 2-methylene function through enolization of the 3-keto group as previously observed for methenolone.     

Studies on anabolic steroids--4. Identification of new urinary metabolites of methenolone acetate (Primobolan) in human by gas chromatography/mass spectrometry

The metabolism of methenolone acetate (17 beta-acetoxy-1-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. After oral administration of a 50 mg dose of the steroid to two male volunteers, twelve metabolites were detected in urine either in the glucuronide, sulfate or free steroid fractions. Methenolone, the parent steroid was detected in urine until 90 h after administration. Its cumulative urinary excretion accounted for 1.63% of the ingested dose. With the exception of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major biotransformation product of methonolone acetate, metabolites were excreted in urine at lower levels, through minor metabolic routes. Most of methenolone acetate metabolites were isolated from the glucuronic acid fraction, namely methenolone, 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, 3 alpha-hydroxy-1 alpha-methyl-5 alpha-androstan-17-one, 17-epimethenolone, 3 alpha,6 beta-dihydroxy-1-methylen-5 alpha-androstan-17-one, 2 xi-hydroxy-1-methylen-5 alpha-androstan-3,17-dione, 6 beta-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione, 16 alpha-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione and 3 alpha,16 alpha-dihydroxy-1-methyl-5 alpha-androst-1-en-17-one. Interestingly, the metabolites detected in the sulfate fraction were isomeric steroids bearing a 16 alpha- or a 16 beta-hydroxyl group, whereas 1-methyl-5 alpha-androst-1-en-3,17-dione was the sole metabolite isolated from the free steroid fraction. Steroids identity was assigned on the basis of the mass spectral features of their TMS ether, TMS enol-TMS ether, MO-TMS, and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. The data indicated that methenolone acetate was metabolized into several compounds resulting from oxidation of the 17-hydroxyl group and reduction of A-ring substituents, with or without concomitant hydroxylation at the C6 and C16 positions.     

Uptake and metabolism of [3H]-Leu-enkephalin following either its intraperitoneal or subcutaneous administration to mice.

The uptake and metabolism of 30 micrograms/kg [3H]-Leu-enkephalin ([3H]-LE) following either intraperitoneal (IP) or subcutaneous (SC) administration to Swiss Webster mice was examined. Uptake of [3H] was rapid, with peak levels of radioactivity in plasma observed at 5 or 10 min following IP or SC peptide injection, respectively. The majority (80-99% +/- 0.8) of plasma radioactivity at all postinjection plasma collection time points was in the form of tyrosine-containing enkephalin metabolites, indicating a substantial and rapid in vivo hydrolysis rate for exogenously administered LE. Leu-enkephalin is metabolized in vivo faster than previously reported in vitro in mouse plasma. However, despite this extensive hydrolysis, levels of intact LE remaining in plasma following its systemic administration are within or above endogenous LE plasma levels.     

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

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

The role of anthocyanins as an antioxidant under oxidative stress in rats

Cyanidin 3-O-beta-D-glucoside (C3G) is included in anthocyanins, and expected to have a potency to scavenge active oxygen species in vivo. Rats were fed a diet containing C3G (2 g/kg diet) for 14 days, and then subjected to hepatic ischemia-reperfusion (I/R) as an oxidative stress model. I/R treatment elevated the liver thiobarbituric acid-reactive substance concentration and the serum activities of marker enzymes for liver injury, and lowered the liver reduced glutathione concentration. Feeding C3G significantly suppressed these changes caused by hepatic I/R. These results indicate that C3G functions as a potent antioxidant in vivo under oxidative stress. To clarify the mechanism of action of C3G, we investigated the absorption and metabolism of C3G in rats. C3G appeared in the plasma immediately after the oral administration of C3G. Protocatechuic acid, which seems to be produced by the degradation of cyanidin, was also present in the plasma. In the liver and kidneys, C3G was metabolized to methylated form.     

Metabolism of thalidomide in human microsomes, cloned human cytochrome P-450 isozymes, and Hansen's disease patients

Previous in vitro studies in rat microsomal preparations suggested that thalidomide is metabolized by the cytochrome P450 system (CYP). In this study, we examined the extent of thalidomide metabolism by preparations of pooled human microsomes, microsomes containing cloned human CYP isozymes (CYPIA2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4), and Hansen's disease patients. Results indicated that thalidomide was a poor substrate for CYP isozymes. Alteration of incubation buffer, pH, incubation time, and microsome and thalidomide concentrations did not increase the production of any metabolites. Thalidomide also did not inhibit metabolism of CYP-specific substrates and therefore any interactions with other drugs that are metabolized by the same enzyme system are unlikely. Hansen's patients were given a single oral dose of thalidomide (400 mg), and their blood and urine were collected at time points up to 72 hours, processed, and analyzed by tandem mass spectrometry. Although thalidomide was present in the plasma and urine, no metabolites were found in the plasma and very low amounts of the 5-OH thalidomide metabolite were present in the urine. These results suggest that thalidomide does not undergo significant metabolism by human CYP and that clinically important interactions between thalidomide and drugs that are also metabolized by this enzyme system are unlikely. The major route of thalidomide breakdown in humans and animals is through spontaneous hydrolysis with subsequent elimination in the urine.     

Structure-activity relationships of a series of novel topical corticosteroids

The effect of various heteroaroyl groups in the 17-position of topical corticosteroids has been studied. The corticosteroids esterified at C17 were of 9 alpha,11 beta-dichloro, 9 alpha-chloro 11 beta-hydroxy and 9 alpha-fluoro 11 beta-hydroxy series. Among the 17-acyl groups 2'-furoates were most extensively investigated, although 2'-thenoates, 3'-thenoates and 3'-furoates were also examined. Many of these esters exhibited enhanced topical anti-inflammatory potencies. The most potent compounds investigated were the 21-chloro 17(2'-furoates) either in the 9 alpha,11 beta-dichloro, or in the 9 alpha-chloro 11 beta-hydroxy series. These compounds were at least 6 times as potent as betamethasone 17-valerate. Among 16-substituents studied 16 alpha-methyl compounds had the highest potency. Topical anti-inflammatory potencies were determined by using a 5-day modification of the croton oil ear assay in mice. The more potent compounds were also evaluated in the P. ovale induced chronic psoriaform lesion in the guinea-pig.     

Plasma uptake and in vivo metabolism of [Leu]enkephalin following its intraperitoneal administration to rats

To understand better how [Leu]enkephalin (LE) acts to modulate learning and memory in rats, the plasma uptake, disappearance, and metabolism of LE were investigated following its intraperitoneal administration. Concentrations of [3H]-LE and its radioactive metabolites were determined by thin layer chromatography in plasma samples withdrawn from rats at various times after injection of peptide. As measured in rats receiving an IP injection of a dose of LE (3 micrograms/kg) that impairs active avoidance conditioning, the LE was very rapidly metabolized, with greater than 95% of plasma [3H] in the form of metabolites by 1 min after injection. Despite this rapid metabolism, low but measurable quantities of intact LE were detectable in plasma at all sampling times. Consistent with a greater potency of D-Ala2-[D-Leu5]enkephalin (DADLE) than of LE in modulating avoidance conditioning, DADLE was less rapidly metabolized than was LE following its IP administration. The metabolism of DADLE and LE in vivo was more rapid than it was in plasma in vitro, suggesting a role for membrane bound enzymes in the metabolism of IP-administered enkephalins. The data demonstrate that, despite a rapid hydrolysis of LE in vivo, sufficient LE is present in plasma following IP administration of a behaviorally active dose to support a role of circulating intact LE in the modulation of avoidance conditioning.     

Hydroxylation of [3H]5 alpha-androstane-3 beta,17 beta-diol by whole tissue, epithelial cells and fibroblasts from the same hyperplastic human prostate

Hydroxylations of 3 beta-hydroxy 5 alpha-dihydro C19-steroids are terminal reactions by which male accessory sex organs dispose of intracellular androgens. Cellular androgen egress is of particular interest in benign prostatic hyperplasia (BPH) where the elevated nuclear 5 alpha-dihydrotestosterone-receptor content may be implicated in the etiology of the disease. We here report substitution of hydroxyl groups at C-6 alpha, C-7 beta and predominantly at C-7 alpha of [3H]5 alpha-androstane-3 beta,17 beta-diol on incubation of 3 and 8.5 nM substrate concentrations with minced and explanted human BPH tissue. Fibroblasts isolated from the same prostatectomy specimen hydroxylated 3 nM radiosubstrate mainly at C-6 alpha, with extensive metabolism to 17-oxosteroids. Epithelial cells from the same tissue source substituted to the same extent at the three positions. Competing 3 beta-hydroxysteroid dehydrogenase exceeded hydroxylase activity only in epithelial-cell cultures. Our findings support previous evidence that prostatic epithelial and stromal cells make different contributions to androgen disposition by the 3 beta-hydroxysteroid pathway.     

3-Hydroxy steroid dehydrogenase activities of cortisone reductase

The behaviour of various C(19) and C(18) steroids as substrates for crystalline preparations of cortisone reductase (EC 1.1.1.53) is described. 3alpha(Axial,3R)-, 3alpha(equatorial,3R)- and 3beta(axial,3S)-hydroxy steroid-NAD oxidoreductase activities are demonstrated. Four pairs of the substrates differed only in the shape of the a/b ring junction, three pairs differed only in substitution at C-10, and four pairs differed only in substitution in ring d. The shape of the substrate molecule and certain substituents (e.g. 10beta-methyl, 17beta-hydroxy, 16-oxo or 17-oxo) altered substrate behaviour, but steroids differing considerably in shape nevertheless acted as substrates, suggesting the possibility of a large or flexible binding site. K(m) values varied about 10-fold, many being approx. 140mum. V(max.) values covered a greater range (about 200-fold) and the good substrates had high V(max.) values rather than low K(m) values.     

Hypoglycemic effects of brassinosteroid in diet-induced obese mice

The prevalence of obesity is increasing globally, and obesity is a major risk factor for metabolic diseases such as type 2 diabetes. Previously, we reported that oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. Therefore, the aim of this study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50-300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 h of treatment. Daily chronic administration of HB (50 mg/kg for 8 wk) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition. These changes were accompanied by lower expression of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), and increased phosphorylation of AMP-activated protein kinase in the liver and muscle tissue. In vitro, HB treatment (1-15 μM) inhibited cyclic AMP-stimulated but not dexamethasone-stimulated upregulation of PEPCK and G-6-Pase mRNA levels in H4IIE rat hepatoma cells. Among a series of brassinosteroid analogs related to HB, only homocastasterone decreased glucose production in cell culture significantly. These results indicate the antidiabetic effects of brassinosteroids and begin to elucidate their putative cellular targets both in vitro and in vivo.     

Preclinical experience with two selective progesterone receptor modulators on breast and endometrium

Org 31710 and Org 33628 are two highly selective progesterone receptor modulators (PRMs) with respect to their anti-progestational and anti-glucocorticoid activity. The compounds have been studied both in vitro and in vivo. Org 33628 has approximately four times stronger anti-progestational activity in vitro than does Org 31710, and in rats it is about 15 times more potent in the pregnancy interruption test. Two main indications for the use of PRMs are breast cancer and fertility regulation. The effects of both Org 31710 and Org 33628 were tested in relevant models for these indications. The effects of the two compounds on breast tumor development were assessed and in rats using the DMBA model. Their potency in menses induction was tested in monkeys on a 4-day regimen in the luteal phase, and after a single dose at day 21 of the normal cycle, and under a continuous progestin treatment using desogestrel. The compounds were also tested alone in a continuous low-dose regimen. The effects on follicular development and ovulation were determined by measuring estradiol and progesterone levels. Cycle control was monitored by daily vaginal swabs. In the DMBA model, Org 31710 at oral doses of 0.8, 2.0, and 5.0 mg/kg showed a clear dose-related reduction in tumor load. With the two highest doses, an even lower tumor load was seen after a 3-week treatment period compared to the tumor load at the start of treatment. Org 33628 showed a similar efficacy as Org 31710 at a dose of 2.0 mg/kg. RU 486 after oral treatment was two times less potent in this model than Org 31710 and Org 33628. The efficacy of menses induction using the 4-day regimen is dependent on the time of administration relative to the progesterone peak in the luteal phase. The highest efficacy is achieved in the descending part of the peak, at which a 100% success rate is found with a dose of 1 mg/kg of either Org 31710 or Org 33628. In Cynomolgus monkeys, at a single dose of 15 mg/kg of Org 31710 or Org 33628 in the luteal phase, menses induction was achieved only in 60% of the treatment cycles. Surprisingly menses induction can be achieved with a single dose that is about a ten-times lower when the monkeys are treated continuously with desogestrel. Cycle control is better at low than at high doses of antiprogestin in combination with daily dosing of 4 microg/kg desogestrel. Despite the difference in receptor affinity, no difference between Org 31710 and Org 33628 was found in menses induction. In the continuous low-dose (1 mg/kg) regimen with the PRMs, follicular development occurs normally while ovulation is inhibited. Ovulation is resumed shortly after stopping treatment, and a normal menses occurs after the first progesterone peak. Both compounds may be interesting options for the prevention and treatment of breast cancer and for fertility control.     

Substrate-binding ability of Escherichia coli ribonucleic acid polymerase in relation to its protein composition.

The metabolism of [3H]progesterone in the rabbit endometrium and myometrium was studied in vitro. The major metabolities identified were 5alpha-pregnane-3,20-dione, 20alpha-hydroxypregn-4-en-3-one, 3beta-hydroxy-5alpha-preganan-20-one and 5alpha-pregnane-3beta,20alpha-diol. Other minor metabolites tentatively identified were 3alpha-hydroxy-5beta-pregnan-20-one,20alpha-hydroxy-5beta-pregnan-3-one and 5beta-pregnane-3alpha,20alpha-diol. The ability of the endometrium to metabolize progesterone on a unit weight bais was about 2.7 times that of the myometrium. The metabolism of [3H]progesterone in the rabbit uterus under the influnce of oestradiol-17beta and progesterone was studied. The ability of the oestradiol-treated rabbit uterus to metabolize progesterone was increased to 3.47 times that of the overiectomized control uterus, whereas the oestradiol-progesterone-treated rabbit uterus metabolized only 1.86 times that of the control. Study of the metabolism of progesterone with uterine subcellular preparations revealed that the 5alpha-reductase enzyme was present mainly in the nuclear fraction; 20alpha-hydroxysteroid dehydrogenase was found in the cytosol fraction and 3beta-hydroxysteroid dehydrogenase in the particulate fraction of the uterus. The metabolic pathways of progesterone in the rabbit uterine tissue are discussed.