Effects of estrogen and progesterone on neuroactive steroids and cytokines in patients with suicidality.

BACKGROUND: In ovulating psychiatric patients experiencing suicidality, suicidal ideation (SI) often peaks perimenstrually. Our recent double-blind, placebo-controlled, crossover randomized clinical trial (RCT; NCT03720847) showed that perimenstrual administration of estradiol and progesterone (EP) can prevent this peak in SI and depressed mood. In this pre-registered follow-up analysis, we studied how the menstrual cycle and experimental manipulation affected two neurobiological systems associated with the menstrual cycle and suicide risk: GABAergic neuroactive steroids (NAS) and peripheral cytokines. METHODS: In 26 psychiatric outpatients with natural menstrual cycles and past-month SI, we analyzed serum samples from three blood draws (midluteal, perimenstrual, midfollicular) per experimental condition (EP vs placebo) timed to a luteinizing hormone-surge ovulation test. Using gas chromatography/mass spectrometry (GC/MS), we measured the progesterone (P4)-derived pregnane NAS (3α,5α)- 3-hydroxypregnan20-one (3α,5α-THP), (3α,5ß)- 3-hydroxypregnan-20-one (3α,5ß-THP), (3α,5α)- 3,21-dihydroxypregnan-20-one (3α,5α-THDOC), (3α,5α)- 3-hydroxyandrostan-17-one (3α,5α-A), the androstane NAS (3α,5ß)- 3-hydroxyandrostan-17-one (3α,5ß-A), (3α,5α,17ß)-androstane-3,17-diol (3α,5α-A-diol), (3α,5ß,17ß)-androstane-3,17-diol (3α,5ß-A-diol), and their precursor pregnenolone. High sensitivity multiplex assay kits quantified peripheral cytokines IL-1ß, IL-6, and TNF-α. RESULTS: P4-derived NAS fluctuated in parallel with P4 and increased with exogenous perimenstrual administration of EP. Conversely, androstane NAS either did not fluctuate or fluctuated inversely from P4, and these NAS decreased with exogenous EP. Peripheral cytokines did not show cyclical patterns, but each significantly predicted SI, depressed mood, or anxiousness. Concomitant SSRI medication use predicted lower androstane NAS. CONCLUSIONS: While preliminary and exploratory, our findings provide critical descriptive context for future studies. Further, our work presents menstrual cycle-related patterns for ten frequently-studied biomarkers, allowing for improved quality of comparisons involving naturally-cycling populations in research.

Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp.

The presence of the vertebrate steroids, testosterone (T) and 17ß-estradiol in mollusks is often cited as evidence that they are involved in the control of their reproduction. In this paper, we show that a likely source of T in at least one species, the common mussel (Mytilus spp.), is from uptake from water. When mussels were exposed to waterborne tritiated T ([3H]-T) in a closed container, the radioactivity decreased rapidly and exponentially until, by 24h, approximately 35% remained in the water. The rate of uptake of radiolabel could not be saturated by concentrations as high as 16.5µgL-1 (mean measured) of non-radiolabeled T, showing that the animals have a very high capacity for uptake of T. At least 30% of the applied radioactivity could be extracted from the tissues of the animals with organic solvents and most of this (26% of the total applied radioactivity) was in the fatty acid ester fraction. Following alkaline hydrolysis, reverse phase HPLC and TLC, this fraction was shown to consist predominantly of 5α-dihydrotestosterone and 5α-androstane-3ß,17ß-diol, while T was a minor component. These steroids were definitively identified in the fatty acid ester fraction by mass spectrometry. Overall, less than 5% of the [3H]-T applied to the system remained untransformed at the end of exposure. After ten days of depuration there was no reduction in the total amount of radioactivity in the tissues, nor any changes in the ratio of the metabolites in the ester fraction. These findings show that any association between T presence and reproductive status or sex is confounded by their significant capacity for uptake, and that T undergoes extensive metabolism in mussels in vivo and therefore may not be representative of the androgenic burden of the animals. Consequently, measurements of T in mussel tissue offer little utility as an indicator of reproductive status or sex.

Validation of a sequential extraction and liquid chromatography-tandem mass spectrometric method for determination of dihydrotestosterone, androstanediol and androstanediol-glucuronide in prostate tissues.

Androgens are key mediators of prostate development and function, a role that extends to the development of prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In prostate, DHT is the major androgen and reduction and glucuronidation are the major metabolic pathways for DHT elimination. A streamlined method for quantitation of dihydrotestosterone (DHT), 5α-androstan-3α,17ß-diol (3α-diol), and 3α-diol glucuronide (diol-gluc) was established and validated for use with archived prostate tissue specimens to facilitate examination of the roles of the underlying metabolism. This involved a sequential 70/30 hexane/ethyl acetate (hex/EtOAc) extraction of steroids, followed by an ethyl acetate extraction for diol-gluc. Derivatization of the hex/EtOAc fraction with2-fluoro-1-methylpyridinium p-toluene-4-sulfonate (FMP) was used to enhance sensitivity for hydroxyl steroids and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was utilized for analysis of both fractions. The method was validated with calibration standards followed by recovery assessment from spiked samples of BPH and normal prostate. Lower limits of quantitation (LLOQ) were 50 pg/g, 20 pg/g and 100 pg/g for DHT, 3α-diol and diol-gluc, respectively for extracts from 50mg equivalents of tissue. Prepared samples were stable for up to three weeks at 4 °C and 37 °C. The method provides excellent sensitivity and selectivity for determination of tissue levels of DHT, 3α-diol, and diol-gluc. Furthermore, this protocol can easily be extended to other hydroxyl steroids, is relatively straightforward to perform and is an effective tool for assessing steroid levels in archived clinical prostate samples.

Endogenous steroid profiling in the athlete biological passport.

The Athlete Biological Passport (ABP) is an individual electronic document that collects data regarding a specific athlete that is useful in differentiating between natural physiologic variations of selected biomarkers and deviations caused by artificial manipulations. A subsidiary of the endocrine module of the ABP, that which here is called Athlete Steroidal Passport (ASP), collects data on markers of an altered metabolism of endogenous steroidal hormones measured in urine samples. The ASP aims to identify not only doping with anabolic-androgenic steroids, but also most indirect steroid doping strategies such as doping with estrogen receptor antagonists and aromatase inhibitors. Development of specific markers of steroid doping, use of the athlete's previous measurements to define individual limits, with the athlete becoming his or her own reference, the inclusion of heterogeneous factors such as the UDPglucuronosyltransferase B17 genotype of the athlete, the knowledge of potentially confounding effects such as heavy alcohol consumption, the development of an external quality control system to control analytical uncertainty, and finally the use of Bayesian inferential methods to evaluate the value of indirect evidence have made the ASP a valuable alternative to deter steroid doping in elite sports. The ASP can be used to target athletes for gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS) testing, to withdraw temporarily the athlete from competing when an abnormality has been detected, and ultimately to lead to an antidoping infraction if that abnormality cannot be explained by a medical condition. Although the ASP has been developed primarily to ensure fairness in elite sports, its application in endocrinology for clinical purposes is straightforward in an evidence-based medicine paradigm.

Studies on neurosteroids XXIV. Determination of neuroactive androgens, androsterone and 5alpha-androstane-3alpha,17beta-diol, in rat brain and serum using liquid chromatography-tandem mass spectrometry.

The development and validation of liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) methods that enable the quantification of neuroactive androgens, androsterone (5alpha-androstan-3alpha-ol-17-one, 3alpha,5alpha-A) and 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), in the rat brain and serum are presented. The androgens were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges, derivatized with an ESI-active reagent, isonicotinoyl azide (INA), and then subjected to LC-ESI-MS/MS. The quantifications were based on selected reaction monitoring mode using the characteristic transitions of the INA derivatives. The methods allowed the reproducible and accurate quantification of the brain and serum neuroactive androgens using a 100 mg or 100 microL sample; the intra- and inter-assay relative standard deviations were below 3.6%, and the percentage accuracy values were 97.1-103.7% for both androgens. The animal study using the methods suggests that most of 3alpha,5alpha-Adiol found in the brain is derived from the periphery, while 3alpha,5alpha-A is not only transported from the periphery into the brain, but also synthesized in the brain by the oxidation of 3alpha,5alpha-Adiol. The androgens in the rats intraperitoneally administered finasteride, a 5alpha-reductatse inhibitor, were also measured; this treatment significantly reduced the brain 3alpha,5alpha-A and 3alpha,5alpha-Adiol levels and increased only the brain level of androstenedione, the precursor of 3alpha,5alpha-A.

Studies on Neurosteroids XXI: an improved liquid chromatography-tandem mass spectrometric method for determination of 5alpha-androstane-3alpha,17beta-diol in rat brains.

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the determination of the rat brain 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol) has been developed and validated. The brain extract was purified using solid-phase extraction cartridges, derivatized with isonicotinoyl azide, and subjected to LC-MS/MS. The method was accurate and reproducible, and the limit of quantitation was 0.1 ng/g tissue when a 100-mg tissue sample was used. The change in the brain 3alpha,5alpha-Adiol level by immobilization stress was also analyzed using the developed method.

Ontogeny and pathway of formation of 5alpha-androstane-3alpha,17beta-diol in the testes of the immature brushtail possum Trichosurus vulpecula.

The testicular androgen 5alpha;-androstane-3alpha,17beta-diol (androstanediol) mediates virilisation in pouch young of a marsupial, the tammar wallaby, and is the principal androgen formed in immature rodent testes. To chart the pattern of androstanediol formation in another marsupial species, the testes or fragments of testes from brushtail possums (Trichosurus vulpecula) that spanned the age range from early pouch young to mature adults were incubated with (3)H-progesterone and the products were identified by high-performance liquid chromatography. The only 19-carbon steroids identified in pouch young and adult testes were the Delta(4)-3-keto-steroids testosterone and androstenedione. However, androstanediol and another 5alpha-reduced androgen (androsterone) were synthesised by testes from Day 87-200 males and these appeared to be formed from the 5alpha-reduction and 3-keto reduction of testosterone and androstenedione. In the prostate and glans penis of the immature male, (3)H-androstanediol was converted to dihydrotestosterone. We conclude that the timing of androstanediol formation in the possum testis resembles the process in rodents rather than in the tammar wallaby and that any androstanediol in the circulation probably acts in target tissues via conversion to dihydrotestosterone.

Simultaneous determination of steroid composition of human testicular fluid using liquid chromatography tandem mass spectrometry.

A rapid, sensitive, and specific method using liquid chromatography tandem mass spectrometry (LC/MS/MS) has been developed for simultaneous determination of testosterone (T), dihydrotestosterone (DHT), estradiol (E2), and 5alpha-androstan-3alpha, -17beta-diol (3alpha-Diol) within human testicular fluid. Sample pretreatment involved a one-step extraction with diethyl ether. The analytes were separated on a Waters X-Terra C18 (150 mm x 2.1 mm i.d., 3.5 microm) analytical column with acetonitrile/water mobile phase (70:30, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 8 min. The column effluent was monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.1-50 ng/ml for T, 0.02-1 ng/ml for DHT, 0.05-2 ng/ml for E2, and 0.2-10 ng/ml for 3alpha-Diol, with values for the coefficient of determination of >0.99. The overall extraction efficiency was greater than 86% for T, 75% for DHT, 66% for E2, and 60% for 3alpha-Diol. The values for within-day and between-day precision and accuracy were <15%. We measured each of the four steroids in testicular sample volumes of only 20 microl, obtained by percutaneous testicular aspiration. The mean intratesticular testosterone concentration found by LC/MS/MS, 572 +/- 102 ng/ml, was similar to that previously obtained by radioimmunoassay (RIA). The mean intratesticular estradiol concentration was 15.7 +/- 2.3 ng/ml, which also correlated well with RIA measurement. Both DHT and 3alpha-Diol were below the limits of detection by RIA, but could be measured accurately by LC/MS/MS. In conclusion, LC/MS/MS represents a sensitive and accurate means by which to measure four separate steroids within small volume samples of testicular fluid.

Analytical strategies for the detection of non-labelled anabolic androgenic steroids in nutritional supplements.

Recent studies showed that non-hormonal supplements such as vitamins, minerals and amino acids can contain anabolic androgenic steroids not declared on the labels of the products. These undeclared substances (often prohormones of testosterone or 19-nortestosterone) can cause health risks to consumers and might lead to positive results in sports doping control, especially for the nandrolone metabolite norandrosterone. The analysis of nutritional supplements for anabolic steroids has proven to be rather difficult due to the different matrices in the various products. To conduct a broad-based analysis, a few robust methods capable of analysing various matrices are needed. To obtain a sensitive gas chromatography-mass spectrometry (GC-MS) analysis, a method including extraction and purification of the analytes followed by GC-MS analysis of the trimethylsilyl (TMS) derivatives of the steroids was developed. The limit of detection was improved by the addition of a mixture of 1-N,N-diisopropylamino-n-alkanes (DIPAs) to the final extract. In pure creatine monohydrate powder the limits of detection were demonstrated to be 0.1 ng microg (-1) for dehydroepiandrosterone (DHEA) and estr-4-ene-3beta,17beta-diol, 0.7 ng g(-1) for 5alpha-androstane-3beta,17beta-diol and androsta-1,4-diene-3,17-dione, 1 ng g(-1) for estr-5-ene-3beta,17beta-diol, estr-4-ene-3,17-dione, 19-nortestosterone, androst-4-ene-3, 17-dione and testosterone, and 2 ng g(-1) for androst-4-ene-3beta,17beta-diol and androst-5-ene-3beta,17beta-diol. The recovery (determined at 200 ng g(-1)) ranged from 32% for 19-nortestosterone to 92% for androst-5-ene-3beta,17beta-diol. During the investigation of different nutritional supplements, several analytical difficulties occurred. Aspects of homogenization, extraction, separation, derivatization and GC-MS measurement as well as strategies for the solution of problems arising were optimized. For quantitative measurements of the steroids in nutritional supplements, deuterated internal standards of the specific steroids or standard addition are necessary to compensate for matrix effects.

Immunization against 5alpha-androstane-3alpha,17beta-diol affects ovarian folliculogenesis in Merino ewes.

The 5alpha-reduced androgens have been implicated as antagonists of follicular development. In this experiment, we examined the effect of active immunization against 5alpha-reduced androgen on follicular development in ewes. During the breeding season, cyclic Merino ewes were either actively immunized three times against 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) or served as controls. Six to nine weeks after the last immunization, they were treated with PGF(2alpha) analog (PG, 125mg cloprostenol i.m.) and luteolysis was induced. Fourteen days after the PG treatment, the ewes were either killed (mid-luteal phase) or treated a second time with PG and killed 24h later (early follicular phase). At slaughter, blood samples were collected and ovaries recovered. All CL and follicles larger than 2mm were dissected and their size and appearance were recorded. Follicular fluid was collected and concentrations of estradiol-17beta (E(2)), progesterone (P), androstenedione (A(4)), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstane-3alpha-ol,17beta-one (androsterone: 3alpha-ol) and 3alpha-diol were determined by RIA. Immunization induced antibodies primarily to DHT and its 5alpha-reduced substrates 3alpha-diol and 3alpha-ol but not to E(2), P, A(4) or T. Immunization increased ovulation rate, size of ovulatory follicles and weight of CL. Immunization appeared to increase ovulation rate by decreasing the incidence of atresia in large preovulatory follicles. Regardless of their physiological status follicles contained only low levels of DHT; 3alpha-ol and 3alpha-diol were not detected in most follicles. Immunization did not appear to affect levels of DHT or other steroids in the follicular fluid. In conclusion, the induction of antibodies to 5alpha-reduced androgens increases ovulation rate by enhancing follicular viability during the preovulatory period in ewes. However, this effect is not brought about by the direct immune-neutralization of DHT or its 5alpha-reduced substrates 3alpha-ol and 3alpha-diol at the ovarian level.

Assessment of the androgen environment within the human testis: minimally invasive method to obtain intratesticular fluid.

Previous studies of the rat have shown that testosterone concentrations within the interstitial and seminiferous tubularfluids of the testes are significantly higher than normal serum levels, and further, that although intratesticular testosterone concentration can be substantially reduced without an effect on spermatogenesis, the concentration that is minimally required to maintain spermatogenesis is also substantially higher than serum levels. The purpose of the present study was to adapt a minimally invasive technique to sample human intratesticular fluid to enable parallel observations in man. To this end, aspiration methods were first developed for the rat testis and then adapted to the human. The testosterone concentration in fluid obtained by unilateral aspiration of rat testes was approximately 50 ng/mL, similar to the known concentration in seminiferous tubular fluid. These aspiration methods were then adapted to obtain intratesticular fluid from human testes. Studies of 12 fertile human subjects demonstrated that percutaneous testicular aspiration could be performed safely and successfully using a 19-gauge needle. Nine additional human subjects had bilateral testicular aspiration and simultaneous measurement of peripheral blood testosterone levels. Testicular aspirations yielded 8 to 117 microL of fluid from each testicle. The mean concentration of testosterone in aspirates obtained from the 21 patients was 609 +/- 50 ng/mL. Dihydrotestosterone and 3alpha-androstanediol concentrations were quite low, below the limits of detection of our assay. The SHBG/ABP concentration in the aspirates was 8.5 +/- 1.1 nM. These results define testosterone as the major androgenic steroid in the human testis, as in the rat testis, and indicate that the testosterone concentration within the human testis is approximately 200-fold greater than that of SHBG/ABP, and more than 100-fold greater than the concentration of testosterone found in normal human serum.

Hormones and diet: low insulin-like growth factor-I but normal bioavailable androgens in vegan men.

Mean serum insulin-like growth factor-I was 9% lower in 233 vegan men than in 226 meat-eaters and 237 vegetarians (P = 0.002). Vegans had higher testosterone levels than vegetarians and meat-eaters, but this was offset by higher sex hormone binding globulin, and there were no differences between diet groups in free testosterone, androstanediol glucuronide or luteinizing hormone.

Changes in the metabolic elimination profile of testosterone following exposure of the crustacean Daphnia magna to tributyltin.

The biocide tributyltin has been found to cause the development of pseudohermaphroditic conditions in some neogastropod species. These abnormalities of the reproductive system have adversely affected the fecundity of some field populations of gastropods, resulting in local population declines. Current evidence suggests that tributyltin elicits these effects by interfering with the biotransformation of testosterone to other steroid derivatives, resulting in an elevation in endogenous testosterone or some of its bioactive derivatives. The purpose of the present study was to determine whether tributyltin altered testosterone metabolism in daphnids (Daphnia magna), a species commonly used in ecotoxicology testing. Exposure of daphnids to 1.2 microg (tin)/L caused a general increase in the rate of elimination of oxido-reduced, hydroxylated, and glucose-conjugated derivatives of testosterone. However, tributyltin exposure had no significant effect on the rate of elimination of the glucose-conjugated forms of the various oxido-reduced and hydroxylated derivatives of testosterone. As a result, the percentage of the oxido-reduced and hydroxylated metabolites of testosterone eliminated as glucose conjugates decreased with increasing tributyltin exposure levels. These results demonstrate that tributyltin causes alterations in testosterone metabolism in daphnids that would result in an increase in the production of oxido-reduced derivatives. These products are preferentially retained in the tissues of daphnids and are variously androgenic in vertebrates. The increased production of oxido-reduced derivatives of testosterone may be mechanically responsible for the masculinizing effects of tributyltin in some species and suggests that daphnids may be a suitable surrogate for evaluating the potential of chemicals to elicit this form of toxicity.

Androgen metabolism in men receiving finasteride before prostatectomy.

Oral administration of finasteride, a 5 alpha-reductase inhibitor, affects intraprostatic androgens by suppressing dihydrotestosterone and increasing testosterone. This study was designed to determine the correlation of these effects of finasteride with changes in serum dihydrotestosterone, testosterone and androstanediol glucuronide. In a double blind, placebo-controlled study, 27 men with symptomatic benign prostatic hyperplasia were treated with placebo or 1 or 5 mg. per day finasteride for 6 to 8 weeks before transurethral resection of the prostate. There was no significant change in serum testosterone in any group, or in serum dihydrotestosterone or androstanediol glucuronide in the placebo group. There was a decrease in serum dihydrotestosterone by 66 +/- 4% and 70 +/- 8% (p = 0.32), and of serum androstanediol glucuronide by 78 +/- 3% and 86 +/- 3% (p = 0.012) in the 1 and 5 mg. finasteride groups, respectively. Intraprostatic dihydrotestosterone in the placebo group decreased from 18.6 +/- 1.4 nmol./kg. to 3.8 +/- 1.0 nmol./kg. and 1.7 +/- 0.7 nmol./kg. with 1 mg. and 5 mg. finasteride, respectively (p = 0.049 between 1 mg. and 5 mg. finasteride). Intraprostatic testosterone in the placebo group increased from 1.1 +/- 0.2 nmol./kg. to 7.6 +/- 1.0 nmol./kg. and 8.3 +/- 0.7 nmol./kg. with 1 mg. and 5 mg. finasteride, respectively (no significant difference between 1 mg. and 5 mg. finasteride). Serum and intraprostatic dihydrotestosterone correlated (p = 0.002). There was no correlation between intraprostatic dihydrotestosterone and serum androstanediol glucuronide. We conclude that 5 mg. of finasteride cause greater inhibition of intraprostatic 5 alpha-reductase than 1 mg. and that serum dihydrotestosterone is a better marker of intraprostatic dihydrotestosterone than androstanediol glucuronide.

[Analysis of steroids. Part 44: Analytical investigation of the intermediates of the synthesis of pipecuronium bromide (Arduan)]. / Adatok a szteránvázas vegyületek analitikájához 44. Pipecuronium bromid (Arduan) szintézis intermedierjeinek analitikája.

The following methods are described for the analytical investigation of the intermediates of the synthesis of pipecuronium bromide (Arduan) (for the numbering of the intermediates and their impurities see Figure 1.). 1. Gas chromatographic methods (capillary GC using fused silica capillaries Ultra-2 and Silar 10C WCOT) for the impurity profiling of intermediates I, II, IV and V including the identification and spectroscopic characterization of their impurities; 2. TLC methods for the similar characterisation of the further intermediates (III, VI, VII and VIII); 3. Gas chromatographic assay methods for IV and V using fused silica capillary technique and internal standards; 4. Potentiometric titration methods for the determination of VII and VIII using 0.1 M hydrochloric acid as the titrant.

[Analysis of steroids. Part 45: Analytical investigation of pipecuronium bromide (Arduan)]. / Adatok a szteránvázas vegyületek analitikájához 45. Pipecuronium bromid (Arduan) analitikai vizsgálata.

The following methods are described for the analytical investigation of pipecuronium bromide. 1. HPLC method. Of the several systems tried for the separation and quantification of impurities and degradation products the best results were obtained using silica as the stationary phase and 43:43:14 mixture of methanol, acetonitrile and concentrated aqueous ammonia containing 0.1 mole/l each of ammonium chloride and ammonium carbonate as the eluent. The validation of this method is presented. The above described aggressive eluent can be successfully replaced by an ion-pairing system using silica as the stationary phase and 96:4 mixture of acetonitrile and water containing 0.1 mole/l sodium perchlorate as the eluent. 2. Thin-layer chromatography. TLC systems are described for the separation and densitometric quantification of the impurities and degradation products of pipecuronium bromide. 3. Spectrophotometry. Two methods are described. The ester groups of the molecule can be determined by the iron(III)-hydroxamate method while for the ion-pair extraction of the quaternary ammonium steroid picric acid or bromthymol blue are used as the reagents. 4. Titrimetry. In addition to the titration with acetous perchloric acid for the assay of the bulk material a microtitration method is described for the determination of pipecuronium bromide in individual lyophylized ampoules (potentiometric titration with 0.1 M silver nitrate).

Effects of the new steroidal antiandrogen TZP-4238 on hormone-induced canine prostatic hyperplasia.

The effects of the new steroidal antiandrogen TZP-4238 on hormone-induced canine prostatic hyperplasia (BPH) were studied in comparison with those of chlormadinone acetate (CMA), a steroidal antiandrogen used in Japan. One- to 2-year-old beagle dogs were castrated and administered 75 mg/week of androstanediol (A-diol) plus 0.75 mg/week of estradiol (E2) for 25 weeks. These dogs were treated orally with placebo, 0.5 mg/kg/day of TZP-4238, 0.1 mg/kg/day of TZP-4238, and 2.5 mg/kg/day of CMA, respectively, for 21 weeks after 4 weeks treatment with A-diol plus E2. Treatment with 0.5 mg/kg/day of TZP-4238 or 2.5 mg/kg/day of CMA suppressed prostatic growth, and treatment with 0.1 mg/kg/day of TZP-4238 suppressed prostatic growth slightly. Treatment with 0.5 mg/kg/day of TZP-4238 decreased 5 alpha-reductase activity, DHT content, and nuclear androgen receptor (AR) content in the prostate, and treatment with 0.1 mg/kg/day of TZP-4238 or 2.5 mg/kg/day of CMA also decreased or tended to decrease these parameters. In conclusion, TZP-4238 and CMA were effective in inhibiting the growth of hormone-induced canine BPH, and TZP-4238 was at least 5 times more potent than CMA. TZP-4238 inhibited prostatic growth by decreasing prostatic androgen content and the androgen-AR complex. TZP-4238 decreased 5 alpha-reductase activity by prevention of the androgen action described above.

Biochemical characterization and seasonal changes in the concentration of testosterone-metabolizing enzymes in the European great tit (Parus major) brain.

Homogenates of diencephala obtained from brains of European great tits (Parus major) were incubated in the presence of tritiated testosterone (T) as precursor, and four metabolites produced from this steroid were formally identified and quantified. Conversion into 5 beta-dihydrotestosterone (5 beta-DHT) constituted the major metabolic pathway of T. Smaller amounts of 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-DIOL), and estradiol (E2) were also produced. The metabolism of T was time-dependent, and it varied as a function of the initial precursor concentration. The kinetics of 5 beta- and 5 alpha-reductases, as well as aromatase, followed the Michaelis-Menten model. It was found that 5 beta-reductase has a low apparent affinity for T, but is present in large concentrations. In contrast, the apparent affinity for T and the concentration of aromatase were approximately 3.9 times higher and 130 times smaller, respectively, than those of 5 beta-reductase. Intermediate values were found for 5 alpha-reductase. The validated assay was used to measure seasonal changes in the in vitro metabolism of T in the anterior (AH) and posterior (PH) hypothalamus and the cerebellum (CER) of free-living juvenile and adult male great tits. The production of 5 beta-DHT was low during the winter period in the PH of adult males, whereas the 5 beta-DIOL production was low in both parts of the hypothalamus at this time of the year. During autumn the production of these metabolites showed a transitory decrease in both parts of the hypothalamus of the juveniles. The production of 5 beta-reduced metabolites by the CER was high at all times of the year. In juveniles, the CER production of 5 beta-DHT did not change seasonally, whereas 5 beta-DIOL production peaked during summer. In the CER of adults, maximum production of both metabolites occurred during summer. Generally, less T was converted into 5 beta-reduced metabolites by the PH than by either the AH or the CER. E2 production was observed only in the AH and PH. With one exception (summer; AH), E2 production was high in both parts of the hypothalamus of adults throughout the year. In both AH and PH of juveniles, E2 production was low during summer. In these birds, it increased between summer and autumn in both parts of the hypothalamus, and also between autumn and winter in the PH. The production of 5 alpha-DHT did not change as a function of the season, the age of the birds, or the brain region.(ABSTRACT TRUNCATED AT 400 WORDS)

Estimation of impurity profiles of drugs and related materials. Part VIII: Combined application of high-performance liquid chromatography and NMR spectroscopy in the impurity profiling of drugs.

The usefulness of the joint application of HPLC and NMR spectroscopy in drug impurity profiling is demonstrated by the following examples: (1) identification of Z and E isomers of 17 alpha-ethynyl-4-oestrene-3 beta, 17-diol-3-acetate-17-(3'-acetoxy-2'-butenoate) in ethynodiol diacetate; (2) identification of the p-tolyl analogue as the impurity of enalapril maleate; (3) identification and quantification of 2'-dehydro-pipecuronium bromide in pipecuronium bromide. The possibilities of utilizing NMR spectroscopy for the identification and quantification of the impurities with and without their isolation are discussed.