Libraries of 2ß-(N-substituted piperazino)-5α-androstane-3α, 17ß-diols: chemical synthesis and cytotoxic effects on human leukemia HL-60 cells and on normal lymphocytes.

Libraries of steroid derivatives with two levels of molecular diversity were prepared to optimize the antiproliferative activity on leukemia HL-60 cells by first varying the amino acid (AA) at R(1) (libraries A, B, C, and D: with 45, 45, 20, and 20 members, respectively) and, subsequently, the capping group at R(2) (library E: 168 members). The screening of these aminosteroids revealed interesting structure-activity relationships. In library A, the compounds bearing a tetrahydroisoquinolone residue as the first element of diversity showed potent cytotoxicity, principally when isovaleric or cyclohexyl acetic acid was used as a capping group (>40% of cell growth inhibition at 1 µM). In library B, the phenylalanine (Phe) derivatives bearing a cyano group induced a higher growth inhibition than the other Phe derivatives. The screening of library C indicated the increase of hydrophobicity of proline (Pro) seems to preserve the cytotoxic effect achieved by the lead compound. However, the synthesis of structural Pro variants (library D) clearly shows weaker activities when compared to L-Pro building blocks. Finally, by incorporating some of the most active AA of libraries A-D in library E, we observed that the amide coupling functionality gave stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines. Six of the most active amide derivatives (E-37P, E-41P, E-42P, E-46P, E-48F, and E-12T) were selected and IC(50) determined on HL-60 cells as well as on normal human lymphocytes. Among this series of new anticancer agents, good to high selectivity indices (SI = IC(50) (lymphocytes)/IC(50) (HL-60 cells) = 5 - 55) were obtained.

The testosterone-derived neurosteroid androstanediol is a positive allosteric modulator of GABAA receptors.

Testosterone modulates seizure susceptibility, but the underlying mechanisms are obscure. Recently, we demonstrated that testosterone affects seizure activity via its conversion to neurosteroids in the brain. Androstanediol (5alpha-androstan-3alpha,17beta-diol) is an endogenous neurosteroid synthesized from testosterone. However, the molecular mechanism underlying the seizure protection activity of androstanediol remains unclear. Here, we show that androstanediol has positive allosteric activity as a GABA(A) receptor modulator. In whole-cell recordings from acutely dissociated hippocampus CA1 pyramidal cells, androstanediol (but not its 3beta-epimer) produced a concentration-dependent enhancement of GABA-activated currents (EC(50) of 5 microM). At 1 microM, androstanediol produced a 50% potentiation of GABA responses. In the absence of GABA, androstanediol has moderate direct effects on GABA(A) receptor-mediated currents at high concentrations. Systemic doses of androstanediol (5-100 mg/kg), but not its 3beta-epimer, caused dose-dependent suppression of behavioral and electrographic seizures in mouse hippocampus kindling, which is a model of temporal lobe epilepsy. The ED(50) value for antiseizure effects of androstanediol was 50 mg/kg, which did not produce sedation/motor toxicity. At high (2x ED(50)) doses, androstanediol produced complete seizure protection that lasted for up to 3 h after injection. The estimated plasma concentrations of androstanediol producing 50% seizure protection in the kindling model (10.6 microM) are within the range of concentrations that modulate GABA(A) receptors. These studies suggest that androstanediol could be a neurosteroid mediator of testosterone actions on neuronal excitability and seizure susceptibility via its activity as a GABA(A) receptor modulator and that androstanediol may play a key role in men with epilepsy, especially during the age-related decline in androgen levels.

Ultrastructural changes in prostate cells during hormone-induced canine prostatic hyperplasia.

Benign prostatic hyperplasia is a prevalent disease that has received relatively little attention in spite of its morbidity and remarkable social impact. There are few animal models of prostatic hyperplasia. The dog is the only species, along with humans, in which prostatic hyperplasia develops spontaneously and almost universally with age. The aim of the present study has been to compare the ultrastructural findings in a model of experimentally induced canine prostatic hyperplasia with those of the spontaneously developed changes in untreated dogs. An experimental group of 5 male beagle dogs were castrated and treated with combined steroids (3 weekly doses for over 30 weeks). Prostate samples were surgically obtained every 42 days (experimental stages 0 through 6). The control group consisted of 3 noncastrated dogs that were treated with vehicle and in which samples were taken only at stages 0, 1, 4, and 6. Changes in the control groups were similar but of lower intensity compared to those of the experimental groups. In luminal cells, crowding with papillary projections, prominent, branching microvilli, and abundant, often compartmentalized granules were observed. The most striking change was the previously unreported finding of caveolae in basal cells. They were mostly located in the basal aspect of basal cells and were more prominent in the experimental group and in advanced stages of treatment. These ultrastructural findings have not been previously reported in canine or human prostatic hyperplasia and merit further research. The model of experimentally induced canine prostatic hyperplasia provides an adequate setting for the understanding of this disease.

Cell kinetics and differentiation after hormonal-induced prostatic hyperplasia in the dog.

BACKGROUND: Our aim was to characterize the immunophenotypical changes in canine prostate epithelium after hormonal-induced benign prostatic hyperplasia (BPH). METHODS: Castrated dogs (aged 1-2 and 9-12 years) were treated with vehicle (group C), androstanediol (group A), or androstanediol plus estradiol (group AE). Surgical prostate biopsies were obtained before and after castration and after hormonal treatment. Tissue sections were stained using antibodies specific for basal cells (34betaE12), transiently proliferating (TP)/amplifying cells (RCK103), and luminal exocrine cells (RGE53). RESULTS: Castration resulted in a marked reduction in specific immunoreactivity associated with luminal secretory cells and basal cells in young dogs. In older dogs the number of basal cells remained constant. Hormonal treatment (AE) resulted in an increased number of cells with an immunophenotype that was associated with the TP/amplifying cell compartment and hyperplastic luminal epithelium. CONCLUSIONS: The relative increase in TP/amplifying cells in hormonally induced BPH in the dog is in line with a stem-cell-derived proliferation. Moreover, the finding of androgen-independent basal cells in the prostate of older dogs may contribute to the enhanced risk of development of BPH with increasing age.

Dose-dependent hormonal induction of benign prostatic hyperplasia (BPH) in castrated dogs.

A model for the dose-dependent hormonal induction of benign prostatic hyperplasia (BPH) in castrated dogs has been established using subcutaneously implanted Silastic capsules containing 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and estradiol-17 beta. In vivo release rates per capsule approximated 122.0 +/- 4.2 micrograms 3 alpha-diol and 22.7 +/- 0.8 micrograms estradiol per day based on in vitro studies and resulted in dose-dependent increases in serum 3 alpha-diol and dihydrotestosterone concentrations. The implantation of castrated dogs with either 10 or 20 Silastic capsules containing 3 alpha-diol and one capsule containing estradiol or the intramuscular injection of 3 alpha-diol (75 mg/week) and estradiol (0.75 mg/week) for 99 days significantly increased (P less than .01) prostatic weights and total prostatic DNA over intact controls. These treatments also resulted in a histomorphological pattern similar to that observed in dogs with the glandular form of spontaneous BPH. In addition, normal prostatic secretory function as determined by semen volume was maintained in these dogs. Although subcutaneous implantation of five Silastic capsules containing 3 alpha-diol and one capsule containing estradiol into castrated dogs resulted in prostatic weights and total prostatic DNA that were similar (P less than .10) to intact controls, these prostates were characterized histomorphologically by glandular atrophy and squamous metaplasia. Furthermore, prostatic secretory function was decreased (P less than .05) in these animals compared with intact controls at 3 months of treatment. This study has led to the development of a model of steroid-induced BPH that will facilitate the evaluation of competitive androgen-receptor antagonists in the dog.

Light-microscopic stereologic analysis of spontaneous and steroid-induced canine prostatic hyperplasia.

A combined light-microscopic and stereologic analysis of the canine prostate was performed under the following experimental conditions: intact and castrated dogs, spontaneous benign prostatic hyperplasia, intact and castrated dogs after treatment with testosterone, 5 alpha-dihydrotestosterone or 3 alpha-androstanediol in combination with estradiol. Regarding the absolute amounts of the glandular and stromal parts of the prostate as well as the glandular cells, no difference was found among the testosterone, 3 alpha-androstanediol and 5 alpha-dihydrotestosterone treated castrated dogs. Treatment with 5 alpha-dihydrotestosterone or 3 alpha-androstanediol in combination with 17 beta-estradiol induced a four-fold increase in glandular and a two-fold increase in stromal tissue. The glandular to stromal tissue ratio equals that found in spontaneous canine hyperplasia, which is indicative of the glandular type of spontaneous canine hyperplasia. Therefore, it can be stated that treatment with 5 alpha-dihydrotestosterone or 3 alpha-androstanediol combined with 17 beta-estradiol not only induces prostatic overgrowth but also leads to prostatic hyperplasia of the glandular type. However, the stereologic analysis of canine prostates following steroid administration shows that canine hyperplasia is primarily a glandular disease, while human benign prostatic hyperplasia shows more stromal activation.

Androgen receptor content of nafoxidine treated experimentally induced canine prostatic hyperplasia.

An estrogen stimulated increase in prostatic androgen receptor content has been postulated as the mechanism by which canine prostatic hyperplasia can be induced in the castrate dog treated with androstanediol and estradiol but not by androstanediol alone. In order to determine if the potent anti-estrogen Nafoxidine would inhibit this estrogen-associated development of prostatic hyperplasia, 2 week castrate young mongrel male dogs were injected for 1 month with either a) carrier solution (group 1), b) Nafoxidine (group 2), c) androstanediol and estradiol (group 3), or d) androstanediol and estradiol and Nafoxidine (group 4). At the termination of the experimental period the prostates of the animals in groups 1 and 2 were small (3.1 +/- 0.3 g (X +/- SEM) and 8.6 +/- 1.4 g respectively) and were atrophic histologically. The prostates in groups 3 and 4 were significantly heavier (24.7 +/- 3.0 g and 23.6 +/- 3.6 g respectively) than those in groups 1 and 2 and displayed glandular hyperplasia histologically. The dihydrotestosterone content of the prostatic tissue in groups 1 and 2 were similar (4.65 +/- 2.01 ng/mg DNA (X +/- SEM) and 3.13 +/- 0.65 ng/mg DNA respectively) and were significantly less than that of groups 3 and 4 (19.98 +/- 4.70 ng/mg DNA and 19.62 +/- 2.42 ng/mg DNA). The prostates of groups 3 and 4 thus displayed gravimetric, histologic and biochemical evidence of prostatic hyperplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological study of a new competitive neuromuscular blocking steroid, pipecurium bromide.

2 beta, 16 beta-Bis-(4'-dimethyl-1'-piperazino)-3 alpha, 17 beta-diacetoxy-5 alpha-androstane dibromide (pipecurium bromide, RGH-1106, Arduan), a newly synthetized bisquaternary steroid, produces competitive neuromuscular blockade in chicks, rats, cats, rabbits and dogs. The onset of paralysis is rapid. Pipecurium bromide is 2-4 times as potent as pancuronium bromide. The duration of action is about twice as long as that of pancuronium bromide in equiactive doses. Neostigmine rapidly and completely antagonises the neuromuscular blockade caused by pipecurium bromide. Even one hundred times higher dose than the effective blocking dose (2-6 microgram/kg) does not influence the cardiovascular system. Higher doses (1-2 mg/kg) cause transient decrease in blood pressure, in 10-20 mg/kg doses pipecurium bromide has ganglion blocking effect. In 1 mg/kg dose pipecurium bromide does not release histamine. Many times higher doses than the effective dose administered for 20 days, do not cause any toxic damage to respirated beagle dogs. According to examinations in rats, the placentary transfer of pipecurium bromide is lower than 0.1%. According to preliminary clinical examinations pipecurium bromide is free from side effects, and elicits as well controllable muscle relaxation.

Summary of safety tests with pipecurium bromide, a new neuromuscular blocking agent.

Acute toxicity studies with 2 beta,16 beta-bis(4'-dimethyl-1'-piperazino)-3 alpha,17 beta-diacetoxy-5 alpha-androstane dibromide (pipecurium bromide, RGH-1106, Arduan), a new neuromuscular blocking agent, revealed an order of sensitivity as follows: rabbits greater than mice greater than rats. Subchronic toxicity was tested on conscious dogs by 20 daily doses of 150 microgram/kg i.v. corresponding to 4-fold the planned clinical dose followed by daily intubation and artificial ventilation until recovery of spontaneous respiration. Dosage was limited by prolonged paralysis time requiring artificial ventilation. Apart from daily stress of paralysis preceding intubation no irreversible toxic changes were detected by laboratory and morphological tests. ECG changes were restricted to paralysis time, they were characterized by transitory arrhythmia and variation of the ST-segment. In additional studies on conscious and unconscious dogs no lethal ECG changes could be induced with cumulative dose totalling 10 mg/kg i.v. Short-term in vivo and in vitro mutagen tests did not reveal either mutagenic and clastogenic effects or increase of chromosomal aberrations. Intravenous local tolerance in rats was satisfactory.

[Effect of 5alpha-androstan-3beta, 17beta-diol on serum lipids and lipoproteins and the development of experimental atherosclerosis]. / Vliianie 5alpha-androstan-3beta, 17beta-diola na lipidy i lipoproteidy syvorotki krovi i na razvitie éksperimental'nogo ateroskleroza.

The author studied the influence of testosterone metabolite--5alpha-androstan-3beta,17beta-diol--on the development of hyperlipidemia and atherosclerosis in chinchilla rabbits given cholesterol-free semisynthetic atherogenic diet. The metabolite under study inhibited the development of hypercholesterolemia and hyperbetalipoproteinemia and decreased blood phospholipid content in the blood serum of experimental animals below the initial level. Lipid content in the sum total fraction of pre-beta and beta-lipoproteins decreased under the effect of the mentioned metabolite; there was also a fall in the amount of lipoproteins of low density and their greater saturation with cholesterol. Development of experimental atherosclerosis was intensified.