Pharmacokinetic parameters and mechanisms of inhibition of rat type 1 and 2 steroid 5alpha-reductases: determinants for different in vivo activities of GI198745 and finasteride in the rat.

The interaction of baculovirus expressed rat steroid 5alpha-reductase types 1 and 2 (r5AR1 and r5AR2) with 17beta-N-(2,5-bis(trifluoromethyl)phenyl)carbamoyl-4-aza-5alpha-androst-1-en-3-one (GI198745) was investigated at pH 7 and 37 degrees. This 5alpha-reductase inhibitor was found previously to be a time-dependent inhibitor of the two human 5alpha-reductase isozymes. In contrast, we demonstrate in the present study that although GI198745 is a potent time-dependent inhibitor of r5AR2, it is a classical rapid-equilibrium inhibitor of r5AR1. This type of behavior with human and rat 5alpha-reductases has been shown for the inhibitor 17beta-(N-tert-butylcarbamoyl)-4-aza-5alpha-androst-1-en-3-one (finasteride), a current therapy for benign prostatic hyperplasia. Inhibition of r5AR1 by GI198745 was competitive with testosterone and followed Michaelis-Menten kinetics with a K(i) value of 0.3 +/- 0.02 nM. Data for the inhibition of r5AR2 by GI198745 were consistent with a two-step mechanism, where K(i) is the dissociation constant for an initial enzyme-inhibitor complex and k(3) is the rate constant for the second slow step. The pseudo-bimolecular rate constant (k(3)/K(i)) for the association of GI198745 with r5AR2 was (2.0 +/- 0.4) x 10(7) M(-1) sec(-1). The high affinity of this inhibitor for r5AR2 was further demonstrated by the inability of the enzyme-inhibitor complex to dissociate after approximately 7 days of dialysis at 4 degrees. Both GI198745 and finasteride appear to inactivate r5AR2 by apparent irreversible modification, but are classical, reversible inhibitors of r5AR1. Therefore, we hypothesize that because of its pharmacokinetic parameters and increased potency against r5AR1, GI198745 is more effective than finasteride in preventing the growth of the rat prostate.

Troglitazone prevents insulin dependent diabetes in the non-obese diabetic mouse.

Troglitazone has recently been introduced in the treatment of Type 2 diabetes. In addition to its anti-diabetic effects it acts as a perixosome proliferator activated receptor-gamma (PPAR-gamma) agonist and has anti-inflammatory properties by inhibiting macrophage tumour necrosis factor-alpha (TNF-alpha) secretion. It also inhibits the production of endothelial selectin (e-selectin). Troglitazone also reduces interleukin-1alpha induced nitric oxide production in pancreatic beta-cells, which may be relevant in preventing nitric oxide mediated damage to these cells in the Type 1 diabetes process. We tested troglitazone in the spontaneous model of autoimmune diabetes, the non-obese diabetic (NOD) mouse, to determine its effect on the disease process. When administered by gavage from weaning at a dose of 400 mg/kg body weight (n = 32), troglitazone reduced the incidence of diabetes by 16 weeks compared to controls (n = 32) in a pattern that was maintained up to the conclusion of the experiment at 31 weeks of age (p < 0.05). Insulitis was unaltered (index = 1.05 +/- 0.71 vs. 1.13 +/- 0.82, treated vs. controls, p = 0.78). The study was repeated using troglitazone in the diet of NOD mice (n = 24) to give a dose of approximately 200 mg/kg body weight in order to provide a more consistent level of troglitazone during the time course of the experiment. There was a reduction of diabetes incidence in this group but it did not reach significance. Insulin levels were reduced in gavage treated mice although such reduction did not reach significance (p < 0.07). We conclude that, in view of its effect on this model of autoimmune diabetes and because of its known function as an insulin sensitiser, troglitazone might be considered for potential use in those patients with Type 1 masquerading as Type 2 diabetes.

Unique preclinical characteristics of GG745, a potent dual inhibitor of 5AR.

Selective inhibition of type 2 5alpha-reductase has been shown to be efficacious in the treatment of benign prostatic hyperplasia. Pharmacokinetic and pharmacodynamic results are reported of treatment with a potent inhibitor of both 5alpha-reductase isozymes, GG745, in rats, dogs and men. In the rat, GG745 has a similar effect on DHT-driven prostatic growth as finasteride, another dual 5alpha-reductase inhibitor in this species. However, GG745 appears to be more potent in the rat, a result that likely reflects the greater inherent potency and terminal half-life of GG745 (14 hr) compared with that of finasteride (1 hr). These pharmacokinetic differences are also maintained in the dog (65 and 4 hr for GG745 and finasteride, respectively). From these results, the literature, and in vitro studies, we estimated doses of GG745 likely to prove efficacious in reducing DHT levels in man. These estimated values were predictive of single-dose effects of GG745 in man. Results from single-dose evaluations in man indicate that GG745 has a terminal half-life of approximately 240 hr, and single doses of >10 mg decreased DHT levels significantly more than did single 5-mg doses of finasteride. These data support the hypothesis that a molecule (GG745) that effectively inhibits both 5alpha-reductases will lower serum DHT levels significantly more than a molecule that inhibits only a single 5alpha-reductase isozyme (e.g., finasteride, a selective inhibitor of the type 2 enzyme in man).

Androgen independence of primary epithelial cultures of the prostate is associated with a down-regulation of androgen receptor gene expression.

BACKGROUND: Epithelial cells cultured from prostatic acini do not demonstrate significant (P > 0.05) growth response to the testosterone metabolite dihydrotestosterone (DHT) at concentrations of 0.001-10.0 nM. In addition, the nonsteroidal antiandrogen hydroxyflutamide (HO-F) does not influence primary epithelial cell proliferation in this concentration range. METHODS: Northern blotting carried out with an androgen reception (AR)-specific cDNA probe indicated that the extent of AR gene expression in six unpassaged primary prostatic epithelial cell cultures was insufficient to elicit a detectable signal upon autoradiography. However, RT/PCR analysis of total RNA using two sets of intron-spanning androgen receptor (AR) primers demonstrates the presence of full-length receptor transcripts in two BPH-derived epithelial cell cultures (BPH1 and BPH2) as well as a carcinoma-derived culture (CaP1). RESULTS: AR-positive LNCaP cells transfected with the AR reporter plasmid pMMTV/SPAP exhibit significant increases (P < 0.05) in SPAP production upon treatment with DHT. pMMTV/SPAP-transfected primary epithelial cells exhibit no such response when pulsed with either androgen or anti-androgen. CONCLUSIONS: These results indicate that the lack of significant AR gene expression underlies the androgen independence of primary prostatic epithelial cell cultures.

Structure-activity relationships for inhibition of type 1 and 2 human 5 alpha-reductase and human adrenal 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase by 6-azaandrost-4-en-3-ones: optimization of the C17 substituent.

A variety of C17 amide-substituted 6-azaandrost-4-en-3-ones were prepared and tested versus human type 1 and 2 steroid 5 alpha-reductase (5AR) and human adrenal 3 beta-hydroxy-delta 5-steroid dehydrogenase/3-keto-delta 5-steroid isomerase (3BHSD) in order to optimize potency versus both isozymes of 5AR and selectivity versus 3BHSD. Two series of potent and selective C17 amides were discovered, 2,5-disubstituted anilides and (arylcycloalkyl)amides. Compounds from each series with picomolar IC50's versus human type 2 5AR and low nanomolar to picomolar IC50's versus human type 1 5AR possessing 100-500-fold selectivity versus 3BHSD were identified. A conformational model to predict 3BHSD potency was developed which could rationalize 3BHSD potency within three different series of compounds. Evaluation of some optimal compounds from this series in a chronic castrated rat model of 5AR inhibitor induced prostate involution, and pharmacokinetic measurements identified compounds (9, 12, 16, and 29) with good in vivo efficacy and half-life in the dog. An intact rat model of in vivo selectivity for 5AR versus 3BHSD inhibition was also developed. Dual inhibitors of both human 5AR's may show advantages over type 2 selective 5AR inhibitors, such as finasteride (1), in the treatment of disease states which depend upon dihydrotestosterone.

The effects of nonidet P40 on the function of rat peritoneal mast cells in vitro.

1 Treatment of purified rat peritoneal mast cells at 37 degrees C with concentrations of the non-ionic detergent nonidet P40 (NP40) up to 0.005% (v/v) failed to reduce their viability. 2 There was a marked reduction in the histamine releasing capacity of NP40-treated mast cells upon challenge with a variety of selective (adrenocorticotrophic hormone 1-24 (Synacthen), rabbit anti-rat IgE antiserum, adenosine triphosphate (ATP) and the calcium ionophore, A 23187) and non-selective (rabbit anti-rat mast cell antiserum plus complement) histamine liberators. 3 Nonidet P40 (0.005%) was found to reduce the activity of a mast cell membrane 'ecto-enzyme', calcium-activated ATPase, by about 45% when presented at the time of its assay.

A comparison of the histamine-releasing properties of rat pleural and peritoneal mast cells.

A comparison has been made of the histamine-releasing characteristics of rat mast cells of pleural and peritoneal origin in response to a wide variety of immunological and non-immunological stimuli, namely, IgE antibody-antigen interaction; rabbit anti-rat antibody; concanvalin A (Con A); ACTH (1-24) polypeptide (Synacthen); a decapeptide comprising an amino acid sequence (497-506) within the human gamma-chain (Stanworth, Kings, Roy, Moran & Moran, 1979); adenosine triphosphate (ATP) and the calcium ionophore. This has provided valuable information about the relative responsiveness of target cells from two different sources within the same species. Pertioneal cells proved to be more responsive to basic polypeptide liberators, whereas pleural cells were considerably more responsive to stimuli mediated through IgE antibody, and slightly more responsive to challenge with non-immunological liberators. Interestingly, the histamine-release studies using an antiserum raised against mast cell IgE receptors have indicated that pleural mast cell membranes contain a higher density of IgE receptors than peritoneal mast cell membranes. Moreover, it was established that the amount of histamine release from either peritoneal or pleural mast cells effected by the polypeptide liberators was not influenced by the prior occupancy of the mast cell Fc receptors by rat IgE antibody.

A basic design of a chair for hemiplegics with special reference to the back shape.

The seating requirements of hemiplegic patients are studied extensively in the light of ergonomic principles and with the co-operation of the staffs and patients of 25 hospitals in the UK. It is suggested that the provision of a suitably contoured back and seat shape would greatly reduce the need to use other supporting mechanisms in order to seat the patient comfortably and effectively. Plans are given for a chair which should provide integrated support for the patient although no constructional plans are given. The back shape is designed with the aid of data from a survey of elderly people's back shapes and it is hoped that this part of the design will find application in chairs for geriatric patients generally. First, however, it is necessary thoroughly to test the design under working conditions.