Further study on the antitumor activity of a homo-aza-steroidal ester of p-N,N-bis(2-chloroethyl)aminophenylacetic acid.
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Structure--anticancer and structure--genetic activity relationships of homo-aza-steroidal esters of N,N-bis(2-chloroethyl)aminocinnamic acid isomers.
Four steroidal lactams of the A- and D-rings were used for the esterification in the C-3 or C-17 positions, respectively, of their nuclei with the N,N-bis(2-chloroethyl)aminocinnamic acid isomers. The condensation reaction of the hydroxylic group of the steroidal lactams with each mustard was effected in dichloromethane in the presence of the catalyst p-dimethylaminopyridine and dicyclohexylcarbodiimide as dehydrating agent. The esters were obtained in pure form after column chromatography, and their structures were verified and confirmed by analytical methods (IR and UV spectra). The 12 esters were tested in vivo against P388, L1210 leukemias, Ehrlich ascites tumor, and melanoma B16. The esters 3 alpha-hydroxy-13 alpha-amino-13,17-seco-5 alpha-androstan-17- oic-13,17-lactam-o,m,p-N,N-bis(2-chloroethyl)aminocinnamates, in which the alkylating agents are linked to the modified steroid in the axial position, are inactive in the above experimental animal tumor systems. The effect of the homo-aza-steroidal esters of N,N-bis(2-chloroethyl)aminocinnamic acid isomers on the incorporation of radioactive precursors into DNA, RNA, and proteins of L1210, P388 leukemias, Ehrlich ascites tumor, and baby hamster kidney cells was investigated. Higher inhibitory effects on the incorporation of the radioactive precursors was obtained with the ortho-derivatives, yielding > 40% inhibition of thymidine incorporation in all tumor lines tested. The effect of four esters in which the m- N,N-bis(2-chloroethyl)aminocinnamic acid is linked to the modified steroids on sister chromatid exchanges in human lymphocyte culture was investigated.
Alkylating Agents↗
Magnetic resonance imaging of the efficacy of specific inhibition of 5 alpha-reductase in canine spontaneous benign prostatic hyperplasia.
A leading role for prostatic levels of dihydrotestosterone (DHT) in the pathogenesis of benign prostatic hyperplasia is well established, if incompletely understood. The present study provides initial confirmation that 5 alpha-reductase inhibition alone is sufficient to prevent prostatic accumulation of DHT and to produce epithelial regression in the canine prostate. In dogs treated with the specific 5 alpha-reductase inhibitor finasteride, prostatic volume decreased to one-third of the baseline volume, while the prostatic concentration of DHT fell fivefold: both were constant in placebo control dogs. Demonstration that MR imaging can serve as accurate modality to assess prostatic volume was provided by serial measurements of the canine prostate and by correlation of the last imaging measurement with the weight of the excised prostate. Significant intensity changes were observed in T2-weighted images measured post-treatment; these changes correlated with the histopathology of the prostate. These results suggest that beyond quantifying regression, multiecho T2 measurements can be useful in probing accompanying changes occurring on the cellular level.
5-alpha Reductase Inhibitors↗
Effect of a new 5 alpha-reductase inhibitor on size, histologic characteristics, and androgen concentrations of the canine prostate.
Mature male beagles were used in studies designed to determine the effect of the steroidal 5 alpha-reductase inhibitor 17 beta-N,N-diethylcarbamoyl-4-aza-4-methyl-5 alpha-androstan-3-one (4-MA) on size, histology, and androgen concentration of the prostate. Subcutaneous administration of 3 or 15 mg/kg/day for 43 days caused a sharp decline in prostate volume. flattening of prostatic epithelial cells, vacuolization of the cytoplasm and pycnosis of the nuclei. Whereas serum testosterone levels remained normal in dogs injected with 3 mg/kg/day, they were lower in those that received 15 mg/kg/day. Concentration of both testosterone and 5 alpha-dihydrotestosterone were reduced in the prostates of dogs that had received either 3 or 15 mg/kg/day of 4-MA. The 15 mg/kg/day level also appeared to adversely affect spermatogenesis. In a 43-day study, 4-MA given orally once each day at levels of 0.1, 0.3, or 1 mg/kg failed to cause a significant decrease in prostate volume. However, daily divided oral doses totaling 1 or 3 mg/kg were given in a 42-day test and both treatment levels produced significant reductions in prostate size.
5-alpha Reductase Inhibitors↗
The effect of 4MA, a potent inhibitor of 5 alpha-reductase, on the growth of androgen-responsive human genitourinary tumors grown in athymic nude mice.
The five alpha-reductase inhibitors are a newly developed family of compounds that block the intracellular conversion of testosterone (T) to dihydrotestosterone (DHT). 17-beta-N, N-Diethylcarbamoyl-4-methyl-4-aza-5-alpha-androstan-3-one (4MA), the most widely studied of these compounds, has been shown to inhibit the androgen-dependent growth of both normal animal tissues and of the Noble tumor, an experimental, hormone-responsive rat neoplasm. 4MA has been found to inhibit androgen-dependent growth without altering plasma levels of T or DHT. In the present study, we assessed the efficacy of 4MA in inhibiting the growth of PC-82 and R198, human androgen-responsive genitourinary malignancies. In the first experiment, 4MA was administered subcutaneously at a dose of 6 mg/kg/day to castrated and hormonally replaced groups of PC-82-bearing male athymic nude mice. 4MA therapy when compared to control therapy caused significant PC-82 growth inhibition in three different groups of hormonally replaced castrated mice: physiologic T-replaced (P less than .001), supraphysiologic T replaced (P less than .001), and supraphysiologic T and DHT replaced (P less than .001). There was no difference between the post therapy plasma levels of T or DHT in the control-treated and 4MA-treated mice. In the second experiment, the effect of 4MA (6 mg/kg/day S.Q.) was compared to that of castration and control therapy on the growth of R198. Both castration and 4MA therapy effectively inhibited R198 growth when compared to control therapy (P = .01 and .02, respectively), but there was no difference in the degree of growth inhibition seen with 4MA or castration (P = .45). Castration and 4MA-therapy significantly lowered plasma T levels when compared to control therapy; castration also significantly lowered plasma DHT levels, while 4MA and control therapy did not. These studies suggest that 4MA is effective in inhibiting the growth of human androgen-responsive tumors grown in athymic nude mice and that further studies with other 5 alpha-reductase inhibitors are indicated.
5-alpha Reductase Inhibitors↗
Effect of a 4-methyl-4-aza steroid on androgen metabolism by rat ventral prostate epithelial and stromal cell cultures: selective inhibition of 5 alpha-reductase activity.
The effect of a potent steroid metabolic inhibitor, 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (DMAA), on androgen metabolism was investigated in primary monolayer cultures of rat ventral prostate epithelial and stromal cells. Using testosterone (T) as substrate, 5 alpha-reductase (5 alpha-R) activity in both cell types was inhibited by greater than 98% at an inhibitor concentration of 1000 nM. The concentrations required to produce a 50% inhibition (IC50) were 7.4 and 9.0 nM for epithelial and stromal cells, respectively. To examine the specificity of this compound, its effect on other steroid-metabolic enzymes was examined. DMAA at a concentration of 1,000 nM had no effect on 3 alpha-hydroxysteroid oxidase (3 alpha-HSORox), 3-ketosteroid reductase (3 alpha-HSORred), and 6/7-hydroxylase (6/7-HSH) activities in both cell types; 17 beta-hydroxysteroid oxidase (17 beta-HSORox) activity, located primarily in epithelial cells, also was not influenced by DMAA. In contrast, epithelial 3 beta-hydroxysteroid oxidase (3 beta-HSORox) and 3-ketosteroid reductase (3 beta-HSORred) activities were inhibited by 65% (P less than .001) and 58% (P greater than .05), respectively, albeit the latter result was not statistically significant. Stromal 3 beta-HSORox and 3 beta-HSORred activities were negligible; hence the effect of the inhibitor of these enzymes could not be assessed. In conclusion, DMAA is a relatively selective and potent inhibitor of 5 alpha-R activity in primary cultures of rat ventral prostate epithelial and stromal cells and should be a useful compound for antagonizing androgen-mediated actions in the prostate and other androgen target tissues.
5-alpha Reductase Inhibitors↗
Effects of androgen and antiandrogen treatment on canine prostatic arginine esterase.
A series of experiments were conducted to investigate the regulation of the primary secretory protein of the canine prostate, arginine esterase, by androgens and/or new antiandrogen under development. In the first experiment, castration decreased (P less than 0.05) prostatic arginine esterase levels relative to intact controls (0.26 +/- 0.1 and 17.0 +/- 0.1 mumole/min/mg protein, respectively). Treatment of castrate dogs with either 5, 10, or 20 silastic capsules (8 cm length) containing the androgen 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) plus 1 capsule containing estradiol-17 beta (E2) or the i.m. injection of 25 mg 3 alpha-diol and 0.25 mg E2 for 12 weeks resulted in a dose-dependent increase (P less than 0.05) in prostatic arginine esterase activity (6.8 +/- 1.7, 19.0 +/- 3.6, 21.3 +/- 0.9, and 14.2 +/- 0.7 mumole/min/mg protein, respectively). In the second experiment, steroid treatment (10 3 alpha-diol plus 1 E2 silastic capsules) of castrate dogs for 12 weeks resulted in prostatic arginine esterase activity of 17.8 +/- 2.3 mu mole/min/mg. Co-administration of the steroidal androgen receptor antagonist. Win 49,596 (WIN) at doses of 0.625, 2.5, 10, or 40 mg/kg/day p.o., dose-dependently inhibited (P less than 0.05) prostatic arginine esterase activity (14.9 +/- 1.1, 14.3 +/- 1.3, 3.4 +/- 1.9, and 0.21 +/- 0.1 mumole/min/mg, respectively) to levels similar to that observed in castrate controls (0.14 +/- 0.03 mumole/min/mg). Administration of the nonsteroidal androgen receptor antagonist flutamide at 10 mg/kg/day p.o. to steroid-induced dogs also inhibited (P less than 0.05) arginine esterase activity (0.07 +/- 0.02 mumole/min/mg). In the last experiment, treatment of intact dogs with WIN at 0.625, 2.5, 10, and 40 mg/kg/day for 16 weeks dose-dependently reduced (P less than 0.05) arginine esterase levels (17.0 +/- 1.0, 16.3 +/- 1.5, 10.2 +/- 1.2, and 3.9 +/- 2.5 mumole/min/mg, respectively) compared to intact controls (14.4 +/- 1.2 mumole/min/mg). Histomorphologic and ultrastructural evaluation of prostates from dogs indicated that antiandrogen treatment resulted in glandular epithelial atrophy as well as a reduction in the number of secretory granules. The results of these experiments support that canine prostatic arginine esterase activity is under androgenic control, can be inhibited by antiandrogen treatment and may serve as a functional marker of the androgenic state of the prostate. Whether the effects of androgen and antiandrogens on prostatic arginine esterase is direct or indirect due to a general inhibitory effect on secretory epithelial cell function requires additional study. Furthermore, subject to further evaluation, the steroidal androgen receptor antagonist.(ABSTRACT TRUNCATED AT 400 WORDS)
5-alpha Reductase Inhibitors↗
Effect of castration, DES, flutamide, and the 5 alpha-reductase inhibitor, MK-906, on the growth of the Dunning rat prostatic carcinoma, R-3327.
Male rats bearing implants of the Dunning rat prostatic carcinoma, R-3327, were used in a 42-day study to determine the effect of castration or orally administered flutamide (FL), DES (diethylstilbestrol) or the 5 alpha-reductase inhibitor, MK-906, on the growth of this androgen-responsive cancer. The rate of growth and final weights of the tumor and the ventral prostate (VP) were all reduced (P less than 0.05) by castration. Flutamide (25 mg/kg/day) significantly decreased tumor and VP weights in intact rats and castrates given 100 micrograms/day (SC) of testosterone propionate (TP) or dihydrotestosterone propionate (DHTP). It also significantly retarded tumor growth rate in TP- or DHTP-treated castrates and was marginally effective in intact animals. DES (100 micrograms/kg/day) reduced (P less than 0.05) tumor and VP weights of intact rats but did not significantly affect tumor growth rate or weight in castrates given TP or DHTP. These results indicated that the effect of DES on tumor growth is caused by its inhibition of the secretion or release of the gonadotropins necessary for testicular androgen production. MK-906 (25 mg/kg/day) affected neither the gross nor the histomorphology of the tumor in intact rats or castrates given TP or DHTP. Further, it caused no histological changes in the testes of intact rats. It did, however, significantly reduce VP weight in intact animals and TP-treated castrates but not in those given DHTP. This illustrates that the anti-androgenicity of MK-906 stems from its inhibition of DHT formation. The failure of MK-906 to influence tumor growth in the TP-treated castrates strongly suggests that the R-3327 tumor can respond to testosterone directly. If that is true, then its growth is unlikely to be affected by a pure 5 alpha-reductase inhibitor such as MK-906. In ancillary experiments, tumors from MK-906-treated animals were found to have reduced levels of DHT and, when assayed in vitro, to have a reduced capacity to convert [3H]-T to [3H]-DHT.
5-alpha Reductase Inhibitors↗
In vivo assay for conversion of testosterone to dihydrotestosterone by rat prostatic steroid 5 alpha-reductase and comparison of two inhibitors.
An in vivo assay for steroid 5 alpha-reductase in rat ventral prostate has been developed and used to compare the inhibitory activity of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and 6-methylene-4-pregnene-3,20-dione (LY207320). Immature rats (70-80 g) received test compounds 30 min prior to s.c. injection of [3H]-T. The rats were sacrificed 30 min later and the ventral prostates were analyzed for [3H]-T metabolites. Intraprostatic [3H]-T and [3H]-DHT reached peak levels within 5 min after injection of [3H]-T and declined to about 25% of peak levels after 2 hr. 4-MA was a very potent inhibitor of [3H]-DHT formation with an estimated IC50 of 0.2 mg/kg. LY207320, an inhibitor of 5 alpha-reductase in vitro, was weakly active in vivo and did not achieve greater than 45% inhibition at high doses (greater than 200 mg/kg, s.c.). Tissue uptake of [3H]-T was also inhibited by LY207320, which may contribute to its inhibitory activity on accessory sex organ growth in the rat.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
The effect of finasteride on prostate-specific antigen in men with benign prostatic hyperplasia.
Finasteride is a specific 5-alpha-reductase inhibitor that has been shown to reduce prostate size and decrease serum levels of prostate specific antigen (PSA). Among men who received finasteride (5 mg/day) for 12 months in North American clinical trials and in whom prostate cancer was not diagnosed the median percentage change in PSA was -50% (5-95% range: -81% to +20%). At baseline 72% had PSA < or = 4.0 ng/ml and 93% had PSA < or = 10.0 ng/ml. After 12 months on finasteride, 75% had PSA < or = 2.0 ng/ml and 95% had PSA < or = 5.0 ng/ml. Thus, the proportion of BPH patients with PSA levels of 2.0 ng/ml and 5.0 ng/ml after 12 months of treatment was comparable to the proportion with pretreatment PSA levels of 4.0 ng/ml and 10.0 ng/ml. Among the 10 men in these trials subsequently diagnosed with prostate cancer while on long-term finasteride therapy (5 mg/day), the median percentage change in PSA was -26% (range: -48% to +12%). Limited experience with finasteride in men with prostate cancer suggests that the reduction in PSA of malignant origin appears to be no greater than the percentage reduction in PSA of benign origin. These effects on PSA have not been shown to confer any therapeutic benefit. Physicians using finasteride should be aware of its effect on PSA levels.
5-alpha Reductase Inhibitors↗
The influence of finasteride on the volume of the peripheral and periurethral zones of the prostate in men with benign prostatic hyperplasia.
To determine the influence of androgen deprivation induced by the potent 5 alpha-reductase inhibitor finasteride on the volume of the zones of the prostate, 20 symptomatic men with established BPH were randomized to one of three groups: placebo, finasteride 1 mg, and finasteride 5 mg/day. The volume of the entire prostate gland, periurethral zone, and peripheral zone and the seminal vesicles were determined by three dimensional reconstructions of magnetic resonance contoured images of the prostate. There was no significant difference between the results achieved with 1 and 5 mg of finasteride per day; thus the results in these two groups were combined. In the placebo group there was no significant change in the volume of any structure. Following treatment for 1 year with finasteride there was a significant (P < 0.02) 17% decrease in total gland size (11.5 +/- 3.2 cc). Similarly, there was a significant (P < 0.02) 17% decrease in total gland size (11.5 +/- 3.2 cc). Similarly, there was a significant (P < 0.03) decrease in the size of the periurethral zone of the prostate (6.2 +/- 3 cc). Although there was also a decrease in the size of the peripheral zone of the prostate (2.8 +/- 1.2 cc) this did not reach statistical significance. There was no significant change in the volume of the seminal vesicles. These findings indicate for the first time that androgen deprivation induces a significant decrease in the size of the periurethral zone of the prostate in men with established BPH.
5-alpha Reductase Inhibitors↗
Comparison of finasteride (Proscar), a 5 alpha reductase inhibitor, and various commercial plant extracts in in vitro and in vivo 5 alpha reductase inhibition.
Human prostate was used as a source of 5 alpha reductase. Compounds were incubated with an enzyme preparation and [3H]testosterone. [3H]-dihydrotestosterone production was measured to calculate 5 alpha reductase activity. IC50 values (ng/ml) were finasteride = 1; Permixon = 5,600; Talso = 7,000; Strogen Forte = 31,000; Prostagutt = 40,000; and Tadenan = 63,000. Bazoton and Harzol had no activity at concentrations up to 500,000 ng/ml. In castrate rats stimulated with testosterone (T) or dihydrotestosterone (DHT), finasteride, but not Permixon or Bazoton, inhibited T stimulated prostate growth, while none of the three compounds inhibited DHT stimulated growth. These results demonstrate that finasteride inhibits 5 alpha reductase, while Permixon and Bazoton have neither anti-androgen nor 5 alpha reductase inhibitory activity. In addition, in a 7 day human clinical trial, finasteride, but not Permixon or placebo, decreased serum DHT in men, further confirming the lack of 5 alpha reductase inhibition by Permixon. Finasteride and the plant extracts listed above do not inhibit the binding of DHT to the rat prostatic androgen receptor (concentrations to 100 micrograms/ml). Based on these results, it is unlikely that these plant extracts would shrink the prostate by inhibiting androgen action or 5 alpha reductase.
5-alpha Reductase Inhibitors↗
Finasteride (MK-906) in the treatment of benign prostatic hyperplasia. The Finasteride Study Group.
The effects of finasteride, a potent 5 alpha-reductase inhibitor, were assessed in 750 patients with benign prostatic hyperplasia (BPH) in a multicenter, double-blind, placebo-controlled study. Patients were randomized to finasteride, 1 or 5 mg, or placebo, daily for 12 months. The following changes were seen with finasteride 5 mg after 12 months: (1) Serum dihydrotestosterone decreased by 62% compared to no change with placebo (P < 0.001); (2) serum prostate-specific antigen (PSA) decreased by 46% compared to no change with placebo (P < 0.001); (3) prostate volume was reduced by 22% compared to a 5% decrease with placebo (P < 0.001); (4) maximum urinary flow rate increased by 1.7 ml/sec compared to a 0.4 ml/sec increase with placebo (P < 0.025); (5) total urinary symptom scores decreased by 3.3 points compared to a 2.0 point decrease with placebo (P = 0.005), and (6) obstructive symptom scores decreased by 2.1 points compared to a 1.4 point decrease with placebo (P = 0.017). The effects of finasteride 1 mg were similar to 5 mg, except in urinary symptoms, where no significant differences between 1 mg and placebo were seen. Patients treated with 5 mg finasteride demonstrated significantly greater improvements in urological status (assessed by the investigator) and the troublesome symptom score, compared to placebo. Treatment with finasteride was well tolerated. We conclude that finasteride is an effective medical therapy for a significant proportion of patients with BPH.
5-alpha Reductase Inhibitors↗
Effects of 4-MAPC, a 5 alpha-reductase inhibitor, and cyproterone acetate on regrowth of the rat ventral prostate.
Inhibitors of 5 alpha-reductase activity cause less involution of the rat ventral prostate (VP) than does castration. Studies were conducted in adult Sprague Dawley rats to evaluate the effects of a potent 5 alpha-reductase inhibitor, 4-MAPC, and the antiandrogen, cyproterone acetate (CA), on DNA synthesis and apoptosis. In experiment 1, VP weight fell 33%, 53%, and 83%, and DNA per ventral prostate was reduced 24%, 46%, and 71%, by 4-MAPC, CA, and castration, respectively. In experiment 2, adult rats were castrated, and the VP involuted for 7 days prior to 3 daily injections of testosterone propionate (TP; 1 mg/kg/d) +/- 10 mg/kg/d of 4-MAPC or CA. 3H-thymidine incorporation into VP DNA was increased in castrated animals treated with TP, and 4-MAPC and CA reduced uptake. In experiment 3, animals were treated for 14 days with the same protocol as that used in experiment 2. VP weight was increased in all animals treated with TP when compared with castration, and was reduced by both 4-MAPC and CA. DNA in rats treated with TP was similar to that in intact animals. DNA was not reduced by 4-MAPC, but was reduced by CA. The mRNA for TRPM-2, a marker of apoptosis, was increased only in untreated castrated rats. It appears that CA has a greater inhibitory effect than 4-MAPC on DNA synthesis. A major reason why castration reduces DNA more than either 4-MAPC or CA is that neither of these agents was able to increase programmed cell death to the degree seen with castration.
5-alpha Reductase Inhibitors↗
Effect of 5-alpha-reductase inhibition and dexamethasone administration on the growth characteristics and intratumor androgen levels of the human prostate cancer cell line PC-3.
The endocrine treatment of metastatic prostate cancer includes castration which reliably lowers the serum testosterone (T); however, the effect on intratumor levels of T and dihydrotestosterone (DHT) is less predictable. In vitro work demonstrated that the human prostate cancer cell line PC-3 had significant 5-a-reductase activity that could be inhibited with 17b-N,N-diethylcarbamoyl-4-aza-5a-androstan-3-one (4MA). In this study, we examined the effect of 5-a-reductase inhibition with 4MA and androgen suppression with dexamethasone on the growth characteristics and intratumor androgen levels in the PC-3 cell line in male athymic nude mice (Balb/c). The mice were randomized into six treatment groups: 1) noncastrate vehicle control, 2) 4MA, 0.25 mg/day, 3) 4MA, 1 mg/day, 4) dexamethasone, 25 micrograms/day, 5) 4MA, 1 mg/day, and dexamethasone, 25 micrograms/day, and 6) castrate control group. After 21 days of treatment the animals were sacrificed, serum collected, and tumors harvested. Each treatment produced intratumor DHT levels equivalent to the castrate group. Only the low dose 4MA caused a reduction in intratumor DHT without producing castrate levels of circulating T. The combination of dexamethasone and 4MA was less effective in lowering the intratumor DHT/T ratio than 4MA alone. No significant differences in tumor growth parameters were noted between intact control animals and any of the treatment arms. Serum T levels correlated poorly with intratumor androgen levels. Five-a-reductase inhibition produced castrate levels of intratumor DHT in the nonandrogen-dependent prostate cancer cell line PC-3. The combination of dexamethasone and 5-a-reductase inhibition with 4MA appears to be less effective in lowering intratumor androgen levels than either therapy alone.
5-alpha Reductase Inhibitors↗
A novel GABAA antagonist [3H]SR 95531: microscopic analysis of binding in the rat brain and allosteric modulation by several benzodiazepine and barbiturate receptor ligands.
Recent reports have demonstrated that the synthetic gamma-aminobutyric acid (GABA)-derivative, SR 95531 [2-(3'-carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-pyrid azinium bromide], possesses selective GABAA antagonistic properties. Because of its potency for recognition of GABAA sites, this agent has been used to identify GABAA receptors. In the present investigation, we studied the binding of [3H]SR 95531 to tissue sections of rat brain using microscopic analysis of receptor localization. The appropriate binding conditions for defining GABAA receptors with this radioligand were obtained by determining the dissociation and association kinetics, and performing saturation and displacement studies. Using membrane preparations from whole rat brain (or brain regions representing cortex, striatum, hippocampus, midbrain-thalamus, medulla-pons and cerebellum), saturation and displacement studies were analyzed, and allosteric modulation of [3H]SR 95531 binding was examined by including several benzodiazepine and barbiturate receptor ligands in the incubation media. To assess the stereoselective properties of [3H]SR 95531 binding in rat membranes, numerous barbiturates were added during the incubation. The binding of [3H]SR 95531 was demonstrated to be saturable, specific and to bind with relatively high affinity to low-affinity GABAA sites. Scatchard analysis performed on saturation data of binding to tissue sections showed a dissociation constant (KD) of 42.4 nM and a maximum number of binding sites (Bmax) of 105.8 fmol/mg tissue. Microscopic analysis showed that intermediate to high densities of [3H]SR 95531 binding occurred in brain regions containing intermediate to high densities of low-affinity GABAA receptor sites. The binding of [3H]SR 95531 to membranes also appeared to occur at low-affinity GABAA sites. Results from competition studies demonstrated that [3H]SR 95531 is displaceable by GABAA agents and displaced preferentially by GABAA antagonists. Scatchard analysis of saturation experiments from membrane preparations indicated that the KD and Bmax from the centrifugation assay was 53.0 nM and 4.26 pmol/mg protein, respectively. Using the filtration assay, binding to membranes yielded a KD value of 45.6 nM and a Bmax of 0.77 pmol/mg protein. The allosteric modulation data demonstrated that numerous benzodiazepine and barbiturate agents inhibited [3H]SR 95531 binding and this varied according to brain region. Several barbiturates included in the incubation media exhibited a stereoselective inhibition of [3H]SR 95531 binding to whole rat brain membranes.(ABSTRACT TRUNCATED AT 400 WORDS)
Allosteric Regulation↗
External genitalia abnormalities in male rats exposed in utero to finasteride, a 5 alpha-reductase inhibitor.
A series of studies was conducted to determine the developmental toxicity of the 5 alpha-reductase inhibitor finasteride (MK-0906) in rats. This compound was administered orally once daily to pregnant rats during various extended treatment periods during gestation. F1 offspring were evaluated on Day 20 of gestation as well as postnatally through mating to produce an F2 generation. MK-0906 treatment induced dosage-related incidences of hypospadias (penischisis) in male offspring with a threshold dosage level near 0.1 mg/kg/day and a 100% effect level of 100 mg/kg/day (with dosing through Day 20 of gestation). MK-0906 also caused decreased anogenital distance in male offspring. The dosage response for this effect (ranging from a 4.2% decrease at 0.003 mg/kg/day to a 38% decrease at 100 mg/kg/day) was more shallow than that for hypospadias. The decreases in anogenital distance were at least partially reversible postnatally with essentially complete recovery at dosages up to 0.1 mg/kg/day. There was also a dosage-related, temporary induction of nipples in F1 males. All of these effects were apparent following treatment on Days 6 through 17 of gestation but were more pronounced when dosing extended to Day 20 of gestation. Slight maternal toxicity consisting of minor decreases in body weight gain occurred only at dosages of 3 mg/kg/day and higher, indicating the selective nature of the developmental toxicity. The 5 alpha-reductase enzyme located in the rat fetal genital tubercle was studied in vitro and compared to that in the adult ventral prostate. The values for Km, Vmax, and IC50 for inhibition by MK-0906 were similar in the two tissues, suggesting that the enzymatic proteins in the genital tubercle and ventral prostate may be similar.
5-alpha Reductase Inhibitors↗
External genitalia of the rat: normal development and the histogenesis of 5 alpha-reductase inhibitor-induced abnormalities.
The normal histogenesis of the rat genital tubercle and the effect of exposure in utero to the 5 alpha-reductase inhibitor finasteride (L-652,931; MK-0906; Proscar) on that process were studied. In normal males and females, the genital tubercle was first seen on Day 14.25 of gestation. It contained a urethral plate which extended from the cloaca (and after Day 15.25, from the urogenital sinus) to the tip of the tubercle. On Day 18.25 the glans lamellae, which would separate the glans penis or the clitoris from the prepuce, began to develop in both sexes. Also on Day 18.25 a dense, midline plate of mesenchymal cells was first evident between the urogenital sinus and the rectum in normal males. This plate acted as a wedge, first increasing the separation between the rectum and the urogenital sinus, and subsequently separating the urethral plate from the surface epithelium in the genital tubercle. As a result, by Day 21.25 the urethra in males followed an "S"-shaped course, extending from the pelvis through the center of the glans penis to an orifice near the tip of the genital tubercle. In females, in which a mesenchymal plate did not develop, the urethral orifice remained at the base of the tubercle, and the clitoris contained the remnants of the urethral plate, extending as an open groove from the urethral orifice to the tip of the tubercle. Finasteride did not affect development of the genital tubercle in females. However, in males exposed to finasteride in utero, there was variable failure of the mesenchymal wedge to develop. As a result, the urethral plate remained in contact with the surface epithelium and eventually opened to form a groove on the ventral surface of the glans penis (hypospadias). Also, the persistence of the urethral plate along the ventral midline in finasteride-treated male fetuses and its subsequent opening as a groove interfered with development of the glans lamellae, causing displacement of the frenulum distally on the glans penis and the development of a cleft in the prepuce.
Androstenes↗