Decreased efficacy of twice-weekly intravaginal dehydroepiandrosterone on vulvovaginal atrophy.

OBJECTIVE: While daily intravaginal administration of 0.50% (6.5 mg) dehydroepiandrosterone (DHEA, prasterone) for 12 weeks has shown clinically and statistically significant effects on moderate to severe (MS) dyspareunia as the most bothersome symptom (MBS), the present study analyzes the effect of a reduced dosing regimen on MBS vaginal dryness. METHOD: Daily intravaginal 0.50% prasterone for 2 weeks followed by twice weekly for 10 weeks versus placebo. RESULTS: Maximal beneficial changes in vaginal parabasal and superficial cells and pH were observed at 2 weeks as observed for intravaginal 10 µg estradiol (E2). This was followed by a decrease or lack of efficacy improvement after switching to twice-weekly dosing. The decrease in percentage of parabasal cells, increase in percentage of superficial cells and decrease in vaginal pH were all highly significant (p < 0.0001 to 0.0002 over placebo) at 12 weeks. In parallel, the statistical significance over placebo (p value) on MBS vaginal dryness at 6 weeks was 0.09 followed by an increase to 0.198 at 12 weeks. For MBS dyspareunia, the p value of 0.008 at 6 weeks was followed by a p value of 0.077 at 12 weeks, thus illustrating a decrease of efficacy at the lower dosing regimen. The improvements of vaginal secretions, color, epithelial integrity and epithelial surface thickness were observed at a p value < 0.01 or 0.05 over placebo at 2 weeks, with a similar or loss of statistical difference compared to placebo at later time intervals. No significant adverse event was observed. Vaginal discharge related to the melting of Witepsol was reported in 1.8% of subjects. CONCLUSION: The present data show that daily dosing with 0.50% DHEA for 2 weeks followed by twice-weekly dosing is a suboptimal treatment of the symptoms/signs of vulvovaginal atrophy resulting from a substantial loss of the efficacy achieved at daily dosing.

Chronic administration of dehydroepiandrosterone (DHEA) to female monkey and rat has no effect on mammary gland histology.

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, has been shown to exert chemopreventive effect on the development of carcinogen-induced rat mammary tumors. Since little is known on the effect of DHEA administration on mammary gland physiology and histology, we have studied the effect of long-term administration of DHEA to normal female monkey and rat on mammary gland histology as well as on serum DHEA, DHEA sulphate (DHEA-S), testosterone and estradiol levels. In monkeys, DHEA treatment (2 or 10 mg/(kg b.w.day)) induced a dose-related increase in serum DHEA and DHEA-S (above 20-fold) levels. At the highest dose of DHEA, serum testosterone levels were significantly increased (three- to fourfold), while serum estradiol concentration was not modified. DHEA treatment did not modify the histological characteristics of monkey mammary glands. In the rat, following DHEA administration (10 or 100 mg/(kg b.w.day)), a dose-related marked increase in serum DHEA and DHEA-S was observed. Serum testosterone was also increased in DHEA-treated animals, while no significant changes in serum estradiol levels were detected. As in the monkey, the histology of the female rat mammary gland remained unchanged following long-term treatment with any of the two doses of DHEA.

Effect of dehydroepiandrosterone and the antiestrogen EM-800 on growth of human ZR-75-1 breast cancer xenografts.

BACKGROUND: In the mammary gland, androgens are formed from the precursor steroid dehydroepiandrosterone (DHEA). Clinical evidence indicates that androgens have inhibitory effects on breast cancer. Estrogens, on the other hand, stimulate the development and growth of breast cancer. We studied the effect of DHEA alone or in combination with the newly described pure antiestrogen EM-800 on the growth of subcutaneous tumor xenografts formed by the human breast cancer cell line ZR-75-1 in ovariectomized nude mice. METHODS: Immediately after ovariectomy, mice received daily subcutaneous injections of 0.5 microg estrone (E1) (an estrogenic hormone). EM-800 (15, 50, or 100 microg) was given orally once daily. DHEA was administered percutaneously twice daily (total dose of 0.3, 1.0, or 3.0 mg) to the dorsal skin either alone or in combination with a 15-microg daily oral dose of EM-800. Changes in tumor size in response to the treatments (in relation to measurements made on the first day of treatment) were assessed periodically. At the end of the experiments, tumors were dissected and weighed. RESULTS: A 9.4-fold increase in tumor size in 9.5 months was observed in ovariectomized mice receiving E1 alone. Administration of 15, 50, or 100 microg EM-800 in E1-supplemented mice led to inhibitions of 87.5%, 93.5%, and 94.0% in tumor size, respectively. DHEA, on the other hand, at doses of 0.3, 1.0, or 3.0 mg inhibited terminal tumor size by 50.4%, 76.8%, and 80.0%, respectively. Comparable inhibitions in tumor size were obtained with a daily 15-microg oral dose of EM-800 with or without different doses of percutaneous DHEA. CONCLUSIONS: DHEA and EM-800 independently suppressed the growth of E1-stimulated ZR-75-1 xenograft tumors in nude mice. Administration of DHEA at the defined doses did not alter the inhibitory effect of EM-800.

Combined effects of dehydroepiandrosterone and EM-800 on bone mass, serum lipids, and the development of dimethylbenz(A)anthracene-induced mammary carcinoma in the rat.

Although treatment with dehydroepiandrosterone (DHEA) and the antiestrogen EM-800 alone decreased dimethylbenz(A)anthracene (DMBA)-induced mammary tumor incidence from 95% to 57% and 38%, respectively, approximately 9 months after DMBA administration, only two tumors developed in the group of animals that received the combination of DHEA and EM-800, and these two tumors disappeared before the end of the experiment (P < 0.01 vs. DHEA or EM-800 alone). Average tumor number per tumor-bearing animal as well as average tumor area per tumor-bearing animal were further decreased in animals that received the combination therapy compared with the effect of each treatment alone (P < 0.01). DHEA induced 6.9% (P < 0.01), 10.6% (P < 0.05), and 8.2% (P < 0.01) increases in bone mineral density of total skeleton, lumbar spine, and femur, respectively. The addition of EM-800 to DHEA did not affect the enhancing effect of DHEA on bone mass. The combination of the two drugs had important inhibitory effects on the urinary excretion of calcium and phosphorus as well as on the urinary hydroxyproline/creatinine ratio. Serum total alkaline phosphatase was stimulated by DHEA. Treatment with EM-800 decreased both serum triglyceride and cholesterol levels, whereas DHEA had an inhibitory effect on serum triglycerides. Although treatment with EM-800 caused a marked atrophy of the mammary gland, DHEA alone reduced lobular hyperplasia seen in aged intact rats while causing an androgen-specific stimulation of the same structures in animals already receiving the antiestrogen EM-800. The combination of DHEA and EM-800 lowered ovarian weight by 24% (P < 0.01) and decreased serum estradiol concentrations to intact control levels, whereas each compound alone had no effect on ovarian weight and stimulated serum estradiol levels by 45% (P < 0.05) and 46% (P < 0.05), respectively. Treatment with EM-800 caused a marked inhibition of uterine and vaginal weight. The present data show the additive inhibitory effects of DHEA and EM-800 on the development of DMBA-induced mammary carcinoma in the rat, thus suggesting the potential benefits of such a combination for the prevention of breast cancer in women while preserving or even increasing bone mass and maintaining a favorable lipid profile.

Effect of dehydroepiandrosterone on bone mass, serum lipids, and dimethylbenz(a)anthracene-induced mammary carcinoma in the rat.

The present study investigated the effect of dehydroepiandrosterone (DHEA) on bone mass and serum lipids in the rat with dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma. The animals received DHEA once daily, percutaneously, at the dose of 5, 10, or 20 mg for 9 months following a single dose of 20 mg DMBA at 50-52 days of age. Bone mineral content (BMC) and bone mineral density (BMD) of total skeleton, lumbar spine, and femur were measured by dual energy x-ray absorptiometry. A 9-month treatment with DHEA increased BMC and BMD of total skeleton by 14.2% to 14.5% (all P < 0.01) and 6.7% to 8.3% (all P < 0.01), respectively. Similarly, femoral BMC and BMD were stimulated by 13.6% to 14.7% (all P < 0.05) and by 8.1% to 9.5% (all P < 0.01), respectively. In addition, BMD of lumbar spine was increased by 10.4% to 10.8% (all P < 0.05), whereas the 9.4% to 11.1% increment in BMC of lumbar spine was not statistically significant. Treatment with DHEA led to 26% (NS), 60% (P < 0.01), and 62% (P < 0.01) decreases in serum triglyceride levels at the same doses. On the other hand, no significant change in serum cholesterol concentrations was observed. Two hundred and seventy-nine days after DMBA administration, the incidence of mammary carcinoma had decreased from 95% in control animals to 73% (P < 0.05), 57% (P < 0.01), and 38% (P < 0.01) at the daily percutaneous doses of 5, 10, and 20 mg of DHEA, respectively. Moreover, the mean tumor number per tumor-bearing animal and the mean tumor area per tumor-bearing animal were also reduced by the same treatments. DHEA increased serum total alkaline phosphatase activity and decreased urinary calcium excretion, but had no effect on the urinary ratio of hydroxyproline to creatinine and urinary phosphorus excretion. These data show that DHEA exerts a stimulatory effect on bone mass and an inhibitory effect on serum triglycerides, as well as a preventive effect on the development of mammary carcinoma induced by DMBA in the rat. Such data suggest that while decreasing the risk of breast cancer, DHEA replacement therapy could also exert beneficial effects on the bone and lipid metabolism in women receiving DHEA replacement therapy.

Daily dosing with flutamide or Casodex exerts maximal antiandrogenic activity.

OBJECTIVES: Because the large increase in luteinizing hormone secretion induced by flutamide in the intact rat is not found in men, we have used castrated rats and mice supplemented with androstenedione (4-dione) instead of intact animals to measure the activity of the pure antiandrogens flutamide and Casodex. METHODS: We first compared the effect of different schedules of administration of various doses of the two antiandrogens on prostate and seminal vesicle weights in the castrated rat and mice models. RESULTS: For both flutamide and Casodex, no consistent difference was found between the effects of once daily and thrice daily oral dosing in the rat. It was observed, however, that flutamide, especially at the high and therapeutically more effective doses, is about three times more potent than Casodex under both schedules of dosing. When flutamide was administered subcutaneously three times a day, twice a day, once a day, or once every second day in rats and mice, no difference was observed in the degree of inhibition achieved on prostate and seminal vesicle weights. CONCLUSIONS: The present data show that Casodex is about three times less potent than flutamide on the well-recognized parameters of androgen responsiveness in the rat, namely prostate and seminal vesicle weights. Another finding is that once daily dosing with flutamide exhibits an effectiveness comparable to thrice daily dosing; such data may have potential significance in facilitating compliance by administration of flutamide once daily instead of the current thrice daily schedule in men. Moreover, these data, if obtained in a reliable in vivo model, should be helpful in determining the choice of an appropriate dose of Casodex for the treatment of prostate cancer.

Transcription factors RFX1/EF-C and ATF-1 associate with the adenovirus E1A-responsive element of the human proliferating cell nuclear antigen promoter.

The proliferating cell nuclear antigen (PCNA) is an adenovirus E1A-inducible factor that is intimately linked to the processes of DNA replication and cell cycle regulation. Previously, we defined a novel cis-acting element, the PCNA E1A-responsive element (PERE), that confers induction by the E1A 243R oncoprotein upon the human PCNA promoter. To better understand the regulation of PCNA expression by E1A 243R, we have identified cellular transcription factors that associate with the PERE. In electrophoretic mobility shift assays, the PERE formed three major complexes (P1, P2 and P3) with proteins in nuclear extracts from HeLa or 293 cells. Formation of complexes P2 and P3, which correlates with PCNA promoter activity in vivo, requires the activating transcription factor (ATF) binding site found within the PERE [Labrie et al. (1993) Mol. Cell. Biol., 13, 1697-1707]. Antibody interference experiments and mobility shift assays performed with in vitro-synthesized protein indicated that the transcription factor ATF-1 is a major component of these complexes. Similar assays demonstrated that the hepatitis B virus enhancer-associated protein RFX1 constitutes a major component of the P1 complex. In addition, we examined the binding of proteins to the minimal E1A-responsive promoter to identify other factors important for transcription from the PCNA promoter. Mobility shift assays revealed that a fragment encompassing the region from -87 to +62 relative to the transcription initiation site forms at least five complexes, EH1-EH5, with HeLa cell nuclear extracts. The transcription factor YY1 associates with the initiator element of the PCNA promoter. The identification of these transcription factors will allow their roles in the activation of PCNA by E1A to be evaluated.

Blockade of androstenedione-induced stimulation of androgen-sensitive parameters in the rat prostate by combination of Flutamide and 4-MA.

In order to mimic the human situation in which adrenal steroid precursors are converted to the active androgen dihydrotestosterone (DHT) in prostatic tissue, we have used castrated rats supplemented with the precursor steroid androstenedione (delta 4-dione) released from Silastic implants. While it is well known that the action of DHT can be partially neutralized by antiandrogens which compete for binding to the androgen receptor, we have used 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA), an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone into DHT, in order to decrease intraprostatic DHT levels and thus facilitate the action of the antiandrogen. Animals were treated for 7 days with Flutamide (FLU, 2 mg) or 4-MA (4 mg) injected subcutaneously, twice daily, alone or in combination. 4-MA administered alone caused a 54% inhibition of delta 4-dione-stimulated ventral prostate weight while FLU exerted a 74% inhibitory effect and 4-MA+FLU further improved inhibition to 81%. We then measured, by in situ hybridization, the levels of prostatic mRNAs encoding the C1 and C3 components of the prostatic binding protein (PBP) which are highly specific and sensitive markers of androgen action. PBP-C3 mRNA levels fell by 95% following castration while treatment with delta 4-dione completely reversed the effect of castration. Administration of FLU or 4-MA independently caused 33% and 10% decreases, respectively, of PBP-C3 mRNA levels stimulated by delta 4-dione while the combination of both compounds further inhibited PBP-C3 mRNA levels to reach a 55% inhibition. Similar effects were observed on PBP-C1 mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of pro-gonadotropin-releasing hormone gene expression by sex steroids in the brain of male and female rats.

The fine modulation of gonadotropin gene expression and secretion is well recognized to be regulated by sex steroids through their direct action both at the anterior pituitary level and on the pulsatile pattern of GnRH secretion at the hypothalamic level. Since the influence of sex steroids on hypothalamic GnRH mRNA levels remains to be elucidated, quantitative in situ hybridization was used to study the effect of sex steroids on cellular levels of pro-GnRH mRNA in adult rats of both sexes. The effects of 14-day gonadectomy as well as administration of 17 beta-estradiol (E2, 0.25 micrograms) or dihydrotestosterone (DHT, 100 micrograms) twice a day during 14 days to gonadectomized animals were evaluated. In addition, the effect of progesterone (P, 2 mg, twice daily) alone or in the presence of E2 was also studied in ovariectomized animals. Hybridization was performed using a 35S-labeled cDNA probe encoding rat pro-GnRH and the corresponding mRNA levels were assessed by counting the number of silver grains overlying labeled neurons. In male rats, castration induced a highly significant 65% increase (compared to intact rats) in the mean number of grains per neuron. Administration of E2 or DHT to castrated animals completely prevented the post castration rise in pro-GnRH mRNA levels. In female animals, the effect of ovariectomy was less striking than in the male, a 25% increase (P less than 0.001) being observed. Treatment with E2 or DHT also completely prevented the increase in pro-GnRH mRNA levels induced by ovariectomy. Moreover, treatment with P in ovariectomized animals markedly potentiated the inhibitory effect of E2 on pro-GnRH mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)