Preventive effect of oral estetrol in a menopausal hot flush model.

OBJECTIVE: To evaluate the efficacy of estetrol (E(4)) in alleviating hot flushes in an experimental animal model considered representative for menopausal vasomotor symptoms. METHODS: Recording of the thermal responses in the tail skin of morphine-dependent ovariectomized rats after morphine withdrawal by administration of naloxone. Six groups of rats were treated orally for 8 days as follows: vehicle (negative) control; E(4): 0.1, 0.3, 1.0 and 3.0 mg/kg/day; and, as active (positive) control, ethinylestradiol 0.3 mg/kg/day. On day 8, tail skin temperature was recorded at baseline and for 60 min at 5-min intervals following naloxone administration. Results In control animals, tail skin temperature increased sharply by about 4.5 degrees C after naloxone treatment and reverted to baseline by 60 min. Estetrol suppressed the tail skin temperature increase in a dose-dependent fashion. The highest dose of E(4) tested (3 mg/kg/day) was equipotent to a 10-fold lower dose of ethinylestradiol. Both fully suppressed tail skin temperature changes. CONCLUSION: Estetrol is effective in alleviating hot flushes in an experimental animal model considered representative for studying menopausal hot flushes (vasomotor symptoms). In this model, the potency of estetrol is 10-fold lower compared to ethinylestradiol.

Estrogenic uterovaginal effects of oral estetrol in the modified Allen-Doisy test.

OBJECTIVE: To evaluate the effect of estetrol (E(4)) on vaginal cornification and uterine wet weight in the ovariectomized rat. METHODS: Adult female rats served as experimental animals. Six groups of ovariectomized rats (eight per group) were treated orally once daily for 7 days as follows: vehicle (negative) control; E(4): 0.1, 0.3, 1.0 and 3.0 mg/kg/day, and ethinylestradiol 0.05 mg/kg/day as active (positive) control. Vaginal lavages were obtained daily and uterine wet weight was determined on day 7. RESULTS: Vaginal cornification was observed by day 5 in all rats at all E(4) doses and in the animals receiving ethinylestradiol, but not in the control rats. The onset of cornification with E(4) was dose-dependent. After 7 days' treatment, the two highest E(4) doses (1.0 and 3.0 mg) induced statistically significant higher uterine wet weight compared to vehicle. CONCLUSION: Estetrol has estrogenic effects on the vagina and on the uterus of ovariectomized rats. The potency of E(4) is approximately 20-fold lower compared to ethinylestradiol.

First human exposure to exogenous single-dose oral estetrol in early postmenopausal women.

OBJECTIVE: To evaluate the safety, tolerability, pharmacokinetics and effect on gonadotropins of a single escalating dose of estetrol (E(4)). METHODS: A first-in-human study with E(4) was performed in healthy early postmenopausal women. Four single doses of 0.1, 1, 10 and 100 mg E(4) were evaluated in study groups of eight subjects each, of whom six received active treatment and two placebo treatment. Safety and tolerability were documented and several pharmacokinetic parameters were determined, as were the plasma levels of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH) (pharmacodynamics). The next higher-dose group was enrolled after pharmacokinetic evaluation and confirmed safety of the previous group. RESULTS: After oral intake, the plasma concentrations of E(4) showed a steep increase, followed by a sharp decline and a secondary increase at all dose levels. Estetrol was distributed and reabsorbed during the first 18 h after oral intake. The terminal elimination phase started at 24 h post-dose and half-life (t((1/2))) ranged in the 10 mg group between 19 and 40 h (mean 28.4 h, median 28.8 h) and in the 100 mg group between 18 and 60 h (mean 28.0 h, median 20 h), indicating a dose independency of the half-life. The pharmacokinetic parameters also demonstrated a high dose-response relationship and showed excellent consistency and low variability within the dose groups. The pharmacodynamic data showed a dose-dependent inhibition of plasma LH levels by E(4). A profound and sustained inhibition of FSH levels, lasting over 168 h, was observed in the 100 mg dose group (FSH was not measured in the other dose groups). Estetrol was well tolerated at all dose levels and no safety problems were encountered. CONCLUSIONS: Estetrol is orally absorbed and bioavailable with a strong dose-response relationship suggesting high oral bioavailability. Interindividual variations of plasma levels are low. The elimination half-life of 28 h suggests slow metabolism of E(4). The pharmacodynamic pattern complies with enterohepatic recirculation. Estetrol has a profound central inhibitory and dose-dependent effect on gonadotropins, confirming its biological potency.

Estetrol review: profile and potential clinical applications.

In this review paper, the existing information on the human fetal steroid estetrol (E4) has been summarized. In the past, E4 was considered as a weak estrogen and interest disappeared. However, recent new research has demonstrated that E4 is a potent, orally bioavailable, natural human fetal selective estrogen receptor modulator, since it acts in the rat as an estrogen on all tissues investigated except breast tumor tissue, where it has estrogen antagonistic properties in the presence of estradiol. Based on its safety data, its pharmacokinetic properties, its pharmacological profile and the results of first human studies, E4 may be suitable as a potential drug for human use in applications such as hormone replacement therapy (vaginal atrophy, hot flushes), contraception and osteoporosis. Additional areas worth exploring are the treatment of breast and prostate cancer, hypoactive sexual desire disorder and topical use (wrinkles) in women, auto-immune diseases, migraine, cardiovascular applications and the treatment of selected obstetric disorders.

Ovulation inhibition by estetrol in an in vivo model.

Background Currently, the synthetic steroid ethinylestradiol is the preferred estrogen in combined oral contraceptives. The aim of the present study was to evaluate the effectiveness of the natural steroid estetrol (E4) as an ovulation inhibitor in rats when compared to ethinylestradiol. Study design Regularly cycling female rats were treated orally twice daily for four consecutive days, starting on the day of estrus, with E4 (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg), ethinylestradiol (0.0003, 0.001, 0.003, 0.01 or 0.03 mg/kg) or vehicle control (eight animals per group). In a second experiment conducted under the same experimental protocol, 2.0 mg/kg of E4 was administered as a single daily dose or as a dose of 1.0 mg/kg twice daily. In both studies, the primary endpoint was the number of ovulated oocytes in the genital tract. Results Estetrol at the twice-daily dose of 0.3 mg/kg and above inhibited ovulation. This effect was statistically significant (p < 0.05). The comparator, ethinylestradiol, significantly inhibited ovulation (p < 0.05) at the highest dose (0.03 mg/kg) administered twice daily. An E4 dose of 2.0 mg/kg administered once daily for four consecutive days inhibited ovulation in four of eight rats. In contrast, when the same dose was administered in two separate doses, that is 1.0 mg/kg twice daily, ovulation was inhibited in eight of eight rats. The ED50 for the ethinylestradiol and the E4 dose-response curves shows that ethinylestradiol is 18 times more potent than E4. Conclusion Twice-daily administration of E4 effectively inhibits ovulation in cycling rats. The effect is dose-dependent. The relative potency of E4 is about 18 times less compared to that of ethinylestradiol. We conclude that, based on these data, combined with available pharmacological and clinical data on the safety and efficacy of E4, the human fetal estrogenic steroid estetrol is a potential candidate to replace ethinylestradiol in combined oral contraceptives.

Oral bioavailability and bone-sparing effects of estetrol in an osteoporosis model.

OBJECTIVES: To measure the oral bioavailability of estetrol (E(4)) in rats relative to its subcutaneous administration and to test the bone-sparing effect of oral E(4) compared to that of ethinylestradiol (EE). METHODS: In the bioavailability study, E(4) was administered as a single dose of 0.05, 0.5 or 5.0 mg/kg orally or subcutaneously to female rats. Plasma was analyzed using an LC-MS/MS method. The bone study was conducted in 3-month-old female rats assigned to the following seven treatment groups of ten animals each: no treatment; sham-operated + vehicle; bilaterally ovariectomized (OVX) + vehicle; OVX + E(4) 0.1, 0.5, or 2.5 mg/kg/day and OVX + EE 0.1 mg/kg/day. Once-daily treatment by oral gavage was given for 4 weeks and the following measurements were performed: serum osteocalcin, bone mineral density, bone mineral content and bone mineral area of lumbar vertebrae L3-L6, peripheral quantitative computed tomography of the left tibiae and the biomechanical properties of the distal femora. RESULTS: Oral bioavailability of E(4), relative to that of subcutaneous dosing, was 70% and above at the 0.05 and 0.5 mg/kg doses based on the AUC(0-t last). Subcutaneous dosing provided significantly higher E(4) levels at the 1-h time point only, and was comparable to oral dosing after 0.5, 2, 4 and 8 h. In the bone study, E(4) dose-dependently and significantly (1) inhibited the OVX-related increase in osteocalcin levels, (2) increased bone mineral density and content, and (3) increased bone strength, all attenuated by ovariectomy. In this rat model, the relative potency of the highest dose of E(4) (2.5 mg/kg/day) was comparable to the EE dose, used as positive control. CONCLUSIONS: Estetrol exhibits high oral bioavailability in the rat, a species considered relevant for pharmacological studies that are predictive for effects on human bone. Oral administration of E(4) conveys dose-dependent bone-sparing effects of high-quality bone in estrogen-depleted OVX rats. Based on its bone-sparing effects, its oral bioavailability and its preclinical safety and efficacy profile, E(4) may be superior to other estrogens and is a potential drug for the prevention of osteoporosis in postmenopausal women.

Estetrol, a pregnancy-specific human steroid, prevents and suppresses mammary tumor growth in a rat model.

Estetrol (E4) is a pregnancy-specific D-ring metabolite of estradiol (E2) and estriol (E3) produced by the human fetal liver and present in both male and female fetuses. In adults, female exposure is restricted to the gestational period. We report that E4, dose-dependently, prevents the growth of chemically induced (7,12-dimethylbenz(a)anthracene, DMBA) mammary tumors in female Sprague-Dawley rats and that E4 has the potential to reduce the number and size of pre-existing mammary tumors. We performed two prevention studies and one intervention study. In the prevention studies, we investigated the effect of oral doses of E4 over a dose range of 0.5-3.0 mg/kg. The intervention study used oral dose levels of 1, 3 and 10 mg/kg E4. The antiestrogen tamoxifen was used as reference compound in all three studies and ovariectomy and ethinylestradiol, at estrogenic doses pharmacologically equipotent to E4, acted as control treatments in the second prevention study and in the intervention study. Rats treated with DMBA develop estrogen-responsive breast tumors. This model has become the standard pharmacological model to investigate the effect of new compounds on breast tumors. When DMBA-induced rats were co-treated with E4 for 8 weeks, this resulted in a dose-dependent reduction in the number and size of tumors, an effect that appeared equally effective as tamoxifen treatment or ovariectomy and was not seen with ethinylestradiol. When E4 was administered to rats in which tumors had already developed, a significant decrease in the number and size of tumors was observed after 4 weeks. This decrease was dose-dependent, comparable to tamoxifen-treated animals, and at high dose levels E4 was as effective as ovariectomy.

Histological evaluation of in vitro responses of endometrial adenocarcinoma to progestins and their relation to progesterone receptor levels.

In vitro responsiveness of endometrial adenocarcinoma to progestins was evaluated histologically by incubation of tissue fragments in medium containing 10(-6) M medroxyprogesterone acetate. In a series of 19 experiments, formation of sub- and supranuclear vacuoles, which reflects accumulation of glycogen in response to medroxyprogesterone acetate added to the medium, was observed in well-preserved glandular epithelial cells only when the level of cytosolic progesterone receptor was above 300 fmol/mg protein. Previously, we have reported similar results obtained in in vivo experiments. The present findings suggest that simple organ culture and histological procedures can be used to identify specimens of endometrial cancer that have functional progesterone receptors and are capable of responding to progestins. They also indicate that levels of progesterone receptor required to obtain responses to progestins are considerably higher than those necessary for analytical detection and that therefore the quantity and not merely the detectability of progesterone receptors must be taken into consideration for the prediction of responses to progestins. In addition, in vitro responses to progestins may indicate the presence in endometrial cancer tissue of functional estrogen receptors and potential responsiveness to antiestrogens, since estrogen stimulation appears to be needed for the synthesis of progesterone receptors.

Effects of steroid hormones and antisteroids on alkaline phosphatase activity in human endometrial cancer cells (Ishikawa line).

Alkaline phosphatase activity in human endometrial cancer cells of the estrogen-responsive Ishikawa line was markedly stimulated (3-20-fold in 4 days) by estrogens, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone but not by testosterone, medroxyprogesterone acetate, glucocorticoids, several peptide hormones, prostaglandins, or growth factors. Maximum responses to estradiol were obtained at concentrations between 10(-9) and 10(-7) M; at 10(-8) M estradiol, the highest activity was reached 48-72 h after addition of the hormone. A linear relationship between enzyme activity at 48 h and the length of exposure to the hormone was observed. Dibutyryl cyclic guanosine 3':5'-monophosphate, but not dibutyryl cyclic adenosine 3':5'-monophosphate enhanced alkaline phosphatase activity and acted synergistically with estradiol. trans-4-Monohydroxytamoxifen completely antagonized the stimulatory effect of estradiol and had no agonistic activity. Dihydrotestosterone and dehydroepiandrosterone appear to exert their effects, at least in part, by interacting with estrogen receptors, since the simultaneous presence in the medium of monohydroxytamoxifen abolished their influence on alkaline phosphatase activity. The specific antiandrogen monohydroxyflutamide partially antagonized the effect of these hormones, suggesting that their action involved androgenic mechanisms as well. Exposure to elevated temperature and to specific inhibitors identified alkaline phosphatase of Ishikawa cells as a placental-type isoenzyme, thus contrasting with the nonplacental type found in glandular epithelial cells of normal endometrium and in another human endometrial cancer cell line, HEC-50. This study extends our previous observations of estrogen responsiveness in the Ishikawa cell line. In addition to the previously reported stimulatory effects on growth and progesterone receptor levels, we are now describing the stimulation by estrogens and C19 steroids of an enzyme, alkaline phosphatase, which can be used as a convenient end point to examine mechanisms of hormonal action.

Stimulatory effects of 4-hydroxytamoxifen on proliferation of human endometrial adenocarcinoma cells (Ishikawa line).

The effects of trans-4-hydroxytamoxifen (OHTam) on proliferation of cells of the Ishikawa human endometrial adenocarcinoma line were studied under serum-free, phenol red-free conditions and compared to those of estradiol. The addition of OHTam (1 microM) to basal medium (BM), consisting of equal parts of Dulbecco's modified Eagle's medium and Ham's F-12 with additional glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, resulted in significant increases in cell numbers relative to controls. These effects were even greater than those obtained with estradiol (10 nM-1 microM) or 1% charcoal-treated fetal bovine serum (ctFBS). Addition of 1% ctFBS to BM containing 1 microM OHTam further increased cell numbers whereas addition of estradiol (10 nM) did not do so. The stimulation of growth was positively correlated with OHTam concentrations in the range of 10 nM to 1 microM. Dissociation of estradiol and OHTam proliferative effects was observed in a variant of Ishikawa cells in which estradiol did not increase proliferation while OHTam had a strong stimulatory effect. The growth-promoting effects of OHTam were also observed in BM containing 5% or 15% ctFBS. In contrast, in parallel experiments in which BM was replaced by minimal essential medium (Eagle's) with Earle's salts, OHTam (1 microM) did not stimulate proliferation under these conditions and acted as an antiestrogen, inhibiting the proliferative effects of estradiol. These results illustrate marked effects of medium composition on proliferation and antiestrogenic actions of OHTam. Alkaline phosphatase activity was strongly stimulated by estradiol (10 nM) but only very weakly affected by OHTam (1 microM); at these concentrations, OHTam inhibited the effect of estradiol, both in serum-free BM and in minimal essential medium plus 15% ctFBS, demonstrating dissociation in its actions on proliferation and on enzymatic activity. These findings suggest that OHTam may stimulate the proliferation of particular clones of endometrial cancer cells in human tumors. They also suggest that OHTam can exert effects not mediated by the estrogen receptor system, or form OHTam-estrogen receptor agonistic complexes unlike those resulting from estradiol-estrogen receptor interactions. Clearly, Ishikawa cells provide a useful model to investigate mechanisms of action of antiestrogens.

Proliferation and responsiveness to estrogen of human endometrial cancer cells under serum-free culture conditions.

Studies of hormonal growth regulation in cultured human endometrial cancer cells are limited by the requirement of exogenous growth factors, usually supplied by addition of serum. The present report provides evidence that estradiol can stimulate proliferation of endometrial cancer cells of the Ishikawa line in the absence of serum or added growth factors. Mitogenic effects of estrogen were demonstrated in two different experimental systems, in cells attached to the substratum of mammalian tissue culture dishes, and in cells forming colonies in soft agar under anchorage-independent conditions. Addition of estradiol to a mixture of serum-free, phenol red-free Dulbecco's minimal essential medium and Ham's F-12 medium, supplemented with L-glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [basal medium: (BM)] significantly increased the proliferation of cells attached to culture dishes. Dose-response experiments revealed maximal estradiol stimulation at 10 nM; significant responses were also observed at 1 nM and at 100 nM concentrations. The mitogenic effect of 10 nM estradiol was comparable to that of 1% charcoal-treated fetal bovine serum and the two effects were additive. The presence of estradiol in serum-free BM resulted in a shortening of the doubling time of exponentially proliferating cells from 38 to 29 h. From the labeling index, measured after exposure to a pulse of [3H]thymidine, and from the mitotic index, both determined in exponentially proliferating cells, the lengths of the S and M phases were calculated to be 11 and 1 h, respectively. From these data it was estimated that estradiol shortened the G1 phase by approximately 40%, from 22 to 13 h. Estradiol doubled the colony formation efficiency of cells plated in BM containing 0.3% agar in the absence of serum as well as in the presence of 1% charcoal-treated fetal bovine serum. The stimulation of colony formation by estradiol was influenced by medium components, since no effects were observed in minimal essential medium. The colony formation efficiency was positively related to the serum concentrations and remained significantly lower in minimal essential medium than in BM at comparable serum levels. The observed positive relationship between colony formation efficiency and cell densities at plating suggests a cooperative mitogenic effect, likely due to autocrine and paracrine action of secreted growth factors. These results define a model to evaluate hormonal growth regulation mediated by autocrine mitogens in human endometrial cancer cells in the absence of interfering exogenous growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Transforming growth factor gene expression in human endometrial adenocarcinoma cells: regulation by progestins.

In an attempt to understand the antiproliferative effects of progestins in endometrial cancer, we have examined the effects of the potent progestin, medroxyprogesterone acetate (MPA), on the cell proliferation and the expression of transforming growth factor (TGF) alpha and beta genes in human endometrial adenocarcinoma cell lines. The two cell lines used were Ishikawa, var 1, and HEC-50. In addition, the effects of exogenous TGF-alpha and anti-epidermal growth factor (EGF) receptor monoclonal antibody on cell proliferation were determined. Incubation of both cell lines with MPA resulted in a time- and dose-dependent inhibition of cell proliferation. Half-maximal growth inhibition was observed at 0.6 nM. In Ishikawa cells, the relative abundance of TGF-alpha was significantly reduced by MPA. A significant decrease in TGF-alpha mRNA was apparent 6 h after exposure to MPA and a further decrease was seen 12-24 h after addition of the progestin. The concentration of TGF-alpha immunoreactivity in conditioned medium of MPA-treated cells was also significantly reduced compared to control cultures. MPA had no effect on TGF-alpha expression by HEC-50 cells. EGF mRNA was not detected by Northern blot analysis in either cell type. MPA had no significant effect on EGF receptor mRNA abundance but resulted in a small increase in EGF receptor number in Ishikawa cells. Anti-EGF receptor monoclonal antibody (0.6-6 nM) inhibited Ishikawa cell growth but had no effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines, but Ishikawa cells were significantly more sensitive to exogenous TGF-alpha than HEC-50 cells. Furthermore, TGF-alpha could reverse the growth inhibitory effects of MPA on Ishikawa cells. A decrease in TGF-beta mRNA abundance was also observed in MPA-treated Ishikawa and HEC-50 cells. This effect was of small magnitude, variable, and only observed after prolonged exposure to MPA. These observations are consistent with the hypothesis that the antiproliferative effects of progestins on Ishikawa cells are mediated by decreased expression and autocrine action of TGF-alpha. Since similar growth inhibition is also seen in the HEC-50 cells in which progestins have no effect on TGF-alpha expression, additional mechanisms are likely to be involved in the antiproliferative effects of progestins in human endometrial cancer.

In vivo uptake of estrone sulfate by rabbit uterus.

The capability of rabbit uterus and liver to hydrolyze estrone sulfate (E1S) to estrone (E1) was demonstrated in vitro by superfusion of tissue slices with mixtures of [3H]-E1S and [14C]E1. However, capillary barriers or binding factors may prevent the utilization of the blood-borne estrogen sulfate. To evaluate the extent of uptake of circulating E1S by uterine cells, a mixture of [3H]E1S and [14C]E1 was injected into the abdominal aorta of anesthetized rabbits. Shortly after the injection, samples of blood, uterine tissue, and liver were simultaneously taken. Isotope ratios in E1 isolated from these samples were measured and compared. The 3H to 14C ratios in E1 isolated from the uterus and from plasma were similar. It was concluded from these results that blood-borne E1S was not taken up directly by the uterus, since, otherwise, uterine E1 would be expected to have a higher 3H to 14C ratio than plasma E1. The isotope ratios in hepatic E1 were higher than those in E1 isolated from plasma or uterus, probably as a result of direct uptake of [3H]E1S by the liver.

Aging and uterine growth during implantation in C57BL/6J mice.

Uterine growth during implantation was compared in C57BL/6J mice aged 3-7 mo vs. 11-12 mo. All mice had given birth to at least one previous litter. Older mice had smaller uteri during early gestation (3 to 10 days post coitum) as measured by DNA content, although wet weight and protein were generally similar in both age groups. However, there were no age effects on uterine growth during implantation, as measured by the increments of DNA, as well as protein and wet weight per implantation site (decidual swelling) on days 6-10. These data are discussed in terms of the major increase in fetal death and resorptions in older mice subsequently observed by day 12-13. Larger age-related impairment of the artificially-induced decidual response is also considered. We conclude that impairments of the artificially induced decidual response do not predict the extent of decidual responses during pregnancy in aging mice.

Growth-promoting effects of progesterone in a human endometrial cancer cell line (Ishikawa-Var I).

Significant growth responses to progesterone of human endometrial adenocarcinoma cells (Ishikawa-Var I) were observed under in vitro culture conditions. Progesterone affected both the rate of exponential proliferation and cell population densities after the exponential phase. In the presence of the hormone, the doubling time of exponentially proliferating cells was reduced from 44 to 35.6 h and cell densities were increased by as much as 2-3 times over those of controls during approx. 2 weeks in culture. The effects of progesterone on cell population growth were dose dependent. Estradiol (10(-8) M) and testosterone (10(-6) M) did not affect cell densities and the effects of dexamethasone (10(-6) M) were small. In contrast, both progesterone and estradiol stimulated colony formation under anchorage-independent conditions in soft agar. These results suggest the possibility that growth of sensitive cell clones in endometrial tumors could be enhanced in some patients during adjuvant progestin therapy.

Effects of transforming growth factors and regulation of their mRNA levels in two human endometrial adenocarcinoma cell lines.

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.

Hormonal contraception. Current status and future perspectives.

PIP: Hormonal contraception was pioneered by Gregory Pincus in the 1950s. Today, hormonal contraception is accepted as having a highly favorable benefit/risk profile. There is, however, a need for the development of new contraceptive methods to broaden the range of choices and enhance motivation and compliance in users. With the staggering rate of increase in the world's population, the number of contraceptive users in developing countries is expected to increase from 381 million in 1990 to 567 million in the year 2000. This will require substantial supplies of inexpensive contraceptives and the development of new and improved methods. The use of contraceptives is an asset to women's health, which can be jeopardized by the risks of pregnancy, as well as to the psychological and social well-being of mother and child. Oral contraceptives also have noncontraceptive health benefits such as protecting against endometrial cancer, uterine fibroids, menorrhagia, benign breast disease, anemia, ovarian cancer, functional ovarian cysts, dysmenorrhea, ectopic pregnancy, salpingitis, and bone loss. The new low-dose formulations are considered to be very safe for most healthy, nonsmoking women of reproductive age. Therefore, current research efforts are focused on new delivery methods, such as vaginal rings, rather than on the development of new hormonally active steroids. Nonoral contraceptive methods which avoid first-pass effects on the liver are being developed or improved. These include implants, vaginal rings, vaginally applied pills, and progestogen-containing IUDs. Contraceptive research is also focusing on immunologic interference with the hypothalamic-pituitary-gonadal axis in both men and women. This may spawn as yet unforseen methods of molecular modulation of sperm-ovum interactions which would result in the inhibition of implantation.^ieng

Aspects of hormone replacement therapy.

The most important benefit of ERT may well be its cardioprotective effect. But unopposed estrogen therapy also carries the risk of inducing endometrial cancer. A compelling body of evidence indicates that adjunctive progestogen protects effectively against estrogen-induced endometrial adenocarcinoma, and that progestogen therapy is effective in the treatment of hyperplasia in most women who have taken unopposed estrogen. Yet there is concern that adjunctive progestogen may attenuate the cardioprotective effects of estrogen. It is therefore agreed that adjunctive progestogen therapy is not indicated for hysterectomized women, and should be given at the lowest effective dose to non-hysterectomized women. Phase II dose-ranging clinical trials using secretory transformation as an efficacy endpoint to estimate protective effects of different doses of progestogen against endometrial hyperplasia/adenocarcinoma are complicated by the possibility that the doses protecting against hyperplasia may differ from those producing secretory changes. Further work is needed to identify one or several progestogen-regulated markers that most closely correlate with protection against estrogen-induced endometrial cancer.

Design and conduct of clinical trials in hormone replacement therapy.

Postmenopausal hormone replacement therapy represents an area of outstanding importance in preventive medicine that greatly affects personal well-being as well as public health. The number of women living in the United States who are 50 years or older has been estimated at nearly 50 million. Many of those women are likely to be eligible for postmenopausal hormone replacement, which may consist either of estrogen replacement therapy (ERT) in women without a uterus or, more frequently, estrogen/progestin combination therapy (HRT) in women with a uterus. This chapter first presents an overview of general regulatory requirements pertaining to the design and conduct of clinical studies in support of marketing approval for a drug product. These requirements include, but are not restricted to, studies in HRT. The chapter next discusses the design and conduct of clinical trials in support of marketing approval for the indications: treatment of moderate to severe vasomotor symptoms and vulvovaginal atrophy; prevention of osteoporosis; and protection by adjunctive progestin against estrogen-induced endometrial hyperplasia/cancer in women with a uterus. Finally, data related to the potential cardioprotective action of HRT and its protection against Alzheimer's disease and colon cancer are discussed.

Interaction of ovarian hormones with normal endometrium and endometrial adenocarcinoma.

PIP: Steroid receptors (tissue specific proteins which bind with high affinity, low capacity and remarkable selectivity to compounds having similar biologic action) have provided the basis for developing tests to predict responsiveness of patients with breast cancer to surgical procedures (eg, ovariectomy, adrenalectomy or hypophysectomy) aiming to eliminate estrogens from circulation. Clinical studies have confirmed the claim that breast tumors without estrogen receptors do not benefit from these major procedures. Besides cytoplasmic and nuclear receptors, 2 types of high affinity, low capacity binders of E2 (estradiol) had been identified, this time in rat uterus cytosol, by resolving saturation curves with H 3 -E2 into 2 components. Heterogeneity in the cytosol receptors for E2 in the rat uterus was found. The specificity of receptors towards different classes of active sterioids has also been considered. A new finding is that some steroids, when their concentrations are sufficiently high, bind receptors that are primarily involved in the action of another group of hormones. The metabolism of E2 in the target cell is an important consideration in analyzing physiologic action of estrogens. Intracellular metabolism of the hormone is an important determinant of its action as is the amount of receptor found in the cell. The relation of estrogens in the development of endometrial cancer has been reported. Women exposed to prolonged estogen stimulation in the absence of progesterone are at higher risk to develop endometrial adenomatous hyperplasia, a precursor of adenocarcinoma.^ieng