Regulatory oversight of human pathogens and toxins in Canada.

From 1994 to 2009, federal oversight of human pathogens and toxins was limited to facilities importing human pathogens and toxins into Canada under the Human Pathogens Importation Regulations (HPIR). This narrow focus of authority restricted the Government of Canada's ability to regulate and monitor a full range of activities, including those involving human pathogens and toxins acquired from domestic sources. In 2009, the Human Pathogens and Toxins Act (the Act) received Royal Assent to establish a national safety and security regime and expand oversight through a national, standardized process to verify safe and secure use of human pathogens and toxins in Canada. The Act and the Human Pathogens and Toxins Regulations (the Regulations), in full force since December 1, 2015, provides legislative and statutory requirements for the comprehensive oversight of the control of human pathogens and toxins in Canada. Expanded regulation and monitoring program activities aim to reduce the risks posed by human pathogens and toxins and strengthen biosafety management systems that serve to protect the health of Canadians.

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.

DHEA and intracrinology at menopause, a positive choice for evolution of the human species.

Menopause has been chosen by evolution as the convergence of three factors, namely cessation of ovarian function (reproduction and estrogen secretion), high circulating dehydroepiandrosterone (DHEA), and intracrine enzymes able to convert DHEA into active sex steroids in peripheral tissues. The arrest of estrogen secretion by the ovaries at menopause causes a decrease of circulating estradiol below the threshold of biological activity, thus eliminating stimulation of the endometrium and risk of endometrial cancer. As much as the arrest of secretion of estradiol by the ovaries is essential to protect the uterus, it is of major importance that sex steroids continue to be made available in most other tissues which need estrogens and/or androgens for their normal functioning. Evolution, through 500 million years, has progressively provided the peripheral tissues with the enzymes able to make androgens and estrogens while high levels of DHEA, the precursor of all sex steroids, have appeared much later with the primates approximately 20 million years ago. All elements were thus in place for the functioning of intracrinology or the cell-specific formation of estrogens and androgens in peripheral tissues from the inactive precursor DHEA, with no significant release of active sex steroids in the circulation, thus eliminating the risks of adverse effects in the other tissues, especially the uterus. The presence of subthreshold levels of circulating estradiol combined with the formation of sex steroids from DHEA in specific peripheral tissues (intracrinology) makes menopause a positive characteristic supporting many years of good-quality postmenopausal life, useful for taking care of children and grandchildren. DHEA, however, decreases with age and is present at very different concentrations between different women, with the consequence that approximately 75% of postmenopausal women have too low circulating DHEA levels and suffer from symptoms/signs of hormone deficiency.

Intravaginal dehydroepiandrosterone (prasterone), a highly efficient treatment of dyspareunia.

OBJECTIVE: To examine the effect of intravaginal dehydroepiandrosterone (DHEA) on pain at sexual activity (dyspareunia) identified as the most bothersome symptom of vaginal atrophy in postmenopausal women at both screening and day 1. METHODS: This prospective, randomized, double-blind and placebo-controlled phase III clinical trial studied the effect of prasterone (DHEA) applied locally in the vagina on the severity of dyspareunia in 114 postmenopausal women who had identified dyspareunia as their most bothersome symptom of vaginal atrophy, while meeting the criteria for superficial cells ≤ 5% and pH > 5.0 at both screening and day 1. RESULTS: At the standard duration of 12 weeks of treatment, increasing doses of 0.25%, 0.5% and 1.0% DHEA decreased the percentage of parabasal cells by 48.6  ±â€Š 6.78%, 42.4  ±  7.36% and 54.9  ±â€Š 6.60% (p < 0.0001 vs. placebo for all) with no change with placebo (p = 0.769). The effects on superficial cells and pH were also highly significant compared to placebo at all DHEA doses. The severity score of pain at sexual activity decreased by 0.5, 1.4, 1.6 and 1.4 units in the placebo and 0.25%, 0.5% and 1.0% DHEA groups, respectively, with the p value of differences from placebo ranging from 0.0017 to < 0.0001. CONCLUSIONS: Intravaginal DHEA, through local estrogen and androgen formation, causes a rapid and highly efficient effect on pain at sexual activity without systemic exposure of the other tissues, thus avoiding the recently reported systemic effects of estrogens.

Pangenomic changes induced by DHEA in the skin of postmenopausal women.

The objective of this study was to explore, for the first time, the changes in the pangenomic profile induced in human skin in women treated with dehydroepiandrosterone (DHEA) applied locally. Sixty postmenopausal women participated in this phase II prospective, randomized, double-blind and placebo-controlled study. Women were randomized to the twice daily local application of 0% (placebo), 0.3%, 1% or 2% DHEA cream. Changes in the pangenomic expression profile were studied using Affymetrix Genechips. Significant changes (p<0.05) in sixty-six DHEA-responsive probe sets corresponding to 52 well-characterized genes and 9 unknown gene sequences were identified. A dose-dependent increase in the expression of several members of the collagen family was observed, namely COL1, COL3 and COL5 as well as the concomitant modulation of SPARC, a gene required for the normal deposition and maturation of collagen fibrils in the dermis. Several genes involved in the proliferation and differentiation of keratinocytes were also modulated. In addition, topical DHEA reduced the expression of genes associated with the terminal differentiation and cornification of keratinocytes. Our results strongly suggest the possibility that DHEA could exert an anti-aging effect in the skin through stimulation of collagen biosynthesis, improved structural organization of the dermis while modulating keratinocyte metabolism.

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.

Bioavailability and metabolism of oral and percutaneous dehydroepiandrosterone in postmenopausal women.

To study the bioavailability of dehydroepiandrosterone (DHEA) administered by the oral and percutaneous routes, three groups of 12 postmenopausal women aged 60-70 years received two capsules of 50mg of DHEA orally before breakfast daily for 14 days or applied 4 g of a 10% DHEA cream or gel at the same time of the day on a 30 cm x 30 cm surface area on the thighs. Detailed serial blood sampling over 24h was performed following 1st and 14th DHEA administration for measurement of DHEA and nine of its metabolites by liquid chromatography tandem mass spectrometry (LC-MS/MS) or gas chromatography mass spectrometry (GC-MS). Serum levels of estrone (E1) and estradiol (E2) did not change following DHEA administration by any of the three formulations, while serum androstenedione (4-dione), testosterone, DHEA sulfate (DHEA-S), E(1)-S, androsterone glucuronide (ADT-G) and 3alpha-androstanediol-G (3alpha-diol-G), increased in all cases, the effect on these parameters being more important after oral than percutaneous administration due to the metabolism of DHEA into these metabolites in the gastrointestinal tract and liver. No qualitative differences in DHEA metabolism are observed between the oral and percutaneous routes of DHEA administration while the levels of all steroids remain on a plateau during the 24h period during chronic percutaneous DHEA administration. The present data show that DHEA is transformed into active androgens and estrogens in peripheral intracrine tissues with no or minimal release of the active steroids E(1), E(2) or testosterone in the circulation. Moreover, DHEA is preferentially transformed into androgens rather than into estrogens. Most importantly, the present data show that changes in serum DHEA following oral or percutaneous DHEA administration are not a valid parameter of DHEA action since the increase in serum DHEA is at least 100% greater than the increase in the formation of active androgens and estrogens and thus much higher than the potential physiological effects.

Is dehydroepiandrosterone a hormone?

Dehydroepiandrosterone (DHEA) is not a hormone but it is a very important prohormone secreted in large amounts by the adrenals in humans and other primates, but not in lower species. It is secreted in larger quantities than cortisol and is present in the blood at concentrations only second to cholesterol. All the enzymes required to transform DHEA into androgens and/or estrogens are expressed in a cell-specific manner in a large series of peripheral target tissues, thus permitting all androgen-sensitive and estrogen-sensitive tissues to make locally and control the intracellular levels of sex steroids according to local needs. This new field of endocrinology has been called intracrinology. In women, after menopause, all estrogens and almost all androgens are made locally in peripheral tissues from DHEA which indirectly exerts effects, among others, on bone formation, adiposity, muscle, insulin and glucose metabolism, skin, libido and well-being. In men, where the secretion of androgens by the testicles continues for life, the contribution of DHEA to androgens has been best evaluated in the prostate where about 50% of androgens are made locally from DHEA. Such knowledge has led to the development of combined androgen blockade (CAB), a treatment which adds a pure anti-androgen to medical (GnRH agonist) or surgical castration in order to block the access of the androgens made locally to the androgen receptor. In fact, CAB has been the first treatment demonstrated to prolong life in advanced prostate cancer while recent data indicate that it can permit long-term control and probably cure in at least 90% of cases of localized prostate cancer. The new field of intracrinology or local formation of sex steroids from DHEA in target tissues has permitted major advances in the treatment of the two most frequent cancers, namely breast and prostate cancer, while its potential use as a physiological HRT could well provide a physiological balance of androgens and estrogens, thus offering exciting possibilities for women's health at menopause.

Effects of dihydrotestosterone on adipose tissue measured by serial analysis of gene expression.

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50,000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBPalpha, cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.

Transcriptome of mouse uterus by serial analysis of gene expression (SAGE): comparison with skeletal muscle.

The aim of this study was to identify the transcriptome of the normal mouse uterus by Serial Analysis of Gene Expression method. mRNA was extracted from the uterus and also from the gastrocnemius muscle of mice. Short sequences (tags), each one usually corresponding to a distinct transcript, were isolated and concatemerized into long DNA molecules which were cloned and sequenced. We detected 44,484 tags for the uterus and 42,518 tags for the muscle, representing 14,543 and 14,958 potential transcript species, respectively. Seventy-five and sixty-nine genes were expressed at more than 0.1%, thus corresponding to 37 and 34% of the mRNA population detected in the respective tissues. In both cases, the most highly expressed genes are especially involved in muscle contraction, energy metabolism, and protein synthesis. Compared to skeletal muscle, some differentially expressed genes in the uterus are likely to correspond to its specific reproductive functions. The majority of these genes remain to be characterized. More than 70% of the different tags detected in the uterus did not match any sequence in the public databases and can represent novel or poorly identified genes. This study is the first quantitative description of the transcriptome of the uterus.

Role of Smad1 and Smad4 proteins in the induction of p21WAF1,Cip1 during bone morphogenetic protein-induced growth arrest in human breast cancer cells.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta family of cytokines. The recent observation that BMPs can inhibit breast cancer cell proliferation in vitro suggests that BMPs or the BMP pathway may hold promise as therapeutic targets for the control of breast tumor growth in women. Better to understand the mechanism of BMP-induced growth arrest we examined the effect of BMP-2 and mediators of BMP-2 action on cell proliferation and p21(Cip1) expression in breast cancer cell lines. We show here that BMP-2 potently inhibited the proliferation of breast cancer cell lines that express both Smad1 and Smad4 (CAMA-1, MCF7, MDA-MB-231, T-47D, ZR-75-1), but not that of cells that only express Smad1 (MDA-MB-468). Growth inhibition correlated with up-regulation of p21 mRNA and protein levels. Up-regulation of p21 was resistant to cycloheximide but not to actinomycin D, suggesting that it occurred at the transcriptional level. Using p21 promoter-luciferase reporter constructs we mapped the BMP-responsive region of the p21 promoter to within 211 base pairs of the transcription start site. Induction of p21 promoter activity was rapid and coincided with up-regulation of p21 mRNA and protein levels. p21 promoter activity required both Smad1 and Smad4 and was induced by either BMP-2 or constitutively active type I BMP receptors. Moreover, the C-terminal SSVS region of Smad1 was necessary for activation of the p21 promoter by BMP-2. Taken together, these results indicate that the mechanism of BMP-induced p21 promoter activation involves BMP receptors and BMP Smads.

Effect of long-term treatment with the antiestrogen EM-652.HCl on pituitary estrogen receptor alpha and prolactin mRNA expression in intact, ovariectomized and gonadotropin-releasing hormone-treated female rats.

Estrogen receptor alpha (ERalpha) is the predominant estrogen receptor subtype in the anterior pituitary gland. In order to assess the influence of the pure antiestrogen EM-652.HCl on ERalpha gene transcription, we have studied the effect of long-term administration of the antiestrogen in ovariectomized rats as well as in intact female rats treated or not with the GnRH-agonist D-trp(6), des-Gly-NH2(10) GnRH ethylamide (GnRH-A), a treatment which induces pharmacological castration. To evaluate the degree of pituitary responsiveness to changes in estrogen exposure, prolactin (PRL) mRNA levels were also measured. ERalpha and PRL mRNA levels were evaluated by quantitative in situ hybridization. It was found that, 49 weeks after ovariectomy (OVX), pituitary ERalpha mRNA levels were decreased by 55%. Long-term administration (49 weeks) of EM-652.HCl to OVX animals resulted in a further 41% decrease in ERalpha mRNA. On the other hand, ovariectomy induced an 82% decrease in PRL mRNA levels while the administration of EM-652.HCl to OVX animals did not further decrease PRL mRNA. The administration of EM.652.HCl or GnRH-A alone to intact rats during 52 weeks did not significantly modify pituitary ERalpha mRNA levels. Concomitant administration of both GnRH-A and EM-652.HCl induced 41 and 47% decreases in ERalpha mRNA levels, when compared to the levels measured in vehicle-treated and GnRH-treated animals respectively. Combined administration of EM.652.HCl and GnRH-A induced 56 and 65% decreases in PRL mRNA, respectively. When EM-652.HCl was administered concomitantly with GnRH-A, the inhibitory effect on PRL mRNA levels was more marked than that observed in GnRH-A-treated animals. The present data demonstrate that when circulating estrogens are absent or maintained at very low levels by GnRH administration, EM-652.HCl can still depress ERalpha gene transcription. It is suggested that estrogens can positively regulate pituitary ERalpha gene transcription and that the antiestrogen EM-652.HCl can downregulate by itself pituitary ERalpha gene transcription.

Role of the cyclin-dependent kinase inhibitor p27(Kip1) in androgen-induced inhibition of CAMA-1 breast cancer cell proliferation.

Androgens are known to inhibit the growth of breast cancer cells, but the molecular mechanism of androgen-induced growth inhibition remains unknown. To address this question, we examined functional and quantitative alterations in cell cycle regulators in the E-responsive CAMA-1 breast cancer cell line. We report here that the androgen 5 alpha-dihydrotestosterone inhibits the proliferation of CAMA-1 breast cancer cells. This inhibition of cell proliferation was dose dependent, and maximal inhibition of E2-stimulated proliferation was observed at the concentration of 1 nM 5 alpha-dihydrotestosterone. 5 alpha-Dihydrotestosterone-induced growth arrest was accompanied by an increase in the proportion of cells in the G(1) phase of the cell cycle. Compared with control cells, 5 alpha-dihydrotestosterone-treated cells showed an increase in the relative proportion of hypophosphorylated retinoblastoma protein consistent with G(1) arrest. In CAMA-1 cells, 5 alpha-dihydrotestosterone caused an accumulation of the cyclin-dependent kinase inhibitor p27(Kip1). Cyclin E-cyclin-dependent kinase-2-associated kinase activity was strongly inhibited in 5 alpha-dihydrotestosterone-treated cells, and immunoprecipitation-Western blot analysis showed an increase in the amount of p27(Kip1) associated with cyclin E-cyclin-dependent kinase-2 complexes. These results suggest that inhibition of breast cancer cell growth by androgens may be mediated at least in part by inactivation of the cyclin E-cyclin-dependent kinase-2 complexes by p27(Kip1).

Role of extra-ovarian oestrogens in the regulation of gonadotropin releasing hormone mRNA expression in the rat brain.

To further understand the role of oestrogens in the regulation of gonadotropin releasing hormone (GnRH) mRNA expression in the female rat brain, the effect of EM-652.HCl, a pure anti-oestrogen, was studied in intact and ovariectomized rats as well as in rats chronically treated with a GnRH agonist D-trp6, des-Gly-NH210 GnRH ethylamide (GnRH-A), a treatment which blocks ovarian steroidogenesis. Quantitative in situ hybridization was used to measure GnRH mRNA at the cellular level in the preoptic-anterior hypothalamic area. It was found that, 49 weeks after ovariectomy (OVX), the number of silver grains per cell corresponding to GnRH mRNA was increased by 34%. Long-term administration (49 weeks) of EM-652.HCl to OVX rats resulted in a further increase (11% over the levels measured in OVX rats) in the hybridization signal. By contrast, in intact female rats, treated during 52 weeks with EM-652.HCl, a 49% increase in the GnRH hybridization signal was detected. In rats treated with GnRH-A during the same period, a 20% decrease in GnRH mRNA was observed. When EM-652.HCl was administered concomitantly with GnRH-A, a further 63% increase over the mRNA levels recorded in GnRH-A treated rats was found. Thus, long-term treatment with the anti-oestrogen EM-652.HCl can upregulate GnRH mRNA expression in intact female rats, OVX rats and female rats chronically treated with a GnRH-A. It is suggested that the pure anti-oestrogen EM-652.HCl can exert an influence on the oestrogen feedback mechanism involved in the regulation of GnRH neuronal activity by neutralizing the action of locally produced or low circulating levels of oestrogens remaining after OVX or GnRH-A treatment.

Androgens induce expression of SPAK, a STE20/SPS1-related kinase, in LNCaP human prostate cancer cells.

Genes that are regulated by androgens in the human prostate are believed to play an essential role in prostate physiology and they may also be involved in the proliferative response of prostate cancer cells to androgens. We used a cDNA subtraction approach to identify novel androgen-regulated transcripts in LNCaP cells that were exposed to 0.1 nM R1881 for 24 h. We report here that SPAK, a recently identified STE20/SPS1-related kinase that modulates p38 MAP kinase activity, exhibited increased expression in androgen-treated LNCaP cells. Androgen regulation of SPAK was both dose- and time-dependent. R1881-induced SPAK expression was completely abrogated by the antiandrogen casodex and by actinomycin D indicating that androgen induction of SPAK requires the androgen receptor and transcription. Cycloheximide caused a partial inhibition of R1881-induced SPAK expression which suggests that androgen induction of SPAK expression may require synthesis of additional proteins. Northern blot and ribonuclease protection assays demonstrated that SPAK is expressed at high levels in normal human testes and prostate, as well as in a number of breast and prostate cancer cell lines. These results identify SPAK, a member of a key cell signalling pathway, as an androgen-responsive gene in LNCaP cells. We hypothesize that SPAK may mediate androgen action in the normal and cancerous prostate gland.

DHEA and its transformation into androgens and estrogens in peripheral target tissues: intracrinology.

A new understanding of the endocrinology of menopause is that women, at menopause, are not only lacking estrogens resulting from cessation of ovarian activity but have also been progressively deprived for a few years of androgens and some estrogens originating from adrenal DHEA and androstenedione (4-dione). In fact, serum DHEA decreases by about 60% between the maximal levels seen at 30 years of age to the age of menopause. This decreased secretion of DHEA and DHEA-S by the adrenals is responsible for a parallel decrease in androgen and estrogen formation in peripheral tissues by the steroidogenic enzymes specifically expressed in each cell type in individual target tissues. This new field of endocrinology, called intracrinology, describes the local synthesis of androgens and estrogens made locally in each cell of each peripheral tissue from the adrenal precursors DHEA and 4-dione. These androgens and estrogens exert their action in the same cells where their synthesis takes place and they are released from these target cells only after being inactivated. To further understand the effect of DHEA in women, DHEA has been administered in postmenopausal women for 12 months. Such treatment resulted in increased bone formation and higher bone mineral density accompanied by elevated levels of osteocalcin, a marker of bone formation. Vaginal maturation was stimulated, while no effect was observed on the endometrium. Preclinical studies, on the other hand, have shown that, due to its predominant conversion into androgens, DHEA prevents the development and inhibits the growth of dimethylbenz(a)anthracene-induced mammary carcinoma in the rat, a model of breast cancer. DHEA also inhibits the growth of human breast cancer ZR-75-1 xenografts in nude mice. The inhibitory effect of DHEA on breast cancer is due to an androgenic effect of testosterone and dihydrotestosterone made locally from DHEA. When used as replacement therapy, DHEA is free of the potential risk of breast and uterine cancer, while it stimulates bone formation and vaginal maturation and decreases insulin resistance. The combination of DHEA with a fourth generation SERM, such as EM-652 (SCH 57068), a compound having pure and potent antiestrogenic activity in the mammary gland and endometrium, could provide major benefits for women at menopause (inhibition of bone loss and serum cholesterol levels) with the associated major advantages of preventing breast and uterine cancer. A widely used application of intracrinology is the treatment of prostate cancer where the testicles are blocked by an LHRH agonist while the androgens made locally in the prostate from DHEA are blocked by a pure antiandrogen. Such treatment, called combined androgen blockade, has led to the first demonstration of a prolongation of life in prostate cancer.

Glucuronidation of the nonsteroidal antiestrogen EM-652 (SCH 57068), by human and monkey steroid conjugating UDP-glucuronosyltransferase enzymes.

EM-652 (SCH 57068) is a new orally active antiestrogen that demonstrates pure antagonistic effects in the mammary gland and endometrium. In vivo studies have shown that EM-652 is primarily glucuronidated at the 7-hydroxy position in rats and that the metabolite is present in the plasma of female monkeys and human subjects after EM-800 (SCH 57050) or EM-652.HCl oral administration. Using hepatic microsomes from rat, monkey, and human, the formation of two EM-652 monoglucuronides at positions 4' and 7 was demonstrated by a liquid chromatographic tandem mass spectrometric method. Although no difference in EM-652 conjugation was observed between male and female monkey livers, an interindividual variation of hepatic EM-652 glucuronidation was shown with female human donors. Using microsome preparations from human embryonic kidney 293 cells stably expressing each of the 12 human and 11 monkey UGT enzymes cloned to date, the two EM-652-monoglucuronides were detected after incubation with microsomes containing human UGT1A1, UGT1A3, UGT1A8, UGT1A9, and monkey monUGT1A01, monUGT1A03, and monUGT1A09. Despite human UGT1A1 and monkey monUGT1A09 favored formation of EM-652-7-glucuronide, other active UGT1A enzymes formed both 4'- and 7-glucuronide derivatives in equal amounts. Kinetic analysis of EM-652 glucuronidation by these enzymes showed Michaelis constant (K(m)) values between 36 and 302 microM for EM-652-4'-glucuronide and 19 and 233 microM for EM-652-7-glucuronide. The present results demonstrate the importance of UGT1A isoforms, mainly UGT1A1, for EM-652 metabolism in humans.

EM-652 (SCH57068), a pure SERM having complete antiestrogenic activity in the mammary gland and endometrium.

In order to minimize the risks of endometrial cancer and the development of resistance to antiestrogen therapy, we have synthesized the orally active antiestrogen EM-652 which is the most potent of the known antiestrogens and exerts pure antiestrogenic activity in the mammary gland and endometrium. EM-652 inhibits the AF-1 and AF-2 functions of both ERalpha and beta while the inhibitory action of OH-TAM is limited to AF-2. EM-652, thus, inhibits Ras-induced transcriptional activity and blocks SRC-1-stimulated activity of the two receptors. The absence of blockade of AF-1 by OH-TAM could explain why resistance develops to Tamoxifen treatment. Not only the development, but also the growth of established DMBA-induced mammary carcinoma is inhibited by treatment with EM-800, the prodrug of EM-652. EM-652 is the most potent antiestrogen to inhibit the growth of human breast cancer ZR-75-1, MCF-7 and T-47D cells in vitro. When incubated with human Ishikawa endometrial carcinoma cells, EM-800 has no stimulatory effect on the estrogen-sensitive parameter alkaline phosphatase activity. When administered to ovariectomized animals, EM-800 prevents bone loss, and lowers serum cholesterol and triglyceride levels. EM-800 has shown benefits in women with breast cancer who had failed Tamoxifen. The above-summarized preclinical and clinical data clearly suggest the interest of studying this compounds in the neoadjuvant and adjuvant settings and, most importantly, for the prevention of breast and uterine cancer.

Prevention of bone loss by EM-800 and raloxifene in the ovariectomized rat.

Some undesirable effects are associated with chronic estrogen and progestin administration used to prevent bone loss in postmenopausal women, thus leading to poor compliance and the need for improved therapeutic and preventive agents. We have thus studied the ability of the new antiestrogen EM-800 (SCH 57050) to prevent bone loss and lower serum cholesterol levels and compared its effects with those of raloxifene. Ovariectomized (OVX) female rats were treated by oral gavage for 37 weeks with increasing daily doses (0.01, 0.03, 0.1, 0. 3 or 1 mg/kg) of EM-800 or raloxifene. At 35 weeks after OVX, lumbar spine bone mineral density (BMD) was 19% lower than in intact animals (P<0.01), while the OVX animals given EM-800 or raloxifene had 90-93 and 85-90%, respectively, of the BMD values observed in intact rats. Similar effects were observed on femoral BMD. Bone histomorphometry measurements were performed on proximal tibia. At the 0.01 mg/kg dose, EM-800 prevented the effect of OVX on TBV by 34% (P<0.01), while raloxifene had no detectable effect. Treatment with 1 mg/kg EM-800 and raloxifene resulted in, respectively, 68% (P<0.01) and 64% (P<0.01) prevention of the OVX-induced decrease in TBV. In addition, the administration of 0.01 and 0.03 mg/kg EM-800 caused, respectively, 54% (P<0.01) and 56% (P<0.01) inhibitions of serum cholesterol levels, while raloxifene administered at the same doses caused, respectively, 24% (P<0.01) and 41% (P<0.01) decreases of the value of the same parameter. At the highest doses used (0.1-1 mg/kg), both compounds lowered serum cholesterol levels by approximately 65% (P<0.01). No stimulatory effect of EM-800 was observed on the endometrial epithelial cells at doses up to 1 mg/kg, while hypertrophy of uterine epithelium was observed with raloxifene. EM-800 and raloxifene achieve the same degree of effectiveness on bone and serum cholesterol at higher doses, but EM-800 is at least three to ten times more potent than raloxifene at lower concentrations and has no stimulatory effect on uterine epithelium. The present data show the potent effect of EM-800 preventing bone loss and lower serum cholesterol levels without the negative effect on the endometrium, thus suggesting the particular interest of this new fully tissue-specific selective estrogen receptor modulator.