Ov D. Slayden

Design, Synthesis, and SAR of New Pyrrole-Oxindole Progesterone Receptor Modulators Leading to 5-(7-Fluoro-3,3-dimethyl-2-oxo-2,3-dihydro-1H-indol-5-yl)-1-methyl-1H-pyrrole-2-carbonitrile (WAY-255348)

We have continued to explore the 3,3-dialkyl-5-aryloxindole series of progesterone receptor (PR) modulators looking for new agents to be used in female healthcare: contraception, fibroids, endometriosis, and certain breast cancers. Previously we reported that subtle structural changes with this and related templates produced functional switches between agonist and antagonist properties ( Fensome et al. Biorg. Med. Chem. Lett. 2002, 12, 3487; 2003, 13, 1317 ). We herein report a new functional switch within the 5-(2-oxoindolin-5-yl)-1 H-pyrrole-2-carbonitrile class of compounds. We found that the size of the 3,3-dialkyl substituent is important for controlling the functional response; thus small groups (dimethyl) afford potent PR antagonists, whereas larger groups (spirocyclohexyl) are PR agonists. The product from our optimization activities in cell-based systems and also for kinetic properties in rodents and nonhuman primates was 5-(7-fluoro-3,3-dimethyl-2-oxo-2,3-dihydro-1 H-indol-5-yl)-1-methyl-1 H-pyrrole-2-carbonitrile 27 (WAY-255348), which demonstrated potent and robust activity on PR antagonist and contraceptive end points in the rat and also in cynomolgus and rhesus monkeys including ovulation inhibition, menses induction, and reproductive tract morphology.

Premenstrual and menstrual changes in the macaque and human endometrium: relevance to endometriosis.

Abstract: According to current theory, endometriosis is initiated during retrograde menstruation when menstrual fragments flow out of the fimbriated end of the fallopian tubes and become established on the ovarian surface or other sites in the peritoneal cavity. In recent years, new data have accumulated on the properties of menstruating tissue itself, and several laboratories agree that this tissue is rich in matrix metalloproteinases (MMPs) that may facilitate endometriotic implantation. Recently, we found that vascular endothelial growth factor (VEGF) and its receptor VEGFR‐2 (KDR) were dramatically upregulated in the stromal cells of the superficial endometrial zones by progesterone (P) withdrawal during the premenstrual phase. A unique role of VEGF at this stage of the cycle may be to stimulate MMP expression in stromal cells because VEGF, KDR, and MMPs were all coordinately induced in these cells in the superficial zone of the primate endometrium by P withdrawal. The rich content of MMPs and VEGF in the menstrual fragments could facilitate attachment and angiogenesis of menstrual fragments in ectopic sites. In addition, a variety of chemokines, cytokines, and cellular regulators are induced by P withdrawal in the premenstrual human endometrium. These include NFκB, prostaglandins, interleukin‐8 (IL‐8), cyclooxygenase‐2 (COX‐2), and monocyte chemotactic peptide‐1 (MCP‐1), among others. The perivascular expression of several of these factors may facilitate the rapid invasion of leukocytes into the endometrium, especially in the superficial zones. Consequently, menstrual fragments may be rich in IL‐8 and MCP‐1, both of which would add to the angiogenic potential of such fragments in ectopic sites. In sum, menstrual tissue is rich in VEGF, KDR, MMPs, leukocytes, chemokines, cytokines, and prostaglandins, all factors that may facilitate attachment and angiogenesis when menstrual fragments exit from the tubes and implant on pelvic sites. Additional research on these and other factors in premenstrual and menstrual endometrium may deepen our understanding of both the establishment and progression of this debilitating disease.

Androgen Effects on Adipose Tissue Architecture and Function in Nonhuman Primates

The differential association of hypoandrogenism in men and hyperandrogenism in women with insulin resistance and obesity suggests that androgens may exert sex-specific effects on adipose and other tissues, although the underlying mechanisms remain poorly understood. Moreover, recent studies also suggest that rodents and humans may respond differently to androgen imbalance. To achieve better insight into clinically relevant sex-specific mechanisms of androgen action, we used nonhuman primates to investigate the direct effects of gonadectomy and hormone replacement on white adipose tissue. We also employed a novel ex vivo approach that provides a convenient framework for understanding of adipose tissue physiology under a controlled tissue culture environment. In vivo androgen deprivation of males did not result in overt obesity or insulin resistance but did induce the appearance of very small, multilocular white adipocytes. Testosterone replacement restored normal cell size and a unilocular phenotype and stimulated adipogenic gene transcription and improved insulin sensitivity of male adipose tissue. Ex vivo studies demonstrated sex-specific effects of androgens on adipocyte function. Female adipose tissue treated with androgens displayed elevated basal but reduced insulin-dependent fatty acid uptake. Androgen-stimulated basal uptake was greater in adipose tissue of ovariectomized females than in adipose tissue of intact females and ovariectomized females replaced with estrogen and progesterone in vivo. Collectively, these data demonstrate that androgens are essential for normal adipogenesis in males and can impair essential adipocyte functions in females, thus strengthening the experimental basis for sex-specific effects of androgens in adipose tissue.

Intrauterine administration of CDB-2914 (Ulipristal) suppresses the endometrium of rhesus macaques.

BACKGROUND Ulipristal (UPA; CDB-2914) is a progesterone receptor modulator with contraceptive potential. To test its effects when delivered by an intrauterine system (IUS), we prepared control and UPA-filled IUS and evaluated their effects in rhesus macaques. STUDY DESIGN Short lengths of Silastic tubing either empty (n=3) or containing UPA (n=5) were inserted into the uteri of 8 ovariectomized macaques. Animals were cycled by sequential treatment with estradiol and progesterone. After 3.5 cycles, the uterus was removed. RESULTS During treatment, animals with an empty IUS menstruated for a mean total of 11.66+/-0.88 days, while UPA-IUS treated animals bled for only 1+/-0.45 days. Indices of endometrial proliferation were significantly reduced by UPA-IUS treatment. The UPA exposed endometria were atrophied with some glandular cysts while the blank controls displayed a proliferative morphology without cysts. Androgen receptors were more intensely stained in the glands of the UPA-IUS treated endometria than in the blank-IUS treated controls. CONCLUSIONS In rhesus macaques, a UPA-IUS induced endometrial atrophy and amenorrhea. The work provides proof of principle that an IUS can deliver effective intrauterine concentrations of Ulipristal.

Role of nonhuman primate models in the discovery and clinical development of selective progesterone receptor modulators (SPRMs)

Selective progesterone receptor modulators (SPRMs) represent a new class of progesterone receptor ligands that exert clinically relevant tissue-selective progesterone agonist, antagonist, partial, or mixed agonist/antagonist effects on various progesterone target tissues in an in vivo situation depending on the biological action studied. The SPRM asoprisnil is being studied in women with symptomatic uterine leiomyomata and endometriosis. Asoprisnil shows a high degree of uterine selectivity as compared to effects on ovulation or ovarian hormone secretion in humans. It induces amenorrhea and decreases leiomyoma volume in a dose-dependent manner in the presence of follicular phase estrogen concentrations. It also has endometrial antiproliferative effects. In pregnant animals, the myometrial, i.e. labor-inducing, effects of asoprisnil are blunted or absent. Studies in non-human primates played a key role during the preclinical development of selective progesterone receptor modulators. These studies provided the first evidence of uterus-selective effects of asoprisnil and structurally related compounds, and the rationale for clinical development of asoprisnil.

Progesterone-induced gene expression in uterine epithelia: a myth perpetuated by conventional wisdom.

Many in the field of reproductive biology, including ourselves, have published on studies of ‘‘progesterone-induced proteins’’ such as uteroferrin (Phosphatase, Acid, Type 5, TartrateResistant; ACP5) expressed by uterine glandular epithelium in several species. Another example is ‘‘progesterone-induced’’ genes expressed by uterine lumenal epithelium, such as plasmintrypsin inhibitor. Publication on ‘‘progesterone-induced’’ uterine proteins is not surprising as progesterone is, after all, the hormone of pregnancy required for establishment and maintenance of pregnancy in all mammals. However, results of research using state-of-the-art molecular techniques and attention to temporal and spatial (cell-specific) aspects of gene expression have revealed a paradox with respect to mechanisms of action of progesterone. The paradox is that uterine endometrial epithelia cease expressing receptors for progesterone and estradiol prior to implantation in all mammals studied, including humans, nonhuman primates, mice, shrew, spotted skunk, and domestic animals. In sheep, for example, loss of progesterone receptors in uterine lumenal and glandular epithelia occurs by Day 11 and Day 13 of the estrous cycle and pregnancy, respectively; however, endometrial stromal cells and myometrial cells express progesterone receptors throughout gestation. Similarly, progesterone receptors are minimal in the glandular epithelium, but retained in the stroma of the functionalis zone of the macaque during the mid-secretory phase corresponding to the window of implantation. The loss of progesterone receptors in uterine epithelia appears to be a prerequisite for implantation and differentiated functions of uterine epithelia directed by factors produced by stromal cells that do express progesterone receptors. In addition, loss of progesterone receptors by uterine epithelia is associated with reduced, modified, or lost expression of genes, such as the anti-adhesive protein MUC1, that may interfere with implantation. Following the discovery that uterine epithelia of ewes do not express progesterone receptors after Days 11 to 13 of pregnancy, we determined that endometrial stromal cells express fibroblast growth factor 10 (FGF10) and hepatocyte growth factor (HGF), while the tunica intima of blood vessels express FGF7. These growth factors, collectively known as progestamedins, exert their effects via their respective receptors, fibroblast growth factor 2 IIIb splice variant (FGFR2(IIIb)) and met proto-oncogene (hepatocyte growth factor receptor) (MET) expressed by all uterine epithelia and conceptus trophectoderm in sheep. The differential spatial patterns of expression for FGF10 and FGF7 in ovine uteri suggest that they have independent roles in endometrial function. Fgf7-null mice lack a phenotype, but Fgf10-null mice have many developmental deficiencies. FGF7 supports epithelial cell proliferation and differentiation to mediate effects of progesterone in the primate endometria, while FGF10 is a mesenchymal-derived growth factor essential for patterning branching and morphogenic events, including embryonic lung and limb bud formation. FGF7 and FGF10 are mitogens for epithelial cells, while HGF induces proliferation, motility, and morphogenesis of cells. Progesterone also suppresses levels of uterine glandular estrogen receptor in most mammals, resulting in decreased expression of estrogen-dependent genes. In women estradiol inhibits integrin beta 3 (ITGB3), a marker of endometrial receptivity. The loss of estrogen receptors during the mid-secretory phase is correlated with increased ITGB3. Progesterone-induced loss of estrogen receptor further delineates the period in the menstrual cycle when glandular proliferation ceases and implantation can occur in women. In primates, progesterone-stimulated reduction of epithelial estrogen receptors occurs concomitantly with loss of epithelial progesterone receptor. Loss of expression of progesterone and estrogen receptors by endometrial epithelia is a prerequesite for implantation, expression of genes for secretory proteins, and selective transport of molecules into the uterine lumen to create histotroph that is essential for conceptus growth and development. So, how does progesterone influence expression of these genes in the uterine epithelium? It seems clear that the effect cannot be via progesterone response elements in the genes by ligand-activated progesterone receptors binding to progesterone response elements in the promoter region of genes. Therefore, attention must be directed toward alternative mechanisms that include the following scenarios: (a) low levels of progesterone receptor, which remain after progesterone-induced down-regulation of progesterone receptor, may still be sufficient to maintain uterine gene expression; (b) progesterone may induce expression of progestamedins (e.g., FGF7, FGF10, HGF) by stromal cells that retain progesterone receptor, which in turn, exert paracrine effects on epithelia expressing FGFR2(IIIb) and MET. Uterine receptivity to implantation involves multiple events, including: (a) silencing of progesterone receptor and estrogen receptor genes, (b) suppression of genes for immune recognition, (c) increased expression of genes for attachment of trophectoderm to the uterine lumenal and superficial glandular epithelia, (d) phenotypic modification/decidualization of uterine stromal cells, (e) alterations in membrane permeability to enhance conceptus-maternal exchange of regulatory factors and the transport of nutrients into the uterine lumen, and (f) a dramatic increase in endometrial vascularity. Ultimately these events culminate in endometrial receptivity as well as signaling for pregnancy recognition. Differential expression of genes by uterine epithelia and stromal cells in response to progesterone is considered essential for successful embryo implantation in most mammals. As reproduc-

A critical period of progesterone withdrawal precedes menstruation in macaques

Macaques are menstruating nonhuman primates that provide important animal models for studies of hormonal regulation in the uterus. In women and macaques the decline of progesterone (P) at the end of the cycle triggers endometrial expression of a variety of matrix metalloproteinase (MMP) enzymes that participate in tissue breakdown and menstrual sloughing. To determine the minimal duration of P withdrawal required to induce menses, we assessed the effects of adding P back at various time points after P withdrawal on both frank bleeding patterns and endometrial MMP expression. Artificial menstrual cycles were induced by treating the animals sequentially with implants releasing estradiol (E2) and progesterone (P). To assess bleeding patterns, P implants were removed at the end of a cycle and then added back at 12, 24, 30, 36, 40, 48, 60, or 72 hours (h) after the initial P withdrawal. Observational analysis of frank bleeding patterns showed that P replacement at 12 and 24 h blocked menses, replacement at 36 h reduced menses but replacement after 36 h failed to block menses. These data indicate that in macaques, a critical period of P withdrawal exists and lasts approximately 36 h. In other similarly cycled animals, we withdrew P and then added P back either during (12–24 h) or after (48 h) the critical period, removed the uterus 24 h after P add back and evaluated endometrial MMP expression. Immunocytochemistry showed that replacement of P during the critical period suppressed MMP-1, -2 and -3 expression along with menses, but replacement of P at 48 h, which failed to suppress mense, suppressed MMP-1 and MMP-3 but did not block MMP-2. We concluded that upregulation of MMPs is essential to menses induction, but that after the critical period, menses will occur even if some MMPs are experimentally blocked.

Alterations in progesterone receptor membrane component 2 (PGRMC2) in the endometrium of macaques afflicted with advanced endometriosis

The hormonally driven expression and cell-specific localization patterns of the progesterone receptor membrane components (PGRMC1 and PGRMC2) in the macaque endometrium during the menstrual cycle are unknown. Additionally, the expression and localization patterns of PGRMC1 and PGRMC2 in the secretory eutopic endometrium of primates afflicted with endometriosis are also unknown. Therefore, we used real-time PCR to quantify transcript expression levels of the PGRMCs in well-defined samples of endometrium collected from artificially cycled macaques during the menstrual cycle, and in the secretory phase endometrium of naturally cycling macaques afflicted with endometriosis. In situ hybridization and immunocytochemistry were used to localize PGRMC1 and PGRMC2 mRNA and protein, respectively. We compared the patterns of expression and localization of the PGRMCs with the expression and localization patterns of nuclear progesterone receptor (PGR). PGRMC1 and PGR were elevated during the proliferative phases of the cycle, and then declined to nearly undetectable levels during the late secretory phase of the cycle. Levels of PGRMC2 were lowest during the proliferative phases of the cycle and then increased markedly during the secretory phases. Strong staining for PGRMC2 was localized to the luminal and glandular epithelia during the secretory phases. When compared with artificially cycled disease-free animals, macaques with endometriosis exhibited no changes in the expression or localization patterns for PGR and PGRMC1 but exhibited strikingly reduced levels of PGRMC2 transcript and altered intracellular staining patterns for the PGRMC2 protein. Collectively, these results suggest that membrane-bound PGRMC2 may provide a pathway of action that could potentially mediate the non-genomic effects of progesterone on the glandular epithelia during the secretory phase of the cycle. Further, reduced levels of membrane-bound PGRMC2 may be associated with the progesterone insensitivity often observed in the endometrium of primates afflicted with endometriosis.

Contrast-enhanced ultrasound reveals real-time spatial changes in vascular perfusion during early implantation in the macaque uterus.

OBJECTIVE To use contrast-enhanced ultrasound (CEU) to quantify blood flow in the macaque uterus during early pregnancy. DESIGN Prospective nonhuman primate study. SETTING Oregon National Primate Research Center. ANIMALS Naturally cycling female rhesus macaques (Macaca mulatta). INTERVENTION(S) Female macaques were mated on days 11-14 of the cycle. Females were then imaged by CEU and Doppler ultrasound once every 3 days from day 21 through day 39 of the fertile cycle. MAIN OUTCOME MEASURE(S) Visualization and quantification of uterine vascular perfusion. RESULT(S) CEU identified the primary placental disc and underlying vessels approximately 2 days earlier than Doppler ultrasound was able to observe endometrial thickening. CEU revealed spatial differences in vascular perfusion between the endometrium, myometrium, and endometrial-myometrial (junctional) zone. Myometrium displayed the highest rate of blood flow (>10 mL/min/g tissue). There was less blood flow in the endometrium and junctional zone (<3 mL/min/g). A brief fall in progesterone was observed during early implantation, which was correlated with reduced blood flow to all three uterine compartments, but did not reduce flow to the placenta. CONCLUSIONS CEU provides a sensitive, noninvasive method to assess vascular perfusion of the uterus during embryo implantation in macaques. We propose CEU as a new diagnostic tool to monitor vascular changes associated with early pregnancy in women.

Blockade of tubal patency following transcervical administration of polidocanol foam: initial studies in rhesus macaques ☆ ☆☆

OBJECTIVE To demonstrate the feasibility of polidocanol foam (PF) as a nonsurgical method of female permanent contraception using a nonhuman primate model. STUDY DESIGN Four groups of adult female rhesus macaques underwent either transcervical treatment with 5% PF directly into the uterine cavity, treatment with inert (methylcellulose, MF) foam or no treatment followed by removal of the reproductive tract for histologic evaluation. Untreated animals were included in Group 1 (n=3). Group 2 animals (n=4) were treated once with MF. Group 3 (n=7) received a single, and Group 4 (n=5) received multiple monthly treatments with PF; in these 2 groups, baseline tubal patency was assessed either laparoscopically by chromopertubation (CP) or by hysterosalpingography. RESULTS Group 1 (untreated) and Group 2 (MF) animals had normal tubal histology. In contrast, Group 3 and 4 females treated with PF showed evidence of tubal damage. In Group 4, bilateral tubal blockade was noted on CP after two (n=2) or three (n=3) treatments. Histologic analysis confirmed complete tubal occlusion (loss of epithelium, fibrosis) in three of these animals, and one showed significant tubal damage localized to the intramural segment. Nontarget (cervix, vagina, endometrium, ovary) reproductive tissues were unaffected. While similar tubal changes were observed after a single treatment (Group 3), endometrial hemorrhage was also noted as an acute change. CONCLUSION PF is a promising candidate agent for nonsurgical permanent female contraception. The histologic features of PF occlusion are confined to the intramural portion of the tube. IMPLICATIONS This study in rhesus macaques supports further development of transcervical administration of PF as a nonsurgical approach to permanent contraception. A nonsurgical method could reduce risks and costs associated with surgical female sterilization and increase access to permanent contraception.