Discovery of Small Molecule Agonist of Gonadotropin-Releasing Hormone Receptor (GnRH1R).

The gonadotrophin-releasing hormone (GnRH) is a central regulator of the human reproductive system and exerts physiological effects by binding to GnRH1R. The GnRH-GnRH1R system is a promising therapeutic target for the maintenance of reproductive function. There are several GnRH1R agonists on the market, but like GnRH, they are all peptide compounds and are limited by their way of administration (subcutaneous or intramuscular injection). To date, no published GnRH1R small molecule agonists have been reported. In this paper, the HTRF-based screening method has been used to screen our in-house chemical library, and we found and confirmed CD304 as a hit compound. Subsequently, structure optimization led to the discovery of compound 6d, exhibited with a certain GnRH1R activation activity (EC50: 1.59 ± 0.38 µM). Further molecular dynamics simulation experiments showed that 6d can well bind to the orthosteric site of GnRH1R through forming a hydrogen-bonding interaction with Y2836.51. Binding of 6d further induces conformational changes in TM6 and TM7, promoting the formation of a continuous water channel in GnRH1R, thereby promoting GnRH1R activation. This well-characterized hit compound will facilitate the further development of novel small molecule agonists of GnRH1R.

Transcriptomic evidence of luteinizing hormone-releasing hormone agonist (LHRH-A) regulation on lipid metabolism in grass carp (Ctenopharyngodon idella).

In this study, RNA sequencing was used to identify the hepatic gene expression profile in grass carp associated with luteinizing hormone-releasing hormone agonist (LHRH-A) treatment. A total of 93,912,172 reads were generated by HiSeq 4000 sequencing platform. After filtering, 83,450,860 clean reads were mapped to the reference genome. By calculating the FPKM of genes, 1475 differentially expressed genes were identified. PPAR signaling pathway was enriched with upregulated genes in LHRH-A injection group showing the regulation of the lipid metabolism by LHRH-A. The expression of eight key genes in PPAR signaling pathway was confirmed by qPCR and the results suggested that ACSL4A, ACSL4B, ANGPTL4, LPL, RXRBA and SLC27A1B were significantly stimulated by LHRH-A injection. This investigation provides the evidence that LHRH-A could play a role in lipid metabolism.

Development of programmable gemcitabine-GnRH pro-drugs bearing linker controllable "click" oxime bond tethers and preclinical evaluation against prostate cancer.

Peptide-drug conjugates (PDCs) are gaining considerable attention as anti-neoplastic agents. However, their development is often laborious and time-consuming. Herein, we have developed and preclinically evaluated three PDCs with gemcitabine as the anticancer cytotoxic unit and D-Lys6-GnRH (gonadotropin-releasing hormone; GnRH) as the cancer-targeting unit. These units were tethered via acid-labile programmable linkers to guide a differential drug release rate from the PDC through a combination of ester or amide and "click" type oxime ligations. The pro-drugs were designed to enable the selective targeting of malignant tumor cells with linker guided differential drug release rates. We exploited the oxime bond responsiveness against the acidic pH of the tumor microenvironment and the GnRH endocytosis via the GnRH-R GPCR which is overexpressed on cancer cells. The challenging metabolic properties of gemcitabine were addressed during design of the PDCs. We developed a rapid (1 hour) and cost-effective "click" oxime bond ligation platform to assemble in one-pot the 3 desired PDCs that does not require purification, surpassing traditional time-ineffective and low yield methods. The internalization of the tumor-homing peptide unit in cancer cells, overexpressing the GnRH-R, was first validated through confocal laser microscopy and flow cytometry analysis. Subsequently, the three PDCs were evaluated for their in vitro antiproliferative effect in prostate cancer cells. Their stability and the release of gemcitabine over time were monitored in vitro in cell culture and in human plasma using LC-MS/MS. We then assessed the ability of the developed PDCs to internalize in prostate cancer cells and to release gemcitabine. The most potent analog, designated GOXG1, was used for pharmacokinetic studies in mice. The metabolism of GOXG1 was examined in liver microsomes, as well as in buffers mimicking the pH of intracellular organelles, resulting in the identification of two metabolites. The major metabolite at low pH emanated from the cleavage of the pH-labile oxime bond, validating our design approach. NMR spectroscopy and in vitro radioligand binding assays were exploited for GOXG1 to validate that upon conjugating the drug to the peptide, the peptide microenvironment responsible for its GnRH-R binding is not perturbed and to confirm its high binding potency to the GnRH-R. Finally, the binding of GOXG1 to the GnRH-R and the associated elicitation of testosterone release in mice were also determined. The facile platform established herein for the rapid assembly of PDCs with linker controllable characteristics from aldehyde and aminooxy units through rapid "click" oxime ligation, that does not require purification steps, could pave the way for a new generation of potent cancer therapeutics, diagnostics and theranostics.

Rescue of mutant gonadotropin-releasing hormone receptor function independent of cognate receptor activity.

Molecules that correct the folding of protein mutants, restoring their functional trafficking, are called pharmacoperones. Most are clinically irrelevant and possess intrinsic antagonist or agonist activity. Here, we identify compounds capable of rescuing the activity of mutant gonadotropin-releasing hormone receptor or GnRHR which, is sequestered within the cell and if dysfunctional leads to Hypogonadotropic Hypogonadism. To do this we screened the E90K GnRHR mutant vs. a library of 645,000 compounds using a cell-based calcium detection system. Ultimately, we identified 399 compounds with EC50 ≤ 5 µM with no effect in counterscreen assays. Medicinal chemistry efforts confirmed activity of 70 pure samples and mode of action studies, including radioligand binding, inositol phosphate, and toxicity assays, proved that we have a series of tractable compounds that can be categorized into structural clusters. These early lead molecules rescue mutant GnRHR function and are neither agonist nor antagonists of the GnRHR cognate receptor, a feature required for potential clinical utility.

Cardiovascular risks and toxicity - The Achilles heel of androgen deprivation therapy in prostate cancer patients.

Androgen deprivation therapy (ADT) is the primary systemic therapy for treating locally advanced or metastatic prostate cancer (PCa). Despite its positive effect on PCa patient survival, ADT causes various adverse effects, including increased cardiovascular risk factors and cardiotoxicity. Lifespans extension, early use of ADT, and second-line treatment with next-generation androgen receptor pathway inhibitors would further extend the duration of ADT and possibly increase the risk of ADT-induced cardiotoxicity. Meanwhile, information on the molecular mechanisms underlying ADT-induced cardiotoxicity and measures to prevent it is limited, mainly due to the lack of specifically designed preclinical studies and clinical trials. This review article compiles up-to-date evidence obtained from observational studies and clinical trials, in order to gain new insights for deciphering the association between ADT use and cardiotoxicity. In addition, potential cardioprotective strategies involving GnRH receptors and second messenger cGMP are discussed.

Efficacy and safety of ASP1707 for endometriosis-associated pelvic pain: the phase II randomized controlled TERRA study.

STUDY QUESTION: Does the GnRH antagonist, ASP1707, reduce endometriosis-associated pelvic pain? SUMMARY ANSWER: ASP1707 significantly reduced endometriosis-associated pelvic pain in a dose-related manner. WHAT IS KNOWN ALREADY: GnRH agonists are an effective therapeutic option for endometriosis that is refractory to non-steroidal anti-inflammatory drugs, oral contraceptives, and progestins. However, GnRH agonists cause complete suppression of estradiol (E2), resulting in hypoestrogenic side-effects such as bone loss that may increase the future risk of osteoporotic fractures. STUDY DESIGN, SIZE, DURATION: This was a Phase II, multicenter, double-blind, randomized, parallel-group, placebo-controlled study conducted in 540 women from 04 December 2012 to 30 July 2015 in Europe and Japan. A sample size of 504 (84 subjects per group) was calculated to provide ≥80% power to detect a dose-related treatment effect among placebo and ASP1707 doses in change from baseline in pelvic pain, assuming different dose-response curves after 12 weeks of treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of 912 women with endometriosis-associated pelvic pain screened, 540 were enrolled, and 532 received ≥1 dose of study drug (placebo, n = 88; ASP1707 3 mg, n = 86; ASP1707 5 mg, n = 91; ASP1707 10 mg, n = 90; ASP1707 15 mg, n = 88; leuprorelin, n = 89) for 24 weeks. MAIN RESULTS AND THE ROLE OF CHANCE: After 12 weeks of treatment with ASP1707, the mean (95% CI) changes in numeric rating score (NRS) for overall pelvic pain (OPP) were -1.56 (-1.91, -1.21), -1.63 (-1.99, -1.27), -1.93 (-2.27, -1.60), -2.29 (-2.64, -1.94), and -2.13 (-2.47, -1.79) for placebo, ASP1707 3 mg, ASP1707 5 mg, ASP1707 10 mg, and ASP1707 15 mg, respectively. Mean (95% CI) changes in NRS for dysmenorrhea were -1.50 (-2.00, -1.00), -2.72 (-3.22, -2.21), -2.85 (-3.33, -2.38), -3.97 (-4.46, -3.48), and -4.18 (-4.66, -3.70), respectively. Mean (95% CI) changes in NRS for non-menstrual pelvic pain (NMPP) were -1.53 (-1.88, -1.19), -1.51 (-1.87, -1.16), -1.80 (-2.14, -1.47), -2.03 (-2.37, -1.68), and -1.86 (-2.20, -1.52), respectively. Statistically significant dose-related treatment effects in reduction in NRS for OPP (P = 0.001), dysmenorrhea (P < 0.001), and NMPP (P = 0.029) were observed after 12 weeks among ASP1707 doses and were maintained through 24 weeks. Serum estradiol and bone mineral density decreased dose dependently with ASP1707 through 24 weeks, however, to a lesser extent than with leuprorelin. LIMITATIONS, REASON FOR CAUTION: This study was not powered for pairwise comparison of each ASP1707 group versus placebo. WIDER IMPLICATIONS OF THE FINDINGS: All doses of ASP1707 reduced serum E2 levels to within the target range and to a lesser extent than leuprorelin. ASP1707 is a potential alternative treatment to leuprorelin for endometriosis-associated pelvic pain with lower impact on bone health. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Astellas Pharma Inc. T.D'.H is Vice President and Head of Global Medical Affairs Fertility at Merck, Darmstadt, Germany since October 1, 2015. At the time that the TERRA study was conducted, he served as Principal Investigator in his role as Coordinator of the Leuven University Fertility Center. Since October 2015, T.D'.H has left Leuven University Hospital Gasthuisberg, but continues to serve as Professor in Reproductive Medicine and Biology at KU Leuven (University of Leuven) Belgium and at the Dept of Obstetrics, Gynecology and Reproduction at Yale University, New Haven, USA. T. Fukaya and Y. Osuga report personal consulting fees from Astellas Pharma Inc. during the conduct of the study and outside the submitted work. G.M. Holtkamp, and L. Skillern are employed by Astellas Pharma Europe B.V.; K. Miyazaki is employed by Astellas Pharma Inc.; B. López, was a biostatistician for Astellas Pharma Europe B.V. during conduct of the study; R. Besuyen was a contract Associate Director of Medical Science for Astellas during conduct of the study. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, www.clinicaltrials.gov, NCT01767090. EudraCT number 2012-002791-14. TRIAL REGISTRATION DATE: 18 December 2012. DATE OF FIRST SUBJECT'S ENROLLMENT: One subject signed informed consent on 04 December 2012; the first subject was randomized on 16 April 2013.

The current status of hormone treatment for prostate cancer patients in Korean real-world practice: a multi-institutional observational study.

We aimed to evaluate the current nationwide trend, efficacy, safety, and quality of life (QoL) profiles of hormone treatment in real-world practice settings for prostate cancer (PCa) patients in Korea. A total of 292 men with any biopsy-proven PCa (TanyNanyMany) from 12 institutions in Korea were included in this multi-institutional, observational study of prospectively collected data. All luteinizing hormone-releasing hormone (LHRH) agonists were allowed to be investigational drugs. Efficacy was defined as (1) the rate of castration (serum testosterone ≤50 ng dl-1) at 4-week visit and (2) breakthrough (serum testosterone >50 ng dl-1 after castration). Safety assessments included routine examinations for potential adverse events, laboratory tests, blood pressure, body weight, and bone mineral density (BMD, at baseline and at the last follow-up visit). QoL was assessed using the Expanded Prostate Cancer Index Composite-26 (EPIC-26). The most common initial therapeutic regimen was LHRH agonist with anti-androgen (78.0%), and the most commonly used LHRH agonist for combination and monotherapy was leuprolide (64.0% for combination and 58.0% for monotherapy). The castration and breakthrough rates were 78.4% and 6.6%, respectively. The laboratory results related to dyslipidemia worsened after 4 weeks of hormone treatment. In addition, the mean BMD T-score was significantly lower at the last follow-up (mean: -1.950) compared to baseline (mean: -0.195). The mean total EPIC-26 score decreased from 84.8 (standard deviation [s.d.]: 12.2) to 78.3 (s.d.: 8.1), with significant deterioration only in the urinary domain (mean: 23.5 at baseline and 21.9 at the 4-week visit). These findings demonstrate the nationwide trend of current practice settings in hormone treatment for PCa in Korea.

Preventing ovarian failure associated with chemotherapy.

Alkylating chemotherapy is often used to treat pre-menopausal women for various malignancies and autoimmune diseases. Chemotherapy-associated ovarian failure is a potential consequence of this treatment which can cause infertility, and increases the risk of other long term adverse health sequelae. Randomised trials, predominantly of women undergoing alkylating chemotherapy for breast cancer, have shown evidence for the efficacy of gonadotropin-releasing hormone agonists (GnRHa) in preventing chemotherapy-associated ovarian failure. The European St Gallen and United States National Comprehensive Cancer Network guidelines recommend the use of concurrent GnRHa to reduce the risk of ovarian failure for pre-menopausal women undergoing chemotherapy for breast cancer. The GnRHa goserelin, a monthly 3.6 mg depot subcutaneous injection, has recently been listed on the Australian Pharmaceutical Benefits Scheme to reduce risk of ovarian failure for pre-menopausal women receiving alkylating therapies for malignancy or autoimmune disease. The first dose of goserelin should ideally be administered at least 1 week before commencement of alkylating treatment and continued 4-weekly during chemotherapy. Concurrent goserelin use should now be considered for all pre-menopausal women due to commence alkylating chemotherapy (except those with incurable cancer), regardless of their childbearing status, in an effort to preserve their ovarian function. For women who have not completed childbearing, consideration of other fertility preservation options, such as cryopreservation of embryos or oocytes, is also important.

Administration of leuprolide acetate, a GnRH agonist, improves urodynamic parameters in ovariectomized rats.

AIM: To evaluate the effects of a treatment with leuprolide acetate (LA) on bladder overactivity as well as the expression of gonadotropin releasing hormone receptor (GnRH-R), and neurofilaments NF68 and NF200 in female rats with overactive bladder induced by castration. METHODS: Changes in the urodynamic parameters were determined in SHAM, ovariectomized (OVX) and ovariectomized rats treated with LA (OVX-LA). A semi-quantitative analysis for the expression pattern of GnRH-R and neurofilaments NF68 and NF200 were determined. RESULTS: Forty-three days after ovariectomy, rats from the OVX group have significant lower values for intercontractile interval (ICI) and compliance (C); as well as higher values for basal bladder pressure (BP) and frequency of non-voiding contractions (NVC). The systemic application of LA increased voiding volume (Vv) and pressure threshold (ThP) in the OVX-LA animals. The application of LA reduced the high frequency of NVC in the OVX rats. No significant differences were found for Vv and NVCs between the OVX-LA vs SHAM groups. At the mid part of the bladder, the presence of GnRH-R was evidenced in the urothelium of the SHAM group. The OVX animals showed different pattern of immunolabeling for GnRH-R as well as for neurofilaments NF200 and NF68, whereas in the OVX-LA group the immunofluorescence pattern was similar to the one seen in SHAM bladders (P < 0.05 for OVX vs OVX + LA). CONCLUSIONS: the results suggest that systemic application of LA can improve bladder dysfunction in castrated rats, and perhaps considered as a treatment for overactive bladder conditions secondary to menopause.

GnRH in the Human Female Reproductive Axis.

Gonadotropin-releasing hormone (GnRH) is recognized as the central regulator of the functions of the pituitary-gonadal axis. The increasing knowledge on the mechanisms controlling the development and the function of GnRH-producing neurons is leading to a better diagnostic and therapeutic approach for hypogonadotropic hypogonadisms and for alterations of the puberty onset. During female life span, the function of the GnRH pulse generator may be affected by a number of inputs from other neuronal systems, offering alternative strategies for diagnostic and therapeutic interventions. Moreover, the identification of a GnRH/GnRH receptor system in both human ovary and endometrium has widened the spectrum of action of the peptide outside its hypothalamic functions. The pharmacological use of GnRH itself or its synthetic analogs (agonists and antagonists) provides a valid tool to either stimulate or block gonadotropin secretion and to modulate the female fertility in several reproductive disorders and in assisted reproduction technology. The use of GnRH agonists in young female patients undergoing chemotherapy is also considered a promising therapeutic approach to counteract iatrogenic ovarian failure.

Mitogen- and stress-activated protein kinase 1 is required for gonadotropin-releasing hormone-mediated activation of gonadotropin α-subunit expression.

Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific ß-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.

"Real-world" pediatric endocrine practice; how much is it influenced by physician's gender and region of practice. Results of an international survey.

OBJECTIVE: To determine whether hormonal treatments for frequent clinical cases (short stature, delayed and precocious puberty) are prescribed strictly according to clinical guidelines or based on personal tendencies, and whether the decisions correlate with physician's personal demographics (age, sex, and place of practice). METHODS: Cross-sectional survey, with made-up clinical cases, distributed to pediatric endocrinologists using 2 web-based professional forums, Israeli and an international. The questionnaire included 8 clinical cases and 5 demographic questions regarding the physician. Differences in practice between Israeli and international endocrinologists were assessed, and correlation between the physician's gender and their decisions regarding treatment. RESULTS: One hundred fifty-five physicians responded, 28% Israeli and 72% international. In girls with early puberty, 60% of international and 26% of Israeli physicians chose not to treat with a gonadotropin-releasing hormone agonist. In girls with short stature, 79% of Israeli and 34% of international physicians offered growth hormone treatment. In girls with early puberty, both male and female physicians responded similarly in the international group, but in the Israeli group 47% of male and 15% of female doctors would not treat. In girls with constitutional growth delay, 67% of Israeli male doctors would not treat with growth hormone compared to 30% of Israeli female physicians. CONCLUSIONS: Our study demonstrated significant practice differences between Israeli and international pediatric endocrinologists. Within the Israeli group, significant practice differences were seen between male and female physicians. Given that Israeli physicians follow the same clinical guidelines it is clear that a large "grey zone" of clinical cases exist and much of the decisions on treatment are personal and influenced by personal beliefs or gender.

Acute gonadotropin-releasing hormone agonist treatment enhances extinction memory in male rats.

Leuprolide acetate (LEU), also known as Lupron, is commonly used to treat prostate cancer in men. As a gonadotropin-releasing hormone (GnRH) receptor agonist, it initially stimulates the release of gonadal hormones, testosterone (T) and estradiol. This surge eventually suppresses these hormones, preventing the further growth and spread of cancer cells. Individuals receiving this treatment often report anxiety and cognitive changes, but LEU's effects on the neural mechanisms that are involved in anxiety during the trajectory of treatment are not well known. In this study, we examined the acute effects of LEU on fear extinction, hypothesizing that increased T levels following a single administration of LEU will facilitate extinction recall by altering neuronal activity within the fear extinction circuitry. Two groups of naïve adult male rats underwent a 3-day fear conditioning, extinction, and recall experiment. The delayed group (n=15) received a single injection of vehicle or LEU (1.2mg/kg) 3weeks before behavioral testing. The acute group (n=25) received an injection one day after fear conditioning, 30min prior to extinction training. Following recall, the brains for all animals were collected for c-fos immunohistochemistry. Blood samples were also collected and assayed for T levels. Acute administration of LEU increased serum T levels during extinction training and enhanced extinction recall 24h later. This enhanced extinction memory was correlated with increased c-fos activity within the infralimbic cortex and amygdala, which was not observed in the delayed group. These results suggest that the elevation in T induced by acute administration of LEU can influence extinction memory consolidation, perhaps through modification of neuronal activity within the infralimbic cortex and amygdala. This may be an important consideration in clinical applications of LEU and its effects on anxiety and cognition.

The potential role of follicle-stimulating hormone in the cardiovascular, metabolic, skeletal, and cognitive effects associated with androgen deprivation therapy.

PURPOSE: To explore how follicle-stimulating hormone (FSH) may contribute to cardiovascular, metabolic, skeletal, and cognitive events in men treated for prostate cancer, with various forms of androgen deprivation therapy (ADT). MATERIALS AND METHODS: A colloquium of prostate cancer experts was convened in May 2015, to discuss the role of FSH in the development of unwanted effects associated with ADT. Subsequently, a literature review (Medline, PubMed, and relevant congress abstract databases) was performed to further explore and evaluate the collected evidence. RESULTS: It has become evident that, in the setting of ADT, FSH can promote the development of atherosclerotic plaque formation, metabolic syndrome, and insulin resistance. Data also suggest that FSH is an important mediator of bone remodeling, particularly bone resorption, and thereby increases the risk for bone fracture. Additional evidence implicates a role for FSH in bone metastasis as well. The influence of FSH on ADT-induced cognitive deficits awaits further elucidation; however, the possibility that FSH may be involved therein cannot be ruled out. CONCLUSIONS: The widespread molecular and physiological consequences of FSH system activation in normal and pathological conditions are becoming better understood. Progress in this area has been achieved by the development of additional investigative and clinical measures to better evaluate specific adverse effects. More research is needed on FSH function in the development of cancer as well as its association with cardiovascular, metabolic, musculoskeletal, and cognitive effects in ADT.

Gonadotropin-releasing hormone agonist (GnRHa) trigger - State of the art.

GnRH agonist (GnRHa) trigger for final oocyte maturation in GnRH antagonist co-treated IVF/ICSI cycles significantly reduces the risk of ovarian hyperstimulation syndrome (OHSS). GnRHa trigger followed by modifications of the standard luteal phase support (modified luteal phase support) secures fresh transfer in the majority of patients with excellent reproductive outcomes. In freeze all cycles (segmented cycles) GnRHa trigger allows oocyte retrieval with a minimal risk of early onset OHSS and good reproductive outcomes in subsequent frozen thaw cycles. Overall, two different luteal phase support strategies have been proposed when a fresh transfer is performed after GnRHa trigger. These involve either boosting the endogenous steroid production or adding exogenous steroids. The present review discusses the advancement of GnRHa trigger in fresh and segmented cycles and how a modified luteal phase support policy in fresh transfer cycles results in good reproductive outcomes as well as a high safety in terms of OHSS reduction. Finally, the new concept of an individualized luteal phase support policy taking the number of pre-ovulatory follicles into account when planning a fresh transfer in GnRHa triggered IVF/ICSI cycle is discussed.

Treatment with a GnRH receptor agonist, but not the GnRH receptor antagonist degarelix, induces atherosclerotic plaque instability in ApoE(-/-) mice.

Androgen-deprivation therapy (ADT) for prostate cancer has been associated with increased risk for development of cardiovascular events and recent pooled analyses of randomized intervention trials suggest that this primarily is the case for patients with pre-existing cardiovascular disease treated with gonadotropin-releasing hormone receptor (GnRH-R) agonists. In the present study we investigated the effects of the GnRH-R agonist leuprolide and the GnRH-R antagonist degarelix on established atherosclerotic plaques in ApoE(-/-) mice. A shear stress modifier was used to produce both advanced and more stable plaques in the carotid artery. After 4 weeks of ADT, increased areas of necrosis was observed in stable plaques from leuprolide-treated mice (median and IQR plaque necrotic area in control, degarelix and leuprolide-treated mice were 0.6% (IQR 0-3.1), 0.2% (IQR 0-4.4) and 11.0% (IQR 1.0-19.8), respectively). There was also evidence of increased inflammation as assessed by macrophage immunohistochemistry in the plaques from leuprolide-treated mice, but we found no evidence of such changes in plaques from control mice or mice treated with degarelix. Necrosis destabilizes plaques and increases the risk for rupture and development of acute cardiovascular events. Destabilization of pre-existing atherosclerotic plaques could explain the increased cardiovascular risk in prostate cancer patients treated with GnRH-R agonists.

Effects of gonadotropin-releasing hormone agonist and antagonist on ovarian activity in a mouse model for polycystic ovary.

PCOS is a major cause of anovulatory infertility in women in their reproductive age. However, its etiology and pathophysiology remain uncertain. The immature mice chronically injected with DHEA, termed as PCO-mice, develop numerous large cystic follicles, high circulating androgen and anovulation similar to PCOS in women. Although PCO-mice show decreased ovarian GnRH I-receptor in immunoblot but show increased immunostaining for GnRH I-receptor in oocytes of cystic follicles. PCO-mice show reduced ovarian LH receptor expression, circulating estradiol and progesterone level compared to normal mice injected with vehicle only. The treatment with low dose of GnRH-Agonist in PCO-mice restores ovarian LH receptor expression to the level of normal mice and promote ovulation and formation of functional corpus luteum. GnRH-Antagonist although cause ovulation in PCO-mice but does not restore LH receptor expression to the level of normal mice, and they show low circulating progesterone and hypertrophied vacuolated corpus luteum. Our study suggests that GnRH-agonist restores ovulation in PCO-mice and produces biphasic and beneficial effect over the use of GnRH-Antagonist.

Persistent GnRH receptor activation in pituitary αT3-1 cells analyzed with a label-free technology.

The gonadotropin-releasing hormone (GnRH) receptor is a drug target for certain hormone-dependent diseases such as prostate cancer. In this study, we examined the activation profiles of the endogenous ligand, GnRH and a well-known marketed analog, buserelin using a label-free assay in pituitary αT3-1 cells with endogenous GnRH receptor expression. This whole cell impedance-based technology allows for the real-time measurement of morphological cellular changes. Both agonists dose-dependently decreased the impedance as a result of GnRH receptor activation with potencies of 9.3 ± 0.1 (pEC50 value, buserelin) and 7.8 ± 0.06 (pEC50 value, GnRH). Subsequently, GnRH receptor activation was completely abolished with a selective Gαq inhibitor, thereby confirming the Gαq-coupling of the GnRH receptor in pituitary αT3-1 cells. Additionally, we observed continued responses after agonist stimulation of αT3-1 cells indicating long-lasting cellular effects. Wash-out experiments demonstrated that the long-lasting effects induced by GnRH were most likely caused by rebinding since over 70% of the original response was abolished after wash-out. In contrast, a long receptor residence time was responsible for the prolonged effects caused by buserelin, with over 70% of the original response remaining after wash-out. In summary, we validated that impedance-based label-free technology is suited for studying receptor-mediated activation in cell lines endogenously expressing the target of interest. Moreover, this real-time monitoring allows the examination of binding kinetics and its influence on receptor activation at a cellular level.