Gonadotropin-releasing Hormone Receptor Agonists Research Articles

Cytotoxic Activity of Novel GnRH Analogs Conjugated with Mitoxantrone in Ovarian Cancer Cells.

The gonadotropin-releasing hormone (GnRH) receptor (GnRH-R) is highly expressed in ovarian cancer cells (OCC), and it is an important molecular target for cancer therapeutics. To develop a new class of drugs targeting OCC, we designed and synthesized Con-3 and Con-7 which are novel high-affinity GnRH-R agonists, covalently coupled through a disulfide bond to the DNA synthesis inhibitor mitoxantrone. We hypothesized that Con-3 and Con-7 binding to the GnRH-R of OCC would expose the conjugated mitoxantrone to the cellular thioredoxin, which reduces the disulfide bond of Con-3 and Con-7. The subsequent release of mitoxantrone leads to its intracellular accumulation, thus exerting its cytotoxic effects. To test this hypothesis, we determined the cytotoxic effects of Con-3 and Con-7 using the SKOV-3 human OCC. Treatment with Con-3 and Con-7, but not with their unconjugated GnRH counterparts, resulted in the accumulation of mitoxantrone within the SKOV-3 cells, increased their apoptosis, and reduced their proliferation, in a dose- and time-dependent manner, with half-maximal inhibitory concentrations of 0.6-0.9 µM. It is concluded that Con-3 and Con-7 act as cytotoxic "prodrugs" in which mitoxantrone is delivered in a GnRH-R-specific manner and constitute a new class of lead compounds for use as anticancer drugs targeting ovarian tumors.

Identification of Environmental Compounds That May Trigger Early Female Puberty by Activating Human GnRHR and KISS1R.

There has been an alarming trend toward earlier puberty in girls, suggesting the influence of an environmental factor(s). As the reactivation of the reproductive axis during puberty is thought to be mediated by the hypothalamic neuropeptides kisspeptin and gonadotropin-releasing hormone (GnRH), we asked whether an environmental compound might activate the kisspeptin (KISS1R) or GnRH receptor (GnRHR). We used GnRHR or KISS1R-expressing HEK293 cells to screen the Tox21 10K compound library, a compendium of pharmaceuticals and environmental compounds, for GnRHR and KISS1R activation. Agonists were identified using Ca2+ flux and phosphorylated extracellularly regulated kinase (p-ERK) detection assays. Follow-up studies included measurement of genes known to be upregulated upon receptor activation using relevant murine or human cell lines and molecular docking simulation. Musk ambrette was identified as a KISS1R agonist, and treatment with musk ambrette led to increased expression of Gnrh1 in murine and human hypothalamic cells and expansion of GnRH neuronal area in developing zebrafish larvae. Molecular docking demonstrated that musk ambrette interacts with the His309, Gln122, and Gln123 residues of the KISS1R. A group of cholinergic agonists with structures similar to methacholine was identified as GnRHR agonists. When applied to murine gonadotrope cells, these agonists upregulated Fos, Jun, and/or Egr1. Molecular docking revealed a potential interaction between GnRHR and 5 agonists, with Asn305 constituting the most conservative GnRHR binding site. In summary, using a Tox21 10K compound library screen combined with cellular, molecular, and structural biology techniques, we have identified novel environmental agents that may activate the human KISS1R or GnRHR.

Profile of Relugolix in the Management of Advanced Hormone-Sensitive Prostate Cancer: Design, Development, and Place in Therapy.

Androgen deprivation therapy, primarily via a gonadotropin-releasing hormone receptor agonist or antagonist together with or without an androgen receptor antagonist, remains the mainstay of medical treatment for advanced prostate cancer. Meanwhile, relugolix has been developed as the first orally active, non-peptide, selective antagonist for the gonadotropin-releasing hormone receptor. Previous randomized studies involving patients with prostate cancer have demonstrated comparable efficacy in androgen suppression between relugolix vs other gonadotropin-releasing hormone antagonists or agonists. This review summarizes available data on the design and development of relugolix and its therapeutic application, and discusses if relugolix represents a promising oral alternative to injectable androgen deprivation therapy. Based on current published evidence, further investigation is likely required to determine the actual clinical benefits of relugolix therapy against prostate cancer.

Case - Leuprolide acetate-induced necrotic skin ulceration.

Androgen deprivation therapy (ADT) has become a well-established modality in the management of locally advanced and metastatic prostate cancer.1 Leuprolide acetate, a synthetically derived gonadotropin-releasing hormone receptor (GnRHR) agonist, is commonly used for ADT in men.2 Despite proven effectiveness, the administration of GnRHR agonists is associated with common adverse reactions, such as impotence, hot flashes, and fatigue. Injection site reactions have also been documented, however, are infrequent, with the most common being localized pain.3,4 Here, we report a case of necrotic skin ulceration following multiple administrations of intramuscular leuprolide acetate.

Discovery of the thieno[2,3-d]pyrimidine-2,4-dione derivative 21a: A potent and orally bioavailable gonadotropin-releasing hormone receptor antagonist

The gonadotropin releasing hormone receptor (GnRH-R) is a G protein-coupled receptor (GPCR) belonging to the rhodopsin family. GnRH-R antagonists suppress testosterone to castrate level more rapidly than gonadotropin releasing hormone agonists but lack the flare phenomenon often seen during the early period of GnRH-R agonist treatment. Recently orgovyx (relugolix) was approved as the first oral GnRH-R antagonist for the treatment of advanced prostate cancer. However, orgovyx has demonstrated poor pharmacokinetic profile with low oral bioavailability and high efflux. Here, we rationally designed and synthesized a series of derivatives (13a-m, 21a-i) through the modification and structure-activity relationship study of relugolix, which led to the discovery of 21a as a highly potent GnRH-R antagonist (IC50 = 2.18 nM) with improved membrane permeability (Papp, A-B = 0.98 × 10−6 cm/s) and oral bioavailability (F % = 44.7). Compound 21a showed high binding affinity (IC50 = 0.57 nM) and potent in vitro antagonistic activity (IC50 = 2.18 nM) at GnRH-R. 21a was well tolerated and efficacious in preclinical studies to suppress blood testosterone levels, which merits further investigation as a candidate novel GnRH-R antagonist for clinical studies.

A case series of androgen receptor (AR) inhibition by bicalutamide in combination with leuprolide acetate and exemestane in recurrent AR positive adult-type ovarian granulosa cell tumor (AGCT) (158)

Objectives: There remains a need to develop targeted therapies for recurrent AGCT that produce measurable tumor responses. The FOXL2C134W mutation is identified in nearly all AGCT. In a genetically engineered mouse model utilized by Cluzet et al., tumorigenesis was associated with the inactivation of p53 and Rb pathways, increased circulating androgens, estradiol, and antimullerian hormone along with decreased FOXL2 abundance [1]. By examining the pathways involved in granulosa cell development, including limitations on GnRH-induced apoptosis, aromatase induction, and increased androgen expression, a novel treatment regimen is proposed for recurrent AGCT: bicalutamide (androgen receptor antagonist), exemestane (aromatase inhibitor), and leuprolide acetate (gonadotropin-releasing hormone receptor agonist) to block excess steroidogenesis seen in AGCT. Methods: Six patients identified at our institution with recurrent AGCT and tumor AR positive (by Leica IHC-AR27 antibody) were treated with bicalutamide 50mg PO once daily, Exemestane 25mg PO once daily, and Leuprolide acetate 3.75mg IM every four weeks. All patients were heavily pre-treated, with two to seven prior lines of therapy and had undergone two to seven surgeries. Prior treatment lines consisted of prior chemotherapy, hormonal therapy, and targeted therapies, including bevacizumab and everolimus. Patients were monitored for toxicities and for response to treatment via surveillance exams, inhibin B levels and CT scans. Results: Based on RECIST v1.1 criteria, of the six patients receiving bicalutamide, exemestane, and leuprolide acetate, two patients had a partial response, four patients had stable disease and one patient had progressive disease. Strong AR expression in 95% of cells was identified in the tumors of patients who had a partial response. This drug combination has been well-tolerated. There has been one hospitalization for disease-related complications; no other grade 3 or higher toxicities reported thus far. Conclusions: Based on the response of our institutional cohort and the rationale behind the antiandrogen/aromatase inhibitor/GnRH agonist combination in recurrent AGCT, this regimen of bicalutamide/exemestane/leuprolide acetate is a promising treatment. An NRG concept has been submitted to proceed with a single-arm, multicenter phase II study of this regimen in patients who meet the criteria of recurrent AGCT, AR expression, measurable disease, and one prior line of therapy. Objectives: There remains a need to develop targeted therapies for recurrent AGCT that produce measurable tumor responses. The FOXL2C134W mutation is identified in nearly all AGCT. In a genetically engineered mouse model utilized by Cluzet et al., tumorigenesis was associated with the inactivation of p53 and Rb pathways, increased circulating androgens, estradiol, and antimullerian hormone along with decreased FOXL2 abundance [1]. By examining the pathways involved in granulosa cell development, including limitations on GnRH-induced apoptosis, aromatase induction, and increased androgen expression, a novel treatment regimen is proposed for recurrent AGCT: bicalutamide (androgen receptor antagonist), exemestane (aromatase inhibitor), and leuprolide acetate (gonadotropin-releasing hormone receptor agonist) to block excess steroidogenesis seen in AGCT. Methods: Six patients identified at our institution with recurrent AGCT and tumor AR positive (by Leica IHC-AR27 antibody) were treated with bicalutamide 50mg PO once daily, Exemestane 25mg PO once daily, and Leuprolide acetate 3.75mg IM every four weeks. All patients were heavily pre-treated, with two to seven prior lines of therapy and had undergone two to seven surgeries. Prior treatment lines consisted of prior chemotherapy, hormonal therapy, and targeted therapies, including bevacizumab and everolimus. Patients were monitored for toxicities and for response to treatment via surveillance exams, inhibin B levels and CT scans. Results: Based on RECIST v1.1 criteria, of the six patients receiving bicalutamide, exemestane, and leuprolide acetate, two patients had a partial response, four patients had stable disease and one patient had progressive disease. Strong AR expression in 95% of cells was identified in the tumors of patients who had a partial response. This drug combination has been well-tolerated. There has been one hospitalization for disease-related complications; no other grade 3 or higher toxicities reported thus far. Conclusions: Based on the response of our institutional cohort and the rationale behind the antiandrogen/aromatase inhibitor/GnRH agonist combination in recurrent AGCT, this regimen of bicalutamide/exemestane/leuprolide acetate is a promising treatment. An NRG concept has been submitted to proceed with a single-arm, multicenter phase II study of this regimen in patients who meet the criteria of recurrent AGCT, AR expression, measurable disease, and one prior line of therapy.

GnRH receptor mediates lipid storage in female adipocytes via AMPK pathway.

Objective: Due to high levels of serum gonadotropin-releasing hormone (GnRH), perimenopausal or menopausal women, girls with central precocious puberty, women of polycystic ovary syndrome, and females receiving long-term GnRH agonist (GnRHa) treatment are at substantially higher risk of developing obesity. However, it remains poorly understood how GnRH affects body weight. Here, we explored whether the gonadotropin-releasing hormone receptor (GnRHR) was expressed in adipocytes and how GnRHR mediated lipid accumulation and the development of obesity.Methods: The samples were from 18 patients with benign tumors operated between 01/2018 and 06/2018 at the Women's Hospital School of Medicine Zhejiang University. Immunofluorescence, Western Blotting, and RT-PCR were used to detect whether the GnRH receptor was expressed in the specimens and human preadipocytes-subcutaneous (HPA-s). The GnRH receptor agonist diphereline with different concentrations was used to stimulate the HPA-s cells for 24, 48, and CCK-8 was used to detect cell proliferation. Oil red-O staining was used to detect lipid droplets in mature adipocytes. The phosphorylation of AMPK-Ser485/Thr172 was detected by Western Blotting.Results: GnRH receptor was expressed in all 18 human subcutaneous adipose tissue specimens. Cultured HPA-s expressed the GnRH receptor, and the expression increased during the process of cell maturation. The GnRH receptor agonist diphereline can stimulate the proliferation of HPA-s cells, which can advance the earliest occurrence of lipid droplets in HPA-s cells and the occurrence of lipid droplets in 50% cells by 1-2 days. Diphereline can stimulate the increase in the number of lipid droplets in mature adipocytes. The phosphorylation level of AMPK-Ser485/Thr172 in mature adipocytes was decreased by diphereline.Conclusion: The GnRH receptor was expressed in adipocytes. As adipocytes mature, GnRH receptor expression gradually increased. GnRHa stimulates the proliferation of HPA-s, promotes adipocyte maturation, increases the formation of lipid droplets in mature adipocytes, and inhibits the activation of the AMPK pathway in adipocytes. Our findings may elucidate the mechanism of obesity in these female populations and provide some evidence on how GnRH contributes to obesity. Additionally, these results provide theoretical support for further research on the mechanisms of obesity, thus enhancing our understanding of the functional diversity of GnRH and establishing a new theoretical basis for the impact of GnRH on metabolism.

Activation of Gonadotropin-releasing Hormone Receptor Impedes the Immunosuppressive Activity of Decidual Regulatory T Cells via Deactivating the Mechanistic Target of Rapamycin Signaling

ABSTRACT Understanding maternal immune tolerance is crucial for the development of therapeutics for immunological pregnancy complications. Decidual regulatory T cells (Tregs) play a pivotal role in the maintenance of maternal immune tolerance. Using a murine allogeneic pregnancy model in the current study, we identified the up-regulation of gonadotropin-releasing hormone receptor (GnRHR) in decidual T cell subsets including CD4+ conventional T cells, CD8+ T cells, and CD4+Foxp3+ Tregs. Using a lentivirus-mediated GnRHR overexpression system and a GnRHR agonist, we found that GnRHR activation decreased the expression of Treg functional molecules such as IL10 (IL-10), IL-35 subunit EBI3 (Ebi3), IL2RA (CD25), TNFRSF18 (GITR), ICOS, and Treg master regulator FOXP3. The functional analysis indicated that GnRHR activation impairs the ability of Tregs to inhibit conventional T cell proliferation. We also revealed that GnRHR activation suppressed the mechanistic target of rapamycin (mTOR) signaling in GnRHR-overexpressing splenic Tregs (Wild type C57BL/6 J background) and decidual Tregs. MHY1485, a potent mTOR activator, effectively abolished the effect of the GnRHR agonist and promoted the immunosuppressive capability of Tregs. Furthermore, in an adoptive transfer model, Treg-specific GnRHR knockdown increased Foxp3 expression in decidual Tregs while decreasing the production of IFN-γ and IL-17 in decidual effector CD4+ T cells and reducing the production of IFN-γ in decidual effector CD8+ T cells. Taken together, the present study unveils a novel mechanism by which the immunosuppressive function of decidual Tregs is modulated, and deepens our understanding of maternal immune tolerance.

Anti-cancer effect of GV1001 for prostate cancer: function as a ligand of GnRHR.

GV1001, a 16-amino acid fragment of the human telomerase reverse transcriptase catalytic subunit (hTERT), has been developed as an injectable formulation of cancer vaccine. Here, we revealed for the first time that GV1001 is a novel ligand for gonadotropin-releasing hormone receptor (GnRHR). The docking prediction for GV1001 against GnRHR showed high binding affinity. Binding of GV1001 to GnRHR stimulated the Gαs-coupled cAMP signaling pathway and antagonized Gαq-coupled Ca2+ release by leuprolide acetate (LA), a GnRHR agonist. Repeated injection of GV1001 attenuated both serum testosterone level and seminal vesicle weight via desensitization of hypothalamic-pituitary-gonadal (HPG) axis. We then tested whether GV1001 has an inhibitory effect on tumor growth of LNCaP cells, androgen receptor-positive human prostate cancer (PCa) cells. GV1001 significantly inhibited tumor growth and induced apoptosis in LNCaP-implanted xenografts. Interestingly, mRNA expressions of matrix metalloproteinase 2 and matrix metalloproteinase 9 were suppressed by GV1001, but not by LA. Moreover, GV1001 significantly inhibited the proliferation and migration of PCa cells and induced apoptosis in a concentration-dependent manner. Our findings suggest that GV1001 functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway, with anti-proliferative and anti-migratory effects on human PCa.

Gemcitabine Based Peptide Conjugate with Improved Metabolic Properties and Dual Mode of Efficacy.

Gemcitabine is a clinically established anticancer agent potent in various solid tumors but limited by its rapid metabolic inactivation and off-target toxicity. We have previously generated a metabolically superior to gemcitabine molecule (GSG) by conjugating gemcitabine to a gonadotropin releasing hormone receptor (GnRH-R) ligand peptide and showed that GSG was efficacious in a castration resistant prostate cancer (CRPC) animal model. The current article provides an in-depth metabolic and mechanistic study of GSG, coupled with toxicity assays that strengthen the potential role of GSG in the clinic. LC-MS/MS based approaches were employed to delineate the metabolism of GSG, its mechanistic cellular uptake, and release of gemcitabine and to quantitate the intracellular levels of gemcitabine and its metabolites (active dFdCTP and inactive dFdU) resulting from GSG. The GnRH-R agonistic potential of GSG was investigated by quantifying the testosterone levels in animals dosed daily with GSG, while an in vitro colony forming assay together with in vivo whole blood measurements were performed to elucidate the hematotoxicity profile of GSG. Stability showed that the major metabolite of GSG is a more stable nonapeptide that could prolong gemcitabine's bioavailability. GSG acted as a prodrug and offered a metabolic advantage compared to gemcitabine by generating higher and steadier levels of dFdCTP/dFdU ratio, while intracellular release of gemcitabine from GSG in DU145 CRPC cells depended on nucleoside transporters. Daily administrations in mice showed that GSG is a potent GnRH-R agonist that can also cause testosterone ablation without any observed hematotoxicity. In summary, GSG could offer a powerful and unique pharmacological approach to prostate cancer treatment: a single nontoxic molecule that can be used to reach the tumor site selectively with superior to gemcitabine metabolism, biodistribution, and safety while also agonistically ablating testosterone levels.

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.

Abstract 9970: Androgen Deprivation Therapy by Surgical Castration, Gonadotropin Releasing Hormone Receptor Agonists or Antagonists and the Development of Atherosclerosis in Apoe-deficient Mice

Introduction: Adjuvant treatment of prostate cancer often involves androgen deprivation therapy (ADT) by orchiectomy (Orx), gonadotropin releasing hormone (GnRH) receptor agonists or GnRH receptor antagonists. Clinical studies of men with preexisting atherosclerotic disease, have indicated that cardiovascular event rates differs between different types of ADT, suggesting that not only testosterone deficiency, but also direct effects of ADT on processes in the atherosclerotic plaque may be involved. Immune cell GnRH receptors have been implicated as potential mediators. In the present study, we compared the effects of common types of ADT on de-novo atherosclerosis in mice. Methods: Chow-fed Apoe-deficient mice (n=121) were allocated to Orx and/or monthly injections with the GnRH receptor agonist leuprolide (0.5 mg s.c.), the GnRH receptor antagonist degarelix (0.5 mg s.c.) or saline starting from 8 weeks of age (n=19-21 in each group). At 26 weeks of age, the burden of atherosclerosis was quantified by en face analysis of the Oil red O-stained aortic arch and in histological sections of the aortic root. Bone marrow-derived macrophages and splenocytes were incubated in the presence of leuprolide (10 -6 mol/L) or degarelix (10 -6 mol/L). Data is shown as mean ± sem, *p<0.05, **p<0.01. Results: All types of ADT augmented hypercholesterolemia and atherosclerosis with no significant differences in the size of the effect (Figure A-B). In Orx mice, the addition of leuprolide, but not degarelix, led to further increases in plasma total cholesterol and in atherosclerosis in the aortic root (D), but not the aortic arch (C). Plaque necrosis was unaffected by ADT. No effects of the drugs on macrophage cytokine secretion or splenocyte proliferation were observed. Conclusions: ADT did not exert differential effects on the development of atherosclerosis in the Apoe-deficient mice. A pro-atherogenic, possibly cholesterol-mediated, effect of leuprolide was seen in Orx mice.

Hypothalamic gonadotropin-releasing hormone (GnRH) receptor neurons fire in synchrony with the female reproductive cycle.

Gonadotropin-releasing hormone (GnRH) controls mammalian reproduction via the hypothalamic-pituitary-gonadal (hpg) axis, acting on gonadotrope cells in the pituitary gland that express the GnRH receptor (GnRHR). Cells expressing the GnRHR have also been identified in the brain. However, the mechanism by which GnRH acts on these potential target cells remains poorly understood due to the difficulty of visualizing and identifying living GnRHR neurons in the central nervous system. We have developed a mouse strain in which GnRHR neurons express a fluorescent marker, enabling the reliable identification of these cells independent of the hormonal status of the animal. In this study, we analyze the GnRHR neurons of the periventricular hypothalamic nucleus in acute brain slices prepared from adult female mice. Strikingly, we find that the action potential firing pattern of these neurons alternates in synchrony with the estrous cycle, with pronounced burst firing during the preovulatory period. We demonstrate that GnRH stimulation is sufficient to trigger the conversion from tonic to burst firing in GnRHR neurons. Furthermore, we show that this switch in the firing pattern is reversed by a potent GnRHR antagonist. These data suggest that endogenous GnRH acts on GnRHR neurons and triggers burst firing in these cells during late proestrus and estrus. Our data have important clinical implications in that they indicate a novel mode of action for GnRHR agonists and antagonists in neurons of the central nervous system that are not part of the classical hpg axis.

Leuprolide-Induced Extrapyramidal Symptoms Successfully Treated With Propranolol

Androgen deprivation therapy is the most widely used systemic treatment for locally advanced and metastatic prostate cancer. Leuprolide acetate, a synthetic gonadotropin-releasing hormone receptor agonist, has proven effective in controlling prostate cancer by suppressing gonadotropic secretion of luteinizing hormone and follicle-stimulating hormone, which subsequently decrease the testosterone level. The most common physical adverse effects of androgen deprivation therapy include fatigue, hot flushes, anemia, decreased muscle mass, osteoporosis, loss of libido, and erectile dysfunction. Adverse neuropsychiatric effects, such as depression, anxiety, and cognitive impairment, are also frequently noted, but the mechanisms underlying these adverse effects of androgen deprivation therapy have not yet been determined.

Degarelix: a guide to its use in advanced prostate cancer

Degarelix (Firmagon®), a gonadotropin-releasing hormone (GnRH) receptor antagonist, is indicated as an androgen-deprivation therapy in men with advanced hormone-dependent prostate cancer. It effectively induces and maintains suppression of serum testosterone to castration levels and has an acceptable tolerability profile. Relative to leuprolide, degarelix is associated with more rapid induction of testosterone and prostate-specific antigen (PSA) suppression without the testosterone surges associated with GnRH receptor agonists, and a potentially lower risk of the combined endpoint of PSA progression and death.

The forkhead transcription factor, FOXP3, is required for normal pituitary gonadotropin expression in mice.

The hypothalamic-pituitary-gonadal axis is central to normal reproductive function. This pathway begins with the release of gonadotropin-releasing hormone in systematic pulses by the hypothalamus. Gonadotropin-releasing hormone is bound by receptors on gonadotroph cells in the anterior pituitary gland and stimulates the synthesis and secretion of luteinizing hormone and, to some extent, follicle-stimulating hormone. Once stimulated by these glycoprotein hormones, the gonads begin gametogenesis and the synthesis of sex hormones. In humans, mutations of the forkhead transcription factor, FOXP3, lead to an autoimmune disorder known as immunodysregulation, polyendocrinopathy, and enteropathy, X-linked syndrome. Mice with a mutation in the Foxp3 gene have a similar autoimmune syndrome and are infertile. To understand why FOXP3 is required for reproductive function, we are investigating the reproductive phenotype of Foxp3 mutant mice (Foxp3(sf/Y)). Although the gonadotroph cells appear to be intact in Foxp3(sf/Y) mice, luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) expression are significantly decreased, demonstrating that these mice exhibit a hypogonadotropic hypogonadism. Hypothalamic expression of gonadotropin-releasing hormone is not significantly decreased in Foxp3(sf/Y) males. Treatment of Foxp3(sf/Y) males with a gonadotropin-releasing hormone receptor agonist does not rescue expression of Lhb or Fshb. Interestingly, we do not detect Foxp3 expression in the pituitary or hypothalamus, suggesting that the infertility seen in Foxp3(sf/Y) males is a secondary effect, possibly due to loss of FOXP3 in immune cells. Pituitary expression of glycoprotein hormone alpha (Cga) and prolactin (Prl) are significantly reduced in Foxp3(sf/Y) males, whereas the precursor for adrenocorticotropic hormone, pro-opiomelanocortin (Pomc), is increased. Human patients diagnosed with IPEX often exhibit thyroiditis due to destruction of the thyroid gland by autoimmune cells. We find that Foxp3(sf/Y) mice have elevated expression of thyroid-stimulating hormone beta (Tshb), suggesting that they may suffer from thyroiditis as well. Expression of the pituitary transcription factors, Pitx1, Pitx2, Lhx3, and Egr1, is normal; however, expression of Foxl2 and Gata2 is elevated. These data are the first to demonstrate a defect at the pituitary level in the absence of FOXP3, which contributes to the infertility observed in mice with Foxp3 loss of function mutations.

Degarelix

Degarelix is a gonadotropin-releasing hormone (GnRH) receptor antagonist that, in common with GnRH receptor agonists (e.g. leuprolide, goserelin and triptorelin), is indicated for use as an androgen-deprivation therapy in patients with advanced prostate cancer. In 1-year, randomized, open-label, phase II or III trials in patients with all stages of prostate cancer, subcutaneous degarelix was associated with rapid, profound and sustained suppression of serum testosterone and prostate-specific antigen (PSA), without evidence of testosterone surges or microsurges. In the phase III trial, degarelix (240 mg initially followed by 80 mg every 28 days) was considered to be effective and noninferior to intramuscular leuprolide (7.5 mg every 28 days) with regard to inducing and maintaining suppression of serum testosterone to castrate levels (i.e. <or=0.5 ng/mL). Degarelix induced testosterone suppression more rapidly than leuprolide. Median serum testosterone levels of <or=0.5 ng/mL were achieved by day 3 in degarelix recipients, but not until day 28 in leuprolide recipients. PSA suppression was also more rapid with degarelix than with leuprolide, with significant between-group differences in serum PSA levels favouring degarelix at 14 and 28 days. Degarelix treatment for 1 year was generally well tolerated; the adverse events reported were mostly related to subcutaneous drug administration (i.e. injection-site reactions) and hormonal androgen deprivation (e.g. hot flushes).

Atrazine Inhibits Pulsatile Luteinizing Hormone Release Without Altering Pituitary Sensitivity to a Gonadotropin-Releasing Hormone Receptor Agonist in Female Wistar Rats1

Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-tri-azine] is one of the most commonly used herbicides in the United States. Atrazine has been shown to suppress luteinizing hormone (LH) release and can lead to a prolongation of the estrous cycle in the rat. The objectives of this study were to examine the effects of atrazine on normal tonic release of LH and to elucidate the site of action of atrazine in the hypothalamic-pituitary-gonadal axis. Episodic release of gonadotropin-releasing hormone (GnRH) and the corresponding release of LH from the anterior pituitary gland are required for normal reproductive function. To determine if atrazine affects pulsatile LH release, ovariectomized adult female Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle control for 4 days. On the final day of atrazine treatment, blood samples were obtained using an indwelling right atrial cannula. In the group receiving 200 mg/kg, there was a significant reduction in LH pulse frequency and a concomitant increase in pulse amplitude. To determine if the effects of atrazine on LH release were due to changes at the level of the pituitary, animals were passively immunized against endogenous GnRH, treated with atrazine, and challenged with a GnRH receptor agonist. Atrazine failed to alter pituitary sensitivity to the GnRH receptor agonist at any dose used. Taken together, these findings demonstrate that high doses of atrazine affect the GnRH pulse generator in the brain and not at the level of gonadotrophs in the pituitary.

Peptidomimetic GnRH receptor antagonists for the treatment of reproductive and proliferative diseases

Gonadotropin releasing hormone (GnRH), also known as luteinising hormone releasing hormone (LHRH), is a decapeptide released from the hypothalamus that binds to the G-protein-coupled GnRH receptor located on the pituitary gland. This receptor plays an important role in human reproduction, reproductive and proliferative diseases such as endometriosis, uterine fibroids, precocious puberty, prostate/breast/ovarian cancers and prostatic hyperplasia, conditions that can be potentially treated by GnRH receptor agonists or antagonists. Peptidic GnRH receptor agonists and antagonists have long been used for the treatment of several disorders, but the problems associated with peptides have encouraged the research and development of orally bioavailable peptidomimetic small-molecule GnRH receptor antagonists. This review summarises the patent literature during 2001 – 2005 on small-molecule GnRH receptor antagonists.

Administration of antisense oligonucleotides to Gα Q/11 reduces the severity of murine lupus

Our principle hypothesis is that the hypothalamic hormone, gonadotropin-releasing hormone (GnRH), is an immunostimulatory hormone and plays a pivotal role in the gender differences in immunity and/or autoimmunity. As a general rule, females display heightened immune responses and heightened endocrinological responsiveness to GnRH compared to males. We have previously demonstrated that GnRH receptor antagonists are effective in ameliorating murine lupus and that GnRH receptor agonists exacerbate murine lupus. GnRH exerts its actions via stimulatory G proteins, specifically via Gα s and the homologous G proteins Gα q and Gα 11 (referred to together as Gα q/11). We have previously demonstrated that females express higher levels of Gα q/11 mRNA and protein compared to males. We hypothesized that antisense inhibition of these specific G proteins would lead to a reduction in inflammatory cytokines and to an amelioration of disease in a mouse model of lupus. We randomized gonadectomized female (NZB × NZW) F1 hybrid mice to treatment with antisense oligonucleotides to Gα q/11 or to missense oligonucleotides. Administration of antisense oligonucleotides to Gα q/11 led to significant reductions in autoantibody levels, serum IgG levels, hematuria, and proteinuria compared to missense oligos. A trend toward prolonged survival was also noted. In vitro co-culture experiments demonstrated that antisense to Gα q/11 significantly inhibited IL–6 production compared to control.