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.     

GnRH agonists and antagonists. Current clinical status

Gonadotrophin-releasing hormone (GnRH) analogues offer a novel approach for the non-steroidal manipulation of the reproductive endocrine axis. GnRH agonists are now effectively employed in the management of precocious puberty, prostate and breast cancer, endometriosis, uterine leiomyoma, polycystic ovarian disease, and various other disorders. Unfortunately, contraceptive applications of GnRH agonists have been disappointing. The availability of slow release depot formulations of GnRH agonists, and the development of GnRH antagonists may further optimise and extend the clinical application of these compounds.     

Design and synthesis of potent hexapeptide and heptapeptide gonadotropin-releasing hormone antagonists by truncation of a decapeptide analogue sequence

A novel strategy for designing reduced-size analogues of the decapeptide gonadotropin-releasing hormone (GnRH) was developed. As opposed to previous attempts to delete residues from either of the peptide's termini, our approach is based upon the known importance of both C- and N-terminals of GnRH analogues for receptor recognition, whereas the central part of the molecule is replaced by a short spacer. The present truncation strategy was successful for generation of reduced-size hexapeptide and heptapeptide antagonists possessing potent antagonistic capacity. The same methodology was not suitable for the generation of reduced-size agonists, suggesting different conformational characteristics for GnRH agonists and antagonists. A heptapeptide antagonist designed by this method was shown to inhibit serum levels of luteinizing hormone in castrated rats in vivo. Structure-activity studies suggested that the structural preferences for GnRH receptor recognition are similar to those reported for decapeptide antagonists. Our studies resulted in a heptapeptide GnRH antagonist (Ac-D-Nal2-D-Cpa-D-Pal-Gly-Arg-Pro-D-Ala-NH2) with high receptor binding affinity (IC50 = 7 nM), as compared to that of GnRH itself (IC50 = 2 nM). The highest affinity of a hexapeptide antagonist that we have synthesized was somewhat lower (IC50 = 45 nM).     

Challenges and opportunities of trapping ligands

Because gonadotropin-releasing hormone (GnRH) analogs constitute an important class of therapeutics for various reproductive and hormone-dependent disorders, many novel compounds have been discovered and studied. Several orally active nonpeptide GnRH antagonists have recently gained increased attention. In the study published in this issue of Molecular Pharmacology, Kohout et al. (p. 238) used small-molecule TAK-013 (sufugolix; developed previously by Takeda Chemical Industries) as a tool to elucidate the mechanism of its insurmountable antagonism. On the basis of receptor mutagenesis combined with molecular modeling, the authors hypothesized that certain amino acid sequences uniquely present in the human GnRH receptor amino terminus and extracellular loop 2 may form a "trap door" retarding dissociation of TAK-013. Such a trapping mechanism could be both ligand- and receptor species-specific. Although analogous models were previously proposed for other G protein-coupled receptors, the study by Kohout et al. (2007) provides an important advance in the GnRH antagonists field and an illustration of the fact that preclinical studies using animal models with nonhuman receptors may have very limited value in predicting drug efficacy in human disease. There are many examples showing that high-affinity protein, peptide, or nonpeptide agonists or antagonists have also enhanced clinical efficacy. However, there are also numerous studies indicating that very high receptor binding affinity is not a guarantee of drug efficacy and that other factors, including pharmacokinetic profile, ligand-induced receptor desensitization, and "trafficking," are critical in design and development of optimal drugs.     

Recent advances in small molecule gonadotrophin-releasing hormone receptor antagonists

Targeting the gonadotrophin-releasing hormone (GnRH) receptor for treatment of a variety of reproductive hormone-dependent diseases has yielded great success so far, with the use of peptide agonists. However, based on the current understanding of GnRH receptor biology, peptide agonists inevitably cause some side effects, such as the ‘flare’ effect and delayed action. By contrast, peptide or small molecule GnRH receptor antagonists should be devoid of the above-mentioned side effects. Furthermore, a small molecule antagonist is more preferable than a peptide antagonist because it also provides the possibility of oral administration, therefore allowing dose flexibility and better patient acceptance. Thus, it is not surprising to see that many patent applications on small molecule GnRH receptor antagonists have emerged over the last 10 years. This review summarises the patent applications of small molecule human GnRH receptor antagonists from 1994 to August 2003. If available, the biological data associated with patent structures are also cited from publications.     

Progress towards the development of non-peptide orally-active gonadotropin-releasing hormone (GnRH) antagonists: therapeutic implications

Gonadotropin-releasing hormone (GnRH) is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2) which is produced from a precursor polypeptide in hypothalamic neurons and secreted in a pulsatile fashion to stimulate the secretion of LH and FSH via its interaction with a cognate receptor on gonadotropes. Low doses of the native peptide delivered in a pulsatile manner to mimic that found in the hypothalamic portal vessels restore fertility in hypogonadal patients, and are also effective in treating cryptorchidism and delayed puberty. Administration of high doses of GnRH, or agonist analogues, causes desensitization of the gonadotrope with consequent decline in gonadal gametogenesis and steroid and peptide hormone synthesis. This phenomenon finds extensive therapeutic application in clinical medicine in a wide spectrum of disease (Table 1). In addition, GnRH analogues have promise as new generation male and female contraceptives in conjunction with steroid hormone replacement. GnRH antagonists inhibit the reproductive system through competition with endogenous GnRH for the receptor and, in view of their rapid effects, are being increasingly used for the above mentioned applications. The peptide agonists and antagonists currently available require parenteral administration, typically in the form of long-acting depots. A new generation of non-peptide GnRH antagonists are beginning to emerge which should allow oral administration and, therefore, may provide greater flexibility of dosing, lower costs and increased patient acceptance.     

Endometriosis 1990. Current treatment approaches

Endometriosis is an extremely common gynaecological disease, affecting between 1 and 5% of women of reproductive age. Women with endometriosis typically present for medical care with one of more of the following problems: pelvic pain, infertility, or a large adnexal mass (an endometrioma). The primary treatment for an endometrioma is surgical. However, long term postoperative hormone therapy may be necessary to prevent new endometriomas from developing. There is no evidence that hormonal therapy of endometriosis will improve fecundability in women with endometriosis and infertility. Pelvic pain due to endometriosis can be successfully treated with hormonal agents in the majority of patients. Four basic hormonal regimens are currently available for the treatment of endometriosis: (a) danazol; (b) gonadotrophin-releasing hormone (GnRH) [luteinising hormone-releasing hormone (LHRH); gonadorelin] agonists; (c) progesterones (progestins); and (d) combined estrogens and progesterones. Randomised, controlled, clinical trials suggest that danazol and the GnRH agonists are equally effective in the treatment of endometriosis. However, the side effects caused by danazol and the GnRH agonists are markedly different. Danazol produces androgenic side effects including weight gain, hirsutism, acne, oily skin and deepening of the voice. GnRH agonists produce side effects due to hypoestrogenism, including hot flushes, osteoporosis and dry vagina. The ideal drug regimen for the treatment of endometriosis remains to be developed.     

Hexapeptide and cyclic pentapeptide endothelin antagonists directly activate pituitary gonadotropin-releasing hormone receptors

In the course of our studies toward the development of novel analogs of the decapeptide gonadotropin releasing hormone (GnRH), we have examined a hexapeptide that is an antagonist of endothelin (ET). It was found that this peptide, Ac-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide 1), binds specifically to the pituitary GnRH receptor. Moreover, peptide 1 exhibits a GnRH agonistic activity (i.e., it induces luteinizing hormone release from rat pituitary). This activity is mediated directly by the GnRH receptor and is suppressed by a GnRH antagonist. Removal of the acetyl group of peptide 1 results in a hexapeptide (peptide 2) with binding properties similar to those of GnRH but with a diminished affinity toward the ET receptor. Several other ET antagonists were screened for a potential interaction with the GnRH receptor. Two of these, the hexapeptide PD145065 and the cyclic pentapeptide BQ-123, expressed GnRH agonistic activity at micromolar concentrations in vitro. BQ-123, previously approved for trials on humans as an ET antagonist, is demonstrated to act in vivo as a GnRH agonist, in a dose that was demonstrated previously as the minimal required dose for significant ET antagonism. The GnRH agonistic activity of ET antagonists may therefore result in interference with the physiological control of the reproductive system. Such effects may be most severe when ET antagonists are used chronically. Thus, the major practical message of this study is the need to circumvent the potential side effects of ET antagonist-based drugs.     

Gonadotropin-releasing hormone antagonists for assisted reproductive techniques: are there clinical differences between agents?

Gonadotropin-releasing hormone (GnRH) antagonists have been tested extensively in ovarian stimulation protocols for assisted reproductive techniques (ART). GnRH antagonists immediately and rapidly inhibit gonadotropin release by the anterior pituitary gland by competitive blockage of the GnRH receptor, preventing and interrupting luteinising hormone surges in controlled ovarian hyperstimulation for infertility treatment. A review of the available literature on GnRH antagonists for ART is presented, focusing on the pharmacological and clinical properties of the two compounds available on the market, cetrorelix and ganirelix. Both cetrorelix and ganirelix are well tolerated and effective drugs for controlled ovarian hyperstimulation and are of comparable value for infertility treatment. Cetrorelix is available as a 0.25mg preparation for daily injections and as a 3mg intermediate depot preparation. Ganirelix is available as a 0.25mg preparation for daily injections.Currently, two treatment protocols are used in clinical practice: the GnRH antagonist multiple-dose protocol and the GnRH antagonist single-dose protocol. Both protocols are effective and well tolerated. Cetrorelix and ganirelix have not yet been directly compared in a clinical trial; nor have the single-dose and the multiple-dose approaches been compared in a randomised, controlled trial. Data to compare these compounds in clinical terms can be extrapolated only from results of phase II dose-finding studies and phase III studies comparing GnRH agonist cycles with GnRH antagonists in single- and multiple-dose protocols. Therefore, all conclusions on clinical differences between cetrorelix and ganirelix should remain tentative, as they are based on a limited amount of available data.Randomised, controlled trials comparing cetrorelix and ganirelix are warranted to further evaluate benefits and drawbacks of individual GnRH antagonists. Furthermore, more data are needed to determine the efficacy and safety of cetrorelix and ganirelix in established treatment protocols in patients other than those included in clinical trials investigating new drugs, such as "poor responders", patients with polycystic ovaries, patients with a history of allergy or overweight patients.     

Emerging drugs in assisted reproduction

Infertility affects approximately 15% of couples of reproductive age. In assisted reproductive technology (ART), medications play a crucial role in stimulating ovaries to produce several oocytes and prepare the endometrium to be receptive after replacing one or more embryos into the uterine cavity. The availability of recombinant human follicle stimulating hormone, luteinising hormone and human chorionic gonadotrophin; of gonadotrophin-releasing hormone (GnRH) agonists and antagonists; and of luteal supplementation with progesterone have allowed the tailoring of several stimulation schemes, which have enhanced the pregnancy outcome after ART treatment. However, the remaining risk of ovarian hyperstimulation syndrome, the still low implantation rates, the unacceptably high rates of multiple pregnancies and the daily parenteral administration of medications do not constitute the features of a patient-friendly procedure. Therefore, a number of molecules with gonadotrophin-like activity, inhibition of GnRH receptor ability, or endometrium receptivity enhancement properties are currently under active investigation. Orally bioactive therapeutic preparations, in particular, may revolutionize in vitro fertilisation (IVF) treatment in the near future. Nevertheless, the implementation of mild ovarian stimulation protocols with single embryo transfer policy and further development of oocyte in vitro maturation techniques may lead to a less drug orientated IVF treatment.     

Peptide ligands for the melanin-concentrating hormone (MCH) receptor 1

The melanin-concentrating hormone receptor 1 (MCH-1R) has been recognized as a receptor which mediates effects of the endogenous melanin-concentrating hormone (MCH) on appetite and body weight gain in rodents. In the last several years, a number of hMCH analogs have been designed which were potent and selective ligands for hMCH-1R. These peptidic agonists and antagonists have served as research tools in animal studies that showed a key role of the MCH-1R in the development of obesity and proved that MCH-1R antagonism can produce anti-obesity effects in rodents.     

Non peptidic urotensin II antagonists: perspectives for a new class of drugs

Urotensin II (U-II) is a cyclic peptide isolated from a fish. Subsequently, human U-II and its receptor were identified. In rat thoracic aorta U-II triggers powerful vasoconstrictor activity. However, the constrictor response to U-II appears to be variable and highly dependent on the vascular bed examined. Vasoconstriction is not its only effect; U-II and its receptor have been demonstrated in the central nervous system, where U-II induces a cardiovascular, behavioural, motor and endocrine response and in the kidney, where it seems to influence renal hemodynamics but also salt and water excretion, in rat pancreas where it inhibits insulin secretion, in the heart where it seems to play a role in cardiac hypertrophy and fibrosis. In humans high plasma or urine levels of U-II have been described in some pathologic conditions. Peptidic and non peptidic UT receptor antagonists have been synthesized and their effects have been evaluated particularly in animal models of diabetes and heart failure. After promising results in animal models, palosuran, a non peptidic U-II antagonist has been administered also in diabetic patients to evaluate its potential nephroprotective activity. This review presents the data available on the U-II system and its role in physiological and pathological conditions, together with data regarding palosuran and other non peptidic and peptidic U-II antagonists.     

Emerging drugs in assisted reproduction

Infertility affects ～ 15% of couples of reproductive age. In assisted reproductive technology (ART), medications play a crucial role in stimulating ovaries to produce several oocytes and prepare the endometrium to be receptive after replacing one or more embryos into the uterine cavity. The availability of recombinant human follicle stimulating hormone, luteinising hormone and human chorionic gonadotrophin; of gonadotrophin-releasing hormone (GnRH) agonists and antagonists; and of luteal supplementation with progesterone have allowed the tailoring of several stimulation schemes, which have enhanced the pregnancy outcome after ART treatment. However, the remaining risk of ovarian hyperstimulation syndrome, the still low implantation rates, the unacceptably high rates of multiple pregnancies and the daily parenteral administration of medications do not constitute the features of a patient-friendly procedure. Therefore, a number of molecules with gonadotrophin-like activity, inhibition of GnRH receptor ability, or endometrium receptivity enhancement properties are currently under active investigation. Orally bioactive therapeutic preparations, in particular, may revolutionise in vitro fertilisation (IVF) treatment in the near future. Nevertheless, the implementation of mild ovarian stimulation protocols with single embryo transfer policy and further development of oocyte in vitro maturation techniques may lead to a less drug orientated IVF treatment.     

New developments in the use of peptide gonadotropin-releasing hormone antagonists versus agonists

Gonadotropin-releasing hormone (GnRH) stimulates the pituitary secretion of both luteinising hormone (LH) and follicle-stimulating hormone (FSH), and thus controls the hormonal and reproductive functions of the gonads. The blockade of the effects of GnRH may be sought for a variety of reasons; for example, to control premature LH surges and to reduce the cancellation rate with the aim of improving the pregnancy rate per treatment cycle or in the treatment of sex hormone-dependent disorders. Selective blockade of LH/FSH secretion and subsequent chemical castration have previously been achieved by desensitising the pituitary to continuously administered GnRH or by giving long-acting GnRH agonists. GnRH analogues are indicated for clinical situations in which the suppression of endogenous gonadotropins (precocious puberty, contraception and controlled ovarian hyperstimulation) or sexual steroids (endometriosis, prostate hyperplasia, cancer and uterine fibroids) is desired. The immediate suppression of the pituitary that is achieved by GnRH antagonists without an initial stimulatory effect is the main advantage of these compounds over the agonists. GnRH antagonists have been developed for clinical use with acceptable pharmacokinetic, safety and commercial profiles. In assisted reproduction, these compounds seem to be as effective as established therapy, but with shorter treatment times, less use of gonadotropic hormones, improved patient acceptance, and fewer follicles and oocytes. All of the current indications for GnRH agonist desensitisation may prove to be indications for a GnRH antagonist, including endometriosis, leiomyoma and breast cancer in women, benign prostatic hypertrophy and prostatic carcinoma in men, and central precocious puberty in children. However, the best clinical evidence has been in assisted reproduction and prostate cancer.     

Growth hormone secretagogue receptor antagonists

The patent claims peptidic/nonpeptidic inhibitors of the ghrelin receptor, the growth hormone secretagogue receptor (GHSR) 1A. Among these compounds, it was disclosed that the addition in some compounds of a GlyMetAla tripeptide at the N-terminus of the ghrelin peptide agonists converts them into ghrelin receptor antagonists. One of these peptides, among the few that have been studied in vivo, was shown to be able to reduce food intake and body weight gain in rats.     

New developments in the use of peptide gonadotropin-releasing hormone antagonists versus agonists

Gonadotropin-releasing hormone (GnRH) stimulates the pituitary secretion of both luteinising hormone (LH) and follicle-stimulating hormone (FSH), and thus controls the hormonal and reproductive functions of the gonads. The blockade of the effects of GnRH may be sought for a variety of reasons; for example, to control premature LH surges and to reduce the cancellation rate with the aim of improving the pregnancy rate per treatment cycle or in the treatment of sex hormone-dependent disorders. Selective blockade of LH/FSH secretion and subsequent chemical castration have previously been achieved by desensitising the pituitary to continuously administered GnRH or by giving long-acting GnRH agonists. GnRH analogues are indicated for clinical situations in which the suppression of endogenous gonadotropins (precocious puberty, contraception and controlled ovarian hyperstimulation) or sexual steroids (endometriosis, prostate hyperplasia, cancer and uterine fibroids) is desired. The immediate suppression of the pituitary that is achieved by GnRH antagonists without an initial stimulatory effect is the main advantage of these compounds over the agonists. GnRH antagonists have been developed for clinical use with acceptable pharmacokinetic, safety and commercial profiles. In assisted reproduction, these compounds seem to be as effective as established therapy, but with shorter treatment times, less use of gonadotropic hormones, improved patient acceptance, and fewer follicles and oocytes. All of the current indications for GnRH agonist desensitisation may prove to be indications for a GnRH antagonist, including endometriosis, leiomyoma and breast cancer in women, benign prostatic hypertrophy and prostatic carcinoma in men, and central precocious puberty in children. However, the best clinical evidence has been in assisted reproduction and prostate cancer.     

Growth hormone secretagogue receptor antagonists

The patent claims peptidic/nonpeptidic inhibitors of the ghrelin receptor, the growth hormone secretagogue receptor (GHSR) 1A. Among these compounds, it was disclosed that the addition in some compounds of a GlyMetAla tripeptide at the N-terminus of the ghrelin peptide agonists converts them into ghrelin receptor antagonists. One of these peptides, among the few that have been studied in vivo, was shown to be able to reduce food intake and body weight gain in rats.     

GnRH agonists and antagonists in cancer therapy

Gonadotropin releasing hormone (GnRH) is a hypothalamic decapeptide that binds to GnRH receptors on pituitary gonadotrope cells to modulate the synthesis and secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins in turn regulate gonadal steroidogenesis and gametogenesis. Chemical characterization and structure-activity analysis of GnRH variants containing systematic amino acid substitutions led to the discovery of GnRH superagonists and antagonists. These peptides are widely used for the treatment of clinical conditions in which modulation of or interference with sex hormone production is beneficial to prevent development or progression of benign conditions (e.g. endometriosis, uterine fibroids) or malignant tumors (e.g. breast, ovarian, endometrial and prostate carcinoma). When compared to native GnRH, GnRH superagonists have increased potency for the short-term release of gonadotropins. However, they show paradoxical action in that chronic treatment with superagonists results in inhibition of gonadotropin production as a result of desensitization of the gonadotropes and down regulation of its receptor. In contrast, GnRH antagonists produce a rapid and dose-dependent suppression of gonadotropin release by competitive blockade of the GnRH receptors without any initial stimulatory effect as seen with superagonists. In recent years, a search for peptidomimetic compounds to replace peptides as therapeutic agents has been undertaken to find compounds with higher affinity for the GnRH receptor but do not have the disadvantages of peptides. Such efforts have resulted in the identification and development of small-molecule non-peptide compounds that are sufficiently stable in vivo and possess favorable pharmacological parameters comparable to peptide antagonists. Some of these compounds are being tested in human volunteers and the preliminary results are very encouraging.     

Structure-activity studies of reduced-size gonadotropin-releasing hormone agonists derived from the sequence of an endothelin antagonist

We have previously determined that Ac-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide I), an endothelin antagonist, binds specifically (Ki = 1.9 microM) to the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Moreover, peptide I exhibits a GnRH agonistic activity, mediated directly by the GnRH receptor. We now report structure-activity studies of peptide I in respect to its interactions with the GnRH receptor. Our studies suggest that the bioactive conformation of peptide I, recognized by the GnRH receptor, is of a cyclic nature. Thus cyclic analogues of peptide I exhibit higher affinity to the GnRH receptor and increased agonistic potencies as compared to peptide I itself. A linear peptide, Ile-Ile-Trp-D-Trp-Leu-Asp, which presumably forms a similar cyclic conformation, was also shown to be a GnRH agonist. Intraperitoneal administration of Ac-Ile-Ile-Trp-D-Trp-Leu-Cys-OH (Ki = 0.32 microM), one of the cyclic hexapeptides that we have synthesized, to rats induces secretion of luteinizing hormone (LH) with a potency which is only 1 order of magnitude less than that of GnRH itself. Moreover, plasma levels of LH remained elevated for a longer period of time following the administration of the cyclic hexapeptide. This novel class of GnRH agonists may prove useful in the development of new therapeutics.     

Small molecule agonists and antagonists for the LH and FSH receptors

Luteinising hormone (LH) and follicle-stimulating hormone (FSH) play a critical role in human reproduction. LH and FSH are secreted from the pituitary and act on their respective G-protein-coupled receptors (GPCRs), LHR and FSHR, in the gonads to either promote follicular growth and differentiation in women or to stimulate the proper progression of spermatogenesis in men. LH and FSH are currently used in the clinic for the treatment of infertility. Small molecule agonists of LHR and FSHR have the potential to become oral therapeutics for infertility treatment, whereas small molecule antagonists of LHR and FSHR may find utility in oral contraception. Advances in molecular biology, high-throughput screening (HTS) and combinatorial chemistry have made significant contributions to the recent discovery of a variety of small molecule LHR and FSHR agonists and antagonists, some of which have shown highly promising efficacy in animal models of fertility control.