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Pharmacological Characterization of a Novel Nonpeptide Antagonist of the Human Gonadotropin-Releasing Hormone Receptor, NBI-42902

Departments of Endocrinology (R.S.S., X.-J.L., Q.X., G.J.R., T.A.K.), Pharmacology (S.K.S.), Medicinal Chemistry (Y.-F.Z., C.C.), Peptide Chemistry (N.L.), Molecular Biology (W.Y., R.A.M.), and Preclinical Development (A.K.B., T.-K.C., H.P.B.), Neurocrine Biosciences Inc., San Diego, California 92130

Address all correspondence and requests for reprints to: Dr. R. Scott Struthers, Department of Endocrinology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, California 92130. E-mail: sstruthers{at}neurocrine.com.

	Abstract

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Suppression of the hypothalamic-pituitary-gonadal axis by peptides that act at the GnRH receptor has found widespread use in clinical practice for the management of sex-steroid-dependent diseases (such as prostate cancer and endometriosis) and reproductive disorders. Efforts to develop orally available GnRH receptor antagonists have led to the discovery of a novel, potent nonpeptide antagonist, NBI-42902, that suppresses serum LH concentrations in postmenopausal women after oral administration. Here we report the in vitro and in vivo pharmacological characterization of this compound. NBI-42902 is a potent inhibitor of peptide radioligand binding to the human GnRH receptor (K_i = 0.56 nM). Tritiated NBI-42902 binds with high affinity (K_d = 0.19 nM) to a single class of binding sites and can be displaced by a range of peptide and nonpeptide GnRH receptor ligands. In vitro experiments demonstrate that NBI-42902 is a potent functional, competitive antagonist of GnRH stimulated IP accumulation, Ca²⁺ flux, and ERK1/2 activation. It did not stimulate histamine release from rat peritoneal mast cells. Finally, it is effective in lowering serum LH in castrated male macaques after oral administration. Overall, these data provide a benchmark of pharmacological characteristics required for a nonpeptide GnRH antagonist to effectively suppress gonadotropins in humans and suggest that NBI-42902 may have clinical utility as an oral agent for suppression of the hypothalamic-pituitary-gonadal axis.

	Introduction

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THE GnRH RECEPTOR is a member of the G protein-coupled receptor superfamily with seven predicted membrane spanning helices. Activation of the receptor stimulates phospholipase C and phosphatidyl inositol turnover by coupling to the GTP binding proteins G_q and G₁₁ (1). Peptide GnRH agonists that down-regulate pituitary gonadotrophic cells have found widespread clinical use for "medical gonadectomy", where ablation of gonadal steroids has proven effective in treating precocious puberty, endometriosis, prostate cancer, uterine fibroids, and breast cancer (2). In addition, GnRH agonists have found important utility in assisted reproductive therapy (3). More recently, peptide GnRH antagonists have been developed (4, 5) to overcome some of the limitations of the agonists. Although agonists require 1–3 wk to reduce estrogen levels (6), antagonists result in an immediate reduction in gonadal steroid levels (7). Furthermore, in patients with prostate cancer, studies comparing the peptide antagonist, abarelix, with leuprolide demonstrated avoidance of an initial "flare" in testosterone and a more rapid reduction in circulating testosterone to castrate levels (8, 9). Similarly, studies with peptide antagonists show maximum reductions in uterine fibroid volume in 2–4 wk (10, 11), which is more rapid than typically observed with agonist therapy. This immediacy of action has also resulted in a smaller number of injections and reduction in the duration of treatment during in vitro fertilization protocols (5). However, the requirement for parenteral administration, often in the form of long-acting depots, of these peptidic drugs, is suboptimal for many indications.

Nonpeptide GnRH antagonists potentially offer several advantages over peptidic GnRH analogs. Of course, an oral route of administration offers improved convenience and perhaps improved patient acceptance for many. Furthermore, injection site reactions observed in peptide depots are avoided and dosing can be rapidly discontinued if necessary—a clinical management option not available with long-acting injectable depots. Perhaps more importantly, it may be possible to vary the level of pituitary suppression by varying dosage and, therefore, allow titration of circulating estrogen levels to a therapeutic window (12) with the hope of enabling long-term treatment without the need for "add-back" therapy. These potential benefits have prompted a number of groups to attempt to develop nonpeptide, orally available GnRH antagonists (see for example Refs. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22).

Since the first publication of an orally active nonpeptide GnRH antagonist (14), many additional examples have emerged, and a few have reported more detailed biological information. The macrolide analog, A-198401 was shown to compete for peptide binding to rat and human GnRH receptors, inhibit GnRH-stimulated LH release in cultured rat anterior pituitary cells, and inhibit LH in castrate male rats and testosterone in intact male rats, without stimulating histamine release (16). Similarly, a low molecular weight furamide derivative was shown to compete for peptide binding to human, rat, and mouse receptors, inhibit GnRH-stimulated inositol phosphate (IP) accumulation, suppress LH in castrate rats, and suppress testosterone in intact male rats (18). The thienopyrimidine derivative, TAK-013, has been administered chronically to regularly cycling female cynomolgus macaques and shown to suppress reversibly the pituitary-ovarian axis (23).

Despite these extensive efforts, as of this writing, only three compounds have been reported at scientific meetings as having been evaluated in humans (24, 25, 26). NBI-42902 (Fig. 1) (27) is the first for which human data are reported in the peer reviewed literature (28). After oral administration of 5–200 mg of this compound to postmenopausal women, circulating LH levels were suppressed in a dose-dependent manner. LH levels recover rapidly after clearance of the compound from the circulation, and a clear pharmacokinetic/pharmacodynamic relationship can be observed. Here we report the detailed pharmacological characterization of this compound in vitro and in castrate macaques. We show that NBI-42902 is a high-affinity, competitive antagonist of the human GnRH receptor. It inhibits GnRH-stimulated signaling and does not stimulate the release of histamine. Furthermore, the compound is able to reduce circulating LH concentrations after oral administration to castrated macaques. These data, together with related chemical (27, 29, 30) and clinical data on this compound (28), provide an increasingly comprehensive picture of an early member of what may eventually be an important new class of agents for control of the hypothalamic-pituitary-gonadal axis.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Chemical structure of NBI-42902, 1-(2,6-difluorobenzyl)-3-[(2R)-amino-2-phenethyl]-5-(2-fluoro-3-methoxyphenyl)-6-methyluracil. The methoxy group (*) was replaced by a fully tritiated methoxy group to prepare radiolabeled material for binding assays.

	Materials and Methods

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Preparation of [³H]NBI-42902
[³H]NBI-42902 was prepared by replacing the three hydrogens on the methoxy group of NBI-42902 with tritium (Fig. 1). NBI-42902 was treated with boron tribromide, followed by di t-butyl dicarbonate to yield the corresponding boc-protected phenol, which was then treated with tritiated iodomethane in the presence of potassium carbonate in DMF, followed by deprotection of Boc group with trifluoroacetic acid to yield the desired product, which was purified by HPLC. The tritiation of NBI-42902 was performed under contract by American Radiolabeled Chemical Inc. (St. Louis, MO). The final material had a specific activity of 80 Ci/mmol, with a compound purity greater than 99% as determined analytic HPLC and was stored at –20 C until use.

Cloning and expression of GnRH receptors
Total RNA was prepared from rabbit pituitary gland using RNeasy (Qiagen, Valencia, CA). RT was performed with the 3' RACE-Tn primer (5'-GCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3') and ThermalScript-RT-PCR kit (Invitrogen Life Technologies). The gene was initially cloned in three segments. The middle portion of the rabbit GnRH receptor was amplified with two primers designed based on the comparison of other GnRH receptors, S-F-1 (5'-TCTGGAAAGATCCGAGTGAC-3') and S-2-R (5'-GGCAGCTGAAGGTGAAAAAGTT-3'). The 5' portion of the receptor was amplified with the GeneRacer 5' primer (5'-CGACTGGAGCACGAGGACACTGA-3') (Invitrogen Life Technologies) plus a gene-specific rabbit 3' primer (5'-ATGACAATCAGAGTCTCCAACAGG-3'). The 3' portion of the gene was amplified with GeneRacer 3' primer (5'-GCTGTCAACGATACGCTACGTAACG-3') (Invitrogen Life Technologies) and a gene-specific rabbit 5' primer (5'-TCCACAATGGTGGCAACAAGCC-3'). The three parts were assembled into the full-length clone by PCR and cloned into pcDNA3, and the sequence was verified by automated sequencing using an ABI 3100 Genetic Analyzer from Applied Biosystems (Foster City, CA) (GenBank accession no. AY781779).

cDNAs of human, rhesus macaque, rabbit, dog, and rat GnRH receptors were cloned into pcDNA3.1 (+) (Invitrogen Life Technologies). Full-length sequences of all receptors were confirmed by DNA sequencing. HEK 293, CHO, or rat basophilic leukemia (RBL) cells were stably transfected with human, rat, or macaque GnRH receptors, and high expressing single cell clones (B_max 0.4 pmol/mg membrane protein) were isolated and maintained in DMEM with following supplements: 10 mM HEPES; 2 mM L-glutamine; 1 mM sodium pyruvate; 50 U/ml penicillin, 50 µg/ml streptomycin; 10% heat-inactivated FBS and 200 µg/ml geneticin (G-418-Sulfate). Nonessential amino acids (0.1 mM) (Irvine Scientific, Santa Ana, CA) were included in the RBL cell media.

In general, initial peptide radioligand binding assays were carried out using membranes from stably transfected HEK 293 cells. RBL stable clones were found to more consistently express high levels of GnRH receptor and, therefore, were used for subsequent binding studies with tritiated NBI-42902, as well as Ca²⁺ flux and IP accumulation assays. Transiently transfected Cos-7 cells were used for preparation of membranes containing GnRH receptors from multiple species (as well as analysis of mutant receptors in other studies) because of the convenience for rapidly analyzing multiple receptors. Stably transfected CHO cells were used for ERK1/2 stimulation assays because of superior signal/noise characteristics in this assay.

Membrane preparation
HEK293 cells stably transfected with the human GnRH receptor were grown for 2 d after achieving confluence then harvested by striking tissue culture flasks against a firm surface. Cells were collected by centrifugation at 1000 x g for 5 min. Cell pellets were resuspended in 5% sucrose and homogenized using a polytron homogenizer for two 15-sec homogenization steps. Cell homogenates were then centrifuged for 5 min at 3000 x g to remove nuclei and the supernatant was subsequently centrifuged for 30 min at 44,000 x g to collect the membrane fraction. The membrane pellet was resuspended in GnRH binding buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, and 0.1% BSA) and aliquots were snap-frozen immediately in liquid nitrogen and stored at –80 C. Protein content of the membrane suspension was determined using the Bio-Rad protein assay kit (Bio-Rad).

RBL cells stably transfected with the human GnRH receptor were grown to 80% confluency before harvesting. The cells were incubated at 37 C for 10 min in 0.5 mM EDTA/PBS (Ca²⁺, Mg²⁺ free), and dislodged from the plate by gentle rapping of the flasks. Cells were collected and pelleted by centrifugation at 1000 x g for 5 min. Cell pellets were resuspended in buffer (Dulbecco’s PBS supplemented with 10 mM MgCl₂, 2 mM EGTA, pH 7.4), and cell lysis was the performed using a pressure cell and applying N₂ at a pressure of 900 for 30 min at 4 C. Unbroken cells and larger debris were removed by centrifugation at 1200 x g for 10 min at 4 C. The cell membrane supernatant was then centrifuged at 45,000 x g, and the resulting membrane pellet was resuspended in assay buffer and homogenized on ice using a tissue homogenizer. Protein concentrations were determined using the Coomassie Plus Protein Reagent kit. Membranes were aliquoted and stored at –80 C until ready for use.

COS-7 cells transiently transfected with GnRH receptors from different species (human, macaque, dog, rabbit, rat) were prepared by bulk electroporation. COS-7 cells were obtained from American Type Cell Culture (Manassas, VA) and were maintained in DMEM (MediaTech Inc., Herndon, VA) containing 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin. COS-7 cells were seeded in 500 cm² tissue culture plates and grown to confluency before cell transfection. A total of 5 x 10⁷ cells were transfected with 50 µg of the appropriate GnRH receptor DNA construct by electroporation in a BTX ElectroCell Manipulator ECM 600 (Fisher Scientific, Pittsburgh, PA) using the following settings: 1000 µF capacitance, 48 resistance, and 300 V/cm charging voltage. Transfected cells were cultured for 36–48 h before membrane preparation. Transiently transfected COS-7 cells were harvested, washed, and resuspended in membrane buffer (20 mM HEPES, pH 7.2, 6 mM MgCl₂, 1 mM EDTA). Cells were centrifuged, and the cell pellets were resuspended in a small volume of membrane buffer. Cells were lysed by release of pressure after incubation at 900 for 30 min at 4 C in a nitrogen chamber. The homogenate was centrifuged at 1000 x g for 10 min at 4 C to remove nuclei and cellular debris. Membranes were collected from the supernatant by centrifugation at 44,000 x g for 45 min at 4 C. Membranes were resuspended in membrane buffer at a concentration of 1 mg/ml, quick-frozen in liquid nitrogen, and stored at –80 C until used.

Radioligand binding assays
Radioligand binding displacement assays using the peptide radioligands were performed in buffer containing 10 mM HEPES, 150 mM NaCl, and 0.1% BSA, pH = 7.5. Radioligand binding assays using [³H]NBI-42902 were run in buffer containing 50 mM Tris, 150 mM NaCl, 5 mM MgCl₂, 0.01% saponin, and 0.5 mM EDTA, pH 7.5. Radioligand displacement assays were performed by incubating radioligand [[¹²⁵I-Tyr⁵,DLeu⁶,NMeLeu⁷,Pro⁹-NEt]GnRH (0.1 nM), [His⁵,¹²⁵I-DTyr⁶]GnRH (0.2 nM) (31), or [³H]NBI-42902 (1 nM), unlabeled competitors at concentrations ranging from 0.3 pM to 10 µM, and membranes for 2 h at room temperature. A total of 10–20 µG protein per well was used from membrane preparations for human, monkey, and rabbit GnRH receptor. Five micrograms per well and 60 µg/well of membranes were used for rat and dog GnRH receptors, respectively. Binding assays were performed in either Millipore 96-well GF/C filtration plates [for [¹²⁵I-Tyr5,DLeu⁶,NMeLeu⁷,Pro⁹-NEt]GnRH assays] or in 96-well low binding plates, which were subsequently filtered onto GF/C Unifilters. Filters were pretreated with 0.5% polyethylenimine for 30 min before use. Reactions were terminated by rapid vacuum filtration, and the filters were washed twice with 250 µl ice cold PBS, pH 7.4 (0.01% Tween 20 was included in wash media for [His⁵,¹²⁵I-DTyr⁶]GnRH and [³H]NBI-42902 radioligands). The filters were dried, and the Millipore filters were monitored for radioactivity using a Cobra II -counter (PerkinElmer Life Sciences). For assays filtered onto the GF/C Unifilter plates, 50 µl scintillation fluid was added to each filter, and radioactivity was monitored using a TopCount NXT. For iodinated radioligands, total radioligand was monitored on a -counter, and for the tritiated radioligand, total radioligand was monitored using a PerkinElmer 1600TR liquid scintillation counter. Total radioligand bound did not exceed 10% of the total radioligand added, a level of depletion which does not appreciably affect the measurement of K_i. (32). Nonspecific binding did not exceed 2% of the total radioligand added in any of the displacement assays. Inhibition of radioligand binding was fit to one-site and two-site competition binding equations, and the best fit was determined using an F test. For all displacement binding experiments, a single-site binding model fit best (P < 0.05). The K_i values were calculated from the IC₅₀ values using the method of Cheng and Prusoff (33) and converted to a pK_i value (negative log of the K_i value). Data are presented as the mean pK_i ± SEM.

Ca²⁺ flux measurement
To determine the inhibition of GnRH-stimulated calcium flux in cells expressing the human GnRH receptor, a 96-well plate was seeded with RBL cells stably transfected with the human GnRH receptor at a density of 50,000 cells per well and allowed to attach overnight. Cells were loaded for 1 h at 37 C in the following medium: DMEM with 20 mM HEPES, 10% FBS, 2 µM Fluo-4, 0.02% pluronic acid, and 2.5 mM probenecid. Cells were washed four times with wash buffer (Hanks’ balanced salt, 20 mM HEPES, 2.5 mM probenecid) after loading, leaving 150 µl in the well after the last wash. GnRH was diluted in 0.1% BSA containing flurometric imaging plate reader (FLIPR) buffer (Hanks’ balanced salt, 20 mM HEPES) to a concentration of 20 nM and dispensed into a low protein binding 96-well plate. Various concentrations of antagonists were prepared in 0.1% BSA/FLIPR buffer in a third 96-well plate. Cell-, agonist-, and antagonist-containing plates were loaded into a FLIPR (FLIPR384 system; Molecular Devices, Sunnyvale, CA) for liquid handling and fluorescence measurements according to the manufacturer’s instructions. The instrument was programmed such that antagonist (50 µl at varying concentrations) was added to cell plates and preincubated for 1 min before addition of agonist (50 µl, or 4 nM final concentration of GnRH).

Measurement of [³H]IP production
The procedure was modified from published protocols (34). Briefly, RBL cells stably transfected with human GnRH receptors were seeded in 24-well plates at a density of 200,000 cells per well for 24 h. Cells were washed once with inositol-free medium containing 10% dialyzed FBS and then labeled with 1 µCi/ml [myo-³H]inositol. After 20–24 h, cells were washed with buffer (140 mM NaCl, 4 mM KCl, 20 mM HEPES, 8.3 mM glucose, 1 mM MgCl₂, 1 mM CaCl₂, and 0.1% BSA) and treated with native GnRH peptide in the same buffer with or without various concentrations of antagonist (NBI-42902) and 10 mM LiCl for 1 h at 37 C. Cells were extracted with 10 mM formic acid at 4 C for 30 min and loaded to the Dowex AG1-X8 column, washed, and eluted with 1 M ammonium formate and 0.1 M formic acid. The eluate was counted in a scintillation counter. Data from inositol phosphate assays were plotted using nonlinear least square regression by Prism program (GraphPad Software, San Diego, CA), from which dose ratio was also calculated. The Schild linear plot was generated from the dose ratios obtained in four independent experiments by linear regression; the X-intercept was used to determine the affinity of NBI-42902.

Activation of ERK1/2
CHO cells stably expressing GnRH receptor were serum-starved for 1 h, incubated for 5 min with various doses of NBI-42902, and stimulated with 1 nM GnRH for 5 min at 37 C. Cells were washed once with PBS and harvested directly into 2x SDS sample buffer. Cell extracts were sonicated, heated at 55 C for 5 min, and subjected to SDS-PAGE. Resolved proteins were transferred onto nitrocellulose membranes. The activated phosphorylated form of ERK1/2 was detected using an anti-phosphoMAPK p42/44 antibody (Cell Signaling Technology, Danvers, MA) diluted at 1:3000 in 1% nonfat dried milk in TBST (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.1% Tween 20). Total ERK1/2 was detected with the anti-ERK2 antibody (K23; Santa Cruz Biotechnology, Santa Cruz, CA). Chemiluminescent detection was performed with SuperSignal West Pico reagent (Pierce, Rockford, IL) and quantified on the VersaDoc3000 (Bio-Rad) imaging system. Dose-response data were plotted and analyzed with GraphPad Prism software.

Histamine release
Rat peritoneal mast cells were obtained in accordance with the current National Institutes of Health guidelines for the humane and ethical use of laboratory animals and animal welfare, and under an International Animal Care and Use Committee-approved protocol. This method has been previously described for the evaluation of mast cell histamine release by peptide GnRH antagonists (35). Briefly, six male Sprague Dawley rats 240–300 g were killed by CO₂ asphyxiation, and 40 ml of cold PIPES buffer (25 mM PIPES; 110 mM NaCl; 5mM KCl; 1 mg/ml glucose; 1 mg/ml BSA; and 20 U/ml heparin, pH 7.4) was injected into the peritoneal cavity and the abdomen was massaged gently. Peritoneal wash was recovered and stored on ice. Cells from the peritoneal wash were washed three times with 5 ml PIPES buffer, pooled, and purified on a Percoll gradient (36). For stimulation assays, approximately 2 x 10⁵ cells in 300 µl PIPES buffer were placed into a 1.5-ml Eppendorf tube, and test compound (100 µl) was added to the cell suspension. The tubes were incubated at 37 C for 15 min, and the reaction was stopped with 600 µl of ice-cold PIPES buffer. After centrifugation at 4 C, the histamine level in the supernatant was determined by histamine EIA kit from SPI-BIO (Cayman Chemical, Ann Arbor, MI) following the manufacturer’s instructions.

LH suppression in castrate macaques
This study in macaques was conducted in accordance with the current National Institutes of Health guidelines for the humane and ethical use of laboratory animals and animal welfare, and under an International Animal Care and Use Committee-approved protocol. A complete orchiectomy (both testes) was performed approximately 4 wk before the first dose on male cynomolgus monkeys approximately 3.7–6.5 yr of age (3.7–4.8 kg). Sexual maturity was verified by testicular volume and testosterone levels before surgery. Blood samples were collected weekly during the 4-wk postsurgery recovery period for measurement of testosterone, FSH, and LH to verify the rise in gonadotropins. NBI-42902 was administered to the stomach by nasogastric gavage or by iv infusion (over 15 min). Blood samples were collected before and after each dose for analysis of serum LH and plasma NBI-42902 concentrations. For the iv infusion dose, samples were collected at 0.25, 0.33, 0.5, 1, 1.5, 4, 8, and 24 h after the initiation of the infusion. Samples were collected at 0.25, 0.5, 1, 1.5, 2, 4, 8, and 24 h postdose for the oral doses. Bioactive LH concentrations in serum samples were measured at the Oregon Regional Primate Center (Beaverton, OR) using a previously reported mouse Leydig cell bioassay, which detects as little as 3 ng LH per milliliter using cynomolgus LH RP-1 as the reference preparation (37). Predose LH levels ranged from 91–525 ng/ml. Plasma NBI-42902 concentrations were determined by liquid chromatography/mass spectrometry using deuterated NBI-42902 as an internal standard.

	Results

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The structure of NBI-42902 is shown in Fig. 1. It is a substituted uracil (m.w. = 495) that has been optimized for high-affinity binding to the human GnRH receptor, as well as for pharmacokinetic properties (27). The ability of the compound to compete for [¹²⁵I-Tyr⁵,DLeu⁶,NMeLeu⁷,Pro⁹-NEt]GnRH binding to membrane preparations of the cloned human GnRH receptor expressed in HEK293 cells is shown in Fig. 2A. The apparent affinity in this assay (K_i = 0.56 nM; pK_i = 9.26 ± 0.003; mean ± SEM) is comparable to that of decapeptide antagonists such as cetrorelix (K_i = 0.25 nM; pK_i = 9.62 ± 0.006; mean ± SEM).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Binding affinity of NBI-42902. A, Inhibition of [125I-Tyr5,DLeu6,NMeLeu7,Pro9-NEt]GnRH binding to human GnRH receptors by increasing concentrations of NBI-42902 () or cetrorelix (). Ki values of NBI-42902 and cetrorelix were 0.56 nM (pKi = 9.26 ± 0.003) and 0.25 nM (pKi = 9.62 ± 0.006), respectively. Membrane fraction from HEK293 cells expressing the human GnRH receptor was prepared as described in Materials and Methods. Data shown are the mean ± SEM of three independent experiments. B, Saturation binding and Scatchard transformation (inset) of [3H]NBI-42902 to the human GnRH receptor. Radioligand saturation was measured as described in Materials and Methods. The radioligand concentration given on the x-axis is the free concentration, calculated by subtracting total binding from the total radioligand added. Kd and Bmax values were determined by fitting the data to a single-affinity state model and to a two-affinity state model, and the best fit was determined using an F test. Nonspecific binding was defined in the presence of 1 µM NBI-42902. Results are the mean of duplicate determinations. Data are representative of four independent experiments.

Competition binding experiments were performed to assess the ability of GnRH agonist and antagonist peptides and small molecules to compete for [³H]NBI-42902. These data were then compared with compound binding activity vs. the peptide radioligand [His⁵,¹²⁵I-DTyr⁶]GnRH. As shown in Fig. 3, the peptides and small molecules tested were all able to completely inhibit the binding of [³H]NBI-42902 and [His^5,125I-DTyr⁶]GnRH, consistent with a competitive interaction of the small molecules and peptides at the human GnRH receptor. The binding of all compounds tested best fit a single-affinity state model and the results are presented in Table 1. The small molecule antagonists NBI-42902 and compound II bound with similar affinities as the peptide antagonists cetrorelix and abarelix. However, the small molecule antagonists are more potent inhibitors of radioligand binding than the agonist peptides [His⁵,DTyr⁶]GnRH and GnRH. The pK_i values obtained for each of the small molecule and peptide competitors were not statistically different from each other with respect to the radioligand used, with the exception of the agonist peptides [His⁵,DTyr⁶]GnRH and GnRH which showed 4- and 14-fold lower potency compared with that measured with a peptide radioligand.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Displacement of [3H]NBI-42902 binding to human GnRH receptor by antagonist and agonist compounds. Competition binding experiments were conducted using [3H]NBI-42902 (1 nM) (A) and [His5,125I-DTyr6]GnRH (0.2 nM) (B) with membranes from RBL cells stably transfected with the hGnRH receptor. Ligands tested were NBI-42902 (), compound II (), abarelix (), cetrorelix (), leuprolide (), His5, D-Tyr6 GnRH (), and GnRH (). Data were analyzed using nonlinear least-squares curve fitting software (GraphPad Software Inc.). Curves were fit to single-site and two-site competition binding equations, and a one-site model provided the best fit in all cases (P value < 0.05). The data points are the mean of duplicate determinations, and are representative of at least three independent experiments. Mean pKi ± SEM values are presented in Table 1.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Inhibition of GnRH stimulated Ca2+ flux by NBI-42902 and cetrorelix. Various concentrations of NBI-42902 () and cetrorelix () were used to inhibit 4 nM GnRH-induced Ca2+ flux in RBL cells stably expressing human GnRH receptor. The cells were loaded with fluorescent dye fluo-4, and the compounds were preincubated with the cells for 1 min before agonist addition. The IC50 for NBI-42902 and cetrorelix are 3.6 nM (pIC50 = 8.48 ± 0.07) and 2.9 nM (pIC50= 8.61 ± 0.15), respectively. Data are the mean ± SEM of three separate experiments for each compound.

View larger version (23K): [in this window] [in a new window]   FIG. 5. NBI-42902 antagonism of IP accumulation in RBL cells stably expressing human or macaque GnRH receptors. A, GnRH dose response curves with the human receptor are shown in the absence of NBI-42902 () or in the presence of 3 x 10–9 M (), 1 x 10–8 M (), or 3 x 10–8 M () NBI-42902. Data shown are means of duplicate measurements in a representative experiment. B, Schild regression plot for NBI-42902 antagonism of the human receptor. The slope is 1.22 ± 0.14 and the pKB is 8.77 (KB = 1.7 nM). Dashed lines indicate the 95% confidence interval for the fitted regression line. Data shown are means ± SEM from four independent experiments. C, GnRH dose response curves with the macaque receptor are shown in the absence of NBI-42902 () or in the presence of 3 x 10–8 M (), 1 x 10–7 M (), 3 x 10–7 M (), or 1 x 10–6 M () NBI-42902. Data points are the mean of duplicate measurements in a representative experiment. D, Schild regression plot for NBI-42902 antagonism of the macaque receptor. The slope is 1.17 ± 0.05 and the pKB is 7.54 (KB = 28.6 nM). Dashed lines indicate the 95% confidence interval for the fitted regression line. Data shown are means ± SEM from three independent experiments.

View larger version (35K): [in this window] [in a new window]   FIG. 6. Inhibition of GnRH-stimulated ERK MAPK activation by NBI-42902. A, CHO-GnRHR cells were serum starved for at least 1 h, incubated for 5 min with various concentrations of NBI-42902, and then stimulated with 1 nM GnRH for 5 min. Representative phospho-ERK1/2 and total ERK immunoblots are shown. B, The levels of phospho-ERK1/2 were quantitated, normalized to the total ERK1/2 in each sample, and graphed as the percentage of the maximal GnRH-stimulated ERK1/2 in the absence of drug. The graph shown is the corresponding quantitation of three independent experiments. The calculated IC50 was 5.22 ± 0.53 nM.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Comparison of histamine release by NBI-42902 (100 µM), cetrorelix (50 µM), and abarelix (50 µM) from rat peritoneal mast cells. The mast cells were incubated with or without the compounds for 15 min, and the amount of histamine released in the supernatant was measured as described in Materials and Methods. The bars represent the mean ± SEM of three independent experiments, analyzed by repeated measures ANOVA using Dunnett’s multiple comparison test to control wells.

View larger version (20K): [in this window] [in a new window]   FIG. 8. Suppression of circulating, bioactive LH in castrate male macaques. Animals were castrated at least 1 month before administration of compound and had achieved high circulating LH levels. Time courses are shown with circulating bioactive LH levels expressed as a percentage of pretreatment LH levels for each individual animal. On the day of the study, they were administered NBI-42902 by nasogastric gavage at doses of 10 mg/kg (), n = 6; 40 mg/kg (), n = 3; 100 mg/kg (), n = 3; iv NBI-42902, 10 mg/kg (), n = 3; or iv vehicle (), n = 3.

View larger version (25K): [in this window] [in a new window]   FIG. 9. Plasma NBI-42902 () and serum LH () levels in castrate male macaques after administration of NBI-42902 at the indicated doses. Data shown are mean ± SEM from three animals.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Overall, NBI-42902 exhibits high affinity for the human GnRH receptor, comparable to peptide drugs such as cetrorelix. In assays of GnRH receptor function, such as IP production, calcium flux, and ERK1/2 phosphorylation, the compound is a potent antagonist of GnRH-stimulated responses. Schild analysis of the inhibition of GnRH-stimulated production of IP indicates that the compound is a potent surmountable, competitive antagonist. However, in vitro potency values for the compound appear to be somewhat dependent upon the details of the assay used. Binding affinity as measured by competition against the peptide radioligand [¹²⁵I-Tyr⁵,DLeu⁶,NMeLeu⁷,Pro⁹-NEt]GnRH is 0.56 nM, whereas direct binding using tritiated NBI-42902 indicates a slightly higher affinity (0.19 nM). Detailed analysis of receptor binding kinetics of [³H]NBI-42902 has subsequently indicated that the assay conditions used above may not have reached complete equilibrium and the kinetically determined disassociation constant (K_d = k_off/k_on) is even more potent still (0.029 nM) (39). Yet, Schild analysis of potency in the functional IP assay carried out with live cells at 37 C indicates a potency (K_b) of 1.7 nM. In the more rapid Ca²⁺ flux and ERK1/2 phosphorylation assays, IC₅₀ values of 3.6 and 5.22 nM were obtained using stimulatory concentrations of GnRH which are in the physiological range (1–4 nM). Furthermore, the observation that [His⁵,DTyr⁶]GnRH and GnRH are significantly less potent competitors of the binding of [³H]NBI-42902 than of [His⁵,¹²⁵I-DTyr⁶]GnRH binding is more difficult to interpret. Differences in the kinetic characteristics of the various assays may contribute to differences in measured potencies. Different peptide radioligands have previously been shown to exhibit different equilibration times in competition binding assays (31). This may contribute to differences in measured affinities in competition binding assays using [³H]NBI-42902 (39). Clearly, the extremely rapid assays such as Ca²⁺ flux and ERK1/2 phosphorylation may be more sensitive to antagonist association kinetics, whereas slower assays such as inhibition of IP accumulation may come closer to equilibrium conditions. Additionally, many of these differences may be attributable to differences in assay conditions such as temperature or buffers used. However, none of these in vitro assays closely mimics the physiological milieu at the extracellular surface of the pituitary gonadotroph, nor are these expressed receptor systems native gonadotrophs. Therefore, these results indicate that, although the compound is highly potent in all in vitro assays used, the actual potency per se is perhaps more meaningfully defined by in vivo pharmacokinetic/pharmacodynamic analysis as discussed below.

The ability of peptide and nonpeptides to compete for binding of radiolabeled peptide and nonpeptide ligands described above is consistent with the existence of a common binding pocket. We previously have demonstrated that species selectivity of several classes of nonpeptide GnRH antagonists is due to differences in receptor sequences of extracellular loops II and III, as well as in the amino terminus (38). The same residues (M24, S203, Q208, and L300) as well as several other residues in the transmembrane region (i.e. K121, Y284, Y290, and F313) are important for high-affinity binding of NBI-42902 (30). Peptide ligand binding has also been shown to require interactions with these same residues, as well as others nearby such as C14, K121, V197, W205, W206, Y283, Y284, Y290, W291, D302, H306, and F309 (1, 30), indicating that NBI-42902 uses a partially overlapping set of interactions for high-affinity binding. Thus, an overall picture of the GnRH receptor ligand binding pocket is emerging where multiple nonpeptide ligands can compete for a common peptide binding pocket, albeit using different sets of specific intermolecular interactions. These interactions can result in an affinity and functional antagonist potency described here, which is sufficient to achieve in vivo LH suppression in castrate macaques and postmenopausal women.

NBI-42902 does not activate the distal downstream effector ERK1/2 and, in fact, inhibits GnRH-stimulated ERK1/2 phosphorylation. Although this result perhaps is not surprising, some peptides that act as antagonists of G_q coupling surprisingly can act as agonists of other signaling pathways such as ERK1/2 (42). Therefore, although we have not evaluated the entire repertoire of signaling pathways previously shown to be able to be activated by the GnRH receptor, NBI-42902 appears to be an antagonist for G_q and whichever pathway is activating ERK1/2 in the cellular context of stable expression in CHO cells.

The development of peptide GnRH antagonists has been hindered by problems associated with histamine release. This peptide-stimulated release of histamine is not believed to be mediated by GnRH receptors (35, 43), but rather is associated with multiple classes of basic peptides. Therefore, although it is unlikely that nonpeptide GnRH antagonists would have a similar effect, we examined the ability of the compound to stimulate histamine release from peritoneal mast cells. Even at very high concentrations (100 µM), NBI-42902 did not stimulate histamine release, whereas the peptide antagonists abarelix and cetrorelix showed significant release at even lower concentrations (50 µM). Furthermore, because this compound is intended for oral administration, it does not present the persistent high local concentrations found at the injection site of peptide GnRH antagonists. Therefore, these data suggest the risk of the histamine-related adverse reactions previously observed for peptides is reduced for this nonpeptide compound.

To characterize the biological activity of NBI-42902 in vivo, its ability to suppress circulating LH in castrate male macaques was explored after oral and iv administration. Activity in rat models was not explored due to the weak affinity of the compound (IC₅₀ > 10 µM) for the rat GnRH receptor. Despite the in vitro data indicating reduced affinity for the macaque receptor compared with the human receptor, the compound was able to suppress LH in a dose-dependent, reversible manner in this species. Although the present data were insufficient for quantitative pharmacokinetic/pharmacodynamic modeling, qualitative inspection of simultaneous concentration-time profiles for NBI-42902 and LH suppression suggested that concentrations of the antagonist of approximately 10–50 ng/ml (20–100 nM) were required to maintain pituitary suppression in the macaque. This value is consistent with the potency determined in the competition binding and functional inhibition assays for the monkey GnRH receptor. However, this circulating concentration in the monkey is comparable to the plasma concentrations required to suppress LH in postmenopausal women (28). This is somewhat surprising given the approximately 10-fold reduced affinity and 17-fold reduced functional potency in the IP assay of the compound for the monkey receptor compared with the human. Of course, a wide range of additional physiological differences between the state of the HPG axis in postmenopausal women and castrate macaques may also contribute to the final in vivo potency of a GnRH antagonist in addition to the compound’s intrinsic effects on the GnRH receptor.

Thus, the present data illustrates the inherent difficulties in quantitatively predicting pharmacological effects in humans from in vitro and nonhuman in vivo data. Nonetheless, the overall qualitative view of the compound from the data presented here is that of a high-affinity functional antagonist of the GnRH receptor that has substantial efficacy in a nonhuman primate model. This conclusion was subsequently borne out by human clinical data. Thus, the preclinical pharmacological data presented in this study provides a benchmark of the pharmacological characteristics of a nonpeptide GnRH antagonist required for efficacy in human gonadotropin suppression. Because of oral bioavailability, lack of histamine releasing activity, and conveniently adjustable level of pituitary suppression, nonpeptide GnRH antagonists may provide novel opportunities for modulation of the hypothalamic-pituitary-gonadal endocrine axis in the treatment of sex-hormone-related diseases.

	Acknowledgments

 
We thank Drs. Bruce Campbell, Paul Conlon, Paul Crowe, and Sam Hoare for helpful advice and discussions; Ms. Robin Lachappell and Ms. Julie K. Meyer for coordinating the primate studies at Sierra Biomedical Inc.; Dr. David Hess (Oregon Regional Primate Center) for performing LH bioassays; and Ms. Jane Wallace for assistance in preparation of this manuscript.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants 1-R43-HD38625-01 and 2-R44-HD38625-02.

Disclosure Summary: All authors are or were employed by and have or had equity interests in Neurocrine Biosciences.

First Published Online November 9, 2006

Abbreviations: FBS, Fetal bovine serum; FLIPR, flurometric imaging plate reader; IP, inositol phosphate; RBL, rat basophilic leukemia.

Received September 6, 2006.

Accepted for publication November 2, 2006.

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