pH Dependence of a GPR4 Selective Antagonist Hampers Its Therapeutic Potential.

Inflammatory bowel disease (IBD) is characterized by chronic mucosal inflammation of the gastrointestinal tract and is associated with extracellular acidification of mucosal tissue. Several extracellular pH-sensing receptors, including G protein-coupled receptor 4 (GPR4), play an important role in the regulation of inflammatory and immune responses, and GPR4 deficiency has been shown to be protective in IBD animal models. To confirm the therapeutic potential of GPR4 antagonism in IBD, we tested Compound 13, a selective GPR4 antagonist, in the interleukin 10-/- mouse model of colitis. Despite good exposures and albeit there was a trend toward improvement for a few readouts, Compound 13 treatment did not improve colitis in this model, and there were no signs of target engagement. Interestingly, Compound 13 behaved as an "orthosteric" antagonist, i.e., its potency was pH dependent and mostly inactive at pH levels lower than 6.8 with preferential binding to the inactive conformation of GPR4. Mutagenesis studies confirmed Compound 13 likely binds to the conserved orthosteric binding site in G protein-coupled receptors, where a histidine sits in GPR4 likely preventing Compound 13 binding when protonated in acidic conditions. While the exact mucosal pH in the human disease and relevant IBD mice models is unknown, it is well established that the degree of acidosis is positively correlated with the degree of inflammation, suggesting Compound 13 is not an ideal tool to study the role of GPR4 in moderate to severe inflammatory conditions. SIGNIFICANCE STATEMENT: Compound 13, a reported selective GPR4 antagonist, has been widely used to assess the therapeutic potential of GPR4, a pH-sensing receptor, for numerous indications. Its pH dependence and mechanism of inhibition identified in this study clearly highlights the limitations of this chemotype for target validation.

Metabolite profiles of degarelix, a new gonadotropin-releasing hormone receptor antagonist, in rat, dog, and monkey.

Degarelix is a novel competitive gonadotropin-releasing hormone receptor blocker (antagonist). In this study, the nonclinical metabolism and excretion of degarelix was investigated in Sprague-Dawley rat, beagle dog, and cynomolgus monkey. Degarelix was found to be stable when incubated in microsomes and cryopreserved hepatocytes from animal liver tissue. Absorption, distribution, metabolism, and excretion studies in male rat, dog, and monkey showed that after a subcutaneous dose of tritium-labeled degarelix, the peptide was rapidly absorbed with C(max) in plasma of 1 to 2 h. The predominant route of excretion was via the kidneys and the bile. In rat and dog, most of the degarelix dose was eliminated within 48 h via urine and feces in equal amounts (40-50% in each matrix), whereas in monkey the major route of excretion was fecal (50%) and renal (22%). In plasma and urine samples from all three species, mainly intact degarelix was detected. In bile and feces samples from rats and dogs, the same truncated peptides of the parent decapeptide were detected. The major metabolites identified represented the N-terminal tetrapeptide, the pentapeptide, and the heptapeptide. From the animal studies, it could be concluded that degarelix is subject to common peptidic degradation in the liver and bile and is fully excreted via metabolic and biliary (as metabolites and parent compound) and urinary (mainly as parent compound) pathways. Systemic exposure to metabolic products seems to be low.

Novel D-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral kappa-opioid receptors.

Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/>1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (>1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.

Additive effect of simultaneous continuous administration of degarelix and TAK-448 on LH suppression in a castrated rat model.

Gonadotropin releasing hormone (GnRH) analogs have long been used in androgen deprivation therapy (ADT) in the treatment of prostate cancer. Chronic administration of either GnRH agonists or antagonists leads to suppression of testosterone production in the testes via either downregulation or direct blockade of the GnRH receptor in the pituitary, respectively. Chronic administration of kisspeptin analogs has more recently been shown to lead to testosterone suppression via desensitization of GnRH neurons in the hypothalamus and an optimized kisspeptin analog, TAK-448, was proven effective in a small phase 1 trial. The current study explored the hypothesis that co-administration of TAK-448 and the GnRH antagonist, degarelix, would have an additive effect on hormonal suppression, as a result of simultaneous intervention in separate steps in the same pathway. TAK-448 or degarelix were first administered individually to castrated rats in order to identify low doses capable of partial or no suppression of luteinizing hormone (LH). In the second step, combinations of the low doses of TAK-448 and degarelix were assessed in a 14 day study and compared to the drugs administered separately. The results showed that simultaneous intervention at the kisspeptin and GnRH receptors caused a more pronounced LH suppression than either drug alone, demonstrating an additive or potentiating effect. These results suggest that such a drug combination may hold promise as novel forms of androgen deprivation therapy in the treatment of prostate cancer.