Pharmacological characterization of the first in class clinical candidate PF-05190457: a selective ghrelin receptor competitive antagonist with inverse agonism that increases vagal afferent firing and glucose-dependent insulin secretion ex vivo.

BACKGROUND AND PURPOSE: Ghrelin increases growth hormone secretion, gastric acid secretion, gastric motility and hunger but decreases glucose-dependent insulin secretion and insulin sensitivity in humans. Antagonizing the ghrelin receptor has potential as a therapeutic approach in the treatment of obesity and type 2 diabetes. Therefore, the aim was to pharmacologically characterize the novel small-molecule antagonist PF-05190457 and assess translational pharmacology ex vivo. EXPERIMENTAL APPROACH: Radioligand binding in filter and scintillation proximity assay formats were used to evaluate affinity, and europium-labelled GTP to assess functional activity. Rat vagal afferent firing and calcium imaging in dispersed islets were used as native tissues underlying food intake and insulin secretion respectively. KEY RESULTS: PF-05190457 was a potent and selective inverse agonist on constitutively active ghrelin receptors and acted as a competitive antagonist of ghrelin action, with a human Kd of 3 nM requiring 4 h to achieve equilibrium. Potency of PF-05190457 was similar across different species. PF-05190457 increased intracellular calcium within dispersed islets and increased vagal afferent firing in a concentration-dependent manner with similar potency but was threefold less potent as compared with the in vitro Ki in recombinant overexpressing cells. The effect of PF-05190457 on rodent islets was comparable with glibenclamide, but glucose-dependent and additive with the insulin secretagogue glucagon-like peptide-1. CONCLUSIONS AND IMPLICATIONS: Together, these data provide the pharmacological in vitro and ex vivo characterization of the first ghrelin receptor inverse agonist, which has advanced into clinical trials to evaluate the therapeutic potential of blocking ghrelin receptors in obesity and type 2 diabetes.

Dapsone-induced hematologic toxicity: comparison of the methemoglobin-forming ability of hydroxylamine metabolites of dapsone in rat and human blood.

The relative methemoglobin (MetHgb) forming ability of two metabolites of dapsone, dapsone hydroxylamine (DDS-NOH) and monoacetyldapsone hydroxylamine (MADDS-NOH), were compared in rat and human whole blood. Concentration-response curves for the two metabolites were generated in vitro in whole blood. Data were fit to both the Emax and Sigmoid Emax models. The Emax values for MetHgb formation in rat blood for MADDS-NOH and DDS-NOH fitted to the Emax model were 83 (8) and 84 (2)%, while the EC50 values were 1087 (283) and 828 (104) microM, respectively (mean +/- SD). Neither these values nor those generated for the Sigmoid Emax model differed significantly between the two metabolites. Similarly, the Emax values in human blood for MADDS-NOH and DDS-NOH fitted to the Emax model were 79 (5) and 80 (2)%, while the EC50 values were 90 (17) and 95 (19) microM, respectively. These values also did not differ between the two metabolites using either pharmacodynamic model. MetHgb was produced at the same rate, reached similar peak concentrations, and exhibited the same rate of decline with both metabolites. The area under the MetHgb content versus time curve did not differ between the two metabolites. These data demonstrate that MADDS-NOH and DDS-NOH are equipotent and equally efficacious in their MetHgb-forming ability. Investigation of the disposition of these metabolites is necessary to assess their relative role in dapsone-induced toxicity in vivo.

Evidence that the biotransformation of dapsone and monoacetyldapsone to their respective hydroxylamine metabolites in rat liver microsomes is mediated by cytochrome P450 2C6/2C11 and 3A1.

The formation of dapsone hydroxylamine (DDS-NOH) and monoacetyldapsone hydroxylamine (MADDS-NOH) was found to be greater in male vs. female rat liver microsomes, suggesting a role for either CYP2C11 or CYP3A2. Preincubation with cimetidine (selective for inhibition of CYP2C11), but not troleandomycin (selective for inhibition of CYP3A1/2), inhibited metabolite formation. Furthermore, incubation with monoclonal antibodies (Mabs) to CYP2C6/2C11 reduced metabolite formation to below the level of detection. Together, these data indicate that N-hydroxylation of DDS and MADDS in rat liver microsomes from untreated male rats is catalyzed by CYP2C6/2C11. Interestingly, dexamethasone pretreatment increased the hydroxylation of both metabolites. Preincubation with cimetidine or Mabs to CYP2C6/2C11 (at an antibody:protein ratio of 26:1) in microsomes from dexamethasone pretreated animals did not reduce the N-hydroxylation of DDS, whereas preincubation with troleandomycin reduced metabolite formation by > or = 50%. Collectively, these data indicate that the constitutive enzymes CYP2C6 and/or CYP2C11, as well as CYP3A1 (nonconstitutive), are capable of catalyzing the hydroxylation of DDS and MADDS.