JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide]: a novel, potent, and selective cholecystokinin 2 receptor antagonist with good oral bioavailability.

JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide] is a representative of a new chemical class of competitive antagonists of cholecystokinin 2 (CCK2) receptors. In this study, the primary in vitro pharmacology of JNJ-26070109 was evaluated along with the pharmacokinetic and pharmacodynamic properties of this compound in rat and canine models of gastric acid secretion. JNJ-26070109 expressed high affinity for human (pK(I) = 8.49 ± 0.13), rat (pK(I) = 7.99 ± 0.08), and dog (pK(I) = 7.70 ± 0.14) CCK2 receptors. The selectivity of JNJ-26070109 at the CCK2 receptor versus the CCK1 receptor was species-dependent, with the greatest degree of selectivity (>1200-fold) measured at the human isoforms of the CCK1 receptor (selectivity at CCK2 versus CCK1 receptors: human, ∼1222-fold; rat, ∼324-fold; dog ∼336-fold). JNJ-26070109 behaved as a surmountable, competitive, antagonist of human CCK2 receptors in a calcium mobilization assay (pK(B) = 8.53 ± 0.05) and in pentagastrin-stimulated gastric acid secretion in the isolated, lumen-perfused, mouse stomach assay (pK(B) = 8.19 ± 0.13). The pharmacokinetic profile of this compound was determined in vivo in rats and dogs. JNJ-26070109 was shown to have high oral bioavailability (%F rat = 73 ± 16; %F dog = 92 ± 12) with half lives of 1.8 ± 0.3 and 1.2 ± 0.1 h in rat and dog, respectively. The pharmacodynamic properties of this compound were investigated using two in vivo models. In conscious rat and dog chronic gastric fistula models of pentagastrin-stimulated acid secretion, JNJ-26070109 had oral EC(50) values of 1.5 and 0.26 µM, respectively. Overall, we have demonstrated that JNJ-26070109 is a high-affinity, selective CCK2 receptor antagonist with good pharmacokinetic properties.

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues.

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand- and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

Rapid assessment of anti-HIV siRNA efficacy using PCR-derived Pol III shRNA cassettes.

Identification of sequences within a target mRNA that are susceptible to potent siRNA knockdown often requires testing several independent siRNAs or shRNA expression cassettes. Using RNAi against HIV RNAs is further complicated by the length of the viral genome, the complexity of splicing patterns, and the propensity for genetic heterogeneity; consequently, it is most important to identify a number of siRNA targets that potently block viral replication. We previously described a facile PCR-based strategy for rapid synthesis of si/shRNA expression units and their testing in mammalian cells. Using this approach, which is rapid and inexpensive, it is possible to screen a number of potential RNAi targets in HIV to identify those that are most susceptible to RNAi. We report that shRNA expression cassettes constructed by PCR and cotransfected directly into mammalian cells with HIV proviral DNA express shRNAs that are inhibitory to HIV-1 replication. Our results also demonstrate that there is a wide range of efficacies among shRNAs targeting different sites throughout the HIV genome. By screening several different targets we were able to identify a sequence in a common tat/rev exon that is exquisitely sensitive to RNAi. Furthermore we relate the efficacies of our PCR product expressed shRNAs to the relative stabilities of the siRNA duplexes and the accessibilities of the target sites to antisense base pairing in cell extracts.

Application of the trak-C HCV core assay for monitoring antiviral activity in HCV replication systems.

The Ortho trak-C immunoassay has recently established detection of the HCV core antigen as a viable indirect marker of HCV replication in clinical samples. In this study, trak-C is used to monitor HCV replication in three pre-clinical models: the cellular HCV replicon system, transient transfection of HCV genomes, and the murine Alb-uPa/SCID HCV infection model. All of these systems utilize full-length HCV genomes that direct the expression of core, facilitating its detection with monoclonal antibodies. When performed with purified protein, the assay detects HCV core with a lower limit of detection at 1.5pg, and exhibits linear detection up to 100pg. When assaying extracts prepared from Huh-7 clone 21-5 cells harboring a full-length HCV replicon, core is detectable from as few as 63 cell equivalents. The assay was used to determine the sensitivity of Huh 21-5 cells to the antiviral effects of interferon (IFN). Inhibition by IFN-alpha using core detection was comparable to that observed using branched-DNA (bDNA 3.0) detection of HCV RNA. Replication of transfected full-length HCV 1a Con1 genomes in Huh-7 cells was also detectable using the trak-C assay. Finally, in the transgenic murine HCV infection model, the course of viral amplification was detected from serum using trak-C with kinetics similar to those observed with RNA detection. Given its ease of use and the lack of requirement for RNA purification, the trak-C assay has several advantages over RNA-based methods of viral monitoring.