Terrye Aigeldinger Delmonte

Multicenter, randomized, double‐blind, active comparator and placebo‐controlled trial of a corticotropin‐releasing factor receptor‐1 antagonist in generalized anxiety disorder

Background: Antagonism of corticotropin‐releasing factor (CRF) receptors has been hypothesized as a potential target for the development of novel anxiolytics. This study was designed to determine the safety and efficacy of pexacerfont, a selective CRF‐1 receptor antagonist, in the treatment of generalized anxiety disorder (GAD). Method: This was a multicenter, randomized, double‐blind, placebo‐controlled and active comparator trial. Two hundred and sixty patients were randomly assigned to pexacerfont 100 mg/day (after a 1 week loading dose of 300 mg/day), placebo or escitalopram 20 mg/day in a 2:2:1 ratio. The primary outcome was the mean change from baseline to end point (week 8) in the Hamilton Anxiety Scale total score. Results: Pexacerfont 100 mg/day did not separate from placebo on the primary outcome measure. The half‐powered active comparator arm, escitalopram 20 mg/day, demonstrated efficacy with significant separation from placebo at weeks 1, 2, 3, 6, and 8 (P<.02). Response rates for pexacerfont, placebo, and escitalopram were 42, 42, and 53%, respectively. Genetic and psychometric rating scale data was obtained in 175 randomized subjects. There was a significant association between a single nucleotide polymorphism (SNP) of the gene encoding plexin A2 (PLXNA2‐2016) with the HAM‐A psychic subscale score for the entire cohort at baseline (FDR‐adjusted P=.015). Conclusions: Pexacerfont did not demonstrate efficacy compared to placebo for the treatment of GAD. Whether these findings are generalizable to this class of agents remains to be determined. Our preliminary genetic finding of an association between a SNP for the gene encoding plexin A2 and an anxiety phenotype in this study merits further exploration. The trial was registered at clinicaltrials.gov (NCT00481325) before enrollment. Depression and Anxiety, 2010.

Evaluation of the Paraoxonases as Candidate Genes for Stroke. Gln192Arg Polymorphism in the Paraoxonase 1 Gene Is Associated With Increased Risk of Stroke

Background and Purpose— The paraoxonases are involved in protecting low-density lipoprotein (LDL) from lipid oxidation. Paraoxonase 1 (PON1) was implicated in susceptibility to coronary artery disease and stroke in previous studies. We evaluated, in a comprehensive way, all 3 paraoxonase genes for association with stroke observed in the Cholesterol and Recurrent Events (CARE) trial. Methods— Over 2500 subjects enrolled in the CARE trial were genotyped for 14 single nucleotide polymorphisms, including 7 newly identified in this study, in the 3 paraoxonase genes. Results— A glutamine (Gln)/arginine (Arg) polymorphism at amino acid residue 192 in PON1 was significantly associated with stroke (P=0.003 in multivariate analysis, including age, sex, LDL, hypertension, diabetes, smoking, and pravastatin treatment as covariates). The odds ratios were 2.28 (95% CI, 1.38 to 3.79) for Gln/Arg heterozygotes and 2.47 (95% CI, 1.18 to 5.19) for Arg/Arg homozygotes compared with Gln/Gln homozygotes. These results are consistent with 2 of 3 other published studies. In combined analysis of all 4 studies, the association between Gln192Arg SNP and stroke was highly significant (&khgr;28df=45.58, P<0.000001). Sequence analysis of the PON1 gene from seventy stroke cases revealed a novel nonsense mutation at codon 32 in one stroke case, which was not detected in over 2500 unaffected individuals. Polymorphisms in the PON2 and PON3 genes were not associated with stroke. Conclusions— These results suggest that Gln192Arg genotype is an important risk factor for stroke.

Genotyping Using the TaqMan Assay

The 5′‐nuclease allelic discrimination assay, or TaqMan assay, is a PCR‐based assay for genotyping single nucleotide polymorphisms (SNPs). The region flanking the SNP is amplified in the presence of two allele‐specific fluorescent probes. The probes do not fluoresce in solution because of a quencher at the 3′ end. The presence of two probes allows the detection of both alleles in a single tube. Moreover, because probes are included in the PCR, genotypes are determined without any post‐PCR processing, a feature that is unavailable with most other genotyping methods. This unit describes probe and primer design and PCR conditions. Curr. Protoc. Hum. Genet. 56:2.10.1‐2.10.8.

Genetic and gene expression studies implicate renin and endothelin-1 in edema caused by peroxisome proliferator-activated receptor γ agonists

Objective Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) agonists can cause peripheral edema in susceptible individuals. To investigate the mechanistic basis underlying this adverse event, we performed a candidate gene analysis of patients enrolled in clinical trials of muraglitazar, an investigational PPAR&agr;/&ggr; dual agonist, and developed a cell culture-based gene expression assay and nonhuman primate model of edema to study the edemagenic properties of PPAR&ggr; agonists. Methods A total of 213 single nucleotide polymorphisms (SNPs) in 63 genes were genotyped in 730 participants. Chi-square and logistic regression analyses were used to test for association with edema. Transcriptional responses to PPAR&ggr; agonists were evaluated in Calu-6 cells using quantitative real-time PCR. Male Cynomolgus monkeys were treated with PPAR agonists and were evaluated for edema using MRI. Results SNPs in renin (rs2368564) and endothelin-1 (rs5370) were associated with reduced risk of edema (P=0.003 and P=0.028, respectively) and an SNP in &bgr;1 adrenergic receptor (rs1801253) was associated with increased susceptibility to edema (P=0.034). Gene expression studies revealed that renin and endothelin-1 were regulated by PPAR&ggr; in Calu-6 cells. A survey of 10 PPAR&ggr; agonists further revealed that a compounds in vitro potency was correlated with its edemagenic potential leading to the prediction that one of three previously uncharacterized PPAR&ggr; agonists would cause less edema. This prediction was validated in a nonhuman primate model of PPAR&ggr; agonist-induced edema. Conclusion Our results implicate a key role for renin and endothelin-1 in the edema caused by PPAR&ggr; agonists and demonstrate how knowledge gained from pharmacogenetic studies can be applied in drug discovery.

Frequency of CYP1A2 Polymorphism in Beagle Dogs

A single nucleotide polymorphism in the dog CYP1A2 gene causes these animals to be CYP1A2 deficient (i.e., lack functional CYP1A2 enzyme activity). Genotyping a colony of 79 dogs revealed 77% wild-type, 19% heterozygous, and 4% homozygous mutant animals. These genetic frequencies are significantly different from those previously reported and illustrate that different sources and populations of dogs can have dramatically different frequencies of this polymorphism.