Louise Hosking

Detection of genotyping errors by Hardy–Weinberg equilibrium testing

Genotyping data sets may contain errors that, in some instances, lead to false conclusions. Deviation from Hardy–Weinberg equilibrium (HWE) in random samples may be indicative of problematic assays. This study has analysed 107 000 genotypes generated by TaqMan, RFLP, sequencing or mass spectrometric methods from 443 single-nucleotide polymorphisms (SNPs). These SNPs are distributed both within genes and in intergenic regions. Genotype distributions for 36 out of 313 assays (11.5%) whose minor allele frequencies were >0.05 deviated from HWE (P<0.05). Some of the possible reasons for this deviation were explored: assays for five SNPs proved nonspecific, and genotyping errors were identified in 21 SNPs. For the remaining 10 SNPs, no reasons for deviation from HWE were identified. We demonstrate the successful identification of a proportion of nonspecific assays, and assays harbouring genotyping error. Consequently, our current high-throughput genotyping system incorporates tests for both assay specificity and deviation from HWE, to minimise the genotype error rate and therefore improve data quality.

Effects of the mu-opioid receptor antagonist GSK1521498 on hedonic and consummatory eating behaviour: a proof of mechanism study in binge-eating obese subjects

The opioid system is implicated in the hedonic and motivational processing of food, and in binge eating, a behaviour strongly linked to obesity. The aim of this study was to evaluate the effects of 4 weeks of treatment with the mu-opioid receptor antagonist GSK1521498 on eating behaviour in binge-eating obese subjects. Adults with body mass index ⩾30 kg m−2 and binge eating scale scores ⩾19 received 1-week single-blind placebo run-in, and were then randomized to 28 days with either 2 mg day−1 GSK1521498, 5 mg day−1 GSK1521498 or placebo (N=21 per arm) in a double-blind parallel group design. The outcome measures were body weight, fat mass, hedonic and consummatory eating behaviour during inpatient food challenges, safety and pharmacokinetics. The primary analysis was the comparison of change scores in the higher-dose treatment group versus placebo using analysis of covariance at each relevant time point. GSK1521498 (2 mg and 5 mg) was not different from placebo in its effects on weight, fat mass and binge eating scores. However, compared with placebo, GSK1521498 5 mg day−1 caused a significant reduction in hedonic responses to sweetened dairy products and reduced calorific intake, particularly of high-fat foods during ad libitum buffet meals, with some of these effects correlating with systemic exposure of GSK1521498. There were no significant effects of GSK1521498 2 mg day−1 on eating behaviour, indicating dose dependency of pharmacodynamics. GSK1521498 was generally well tolerated and no previously unidentified safety signals were detected. The potential for these findings to translate into clinically significant effects in the context of binge eating and weight regain prevention requires further investigation.

Pharmacokinetic and pharmacodynamic profiling of a P2X7 receptor allosteric modulator GSK1482160 in healthy human subjects

AIMS This paper describes findings from the first-in-human study for GSK1482160, an orally available allosteric P2X7 receptor modulator. The study aimed to assess the pharmacokinetics (PK), pharmacodynamics (PD), safety and tolerability of the compound in healthy subjects. METHODS Escalating single doses of up to 1 g were administered to healthy subjects in a single-blind and placebo-controlled fashion. Safety, tolerability, blood drug concentrations and ex vivo Il-1β production in blood were evaluated. RESULTS Drug concentration peaked within 3.5 h of dosing under fasting conditions and declined thereafter with a relatively short half-life of less than 4.5 h. Exposure was proportional to dose with between subject variability of less than 60%. A PK/PD model quantified Il-1β as a function of drug exposure. The model allowed simulation of in vivo pharmacology for various untested dose levels and regimens. Furthermore, the mechanistic model supported the hypothesis that the compound reduces the efficacy of ATP at the P2X7 receptor without affecting its affinity. No major safety or tolerability concerns were identified in this small study (n = 29), except for one case of asymptomatic accelerated idioventricular rhythm at the top dose. CONCLUSION The model-based approach maximized analysis power by integrating all biomarker data and revealed mechanistic insight into the pharmacology of P2X7 modulation by GSK1482160. Simulations by this model ultimately led to the discontinuation of the development of this compound. The therapeutic relevance of the P2X7 receptor remains to be tested in patients. The mechanistic-model-based approach can be applied widely to drug development.

A coding polymorphism in the CYSLT2 receptor with reduced affinity to LTD4 is associated with asthma

BACKGROUND Cysteinyl leukotrienes (CYSLTR) are potent biological mediators in the pathophysiology of asthma for which two receptors have been characterized, CYSLTR1 and CYSLTR2. The leukotriene modifying agents currently used to control bronchoconstriction and inflammation in asthmatic patients are CYSLTR1-specific leukotriene receptor antagonists. In this report, we investigated a possible role for therapeutic modulation of CYSLTR2 in asthma by investigating genetic association with asthma and further characterization of the pharmacology of a coding polymorphism. METHODS The association of CYSLTR2 polymorphisms with asthma was assessed by transmission disequilibrium test in two family-based collections (359 families from Denmark and Minnesota, USA and 384 families from the Genetics of Asthma International Network). RESULTS A significant association of the coding polymorphism, 601A>G, with asthma was observed (P = 0.003). We replicated these findings in a collection of 384 families from the Genetics of Asthma International Network (P = 0.04). The G allele is significantly under-transmitted to asthmatics, indicating a possible role for this receptor in resistance to asthma. The potency of cysteinyl leukotrienes at the wild-type CYSLTR2 and the coding polymorphism 601A>G were assessed using a calcium mobilization assay. The potency of LTC4 and LTE4 was similar for both forms of the receptor and LTB4 was inactive, however, LTD4 was approximately five-fold less potent on 601A>G compared to wild-type CYSLTR2. CONCLUSIONS Since 601A>G alters the potency of LTD4 and this variant allele may be associated with resistance to asthma, it is possible that modulation of the CYSLTR2 may be useful in asthma pharmacotherapy.

GLCCI1 rs37973 does not influence treatment response to inhaled corticosteroids in white subjects with asthma

To The Editor: Inhaled corticosteroids (ICS) are the primary anti-inflammatory therapy for the control and management of asthma, but their effects are characterised by some inter-individual variability that may have a genetic basis1–4. Identification of genetic markers that predict ICS treatment response will facilitate individualised treatment for asthma patients in the future, particularly in those with more severe disease. An association between genetic variation in GLCCI1 and response to ICS therapy in non-Hispanic white asthma subjects was observed by Tantisira et al.5 Genome wide association analysis of 118 trios (one asthmatic child and two parents) from the NHLBI Childhood Asthma Management Program (CAMP) identified 13 single nucleotide polymorphisms (SNPs) with evidence for association with the level of ICS treatment response. These 13 SNPs which included the GLCCI1 promoter polymorphism rs37972, were genotyped and evaluated in four additional independent collections: adult studies SOCS (Salmeterol Or CorticosteroidS) and SLIC (SalmeteroL ± Inhaled CorticosteroidS) (n=264); a second adult study (n=385); LOCCS (Leukotriene modifier Or Corticosteroid or Corticosteroid-Salmeterol) (n=185) and CARE (Childhood Asthma Research and Education) (n=101). In three of the four replicate populations GLCCI1 rs37973, a functional promoter polymorphism5 in complete linkage disequilibrium with rs37972 in white populations, was associated (P<0.05) with change in FEV1 (forced expiratory volume in one second) after 4–8 weeks of ICS treatment. The combined P value measuring association between rs37973 and ICS response over the four collections (n=935) was 0.0007. Tantisira et al. concluded that this functional GLCCI1 polymorphism, rs37973, was associated with response to ICS in asthma patients. Using data from a recently completed genome wide association study of response to steroid therapy, we sought to confirm this observation in a homogeneous, well-characterised population of n=1,924 non-Hispanic white subjects by testing for association between rs37973 and measures of corticosteroid response in subjects treated with either fluticasone furoate (FF) or fluticasone propionate (FP) in seven GSK-sponsored clinical trials (NCT01165138; NCT01134042; NCT01086384; NCT01159912; NCT00603382; NCT00603278; NCT00603746). All seven studies were randomised, double blind, placebo controlled, parallel group multicentre studies in adolescent and adult subjects and employed change from baseline in FEV1 as their primary end-point, apart from HZA106837 which also investigated asthma exacerbations. HZA106837 (N=616) required each subject to have an asthma exacerbation within the 12 months prior to enrolment, whereas the other studies excluded subjects with previous asthma exacerbations. FEV1 was assessed at week 8 for all studies except HZA106837, which used week 12 data. Overall, except for FF and FP dose, baseline demographics and study characteristics were similar across all seven studies (Table 1). TABLE 1 Baseline, demographic and steroid response characteristics in seven GSK-sponsored clinical studies Germline DNA was extracted from peripheral blood collected from all 1,924 subjects all of whom provided consent for genetic analysis. Genotyping used either the KBiosciences Competitive Allele Specific PCR SNP genotype System (KASPar) (Hoddesdon, Herts, UK) or the Illumina Omni1-Quad panel (Expression Analysis, Durham, NC, USA). Analysis was undertaken in 1,916 subjects, including four with imputed data from rs37972. Eight subjects had missing covariate data. Genetic association between rs37973 and ICS response was evaluated, with ICS treatment response defined as change from baseline in trough FEV1 using the last observation carried forward (in any subject who did not complete the specific trial), to week 8 or week 12 of FF or FP treatment. Change in trough FEV1 was regressed against covariates identified in this asthma population: age, percent of predicted baseline FEV1, study, height, asthma duration and drug (FF versus FP). We also evaluated the influence of rs37973 on subject placement within the highest and lowest response quartiles. Subjects who fell into the lowest (n=479) and highest (n=479) response quartiles were identified. Logistic regression was used to fit a model with quartile of response as the dependent variable, and genotype and relevant covariates as the independent variables. The P value measuring heterogeneity among the seven studies was 0.98, allowing pooling of data at the subject level. The minor allele frequency of rs37973 was 0.44 and its genotype frequencies were consistent with Hardy-Weinberg equilibrium (P=0.48). Covariate-adjusted FEV1 change was regressed on rs37973 genotype (Figure 1). Rs37973 did not influence change from baseline in FEV1 in this sample of 1,916 non-Hispanic white subjects treated with either FF or FP (P=0.15). However, this regression analysis suggested a trend toward a slightly lower ICS response for each additional copy of the rs37973 G allele. This direction of effect was consistent with that observed by Tantisira et al. In addition, the percentage change from baseline FEV1 (unadjusted for covariates, results not shown) was 10.4±0.8% in AA homozygotes and 8.8±1.0% in GG homozygotes, while the overall mean response was 9.8±0.4%. FIGURE 1 GLCCI1 rs37973 genotype does not significantly influence steroid response in 1,916 asthma patients This genetic marker did not influence subject membership within response quartiles (P=0.08, Odds Ratio (OR) =1.39, 95% CI 0.96–2.00). The ORs in each clinical study ranged from 0.61 (HZA106827) (N=616) to 4.42 (FFA112059), which is one of two smallest clinical trials, N=143 (Table 1). Meta-analysis of the influence of rs37973 on subject placement within response quartile across all seven clinical studies, revealed similar results to the subject level pooled data, suggesting a non-significant trend towards lower ICS response in GLCCI1 rs37973 GG homozygotes, compared to AA homozygotes (P=0.11, OR 1.32, 95% CI 0.94–1.87). In order to closely mimic the analyses of Tantisira et al 5, change in trough FEV1 was also regressed against age, sex and height. All analyses in our sample were repeated using these covariates; there were no qualitative differences between the two sets of results. Asthma is currently estimated to affect ~315 million people worldwide6. A robust genetic predictor of ICS response in asthma patients would provide clinical value7 as inter-individual variability in ICS treatment response is commonly observed. Tantisira et al 5 reported an association (P=0.0007) in a pooled analysis between GLCCI1 rs37973 and ICS treatment response as measured over 4–8 weeks in 935 white non-Hispanic adults and children, and an OR of 2.36 in a subject level pooled analysis evaluating subject placement with response quartiles. In this larger sample set, n=1,916, drawn from seven clinical studies, we did not confirm GLCCI1 rs37973 as a predictor of ICS response. However, the discrepant outcomes might have been due to various factors: the GSK studies were clinical trials specifically designed with FEV1 change as the primary endpoint for all studies except one, whereas those of Tantisira were designed around a range of other primary endpoints, including lung growth, time to treatment failure and the percentage of asthma control days; paediatric study participants were only included in the initial Tantisira5 cohort; and the duration of ICS treatment at the time of FEV1 assessment varied between the two evaluations. Further genetic studies will be required to fully elucidate the potential role of GLCCI1 in ICS treatment response in asthma patients.

Opioid Antagonists and the A118G Polymorphism in the μ-Opioid Receptor Gene: Effects of GSK1521498 and Naltrexone in Healthy Drinkers Stratified by OPRM1 Genotype.

The A118G single-nucleotide polymorphism (SNP rs1799971) in the μ-opioid receptor gene, OPRM1, has been much studied in relation to alcohol use disorders. The reported effects of allelic variation at this SNP on alcohol-related behaviors, and on opioid receptor antagonist treatments, have been inconsistent. We investigated the pharmacogenetic interaction between A118G variation and the effects of two μ-opioid receptor antagonists in a clinical lab setting. Fifty-six overweight and moderate–heavy drinkers were prospectively stratified by genotype (29 AA homozygotes, 27 carriers of at least 1 G allele) in a double-blind placebo-controlled, three-period crossover design with naltrexone (NTX; 25 mg OD for 2 days, then 50 mg OD for 3 days) and GSK1521498 (10 mg OD for 5 days). The primary end point was regional brain activation by the contrast between alcohol and neutral tastes measured using functional magnetic resonance imaging (fMRI). Secondary end points included other fMRI contrasts, subjective responses to intravenous alcohol challenge, and food intake. GSK1521498 (but not NTX) significantly attenuated fMRI activation by appetitive tastes in the midbrain and amygdala. GSK1521498 (and NTX to a lesser extent) significantly affected self-reported responses to alcohol infusion. Both drugs reduced food intake. Across all end points, there was less robust evidence for significant effects of OPRM1 allelic variation, or for pharmacogenetic interactions between genotype and drug treatment. These results do not support strong modulatory effects of OPRM1 genetic variation on opioid receptor antagonist attenuation of alcohol- and food-related behaviors. However, they do support further investigation of GSK1521498 as a potential therapeutic for alcohol use and eating disorders.

No evidence of large genetic effects on steroid response in asthma patients

Background: Inhaled corticosteroids (ICSs) are considered the most effective anti‐inflammatory therapy for asthma control and management; however, there is substantial treatment response variability. Objective: We sought to identify genetic markers of ICS response by conducting the largest pharmacogenetic investigation to date in 2672 ICS‐treated patients with asthma. Methods: Genotyping and imputation was performed in fluticasone furoate (FF) or fluticasone propionate–treated patients with asthma from 3 phase IIB and 4 phase IIIA randomized, double‐blind, placebo‐controlled, parallel group, multicenter studies. The primary end point analyzed was change in trough FEV1 (&Dgr;FEV1) from baseline to 8 to 12 weeks of treatment. Results: More than 9.8 million common genetic variants (minor allele frequency ≥ 1%) were analyzed to test for association with &Dgr;FEV1. No genetic variant met the prespecified threshold for statistical significance. Conclusions: This study provides no evidence to confirm previously reported associations between candidate genetic variants and ICS response (&Dgr;FEV1) in patients with asthma. In addition, no variant satisfied the criterion for genome‐wide significance in our study. Common genetic variants are therefore unlikely to prove useful as predictive biomarkers of ICS response in patients with asthma.

The effects of alcohol on the pharmacokinetics and pharmacodynamics of the selective mu-opioid receptor antagonist GSK1521498 in healthy subjects.

The mu‐opioid system has a key role in hedonic and motivational processes critical to substance addiction. However, existing mu‐opioid antagonists have had limited success as anti‐addiction treatments. GSK1521498 is a selective and potent mu‐opioid antagonist being developed for the treatment of overeating and substance addictions. In this study, 28 healthy participants were administered single doses of GSK1521498 20 mg, ethanol 0.5 g/kg body weight, or both in combination, in a double blind placebo controlled four‐way crossover design. The primary objective was to determine the risk of significant adverse pharmacodynamic and pharmacokinetic (PK) interactions. The effects of GSK1521498 on hedonic and consummatory responses to alcohol and the attentional processing of alcohol‐related stimuli, and their modulation by the OPRM1 A118G polymorphism were also explored. GSK1521498 20 mg was well tolerated alone and in combination with ethanol. There were mild transient effects of GSK1521498 on alertness and mood that were greater when it was combined with ethanol. These effects were not of clinical significance. There were no effects of GSK1521498 on reaction time, hedonic or consummatory responses. These findings provide encouraging safety and PK data to support continued development of GSK1521498 for the treatment of alcohol addiction.