Effects of genetic variation in the P2RX7 gene on pharmacodynamics of a P2X(7) receptor antagonist: a prospective genotyping approach.

AIMS: To investigate the effects of two single nucleotide polymorphisms (SNPs) in the human P2X7 receptor gene (P2RX7)--1068G>A (A348T) and 1513A>C (E496A)--on P2X7 receptor function, using a specific receptor antagonist (GSK1370319A) and prospective genetic stratification. METHODS: Lipopolysaccharide- and ATP-stimulated interleukin-1ß production was determined in the presence or absence of GSK1370319A in blood culture from 32 prospectively genotyped subjects. RESULTS: There was approximately 6.7-fold difference (P < 0.0001) in IC50 for inhibition of ATP-stimulated interleukin-1ß release by GSK1370319A between individuals with the homozygous gain--(1068A) and loss-of-function (1513C) genotypes (expressing the 348T, 496E and 348A, 496A alleles, respectively). CONCLUSIONS: Leukocyte P2X7 receptors had significantly altered pharmacodynamic responses to a specific antagonist (GSK1370319A), directly related to SNP genotype.

Use of Entero-Test, a simple approach for non-invasive clinical evaluation of the biliary disposition of drugs.

AIM: To evaluate the non-invasive collection of bile from healthy human subjects for the qualitative characterization of the biliary disposition of a drug, using spectrometric techniques. METHODS: Twenty subjects underwent non-invasive bile capture using a peroral string test (Entero-Test) device prior to and following a single oral dose of simvastatin (80 mg). The device, consisting of a weighted gelatin capsule containing a highly absorbent nylon string, was swallowed by each subject with the proximal end of the string taped to the face. Once the weighted string was judged to have reached the duodenum, gallbladder contraction was stimulated in order to release bile. The string was then retrieved via the mouth, and bile samples were analysed for drug-related material using spectrometric and spectroscopic techniques following solvent extraction. RESULTS: Numerous metabolites of simvastatin were detected, and the major metabolites were consistent with those reported from studies where bile was collected using invasive techniques from patients dosed with [(14) C]-simvastatin. CONCLUSIONS: The results from this study demonstrate the utility of deploying the Entero-Test in human studies to provide structural information on biliary metabolites. This can be readily applied in drug development studies, including those in the target patient population and may eliminate the need for more invasive sampling techniques.

Transcriptomic effects of rs4845604, an IBD and allergy-associated RORC variant, in stimulated ex vivo CD4+ T cells.

RORγt is an isoform of RORC, preferentially expressed in Th17 cells, that functions as a critical regulator of type 3 immunity. As murine Th17-driven inflammatory disease models were greatly diminished in RORC knock-out mice, this receptor was prioritised as an attractive therapeutic target for the treatment of several autoimmune diseases. Human genetic studies indicate a significant contributory role for RORC in several human disease conditions. Furthermore, genome-wide association studies (GWAS) report a significant association between inflammatory bowel disease (IBD) and the RORC regulatory variant rs4845604. To investigate if the rs4845604 variant may affect CD4+ T cell differentiation events, naïve CD4+ T cells were isolated from eighteen healthy subjects homozygous for the rs4845604 minor (A) or major (G) allele). Isolated cells from each subject were differentiated into distinct T cell lineages by culturing in either T cell maintenance medium or Th17 driving medium conditions for six days in the presence of an RORC inverse agonist (to prevent constitutive receptor activity) or an inactive diastereomer (control). Our proof of concept study indicated that genotype had no significant effect on the mean number of naïve CD4 T cells isolated, nor the frequency of Th1-like and Th17-like cells following six days of culture in any of the four culture conditions. Analysis of the derived RNA-seq count data identified genotype-driven transcriptional effects in each of the four culture conditions. Subsequent pathway enrichment analysis of these profiles reported perturbation of metabolic signalling networks, with the potential to affect the cellular detoxification response. This investigation reveals that rs4845604 genotype is associated with transcriptional effects in CD4+ T cells that may perturb immune and metabolic pathways. Most significantly, the rs4845604 GG, IBD risk associated, genotype may be associated with a differential detoxification response. This observation justifies further investigation in a larger cohort of both healthy and IBD-affected individuals.

A pilot study to assess the feasibility of measurement of adrenal gland volume by magnetic resonance imaging.

BACKGROUND: Repeated computed tomography (CT) assessment of the adrenal glands is associated with a significant radiation burden. The increasing capabilities of magnetic resonance (MR) volumetric analysis of the adrenals make this a potentially alternative technique in man. PURPOSE: To determine whether MR imaging could be used to measure adrenal volume, and to determine the intra- and interobserver variation and repeatability of MR volume imaging of adrenals in healthy human subjects. MATERIAL AND METHODS: This was a single-cohort, sequential design, three-part study involving four MRI examinations per subject following ethical approval and informed consent. Information was collected on four healthy subjects (three male and one female). Two different investigators estimated the area of the adrenal gland for each of the 3-mm contiguous slices (and consequently adrenal volume). In order to estimate inter- and intrareader variability, a repeated-measures mixed model was fitted with adrenal volume as the dependent variable. In order to estimate any bias between readers, Bland-Altman methodology was applied. RESULTS: Intraobserver variation for adrenal gland volume is approximately 5% of a 3-cm(3 )adrenal gland. Interobserver variation is approximately 9% of a 3-cm(3) adrenal gland. Potential variation in measurement for adrenal volume from all sources equates to approximately 14% of a 3-cm(3) adrenal gland. Verification of image reading by a second investigator (consensus reading) reduces variability. CONCLUSION: Analysis of adrenal gland volume using MRI is a potentially reliable technique that could be used to assess a pathological change in adrenal size.

Identification of BDNF sensitive electrophysiological markers of synaptic activity and their structural correlates in healthy subjects using a genetic approach utilizing the functional BDNF Val66Met polymorphism.

Increasing evidence suggests that synaptic dysfunction is a core pathophysiological hallmark of neurodegenerative disorders. Brain-derived neurotropic factor (BDNF) is key synaptogenic molecule and targeting synaptic repair through modulation of BDNF signalling has been suggested as a potential drug discovery strategy. The development of such "synaptogenic" therapies depend on the availability of BDNF sensitive markers of synaptic function that could be utilized as biomarkers for examining target engagement or drug efficacy in humans. Here we have utilized the BDNF Val66Met genetic polymorphism to examine the effect of the polymorphism and genetic load (i.e. Met allele load) on electrophysiological (EEG) markers of synaptic activity and their structural (MRI) correlates. Sixty healthy adults were prospectively recruited into the three genetic groups (Val/Val, Val/Met, Met/Met). Subjects also underwent fMRI, tDCS/TMS, and cognitive assessments as part of a larger study. Overall, some of the EEG markers of synaptic activity and brain structure measured with MRI were the most sensitive markers of the polymorphism. Met carriers showed decreased oscillatory activity and synchrony in the neural network subserving error-processing, as measured during a flanker task (ERN); and showed increased slow-wave activity during resting. There was no evidence for a Met load effect on the EEG measures and the polymorphism had no effects on MMN and P300. Met carriers also showed reduced grey matter volume in the anterior cingulate and in the (left) prefrontal cortex. Furthermore, anterior cingulate grey matter volume, and oscillatory EEG power during the flanker task predicted subsequent behavioural adaptation, indicating a BDNF dependent link between brain structure, function and behaviour associated with error processing and monitoring. These findings suggest that EEG markers such as ERN and resting EEG could be used as BDNF sensitive functional markers in early clinical development to examine target engagement or drug related efficacy of synaptic repair therapies in humans.