20 research outputs found
Deducing the Temporal Order of Cofactor Function in Ligand-Regulated Gene Transcription: Theory and Experimental Verification
Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available
Impact of glucocorticoid receptor density on ligand-independent dimerization, cooperative ligand-binding and basal priming of transactivation: a cell culture model
Glucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoter-reporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs
Safety and efficacy of GABAA α5 antagonist S44819 in patients with ischaemic stroke: a multicentre, double-blind, randomised, placebo-controlled trial
Background: S44819, a selective GABAA α5 receptor antagonist, reduces tonic post-ischaemic inhibition of the peri-infarct cortex. S44819 improved stroke recovery in rodents and increased cortical excitability in a transcranial magnetic stimulation study in healthy volunteers. The Randomized Efficacy and Safety Trial of Oral GABAA α5 antagonist S44819 after Recent ischemic Event (RESTORE BRAIN) aimed to evaluate the safety and efficacy of S44819 for enhancing clinical recovery of patients with ischaemic stroke. Methods: RESTORE BRAIN was an international, randomised, double-blind, parallel-group, placebo-controlled, multicentre phase 2 trial that evaluated the safety and efficacy of oral S44189 in patients with recent ischaemic stroke. The study was done in specialised stroke units in 92 actively recruiting centres in 14 countries: ten were European countries (Belgium, Czech Republic, France, Germany, Hungary, Italy, Netherlands, Poland, Spain, and the UK) and four were non-European countries (Australia, Brazil, Canada, and South Korea). Patients aged 18–85 years with acute ischaemic stroke involving cerebral cortex (National Institute of Health Stroke Scale [NIHSS] score 7–20) without previous disability were eligible for inclusion. Participants were randomly assigned to receive 150 mg S44819 twice a day, 300 mg S44819 twice a day, or placebo twice a day by a balanced, non-adaptive randomisation method with a 1:1:1 ratio. Treatment randomisation and allocation were centralised via the interactive web response system using computer-generated random sequences with a block size of 3. Blinding of treatment was achieved by identical appearance and taste of all sachets. Patients, investigators and individuals involved in the analysis of the trial were masked to group assignment. The primary endpoint was the modified Rankin Scale (mRS) score 90 days from onset of treatment, evaluated by shift analysis (predefined main analysis) or by dichotomised analyses using 0–1 versus 2–6 and 0–2 versus 3–6 cutoffs (predefined secondary analysis). Secondary endpoints were the effects of S44819 on the NIHSS and Montreal Cognitive Assessment (MoCA) scores, time needed to complete parts A and B of the Trail Making Test, and the Barthel index. Efficacy analyses were done on all patients who received at least one dose of treatment and had at least one mRS score taken after day 5 (specifically, on or after day 30). Safety was compared across treatment groups for all patients who received at least one dose of treatment. The study was registered at ClinicalTrials.gov, NCT02877615. Findings: Between Dec 19, 2016, and Nov 16, 2018, 585 patients were enrolled in the study. Of these, 197 (34%) were randomly assigned to receive 150 mg S44819 twice a day, 195 (33%) to receive 300 mg S44819 twice a day, and 193 (33%) to receive placebo twice a day. 189 (96%) of 197 patients in the 150 mg S44819 group, 188 (96%) of 195 patients in the 300 mg S44819 group, and 191 (99%) patients in the placebo group received at least one dose of treatment and had at least one mRS score taken after day 5, and were included in efficacy analyses. 195 (99%) of 197 patients in the 150 mg S44819 group, 194 (99%) of 195 patients in the 300 mg S44819 group, and 193 (100%) patients in the placebo group received at least one dose of treatment, and were included in safety analyses. The primary endpoint of mRS at day 90 did not differ between each of the two S44819 groups and the placebo group (OR 0·91 [95% CI 0·64–1·31]; p=0·80 for 150 mg S44819 compared with placebo and OR 1·17 [95% CI 0·81–1·67]; p=0·80 for 300 mg S44819 compared with placebo). Likewise, dichotomised mRS scores at day 90 (mRS 0–2 vs 3–6 or mRS 0–1 vs 2–6) did not differ between groups. Secondary endpoints did not reveal any significant group differences. The median NIHSS score at day 90 did not differ between groups (4 [IQR 2–8] in 150 mg S44819 group, 4 [2–7] in 300 mg S44819 group, and 4 [2–6] in placebo group), nor did the number of patients at day 90 with an NIHSS score of up to 5 (95 [61%] of 156 in 150 mg S44819 group, 106 [66%] of 161 in 300 mg S44819 group, and 104 [66%] of 157 in placebo group) versus more than 5 (61 [39%] in 150 mg S44819 group, 55 [34%] in 300 mg S44819 group, and 53 [34%] in placebo group). Likewise, the median MoCA score (22·0 [IQR 17·0–26·0] in 150 mg S44819 group, 23·0 [19·0–26·5] in 300 mg S44819 group, and 22·0 [17·0–26·0] in placebo group), time needed to complete parts A (50 s [IQR 42–68] in 150 mg S44819 group, 49 s [36–63] in 300 mg S44819 group, and 50 s [38–68] in placebo group) and B (107 s [81–144] in 150 mg S44819 group, 121 s [76–159] in 300 mg S44819 group, and 130 s [86–175] in placebo group) of the Trail Making Test, and the Barthel index (90 [IQR 60–100] in 150 mg S44819 group, 90 [70–100] in 300 mg S44819 group, and 90 [70–100] in placebo group) were similar in all groups. Number and type of adverse events were similar between the three groups. There were no drug-related adverse events and no drug-related deaths. Interpretation: There was no evidence that S44819 improved clinical outcome in patients after ischaemic stroke, and thus S44819 cannot be recommended for stroke therapy. The concept of tonic inhibition after stroke should be re-evaluated in humans. Funding: Servier
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P311 Pharmacokinetics and exposure–response relationships of intravenously administered ustekinumab during induction treatment in patients with ulcerative colitis: Results from the UNIFI induction study
Abstract
Background
Pharmacokinetic (PK) and exposure–response (ER) data for ustekinumab (UST) from the UNIFI study in ulcerative colitis (UC)1 were evaluated.
Methods
PK, efficacy, and safety data were obtained from this Phase 3, double-blind, placebo-controlled induction trial which enrolled 961 patients with moderate–severe UC. Pts who previously failed biologics (1 or more TNF-blockers or vedolizumab) or conventional therapy (corticosteroid and/or 6-MP/AZA) were included. Pts were randomised 1:1:1 to receive an IV induction dose of UST 130 mg or a weight-range based dose of ~6 mg/kg, or placebo at Week 0. Serum UST concentrations and antibodies to UST were evaluated with validated assays. Clinical efficacy outcomes based on the Mayo score were assessed at Wk8; C-reactive protein (CRP) and faecal markers were evaluated as efficacy biomarkers. The relationships between serum UST concentrations and efficacy, as well as the incidence of infections, serious infection and serious adverse events (SAE) during induction were evaluated.
Results
Serum UST concentrations over time through Wk8 were dose proportional and similar between biofailure and non-biofailure patients, and patients receiving immunomodulators (IMM) at baseline and those not receiving IMM. Median peak serum UST concentrations for UST 130 mg and ~6 mg/kg dose groups were 43.2 μg/ml and 127.0 μg/ml, respectively; median Wk8 UST concentrations were 2.5 μg/ml and 8.6 μg/ml, respectively. The incidence of antibodies to UST through 8 weeks was 0.6% based on a drug-tolerant assay. Wk8 serum UST concentrations were positively associated with the proportions of patients achieving clinical response, clinical remission, and endoscopic healing (Figure 1), and inversely related to CRP and faecal calprotectin/lactoferrin levels. Greater proportions of patients in the ~6 mg/kg group achieved UST exposures in the upper quartiles of UST exposure associated with higher efficacy. Serum UST concentrations were not associated with the incidence of infections, serious infections or SAEs.
Figure 1. Relationship between serum ustekinumab concentrations at Week 8 and clinical efficacy outcomes at Week 8.
Conclusions
Serum UST concentrations were approximately dose-proportional and a positive E-R relationship for efficacy was observed during UST induction treatment in patients with UC. No associations were observed between systemic UST exposure and selected safety events at the IV doses evaluated. These results are consistent with those reported for patients with Crohn's disease.
Reference
1. Sands BE, Sandborn WJ, Panaccione P, et al. Safety and efficacy of ustekinumab induction therapy in patients with moderate to severe ulcerative colitis: results from the Phase 3 UNIFI Study. Oral Presentation at ACG 2018, Philadelphia, PA