Christopher P. Regan

Effects of the prototype serotonin 5-HT1B/1D receptor agonist sumatriptan and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP8-37 on myocardial reactive hyperemic response in conscious dogs

The triptans, serotonin 5-HT(1B/1D) receptor agonists exemplified by sumatriptan, are a mainstay migraine therapy but have class labeling contraindicating their use in patients with coronary artery disease. Triptans constrict human coronary artery in vitro, and there are case reports of myocardial infarction in patients using sumatriptan. However, preclinical studies with sumatriptan in normal dogs have failed to demonstrate effects on resting coronary flow. Calcitonin gene-related peptide (CGRP) receptor antagonism, exemplified by the prototype CGRP receptor antagonist peptide CGRP(8-37), is a new antimigraine mechanism which also has been reported to have no effect on coronary flow in normal, non-stressed animals. The goal of the present studies was to compare the effects of sumatriptan (10microg/kg/min i.v.) and CGRP(8-37) (30microg/kg/min i.v.) on systemic and coronary hemodynamics in conscious dogs under resting conditions and during myocardial reactive hyperemia following a brief 15s of coronary artery occlusion. Neither CGRP(8-37) nor sumatriptan affected resting coronary flow. However, whereas CGRP(8-37) had no effect on myocardial reactive hyperemic response, sumatriptan reduced peak reactive hyperemic coronary artery blood flow (baseline vs treatment: 75.4+/-12.7 vs 60.0+/-10.3ml/min, P<0.05), reactive hyperemic flow (16.7+/-5.2 vs 11.6+/-3.3ml, P<0.05) and the repayment of coronary blood flow debt following coronary artery occlusion (484+/-76 vs 369+/-57%, P<0.05), indicating an impairment in coronary blood flow reserve. The positive control nitric oxide synthase inhibitor L-NNA (30mg/kg/30min i.v.) likewise significantly attenuated myocardial reactive hyperemic response. These findings provide evidence for a differentiation between CGRP receptor antagonism and triptan effects on coronary vascular function.

Renin inhibitors for the treatment of hypertension: Design and optimization of a novel series of pyridone-substituted piperidines

An SAR campaign aimed at decreasing the overall lipophilicity of renin inhibitors such as 1 is described herein. It was found that replacement of the northern appendage in 1 with an N-methyl pyridone and subsequent re-optimization of the benzyl amide handle afforded compounds with in vitro and in vivo profiles suitable for further profiling. An unexpected CV toxicity in dogs observed with compound 20 led to the employment of a time and resource sparing rodent model for in vivo screening of key compounds. This culminated in the identification of compound 31 as an optimized renin inhibitor.

Attenuation of edema and infarct volume following focal cerebral ischemia by early but not delayed administration of a novel small molecule KDR kinase inhibitor.

Vascular endothelial growth factor (VEGF) may mediate increases in vascular permeability and hence plasma extravasation and edema following cerebral ischemia. To better define the role of VEGF in edema, we examined the effectiveness of a novel small molecule KDR kinase inhibitor Compound-1 in reducing edema and infarct volume following focal cerebral ischemia in studies utilizing treatment regimens initiated both pre- and post-ischemia, and with study durations of 24-72 h. Rats were subjected to 90 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. Pretreatment with Compound-1 (40 mg/kg p.o.) starting 0.5h before occlusion significantly reduced infarct volume at 72 h post-MCAO (vehicle, 194.1+/-22.9 mm(3) vs. Compound-1, 127.6+/-22.8mm(3) and positive control MK-801, 104.4+/-22.6mm(3), both p<0.05 compared to vehicle control), whereas Compound-1 treatment initiated at 2h after occlusion did not affect infarct volume. Compound-1 pretreatment also significantly reduced brain water content at 24h (vehicle, 80.3+/-0.2% vs. Compound-1, 79.7+/-0.2%, p<0.05) but not at 72 h after MCAO. These results demonstrate that early pretreatment administration of a KDR kinase inhibitor elicited an early, transient decrease in edema and subsequent reduction in infarct volume, implicating VEGF as a mediator of stroke-related vascular permeability and ischemic injury.

Assessment of the clinical cardiac drug-drug interaction associated with the combination of hepatitis C virus nucleotide inhibitors and amiodarone in guinea pigs and rhesus monkeys.

In 2015, European and U.S. health agencies issued warning letters in response to 9 reported clinical cases of severe bradycardia/bradyarrhythmia in hepatitis C virus (HCV)‐infected patients treated with sofosbuvir (SOF) in combination with other direct acting antivirals (DAAs) and the antiarrhythmic drug, amiodarone (AMIO). We utilized preclinical in vivo models to better understand this cardiac effect, the potential pharmacological mechanism(s), and to identify a clinically translatable model to assess the drug‐drug interaction (DDI) cardiac risk of current and future HCV inhibitors. An anesthetized guinea pig model was used to elicit a SOF+AMIO‐dependent bradycardia. Detailed cardiac electrophysiological studies in this species revealed SOF+AMIO‐dependent selective nodal dysfunction, with initial, larger effects on the sinoatrial node. Further studies in conscious, rhesus monkeys revealed an emergent bradycardia and bradyarrhythmia in 3 of 4 monkeys administered SOF+AMIO, effects not observed with either agent alone. Morever, bradycardia and bradyarrhythmia were not observed in rhesus monkeys when intravenous infusion of MK‐3682 was completed after AMIO pretreatment. Conclusions: These are the first preclinical in vivo experiments reported to replicate the severe clinical SOF+AMIO cardiac DDI and provide potential in vivo mechanism of action. As such, these data provide a preclinical risk assessment paradigm, including a clinically relevant nonhuman primate model, with which to better understand cardiovascular DDI risk for this therapeutic class. Furthermore, these studies suggest that not all HCV DAAs and, in particular, not all HCV nonstructural protein 5B inhibitors may exhibit this cardiac DDI with amiodarone. Given the selective in vivo cardiac electrophysiological effect, these data enable targeted cellular/molecular mechanistic studies to more precisely identify cell types, receptors, and/or ion channels responsible for the clinical DDI. (Hepatology 2016;64:1430‐1441)

Lack of Protection with a Novel, Selective Melanocortin Receptor Subtype-4 Agonist RY767 in a Rat Transient Middle Cerebral Artery Occlusion Stroke Model

Previous studies utilizing α-melanocyte-stimulating hormone (α-MSH) or the synthetic analog [Nle4, D-Phe7] α-MSH have reported beneficial effects in animal models of ischemic stroke, with the latter studies suggesting melanocortin receptor subtype-4 (MC4R) activation as a protective mechanism. The present study directly addresses the hypothesis that MC4R activation may ameliorate ischemic brain injury by assessing the efficacy of a novel small molecule MC4R agonist RY767, administered in a pharmacokinetically guided and pharmacologically validated dosing regimen, in a rat stroke model of transient middle cerebral artery occlusion (tMCAO). Male Wistar rats were subjected to 90-min tMCAO followed by 72 h of reperfusion. Treatments were i.p. pretreatment with MK-801 (15 min prior to occlusion, positive control), or combined i.v. and p.o. daily administrations of vehicle, dextrose (negative control) or RY767 in blinded fashion initiated 2 h after occlusion. Infarct volume in MK-801-treated rats (158.7 ± 22.3 mm3) was reduced significantly compared to vehicle infarct volume (243.4 ± 12.5 mm3), whereas infarct volumes in dextrose- (224.3 ± 16.5 mm3) and RY767- (262.1 ± 19.2 mm3) treated rats did not differ from vehicle infarct volume. These results indicate that selective MC4R activation provides no significant neuroprotection, as reflected by infarct volume, in a rat stroke model utilizing a 90-min ischemic insult.

Long term assessment of blood pressure transducer drift in rhesus monkeys chronically instrumented with telemetry implants.

INTRODUCTIONnThe accurate assessment of blood pressure is often a key component of preclinical cardiovascular disease/efficacy models and of screening models used to determine the effects of test agents on cardiovascular physiology. Of the many methods utilized in large animals, telemetry is becoming more widely used throughout preclinical testing, and non-human primates are playing an ever increasing role as a large animal model to evaluate the cardiovascular effect of novel test agents. Therefore, we sought to characterize pressure transducer drift of a telemetry implant in primates over an extended duration.nnnMETHODSnWe instrumented ten rhesus monkeys with a Konigsberg T27F implant and a chronic indwelling arterial catheter and cross calibrated the diastolic pressure recorded by the implant to the diastolic pressure that was simultaneously recorded through the arterial catheter using a calibrated external transducer/amplifier system.nnnRESULTSnWhile all implanted pressure transducers experienced drift to some degree, magnitude of drift varied across animals (range of average drift 0.7-20.5 mmHg/month). Specifically, we found that all implants could be calibrated within the voltage range of the instrument up to 6 months after implantation despite the drift observed. Between 6 and 12 months, 3 of the 10 implants studied drifted outside the defined voltage range and were unusable, two more drifted off scale within 2 years, while the remainder remained within the operating voltage range.nnnDISCUSSIONnGiven that pressure transducer drift was not consistent across implants or time, these data suggest careful assessment and quantitative correction for in vivo drift of telemetry blood pressure transducers implanted for extended duration should be considered.

Shortening of the electromechanical window in the ketamine/xylazine-anesthetized guinea pig model to assess pro-arrhythmic risk in early drug development

BACKGROUNDnA negative electromechanical window (EMw) was recently proposed as a better preclinical tool than QTc interval to predict clinical pro-arrhythmic potential. As such, we utilized the ketamine/xylazine anesthetized guinea pig to characterize the EMw and QTc interval for a diverse set of reference agents with known clinical pro-arrhythmic potential. Then we determined the clinical proarrhythmia predictive capacity of EMw shortening compared to hERG inhibition or QTc interval prolongation alone.nnnMETHODSnChanges in EMw and QTc interval by 26 reference agents were evaluated in the ketamine/xylazine-anesthetized guinea pig. Confusion matrix analysis using the hERG, QTc and EMw indexes (hERG IC50, QTc EC5 or the EMw EC-10 divided by their respective free therapeutic maximal plasma concentration) at various folds the therapeutic concentrations was conducted to assess the concordance of each index to predict clinical pro-arrhythmic risk.nnnRESULTSnShortening of the EMw concomitant to an increase in QTc interval was observed in the GP with known pro-arrhythmic drugs. Non-torsadogenic compounds did not cause EMw shortening, although some prolonged the QTc interval. The preclinical:clinical concordance of the EMw index (88%) was similar (p>0.05) to using QTc interval prolongation alone (85%) but significantly greater (p<0.05) than using hERG inhibition alone (69%). In addition, the specificity when using the EMw (87%) was largely greater (p<0.05) than using QTc interval (73%) or hERG inhibition (60%) alone. When the components of the response (duration of left ventricular pressure (LVP) cycle (QLVPend) or QT interval) that caused EMw shortening were considered, the concordance is further improved (>95%).nnnCONCLUSIONnEMw shortening improves QTc interval prolongation recording in early drug development and increases the translatability over existing preclinical tools in predicting clinical arrhythmias.

P4-332: In vivo pharmacodynamic effects of BQCA, a novel selective allosteric M1 receptor modulator

Background: The loss of cholinergic neurons in the basal forebrain is a hallmark of the pathophysiology of Alzheimer’s disease (AD) and is believed to underlie memory loss and cognitive decline in AD patients. Based on this hypothesis, cholinesterase inhibitors have been developed and shown to be efficacious in patients. However, their efficacy is limited due to severe side effects. Therefore, novel cholinergic approaches are being broadly investigated. M1 muscarinic receptors are expressed in neurons in the cortex and hippocampus and are believed to play a central role in cognition. Therefore, we developed a selective allosteric M1 potentiator, BQCA, as a novel treatment approach for AD. Here we demonstrate that BQCA activates M1 receptors in vivo in three pharmacodynamic assays with relevance for cognition. Methods: EEG, Laser Doppler blood flow, and sleep telemetry were used to evaluate efficacy of BQCA in rodents. Results: 1) Cognition enhancing drugs, including donepezil, have been shown to lead to an increase in theta activity in a stimulated EEG model in anesthetized rats (Kinney et al. 1998). Acute high frequency stimulation of the brainstem leads to induction of theta and gamma oscillations in the CA1 region of the hippocampus. In this model, IP dosing of 10mg/kg BQCA leads to a distinct change of EEG oscillations, mainly affecting higher EEG frequencies (20-80Hz). This effect is blocked by application of the muscarinic antagonist scopolamine (1mg/kg). 2) Non-selective muscarinic agonists have been shown to increase cerebral blood flow. We show that BQCA (10mg/kg, IV) can significantly increase cerebral blood flow measured by Laser Doppler flowmetry in anesthetized rats (20% increase) without significantly affecting mean arterial pressure. 3) Finally, we tested BQCA in a 7 day cross over sleep architecture study in awake rats. BQCA significantly affects sleep architecture at 10mg/kg IP leading to an increase in active wake and light sleep, with corresponding decreases in delta sleep and REM sleep. Conclusions: Taken together, these data suggest that BQCA activates M1 muscarinic receptors in the brain in a way that indicates a cognitive enhancing effect of the compound making BQCA a promising new approach for the treatment of AD.