Cleveland, OH, United States
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Kuryshev Y.A.,Chantest | Bruening-Wright A.,Chantest | Brown A.M.,Chantest | Kirsch G.E.,Chantest
Journal of Cardiovascular Pharmacology | Year: 2010

Methadone, a synthetic opioid for treatment of chronic pain and withdrawal from opioid dependence, has been linked to QT prolongation, potentially fatal torsades de pointes, and sudden cardiac death. Concomitant use of diazepam or other benzodiazepines in methadone maintenance treatment can increase the risk of sudden death. Therefore, we determined the effects of methadone and diazepam singly and in combination on cardiac action potentials (APs) and on the major ion channels responsible for cardiac repolarization. Using patch clamp recording in human stem cell-derived cardiomyocytes and stably transfected mammalian cells, we found that methadone produced concentration-dependent AP prolongation and ion channel block at low micromolar concentrations: hERG (IC50 = 1.7 μM), hNav1.5 (11.2 μM tonic block; 5.5 μM phasic block), and hCav1.2 (26.7 μM tonic block; 7.7 μM phasic block). Methadone was less potent in hKv4.3/hKChIP2.2 (IC50 = 39.0 μM) and hKvLQT1/hminK (53.3 μM). In contrast, diazepam blocked channels only at much higher concentrations and had no effect on AP duration at 1 μM. However, coadministration of 1-μM diazepam with methadone caused a statistically significant increase in AP duration and a 4-fold attenuation of hNav1.5 block (IC50 values were 44.2 μM and 26.6 μM, respectively, for tonic and phasic block), with no significant effect on methadone-induced block of hERG, hCav1.2, hKv4.3/hKChIP2.2, and hKvLQT1/hminK channels. Thus, although diazepam alone does not prolong the QT interval, the relief of methadone-induced Na+ channel block may leave hERG K+ channel block uncompensated, thereby increasing cardiac risk. Copyright © 2010 by Lippincott Williams & Wilkins.


Kramer J.,Chantest | Obejero-Paz C.A.,Chantest | Myatt G.,Leadscope, Inc. | Kuryshev Y.A.,Chantest | And 3 more authors.
Scientific Reports | Year: 2013

Drug-induced block of the cardiac hERG (human Ether-à-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Nav1.5 and Cav1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Cav1.2.


Kuryshev Y.A.,Chantest | Brown A.M.,Chantest | Duzic E.,Chantest | Kirsch G.E.,Chantest
Assay and Drug Development Technologies | Year: 2014

Voltage-gated Ca2+ channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks™ Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCa v1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug-channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB platform. The assays can be used to screen focused compound libraries for state-dependent Cav channel antagonists, to prioritize compounds for potency or to counterscreen for Cav subtype selectivity. © Copyright 2014, Mary Ann Liebert, Inc. 2014.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.18M | Year: 2012

DESCRIPTION (provided by applicant): Stem Cell-derived Human Cardiomyocytes (SC-hCMs) offer great potential for improving the accuracy of pre- clinical cardiac safety testing. We have characterized a population of SC-hCMs and have demonstrated that these cells show sensitive pharmacology that accurately predicts clinical responses. However, due to low assay throughput and limited resources, only 15 reference compounds were tested. We now propose to increase throughput in preclinical electrophysiology (EP) screens by utilizing a higher-throughput automated EP instrument. We will expand our preclinical in vitro testing to 77 compounds that have been carefully selected based on their known torsadogenic and/or QT prolonging effects. Results from SC-hCM-based assays will be referenced against complete (8 concentration, 8 replicate) concentration-curves of the same compounds generated from high-throughput screens of cell lines expressing each of the major cardiac ion channels. A statistics-based model will be created in collaboration with the U.S. Food and Drug Administration and a leading in silico modeling firm, Leadscope. This model will be based on the unique databases we create which, together the development of software dedicated to mining public and proprietary cardiac databases will dramatically increase productivity of pre-clinical cardiac safet screening. The set of services and products that will result from this project have the potential t save millions of dollars annually by reducing attrition of marketed but cardiotoxic drugs, to improve the safety of drugs in development, and to increase efficiency of drug development by allowing companies to focus on the most promising and safe drug candidates. PUBLIC HEALTH RELEVANCE: The recent availabilityof human myocytes derived from stem cells (SC-hCMs) provides an opportunity to develop pre-clinical cardiac safety assays with better predictive value compared to conventional assays. The benefits to public health are: 1) Improved productivity in pre-clinical cardiac safety screening, thereby reducing risk of adverse cardiac events in clinical trials, and 2) Added cost-efficiencies in the pharmaceutical drug development process.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.28K | Year: 2010

DESCRIPTION (provided by applicant): Stem Cell-derived Human Cardiomyocytes (SC-hCMs) offer great potential for improving the accuracy of pre-clinical cardiac safety screening. We have recently characterized a population of SC-hCMs that will be made commercially available in 2010 by our collaborator GE Healthcare, and have demonstrated that these cells show sensitive pharmacology that accurately predicts clinical responses. However, the utility of SC-hCMs in cardiac safety testing, and more broadly, in regenerative medicine, is partially limited by the fact that they are functionally immature. The resting and action potential properties of the cells resemble embryonic or neonatal CMs rather than adult CMs, and SC-hCMs spontaneously beat in culture which limits their utility for detection of rate-dependent compound effects. To address these shortcomings, we propose to genetically engineer functionally mature SC-hCMs by selectively supplementing under- expressed ionic currents. The functionally mature SC-hCMs will then be validated for use in improved commercial cardiac safety screens. More broadly, these experiments will serve a proof-of-concept function by identifying ion channels that are under-expressed in terminally differentiated SC-hCMs. Once these ion channels are identified, future experiments may focus on inducing stable, elevated ion channel expression early in the differentiation process. PUBLIC HEALTH RELEVANCE: The recent availability of human myocytes derived from stem cells (SC-hCMs) provides an opportunity to develop pre-clinical cardiac safety assays with better predictive value compared to conventional assays. The benefits to public health are: 1) Improved predictivity in pre-clinical cardiac safety screening, thereby reducing risk of adverse cardiac events in clinical trials 2) Added cost-efficiencies in the pharmaceutical drug development process, and 3) Long-term potential to increase the utility of SC-hCMs in the field of regenerative medicine.


Trademark
Chantest | Date: 2013-04-02

Medical diagnostic assays for testing of body fluids; assays. Conducting early evaluations in the field of new pharmaceuticals; Consulting services in the fields of biotechnology, pharmaceutical research and development, laboratory testing, diagnostics, and pharmacogenetics; Pharmaceutical drug development services; Pharmaceutical product evaluation; Pharmaceutical research and development; Pharmaceutical research services; Providing medical and scientific research information in the field of pharmaceuticals and clinical trials; Testing, inspection or research of pharmaceuticals, cosmetics or foodstuff; custom design and development of chemical reagents and biochemical assays.


Trademark
Chantest | Date: 2013-05-20

Medical diagnostic assays for testing of body fluids; assays. Biochemical research and development; Biomedical research services; Chemical research; Chemical, biochemical, biological and bacteriological research and analysis; Conducting early evaluations in the field of new pharmaceuticals; Consulting services in the fields of biotechnology, pharmaceutical research and development and genetic science; Consulting services in the fields of biotechnology, pharmaceutical research and development, laboratory testing, diagnostics, and pharmacogenetics; Drug discovery services; Medical research; Pharmaceutical drug development services; Pharmaceutical research and development; Pharmaceutical research services; Providing medical and scientific research information in the field of clinical trials; Providing medical and scientific research information in the field of pharmaceuticals and clinical trials; Research and development in the pharmaceutical and biotechnology fields; Research and development of pharmaceuticals for the treatment of age-related diseases and cancer; Research on the subject of pharmaceuticals; Scientific research; Scientific research and development; Testing, inspection or research of pharmaceuticals, cosmetics or foodstuff.


Trademark
Chantest | Date: 2012-06-05

Cells for scientific, laboratory or medical research.


Trademark
Chantest | Date: 2013-04-05

Medical diagnostic assays for testing of body fluids; nonclinical assays; assays. Conducting clinical trials for others; Conducting early evaluations in the field of new pharmaceuticals; Consulting services in the fields of biotechnology, pharmaceutical research and development, laboratory testing, diagnostics, and pharmacogenetics; Pharmaceutical drug development services; Pharmaceutical product evaluation; Pharmaceutical research and development; Pharmaceutical research services; Providing medical and scientific research information in the field of pharmaceuticals and clinical trials; Testing, inspection or research of pharmaceuticals, cosmetics or foodstuff; custom design and development of chemical reagents and biochemical assays.

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