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Cleveland, OH, United States

Lopin K.V.,Case Western Reserve University | Gray I.P.,Case Western Reserve University | Obejero-Paz C.A.,Case Western Reserve University | Obejero-Paz C.A.,Chantest | And 2 more authors.
Molecular Pharmacology | Year: 2012

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe2+ on permeation and gating of CaV3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe2+ state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe2+ rapidly blocked currents with 2 mM extracellular Ca2+ in a voltage-dependent manner, as described by a Woodhull model with KD = 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe2+ also shifted activation to more-depolarized voltages (by ∼10 mV with 1.8 mM extracellular Fe2+) somewhat more strongly than did extracellular Ca2+or Mg2+, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e-/98 Å2 and KFe = 4.5 M-1 for extracellular Fe2+. In the absence of extracellular Ca 2+ (and with extracellular Na+ replaced by TEA), Fe 2+ carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at -60 mV with 10 mM extracellular Fe 2+). With a two-site/three-barrier Eyring model for permeation of CaV3.1 channels, we estimated a transport rate for Fe2+ of ∼20 ions/s for each open channel at -60 mV and pH 7.2, with 1 μM extracellular Fe2+ (with 2 mM extracellular Ca2+). Because CaV3.1 channels exhibit a significant "window current" at that voltage (open probability, ∼1%), CaV3.1 channels represent a likely pathway for Fe2+ entry into cells with clinically relevant concentrations of extracellular Fe2+. Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics.

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.

Chantest | Date: 2013-04-05

Medical diagnostic assays for testing of body fluids. 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.

Borsini F.,Sigma tau Industrie Farmaceutiche Riunite S.p.A. | Crumb W.,Zenas Technologies LLC | Pace S.,Sigma tau Industrie Farmaceutiche Riunite S.p.A. | Ubben D.,Medicine for Malaria Venture MMV | And 4 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2012

The in vitro cardiac properties of dihydroartemisinin (DHA) plus piperaquine phosphate (PQP) were compared with those of other antimalarial compounds. Results with antimalarial drugs, chosen on the basis of their free therapeutic maximum concentration in plasma (Cmax), were expressed as the fold of that particular effect with respect to their Cmax. The following tests were used at 37°C: hERG (human ether-à-go-go-related gene) blockade and trafficking, rabbit heart ventricular preparations, and sodium and slow potassium ion current interference (INa and I Ks, respectively). Chloroquine, halofantrine, mefloquine, and lumefantrine were tested in the hERG studies, but only chloroquine, dofetilide, lumefantrine, and the combination of artemetherlumefantrine were used in the rabbit heart ventricular preparations, hERG trafficking studies, and I Na and IKs analyses. A proper reference was used in each test. In hERG studies, the high 50% inhibitory concentration (IC50) of halofantrine, which was lower than its Cmax, was confirmed. All the other compounds blocked hERG, with IC50s ranging from 3- to 30-fold their Cmaxs. In hERG trafficking studies, the facilitative effects of chloroquine at about 30-fold its Cmax were confirmed and DHA blocked it at a concentration about 300-fold its Cmax. In rabbit heart ventricular preparations, dofetilide, used as a positive control, revealed a high risk of torsades de pointes, whereas chloroquine showed a medium risk. Neither DHA-PQP nor artemether-lumefantrine displayed an in vitro signal for a significant proarrhythmic risk. Only chloroquine blocked the INa ion current and did so at about 30-fold its Cmax. No effect on I Ks was detected. In conclusion, despite significant hERG blockade, DHA-PQP and artemether-lumefantrine do not appear to induce potential torsadogenic effects in vitro, affect hERG trafficking, or block sodium and slow potassium ion currents. Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 580.45K | Year: 2003

DESCRIPTION (provided by applicant): Atrial fibrillation (AF) is the most common cause of arrhythmias in the elderly; it has an incidence of more than 5 percent in people > 69 years of age. At present, there is no satisfactory treatment of this disease. The ChanTest Phase I SBIR was directed towards the discovery of novel drugs for this disease and in these experiments, the investigators identified a substituted piperidine compound that promises to be an effective antiarrhythmic agent. They found that this drug blocks the hERG/IKr current at low nanomolar concentrations, yet does not prolong the action potential duration in canine Purkinje fibers at micromolar concentrations as might be expected. The investigators hypothesized that the drug also blocked cardiac Na and Ca currents at nanomolar concentrations and, as a result, the hERG/IKr block was offset and there was no change in action potential duration. The drug had another useful characteristic, namely the forward use-dependence of a drug that is most effective at faster heart rates. This drug was in clinical trials in the late 1970s as an antidepressant and although it was safe, did not have the desired efficacy. It is now in clinical trials as a treatment for substance abuse. In neither of these trials were proarrhythmic tendencies noted and the ECGs in both sets of trials were unaffected. Because its properties are so favorable, ChanTest has filed a use patent on the drug for treatment of cardiac arrhythmias in general, and AF in particular. Given its very high affinity for hERG, a radioactive derivative can be used in high throughput displacement studies to test for non-cardiac drugs that may bind to hERG. Identifications of such drugs are of considerable importance for safety pharmacology. The specific aims of this proposal are to: 1) complete in vitro tests of the effects of the drug on other cardiac membrane currents ITo, IKs and IK1; 2) test the drug's efficacy in animal models of AF; 3) test the drug's safety in the cardiac muscle wedge preparation that is presently the best predictor of the potentially lethal ventricular arrhythmia torsade de pointes (TdP); and 4) characterize the drug congeners as tools for HTS displacement studies of drugs that bind hERG. After the drug passes the hurdles of the specific aims, ChanTest will file a 355(b)(2) NDA application with the FDA to go forward with the Phase II and III clinical trials. ChanTest believes that this drug will offer great relief to the many people who are debilitated by atrial fibrillation.

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