A computational method for identifying an optimal combination of existing drugs to repair the action potentials of SQT1 ventricular myocytes.

Mutations are known to cause perturbations in essential functional features of integral membrane proteins, including ion channels. Even restricted or point mutations can result in substantially changed properties of ion currents. The additive effect of these alterations for a specific ion channel can result in significantly changed properties of the action potential (AP). Both AP shortening and AP prolongation can result from known mutations, and the consequences can be life-threatening. Here, we present a computational method for identifying new drugs utilizing combinations of existing drugs. Based on the knowledge of theoretical effects of existing drugs on individual ion currents, our aim is to compute optimal combinations that can 'repair' the mutant AP waveforms so that the baseline AP-properties are restored. More specifically, we compute optimal, combined, drug concentrations such that the waveforms of the transmembrane potential and the cytosolic calcium concentration of the mutant cardiomyocytes (CMs) becomes as similar as possible to their wild type counterparts after the drug has been applied. In order to demonstrate the utility of this method, we address the question of computing an optimal drug for the short QT syndrome type 1 (SQT1). For the SQT1 mutation N588K, there are available data sets that describe the effect of various drugs on the mutated K+ channel. These published findings are the basis for our computational analysis which can identify optimal compounds in the sense that the AP of the mutant CMs resembles essential biomarkers of the wild type CMs. Using recently developed insights regarding electrophysiological properties among myocytes from different species, we compute optimal drug combinations for hiPSC-CMs, rabbit ventricular CMs and adult human ventricular CMs with the SQT1 mutation. Since the 'composition' of ion channels that form the AP is different for the three types of myocytes under consideration, so is the composition of the optimal drug.

Safety assessment of metarrestin in dogs: A clinical candidate targeting a subnuclear structure unique to metastatic cancer cells.

Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50 mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3 h (overall median Tmax) across all doses with a mean t1/2 over 168 h of 55.5 h. Dose dependent increase in systemic exposure (Cmax and AUClast) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female Cmax = 82.5 ng/mL and AUClast = 2521 h*ng/mL, on Day 27.

LUF7244 plus Dofetilide Rescues Aberrant Kv11.1 Trafficking and Produces Functional IKv11.1.

Voltage-gated potassium 11.1 (Kv11.1) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug-mediated Kv11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal Kv11.1 forward trafficking and thus reduce functional Kv11.1 channel densities. Although class III antiarrhythmics, e.g., dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use because of their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a Kv11.1 allosteric modulator/activator, to rescue Kv11.1 trafficking and produce functional Kv11.1 current. LUF7244 treatment by itself did not disturb or rescue wild type (WT) or G601S-Kv11.1 trafficking, as shown by Western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT Kv11.1 levels was rescued by 10 µM dofetilide or 10 µM dofetilide + 5 µM LUF7244. In trafficking defective G601S-Kv11.1 cells, dofetilide (10 µM) or dofetilide + LUF7244 (10 + 5 µM) also restored Kv11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 µM) increased IKv 11.1 despite the presence of dofetilide (1 µM) in WT Kv11.1 cells. In G601S-expressing cells, long-term treatment (24-48 hour) with LUF7244 (10 µM) and dofetilide (1 µM) increased IKv11.1 compared with nontreated or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues Kv11.1 trafficking and produces functional IKv11.1 Thus, combined administration of LUF7244 and an IKv11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced Kv11.1 trafficking defects. SIGNIFICANCE STATEMENT: Decreased levels of functional Kv11.1 potassium channel at the plasma membrane of cardiomyocytes prolongs action potential repolarization, which associates with cardiac arrhythmia. Defective forward trafficking of Kv11.1 channel protein is an important factor in acquired and congenital long QT syndrome. LUF7244 as a negative allosteric modulator/activator in combination with dofetilide corrected both congenital and acquired Kv11.1 trafficking defects, resulting in functional Kv11.1 current.

The macrolide drug erythromycin does not protect the hERG channel from inhibition by thioridazine and terfenadine.

The macrolide antibiotic erythromycin has been associated with QT interval prolongation and inhibition of the hERG-encoded channels responsible for the rapid delayed rectifier K+ current I(Kr ). It has been suggested that low concentrations of erythromycin may have a protective effect against hERG block and associated drug-induced arrhythmia by reducing the affinity of the pore-binding site for high potency hERG inhibitors. This study aimed to explore further the notion of a potentially protective effect of erythromycin. Whole-cell patch-clamp experiments were performed in which hERG-expressing mammalian (Human Embryonic Kidney; HEK) cells were preincubated with low to moderate concentrations of erythromycin (3 or 30 µM) prior to whole-cell patch clamp recordings of hERG current (IhERG ) at 37°C. In contrast to a previous report, exposure to low concentrations of erythromycin did not reduce pharmacological sensitivity of hERG to the antipsychotic thioridazine and antihistamine terfenadine. The IC50 value for IhERG tail inhibition by terfenadine was decreased by ~32-fold in the presence of 3 µM erythromycin (p < .05 vs. no preincubation). Sensitivity to thioridazine remained unchanged (p > .05 vs. no preincubation). The effects of low concentrations of erythromycin were investigated for a series of pore blocking drugs, and the results obtained were consistent with additive and/or synergistic effects. Experiments with the externally acting blocker BeKm-1 on WT hERG and a pore mutant (F656V) were used to explore the location of the binding site for erythromycin. Our data are inconsistent with the use of erythromycin for the management of drug-induced QT prolongation.

Identification of a proton sensor that regulates conductance and open time of single hERG channels.

The hERG potassium channel influences ventricular action potential duration. Extracellular acidosis occurs in pathological states including cardiac ischaemia. It reduces the amplitude of hERG current and speeds up deactivation, which can alter cardiac excitability. This study aimed to identify the site of action by which extracellular protons regulate the amplitude of macroscopic hERG current. Recordings of macroscopic and single hERG1a and 1b channel activity, mutagenesis, and the recent cryoEM structure for hERG were employed. Single hERG1a and 1b channels displayed open times that decreased with membrane depolarization, suggestive of a blocking mechanism that senses approximately 20% of the membrane electric field. This mechanism was sensitive to pH; extracellular acidosis reduced both hERG1a and1b channel open time and conductance. The effects of acidosis on macroscopic current amplitude and deactivation displayed different sensitivities to protons. Point mutation of a pair of residues (E575/H578) in the pore turret abolished the acidosis-induced decrease of current amplitude, without affecting the change in current deactivation. In single hERG1a channel recordings, the conductance of the double-mutant channel was unaffected by extracellular acidosis. These findings identify residues in the outer turret of the hERG channel that act as a proton sensor to regulate open time and channel conductance.

Histidine at position 462 determines the low quinine sensitivity of ether-à-go-go channel superfamily member Kv 12.1.

BACKGROUND AND PURPOSE: The ether-à-go-go (Eag) Kv superfamily comprises closely related Kv 10, Kv 11, and Kv 12 subunits. Kv 11.1 (termed hERG in humans) gained much attention, as drug-induced inhibition of these channels is a frequent cause of sudden death in humans. The exclusive drug sensitivity of Kv 11.1 can be explained by central drug-binding pockets that are absent in most other channels. Currently, it is unknown whether Kv 12 channels are equipped with an analogous drug-binding pocket and whether drug-binding properties are conserved in all Eag superfamily members. EXPERIMENTAL APPROACH: We analysed sensitivity of recombinant Kv 12.1 channels to quinine, a substituted quinoline that blocks Kv 10.1 and Kv 11.1 at low micromolar concentrations. KEY RESULTS: Quinine inhibited Kv 12.1, but its affinity was 10-fold lower than for Kv 11.1. Contrary to Kv 11.1, quinine inhibited Kv 12.1 in a largely voltage-independent manner and induced channel opening at more depolarised potentials. Low sensitivity of Kv 12.1 and characteristics of quinine-dependent inhibition were determined by histidine 462, as site-directed mutagenesis of this residue into the homologous tyrosine of Kv 11.1 conferred Kv 11.1-like quinine block to Kv 12.1(H462Y). Molecular modelling demonstrated that the low affinity of Kv 12.1 was determined by only weak interactions of residues in the central cavity with quinine. In contrast, more favourable interactions can explain the higher quinine sensitivity of Kv 12.1(H462Y) and Kv 11.1 channels. CONCLUSIONS AND IMPLICATIONS: The quinoline-binding "motif" is not conserved within the Eag superfamily, although the overall architecture of these channels is apparently similar. Our findings highlight functional and pharmacological diversity in this group of evolutionary-conserved channels.

Histone deacetylase inhibition by Entinostat for the prevention of electrical and structural remodeling in heart failure.

BACKGROUND: The development of heart failure is accompanied by complex changes in cardiac electrophysiology and functional properties of cardiomyocytes and fibroblasts. Histone deacetylase (HDAC) inhibitors hold great promise for the pharmaceutical therapy of several malignant diseases. Here, we describe novel effects of the class I HDAC inhibitor Entinostat on electrical and structural remodeling in an in vivo model of pacing induced heart failure. METHODS: Rabbits were implanted a pacemaker system, subjected to rapid ventricular pacing and treated with Entinostat or placebo, respectively. Following stimulation, rabbit hearts were explanted and subsequently subjected to electrophysiological studies and further immunohistological analyses of left ventricles. RESULTS: In vivo, rapid ventricular stimulation caused a significant prolongation of monophasic action potential duration compared to sham hearts (from 173 ± 26 ms to 250 ± 41 ms; cycle length 900 ms; p < 0.05) and an increased incidence of Early afterdepolarisations (+ 150%), while treatment with Entinostat in failing hearts could partially prevent this effect (from 250 ± 41 ms to 170 ± 53 ms, p < 0.05; reduction in EAD by 50%). Entinostat treatment partially restored KCNH2 and Cav1.3 gene expressions in failing hearts, and inhibited the development of cardiac fibrosis in vivo. CONCLUSION: In a rabbit model of heart failure, Entinostat diminishes heart failure related prolongation of repolarization and partially restores KCNH2 and Cav1.3 expression. In addition, Entinostat exerts antifibrotic properties both in vitro and in vivo. Thus, Entinostat might be an interesting candidate for the pharmaceutical therapy of heart failure directed against structural and electrical remodeling.

Lipid roles in hERG function and interactions with drugs.

Human-ether-a-go-go-related channel (hERG) is a voltage gated potassium channel (Kv11.1) abundantly expressed in heart and brain tissues. In addition to playing an important role in mediation of repolarizing K+ currents (IKr) in Action Potential (AP), hERG is notorious for its propensity to interact with various medications. The drug-induced block of K+ currents across hERG channel are strongly associated with dysrhythmic conditions collectively known as drug-induced long-QT-syndrome. The recent availability of the high-resolution Cryo-EM structures for the hERG channel has provided unique opportunity to resolve structural mechanisms involved into the process of voltage-gating of hERG channels, map various roles played by components of ventricular and neuronal membranes and then to connect it to cellular pathways through which diverse chemical compounds might be affecting function of the channel. Specifically, lipids and lipid derivatives such as polyunsaturated fatty acids (PUFAs), ceramides and steroids have been shown to directly interact with the lipid facing amino acids in various Kv channels including hERG. In this review, possible lipophilic pathways of hERG activators and blockers, together with the existence of fenestration windows and effects of PUFAs, ceramides and steroids are explored throughout different sections. Finally, the interplay between long QT inducing drugs and phospholipidosis is briefly discussed.

CiPA challenges and opportunities from a non-clinical, clinical and regulatory perspectives. An overview of the safety pharmacology scientific discussion.

The Safety Pharmacology Society organized a scientific session at its annual conference in 2017 to discuss the challenges and opportunities of the Comprehensive In-Vitro Proarrhythmia Assay (CiPA) paradigm. Our intention was to raise awareness of this initiative with its members and also to gauge the extent to which safety pharmacologists have incorporated the CiPA testing strategy within the pharmaceutical industry. CiPA offers many potential opportunities including 1) a focus on proarrhythmic risk (as opposed to QTc prolongation), 2) providing scientific rationale to support the continued development of compounds that may have a poor selectivity over hERG whilst also blocking other inward currents and 3) reducing the extent of ECG monitoring in clinical trials with a greater influence of the non-clinical studies. Such opportunities may speed drug development and reduce costs. However, there are also challenges for CiPA implementation. For example, the mixed ion channel paradigm does not easily lend itself to a prospective drug discovery strategy although testing for such effects can be achieved with assays with good throughput. However, it should also be recognized that compounds with a mixed ion channel profile might also have properties that are undesirable to treat non-life threatening indications. All components of CiPA (nonclinical and clinical) require validation, particularly as a composite package to impact drug development and evaluation. One of the significant discussion points was that the existing regulatory guidance supports the use of components of CiPA through follow-up studies. A survey of the conference audience showed that the level of awareness of CiPA is quite high and that companies are already conducting some testing against a wider panel of cardiac ion channels beyond hERG. However, the adoption of other technologies (stem cell derived cardiac myocytes and in silico modeling) is less well developed. Taken together, the session demonstrated the potential advantages of CiPA, but also some significant challenges.

Common variants in the hERG (KCNH2) voltage-gated potassium channel are associated with altered fasting and glucose-stimulated plasma incretin and glucagon responses.

BACKGROUND: Patients with long QT syndrome due to rare loss-of-function mutations in the human ether-á-go-go-related gene (hERG) have prolonged QT interval, risk of arrhythmias, increased secretion of insulin and incretins and impaired glucagon response to hypoglycemia. This is caused by a dysfunctional Kv11.1 voltage-gated potassium channel. Based on these findings in patients with rare variants in hERG, we hypothesized that common variants in hERG may also lead to alterations in glucose homeostasis. Subsequently, we aimed to evaluate the effect of two common gain-of-function variants in hERG (rs36210421 and rs1805123) on QT interval and plasma levels of glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), insulin and glucagon during an oral glucose tolerance test (OGTT). We used two population-based cohorts for evaluation of the effect of common variants in hERG on QT-interval and circulation levels of incretins, insulin and glucagon. The Danish population-based Inter99 cohort (n = 5895) was used to assess the effect of common variants on QT-interval. The Danish ADDITION-PRO cohort was used (n = 1329) to study genetic associations with levels of GLP-1, GIP, insulin and glucagon during an OGTT. RESULTS: Carriers of either the minor A-allele of rs36210421 or the minor G-allele of rs1805123 had ~ 2 ms shorter QT interval per risk allele (p = 0.025 and p = 1.9 × 10- 7). Additionally, both variants were associated with alterations in pancreatic and gut hormone release among carriers. The minor A- allele of rs36210421 was associated with increased GLP-1 and decreased GIP response to oral glucose stimulation, whereas the minor G-allele of rs1805123 is associated with decreased fasting plasma insulin and glucagon release. A genetic risk score combining the two gene variants revealed reductions in glucose-stimulated GIP, as well as suppressed glucagon response to increased glucose levels during an OGTT. CONCLUSIONS: Two common missense polymorphisms of the Kv11.1 voltage-gated hERG potassium channel are associated with alterations in circulating levels of GIP and glucagon, suggesting that hERG potassium channels play a role in fasting and glucose-stimulated release of GIP and glucagon. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT00289237 ). Trial retrospectively registered at February 9, 2006. Studies were approved by the Ethical Committee of the Central Denmark Region (journal no. 20080229) and by the Copenhagen County Ethical Committee (KA 98155).

Effects of norquetiapine, the active metabolite of quetiapine, on cloned hERG potassium channels.

Quetiapine is an atypical antipsychotic drug that is widely used for the treatment of schizophrenia. It is mainly metabolized by a cytochrome P450 system in the liver. Norquetiapine is a major active metabolite in humans with a pharmacological profile that differs distinctly from that of quetiapine. We used the whole-cell patch-clamp technique to investigate the effects of norquetiapine on hERG channels that are stably expressed in HEK cells. Quetiapine and norquetiapine inhibited the hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 8.3 and 10.8µM, respectively, which suggested equal potency. The block of hERG currents by norquetiapine was voltage-dependent with a steep increase over a range of voltages for channel activation. However, at more depolarized potentials where the channels were fully activated, the block by norquetiapine was voltage-independent. The steady-state inactivation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of norquetiapine. Norquetiapine did not produce a use-dependent block. A fast application of norquetiapine inhibited the hERG current elicited by a 5s depolarizing pulse to +60mV, which fully inactivated the hERG currents, suggesting an inactivated-state block. During a repolarizing pulse wherein the hERG current was slowly deactivated, albeit remaining in an open state, a fast application of norquetiapine rapidly and reversibly inhibited the open state of the hERG current. Our results indicated that quetiapine and norquetiapine had equal potency in inhibiting hERG tail currents. Norquetiapine inhibited the hERG current by preferentially interacting with the open and/or inactivated states of the channels.

Nonclinical safety assessment of SPX-101, a novel peptide promoter of epithelial sodium channel internalization for the treatment of cystic fibrosis.

BACKGROUND: ENaC inhibition has long been an attractive therapeutic target for the treatment of cystic fibrosis. However, previous attempts at developing ENaC inhibitors have been unsuccessful due to complications arising from systemic circulation of the compounds. Here, we describe the preclinical toxicology assessment of a new inhaled peptide promoter of ENaC internalization delivered as a nebulized aerosol. METHODS: Preclinical assessment of SPX-101 safety was determined using an in vitro hERG assay, bolus injection of SPX-101 in a canine cardiovascular and respiratory safety pharmacology model and 28-day inhalation toxicology studies of nebulized drug in rats and dogs. RESULTS: SPX101 had no effects on the respiratory, cardiac or central nervous systems. The 28-day inhalation toxicology studies of nebulized SPX-101 in rats and dogs revealed no drug-related adverse events. Plasma levels of SPX-101 peaked less than 1 h after the end of treatment in rats and were below the limit of detection in canine models. CONCLUSIONS: SPX-101, a novel peptide promoter of ENaC internalization, elicited no adverse effects at doses up to the MFD and in excess of the highest preclinical efficacious and expected clinical doses. In contrast to channel blockers like amiloride and derivative small molecules, SPX-101 does not achieve significant systemic circulation, thus doses are not limited due to toxic side effects like hyperkalemia and weight loss.

Gene Signature of High White Blood Cell Count in B-Precursor Acute Lymphoblastic Leukemia.

In this study we sought to identify genetic factors associated with the presenting white blood cell (WBC) count in B-precursor acute lymphoblastic leukemia (BP-ALL). Using ETV6-RUNX1-positive BP-ALL patient samples, a homogeneous subtype, we identified 16 differentially expressed genes based on the presenting WBC count (< 50,000/cumm vs > 50,000). We further confirmed that IL1R1, BCAR3, KCNH2, PIR, and ZDHHC23 were differentially expressed in a larger cohort of ETV6-RUNX1-negative BP-ALL patient samples. Statistical analysis demonstrated that expression levels of these genes could accurately categorize high and low WBC count subjects using two independent patient sets, representing positive and negative ETV6-RUNX1 cases. Further studies in leukemia cell line models will better delineate the role of these genes in regulating the white blood cell count and potentially identify new therapeutic targets.

Mitragynine and its potential blocking effects on specific cardiac potassium channels.

Mitragyna speciosa Korth is known for its euphoric properties and is frequently used for recreational purposes. Several poisoning and fatal cases involving mitragynine have been reported but the underlying causes remain unclear. Human ether-a-go-go-related gene (hERG) encodes the cardiac IKr current which is a determinant of the duration of ventricular action potentials and QT interval. On the other hand, IK1, a Kir current mediated by Kir2.1 channel and IKACh, a receptor-activated Kir current mediated by GIRK channel are also known to be important in maintaining the cardiac function. This study investigated the effects of mitragynine on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells and Xenopus oocytes. The effects on Kir2.1 and GIRK channels currents were also determined in the oocytes. The hERG tail currents following depolarization pulses were inhibited by mitragynine with an IC50 value of 1.62µM and 1.15µM in the transfected cell line and Xenopus oocytes, respectively. The S6 point mutations of Y652A and F656A attenuated the inhibitor effects of mitragynine, indicating that mitragynine interacts with these high affinity drug-binding sites in the hERG channel pore cavity which was consistent with the molecular docking simulation. Interestingly, mitragynine does not affect the hERG expression at the transcriptional level but inhibits the protein expression. Mitragynine is also found to inhibit IKACh current with an IC50 value of 3.32µM but has no significant effects on IK1. Blocking of both hERG and GIRK channels may cause additive cardiotoxicity risks.

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

KEY POINTS: Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. ABSTRACT: In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.

Differentiation of ROMK potency from hERG potency in the phenacetyl piperazine series through heterocycle incorporation.

Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.

Nonclinical Safety Profile of Etelcalcetide, a Novel Peptide Calcimimetic for the Treatment of Secondary Hyperparathyroidism.

Etelcalcetide is a novel d-amino acid peptide that functions as an allosteric activator of the calcium-sensing receptor and is being developed as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on hemodialysis. To support clinical development and marketing authorization, a comprehensive nonclinical safety package was generated. Primary adverse effects included hypocalcemia, tremoring, and convulsions. Other adverse effects were considered sequelae of stress associated with hypocalcemia. Cardiovascular safety evaluations in the dog revealed an anticipated prolongation of the corrected QT interval that was related to reductions in serum calcium. Etelcalcetide did not affect the human ether-a-go-go gene ion channel current. Etelcalcetide was mutagenic in some strains of Salmonella, however, based on the negative results in 2 in vitro and 2 in vivo mammalian genotoxicity assays, including a 28-day Muta mouse study, etelcalcetide is considered nongenotoxic. Further support for a lack of genotoxicity was provided due to the fact that etelcalcetide was not carcinogenic in a 6-month transgenic rasH2 mouse model or a 2-year study in rats. There were no effects on fertility, embryo-fetal development, and prenatal and postnatal development. All of the adverse effects observed in both rat and dog were considered directly or secondarily related to the pharmacologic activity of etelcalcetide and the expected sequelae associated with dose-related reductions in serum calcium due to suppression of parathyroid hormone secretion. These nonclinical data indicate no safety signal of concern for human risk beyond that associated with hypocalcemia and associated QT prolongation.

Why Can dl-Sotalol Prolong the QT Interval In Vivo Despite Its Weak Inhibitory Effect on hERG K(+) Channels In Vitro? Electrophysiological and Pharmacokinetic Analysis with the Halothane-Anesthetized Guinea Pig Model.

In order to bridge the gap of action of dl-sotalol between the human ether-a-go-go-related gene (hERG) K(+) channel inhibition in vitro and QT-interval prolongation in vivo, its electropharmacological effect and pharmacokinetic property were simultaneously studied in comparison with those of 10 drugs having potential to prolong the QT interval (positive drugs: bepridil, haloperidol, dl-sotalol, terfenadine, thioridazine, moxifloxacin, pimozide, sparfloxacin, diphenhydramine, imipramine and ketoconazole) and four drugs lacking such property (negative drugs: enalapril, phenytoin, propranolol or verapamil) with the halothane-anesthetized guinea pig model. A dose of each drug that caused 10 % prolongation of Fridericia-corrected QT interval (QTcF) was calculated, which was compared with respective known hERG K(+) IC50 value and currently obtained heart/plasma concentration ratio. Each positive drug prolonged the QTcF in a dose-related manner, whereas such effect was not observed by the negative drugs. Drugs with more potent hERG K(+) channel inhibition showed higher heart/plasma concentration ratio, resulting in more potent QTcF prolongation in vivo. The potency of dl-sotalol for QTcF prolongation was flat middle, although its hERG K(+) channel inhibitory property as well as heart/plasma concentration ratio was the smallest among the positive drugs, which may be partly explained by its lack of binding to plasma protein.