Homologation of the Alkyl Side Chain of Antimitotic Phenyl 4-(2-Oxo-3-alkylimidazolidin-1-yl)benzenesulfonate Prodrugs Selectively Targeting CYP1A1-Expressing Breast Cancers Improves Their Stability in Rodent Liver Microsomes.

Phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) are a new family of antimitotic prodrugs bioactivated in breast cancer cells expressing CYP1A1. In this study, we report that the 14C-labeled prototypical PAIB-SO [14C]CEU-818 and its antimitotic counterpart [14C]CEU-602 are distributed in whole mouse body and they show a short half-life in mice. To circumvent this limitation, we evaluated the effect of the homologation of the alkyl side chain of the imidazolidin-2-one moiety of PAIB-SOs. Our studies evidence that PAIB-SOs bearing an n-pentyl side chain exhibit antiproliferative activity in the nanomolar-to-low-micromolar range and a high selectivity toward CYP1A1-positive breast cancer cells. Moreover, the most potent n-pentyl PAIB-SOs were significantly more stable toward rodent liver microsomes. In addition, PAIB-SOs 10 and 14 show significant antitumor activity and low toxicity in chorioallantoic membrane (CAM) assay. Our study confirms that homologation is a suitable approach to improve the rodent hepatic stability of PAIB-SOs.

Development and Evaluation of a Height-Based Tobramycin Initial Dosing Nomogram for the Treatment of Adult Cystic Fibrosis Pulmonary Exacerbations.

ABSTRACT: Tobramycin is widely used to treat pulmonary exacerbations of cystic fibrosis. Height has been previously found to be significantly more predictive of tobramycin pharmacokinetics than body weight. This study aimed to develop a height-based initial dosing nomogram and evaluate its performance in peak concentration (Cmax) precision relative to standard and fixed dosing. Monte Carlo simulations were performed to develop a nomogram representing the doses required to reach Cmax targets at different heights. Cmax data observed at 2 clinical centers [McGill University Health Centre (MUHC) and Institut universitaire de cardiologie et pneumologie de Québec (IUCPQ-UL)] were compared with population-predicted Cmax using the doses derived from the nomogram alongside a fixed dose. Height-based dosing resulted in significantly less variable-predicted Cmax values [coefficient of variation (CV) MUHC = 15.7% and IUCPQ-UL = 10.8%] than the Cmax values observed in clinical practice (CV MUHC = 30.0% and CV IUCPQ-UL = 26.9%) and predicted Cmax values obtained from a fixed dose (CV MUHC = 21.2% and CV IUCPQ-UL = 16.3%). An initial dosing nomogram was developed to help reduce pharmacokinetic variability in the observed Cmax. More precise dosing would allow for better clinical outcomes in adult patients with cystic fibrosis.

Remdesivir (VEKLURY) for Treating COVID-19: Guinea Pig Ex Vivo and In Vivo Cardiac Electrophysiological Effects.

ABSTRACT: Bradycardia and QTc interval prolongation on the ECG have been reported with remdesivir (Veklury), an antiviral drug recently approved for treating severely ill patients with COVID-19. The objective was to evaluate the effects of remdesivir on cardiac electrophysiology ex vivo and in vivo. Ex vivo: Langendorff retroperfusion experiments were performed on isolated hearts from male Hartley guinea pigs (n = 23, total) exposed to either remdesivir 3, 10, or 30 µmol/L to assess drug-induced prolongation of the monophasic action potential duration measured at 90% repolarization (MAPD 90 ). In vivo: ECG recordings using wireless cardiac telemetry were performed in guinea pigs (n = 6) treated with daily i.p. doses of remdesivir 5 mg/kg on day 1 and 2.5 mg/kg on days 2-10. Ex vivo remdesivir (3, 10, and 30 µmol/L) had no statistically significant effect on MAPD 90 , while pacing the hearts at basic stimulation cycle lengths of 200 or 250 milliseconds, or when the hearts were not paced and beating at their intrinsic heart rate. In a second set of similar ex vivo experiments, remdesivir 10 µmol/L did not potentiate the MAPD 90 -prolonging effects of dofetilide 20 nmol/L (n = 4) hearts. In vivo remdesivir caused small but statistically significant prolongations of the RR and QTc F intervals at day 1 (5 mg/kg) and at day 10 (2.5 mg/kg). No ventricular arrhythmias were ever observed under the effect of remdesivir. Remdesivir causes bradycardia, and mild QTc prolongation, which nonetheless, could be of clinical relevance in many hospitalized patients with COVID-19 concomitantly treated with multiple drugs.

Consideration of height-based tobramycin dosing regimens for the treatment of adult cystic fibrosis pulmonary exacerbations.

AIMS: Some population pharmacokinetic models have been developed using height to explain some of the interindividual variability in tobramycin pharmacokinetics in cystic fibrosis patients. However, their predictive performance when extrapolated to other clinical centres is unclear. Therefore, the aim of this study was to externally evaluate the predictability of tobramycin population pharmacokinetic models with an independent dataset and perform simulations using previously recommended height-based dosing regimens. METHODS: A literature search was conducted through the PubMed database to identify relevant population pharmacokinetic models. Tobramycin plasma concentration data from April 2014 to November 2019 were retrospectively collected from the Institut universitaire de cardiologie et de pneumologie de Québec, Canada. External evaluations were performed using NONMEM® v7.5 and RStudio® v1.3.1073. Monte Carlo simulations were performed to evaluate the probability of target attainment of Cmax /MIC ratios for several dosing regimens. RESULTS: The validation dataset included 27 patients and 143 concentration samples. Three models were evaluated. Only the ones by Crass et al. and Alghanem et al. performed satisfactorily in terms of prediction-based diagnostics with MDPE values of -3.4% and 29.3% and MDAPE values of 19.0 and 29.5%, respectively. In simulation-based evaluations, both pcVPC and NPDE showed no evidence of model misspecification. Our simulations suggest that patients treated with a once-daily dose of 3.4 mg/cm should produce peak and trough levels consistent with current guidelines. CONCLUSION: Our results show that the models by Crass et al. and Alghanem et al. are appropriate for simulation-based applications to aid individualized dosing in our population and that height-based dosing regimens could be considered in cystic fibrosis patients.

SCN5A-C683R exhibits combined gain-of-function and loss-of-function properties related to adrenaline-triggered ventricular arrhythmia.

NEW FINDINGS: What is the role of SCN5A-C683R? SCN5A-C683R is a novel variant associated with an uncommon phenotype of adrenaline-triggered ventricular arrhythmia in the absence of a distinct ECG phenotype. What is the main finding and its importance? Functional studies demonstrated that NaV 1.5/C683R results in a mixed electrophysiological phenotype with gain-of-function (GOF) and loss-of-function (LOF) properties compared with NaV 1.5/wild type. Gain-of-function properties are characterized by a significant increase of the maximal current density and a hyperpolarizing shift of the steady-state activation. The LOF effect of NaV 1.5/C683R is characterized by increased closed-state inactivation. Electrophysiological properties and clinical manifestation of SCN5A-C683R are different from long-QT-3 or Brugada syndrome and might represent a distinct inherited arrhythmia syndrome. ABSTRACT: Mutations of SCN5Ahave been identified as the genetic substrate of various inherited arrhythmia syndromes, including long-QT-3 and Brugada syndrome. We recently identified a novel SCN5A variant (C683R) in two genetically unrelated families. The index patients of both families experienced adrenaline-triggered ventricular arrhythmia with cardiac arrest but did not show a specific ECG phenotype, raising the hypothesis that SCN5A-C683R might be a susceptibility variant and the genetic substrate of distinct inherited arrhythmia. We conducted functional cellular studies to characterize the electrophysiological properties of NaV 1.5/C683R in order to explore the potential pathogenicity of this novel variant. The C683R variant was engineered by site-directed mutagenesis. NaV 1.5/wild type (WT) and NaV 1.5/C683R were expressed in tsA201 cells. Electrophysiological characterization of C683R was performed using the whole-cell patch-clamp technique. Adrenergic stimulation was mimicked by exposure to the protein kinase A activator 8-CPT-cAMP. The impact of ß-blockers was tested by exposing NaV 1.5/WT and NaV 1.5/C683R currents to propranolol and nadolol. C683R resulted in a co-association of gain-of-function and loss-of-function properties of NaV 1.5. Gain-of-function properties were characterized by a significant increase of the maximal NaV 1.5 current density compared with NaV 1.5/WT (861 ± 309 vs. 627 ± 489 pA/pF; P < 0.05, n ≥ 9) that was potentiated in NaV 1.5/C683R with 8-CPT-cAMP stimulation (869 ± 287 vs. 607 ± 320 pA/pF; P < 0.05, n ≥ 12). C683R also resulted in a significant hyperpolarizing shift in the voltage of steady-state activation (-65.4 ± 3.0 vs. -57.2 ± 4.8 mV; P < 0.001), resulting in an increased window current compared with WT. The loss-of-function effect of NaV 1.5/C683R was characterized by significantly increased closed-state inactivation compared with NaV 1.5/WT (P < 0.05). C683R is a novel SCN5A variant resulting in a co-association of gain-of-function and loss-of-function properties of the cardiac sodium channel NaV 1.5. The phenotype is characterized by adrenaline-triggered ventricular arrhythmias. Electrophysiological properties and clinical manifestations are different from long-QT-3 or Brugada syndrome and might represent a distinct inherited arrhythmia syndrome.

Altered Protein Expression of Cardiac CYP2J and Hepatic CYP2C, CYP4A, and CYP4F in a Mouse Model of Type II Diabetes-A Link in the Onset and Development of Cardiovascular Disease?

Arachidonic acid can be metabolized by cytochrome P450 (CYP450) enzymes in a tissue- and cell-specific manner to generate vasoactive products such as epoxyeicosatrienoic acids (EETs-cardioprotective) and hydroxyeicosatetraenoic acids (HETEs-cardiotoxic). Type II diabetes is a well-recognized risk factor for developing cardiovascular disease. A mouse model of Type II diabetes (C57BLKS/J-db/db) was used. After sacrifice, livers and hearts were collected, washed, and snap frozen. Total proteins were extracted. Western blots were performed to assess cardiac CYP2J and hepatic CYP2C, CYP4A, and CYP4F protein expression, respectively. Significant decreases in relative protein expression of cardiac CYP2J and hepatic CYP2C were observed in Type II diabetes animals compared to controls (CYP2J: 0.80 ± 0.03 vs. 1.05 ± 0.06, n = 20, p < 0.001); (CYP2C: 1.56 ± 0.17 vs. 2.21 ± 0.19, n = 19, p < 0.01). In contrast, significant increases in relative protein expression of both hepatic CYP4A and CYP4F were noted in Type II diabetes mice compared to controls (CYP4A: 1.06 ± 0.09 vs. 0.18 ± 0.01, n = 19, p < 0.001); (CYP4F: 2.53 ± 0.22 vs. 1.10 ± 0.07, n = 19, p < 0.001). These alterations induced by Type II diabetes in the endogenous pathway (CYP450) of arachidonic acid metabolism may increase the risk for cardiovascular disease by disrupting the fine equilibrium between cardioprotective (CYP2J/CYP2C-generated) and cardiotoxic (CYP4A/CYP4F-generated) metabolites of arachidonic acid.

Fingolimod (Gilenya® ) in multiple sclerosis: bradycardia, atrioventricular blocks, and mild effect on the QTc interval. Something to do with the L-type calcium channel?

Cardiac arrhythmias and ECG abnormalities including bradycardia, prolongation of the QT interval, and atrioventricular (AV) conduction blocks have been extensively observed with fingolimod, the first marketed oral drug for treating the relapsing-remitting form of multiple sclerosis. This study was aiming to further elucidate the effects of fingolimod on cardiac electrophysiology at three different levels: (i) in vitro, (ii) ex vivo, and (iii) in vivo. (i) Patch-clamp experiments in whole cell configuration were performed on Cav 1.2-transfected tsA201 cells exposed to fingolimod-phosphate 100 or 500 nmol/L (n = 27 cells, total) to measure drug effect on L-type calcium current (ICaL ). (ii) Langendorff perfusion experiments were undertaken on male Hartley guinea-pigs isolated hearts (n = 4) exposed to fingolimod 10 and 100 nmol/L to evaluate drug-induced effects on monophasic action potential duration measured at 90% repolarization (MAPD90 ). (iii) Implanted cardiac telemeters were used to record ECGs in guinea-pigs (n = 7) treated with a single dose of fingolimod 0.0625 mg/kg suspension, administered as an oral gavage. (i) In vitro cellular experiments showed that fingolimod-phosphate causes a concentration-dependent reduction in ICaL . (ii) Ex vivo Langendorff experiments revealed that fingolimod had no significant effect on MAPD90 . (iii) Fingolimod caused significant prolongations of the RR, PR, QT, and QTcF intervals in vivo. Reversible AV blocks were also observed in 7/7 animals. Fingolimod possesses ICaL -blocking properties, further contributing to its AV conduction-slowing effects. These properties are also consistent with its mitigated effect on the QT interval in humans, despite previously shown HERG-blocking effect.

The guinea-pig expresses functional CYP2C and P-glycoprotein: further validation of its usefulness in drug biotransformation/transport studies.

BACKGROUND: The guinea-pig is an excellent animal model for studying cardiopulmonary physiology/pharmacology. Interestingly, it also possesses a number of drug-metabolizing enzymes found in humans, such as CYP1A, CYP2D and CYP3A. OBJECTIVE: To evaluate the hypothesis that the guinea-pig also expresses a functional CYP2C drug-metabolizing enzyme and the P-glycoprotein (P-gp) drug transporter in various tissues. METHODS: cDNAs encoding CYP2C and P-gp were obtained from guinea-pig liver or small intestine and sequenced. Western blotting was performed to confirm the expression of CYP2C and P-gp. The functional enzymatic activity of guinea-pig CYP2C was evaluated with microsomal preparations using diclofenac and tolbutamide as specific drug substrates in HPLC analyses. To further study both P-gp and CYP2C functional activities, the guinea-pig ABCB1/MDR1 and CYP2C genes were cloned. The recombinant plasmids were then transfected in HEK293 (human embryonic kidney) cells and either calcein-acetoxymethyl ester (AM) accumulation assays or 14,15-EET/DHET formation experiments were performed to evaluate either P-gp transport activity or CYP2C epoxygenase activity, respectively. The guinea-pig tissue distribution of P-gp was studied by Western blotting. RESULTS: Functional expression of CYP2C was demonstrated in guinea-pig liver microsomal preparations. CYP2C-mediated biotransformation of diclofenac and tolbutamide were shown. Expression of P-gp protein was detected in guinea-pig liver and small intestine. Functional activity of guinea-pig P-gp was demonstrated in ABCB1/MDR1-transfected cells. GP-CYP2C-transfected cells also showed functional epoxygenase activity. CONCLUSION: The guinea-pig expresses functional CYP2C and P-gp, thus suggesting its usefulness for further validating data obtained with other animal models in drug biotransformation/transport studies.

Modulation of CYP3a expression and activity in mice models of type 1 and type 2 diabetes.

CYP3A4, the most abundant cytochrome P450 enzyme in the human liver and small intestine, is responsible for the metabolism of about 50% of all marketed drugs. Numerous pathophysiological factors, such as diabetes and obesity, were shown to affect CYP3A activity. Evidences suggest that drug disposition is altered in type 1 (T1D) and type 2 diabetes (T2D). The objective was to evaluate the effect of T1D and T2D on hepatic and intestinal CYP3a drug-metabolizing activity/expression in mice. Hepatic and intestinal microsomes were prepared from streptozotocin-induced T1D, db/db T2D and control mice. Domperidone was selected as a probe substrate for CYP3a and formation of five of its metabolites was evaluated using high performance liquid chromatography. Hepatic CYP3a protein and mRNA expression were assessed by Western blot and reverse-transcription quantitative polymerase chain reaction respectively. Hepatic microsomal CYP3a activity was significantly increased in both T1D and T2D groups versus control group. Intestinal CYP3a activity was also significantly increased in both T1D and T2D groups. Moreover, significant increases of both hepatic CYP3a mRNAs and protein expression were observed in both T1D and T2D groups versus control group. Additional experiments with testosterone further validated the increased activity of CYP3a under the effect of both T1D and T2D. Although differences exist in the pathophysiological insults associated with T1D and T2D, our results suggest that these two distinct diseases may have the same modulating effect on the regulation of CYP3a, ultimately leading to variability in drug response, ranging from lack of effect to life-threatening toxicity.

Decreased CYP3A expression and activity in guinea pig models of diet-induced metabolic syndrome: is fatty liver infiltration involved?

BACKGROUND: In humans, CYP3A drug-metabolizing enzyme subfamily is the most important. Numerous pathophysiological factors, such as diabetes and obesity, were shown to affect CYP3A activity. Often considered a precursor state for type II diabetes, metabolic syndrome exerts a modulating role on CYP3A, in our hypothesis. OBJECTIVE: To evaluate the effect of metabolic syndrome on CYP3A drug-metabolizing activity/expression in guinea pigs. METHODS: Hepatic microsomes were prepared from male Hartley guinea pigs fed with a control, a high-fat high sucrose (HFHS) or a high-fat high fructose diet (HFHF). Domperidone was selected as a probe substrate of CYP3A and formation of four of its metabolites was evaluated using high-performance liquid chromatography. CYP3A protein and mRNA expression were assessed by Western blot and reverse-transcription quantitative polymerase chain reaction, respectively. Hepatic fatty infiltration was evaluated using standard Oil Red O staining. Triglyceride and free fatty acid liver content were also quantified. RESULTS: Microsomal CYP3A activity was significantly decreased in both HFHS and HFHF diet groups versus the control diet group. Significant decreases of CYP3A mRNA and protein expression were observed in both HFHS and HFHF diet groups. Oil Red O staining showed a massive liver fatty infiltration in the HFHS and HFHF diet groups, which was not observed in the control diet group. Both triglyceride and free fatty acid liver content were significantly increased in the HFHS and HFHF diet groups. CONCLUSION: Diet-induced metabolic syndrome decreases CYP3A expression/activity in guinea pigs. This may ultimately lead to variability in drug response, ranging from lack of effect to life-threatening toxicity.

Iloperidone (Fanapt®), a novel atypical antipsychotic, is a potent HERG blocker and delays cardiac ventricular repolarization at clinically relevant concentration.

QT interval prolongation on the electrocardiogram (ECG) has extensively been reported with iloperidone, a novel antipsychotic drug. The objective of the present study was to evaluate the effects of iloperidone on cardiac ventricular repolarization at three different levels; in vitro, ex vivo and in vivo. (1) In vitro level: whole-cell patch-clamp experiments were performed on HERG-transfected HEK293 cells exposed to iloperidone 0.01-1 µmol/L (n = 35 cells, total) to assess drug effect on HERG current. (2) Ex vivo level: Langendorff retroperfusion experiments were performed on isolated hearts from male Hartley guinea pigs (n = 7) exposed to iloperidone 100 nmol/L with/without chromanol 293B 10 µmol/L to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization (MAPD(90)). (3) In vivo level: ECG recordings using wireless cardiac telemetry were performed in guinea pigs (n = 5) implanted with radio transmitters and treated with a single oral gavage dose of iloperidone 3 mg/kg. (1) Patch-clamp experiments revealed an estimated IC50 for iloperidone on HERG current of 161 ± 20 nmol/L. (2) While pacing the hearts at stimulation cycle lengths of 200 or 250 ms, or during natural sinus rhythm (no external pacing), iloperidone 100 nmol/L prolonged MAPD(90) by respectively 9.2 ± 0.9, 11.2 ± 1.6 and 21.4 ± 2.3 ms. After adding chromanol 293B, MAPD(90) was further prolonged by 7.3 ± 3.3, 11.5 ± 2.3 and 29.2 ± 6.7 ms, respectively. (3) Iloperidone 3mg/kg p.o. caused a maximal 42.7 ± 10.2 ms prolongation of corrected QT interval (QTc(F)), 40 min after administration. Iloperidone prolongs the QT interval, the cardiac action potentials and is a potent HERG blocker. Patients are at increased risk of cardiac proarrhythmia during iloperidone treatment, as this drug possesses significant cardiac repolarization-delaying properties at clinically relevant concentration.

Galantamine (Reminyl) delays cardiac ventricular repolarization and prolongs the QT interval by blocking the HERG current.

Galantamine is a reversible inhibitor of acetylcholinesterase and an allosteric-potentiating ligand of the nicotinic acetylcholine receptors. It is used for treating mild-to-moderate Alzheimer's disease. Interestingly, QT interval prolongation on the electrocardiogram (ECG), malignant ventricular arrhythmias and syncope have been reported with galantamine. Our objective was to evaluate the effects of galantamine on cardiac ventricular repolarization. Three sets of experiments were undertaken: 1) Whole cell patch-clamp experiments: HERG- or KCNQ1+KCNE1-transfected cells were exposed to galantamine 0.1-1000 µmol/l (n=25 cells, total) to assess drug effect on HERG and KCNQ1+KCNE1 currents. 2) Langendorff perfusion experiments: Isolated hearts from male Hartley guinea pigs (n=9) were exposed to galantamine 1 µmol/l to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization (MAPD(90)). 3) Cardiac telemetry experiments: Guinea pigs (n=7) implanted with wireless transmitters were injected a single intraperitoneal (i.p.) dose of galantamine 3mg/kg and 24h ECG recordings were made. 1) The estimated IC(50) for galantamine on HERG current was 760.2 µmol/l. Moreover, galantamine 10 µmol/l had a small inhibiting effect on KCNQ1+KCNE1 current (12.17 ± 2.19% inhibition, n=10 cells). 2) While pacing at cycle lengths of 150, 200 or 250 ms, galantamine 1 µmol/l prolonged MAPD(90) by respectively 5.1 ± 1.6 ms, 9.4 ± 1.9 ms and 12.1 ± 2.1 ms. 3) Galantamine 3 mg/kgi.p. caused a maximal 11.9 ± 2.7 ms prolongation of the corrected QT (QTc). Galantamine is a weak HERG blocker. This contributes to its mild QT-prolonging effect. Patients could be at risk of cardiac proarrhythmia during drug overdosage or interactions involving cytochrome 2D6 drug-metabolizing enzyme.

Metabolic syndrome potentiates the cardiac action potential-prolonging action of drugs: a possible 'anti-proarrhythmic' role for amlodipine.

Type II diabetes was shown to prolong the QT interval on the ECG and to promote cardiac arrhythmias. This is not so clear for metabolic syndrome, a precursor state of type II diabetes. The objectives of the present study were to generate a guinea pig model of metabolic syndrome by long-term exposure to diabetogenic diets, and to evaluate the monophasic action potential duration (MAPD)-modulating effects of drugs in these animals. Male Hartley guinea pigs were fed with either the control, the High Fat High Sucrose (HFHS) or the High Fat High Fructose (HFHF) diet for 150 days. Evolution of weight, blood cholesterol, triglycerides, urea and glucose tolerance were regularly monitored. Histopathological evolution was also evaluated in target organs such as pancreas, heart, liver and kidneys. Ex vivo experiments using the Langendorff retroperfusion technique, isolated hearts from guinea pigs either fed with the control, the HFHS or the HFHF diet were exposed to dofetilide 20 nM (D), chromanol 293B 10 µM (C) and amlodipine 100 nM (A) in different drug combinations and monophasic action potential duration was measured at 90% repolarization (MAPD90). Our data show that it is possible to generate a guinea pig model of metabolic syndrome by chronic exposure to diabetogenic diets. Minor histopathological abnormalities were observed, mainly in the pancreas and the liver. Metabolic syndrome potentiates the MAPD-prolonging actions of I(Kr)-blocking (dofetilide) and I(Ks)-blocking (chromanol 293B) drugs, an effect that is reversible upon administration of the calcium channel blocker amlodipine.

QRS widening and QT prolongation under bupropion: a unique cardiac electrophysiological profile.

QRS widening and QT prolongation are associated with bupropion. The objectives were to elucidate its cardiac electrophysiological properties. Patch-clamp technique was used to assess the I(Kr) -, I(Ks) -, and I(Na) -blocking effects of bupropion. Langendorff retroperfusion technique on isolated guinea-pig hearts was used to evaluate the MAPD(90) -, MAP amplitude-, phase 0 dV/dt-, and ECG-modulating effects of bupropion and of two gap junction intercellular communication inhibitors: glycyrrhetinic acid and heptanol. To evaluate their effects on cardiac intercellular communication, fluorescence recovery after photobleaching (FRAP) technique was used. Bupropion is an I(Kr) blocker. IC(50) was estimated at 34 µm. In contrast, bupropion had hardly any effect on I(Ks) and I(Na) . Bupropion had no significant MAPD(90) -modulating effect. However, as glycyrrhetinic acid and heptanol, bupropion caused important reductions in MAP amplitude and phase 0 dV/dt. A modest but significant QRS-widening effect of bupropion was also observed. FRAP experiments confirmed that bupropion inhibits gap junctional intercellular communication. QT prolongation during bupropion overdosage is due to its I(Kr) -blocking effect. QRS widening with bupropion is not related to cardiac sodium channel block. Bupropion rather mimics the QRS-widening, MAP amplitude- and phase 0 dV/dt -reducing effect of glycyrrhetinic acid and heptanol. Unlike class I anti-arrhythmics, bupropion causes cardiac conduction disturbances by reducing cardiac intercellular coupling.

Tizanidine (Zanaflex): a muscle relaxant that may prolong the QT interval by blocking IKr.

BACKGROUND: Tizanidine (Zanaflex) is a centrally acting imidazoline muscle relaxant that is structurally similar to clonidine (α(2)-adrenergic agonist) but not to other myorelaxants such as baclofen or benzodiazepines. Interestingly, cardiac arrhythmias and QT interval prolongation have been reported with tizanidine. OBJECTIVE: To evaluate the effects of tizanidine on cardiac ventricular repolarization. METHODS: (1) Whole-cell patch-clamp experiments: HERG- or KCNQ1+KCNE1-transfected cells were exposed to tizanidine 0.1-100 µmol/L (n = 29 cells, total) to assess drug effect on the rapid (I(Kr)) and slow (I(Ks)) components of the delayed rectifier potassium current. (2) Langendorff retroperfusion experiments: isolated hearts from male Hartley guinea pigs (n = 6) were exposed to tizanidine 1 µmol/L to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization (MAPD(90)). (3) In vivo wireless cardiac telemetry experiments: guinea pigs (n = 6) implanted with radio transmitters were injected a single intraperitoneal (ip) dose of tizanidine 0.25 mg/kg and 24 hours electrocardiography (ECG) recordings were made. RESULTS: (1) Patch-clamp experiments revealed an estimated IC(50) for tizanidine on I(Kr) above 100 µmol/L. Moreover, tizanidine 1 µmol/L had hardly any effect on I(Ks) (5.23% ± 4.54% inhibition, n = 5 cells). (2) While pacing the hearts at stimulation cycle lengths of 200 or 250 ms, tizanidine 1 µmol/L prolonged MAPD(90) by 8.22 ± 2.03 (6.7%) and 11.70 ± 3.08 ms (8.5%), respectively (both P < .05 vs baseline). (3) Tizanidine 0.25 mg/kg ip caused a maximal 11.93 ± 1.49 ms prolongation of corrected QT interval (QTc), 90 minutes after injection. CONCLUSION: Tizanidine prolongs the QT interval by blocking I(Kr). Patients could be at risk of cardiac proarrhythmia during impaired drug elimination, such as in case of CYP1A2 inhibition during drug interactions.

A novel KCNQ1 variant (L203P) associated with torsades de pointes-related syncope in a Steinert syndrome patient.

BACKGROUND: A 43-year-old woman suffering from Steinert syndrome was admitted after experiencing multiple episodes of torsades de pointes-related syncope. OBJECTIVES: To elucidate the pathophysiology of these arrhythmic events. METHODS AND RESULTS: We obtained DNA from the patient and sequenced the coding region of KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. A single nucleotide change was identified in the KCNQ1 gene at position 608 (T608C), resulting in a substitution from leucine to proline at position 203 (L203P). CHO cells were used to express either wild-type KCNQ1, wild-type KCNQ1+L203P KCNQ1 (50:50), or L203P KCNQ1, along with KCNE1 to recapitulate the slow cardiac delayed rectifier potassium current (I(Ks)). Patch-clamp experiments showed that the variant L203P causes a dominant negative effect on I(Ks). Coexpression of wild-type KCNQ1 and L203P KCNQ1 (50:50) caused a ~75% reduction in current amplitude when compared to wild-type KCNQ1 alone (131.40 ± 23.27 vs 567.25 ± 100.65 pA/pF, P < .001). Moreover, when compared with wild-type KCNQ1 alone, the coexpression of wild-type KCNQ1 and L203P KCNQ1 (50:50) caused a 7.5-mV positive shift of midpoints of activation (from 27.5 ± 2.4 to 35.1 ± 1.2 mV, P < .05). The wild-type KCNQ1 and L203P KCNQ1 (50:50) coexpression also caused alteration of I(Ks) kinetics. The activation kinetics of the L203P variant (50:50) were slowed compared with wild-type KCNQ1, while the deactivation kinetics of L203P (50:50) were accelerated compared with wild type, all these further contributing to the "loss-of-function" phenotype of I(Ks) associated with the variant L203P. CONCLUSION: Torsades de pointes and episodes of syncope are very likely to be due to the KCNQ1 variant L203P found in this patient.

Prolongation of cardiac ventricular repolarization under paliperidone: how and how much?

INTRODUCTION: Paliperidone (9-hydroxyrisperidone) is a second-generation antipsychotic. As observed with risperidone, QT interval prolongation was reported with paliperidone. OBJECTIVE: The aim was to evaluate the effects of paliperidone on cardiac ventricular repolarization. METHODS: (1) Patch-clamp experiments: Human ether-a-go-go-related gene (HERG)- or KCNQ1 + KCNE1-transfected cells were exposed to 0.1-100 µmol/L paliperidone (N = 39 cells, total) to assess the drug effect on HERG and KCNQ1 + KCNE1 currents. (2) Langendorff perfusion experiments: Hearts isolated from male Hartley guinea pigs (N = 9) were exposed to 0.1 µmol/L paliperidone to assess drug-induced prolongation of monophasic action potential duration measured at 90% repolarization. (3) In vivo cardiac telemetry experiments: Guinea pigs (N = 8) implanted with transmitters were injected a single intraperitoneal dose of 1 mg/kg of paliperidone, and 24-hour electrocardiogram recordings were made. RESULTS: (1) The estimated concentration at which 50% of the maximal inhibitory effect is observed (IC(50)) for paliperidone on HERG current was 0.5276 µmol/L. In contrast, 1 µmol/L paliperidone had hardly any effect on KCNQ1 + KCNE1 current (4.0 ± 1.6% inhibition, N = 5 cells). (2) While pacing the hearts at cycle lengths of 150, 200, or 250 milliseconds, 0.1 µmol/L paliperidone prolonged monophasic action potential duration measured at 90% repolarization by, respectively, 6.1 ± 3.1, 9.8 ± 2.7, and 12.8 ± 2.7 milliseconds. (3) Paliperidone (1 mg/kg) intraperitoneal caused a maximal 15.7 ± 5.3-millisecond prolongation of QTc. CONCLUSIONS: Paliperidone prolongs the QT interval by blocking HERG current at clinically relevant concentrations and is potentially unsafe.

Toward optimal treatment in women: the effect of sex on metoprolol-diphenhydramine interaction.

The objective of this study was to determine if sex influences the pharmacokinetics and hemodynamics of the CYP2D6 substrate metoprolol and its interaction with diphenhydramine (CYP2D6 inhibitor) in healthy young participants with high (extensive metabolizer [EM]) or low (poor metabolizer [PM]) CYP2D6 activities. A prespecified comparative analysis of data from 2 sequential clinical trials that included 16 EM and 4 PM women and 10 EM and 6 PM men was performed. The participants in the 2 trials were administered a single oral dose of 100 mg metoprolol in the presence of steady-state diphenhydramine or placebo. Serial plasma and urine samples were obtained for 48 hours, and hemodynamic data was obtained for 12 hours after metoprolol. In the placebo arm, EM and PM women had 62% and 59% higher S-metoprolol AUC(0-infinity) and 26% and 71% lower CL/F, respectively, compared to men with the same phenotype (all Ps < .05 women compared to men). These differences dissipated on body weight (WT) correction. Women (especially PMs) experienced greater negative chronotropic effects of metoprolol than men (P < .0001 women compared to men). Diphenhydramine coadministration increased S-metoprolol AUC by 84% in EM women and 45% in EM men (P < .009 women compared to men). In the diphenhydramine arm, sex differences in pharmacokinetics persisted even after WT correction, resulting in greater negative chronotropic effects in women (all Ps < .05 women compared to men). Metoprolol dose should be adjusted for body weight, particularly in women. Coadministration of a CYP2D6 inhibitor such as diphenhydramine, by a patient at similar doses in the 2 sexes, could result in a greater inhibition of clearance of CYP2D6 substrates with a resulting higher risk of pronounced pharmacological and adverse effects in women compared to men.

Acidic residues on the voltage-sensor domain determine the activation of the NaChBac sodium channel.

The voltage-sensing domain of voltage-gated ion channels is characterized by specific, conserved, charged residues. Positively charged residues on segment S4 are the main contributors to voltage-sensing and negatively charged residues on the S2 and S3 segments are believed to participate to the process. However, their function in the voltage sensor is not well understood. To probe the role of three acidic residues in NaChBac (D-58 and E-68 in S2, and D-91 in S3), we employed site-directed mutagenesis to substitute native acidic residues with cysteine (neutral), lysine (positive charge), or either aspartate or glutamate (negative charge). We used a combination of the patch-clamp technique to record Na+ currents and molecular modeling to visualize interacting amino acid residues. We suggest that the acidic residues on the S2 and S3 segments form specific interactions with adjacent amino acids in the voltage-sensor domain. The main interactions in NaChBac are D-58 (S2) with A-97-G-98 (S3) and R-120 (S4), E-68 (S2) with R-129 (L4-5), and D-91 (S3) with R-72 (S2). Changing these acidic residues modified the interactions, which in turn altered the sensitivity of the voltage sensor.

A novel nonsense mutation in the SCN5A gene leads to Brugada syndrome and a silent gene mutation carrier state.

BACKGROUND: Brugada syndrome (BS) is an electrical cardiac disorder with a right bundle branch block and ST segment elevation in leads V1 to V3 on surface electrocardiograms (ECGs), and is a syndrome that may lead to sudden cardiac death. PURPOSE: The aim of the present study was to screen for mutations in the SCN5A gene in a family with BS, and to characterize the consequences of the mutation on channel function. RESULTS: A heterozygous nonsense SCN5A mutation (W822X) was identified in the index patient. The mutation was confirmed in the patient's asymptomatic 16-year-old brother and 48-year-old father. The mutation was absent in the index patient's sister and mother. The ECG of the index patient showed a BS type 2 ECG phenotype, which converted into a type 1 ECG phenotype in the presence of flecainide. The ECG of the patient's brother was not typical for BS, but ajmaline treatment unmasked a type 1 ECG phenotype. The ECG of the asymptomatic father was normal at baseline and in the presence of ajmaline. No Na+ currents could be measured in tsA201 cells transfected with W822X mutant channels. Heterozygote expression showed a nearly 50% reduction in Na+ current amplitude with no significant alterations of biophysical properties, indicating a loss of functional Na+ channels, obviously without any dominant-negative activity on wild type channels. CONCLUSIONS: The haploinsufficiency of the Nav1.5 protein is the plausible explanation for the clinical BS phenotype in this family. Because the heterozygous W822X mutation theoretically leads to channel expression at one-half of the normal level, the authors suggest that a modifier gene may influence or rescue the phenotype in the asymptomatic family members.