Dany Patoine

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 (IC50) 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.

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.

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 IKr‐, IKs‐, and INa‐blocking effects of bupropion. Langendorff retroperfusion technique on isolated guinea‐pig hearts was used to evaluate the MAPD90‐, 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 IKr blocker. IC50 was estimated at 34 μm. In contrast, bupropion had hardly any effect on IKs and INa. Bupropion had no significant MAPD90‐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 IKr‐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.

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.

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.

Simultaneous Determination of Dofetilide and Amlodipine in Plasma by HPLC

Dofetilide is a Class III antiarrhythmic drug useful in the management of atrial fibrillation but also known to prolong the QT interval on the ECG and to induce malignant ventricular tachyarrhythmias such as torsades de pointes (TDP). Drugs with calcium channel-blocking properties contribute to decrease the incidence of TDP. Amlodipine is a dihydropyridine calcium channel antagonist. Considering the potentially safe and useful combined use of dofetilide and amlodipine, we undertook drug biotransformation studies. To perform studies aiming to evaluate the possible pharmacokinetic interaction between these drugs, a unique sensitive HPLC assay able to quantify both drugs simultaneously was required. A sensitive and specific HPLC method using a tandem of UV/fluorescence detection was described for the analysis of amlodipine and dofetilide in plasma. The within-day and between-day precision studies showed good reproducibility with coefficients of variation less than 10% for all the analytes. The limits of detection were 0.5 ng/mL and 0.25 ng/mL and the limit of quantification were 1.7 ng/mL and 0.8 ng/mL for dofetilide and amlodipine respectively. This new method could be of great value in many applications such as in vitro and in vivo pharmacokinetic and drug-drug interactions studies, as well as therapeutic drug monitoring.

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 (α2-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 (IKr) and slow (IKs) 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 (MAPD90). (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 IC50 for tizanidine on IKr above 100 µmol/L. Moreover, tizanidine 1 µmol/L had hardly any effect on IKs (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 MAPD90 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 IKr. Patients could be at risk of cardiac proarrhythmia during impaired drug elimination, such as in case of CYP1A2 inhibition during drug interactions.

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.

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. Copyright

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.