P-Glycoprotein-Mediated Pharmacokinetic Interactions Increase Pimozide hERG Channel Inhibition.

Pimozide, an antipsychotic drug, is a potent inhibitor of the hERG channel. A case of death due to cardiac arrest has been reported in a boy who received pimozide together with sertraline and aripiprazole. In this study, we focused on drug-drug interactions and investigated the relationships between transporter-mediated intracellular accumulation and the hERG inhibitory effect of pimozide. The accumulation of pimozide in cardiomyocyte-derived AC16 cells was significantly increased by sertraline and aripiprazole, which are thought to have a P-glycoprotein (P-gp) inhibitory effect, and under P-gp siRNA conditions. These results suggest P-gp inhibition increases pimozide accumulation in AC16 cells. We introduced the hERG plasmid into AC16 cells and investigated the concentration-dependent hERG inhibitory effect of pimozide from within AC16 cells. Addition of 10 nM or more pimozide significantly inhibited the hERG current with concentration dependence. These results indicate P-gp-mediated pharmacokinetic interaction increases pimozide accumulation in AC16 cells, and the subsequent elevated pimozide levels within the cells may result in an increased risk of hERG channel inhibition. Our present study calls attention to the risks associated with the combined use of cardiotoxic P-gp substrate(s) and P-gp inhibitory medicines.

Gastrointestinal absorption of pimozide is enhanced by inhibition of P-glycoprotein.

Drug-drug interaction was suggested to have played a role in the recent death due to cardiac arrest of a patient taking pimozide, sertraline and aripiprazole antipsychotic/antidepressant combination therapy. Here, we investigated the possible involvement of P-glycoprotein (P-gp)-mediated interaction among these drugs, using in vitro methods. ATPase assay confirmed that pimozide is a P-gp substrate, and might act as a P-gp inhibitor at higher concentrations. The maximum transport rate (Jmax) and half-saturation concentration (Kt) for the carrier-mediated transport estimated by means of pimozide efflux assay using P-gp-overexpressing LLC-GA5-CoL150 cells were 84.9 ± 8.9 pmol/min/mg protein, and 10.6 ± 4.7 µM, respectively. These results indicate that pimozide is a good P-gp substrate, and it appears to have the potential to cause drug-drug interactions in the digestive tract at clinically relevant gastrointestinal concentrations. Moreover, sertraline or aripiprazole significantly decreased the efflux ratio of pimozide in LLC-GA5-CoL150 cells. Transport studies using Caco-2 cell monolayers were consistent with the results in LLC-GA5-CoL150 cells, and indicate that P-gp-mediated drug-drug interaction may occur in the gastrointestinal tract. Thus, P-gp inhibition by sertraline and/or aripiprazole may increase the gastrointestinal permeability of co-administered pimozide, resulting in an increased blood concentration of pimozide, which is known to be associated with an increased risk of QT prolongation, a life-threatening side effect.

The Respective Roles of CYP3A4 and CYP2D6 in the Metabolism of Pimozide to Established and Novel Metabolites.

Pimozide is a dopamine receptor antagonist indicated for the treatment of Tourette syndrome. Prior in vitro studies characterized N-dealkylation of pimozide to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI) via CYP3A4 and, to a lesser extent, CYP1A2 as the only notable routes of pimozide biotransformation. However, drug-drug interactions between pimozide and CYP2D6 inhibitors and CYP2D6 genotype-dependent effects have since been observed. To reconcile these incongruities between the prior in vitro and in vivo studies, we characterized two novel pimozide metabolites: 5-hydroxypimozide and 6-hydroxypimozide. Notably, 5-hydroxypimozide was the major metabolite produced by recombinant CYP2D6 (Km ∼82 nM, V max ∼0.78 pmol/min per picomoles), and DHPBI was the major metabolite produced by recombinant CYP3A4 (apparent Km ∼1300 nM, V max ∼2.6 pmol/min per picomoles). Kinetics in pooled human liver microsomes (HLMs) for the 5-hydroxylation (Km ∼2200 nM, V max ∼59 pmol/min per milligram) and N-dealkylation (Km ∼3900 nM, V max ∼600 pmol/min per milligram) reactions were also determined. Collectively, formation of DHPBI, 5-hydroxypimozide, and 6-hydroxypimozide accounted for 90% of pimozide depleted in incubations of NADPH-supplemented pooled HLMs. Studies conducted in HLMs isolated from individual donors with specific cytochrome P450 isoform protein abundances determined via mass spectrometry revealed that 5-hydroxypimozide (r 2 = 0.94) and 6-hydroxypimozide (r 2 = 0.86) formation rates were correlated with CYP2D6 abundance, whereas the DHPBI formation rate (r 2 = 0.98) was correlated with CYP3A4 abundance. Furthermore, the HLMs differed with respect to their capacity to form 5-hydroxypimozide relative to DHPBI. Collectively, these data confirm a role for CYP2D6 in pimozide clearance via 5-hydroxylation and provide an explanation for a lack of involvement when only DHPBI formation was monitored in prior in vitro studies. SIGNIFICANCE STATEMENT: Current CYP2D6 genotype-guided dosing information in the pimozide label is discordant with available knowledge regarding the primary biotransformation pathways. Herein, we characterize the CYP2D6-dependent biotransformation of pimozide to previously unidentified metabolites. In human liver microsomes, formation rates for the novel metabolites and a previously identified metabolite were determined to be a function of CYP2D6 and CYP3A4 content, respectively. These findings provide a mechanistic basis for observations of CYP2D6 genotype-dependent pimozide clearance in vivo.

In silico repurposing of antipsychotic drugs for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most prevalent form of dementia and represents one of the highest unmet requirements in medicine today. There is shortage of novel molecules entering into market because of poor pharmacokinetic properties and safety issues. Drug repurposing offers an opportunity to reinvigorate the slowing drug discovery process by finding new uses for existing drugs. The major advantage of the drug repurposing approach is that the safety issues are already investigated in the clinical trials and the drugs are commercially available in the marketplace. As this approach provides an effective solution to hasten the process of providing new alternative drugs for AD, the current study shows the molecular interaction of already known antipsychotic drugs with the different protein targets implicated in AD using in silico studies. RESULT: A computational method based on ligand-protein interaction was adopted in present study to explore potential antipsychotic drugs for the treatment of AD. The screening of approximately 150 antipsychotic drugs was performed on five major protein targets (AChE, BuChE, BACE 1, MAO and NMDA) by molecular docking. In this study, for each protein target, the best drug was identified on the basis of dock score and glide energy. The top hits were then compared with the already known inhibitor of the respective proteins. Some of the drugs showed relatively better docking score and binding energies as compared to the already known inhibitors of the respective targets. Molecular descriptors like molecular weight, number of hydrogen bond donors, acceptors, predicted octanol/water partition coefficient and percentage human oral absorption were also analysed to determine the in silico ADME properties of these drugs and all were found in the acceptable range and follows Lipinski's rule. CONCLUSION: The present study have led to unravel the potential of leading antipsychotic drugs such as pimozide, bromperidol, melperone, anisoperidone, benperidol and anisopirol against multiple targets associated with AD. Benperidol was found to be the best candidate drug interacting with different target proteins involved in AD.

The Influence of the CYP3A4*22 Polymorphism and CYP2D6 Polymorphisms on Serum Concentrations of Aripiprazole, Haloperidol, Pimozide, and Risperidone in Psychiatric Patients.

INTRODUCTION: Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of greater than 50% of the prescribed drugs. Recently, the CYP3A4*22 allele was reported to be associated with lower CYP3A4 expression and activity. Quetiapine, an antipsychotic metabolized by only CYP3A4, displayed higher serum levels in CYP3A4*22 carriers. Aripiprazole, haloperidol, pimozide, and risperidone are antipsychotics that are metabolized by CYP3A4 and CYP2D6. We investigated to which degree the CYP3A4*22 single-nucleotide polymorphism affects serum concentrations of patients receiving these drugs and compared this with the influence of CYP2D6 polymorphisms. METHODS: Eight hundred thirty-four adult patients were included in this study, of whom 130 used aripiprazole, 312 used haloperidol, 86 used pimozide, and 396 used risperidone. Serum levels of the drug and, if available, their active metabolites were collected as well as information on dose. Patients were genotyped for CYP3A4*22 using restriction fragment length polymorphism analysis. Genotyping for CYP2D6 was done with allele-specific polymerase chain reaction. RESULTS: No differences were found in serum (dose-corrected) concentrations of the antipsychotics between CYP3A4*22 wild-type and carrier groups. In contrast, CYP2D6 genotype did affect dose-corrected concentrations of the antipsychotics: for example, median dose-corrected concentrations were 56%, 86%, and 400% higher in predicted poor metabolizers versus extensive metabolizers for aripiprazole (P = 0.004), haloperidol (P > 0.001), and risperidone (P < 0.001), respectively, although a multiple regression analysis showed that only 4% to 17% of the variation in these concentrations could be explained by CYP2D6 status. CONCLUSIONS: Heterozygous presence of CYP3A4*22 does not increase serum levels of antipsychotics metabolized by both CYP3A4 and CYP2D6, whereas CYP2D6 polymorphisms do affect serum levels to a limited extent.

CYP2D6 genotype information to guide pimozide treatment in adult and pediatric patients: basis for the U.S. Food and Drug Administration's new dosing recommendations.

OBJECTIVE: The occurrence of pimozide-induced arrhythmias is concentration dependent. Hence, it is important for prescribers to consider causes of increased pimozide exposure. This article summarizes the U.S. Food and Drug Administration's (FDA's) review of drug interaction and pharmacogenomic studies and discusses pharmacokinetic simulations we performed to develop new cytochrome P450 2D6 (CYP2D6) genotype-guided dosing recommendations for pimozide. METHOD: Pharmacokinetic parameters by CYP2D6 genotype were derived from a published single-dose pharmacogenomic study of pimozide. We simulated what pimozide exposures would result from a multiple-dose scenario in different CYP2D6 genotype groups: extensive, intermediate, and poor metabolizers. The maximum dose for poor metabolizers was defined as the dose that would not exceed pimozide concentrations following 10 mg daily in extensive metabolizers and intermediate metabolizers (the current maximum dose in an unselected population). RESULTS: Dose-ranging analyses revealed that 4 mg daily in CYP2D6 poor metabolizers was the maximum dose that would not result in plasma concentrations in excess of those observed in extensive metabolizer and intermediate metabolizer patients receiving 10 mg daily. CYP2D6 genotyping is now consequently recommended in the pimozide product label before exceeding 4 mg of pimozide daily in adults or 0.05 mg/kg/d in children. Previously, dose adjustment was recommended every 3 days to achieve the desired clinical response for all patients. The label was modified to subsequently reflect that pimozide doses should not be increased earlier than 14 days in patients who are known CYP2D6 poor metabolizers. CONCLUSIONS: Given the risk of increased pimozide concentrations and longer time to steady state in CYP2D6 poor metabolizers, the FDA has revised the pimozide label to provide clinicians with clearer dosing, titration, and genotype testing recommendations. The new information is intended to enhance therapeutic individualization of pimozide in pediatric and adult patients.

Quantitative determination of pimozide in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study.

A simple, sensitive and specific LC-ESI/MS method was developed for the determination of pimozide in human plasma. Pimozide and cinnarizine (internal standard) were isolated from plasma samples by liquid-liquid extraction. The chromatographic separation was accomplished on a Thermo Hypersil-HyPURITY C18 reversed-phase column (150mmx2.1mm, i.d., 5microm) with the mobile phase consisting of 5mM ammonium acetate (pH 3.5, adjusted with acetic acid)-methanol-acetonitrile (39:5:56, v/v/v). The lower limit of quantification was 0.02ng/mL, and the assay exhibited a linear range of 0.025-12.800ng/mL. The established method has been successfully applied to a bioequivalence study of 2 pimozide formulations in 32 healthy male Chinese volunteers.

Coadministration of sertraline with cisapride or pimozide: an open-label, nonrandomized examination of pharmacokinetics and corrected QT intervals in healthy adult volunteers.

BACKGROUND: Sertraline hydrochloride is a selective serotonin reuptake inhibitor with demonstrated efficacy and safety for the treatment of the following disorders: major depressive, obsessive-compulsive, panic, premenstrual dysphoric, social anxiety, and posttraumatic stress. Although sertraline is unlikely to cause clinically significant inhibition of cytochrome P450 (CYP) 3A4 substrates, even modest concentration increases for narrow therapeutic index drugs, such as pimozide or cisapride, are potentially important. OBJECTIVE: The goal of this study was to determine whether there is a pharmacokinetic interaction, as shown by plasma concentrations and electrocardiographic evidence of QTc intervals, between sertraline 200 mg QD and cisapride 10 mg QID, and between sertraline 200 mg QD and pimozide (single 2-mg dose). METHODS: Patients in group A were administered cisapride on days 1 and 2 (10 mg QID), day 3 (10 mg/d), days 25 through 29 (10 mg QID), and day 30 (10 mg/d). Sertraline was administered on days 4 through 29 at a starting dose of 50 mg/d, which was titrated upward in 50-mg increments every third day to a maximum of 200 mg/d. Patients in group B were treated with 2 mg of pimozide on days 1 and 39. Sertraline was administered on days 18 through 46 at a starting dose of 50 mg/d, which was titrated upward in 50-mg increments every third day to a maximum of 200 mg/d. RESULTS: There were 9 males and 6 females in group A (sertraline + cisapride) (mean age, 34.4 years for males, 41.7 years for females; mean weight, 78.7 kg for males, 66.6 kg for females; 14 Hispanic, 1 white), and 8 males and 7 females in group B (sertraline + pimozide) (mean age, 26.1 years for males, 33.4 years for females; mean weight, 70.8 kg for males, 61.4 kg for females; 15 Hispanic). Coadministration of sertraline and cisapride resulted in statistically significant reductions of 29% and 36% in cisapride C(max) and AUC from time 0 to 6 hours, respectively, compared with cisapride alone. Coadministration of sertraline and pimozide resulted in statistically significant increases of 35% and 37% in pimozide Cmax and AUC(0-infinity), respectively, compared with pimozide alone. No subject exhibited a prolongation of the QTc interval > or =15% with coadministration of sertraline and cisapride, or sertraline and pimozide. CONCLUSIONS: This study found that coadministration of sertraline with cisapride resulted in decreases in cisapride concentrations, and no significant effects on QTc intervals. Coadministration of sertraline 200 mg/d and a single dose of pimozide 2 mg produced significant increases in pimozide concentrations but no prolongation of the QTc interval > or =15%. This opposite effect for pimozide compared with cisapride, as well as other previously tested CYP3A4 substrates, suggests that there are mechanisms other than CYP3A4 involved in the sertraline-pimozide interaction.

Practical application of guinea pig telemetry system for QT evaluation.

The purpose of this study was to evaluate a telemetry system for examining QT evaluation in the conscious free-moving guinea pig using 10 reference compounds whose effects on human QT interval are well established: 8 positive references (bepridil, terfenadine, cisapride, haloperidol, pimozide, quinidine, E-4031 and thioridazine), and 2 negative references (propranolol and nifedipine). Pharmacokinetic experiments were also performed for the 8 positive references. Telemetry transmitters were implanted subcutaneously in male Hartley guinea pigs, and the RR and QT intervals were measured. All 8 positive references prolonged QTc (QTc = k x QT/RR(1/2)) 10% or more during the 60 min observation period. When the values of the QTc changes were plotted against the serum concentrations, the resulting curves exhibited an anticlockwise hysteresis loop for all 8 references. In guinea pigs treated with haloperidol, changes of the T-wave shape from positive to flat were observed. The 2 negative references did not prolong the QTc. These findings suggest that the present telemetry guinea pig model is useful for QT evaluation in the early stages of drug development, because of the small body size of guinea pigs and their action potential configuration, which is similar to that of humans.

In vitro inhibition of pimozide N-dealkylation by selective serotonin reuptake inhibitors and azithromycin.

Pimozide is often coprescribed with serotonin reuptake inhibitor (SSRI) antidepressants to treat depression in patients with Tourette's syndrome. In human liver microsomes (HLMs), the inhibition of the primary route of pimozide metabolism, N-dealkylation to 1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one (DHPBI), by four SSRIs (fluoxetine, sertraline, paroxetine, and fluvoxamine) and azithromycin was tested. Inhibition constants (K(i) values) were estimated from Dixon plots (three HLMs for each inhibitor) using the appropriate enzyme inhibition model by nonlinear regression. At 10 microM paroxetine, sertraline, fluoxetine, or fluvoxamine, the formation of DHPBI from pimozide (10 microM) in HLMs was inhibited by an average (three HLMs) of 7%, 7.7%, 8%, and 16%, respectively, whereas this inhibition did not exceed 55% at the maximum concentrations (100 microM) of the SSRIs tested. Azithromycin had negligible effect on pimozide (10 microM) N-dealkylation (19% at 100 microM azithromycin). These inhibition data were compared with ketoconazole, which was included as a positive control of CYP3A inhibition. At 0.1 microM and 0.5 microM ketoconazole, the formation of DHPBI from 10 microM pimozide was inhibited by 32% and 62%, respectively. The K(i) values (+/- SD) of ketoconazole, sertraline, fluvoxamine, azithromycin, fluoxetine, and paroxetine were 0.07 microM, 89 +/- 44 microM, 89 +/- 24 microM, 103 +/- 52 microM, 117 +/- 27 microM, and 129 +/- 33 microM, respectively. These values are least 100-fold higher than the expected plasma concentrations after the usual daily doses of the SSRIs and azithromycin, suggesting that coadministration of SSRIs and azithromycin are unlikely to markedly diminish the elimination of pimozide in patients. However, in vivo predictions from in vitro data are not always perfect. In vivo, the SSRIs or azithromycin may concentrate in the liver relative to plasma. In addition, the possibility that these drugs could alter pimozide disposition through effects on transport proteins or via promoter repression cannot be ruled out.

Studies on the mechanism of a fatal clarithromycin-pimozide interaction in a patient with Tourette syndrome.

The authors report in detail the case of a 27-year-old man who experienced sudden cardiac death 2 days after coprescription of the neuroleptic pimozide and the macrolide antibiotic clarithromycin after the documentation of a prolonged QT interval. To determine the prevalence of this interaction, the authors referred to the Spontaneous Reporting System of the Food and Drug Administration and identified one similar case in which clarithromycin was coprescribed with pimozide and sudden cardiac death occurred shortly thereafter. In addition, the search identified 39 cases of cardiac arrhythmia associated with pimozide, 11 with pimozide alone, and 6 with clarithromycin alone, 1 of which had a positive rechallenge. The mechanism of the interaction between clarithromycin and pimozide seems to involve the inhibition of the hepatic metabolism of pimozide by the macrolide. The authors demonstrated that clarithromycin is able to inhibit the metabolism of pimozide in human liver microsomal preparations (K(i) = 7.65 +/- 1.18 microM) and that pimozide, but not clarithromycin or its primary metabolite, is able to prolong the electrocardiac QT interval in a dose-dependent manner in the isolated perfused rabbit heart. The increase was 9.6 +/- 1.1% in male hearts (N = 5) and 13.4 +/- 1.2% in female hearts (N = 4) (p < 0.05).

Effect of clarithromycin on the pharmacokinetics and pharmacodynamics of pimozide in healthy poor and extensive metabolizers of cytochrome P450 2D6 (CYP2D6).

BACKGROUND: The use of pimozide is associated with prolongation of the QT interval and fatal ventricular arrhythmia. We recently reported 2 fatal cases in patients taking pimozide and clarithromycin and we have shown that clarithromycin inhibits CYP3A-mediated metabolism of pimozide in vitro. In this study, we examined the effect of clarithromycin on pimozide pharmacokinetics and QT interval changes in a total of 12 healthy subjects (7 men and 5 women), documented as extensive metabolizers or poor metabolizers of CYP2D6. METHODS: In a randomized, double-blind placebo-controlled crossover design, subjects were given a single 6-mg oral dose of pimozide after 5 days of treatment with clarithromycin (500 mg twice a day) or a placebo pill. Blood samples were obtained before and for 96 hours after pimozide administration, and plasma pimozide and clarithromycin concentrations were measured by HPLC. Electrocardiograms for the analysis of the QTc intervals were recorded immediately before each blood sample. RESULTS: Pimozide significantly lengthened QTc interval in the first 20 hours in both the placebo-treated groups (delta QTcmax = 13.3 +/- 5.3 ms; P = .003) and clarithromycin-treated groups (delta QTcmax = 15.7 +/- 9.5 ms; P = .005) compared with baseline values. This is consistent with an effect of the parent drug. Clarithromycin caused a significant increase in the peak plasma concentration (P = .015), terminal elimination half-life (P = .003), and area under the plasma concentration-time curve (P = .024) and a decrease in the clearance (P = .029) of pimozide. Mean QTcmax observed within 20 hours of pimozide administration was significantly greater in the clarithromycin-treated group (23.8 +/- 12.2 ms; P = .0397) than in the placebo-treated group (16.8 +/- 6 ms). There was no significant effect of CYP2D6 or gender on the pharmacokinetics or pharmacodynamics of pimozide. CONCLUSIONS: A single 6-mg oral dose of pimozide resulted in measurable QT interval changes. Clarithromycin inhibited CYP3A-mediated pimozide metabolism and the resulting elevation in plasma concentrations may increase the risk of pimozide cardiotoxicity.

Sensitive assay for pimozide in human plasma using high-performance liquid chromatography with fluorescence detection: application to pharmacokinetic studies.

A method is described for the measurement of pimozide in human plasma using HPLC with fluorescence detection. The method is specific and sensitive in the range of concentrations seen in human plasma after conventional dosing (1-15 ng/ml) with a limit of quantification of 1 ng/ml. The calibration curves are linear for concentrations between 1 and 50 ng/ml. Within-day and inter-day coefficients of variation are less than 7.4% and 15.5%, respectively, at three concentrations of pimozide (2, 10 and 20 ng/ml). Intra-day and inter-day bias are less than 18.5% and 12.5%, respectively. A pharmacokinetic study conducted in a healthy volunteer administered 6 mg of pimozide orally demonstrates the utility of this method.

Modelling drug kinetics with brain stimulation: dopamine antagonists increase self-stimulation.

The rewarding effects of brain stimulation and drugs are believed to depend on a common neural system. However, the pattern of responding produced by drug reinforcers is different from the pattern produced by conventional brain stimulation. Furthermore, pharmacological antagonists of reinforcement increase the rate of drug self-administration but depress self-stimulation. To test the hypothesis that the differences in the characteristics of brain stimulation and drugs as reinforcers are due to differences in the kinetics of drugs and brain stimulation, we modelled drug kinetics with frequency-modulated trains of brain stimulation. We report that animals will self-administer such brain stimulation in a manner that resembles drug self-administration and that, under these conditions, dopamine antagonists can increase the rate of self-stimulation.

Pharmacokinetics of pimozide in adults and children with Tourette's syndrome.

Pimozide is a neuroleptic drug with dopamine receptor and calcium channel blocking activity that is used in the treatment of Tourette's syndrome. A comparison of the pharmacokinetics of pimozide was made in children and adults with Tourette's syndrome. Seven adults (ages 23-39) and four children (ages 6-13) received a single 2-mg oral dose of pimozide and a minimum of nine blood samples were collected over a four-day period. Mean elimination half-life of pimozide in children was 66 hours compared with 111 hours in adults with Tourette's syndrome. Significant interindividual variability of pimozide pharmacokinetics was found in both adults and children with Tourette's syndrome. The pharmacokinetics of pimozide in patients with Tourette's syndrome differs from that reported in adult populations with chronic schizophrenia.