Metabolic Activation and Cytotoxicity of Propafenone Mediated by CYP2D6.

Propafenone (PPF) is a class IC antidysrhythmic drug, which is commonly used for the treatment of atrial fibrillation and other supraventricular arrhythmias. It is also a ß-adrenoceptor antagonist that can cause bradycardia and bronchospasm. Hepatotoxicity is one of the adverse reactions reported, with clinical manifestations including acute cholestasis and hepatocyte necrosis. However, the mechanism of PPF-induced hepatotoxicity remains unclear. The present study was conducted to identify reactive metabolite(s) to determine related metabolic pathways and define the possible association of the bioactivation with PPF cytotoxicity. An O-demethylation phase I metabolite (M1), a further position C5 hydroxylation (para-position of the benzene ring) metabolite (M2), glutathione (GSH) conjugates (M3 and M4), and N-acetylcysteine (NAC) conjugates (M5 and M6) were detected in rat liver microsomal incubations containing PPF and GSH or NAC as trapping agents. The corresponding GSH conjugates and NAC conjugates were found in the bile and urine of rats after PPF administration, respectively. The observed GSH and NAC conjugates indicate that a quinone metabolite was generated in vitro and in vivo. Recombinant P450 enzyme incubations showed that CYP2D6 was the principal enzyme catalyzing this metabolic activation. Quinidine, a selective inhibitor of CYP2D6, attenuated the susceptibility of hepatocytes to the cytotoxicity of PPF. The results suggest that PPF was metabolized to a p-quinone intermediate which may be involved in PPF-induced hepatotoxicity.

Cytochrome P450-dependent drug oxidation activities and their expression levels in liver microsomes of chimeric TK-NOG mice with humanized livers.

Hepatic cytochrome P450 (P450)-dependent drug oxidation activity has not been completely characterized in chimeric TK-NOG mice with humanized livers (humanized liver mice). In this study, we examined several drug oxidation activities catalyzed by liver microsomes from humans, humanized liver mice, and TK-NOG mice using 9 P450 substrates. The catalytic activities of liver microsomes from humans and humanized liver mice showed relatively similar rates of oxidation of 7-ethoxyresorufin, coumarin, 7-pentoxyresorufin, flurbiprofen, S-mephenytoin, chlorzoxazone, and midazolam, whereas bufuralol 1'-hydroxylation and propafenone 4'-hydroxylation (rodent-specific propafenone oxidation activity) were higher in humanized liver mice than in humans. In addition, P450 protein expression levels in the humanized mouse liver were quantified using a liquid chromatography-tandem mass spectrometry-based protein quantification method. Quantification of P450 enzymes showed a 3-fold difference between human and humanized liver mouse livers, except for CYP2B6, which showed an approximately 6-fold difference. Overall, most P450-dependent drug oxidation activities were comparable between liver microsomes from human and humanized liver mice based on the similar expression levels of human P450 enzymes. However, some differences were observed between both species, including considerable differences in bufuralol 1'-hydroxylation and propafenone 4'-hydroxylation activities.

Serotonin Regulates Adult ß-Cell Mass by Stimulating Perinatal ß-Cell Proliferation.

A sufficient ß-cell mass is crucial for preventing diabetes, and perinatal ß-cell proliferation is important in determining the adult ß-cell mass. However, it is not yet known how perinatal ß-cell proliferation is regulated. Here, we report that serotonin regulates ß-cell proliferation through serotonin receptor 2B (HTR2B) in an autocrine/paracrine manner during the perinatal period. In ß-cell-specific Tph1 knockout (Tph1 ßKO) mice, perinatal ß-cell proliferation was reduced along with the loss of serotonin production in ß-cells. Adult Tph1 ßKO mice exhibited glucose intolerance with decreased ß-cell mass. Disruption of Htr2b in ß-cells also resulted in decreased perinatal ß-cell proliferation and glucose intolerance in adulthood. Growth hormone (GH) was found to induce serotonin production in ß-cells through activation of STAT5 during the perinatal period. Thus, our results indicate that GH-GH receptor-STAT5-serotonin-HTR2B signaling plays a critical role in determining the ß-cell mass by regulating perinatal ß-cell proliferation, and defects in this pathway affect metabolic phenotypes in adults.

Regioselective hydroxylation of an antiarrhythmic drug, propafenone, mediated by rat liver cytochrome P450 2D2 differs from that catalyzed by human P450 2D6.

1. Propafenone, an antiarrhythmic drug, is a typical human cytochrome P450 (P450) 2D6 substrate used in preclinical studies. Here, propafenone oxidation by mammalian liver microsomes was investigated in vitro. 2. Liver microsomes from humans and marmosets preferentially mediated propafenone 5-hydroxylation, minipig, rat and mouse livers primarily mediated 4'-hydroxylation, but cynomolgus monkey and dog liver microsomes differently mediated N-despropylation. 3. Quinine, ketoconazole or anti-P450 2D antibodies suppressed propafenone 4'/5-hydroxylation in human and rat liver microsomes. Pretreatments with ß-naphthoflavone or dexamethasone increased N-despropylation in rat livers. 4. Recombinant rat P450 2D2 efficiently catalysed propafenone 4'-hydroxylation in a substrate inhibition manner, comparable to rat liver microsomes, while human P450 2D6 displayed propafenone 5-hydroxylation. Human and rat P450 1A, 2C and 3A enzymes mediated propafenone N-despropylation with high capacities. 5. Carbon-4' of propafenone docked favourably into the active site of P450 2D2 based on an in silico model; in contrast, carbon-5 of propafenone docked into human P450 2D6. 6. These results suggest that the major roles of individual P450 2D enzymes in regioselective hydroxylations of propafenone differ between human and rat livers, while the minor roles of P450 1A, 2C and 3A enzymes for propafenone N-despropylation are similar in livers of both species.

Simultaneous determination of serum propafenone and its metabolites using high-performance liquid chromatography.

Propafenone, a class Ic antiarrhythmic agent, is metabolized to 5-hydroxypropafeone (5-OHP) and N-depropylpropafenone (NDPP). Simultaneous determination of serum propafenone and its metabolites was performed using HPLC equipped with a conventional octadecylsilyl silica column and ultraviolet detector. The wavelength was set at 250 nm. Propafenone and its metabolites in the serum were extracted using diethyl ether. The mobile phase solution, comprising 1-pentanesulfonic acid sodium salt (0.1 m), acetonitrile and acetic acid (280:185:2.5, v/v/v), was pumped at a flow rate of 1 mL/min. The recoveries of propafenone, 5-OHP and NDPP were greater than 85, 82 and 60%, respectively, with the coefficients of variation (CVs) less than 5.4, 1.9 and 2.9%, respectively. The calibration curves were linear for a concentration range of 12.5-1500 ng/mL for propafenone and 2-500 ng/mL for 5-OHP and NDPP (r > 0.999). CVs in the intraday assays were 1.0-3.8% for propafenone, 0.6-2.0% for 5-OHP and 0.6-1.7% for NDPP. CVs in interday assays were 1.3-7.7% for propafenone, 1.1-6.5% for 5-OHP and 5.4-8.0% for NDPP. The present HPLC method can be used to assess the disposition of propafenone and its metabolites for pharmacokinetic studies and therapeutic drug monitoring of propafenone.

Anti-arrhythmic Medication Propafenone a Potential Drug for Alzheimer's Disease Inhibiting Aggregation of Aß: In Silico and in Vitro Studies.

Alzheimer's disease (AD) is the most common form of dementia caused by the formation of Aß aggregates. So far, no effective medicine for the treatment of AD is available. Many efforts have been made to find effective medicine to cope with AD. Curcumin is a drug candidate for AD, being a potent anti-amyloidogenic compound, but the results of clinical trials for it were either negative or inclusive. In the present study, we took advantages from accumulated knowledge about curcumin and have screened out four compounds that have chemical and structural similarity with curcumin more than 80% from all FDA-approved oral drugs. Using all-atom molecular dynamics simulation and the free energy perturbation method we showed that among predicted compounds anti-arrhythmic medication propafenone shows the best anti-amyloidogenic activity. The in vitro experiment further revealed that it can inhibit Aß aggregation and protect cells against Aß induced cytotoxicity to almost the same extent as curcumin. Our results suggest that propafenone may be a potent drug for the treatment of Alzheimer's disease.

Assessment of 25 CYP2D6 alleles found in the Chinese population on propafenone metabolism in vitro.

Cytochrome P450 enzyme 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily, with more than 100 CYP2D6 allelic variants being previously reported. The aim of this study was to assess the catalytic characteristics of 25 alleles (CYP2D6.1 and 24 CYP2D6 variants) and their effects on the metabolism of propafenone in vitro. Twenty-five CYP2D6 alleles were expressing in 21 Spodoptera frugiperda (Sf) insect cells, and each variant was evaluated using propafenone as the substrate. Reactions were performed at 37 °C with 1-100 µmol/L propafenone for 30 min. After termination, the product 5-OH-propafenone was extracted and used for signal collection by ultra-performance liquid chromatography (UPLC). Compared with wild type CYP2D6.1, the intrinsic clearance (Vmax and Km) values of all variants were significantly altered. Three variants (CYP2D6.87, CYP2D6.90, CYP2D6.F219S) exhibited markedly increased intrinsic clearance values (129% to 165%), whereas 21 variants exhibited significantly decreased values (16% to 85%) due to increased Km and (or) decreased Vmax values. These results indicated that the majority of tested alleles had significantly altered catalytic activity towards propafenone hydroxylation in this expression system. Attention should be paid to subjects carrying these rare alleles when treated with propafenone.

Structural basis of drugs that increase cardiac inward rectifier Kir2.1 currents.

AIMS: We hypothesize that some drugs, besides flecainide, increase the inward rectifier current (IK1) generated by Kir2.1 homotetramers (IKir2.1) and thus, exhibit pro- and/or antiarrhythmic effects particularly at the ventricular level. To test this hypothesis, we analysed the effects of propafenone, atenolol, dronedarone, and timolol on Kir2.x channels. METHODS AND RESULTS: Currents were recorded with the patch-clamp technique using whole-cell, inside-out, and cell-attached configurations. Propafenone (0.1 nM-1 µM) did not modify either IK1 recorded in human right atrial myocytes or the current generated by homo- or heterotetramers of Kir2.2 and 2.3 channels recorded in transiently transfected Chinese hamster ovary cells. On the other hand, propafenone increased IKir2.1 (EC50 = 12.0 ± 3.0 nM) as a consequence of its interaction with Cys311, an effect which decreased inward rectification of the current. Propafenone significantly increased mean open time and opening frequency at all the voltages tested, resulting in a significant increase of the mean open probability of the channel. Timolol, which interacted with Cys311, was also able to increase IKir2.1. On the contrary, neither atenolol nor dronedarone modified IKir2.1. Molecular modelling of the Kir2.1-drugs interaction allowed identification of the pharmacophore of drugs that increase IKir2.1. CONCLUSIONS: Kir2.1 channels exhibit a binding site determined by Cys311 that is responsible for drug-induced IKir2.1 increase. Drug binding decreases channel affinity for polyamines and current rectification, and can be a mechanism of drug-induced pro- and antiarrhythmic effects not considered until now.

Propafenone associated severe central nervous system and cardiovascular toxicity due to mirtazapine: a case of severe drug interaction.

We describe a rare case of severe drug-drug interaction between propafenone and mirtazapine leading to propafenone toxicity. A 69-year-old Caucasian male taking propafenone for atrial fibrillation was prescribed mirtazapine for insomnia. Subsequent to the first dose of mirtazapine the patient experienced seizures, bradycardia and prolonged QRS as well as QTc intervals on EKG. The patient was admitted to the ICU and recovered after supportive management. Propafenone is an established class IC antiarrhythmic drug commonly used in the treatment of atrial fibrillation. It is metabolized through the CYP4502D6 pathway. Five to 10 percent of Caucasians are poor metabolizers. Mirtazapine is a commonly prescribed antidepressant drug, which is also metabolized through and may modulate the CYP4502D6 pathway leading to altered metabolism of propafenone and possible adverse effects. In this case, toxicity was reversed once the offending drugs were discontinued. An extensive review of the literature revealed this to be the first described case of drug interaction between propafenone and mirtazapine.

Pore-exposed tyrosine residues of P-glycoprotein are important hydrogen-bonding partners for drugs.

The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.

Exhaustive sampling of docking poses reveals binding hypotheses for propafenone type inhibitors of P-glycoprotein.

Overexpression of the xenotoxin transporter P-glycoprotein (P-gp) represents one major reason for the development of multidrug resistance (MDR), leading to the failure of antibiotic and cancer therapies. Inhibitors of P-gp have thus been advocated as promising candidates for overcoming the problem of MDR. However, due to lack of a high-resolution structure the concrete mode of interaction of both substrates and inhibitors is still not known. Therefore, structure-based design studies have to rely on protein homology models. In order to identify binding hypotheses for propafenone-type P-gp inhibitors, five different propafenone derivatives with known structure-activity relationship (SAR) pattern were docked into homology models of the apo and the nucleotide-bound conformation of the transporter. To circumvent the uncertainty of scoring functions, we exhaustively sampled the pose space and analyzed the poses by combining information retrieved from SAR studies with common scaffold clustering. The results suggest propafenone binding at the transmembrane helices 5, 6, 7 and 8 in both models, with the amino acid residue Y307 playing a crucial role. The identified binding site in the non-energized state is overlapping with, but not identical to, known binding areas of cyclic P-gp inhibitors and verapamil. These findings support the idea of several small binding sites forming one large binding cavity. Furthermore, the binding hypotheses for both catalytic states were analyzed and showed only small differences in their protein-ligand interaction fingerprints, which indicates only small movements of the ligand during the catalytic cycle.

Stereoselective glucuronidation of propafenone and its analogues by human recombinant UGT1A9.

Stereoselective glucuronidation of propafenone and its beta-blocker analogues by human recombinant UGT1A3 and UGT1A9 from the recombinant baculovirus in insect sf9 cells was studied. The glucuronides produced in incubation mixtures were assayed by HPLC equipped with UV detector, and identified by beta-glucuronidase. The stereoselective glucuronidation was measured by pre-column 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocynate (GITC) derivatization HPLC method for propafenone and esomolol. In all of ten beta-blocker drugs studied, six showed the glucuronidation activity with UGT1A9, while four with UGT1A3. From roughly quantitative stereoselective glucuronidation study of racemic beta-blocker analogues by UGT1A9, propranolol had a high ratio of the ratios of S- to R-isomer glucuronide (S-G/R-G), about 4.3, the ratios of terbutaline, atenolol and esomolol were 3.3, 3.1 and 2.8 respectively, sotalol and propafenone were 2.3 and 2.0. In a word, S-isomers of these drugs were glucuronidated by human UGT1A9 much faster than their antipodes.

A natural variant of the heme-binding signature (R441C) resulting in complete loss of function of CYP2D6.

A new variant allele CYP2D6*62 (g.4044C>T; R441C) of the drug-metabolizing cytochrome P450 (P450) CYP2D6 was identified in a person with reduced sparteine oxidation phenotype, which was unexpected based on a genetic CYP2D6*1A/*41 background. The recombinantly expressed variant protein had no activity toward propafenone as a result of missing heme incorporation. Sequence alignment revealed that the positively charged R441 residue is part of the heme-binding signature but not strictly conserved among all the P450s. A compilation of described P450 monooxygenase variants revealed that other enzymes can functionally tolerate even nonconservative amino acid changes at the corresponding position (i.e., the invariant cysteine 2). This suggests that heme binding in mammalian P450s depends not only on the integrity of the heme-binding signature but also on other family- and subfamily-specific sequence determinants.

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4.

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples.

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.

Usefulness of hepatocytes for evaluating the genetic polymorphism of CYP2D6 substrates.

The usefulness of human hepatocytes for assessing CYP2D6-related genetic polymorphisms was investigated. Propranolol and propafenone, which undergo phase I and II biotransformations, were used as model substrates alongside metoprolol, which is only metabolized via oxidative pathways. The contributions of CYP2D6 to the primary metabolisms of the substrates were estimated from the quinidine-mediated inhibition of their depletion rate constants in human hepatocytes and liver microsomes. The contributions in hepatocytes were 19.2% for propranolol at 0.05 microM and 36.7--76.3% for propafenone at 0.05--1.0 microM, and smaller than the contribution in microsomes, unlike the case for metoprolol. The differences between microsomes and hepatocytes were attributable to conjugate formation. The CYP2D6 contributions in hepatocytes reflected the in vivo data. The relevance of the concentration-dependent involvement of CYP2D6 in propafenone metabolism in hepatocytes to the in vivo polymorphic profile and the applicability of hepatocytes for evaluating these polymorphisms are discussed.

[Pharmacogenetic study of the response to flecainide and propafenone in patients with atrial fibrillation]. / Estudio farmacogenético de la respuesta a flecainida y propafenona en pacientes con fibrilación auricular.

We analyzed cytochrome P450 2D6 polymorphism by determining phenotype as the metabolic ratio between dextromethorphan and its main metabolite, dextrorphan. We studied 18 men and 22 women in whom mean age was 54.6+/-11.9 years. In 9 patients metabolic ratio was determined before antiarrhythmic treatment and again during treatment, with a mean increase of 0.13+/-0.15 (P=.03). We found 19 poor metabolizers and 21 extensive metabolizers. Adverse effects were more frequent in poor metabolizers (21.1%) than in extensive metabolizers (4.8%; P=.12). Antiarrhythmic treatment was effective in 27 patients (67.5%), with no difference between poor and extensive metabolizers.

Estudio farmacogenético de la respuesta a flecainida y propafenona en pacientes con fibrilación auricular / Pharmacogenetic study of the response to flecainide and propafenone in patients with atrial fibrillation

Se analizó el polimorfismo del citocromo P450-2D6 mediante la determinación del fenotipo, utilizando la ratio entre dextrometorfano y su metabolito dextrorfano. Estudiamos a 18 varones y 22 mujeres, con una edad media de 54,6 ± 11,9 años. En 9 pacientes se realizó una determinación de la ratio metabólica antes de iniciar el tratamiento antiarrítmico y una segunda determinación bajo tratamiento, con un incremento promedio de 0,13 ± 0,15 (p = 0,03). De los 40 pacientes, 19 eran metabolizadores lentos y 21 metabolizadores rápidos. Los efectos secundarios fueron más frecuentes en los metabolizadores lentos (21,1%) que en los metabolizadores rápidos (4,8%) (p = 0,12). El tratamiento antiarrítmico fue eficaz en 27 pacientes (67,5%), con un porcentaje similar en metabolizadores lentos y rápidos

We analyzed cytochrome P450 2D6 polymorphism by determining phenotype as the metabolic ratio between dextromethorphan and its main metabolite, dextrorphan. We studied 18 men and 22 women in whom mean age was 54.6±11.9 years. In 9 patients metabolic ratio was determined before antiarrhythmic treatment and again during treatment, with a mean increase of 0.13±0.15 (P=.03). We found 19 poor metabolizers and 21 extensive metabolizers. Adverse effects were more frequent in poor metabolizers (21.1%) than in extensive metabolizers (4.8%; P=.12). Antiarrhythmic treatment was effective in 27 patients (67.5%), with no difference between poor and extensive metabolizers

P-glycoprotein substrate binding domains are located at the transmembrane domain/transmembrane domain interfaces: a combined photoaffinity labeling-protein homology modeling approach.

P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino- and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.