Human iPSC-derived cardiomyocytes and pyridyl-phenyl mexiletine analogs.

In the United States, approximately one million individuals are hospitalized every year for arrhythmias, making arrhythmias one of the top causes of healthcare expenditures. Mexiletine is currently used as an antiarrhythmic drug but has limitations. The purpose of this work was to use normal and Long QT syndrome Type 3 (LQTS3) patient-derived human induced pluripotent stem cell (iPSC)-derived cardiomyocytes to identify an analog of mexiletine with superior drug-like properties. Compared to racemic mexiletine, medicinal chemistry optimization of substituted racemic pyridyl phenyl mexiletine analogs resulted in a more potent sodium channel inhibitor with greater selectivity for the sodium over the potassium channel and for late over peak sodium current.

Rapid Characterization of Formulated Pharmaceuticals Using Fast MAS 1H Solid-State NMR Spectroscopy.

Active pharmaceutical ingredients (APIs) can be prepared in many different solid forms and phases that affect their physicochemical properties and suitability for oral dosage forms. The development and commercialization of dosage forms require analytical techniques that can determine and quantify the API phase in the final drug product. 13C solid-state NMR (SSNMR) spectroscopy is widely employed to characterize pure and formulated solid APIs; however, 13C SSNMR experiments on dosage forms with low API loading are often challenging due to low sensitivity and interference from excipients. Here, fast magic angle spinning 1H SSNMR experiments are shown to be applicable for the rapid characterization of low drug load formulations. Diagnostic 1H SSNMR spectra of APIs within tablets are obtained by using combinations of frequency-selective saturation and excitation pulses, two-dimensional experiments, and 1H spin diffusion periods. Selective saturation pulses efficiently suppress the broad 1H SSNMR signals from the most commonly encountered excipients such as lactose and cellulose, allowing observation of high-frequency API 1H NMR signals. 1H SSNMR provides a 1-3 orders of magnitude reduction in experiment time compared to standard 13C SSNMR experiments, enabling diagnostic SSNMR spectra of dilute APIs within tablets to be obtained within minutes. The 1H SSNMR spectra can be used for quantification, provided calibrations are performed on a standard sample with known API loading.

Different electrophysiological effects of the levo- and dextro-rotatory isomers of mexiletine in isolated rabbit cardiac muscle.

Racemic mexiletine is a widely used antiarrhythmic agent that blocks sodium channels. The effects of R-(-) and S-(+) mexiletine stereoisomers on maximum rate of depolarization ([Formula: see text]), conduction time, and repolarization have not yet been investigated in isolated cardiac preparations. We studied the effect of the R-(-) and S-(+) mexiletine on rabbit cardiac action potential parameters by using the conventional microelectrode technique. Both enantiomers at 20 µmol/L of therapeutically and experimentally relevant concentration, significantly depressed the [Formula: see text] at fast heart rates (BCLs 300-700 ms). R-(-) mexiletine has more potent inhibitory effect than S-(+) mexiletine. Both R-(-) and S-(+) mexiletine significantly inhibited the [Formula: see text] of early extrasystoles measured at 70 ms diastolic interval induced by S1-S2 stimuli. R-(-) mexiletine has more pronounced inhibitory effect than S-(+) mexiletine. Both R-(-) and S-(+) mexiletine increased significantly the ERP/APD90 ratio. The time constant (τ) of recovery of [Formula: see text] was found to be τ = 376.0 ± 77.8 ms for R-(-) mexiletine and τ = 227.1 ± 23.4 ms for S-(+) mexiletine, which indicates a slower offset kinetics for R-(-) mexiletine from sodium channels than that of the S-(+) enantiomer. These data suggest that R-(-) mexiletine might be a more potent antiarrhythmic agent than S-(+) mexiletine.

Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism.

Discovery of (phenoxy-2-hydroxypropyl)piperidines as a novel class of voltage-gated sodium channel 1.7 inhibitors.

A novel class of NaV1.7 inhibitors has been identified by high-throughput screening followed by structure activity relationship studies. Among this series of compounds, piperidine 9o showed potent human and mouse NaV1.7 inhibitory activities with fair subtype selectivity over NaV1.5. Compound 9o successfully demonstrated analgesic efficacy in mice comparable to that of the currently used drug, mexiletine, but with an expanded central nervous system safety margin.

Liquid chromatographic resolution of mexiletine and its analogs on crown ether-based chiral stationary phases.

Mexiletine, an effective class IB antiarrhythmic agent, and its analogs were resolved on three different crown ether-based chiral stationary phases (CSPs), one (CSP 1) of which is based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and the other two (CSP 2 and CSP 3) are based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6. Mexiletine was resolved with a resolution (R(S)) of greater than 1.00 on CSP 1 and CSP 3 containing residual silanol group-protecting n-octyl groups on the silica surface, but with a resolution (R(S)) of less than 1.00 on CSP 2. The chromatographic behaviors for the resolution of mexiletine analogs containing a substituted phenyl group at the chiral center on the three CSPs were quite dependent on the phenoxy group of analytes. Namely, mexiletine analogs containing 2,6-dimethylphenoxy, 3,4-dimethylphenoxy, 3-methylphenoxy, 4-methylphenoxy, and a simple phenoxy group were resolved very well on the three CSPs even though the chiral recognition efficiencies vary with the CSPs. However, mexiletine analogs containing 2-methylphenoxy group were not resolved at all or only slightly resolved. Among the three CSPs, CSP 3 was found to show the highest chiral recognition efficiencies for the resolution of mexiletine and its analogs, especially in terms of resolution (R(S)).

Influence of experimental diabetes and insulin treatment on the enantioselective pharmacokinetics of mexiletine and its metabolites.

This study evaluates the influence of streptozotocin-induced diabetes on the kinetic disposition and metabolism of mexiletine (MEX) enantiomers in rats. Animals in the control (n = 6 for each blood collection time), diabetic (single intravenous dosage of 45 mg·(kg body mass)(-1) of streptozotocin), and insulin-treated groups (diabetic rats treated daily with 2 IU insulin) received by gavage a single dose of 10 mg·(kg body mass)(-1) racemic MEX. MEX enantiomers and the metabolites hydroxymethylmexiletine (HMM) and p-hydroxymexiletine PHM) were analyzed by LC-MS/MS. Statistical analysis was based on a serial sacrifice design, and parameter estimation was performed using a Bayesian modeling procedure. Area under the curve (AUC) for the (-)-(R) enantiomers of MEX, HMM, and PHM did not differ between the control and diabetic groups. However, AUC for (+)-(S)-MEX and (+)-(S)-HMM were lower in the diabetic than in the control group. Insulin treatment recovered glucose levels to normal and the (+)-(S)-MEX AUC and (+)-(S)-HMM AUC became similar to the AUCs observed in the nondiabetic animals.

Liquid chromatographic enantioseparation of (RS)-mexiletine and (RS)-fluoxetine using chiral derivatizing reagents synthesized with (S)-naproxen moiety.

Enantiomeric separation of racemic mexiletine and fluoxetine was achieved using three chiral derivatizing reagents (CDRs) based on (S)-naproxen. Diastereomers were synthesized by reaction of mexiletine or fluoxetine with the CDRs and were separated on a C18 column under reversed-phase conditions using a binary mixture of acetonitrile and triethylammonium phosphate/water, with UV detection at 230 and 226 nm. The results obtained for enantioseparation of the two drugs using the three CDRs were compiled and compared. The conditions for derivatization and chromatographic separation were optimized. The method was validated for linearity, repeatability, limit of detection and limit of quantification.

Development, validation and long-term evaluation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of amiodarone, desethylamiodarone and mexiletine in human plasma and serum.

Amiodarone and mexiletine are used for ventricular arrhythmias, for which a combination therapy of both anti-arrhythmic drugs (AADs) is not uncommon. Therapeutic drug monitoring (TDM) can be beneficial for clinical guidance of therapy, especially to correctly identify adverse events. Desethylamiodarone, the active metabolite of amiodarone, accumulates over time and is associated with serious adverse events. Therefore, simultaneous TDM for amiodarone, desethylamiodarone and mexiletine is advantageous in clinical practice. The presented LC-MS/MS method was validated for selectivity, matrix effect, linearity, accuracy, precision, carry-over and stability. The method was continuously evaluated during eight months of clinical use. The method was shown to be linear within the measured range of 0.1 to 10 mg/L for each component. The matrix effect was considered negligible. No interfering responses were found for amiodarone, desethylamiodarone and the isotopic-labeled internal standards. A constant and reproducible within-run contribution of 45.3 %, originating from the system, was identified for mexiletine. The systemic contribution to the peak area of the lowest quantifiable concentration of mexiletine affected the selectivity and carry-over effect measurements. Multiple measurements showed that regression adjusted concentrations were accurate and reproducible, indicating calibration correction was applicable. Sample stability was found to be within limits for all storage conditions and freeze-thaw cycles. Furthermore, long-term method evaluation with external controls resulted in stable measurements with a percentage coefficient of variance between 1.3 % and 6.3 %. The presented practical and reliable method is applicable for clinical TDM and will allow clinical practitioners to guide drug therapy of amiodarone and mexiletine.

Combined modifications of mexiletine pharmacophores for new lead blockers of Na(v)1.4 channels.

Previously identified potent and/or use-dependent mexiletine (Mex) analogs were used as template for the rational design of new Na(v)-channel blockers. The effects of the novel analogs were tested on sodium currents of native myofibers. Data and molecular modeling show that increasing basicity and optimal alkyl chain length enhance use-dependent block. This was demonstrated by replacing the amino group with a more basic guanidine one while maintaining a proper distance between positive charge and aromatic ring (Me13) or with homologs having the chirality center nearby the amino group or the aromatic ring. Accordingly, a phenyl group on the asymmetric center in the homologated alkyl chain (Me12), leads to a further increase of use-dependent behavior versus the phenyl Mex derivative Me4. A fluorine atom in paraposition and one ortho-methyl group on the xylyloxy ring (Me15) increase potency and stereoselectivity versus Me4. Charge delocalization and greater flexibility of Me15 may increase its affinity for Tyr residues influencing steric drug interaction with the primary Phe residue of the binding site. Me12 and Me15 show limited selectivity against Na(v)-isoforms, possibly due to the highly conserved binding site on Na(v). To our knowledge, the new compounds are the most potent Mex-like Na(v) blockers obtained to date and deserve further investigation.

Asymmetric hydrolytic kinetic resolution with recyclable macrocyclic Co(III)-salen complexes: a practical strategy in the preparation of (R)-mexiletine and (S)-propranolol.

A chiral cobalt(III) complex (1e) was synthesized by the interaction of cobalt(II) acetate and ferrocenium hexafluorophosphate with a chiral dinuclear macrocyclic salen ligand that was derived from 1R,2R-(-)-1,2-diaminocyclohexane with trigol bis-aldehyde. A variety of epoxides and glycidyl ethers were suitable substrates for the reaction with water in the presence of chiral macrocyclic salen complex 1e at room temperature to afford chiral epoxides and diols by hydrolytic kinetic resolution (HKR). Excellent yields (47% with respect to the epoxides, 53% with respect to the diols) and high enantioselectivity (ee>99% for the epoxides, up to 96% for the diols) were achieved in 2.5-16 h. The Co(III) macrocyclic salen complex (1e) maintained its performance on a multigram scale and was expediently recycled a number of times. We further extended our study of chiral epoxides that were synthesized by using HKR to the synthesis of chiral drug molecules (R)-mexiletine and (S)-propranolol.

Stereoselective binding of mexiletine and ketoprofen enantiomers with human serum albumin domains.

AIM: To investigate the stereoselective binding of mexiletine or ketoprofen enantiomers with different recombinant domains of human serum albumin (HSA). METHODS: Three domains (HSA DOM I, II and III) were expressed in Pichia pastoris GS115 cells. Blue Sepharose 6 Fast Flow was employed to purify the recombinant HSA domains. The binding properties of the standard ligands, digitoxin, phenylbutazone and diazepam, and the chiral drugs to HSA domains were investigated using ultrafiltration. The concentrations of the standard ligands, ketoprofen and mexiletine were analyzed with HPLC. RESULTS: The recombinant HSA domains were highly purified as shown by SDS-PAGE and Western blotting analyses. The standard HSA ligands digitoxin, phenylbutazone and diazepam selectively binds to DOM I, DOM II and DOM III, respectively. For the chiral drugs, R-ketoprofen showed a higher binding affinity toward DOM III than S-ketoprofen, whereas S-mexiletine bound to DOM II with a greater affinity than R-mexiletine. CONCLUSION: The results demonstrate that HSA DOM III possesses the chiral recognition ability for the ketoprofen enantiomers, whereas HSA DOM II possesses that for the mexiletine enantiomers.

High performance liquid chromatography enantioseparation of the novel designed mexiletine derivatives and its analogs.

A series of novel designed mexiletine derivatives and its analogs were prepared, the structures were confirmed by Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared Spectroscopy (FTIR), and Electrospray Ionization-Mass Spectrometry (ESI-MS), and the enantioseparations were performed on polysaccharide-based chiral stationary phase (CSP), Chiralcel OD-H, and Chiralcel OJ-H, under normal-phase mode. The effects of the concentration of isopropanol in the mobile phase were studied, seven of the eight enantiomers got baseline separation on Chiralcel OD-H, and five of the eight enantiomers got successfully separation on Chiralcel OJ-H. The effects of structural features were also discussed.

Indirect enantioresolution of (R,S)-mexiletine by reversed-phase high-performance liquid chromatography via diastereomerization with [(S,S)-O,O'-di-p-toluoyl tartaric acid anhydride], (S)-naproxen and nine chiral reagents synthesized as variants of Marfey's reagent.

Eleven chiral derivatizing reagents (CDRs) were used for preparation of diastereomers of (R,S)-mexiletine containing a primary amino group in close proximity to the stereogenic center. One anhydride, namely [(S,S)-O,O'-di-p-toluoyl tartaric acid anhydride] was synthesized and (S)-naproxen was used as such as the chiral derivatizing reagent. The other nine CDRs were synthesized by substituting one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene with six amino acid amides and three amino acids. The diastereomers were separated by reversed-phase high-performance liquid chromatography. The method was validated for linearity, accuracy, limit of detection and limit of quantification. The limit of detection was found in the range of 10-30 pmol.

Hydroxylated analogs of mexiletine as tools for structural-requirements investigation of the sodium channel blocking activity.

[2-(2-Aminopropoxy)-1,3-phenylene]dimethanol 1 and 4-(2-aminopropoxy)-3-(hydroxymethyl)-5-methylphenol 2, two dihydroxylated analogs of mexiletine - a well known class IB anti-arrhythmic drug - were synthesized and used as pharmacological tools to investigate the blocking-activity requirements of human skeletal muscle, voltage-gated sodium channel. The very low blocking activity shown by newly synthesized compounds corroborates the hypothesis that the presence of a phenolic group in the para-position to the aromatic moiety and/or benzylic hydroxyl groups on the aromatic moiety of local anesthetic-like drugs impairs either the transport to or the interaction with the binding site in the pore of Na(+) channels.

Enantiodivergent syntheses of (+)- and (-)-1-(2,6-dimethylphenoxy)propan-2-ol: A way to access (+)- and (-)-mexiletine from D-(+)-mannitol.

Chiron approach was used to acquire optically pure (R)- and (S)-1-(2,6-dimethylphenoxy)propan-2-ol, immediate precursors of (S)- and (R)-mexiletines, respectively. Two different routes were followed from a D-mannitol-derived optically pure common precursor to get the enantiomeric alcohols separately. Comparison of their specific rotation values with the corresponding literature values as well as exact mirror-image relationship between their CD curves proved their high enantiopurity. These alcohols were then transformed to the corresponding amine-drugs in an efficient one-step process instead of two steps described in the literature.

Fragment-based discovery of mexiletine derivatives as orally bioavailable inhibitors of urokinase-type plasminogen activator.

Fragment-based lead discovery has been applied to urokinase-type plasminogen activator (uPA). The (R)-enantiomer of the orally active drug mexiletine 5 (a fragment hit from X-ray crystallographic screening) was the chemical starting point. Structure-aided design led to elaborated inhibitors that retained the key interactions of (R)-5 while gaining extra potency by simultaneously occupying neighboring regions of the active site. Subsequent optimization led to 15, a potent, selective, and orally bioavailable inhibitor of uPA.

Novel automated assay for the quality control of mexiletine hydrochloride formulations using sequential injection and on-line dilution.

The first automated method for the determination of mexiletine hydrochloride - an antiarrhythmic agent - is reported. The method is based on the reaction of the analyte with o-phthalaldehyde (OPA) in the presence of sulfite in basic medium using a sequential injection (SI) manifold. The reaction product was monitored spectrofluorimetrically (lambda(ex)=350 nm/lambda(em)=446 nm). A simple and effective on-line dilution approach was adopted in order to expand the linearity and apply the method to assay, dosage uniformity and dissolution tests with minimum sample preparation. Chemical (pH, amount concentrations of OPA and sulfite) and instrumental variables (temperature, flow rate, injection volumes, etc.) that affected the determination were studied. The developed assay was validated in terms of linearity, range, limits of detection (LOD=3.4 mg L(-1)) and quantitation (LOQ=10 mg L(-1)), accuracy, precision (R.S.D.<3.4%) and selectivity. The method was applied successfully to the quality control of a mexiletine-containing formulation. The results were in good agreement with the US pharmacopoeia HPLC method.

Simultaneous determination of ten antiarrhythic drugs and a metabolite in human plasma by liquid chromatography--tandem mass spectrometry.

A simple, accurate and selective LC-MS/MS method was developed and validated for simultaneous quantification of ten antiarrhythic drugs (diltiazem, amiodarone, mexiletine, propranolol, sotalol, verapamil, bisoprolol, metoprolol, atenolol, carvedilol) and a metabolite (norverapamil) in human plasma. Plasma samples were simply pretreated with acetonitrile for deproteinization. Chromatographic separation was performed on a Capcell C(18) column (50mmx2.0mm, 5microm) using a gradient mixture of acetonitrile and water (both containing 0.02% formic acid) as a mobile phase at flow rate of 0.3ml/min. The analytes were protonated in the positive electrospray ionization (ESI) interface and detected in multiple reaction monitoring (MRM) mode. Calibration curves were linear over wide ranges from sub- to over-therapeutic concentration in plasma for all analytes. Intra- and inter-batch precision of analysis was <12.0%, accuracy ranged from 90% to 110%, average recovery from 85.0% to 99.7%. The validated method was successfully applied to therapeutic drug monitoring (TDM) of antiarrhythic drugs in routine clinical practice.

[Determination of mexiletine in human blood by liquid chromatography-tandem mass spectrometry].

OBJECTIVE: To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of mexiletine by liquid chromatography tandem mass spectrometry. METHODS: After simple protein precipitation of the blood sample with acetonitrile, the organic solvent layer diluted with LC mobile solvent was separated by Allure PFP Propyl column, confirmed and quantified by MS/MS in the multi-reaction monitoring (MRM) mode via positive electrospray ionization. RESULTS: Mexiletine and naloxone (internal standard) got ideal resolution under the selected analytical condition. The correlation coeficient of linear calibration curve was over 0.9999 within the mexiletine concentration range 0.02-10 microg/mL. The relative standard deviations were under 10% for intra-day and under 15% for inter-day, and the detection limit was 0.01 microg/mL. CONCLUSION: The established LC-MS/MS method is simple, rapid, sensitive, unaffected by matrix effect and appropriate for detection of mexiletine in blood in the field of therapeutic drug monitoring and forensic toxicology.