Isolation and structural characterization by spectroscopic methods of two glucuronide metabolites of mexiletine after N-oxidation and deamination.

Urine samples from control and mexiletine-treated human subjects or rabbits (test group) were collected and passed through an ion exchange resin to isolate polar compounds. Methanolic eluates from control and test urines were analyzed by TLC. Exposure to p-dimethylaminocinnamaldehyde gave an additional intense pink band at Rt 0.40-0.45 in TLC analysis of test urine eluate when compared to control urine eluate. Non-exposed silica at this Rt was scraped and metabolites were extracted with methanol. Hydrolysis of this methanolic extract at 100 degrees C with hydrochloric acid released mexiletine. GC/MS and fast atom bombardment mass spectrometry analyses of nonhydrolyzed methanolic extracts evidenced the presence of two conjugated metabolites of mexiletine, namely, N-hydroxymexiletine glucuronide and mexiletine alcohol glucuronide. Synthetic compounds corresponding to these metabolites were obtained and spectra compared with those of isolated metabolites from urine. Definite structure assignment of N-hydroxymexiletine glucuronide was obtained from NMR spectrometry which confirmed the structure to be a hydoxylamine glucuronide (N-O-C link) and showed that the glycoside moiety was in the beta configuration. Thus, it is proposed that N-hydroxymexiletine glucuronide corresponds to mexiletine acid-labile conjugate and represents a major metabolic pathway in the disposition of mexiletine.

Influence of debrisoquine phenotype and of quinidine on mexiletine disposition in man.

Mexiletine is a low clearance drug which undergoes extensive metabolism in man. In vitro studies with human liver microsomes have suggested that major oxidation pathways of mexiletine are predominantly catalyzed by the genetically determined debrisoquine 4-hydroxylase (cytochrome P450IID6) activity. In this study, we investigated the role of debrisoquine polymorphism and the effects of low dose quinidine, a selective inhibitor of cytochrome P450IID6, on the disposition of mexiletine. Fourteen healthy volunteers, 10 with the extensive metabolizer (EM) and 4 with the poor metabolizer (PM) phenotype, received a single 200-mg dose of mexiletine hydrochloride orally on two occasions (1 week apart), once alone and once under steady-state conditions for quinidine (50 mg QID). During the phase mexiletine alone, total clearance, nonrenal clearance and partial metabolic clearance of mexiletine to hydroxymethylmexiletine, to m-hydroxymexiletine and to p-hydroxymexiletine were decreased in PM compared to EM (all P less than .05). In EM, quinidine decreased mexiletine total clearance from 621 +/- 298 to 471 +/- 214 ml/min (mean +/- S.D.; P less than .05) and mexiletine nonrenal clearance from 583 +/- 292 to 404 +/- 188 ml/min (P less than .05). Moreover, quinidine increased mexiletine elimination half-life in EM from 9 +/- 1 to 11 +/- 2 h (P less than .05). In these subjects, partial metabolic clearance to hydroxymethylmexiletine, m-hydroxymexiletine and p-hydroxymexiletine were decreased by quinidine coadministration 5-, 4- and 7-fold, respectively, whereas partial metabolic clearance to N-hydroxymexiletine was unaffected. Changes induced by quinidine in EM were correlated to their debrisoquine metabolic ratio. Thus, genetically determined or pharmacologically induced modulation of cytochrome P450IID6 activity represents a major determinant of mexiletine disposition.

Resolution and electrophysiological effects of mexiletine enantiomers.

Resolution of mexiletine enantiomers from the racemic mixture has been achieved by fractional crystallization through the formation of diastereoisomeric p-toluoyl tartrate salts. Following three crystallization steps in methanol, R-(-)- and S-(+)-mexiletine were resolved with an optical purity greater than 98% (yield approximately 30%) and their hydrochloride salts formed. Incremental doses of mexiletine enantiomers were administered to dogs with experimentally-induced arrhythmias to investigate the stereoselective antiarrhythmic and electrophysiological effects of these compounds. Using up to three extrastimuli, programmed electrical stimulation was performed in conscious animals 7-30 days after coronary ligation. R-(-)-Mexiletine prevented ventricular tachycardia in 3/6 dogs (2 after 0.5 mg kg-1, 1 after 8 mg kg-1); two animals died after 1 and 8 mg kg-1, respectively; one remained unchanged even at the highest dosage (16 mg kg-1). S-(+)-Mexiletine prevented ventricular tachycardia in only one dog (after 1 mg kg-1); two died after 4 and 8 mg kg-1, respectively; 2/5 remained unchanged even after the administration of 16 mg kg-1. No significant changes in any electrocardiographic intervals (PR, QRS, QTc) or refractory periods were induced by mexiletine enantiomers at any doses used (0.5-16.0 mg kg-1). These results suggest that R-(-)-mexiletine possesses greater antiarrhythmic properties than the opposite enantiomer.

Meta-hydroxymexiletine, a new metabolite of mexiletine. Isolation, characterization, and species differences in its formation.

Meta-hydroxymexiletine [1-(3-hydroxy,2,6-dimethyl)phenoxy-2-amino-propane], a novel metabolite of the antiarrhythmic drug mexiletine, was isolated from urine of rats given mexiletine. The structure of the metabolite was elucidated by 1H-NMR and mass spectrometry and by IR spectrophotometry. The metabolite is produced in vitro by hepatic microsomes of various laboratory animals including rat, guinea-pig, hamster, rabbit, and mouse. In humans, meta-hydroxymexiletine accounts for approximately 2% of the administered dose of mexiletine.

The effect of fleroxacin on hepatic drug metabolism in the rat.

Pretreatment of rats with repeated i.p. doses of fleroxacin had no effect on the hepatic O- and N-demethylation of p-nitroanisole (PN) and aminopyrine (A) respectively, on the p-hydroxylation of aniline or on the hepatic levels of cytochrome P-450 and protein content. Addition of fleroxacin directly to incubation mixtures reduced PN O-demethylase activity significantly but did not affect the other two oxidative reactions. The N-oxide metabolite increased PN O-demethylation by 16% whereas the N-demethylated metabolite decreased the N-demethylation of A by 10%. Concomitant administration or pretreatment with fleroxacin had no effect on antipyrine's elimination kinetic parameters.

Pharmacokinetics of mexiletine in the elderly.

The effect of advancing age on the kinetics of the antiarrhythmic agent mexiletine was studied by comparing various kinetic parameters calculated after administration of a single oral dose of mexiletine hydrochloride to seven elderly and eight young healthy volunteers. The rate of absorption of the drug from the gastrointestinal tract was significantly slower in the elderly (1.37 +/- 0.51 hr-1) than in the young group (2.25 +/- 0.79 hr-1). The mean values for elimination half-life and oral clearance were 12.3 +/- 3.7 hr and 10.3 +/- 5.4 mL/min/kg respectively in the young group and 14.4 +/- 4.5 hr and 8.5 +/- 2.9 mL/min/kg respectively in the elderly group. Neither of these parameters was significantly different between the two groups. The amount of mexiletine eliminated in urine up to 48 hours postdose was identical in both groups and represented less than 5% of the administered dose. It is concluded that the age-related modifications in the kinetics of mexiletine are not clinically important during chronic administration of the drug.

Pharmacokinetics of diltiazem in patients undergoing continuous ambulatory peritoneal dialysis.

The disposition of a single oral dose of diltiazem hydrochloride was studied in six male patients treated by continuous ambulatory peritoneal dialysis. Peak concentrations were obtained 2 to 4 hours postdose. The mean absorption rate constant was 0.94 +/- 0.21 (sd) hr-1, and the mean elimination half-life was 3.09 +/- 1.16 hr. Serum levels of deacetyldiltiazem, a metabolite of diltiazem, were always below 10 ng/mL. The amounts of diltiazem and deacetyldiltiazem eliminated in dialysate over 24 hours represent less than 0.1% of the administered dose. The pharmacokinetic parameters of diltiazem determined in these patients did not differ from those determined in healthy volunteers and in patients suffering from end-stage renal disease.

2,6-Dimethylphenol: a new metabolite of mexiletine.

A new metabolite, 2,6-dimethylphenol obtained by O-dealkylation of mexiletine by rabbit liver was identified. A g.c.-f.i.d. method was developed for its analysis in hepatic homogenates and some of the characteristics of the metabolic reaction were studied. Formation of 2,6-dimethylphenol is microsomal and is optimal under aerobic conditions. Both carbon monoxide and n-octylamine significantly depress this O-dealkylation reaction.

Assay of diltiazem and deacetyldiltiazem by capillary gas chromatography.

A highly sensitive gas chromatographic method for the analysis of diltiazem and deacetyldiltiazem in plasma or serum is reported. After silylation with bis (trimethylsilyl) trifluoroacetamide, separation was obtained on a cross-linked fused-silica column and detection was by electron-capture. The minimum measurable concentrations were 3 and 1 ng/ml for diltiazem and deacetyldiltiazem, respectively. Intra- and inter-day coefficients of variation were less or equal to 6.0 and 8.0%, respectively, for both compounds. The method was used to study the kinetics of a single oral dose of 60 mg of diltiazem hydrochloride in a patient with renal failure.

Deacetylation of diltiazem by rat liver.

The enzyme system mediating the hydrolysis of the calcium antagonist diltiazem to give deacetyldiltiazem was characterized in the rat. Tissue distribution studies showed that the highest level of activity was mainly localized in the microsomal fraction of the liver. Some activity was also detected in the red blood cells. The kinetics of the enzymatic reaction demonstrated that the formation of deacetyldiltiazem increased linearly with time up to 60 min and with protein content up to 7.8 mg. Apparent Km and Vmax values calculated from a Lineweaver-Burk plot were 0.17 X 10(-3) M and 0.013 mumol/mg of protein/min. Mercuric chloride, silver nitrate, and cupric chloride at concentrations of 0.6 X 10(-3) M decreased the diltiazem deacetylase activity to 47%, 24%, and 19%, respectively, as compared to control incubations. At a concentration of 6.7 X 10(-8) M, cadmium sulfate decreased the hydrolysis of diltiazem by 40%, whereas cobaltous sulfate at concentrations of 10(-3) M did not affect the deacetylation activity. The hydrolysis reaction was depressed by the organophosphorus compounds, bis-p-nitrophenylphosphate and diisopropyl fluorophosphate to 31% and 0%, respectively, at concentrations of 10(-6) M. Eserine sulfate at a concentration of 2.2 X 10(-4) M, and disulfiram and aspirin at concentrations of 10(-3) M decreased the diltiazem deacetylase activity to 17%, 71%, and 79%, respectively. Rifampicin and phenacetin at concentrations of 10(-3) M did not inhibit the hydrolysis reaction. In vivo pretreatment of the rats with phenobarbital increased the in vitro diltiazem deacetylase activity 3.2-fold, whereas 3-methylcholanthrene did not affect the enzymatic hydrolysis of diltiazem.

Stereoselective disposition of mexiletine in man.

The pharmacokinetics of S-(+)- and R-(-)-mexiletine and of the corresponding conjugates were investigated in six healthy young volunteers after administration of a single 200 mg oral dose of racemic mexiletine hydrochloride. The values for the distribution rate constants as well as for the elimination half-lives of the two enantiomers were similar but the AUC of the S-(+)-enantiomer was always significantly higher (P less than 0.01) than that of the opposite enantiomer. The mean R/S ratios for unchanged mexiletine in serum and in urine were 0.78 +/- 0.12 (s.d.) and 0.80 +/- 0.21, respectively. Urinary excretion of mexiletine conjugates consisted mainly of the R-(-)-enantiomer; the mean R/S enantiomeric ratio over 48 h was 9.65 +/- 3.10. Serum concentrations of the conjugates were measured in three subjects. The mean R/S AUC ratio was 2.94 +/- 0.48 and the renal clearance of the R-(-)-enantiomer was significantly higher (P less than 0.02) than that of the S-(+)-enantiomer.

Effect of cigarette smoking on mexiletine kinetics.

The effect of cigarette smoking on the kinetics of a single, 200 mg, oral dose of the antiarrhythmic drug mexiletine was investigated in healthy subjects. Cigarette smoking had no effect on absorption or distribution of the drug, but it significantly reduced the elimination t1/2 from 11.1 +/- 3.4 to 7.2 +/- 1.8 hours; the effect on clearance was less significant. Determination of the urinary concentrations of the three major metabolites of mexiletine (mexiletine glucuronide conjugate, hydroxymethylmexiletine, and p-hydroxymexiletine) indicated that cigarette smoking selectively induced conjugation of mexiletine with glucuronic acid as well as aliphatic hydroxylation to yield hydroxymethylmexiletine, but that it had no effect on the formation of p-hydroxymexiletine.

High pressure liquid chromatographic assay for mexiletine in serum.

A highly sensitive, rapid, and specific high pressure liquid chromatographic assay for the analysis of the antiarrhythmic agent mexiletine is reported. The method involves extraction of mexiletine with organic solvent followed by analysis using fluorescence detection. The minimum measurable limit is 1 ng and inter- and intra-day coefficients of variation are less than 5.8%. The method is useful for pharmacokinetic studies and routine serum monitoring of mexiletine.

Depressive effect of amiodarone on hepatic drug metabolism in the rat.

The effect of pretreatment of rats with three daily i.p. doses of 25, 50 and 100 mg/kg of the antiarrhythmic agent, amiodarone, on the activity of some hepatic drug metabolizing enzymes, on the levels of cytochromes P-450 and b5 as well as on lipid peroxidation is reported. Amiodarone at doses of 50 and 100 mg/kg significantly reduced the hydroxylation of aniline, the 0- and N-demethylations of p-nitroanisole and aminopyrine respectively and the level of cytochromes P-450. No inhibition in enzymic activity was observed with the 25 mg/kg dose. Testosterone hydroxylation was only depressed with the 100 mg/kg dose; in this case, all four hydroxylation reactions were reduced. Cytochrome b5 content and lipid peroxidation were significantly decreased with doses of 25 and 50 mg/kg. Addition of 1 mM of amiodarone to incubation mixtures containing either aminopyrine or p-nitroanisole or aniline only decreased aniline hydroxylation indicating that at high doses amiodarone acts as a competitive inhibitor of aniline hydroxylase but that the depressive effect observed on the 0- and N-demethylations of p-nitroanisole and aminopyrine is mediated via an indirect mechanism. The inhibitory effect of amiodarone was also shown in vivo by an increase in the elimination half-life and a decrease in the clearance of antipyrine. The results reported herein may explain, in part at least, the drug interactions recently reported in man with amiodarone.

Inhibitory effect of isoniazid on antipyrine clearance in man.

The effect of both concomitant administration and pretreatment with isoniazid on the activity of the hepatic drug-metabolizing enzymes of healthy young volunteers, as indicated by the antipyrine clearance test, is reported. Concomitant administration of isoniazid with antipyrine results in a significant decrease in the hepatic clearance of the latter compound. In contrast, pretreatment for 14 days with isoniazid had no effect on antipyrine elimination kinetics. It is concluded that isoniazid depresses hepatic drug metabolism only when present in significant amounts at the hepatic site of drug oxidation.