A rapid and sensitive liquid chromatography/tandem mass spectrometry assay for simultaneous quantitation of disopyramide and its major metabolite, mono-isopropyl-disopyramide, in rat plasma and its application to a pharmacokinetic study.

Disopyramide as an antiarrhythmic agent has been used for treating ventricular tachycardia and metabolized into its major metabolite, mono-isopropyl-disopyramide, by CYP3A4. We developed a novel, selective, highly sensitive, accurate, rapid method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of disopyramide and mono-isopropyl-disopyramide in rat plasma. This study is the first report for the assay validation using LC-MS/MS in biological fluids after simple protein-precipitation method. The most sensitive signals by multiple reaction monitoring (MRM) showed at m/z 340.2 → 239.2 and 298.2 → 239.2 with same fragment ion for disopyramide and mono-isopropyl-disopyramide, respectively. The lower limit of quantification (LLOQ) was determined at 2 ng/mL for both analytes and the linear concentration ranges were found to be 2-2000 ng/mL for disopyramide and 2-1000 ng/mL for mono-isopropyl-disopyramide. Finally, this assay was successfully applied to pharmacokinetic analysis of disopyramide and mono-isopropyl-disopyramide after oral and intravenous administration of disopyramide.

Characterization of Disopyramide derivative ADD424042 as a non-cardiotoxic neuronal sodium channel blocker with broad-spectrum anticonvulsant activity in rodent seizure models.

It was reported that antiarrhythmic drugs (AADs) can be useful in controlling refractory seizures in humans or in enhancing the action of antiepileptic drugs (AEDs) in animal models. Disopyramide phosphate (DISO) is an AAD that blocks sodium channels in cardiac myocytes. We evaluated a DISO derivative, 2-(2-chlorophenyl)-2-(pyridin-2-yl)acetamide (ADD424042) for its anticonvulsant activity in a battery of rodent models of epileptic seizures. The compound displayed a broad spectrum of activity in the 'classical' models as well as in the models of pharmacoresistant seizures. Furthermore, ADD424042 showed good therapeutic indices between the anticonvulsant activity and the motor impairment. On the contrary, no anticonvulsant effects but severe lethality were observed in the primary anticonvulsant testing of the parent DISO. By performing the whole-cell voltage-clamp experiments in dispersed cortical neurons we demonstrated that ADD424042 decreased the maximal amplitude of voltage-gated sodium channels with an IC50 value in nM range. Moreover, the compound enhanced use-dependent block and decreased excitability in pyramidal neurons in the current-clamp experiments in cortical slices. Importantly, we found that ADD424042 possessed either no, or very small cardiotoxic effect. In contrast to DISO, ADD424042 did not produce any apparent changes in electrocardiogram (ECG) and arterial blood pressure recordings. ADD424042 had no effect on QT and corrected QT intervals, at a dose which was 15 times higher than ED50 for the anticonvulsant effect in the MES model. Taken together, these data suggest that ADD424042 has the potential to become a lead structure for novel broadly acting AEDs with wide margin of cardiac safety.

Effects of serum concentrations of disopyramide and its metabolite mono-N-dealkyldisopyramide on the anticholinergic side effects associated with disopyramide.

The aim of this study was to evaluate the relationship between the anticholinergic side effects associated with disopyramide (DP) and serum DP or mono-N-dealkyldisopyramide (MND) concentrations and the safety range of DP or MND for prevention of anticholinergic side effects in 141 inpatients. The serum DP and MND concentrations were determined by high-performance liquid chromatography. No correlation was observed between creatinine clearance (Ccr) and the ratio of the serum concentration to the dose (C/D) of DP, but a significant inverse correlation was observed between Ccr and the C/D of MND. It was observed that the ratio of MND concentration to DP concentration in the group, whose Ccr was below 20 ml/min, was higher than that of the other groups. Although no significant difference was observed in the DP concentration between the patients without (Group 1) or with (Group 2) anticholinergic side effects, significant differences were observed in the MND concentration, Ccr, and the ratio of MND/DP. The DP concentrations of both Groups 1 and 2 were distributed from 0.13 to about 5 microg/ml. On the other hand, although the MND concentrations of Group 1 were below about 1 microg/ml, the MND concentrations of Group 2 were above about 1 microg/ml. These results suggest that not only DP concentration but also MND concentration should be monitored in patients whose renal function is decreased to prevent anticholinergic side effects associated with DP, and that when serum MND concentration was over approximately 1 microg/ml, the dose should be decreased or discontinued.

Disopyramide and its metabolite enhance insulin release from clonal pancreatic beta-cells by blocking K(ATP) channels.

In an insulin-secreting pancreatic beta-cell line (MIN6), insulin release was caused by disopyramide, an antiarrhythmic drug with Na-channel blocking action, and its main metabolite mono-isopropyl disopyramide (MIP). Insulin secretion, measured as immunoreactive insulin (IRI), was accelerated to 265.7% of the control by disopyramide and to 184.4% by MIP, with half-effective concentrations (EC50) of 30.9 +/- 1.5 microM and 92.4 +/- 2.2 microM. We tested the possibility that these drugs induce insulin release by inhibiting ATP-sensitive K+ (K(ATP)) channels of MIN6 cells. In the cell-attached or ATP-free inside-out mode with patch membranes on MIN6 cells, K-selective channels were recorded with unitary conductance of 70.5 +/- 3.5 pS (150 mM external K+ ions at room temperature). The channels were concluded to be MIN6-K(ATP) channels because they were closed by extracellular high glucose (11.0 mM) or glibenclamide (200 nM) and were reversibly activated by diazoxide (50 microM). In the inside-out patch mode, they were inhibited by micromolar ATP. In both cell-attached and insideout mode, disopyramide and MIP inhibited single MIN6-K(ATP) channels. In the inside-out mode, they produced a dose-dependent inhibition of channel activity: the half-blocking concentrations (IC50) were 4.8 +/- 0.2 microM for disopyramide and 40.4 +/- 3.1 microM for MIP. It was therefore concluded that both agents exert insulinotrphic effect through the inhibition of membrane K(ATP) channels in MIN6 cells.

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in human plasma by capillary electrophoresis.

In this paper, a rapid method for the enantioselective analysis of the antiarrhythmic drug disopyramide and its main metabolite mono-N-dealkyldisopyramide in human plasma by capillary electrophoresis employing the cyclodextrin-modified electrokinetic chromatography mode is described. Sample clean-up was carried out by alkalinization with sodium hydroxide followed by liquid-liquid extraction with toluene. The complete enantioselective analysis was performed within less than 5 min using 20 mmol/L sodium acetate buffer, pH 5.0, containing 0.2% w/v sulfated beta-cyclodextrin as chiral selector. A 40 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 15 kV and at 20 degrees C. The calibration curves were linear over the concentration range of 62.5-1850 ng/mL and 125-1850 ng/mL for each enantiomer of disopyramide and mono-N-dealkyldisopyramide. The mean recoveries for disopyramide and mono-N-dealkyldisopyramide enantiomers were up to 87 and 69%, respectively. All four enantiomers studied could be quantified at three different concentrations (200, 400 and 600 ng/mL) with coefficient of variation and % relative error not higher than 15%. The quantitation limit was 62.5 ng/mL for (+)-(S)-and (-)-(R)-disopyramide and (-)-(R)-mono-N-dealkyldisopyramide and 125 ng/mL for (+)-(S)-mono-N-dealkyldisopyramide, using 1 mL of human plasma.

[Accumulation of a disopyramide metabolite in renal failure].

Twenty-nine cases of hypoglycemia induced by disopyramide (DP) have been reported in the literature to date. Twenty of the reported cases showed hypo-renal function and a high concentration was rare. DP is metabolized to mono-N-dealkyldisopyramide (MND) in the liver and accumulation of MND is to be expected in renal failure. Both DP and MND bind mainly to alpha-1-acid glycoprotein (AAG) in the plasma. In 10 hemodialysis (HD) patients with normal liver function receiving DP therapy in the steady state. DP, MND and AAG were measured pre- and post-HD. Ten patients with normal renal and liver function were selected as the controls. The DP concentration was 2.08 +/- 0.39 micrograms/ml (mean +/- SD) in the control group, and the pre- and post-HD levels were 2.40 +/- 1.08 micrograms/ml and 1.73 +/- 0.87 micrograms/ml, respectively, in the HD group. The MND concentration was 0.42 +/- 0.23 micrograms/ml in the controls, 1.53 +/- 0.52 micrograms/ml in pre-HD and 1.08 +/- 0.32 micrograms/ml in post-HD. Although DP and MND are both classified as substances of small molecular weight, the average decrease in plasma concentration from pre- to post-HD was under 30% with both agents. The MND/DP ratio in the HD group was higher than in the controls, but there was no significant difference between pre- and post-HD. The AAG level was 75 +/- 5mg/dl in the controls and 109 +/- 11mg/dl before HD in the HD group (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of a disopyramide metabolite in renal failure.

Ten hemodialysis (HD) patients with normal liver function received disopyramide (DP) therapy in the steady state. DP, mono-N-dealkyldisopyramide (MND) and alpha-1-acid glycoprotein (AAG) were measured before and after HD. Ten patients with normal renal and liver function were selected as controls. The DP concentration was 2.08 +/- 0.39 micrograms/ml (mean +/- SD) in the control group, and the pre and post HD levels were 2.40 +/- 1.08 micrograms/ml and 1.73 +/- 0.87 micrograms/ml, respectively, in the HD group. The MND concentration was 0.42 +/- 0.23 microgram/ml in controls, 1.53 +/- 0.52 micrograms/ml in pre HD and 1.08 +/- 0.32 microgram/ml in post-HD. Although DP and MND are both classified as small molecular weight substances, the average decrease in plasma concentration from pre to post HD was under 30% with both agents. The MND/DP ratio in the HD group was higher than in controls, but there was no significant difference between pre and post HD. The AAG level was 75 +/- 5 mg/dl in controls and 109 +/- 11 mg/dL before HD in the HD group (p < 0.001). In conclusion, an MND accumulation was observed in HD patients receiving DP therapy. Because the anticholinergic effect of MND is 24 times that of DP, MND is thought to contribute in some way to the hypoglycemia induced by DP in renal failure.

Comparative class 1 electrophysiologic and anticholinergic effects of disopyramide and its main metabolite (mono-N-dealkylated disopyramide) in healthy humans.

During steady-state treatment with disopyramide, its main and active metabolite, mono-N-dealkylated disopyramide, was reported to reach concentrations that were equal to or higher than the parent drug in 25% of 70 evaluated patients. This metabolite has been found to have a more pronounced anticholinergic action than the parent drug on in vitro evaluation, but neither its anticholinergic nor its direct electrophysiologic effects on the human heart have been properly assessed. We therefore compared the acute electrophysiologic and anticholinergic effects (the standard being atropine, 0.04 mg/kg) of disopyramide and its main metabolite, given 2 mg/kg body weight intravenously to 10 healthy individuals in a double-blind, randomized, crossover design. The anticholinergic effect of these substances on sinus and atrioventricular node function was unexpectedly found to be of similar magnitude and more pronounced than previously thought (at least one-third the effects of the atropine dose). The class 1 electrophysiologic effects were as follows: intra-atrial and His-Purkinje conduction (the PA and the HV interval, respectively) was prolonged 33% (95% CI: 18-47%) and 27% (21-32%) by disopyramide, and 15% (10-19%) and 13% (10-17%), respectively, by the metabolite. Disopyramide also prolonged the QRS, JT, and QT intervals by 15% (9-21%), 10% (8-13%), and 10% (7-12%), respectively. The metabolite caused a 9% (7-12%) prolongation of the QRS interval (significantly less than disopyramide), but shortened repolarization (as reflected by the JT interval) by -7% (-2 to -11%; p < 0.01), which is similar to the acute effects of lidocaine 2 mg/kg body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Automated determination of disopyramide and N-monodealkyldisopyramide in plasma by reversed-phase liquid chromatography with a column switching system.

A rapid and simple column liquid chromatographic method involving a column switching system for the determination of disopyramide and its N-monodealkyl metabolite (NMD) in plasma is described. The deproteinized plasma is applied to an automated system. Purification and concentration were performed using a precolumn connected to a six-position valve; analytical separation was done on-line using a cyano reversed-phase column with a mobile phase consisting of 10 mmol/l trimethylamine (pH 2.5, adjusted with phosphoric acid)-acetonitrile-tetrahydrofuran (78:20:2, v/v/v). Absorbance was measured at 265 nm, with a minimum detectable amount of disopyramide and NMD of 0.1 micrograms/ml. The method can be applied to drug monitoring and pharmacokinetic studies.

Stereoselective biliary elimination of disopyramide and mono-N-desisopropyldisopyramide in humans.

The results of a previous pharmacokinetic study of disopyramide (DP) enantiomers in humans suggested that DP and/or mono-N-desisopropyldisopyramide (MND) may show stereoselective extrarenal elimination. Thus, the present study investigates the biliary elimination of DP and MND enantiomers in three patients who had undergone cholecystectomy for cholelithiasis. DP and MND enantiomers displayed biliary elimination. In both subjects, this elimination pathway showed the same characteristics: (1) biliary elimination of DP and MND was stereoselective, (2) the stereoselectivity was opposite to that observed for the metabolic and renal elimination pathways, i.e., the elimination of the (-)-(R)-enantiomer was higher than that of the (+)-(S)-enantiomer, and (3) biliary elimination of MND was higher than that of DP, for both enantiomers. Estimates of the relative contribution of the biliary clearance in the total clearance of DP and MND indicated that this elimination pathway was secondary, especially for DP. The biliary clearance (expressed as % of total clearance) was 1.9 to 4.0% for (-)-(R)-DP, 1.2 to 1.7% for (+)-(S)-DP, 7.8 to 22.9% for (-)-(R)-MND, and 5.2 to 10.5% for (+)-(S)-MND.

Stereoselective disposition and metabolism of disopyramide in pediatric patients.

Pharmacokinetics of disopyramide (DP) enantiomers was studied in six pediatric patients, 5 to 12 years old, with arrhythmias after i.v. and p.o. administrations of racemic DP. The enantiomers of DP and its active metabolite, mono-N-dealkyldisopyramide, in plasma and urine were determined using a chiral, high-performance liquid chromatography. Plasma protein binding of DP was measured by ultrafiltration. Because the protein binding of DP was not only concentration-dependent but also stereoselective (i.e., S-DP binds to protein more extensively than R-DP), unbound pharmacokinetic parameters were used for evaluating the kinetic behaviors of DP enantiomers. The pediatric age patients had the mean (+/- S.D.) systemic clearance of 15.0 +/- 3.8 and 12.7 +/- 3.9 ml/min/kg for unbound S- and R-DP, respectively, which were not only stereoselectively different (P less than .05) but also at least about twice greater than the reported normal adult values. The mean postinfusion elimination half-life values for unbound S- and R-DP (2.7 +/- 0.5 and 2.8 +/- 0.4 hr, respectively) in pediatric patients were shorter than those reported from normal adults (approximately equal to 4 to 5 hr). The mean nonrenal (i.e., hepatic) clearance for unbound S- and R-DP (11.1 +/- 4.1 and 8.1 +/- 3.9 ml/min/kg, respectively) were also stereoselectively different (P less than .01) and accounted for approximately equal to 70% of the unbound systemic clearance of the respective enantiomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Simplified, rapid and inexpensive extraction procedure for a high-performance liquid chromatographic method for determination of disopyramide and its main metabolite mono-N-dealkylated disopyramide in serum.

A simplified, rapid and inexpensive extraction procedure for the determination of the antiarrhythmic drug disopyramide and its main metabolite mono-N-desalkylated disopyramide in serum by high-performance liquid chromatography has been developed. The analysis uses ultraviolet detection at 254 nm, and a 5 micron reversed-phase column with a mobile phase of water-triethylamine-acetonitrile-PIC-B8 reagent. Serum extraction is performed with dichloromethane and 1 M sodium hydroxide. p-Chlorodisopyramide is used as internal standard. Recovery rates were 94.5% (S.D. 5.7%) for disopyramide, 96.8% (S.D. 2.2%) for mono-N-desalkylated disopyramide and 97.9% (S.D. 2.8%) for the internal standard.

In vitro assessment of stereoselective hepatic metabolism of disopyramide in humans: comparison with in vivo data.

Metabolism of disopyramide (DP) enantiomers has been investigated in primary cultures of adult human hepatocytes. Results were compared with in vivo data obtained from a previous pharmacokinetic study (Le Corre et al. Drug Metab. Dispos. 16:858-864 1988). Metabolism of DP enantiomers as a function of incubation time showed constant velocity over time. The intracellular/extracellular distribution of both DP and mono-N-desisopropyldisopyramide did not appear to be stereoselective. Metabolism of DP enantiomers as a function of substrate concentration followed a first order kinetics. The average fractions of (-)-(R)-DP and (+)-(S)-DP metabolized in vitro (4.7 +/- 2.7 and 7.1 +/- 4.2%, respectively, n = 4) were about 5-fold lower than the fractions metabolized in vivo (26.0 +/- 6.0 and 40.2 +/- 8.8%, respectively, n = 6). The stereoselective index [(+)-(S)/(-)-(R)] of the N-dealkylation pathway obtained in vitro (1.51 +/- 0.11, n = 4) was very close to the one obtained in vivo (1.55 +/- 0.10, n = 6). These results highlight the interest of hepatocyte cultures in the evaluation of drug metabolism and especially in the assessment of stereoselectivity.

Simultaneous determination of disopyramide and its mono-N-dealkylated metabolite enantiomers in human plasma and urine by enantioselective high-performance liquid chromatography.

Enantiomers of disopyramide (DP) and its mono-N-dealkylated metabolite (MND) were determined in human plasma and urine by enantioselective high-performance liquid chromatography using a chiral stationary-phase column. This method was precise and sensitive: the mean recoveries from plasma at a concentration of 0.5 microgram/ml were 101.1% for (+)-DP, 98.0% for (-)-DP, 94.4% for (+)-MND and 82.9% for (-)-MND; the within- and between-day coefficients of variation at the same concentration were 4.4 and 3.3% for (+)-DP, 4.7 and 4.1% for (-)-DP, 6.5 and 4.1% for (+)-MND and 7.8 and 2.4% for (-)-MND for plasma; the lower detection limits were 40 ng/ml for (+)-DP, 80 ng/ml for (-)-DP, 100 ng/ml for (-)-MND and 200 ng/ml for (+)-MND, for 0.5 ml of plasma and 0.2 ml of urine. The ultrafiltration technique was used for determination of the unbound concentration of DP enantiomers in plasma. A preliminary study of the determination of DP and MND enantiomers in plasma and urine samples from a healthy subject given racemic DP demonstrated the clinical applicability of the present method for therapeutic monitoring and pharmacokinetic studies.

Frequency and voltage-dependent effects of mono-N-dealkyldisopyramide, the major metabolite of disopyramide, in canine ventricular tissue.

Mono-N-dealkyldisopyramide (MND), the major metabolite of disopyramide, reaches significant concentrations in patients; however, the contribution of MND to the antiarrhythmic or toxic effects of disopyramide is not known. We assessed the kinetics and magnitude of interaction of MND with the sodium channel in canine ventricular tissue superfused in vitro using Vmax as an index of sodium channel block. At a basic cycle length of 1000 msec, MND (4-32 micrograms/ml) produced a concentration-dependent depression of both Vmax and amplitude of the action potential and accelerated all phases of repolarization in Purkinje fibers. To assess rate-dependent block, Purkinje fibers were stimulated with pulse trains at interstimulus intervals of 400 to 2000 msec. MND produced a concentration- and rate-dependent increase in the magnitude of rate-dependent block. There was also a concentration-dependent increase in the kinetics of onset of block (decrease in rate constant). The rate constant increased with faster stimulation rates. Minimal tonic block occurred at clinically relevant concentrations. Recovery from rate-dependent block followed a single exponential time course with time constants of 5.23 +/- 0.90 and 4.88 +/- 0.94 sec for Vmax and activation time, respectively. There was no shift of the normalized Vmax-membrane potential relationship except at the highest concentration, 32 micrograms/ml. At cycle lengths of 250 to 1000 msec, MND (4 micrograms/ml) shortened all phases of repolarization in Purkinje fibers, the greatest shortening occurring at the longest cycle length. Prolongation of effective refractory period occurred only at rapid heart rates. Both action potential duration and effective refractory period were prolonged in ventricular muscle which was independent of rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in plasma and urine by use of a chiral cellulose-derivative column.

This assay allows simultaneous determination of the enantiomers of both disopyramide and its active metabolite, mono-N-dealkyldisopyramide, in 1 mL of plasma or 0.1 mL of urine within approximately 35 min by HPLC with a chiral cellulose-derivative column and ultraviolet detection. Recoveries for the analytes and the internal standard (racemic verapamil) with an extraction from alkalinized plasma or urine into diethyl ether were greater than 90%. Intra- and interassay CVs for disopyramide enantiomers were less than 5.5% at 2.5 mg/L in plasma and less than 6.5% at 25 mg/L in urine; for mono-N-dealkyldisopyramide enantiomers they were less than 6.3% and less than 8.9%, respectively. Intra- and interassay relative errors for determining these analytes in plasma and urine at 2.5 and 25 mg/L, respectively, ranged from -5.9% to +2.5%. The calibration curves for the respective analytes were linear (r = 0.995 or greater, P less than 0.01) from 0.025 to 5.0 mg/L in plasma and from 0.5 to 10 mg/L in urine. The lower detection limits (signal-to-noise ratio of 3) for S(+)-disopyramide and the other analytes were 0.010 and 0.025 mg/L, respectively. We evaluated clinical applicability of this method by determining steady-state plasma concentrations and urinary excretions of the respective analytes in a pediatric patient being treated with racemic disopyramide.

Possible role of drug interactions in bupivacaine-induced problems related to intraventricular conduction disorders.

Clinically, bupivacaine has depressant effects on intraventricular conduction that may lead to serious atrioventricular blocks or reentrant arrhythmias at plasma levels below those required to produce these effects experimentally (2-3 micrograms/ml instead of 8-10 micrograms/ml). The difference could be due to drugs present in the blood at the time of regional anesthesia that similarly inhibit conduction. This hypothesis was examined in 30 anesthesized, closed-chest dogs by measuring conduction time in the ventricular contractile fibers as well as effective refractory period under pacing at a constant, relatively high (180 beats/minute) rate. Changes in sinus rate were limited, as well as changes in ventricular effective refractory period and blood pressure regardless of the drug tested. In contrast, cibenzoline, disopyramide, and propranolol increased conduction time and lengthened QRS duration. Clomipramine appeared to prolong conduction time and widen QRS only moderately in therapeutic doses, whereas verapamil did not manifest noticeable effects on conduction. Caution is therefore recommended in regional anesthesia with bupivacaine in subjects being treated with cardiovascular drugs, such as cibenzoline, disopyramide, and propranolol and their congeners, or even by tricyclic antidepressants.

[Assessment of anti-arrhythmic activity of palpitin and cardio-hemodynamic effects of its administration]. / Otsenka antiaritmicheskoi aktivnosti pal'pitina i kardiogemodinamicheskie éffekty ego primeneniia.

Palpitin, an agent of the disopyramide series, was evaluated for its antiarrhythmic potency in the treatment of ventricular and supraventricular arrhythmias and for its effects of ECG and central hemodynamic parameters in 108 patients (91 had coronary heart disease, 8 presented with postmyocardial cardiosclerosis, 5 had hypertensive disease, and 4 suffered from neurocirculatory dystonia). 24-hour ECG monitoring and echocardiography were employed in the study. It was concluded that the drug possessed a high antiarrhythmic potency. No substantial changes were found in the mean values of echocardiographic parameters, as well as of atrioventricular and intraventricular conduction.