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.

Kinetics and dynamics of disopyramide and its dealkylated metabolite in healthy subjects.

The kinetics and dynamics of total and free (unbound) disopyramide (D) after dosing with D, 1.5 and 2 mg/kg iv, were compared with those of the dealkylated metabolite (MND) after dosing with MND, 0.5 and 1.5 mg/kg iv, in six healthy subjects. Dynamic parameters included ECG with measurement of the QT interval corrected for heart rate (QTc), systolic time intervals, vitamin C-stimulated saliva secretion, pupil size, and maximum accommodation capacity. Mean values of total clearance, apparent volume of distribution, and elimination t1/2 of MND were 5.9, 2.3, and 0.4 times those of total D, respectively. D significantly prolonged the QTc and systolic time intervals and induced transient inhibition of stimulated saliva secretion. In contrast, MND induced no substantial change in either the QTc or systolic time intervals, but did induce more persistent inhibition of salivary secretion. If anticholinergic potency is determined as the degree of inhibition of stimulated saliva flow per plasma concentration unit, MND was three times as potent as its parent when measured at maximum inhibition. There were no consistent drug effects on the ocular parameters. The effect of D on QTc correlated with both total and free plasma concentrations. Furthermore, its transient salivary inhibitory effect paralleled its initial rapid decline in plasma concentration. There was no relationship between the MND plasma concentration and its salivary inhibitory effect. We conclude that disopyramide significantly affected the QTc and systolic time intervals in healthy subjects, while MND in a similar dose had no such effects. MND more strongly inhibited stimulated saliva flow, indicating a more potent anticholinergic effect than D.

Synthesis and anticholinergic properties of the enantiomers of 4-(isopropylamino)-2-(2-pyridyl)-2-phenylbutyramide, the mono-N-dealkylated metabolite of disopyramide.

The 2R and 2S enantiomers of 4-isopropyl-2-(2-pyridyl)-2-phenylbutyramide [(2R)-2 and (2S)-2] were prepared from the respective 2R and 2S enantiomers of disopyramide [(2R)-1 and (2S)-1] by oxidation with peracid, Cope elimination, and subsequent zinc/HCl reduction of the resulting hydroxylamines (2R)-3 and (2S)-3. The enantiomers were tested as antagonists to the contraction of guinea pig ileum longitudinal muscle produced in response to electrically stimulated release of acetylcholine. The enantiomers showed IC50 values of 5.0 X 10(-6) and 14 X 10(-6) M for (2S)-2 and (2R)-2 respectively, about a 3-fold difference between enantiomers. Data are presented showing direct antagonism of acetylcholine in the guinea pig ileum assay. In a comparison of the anticholinergic effects of 2 and 1, the metabolite (2) was slightly less potent than disopyramide (1).

Effects of disopyramide on left ventricular function: assessment by radionuclide cineangiography.

To determine the effects of disopyramide on resting systolic left ventricular (LV) function and LV functional reserve, gated equilibrium radionuclide cineangiography was performed at rest and during maximal symptom-limited supine bicycle exercise in 12 patients after a single 300 mg oral loading dose of disopyramide, and in 22 patients (including the 12 patients just mentioned) after they received disopyramide 150 mg 4 times daily for 5 to 10 days (average 7). The oral loading dose (average serum level 3.6 +/- 1.3 micrograms/ml [standard deviation] produced decreases in ejection fraction in 9 of 12 patients with a decrease in average resting ejection fraction from 40 +/- 15% to 33 +/- 11% (p less than 0.005). However, the lower, sustained dosage of disopyramide was associated with a lower average serum level of 2.5 +/- 0.8 micrograms/ml and with smaller but significant decreases in ejection fraction in 3 of 22 patients during exercise only. At this dosage there was no significant decrease in average ejection fraction for the group at rest or during exercise. Adverse effects of disopyramide on ejection fraction occurred even in patients with previously normal LV function at rest. Hence, disopyramide may be associated with significant decreases in LV systolic function, particularly when given in high, oral "loading" doses. However, sustained therapy with lower dosages as well as lower drug levels is also associated with less depression of LV function.

A double-blind, crossover comparison of flecainide acetate and disopyramide phosphate in the treatment of ventricular premature complexes.

The efficacy and safety of flecainide, 200 mg twice daily, was compared with disopyramide, 150 mg 4 times daily, in a randomized, double-blind, crossover study in 25 patients (19 men and 6 women, aged 20 to 71 years, mean 52.5) with more than 1,000 ventricular premature complexes (VPCs) in a pretrial 24-hour Holter monitoring screen. Each 14-day active treatment period was preceded and followed by a 7-day placebo period. Ambulatory ECGs were recorded at the end of each study week and analyzed blindly. Average VPCs recorded during each of the 2 active periods were compared with average VPCs in the placebo periods. Twenty-two of 25 patients attained therapeutic plasma levels of both drugs. The occurrence of VPCs was significantly less during flecainide than during disopyramide treatment, 92 and 39%, respectively (p less than 0.01). Complex arrhythmic events were significantly more suppressed with flecainide than with disopyramide. No difference was observed between the 2 drugs in the incidence or severity of reported side effects. PQ, QRS and QT intervals increased beyond normal limits on both drugs in some patients, significantly more with flecainide than with disopyramide. The JT interval did not change or decrease; hence, all changes in the QT interval were attributable to a widening of the QRS complex. Neither drug showed any significant effect on blood pressure or heart rate. Flecainide may be a well-tolerated and valuable alternative to currently available antiarrhythmic agents.

A kinetic analysis of the endplate ion channel blocking action of disopyramide and its optical isomers.

The effects of the antiarrhythmic agent disopyramide was studied on responses from voltage-clamped endplates at the neuromuscular junction of the garter snake. Disopyramide reduced endplate current amplitude and decay time constant in a concentration- and voltage-dependent manner. Endplate current decays remained monophasic in the presence of the drug. These results were interpreted in terms of the drug blocking the open form of the acetylcholine receptor-ion channel complex. Disopyramide produced a greater reduction of the amplitude of endplate currents than of miniature endplate currents. The reduction in miniature endplate current amplitude was not voltage-dependent. Analysis of endplate current driving functions showed that this was due to the rapid occurrence of channel block during the rising phase of the endplate current. The residual reduction, apart from that produced by channel block, is most probably due to receptor block. Disopyramide had a voltage-dependent blocking rate constant of about 10(7) M-1 S-1 at -90 mV. The unblocking rate constant was estimated from the results of experiments using paired ionophoretically applied pulses of acetylcholine. This value was again voltage-dependent and approximately 1 s-1. The actions of the (+)- and (-)-stereoisomers of disopyramide on endplate current decay were identical, indicating that the channel binding site at the neuromuscular junction is not stereoselective.

Some effects of disopyramide and its N-dealkylated metabolite on isolated nerve and cardiac muscle.

In animals and man the antidysrhythmic agent disopyramide in primarily metabolised by mono-N-dealkylation. The effects of disopyramide and its N-dealkylated metabolite (MIP) have been investigated using isolated cardiac and nervous tissue, and their effects have been compared with the effects of other antidysrhythmic agents. Disopyramide, d,l-propranolol and quinidine all decreased both maximum driving frequency and developed tension in electrically driven guinea pit atria. MIP and procaine amide also decreased maximum driving frequency, but had a positive intropic effect. MIP was only 4 times less active than disopyramide in decreasing maximum driving frequency. There was no evidence that either disopyramide or MIP possessed beta-adrenoceptor antagonist properties. In superfused rat sciatic nerves, it has been shown that neither disopyramide nor MIP possesses significant local anaesthetic properties. Procaine amide and lignocaine were highly active in this test. The possible contribution of MIP to the actions of disopyramide in vivo is discussed.

High performance liquid chromatographic analysis of the antiarrhythmic drugs procainamide, disopyramide, quinidine, propranolol and metabolites from serum extracts.

We describe a method for the simultaneous high performance liquid chromatographic determination of several antiarrhythmic drugs and some of their metabolites after extraction from 2.5 mL of spiked pooled sera. The extracts were applied to a C8 reversed phase column. Nine compounds of interest were resolved within the 30 minute run. An initial mobile phase of 80% phosphate (25 mmol/L, pH 3.5), 20% organic (acteonitrile:methanol, 2:3) was maintained for 2 min at which time a linear gradient was used to change the mobile phase to 30% phosphate, 70% organic at 20 min after injection. This composition was maintained from an additional 5 min. Absorbance at 212 nm was used for detection. Peak area ratios of drug to internal standard (N-propionylprocainamide) were used for quantitation. The relative standard deviations (and mean solute concentrations) of daily duplicate determinations for 15 days are: procainamide, 5.1% (5.9 mg/L); N-acetylprocainamide, 9.3% (6.0 mg/L); Mono-N-dealkyldisopyramide, 3.7% (4.1 mg/L); disopyramide, 4.3% (4.0 mg/L); quinidine, 4.5% (6.5 mg/L); and propranolol, 5.1% (0.097 mg/L). Dihydroquinidine and 4-hydroxypropranolol were also resolved but not quantitated.

Single column high pressure liquid chromatographic determination of drugs in blood.

1. Analysis of anticonvulsants (phenobarbital, diphenylhydantoin and carbamazepine), theophylline and an antiarrhythmic agent (disopyramide) in blood using a simple high pressure liquid chromatography apparatus equipped with a reversed -- phase column is described. A simple extraction of plasma or serum with organic solvent is used to isolate the anticonvulsants and theophylline. Disopyramide is extracted with ether and is further purified by a back extraction into acid. 2. Hexanesulfonic acid -- methanol solutions are used for chromatography of the anticonvulsants and disopyramide while the mobile phase for theophylline is a NH4H2PO4 -- methanol mixture. Chromatographic analysis time for the drugs is approximately 15 minutes. The drugs are monitored by a UV detector at 254 nm except for theophylline which is measured at 280 nm. Quantitation is accomplished by comparison of peak heights with those of internal standards. Quantities of serum or plasma routinely used for analysis are: 200 ul for the anticonvulsants, 100 ul for theophylline and 0.5 ml for disopyramide. Detection limits are less than 1 ug/ml for these quantities.

Effect of pKb on lipophilic binding of disopyramide derivatives to human plasma.

The extent of plasma binding, the partition coefficient, and the pKb of 13 disopyramide derivatives were determined. The structural variation on the diisopropylaminoethyl group of disopyramide molecules influenced these physical parameters to varying degrees. Results demonstrated that the extent of interaction between drugs and human plasma was a linear function of their lipophilicity and inversely proportional to the magnitude of the pKb value.

Pharmacologically active conformation of disopyramide: evidence from apparent pKa measurements.

We have found evidence that an intramolecular hydrogen bond exists between the amido and pyridine groups of disopyramide in aqueous solutions. This conclusion was reached by a comparison of the pKa values for the basic nitrogen atoms of certain analogues of disopyramide. By comparing the pharmacological actions of disopyramide with those of pheniramine, which lacks an amido group, we have concluded that the constraint on the rotation of the pyridine ring which is imposed by the hydrogen bond is a major determinant of the antiarrhythmic activity. That constraint is, at the same time, a suppressor of the anticholinergic activity. We then concluded that a covalent link would hold the pyridine ring, and the amido group, in the desirable conformation permanently, which would lead to better antiarrhythmic activity and a lower degree of anticholinergic activity. Pharmacological studies on such new molecules are in general agreement with these concepts.

Linear relationships between plasma binding and lipophilicity of disopyramide derivatives.

The extent of plasma binding and the partition coefficient of disopyramide and 20 disopyramide derivatives were determined. Structural variations on the four functional groups around the tetrahedral carbon in the disopyramide molecule was found to influence both parameters to varying degrees. Three linear equations were developed to correlate the observed effects, depending on the type of chemical modification. The linear correlation between drug-plasma interaction and lipophilic character was analyzed theoretically. A simple model was derived to relate quantitatively the variation in the extent of plasma binding to the change in lipophilicity of disopyramide derivatives.

Protein binding of disopyramide--displacement by mono-N-dealkyldisopyramide and variation with source of alpha-1-acid glycoprotein.

The binding of disopyramide to human serum proteins and human alpha-1-acid glycoprotein (AAG) was determined over a wide drug concentration range. Addition of 3.7 X 10(-6) mol litre-1 mono-N-dealkyldisopyramide caused a 20-100% increase in disopyramide free fraction. The disopyramide free fraction in AAG solutions prepared from various commercially available sources of alpha-1-acid glycoprotein varied up to 2.5 fold at corresponding disopyramide concentrations. Pronounced differences in the calculated binding constants (affinity and capacity) were observed among the commercially available AAG preparations. These findings suggest that binding studies should be performed in appropriately harvested human serum or plasma to avoid possible artifacts associated with the use of commercial preparations of alpha-1-acid glycoprotein for binding studies.

Study on transport of disopyramide into the intestinal lumen aimed at gastrointestinal dialysis by activated charcoal in rats.

The characteristics of exsorption and/or excretion of disopyramide into the gastrointestinal lumen have been investigated after intravenous administration of the drug at doses of 10 and 30 mg kg-1 to rats by the in-situ single pass perfusion technique. Disopyramide was appreciably excreted into the bile where its levels were approximately ten-fold higher than those in the serum. The exsorption rate of disopyramide and mono-N-dealkyldisopyramide (MND) into the perfusate was increased with an increase in the serum level following an increase from 10 to 30 mg kg-1 in the dose of disopyramide. The average amounts of disopyramide exsorbed into the perfusate were 17.0 and 18.4% at the dose of 10 and 30 mg kg-1, respectively, whereas those of MND were less than 1% at both doses of disopyramide. Oral administration of activated charcoal reduced the serum disopyramide levels after intravenous administration of the drug (20 mg kg-1) compared with the control treatment. By oral administration of activated charcoal, t 1/2 and AUC were decreased to 89 and 82%, respectively, and Cltot was increased to 122% compared with the corresponding control treatment. Vd was not different between the treated rats and control rats. These results suggest that the oral administration of activated charcoal can enhance the clearance of disopyramide and MND from the blood.

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.

The pharmacokinetic and pharmacodynamic effects of varying the free fraction of disopyramide.

We have evaluated the influence of several factors on the binding of disopyramide to protein in human serum using a new ultrafiltration system and the enzyme multiplied immunoassay technique (EMIT) for disopyramide immunoassay. From these studies and those of other investigators, we conclude that the most significant in vivo factors influencing disopyramide binding in human serum are total disopyramide concentration, the concentration of alpha 1-acid glycoprotein, and the concentration of mono-N-dealkyldisopyramide. Significant variation in binding occurs in patients whose total disopyramide concentration is within the therapeutic range. The proportion of free disopyramide ranged from 16% to 54% in the sera of 50 cardiac patients whose total disopyramide concentration ranged from 0.5 to 5.8 mcg/ml. Since there is so much variation in the proportion of disopyramide that is free, and since it is the unbound form of the drug that is pharmacologically active, it is likely in patients whose arrhythmia responds to disopyramide therapy that unbound drug concentration will be a better indicator of drug efficacy and toxicity than will be total drug concentration. To test this hypothesis, we are investigating the relationship between steady state total and unbound disopyramide plasma levels in patients with supraventricular or ventricular arrhythmias, and pharmacologic response as determined by standard intracardiac electrophysiologic studies.

Pharmacologic evaluation of standard and controlled-release disopyramide.

Controlled-release disopyramide offers many potential advantages over the standard formulation for improved patient compliance, possible reduction of concentration-related adverse effects, and predictability of pharmacologic effect. The pharmacology of disopyramide, potential advantages and disadvantages of the use of sustained- (or controlled-)release formulations of drugs, and the preliminary finding of our use of controlled-release disopyramide are described. Controlled-release disopyramide is a promising addition to the antiarrhythmic formulary that may increase the clinical utility of disopyramide.

The clinical scope of disopyramide seven years after introduction--an overview.

Disopyramide phosphate, seven years after its introduction, has proved to be a useful and effective Type IA oral agent for treatment of ventricular arrhythmias. The experience of these seven years has amplified and more sharply defined the initial efficiency and safety issues related to the use of the agent. This experience now shows that disopyramide is at least as effective as other agents in its class and that it can be used safely in the majority of patients for whom treatment is indicated if therapeutic guidelines are followed. The areas of effectiveness, comparative effectiveness, side effects and toxicity of this agent are summarized and guides to patient selection are presented in this review article.

Transfer from immediate-release disopyramide to controlled-release disopyramide.

Simulation of serum disopyramide concentrations during transfer from steady-state immediate-release (IR) disopyramide to a sustained-release disopyramide preparation was performed based on pharmacokinetic parameters obtained from IR disopyramide and serum concentrations measured following an initial dose of controlled-release (CR) disopyramide phosphate. Based on the results of simulation, a typical patient with good cardiac, renal, and hepatic function can be transferred from a q 6 h IR disopyramide to an equivalent daily dose of controlled-release disopyramide administered q 12 h beginning at 6 hours after the final IR disopyramide dose.