Liquid chromatographic assay with fluorescence detection to determine ajmaline in serum from patients with suspected Brugada syndrome.

Ajmaline is a sodium channel blocking, class 1A anti-arrhythmic drug. It has gained renewed interest in the field of cardiology as a diagnostic agent to reveal the electrocardiographic characteristics in patients with suspected Brugada syndrome. We developed a simple and precise high-performance liquid chromatographic assay to determine ajmaline in serum of patients. The samples were pre-treated using protein precipitation with perchloric acid and the extract was injected into the chromatographic system. The system consisted of an end-capped octadecyl silica column with isocratic elution using perchloric acid in a water-acetonitrile mixture. Ajmaline was detected by fluorescence at 290 and 355 nm for excitation and emission, respectively. The assay was validated in a 21-5300 ng/ml concentration range, the lower limit of quantification was 25 ng/ml. Within day precisions were 1.3-3.9%, between day precisions 2-7% and accuracies were between 95 and 99% for the whole calibration range. The drug was shown to be chemically stable under all relevant conditions. This assay has been successfully applied to pharmacokinetic-pharmacodynamic evaluations of intravenous ajmaline administration to patients with suspected Brugada syndrome.

Effect of experimental renal dysfunction on bioavailability of ajmaline in rats.

The effect of renal dysfunction on the bioavailability of ajmaline has been investigated in rats, where experimental renal dysfunction was induced by subcutaneous injection of uranyl nitrate (10 mg kg(-1)). Renal dysfunction did not cause any change in the blood ajmaline concentration after intravenous administration (2 mg kg(-1)), but it increased the blood ajmaline concentration by approximately 2.8-fold after intraduodenal administration (10 mg kg(-1)). The availability of ajmaline in control rats was 16.7%, whereas the availability was increased to 41.1% in rats with renal dysfunction. The unbound fraction in the blood and the metabolic activity in the liver, was assessed with the 10000-g supernatant fraction and with isolated hepatocytes, respectively. The values were found to be similar in both groups. The blood concentration following intraportal infusion was only slightly increased in rats with renal dysfunction, but the hepatic first-pass extraction was infusion rate-dependent and saturable. The initial absorption rate of ajmaline from the small intestine in rats with renal dysfunction was significantly greater compared with control rats. These results indicated that the increased availability of ajmaline in renal dysfunction was mainly a result of partially saturated extraction in the liver, which was caused by an increased absorption rate in the intestine and non-linear extraction in the liver.

The structure of the ring-opened N beta-propyl-ajmaline (Neo-Gilurytmal) at physiological pH is obviously responsible for its better absorption and bioavailability when compared with ajmaline (Gilurytmal).

Prajmaline, the semisynthetic propyl derivative of ajmaline, shows a much better bioavailability when compared with the Rauvolfia alkaloid ajmaline. Early NMR and IR-studies, fluorescence spectroscopic investigations and extraction experiments combined with ion-pair chromatography proved the thesis of a tautomeric equilibrium between an aldehyde-amine and a quaternary carbinol-ammonium component. The aim of this study was to confirm this thesis by HPLC-separation and by structure-determination of both tautomeric compounds.

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1.

The present study was performed to investigate the ability of the multidrug resistance protein (MRPI) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4',4'-azo-in-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations > or = 10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 microM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1.

Pharmacokinetics and electrophysiological effects of intravenous ajmaline.

The pharmacokinetics of ajmaline were studied in 10 patients with suspected paroxysmal atrioventricular block who received a 1 mg/kg intravenous dose over 2 minutes for diagnostic purposes (ajmaline test). Plasma concentration decay followed a triexponential time course with a final half-life much longer (7.3 +/- 3.6 hours) than that previously found by other investigators (about 15 minutes). Mean total plasma clearance and renal clearance were 9.76 ml/min/kg and 0.028 ml/min/kg, respectively. Although most of the dose was eliminated through the extrarenal route (only 3.5% of the intravenous dose was recovered in urine), no fluorescent metabolites could be detected either in plasma or urine. The steady-state volume of distribution averaged 6.17 L/kg, and plasma protein binding ranged between 29 and 46%. Three patients developed a transient atrioventricular block after ajmaline administration. In the remainder, the drug prolonged atrio-His bundle (AH interval), His bundle-ventricular (HV interval) and intraventricular (QRS interval) conduction times. Corrected ventricular repolarisation time (QTc interval) showed less marked changes, which were biphasic at times. The mean maximum ajmaline-induced increase in HV interval was 98%, in QRS was 58%, in AH was 30%, and in QTc was 17%. In most cases the time course of electrocardiographic changes lagged behind that of plasma concentrations, suggesting a delayed equilibrium of plasma concentrations with the site of action (hysteresis). Despite that, the pharmacokinetic-pharmacodynamic model, which accounted for hysteresis, failed to fit the experimental data adequately.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of lorajmine and its metabolite ajmaline in plasma and urine by a new high-performance liquid chromatographic method.

Lorajmine is a monochloroacetyl derivative of ajmaline with electrophysiological properties somewhat different from those of the compound of origin. Since lorajmine is rapidly hydrolyzed to ajmaline by plasma and tissue esterases, it is crucial to measure plasma levels of both drugs separately. A major problem in assaying lorajmine is its chemical instability in plasma both after blood sampling and during the extraction procedure. Furthermore, lorajmine (unlike ajmaline) is not fluorescent and has a very low UV absorbance, so the standard detectors for high-performance liquid chromatography cannot be used. We describe a new method that solves the problems of instability and sensitivity. Plasma esterases are first blocked pharmacologically (neostigmine); ajmaline is then measured by direct on-column injection of samples. Last, lorajmine is completely converted to ajmaline, extracted, and measured with a fluorescence detector. The molar concentration of ajmaline obtained in the last step, minus that found by direct injection, gives the concentration of lorajmine. Some examples of pharmacokinetic applications are also given.

Ajmaline test in a patient with chronic renal failure. A pharmacokinetic and pharmacodynamic study.

Pharmacokinetic and pharmacodynamic properties were studied after intravenous administration of ajmaline 1 mg/kg in an anuric patient, who underwent the electrophysiological ajmaline test. The magnitude and rate of onset of the typical electrophysiological effects of ajmaline (prolongation in atrio-Hisian and His-ventriculum conduction times) were within the range of normal values. The plasma concentration curve showed a triexponential decay with half-lives as follows: initial phase (t1/2 alpha) 1.34 min, fast elimination phase (t1/2 beta) 10.13 min and terminal (slow) phase (t1/2 gamma) 258.6 min. Other relevant pharmacokinetic parameters calculated were: total plasma clearance 45.91 L/h; volume of distribution 285.6L; protein binding 47%. Five hours after administration the patient underwent a 3.5h haemodialysis without any substantial increase in the slope of the final elimination phase of the curve. A major problem in interpreting the pharmacokinetic results is the lack of reliable reference data in healthy subjects. It is likely that the ajmaline t1/2 reported in the literature (13.4 min) does not reflect the true terminal t1/2 of the drug, because it was determined during an unduly short sampling period (30 min). Nevertheless, if we compare just the first 30 min of the concentration-time curves, our results are nearly superimposable on those found in healthy subjects.

Efecto de la reinfeccion sobre la evalucion de ratas infectadas con Trypanosoma cruzi / Effect of reinfection on the evolution of rats infected with Trypanosoma cruzi

El objetivo de este trabajo fue comprobar si una de las variables medio-ambientales, la reinfeccion, puede modificar el comportamiento observado en un modelo de rata a nivel de parasitemia, anticuerpos sericos, manifestaciones electrocardiograficas y/o lesion miocardica. Los grupos experimentales fueron: GI: ratas infectadas al destete com 1 x "10 POT 6" T. cruzi; GR: igual a GI mas reinfecciones cada 30 dias hasta los 150 dias post-infeccion inicial (p.i.i.); "GI IND 1". Los xenodiagnosticos fueron negativos en los tres grupos. Los anticuerpos sericos no se modificaron significativamente en GR respecto de GI, salvo en los anticuerpos 7S, pues los del GR presentaron titulos superiores en algunos de los dias estudiados. Los ECG basales no mostraron cambios distintivos en las ratas infectadas. La pruieba de ajmalina mostro una disminucion de la FC independiente del tratamiento; el PR, QaT y QRS se prolongaron significativamente en todos los grupos respecto del basal (p < 0.05), salvo el QaT en el GT; ademas el cambio de PR y QaT fue mayor en los infectados (p < 0.05). En los grupos infectados hubo tambien una amplia variacion en la orientacion del eje electrico respecto del valor basal, acompanado de cambios morfologicos mas manifiestos emGR. La proporcion de lesion cardiaca detectada histologicamente en los grupos

Metabolic disposition of ajmaline.

Urine was collected from six patients receiving a continuous infusion of 20 mg/h ajmaline. Pooled urine was extracted with and without enzymatic conjugate cleavage or hydrolysis with concentrated hydrochloric acid. The extracts were analyzed by gas chromatography/mass spectrometry. Ajmaline and its metabolites in urine were identified in the form of their acetylated derivatives. Twenty two different acetylated derivatives of ajmaline and its metabolites could be detected. Three of these derivatives were artifacts generated by acetylation and/or thermal decomposition. The major metabolic pathways were mono- and di-hydroxylation of the benzene ring with subsequent O-methylation, reduction of the C-21, oxidation of the C-17 and C-21-hydroxyl function, N-oxidation, and a combination of these metabolic steps. Ajmaline and its metabolites were mainly excreted in the form of their conjugates. Furthermore, the interference of sparteine, debrisoquine, quinidine, and nifedipine with ajmaline metabolism was studied with semiquantitative thin-layer chromatography. Ajmaline metabolism was inhibited by co-administration of sparteine or quinidine, but not by debrisoquine or nifedipine. Sparteine most likely competed with ajmaline metabolism. Quinidine probably bound competitively to ajmaline-metabolizing enzymes without being metabolized itself. Additionally, the metabolic ratio of hydroxyajmaline/ajmaline in urine was determined in 9 extensive metabolizers and one poor metabolizer of dextromethorphan. The poor metabolizer had a significantly reduced metabolic ratio of hydroxyajmaline/ajmaline, which indicates that ajmaline metabolism probably co-segregates with polymorphic sparteine/debrisoquine/dextromethorphan metabolism.

Pharmacokinetics and antiarrhythmic efficacy of intravenous ajmaline in ventricular arrhythmia of acute onset.

21 patients with acute myocardial infarction and ventricular arrhythmia of Lown class II-IIIB of acute onset received a short infusion of (50 mg/5 min) ajmaline (Gilurytmal). 6 of the patients had normal kidney and liver function (Group 1), 4 patients had acute renal failure and hemodialysis treatment (Group 2), 4 patients had impaired hepatic function (Group 3), 3 patients had cardiogenic shock (Group 4), and 4 patients had been pretreated with phenobarbital for seizures for at least 5 days (Group 5). A distribution half-life of 6 +/- 1 min and an elimination half-life of 95 +/- 6 min was determined in Group 1. The total plasma clearance was significantly lower in patients with impaired liver or cardiac function and significantly higher in Group 5, whereas impaired renal function did not affect total plasma clearance. After short infusion, ventricular arrhythmia of Lown II-IIIB completely disappeared for at least 16 to 36 min (mean: 19 min), which was associated with an ajmaline plasma level of 0.1-0.45 micrograms/ml. Additionally, steady-state plasma levels of ajmaline were determined after continuous infusion of 10-50 mg/h to 16 patients (Group 6) with ventricular arrhythmia of acute onset (Lown class IVA-V). Ventricular arrhythmia completely disappeared or at least changed to lower Lown classes at ajmaline plasma levels of 0.4-2.0 micrograms/ml. The ajmaline plasma protein binding was 76 +/- 9%. Ajmaline had a special affinity to alpha 1-acid glycoprotein.

Pharmacokinetics and dromotropic activity of ajmaline in rats with hyperthyroidism.

1. The pharmacokinetics and the dromotropic action (increased PQ interval) of intravenously administered ajmaline (2 mg kg-1) were studied in hyperthyroid rats with sinus tachycardia. The hyperthyroidism was induced by intraperitoneal injection of 3,5,3'-triiodo-L-thyronine (0.5 mg kg-1) for 4 days. 2. The change in the ajmaline concentration in whole blood could be described by a biexponential equation. The steady state distribution volume of ajmaline decreased from 4.81 l kg-1 in control rats to 3.80 l kg-1 in hyperthyroid rats and the total body blood clearance was slightly higher in hyperthyroid rats than in control rats. 3. Ajmaline exhibited a saturable binding to rat plasma proteins, and one kind of binding site was found in the observed range of concentrations. The binding capacity was 2 fold higher in hyperthyroid rats than in control rats. 4. On the basis of the plasma unbound concentration, ajmaline exhibited an increased negative dromotropic activity in hyperthyroid rats compared with control rats. 5. A positive correlation was found between the pacing rate and the dromotropic action of ajmaline on atrioventricular conduction in isolated perfused hearts. There was no significant difference in the rate-dependence of the effect of ajmaline on the heart between control and hyperthyroid rats. 6. Our findings suggest that the increased dromotropic activity of ajmaline is mainly due to the increased heart rate in hyperthyroid rats.