Quantification of urinary excretion of ethosuximide enantiomers and their major metabolites in the rat

Abstract

A chiral gas chromatographic assay has been developed for quantitative analysis of ethosuximide and its major metabolites in rat urine. The extraction procedure was found to be precise and reproducible. Recovery was in the range of 94-98%, intraday CV(%) was 0.92% for (S)-ethosuximide (50 microg/ml) and 0.51% for (R)-ethosuximide (50 microg/ml). Interday CV(%) was 1.12% for (S)-ethosuximide and 0.72% for (R)-ethosuximide. The limit of detection was determined to be around 0.01 microg/ml for each enantiomer. Following administration of rac-ethosuximide by i.v., i.p. and oral routes, unchanged ethosuximide was detected in urine up to 72 h after drug administration. The appearance of all detected metabolites occurred within 24 h of drug administration. Significantly more (S)-ethosuximide was excreted unchanged than (R)-ethosuximide with all three routes studied. A substantial amount of the drug was eliminated as the 2-(1-hydroxyethyl)-2-methylsuccinimide (2 pairs of diastereoisomers). Much less drug was eliminated as the 2-ethyl-3-hydroxy-2-methylsuccinimide with only one diastereoisomer observed. Examination of the one pair of diastereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide that was resolved showed preferential excretion of one isomer. Comparison of both pairs of diastereoisomers showed that one pair was formed in preference to the other with a ratio of approximately 0.8:1. It is concluded that stereoselective metabolism of ethosuximide occurs.

Comparison of the antiepileptogenic effects of an early long‐term treatment with ethosuximide or levetiracetam in a genetic animal model of absence epilepsy

“…Blumenfeld et al (2008) used a protocol almost identical to the present one; the main difference was that the dose of ETH they used was considerably higher (300 mg/kg) than our dose of 80 mg/kg, which attained a mean plasma concentration of 36.53 € 0.82 lg/ml, in line with the human Epilepsia ILAE therapeutic range (40-100 lg/ml;McNamara, 2003). However, we observed that ETH (but not LEV) plasma concentration was higher at the beginning of treatment and significantly lower at the end, which could be explained by an increased metabolism and elimination (Sarver et al, 1998;Mifsud et al, 2001). By comparison, LEV metabolism may be slower in these animals (c.f., Benedetti et al, 2004).…”

The pharmacokinetics of ethosuximide enantiomers in the rat

“…Also, for each enantiomer, the lack of any significant difference between estimates of clearance when administered as part of a racemic mixture and when administered separately indicates that neither enantiomer Other work published by our group [12] has shown that larger amounts of (S)-ethosuximide than (R)-ethosuximide are excreted unchanged in rat urine, whereas more of the (R)-enantiomer is eliminated in the form of metabolites. Higher plasma concentrations of (S)-ethosuximide would account for the greater excretion of this enantiomer in rat urine, as seen previously [12], without (S)-ethosuximide necessarily having a greater renal clearance value. Therefore, the difference in total clearance observed in the present study probably reflects a larger metabolic clearance of the (R)-enantiomer.…”

Chiral chromatographic resolution of antiepileptic drugs and their metabolites: a challenge from the optimization to the application

“…The original methodology developed by Mifsud et al . (, ) was demonstrated to be selective for the analytes with no endogenous interferences from rat plasma and urine at their retention times and linear and precise within the concentration ranges of 5.0–125 and 2.5–150 µg/mL, respectively (Table ). These validation findings are in agreement with those defined in the guidelines and allowed the techniques to be successfully implemented in pre‐clinical pharmacokinetic studies after intra‐peritoneal, oral and intravenous administrations to rats.…”

“…Only GC methodologies have been employed for the quantification of both enantiomers and their main metabolites in the biological fluids, plasma and urine after intravenous, intraperitoneal or oral administration. Mifsud et al (2001b) developed and fully validated a chiral GC-FID method for the quantification of ethosuximide enantiomers and its major metabolites in rat urine and subsequently adapted it for the analysis of the drug in plasma (Mifsud et al, 2001a). In both studies, the chromatographic conditions remained unchanged and briefly consisted of using helium as the carrier gas, maintaining the injector and detector temperature at 230°C while the oven was set at 100°C for 1 min and then ramped at 5°C per minute up to 230°C; the injector volume was only 1.0 μL.…”

“…In both studies, the chromatographic conditions remained unchanged and briefly consisted of using helium as the carrier gas, maintaining the injector and detector temperature at 230°C while the oven was set at 100°C for 1 min and then ramped at 5°C per minute up to 230°C; the injector volume was only 1.0 μL. The original methodology developed by Mifsud et al (2001aMifsud et al ( , 2001b was demonstrated to be selective for the analytes with no endogenous interferences from rat plasma and urine at their retention times and linear and precise within the concentration ranges of 5.0-125 and 2.5-150 μg/mL, respectively (Table 4). These validation findings are in agreement with those defined in the guidelines and allowed the techniques to be successfully implemented in pre-clinical pharmacokinetic studies after intra-peritoneal, oral and intravenous administrations to rats.…”