Nina Isoherranen

Pharmacokinetics of Levetiracetam and Its Enantiomer (R)‐α‐ethyl‐2‐oxo‐pyrrolidine acetamide in Dogs

Summary:  Purpose: The new antiepileptic drug, levetiracetam (LEV, ucb LO59), is a chiral molecule with one asymmetric carbon atom whose anticonvulsant activity is highly enantioselective. The purpose of this study was to evaluate and compare the pharmacokinetics (PK) of LEV [(S)‐α‐ethyl‐2‐oxo‐pyrrolidine acetamide] and its enantiomer (R)‐α‐ethyl‐2‐oxo‐pyrrolidine acetamide (REV) after i.v. administration to dogs. This is the first time that the pharmacokinetics of both enantiomers has been evaluated.

Enantioselective analysis of levetiracetam and its enantiomer R-α-ethyl-2-oxo-pyrrolidine acetamide using gas chromatography and ion trap mass spectrometric detection

A gas chromatographic-mass spectrometric method was developed for the enantioselective analysis of levetiracetam and its enantiomer (R)-alpha-ethyl-2-oxo-pyrrolidine acetamide in dog plasma and urine. A solid-phase extraction procedure was followed by gas chromatographic separation of the enantiomers on a chiral cyclodextrin capillary column and detection using ion trap mass spectrometry. The fragmentation pattern of the enantiomers was further investigated using tandem mass spectrometry. For quantitative analysis three single ions were selected from the enantiomers, enabling selected ion monitoring in detection. The calibration curves were linear from 1 microM to 2 mM for plasma samples and from 0.5 mM to 38 mM for urine samples. In plasma and urine samples the inter-day precision, expressed as relative standard deviation was around 10% in all concentrations. Selected ion monitoring mass spectrometry is suitable for quantitative analysis of a wide concentration range of levetiracetam and its enantiomer in biological samples. The method was successfully applied to a pharmacokinetic study of levetiracetam and (R)-alpha-ethyl-2-oxo-pyrrolidine acetamide in a dog.

Developmental Outcome of Levetiracetam, Its Major Metabolite in Humans, 2-Pyrrolidinone N-Butyric Acid, and Its Enantiomer (R)-α-ethyl-oxo-pyrrolidine Acetamide in a Mouse Model of Teratogenicity

Summary:  Purpose: The purpose of this study was to test the teratogenic potential of the antiepileptic drug (AED) levetiracetam (LEV), its major metabolite in humans, 2‐pyrrolidone‐N‐butyric acid (PBA), and enantiomer, (R)‐α‐ethyl‐oxo‐pyrrolidine acetamide (REV), in a well‐established mouse model.

Anticonvulsant profile and teratogenicity of N-methyl-tetramethylcyclopropyl carboxamide: A new antiepileptic drug

Summary:  Purpose: The studies presented here represent our efforts to investigate the anticonvulsant activity of N‐methyl‐tetramethylcyclopropyl carboxamide (M‐TMCD) and its metabolite tetramethylcyclopropyl carboxamide (TMCD) in various animal (rodent) models of human epilepsy, and to evaluate their ability to induce neural tube defects (NTDs) and neurotoxicity.

Anticonvulsant Profile of Valrocemide (TV1901): A New Antiepileptic Drug

Summary:  Purpose: We sought to investigate the anticonvulsant activity of the new antiepileptic drug (AED), valrocemide or TV1901 (VGD) in various animal (rodent) models of human epilepsy to determine its anticonvulsant profile and safety margin.

Pharmacokinetic-Pharmacodynamic Relationships of (2S,3S)-Valnoctamide and Its Stereoisomer (2R,3S)-Valnoctamide in Rodent Models of Epilepsy

AbstractPurpose. Racemic valnoctamide (VCD) is a central nervous system- active drug commercially available in Europe. VCD possesses two chiral centers and, therefore, it exists as a mixture of four stereoisomers. The purpose of this study was to evaluate the anticonvulsant activity of two VCD stereoisomers in comparison with VCD (racemate), valpromide (VPD), and valproic acid (VPA) and to study their pharmacokinetic-pharmacodynamic relationships. Methods. The ability of racemic VCD, (2S,3S)-VCD, (2R,3S)-VCD and VPD to block partial seizures was studied in the 6Hz psychomotor seizure model in mice and in the hippocampal kindled rat. The ability of (2S,3S)-VCD and (2R,3S)-VCD to prevent generalized seizures was evaluated in the maximum electroshock (MES) and subcutaneous metrazole (sc Met) seizure tests. The PK of (2S,3S)-VCD, (2R,3S)-VCD, and VPD was studied in the mice utilized in the 6Hz model. Results. All of the tested compounds were effective in the models tested. No significant difference in ED50 values was observed but the plasma and brain EC50 values of (2R,3S)-VCD in the 6Hz model at 32 mA stimulation were 2-fold higher than the EC50 values of (2S,3S)-VCD. An excellent pharmacokinetic-pharmacodynamic correlation was found between the plasma and brain concentrations of the VCD stereoisomers and their anticonvulsant effect in mice. Stereoselectivity was observed in clearance, volume of distribution, and in brain-to-plasma AUC ratio at a dose of 25 mg/kg, but the difference disappeared at higher doses as the clearance of the stereoisomers decreased and their half-life increased. For (2R,3S)-VCD the brain-to-plasma AUC ratio doubled at the tested dose range, while it remained constant for (2S,3S)-VCD. Conclusions. Racemic VCD, VPD, (2R,3S)-VCD, and (2S,3S)-VCD are effective anticonvulsants in animal models of partial seizures and are more potent than VPA. The more favorable brain penetration of (2S,3S)-VCD and its lower EC50 value in the 6Hz test provides one advantage over (2R,3S)-VCD as a new antiepileptic drug.

Characterization of the anticonvulsant profile and enantioselective pharmacokinetics of the chiral valproylamide propylisopropyl acetamide in rodents

Propylisopropyl acetamide (PID) is a new chiral amide derivative of valproic acid. The purpose of this study was to evaluate the anticonvulsant activity of PID in rodent models of partial, secondarily generalized and sound‐induced generalized seizures which focus on different methods of seizure induction, both acute stimuli, and following short‐term plastic changes as a result of kindling, and to assess enantioselectivity and enantiomer–enantiomer interactions in the pharmacokinetics and pharmacodynamics of racemic PID and its pure enantiomers in rodents. Anticonvulsant activity of (S)‐PID, (R)‐PID and racemic PID was evaluated in the 6 Hz psychomotor seizure model in mice, in the hippocampal kindled rat, and in the Frings audiogenic seizure susceptible mouse. The pharmacokinetics of (S)‐PID and (R)‐PID was studied in mice and rats. In mice (S)‐PID, (R)‐PID and racemic PID were effective in preventing the 6 Hz seizures with (R)‐PID being significantly (P<0.05) more potent (ED50 values 11 mg kg−1, 46 mg kg−1 and 57 mg kg−1 at stimulation intensities of 22, 32 and 44 mA, respectively) than (S)‐PID (ED50 values 20 mg kg−1, 73 mg kg−1 and 81 mg kg−1 at stimulation intensities of 22, 32 and 44 mA, respectively). (S)‐PID, (R)‐PID and racemic PID also blocked generalized seizures in the Frings mice (ED50 values 16 mg kg−1, 20 mg kg−1 and 19 mg kg−1 respectively). In the hippocampal kindled rat a dose of 40 mg kg−1 of (R)‐ and (S)‐PID prevented the secondarily generalized seizure, whereas racemic PID also blocked the expression of partial seizures following an i.p. dose of 40 mg kg−1. Racemic PID also significantly increased the seizure threshold in this model. Mechanistic studies showed that PID did not affect voltage‐sensitive sodium channels or kainate‐, GABA‐ or NMDA‐ evoked currents. The pharmacokinetics of PID was enantioselective following i.p. administration of individual enantiomers to mice, with (R)‐PID having lower clearance and longer half‐life than (S)‐PID. In rats and mice, no enantioselectivity in the pharmacokinetics of PID was observed following administration of the racemate, which may be due to enantiomer–enantiomer interaction. This study demonstrated that PID has both enantioselective pharmacokinetics and pharmacodynamics. The better anticonvulsant potency of (R)‐PID in comparison to (S)‐PID may be due to its more favorable pharmacokinetic profile. The enhanced efficacy of the racemate over the individual enantiomers in the kindled rat may be explained by a pharmacokinetic enantiomer–enantiomer interaction in rats. This study also showed the importance of studying the pharmacokinetics and pharmacodynamics of chiral drugs following administration of the individual enantiomers as well as the racemic mixture.

Determination of gentamicin after trimethylsilylimidazole and trifluoroacetic anhydride derivatization using gas chromatography and negative ion chemical ionization ion trap mass spectrometry

A gas chromatographic method for the determination of gentamicin, an aminoglycoside antibiotic, was developed. A two step derivatization method utilizing trimethylsilylimidazole for silylation of the hydroxyl groups and trifluoroacetic anhydride for acetylation of the amino groups was used. Chemical ionization and negative ion monitoring resulted in greatly improved sensitivity and more informative fragmentation of the gentamicin components compared with electron ionization and positive ion monitoring. Tandem mass spectrometry was used for the identification of the gentamicin components and for characterization of the derivatized groups. The optimized method also allowed the use of three ions for selected ion monitoring. The method significantly improved the determination of gentamicin compared with previously described gas chromatographic methods. Chemical ionization and negative ion monitoring with the use of an ion trap analyzer appeared superior to other detection modes.

Anticonvulsant activity, teratogenicity and pharmacokinetics of novel valproyltaurinamide derivatives in mice

The purpose of this study was to synthesize novel valproyltaurine (VTA) derivatives including valproyltaurinamide (VTD), N‐methyl‐valproyltaurinamide (M‐VTD), N,N‐dimethyl‐valproyltaurinamide (DM‐VTD) and N‐isopropyl‐valproyltaurinamide (I‐VTD) and evaluate their structure–pharmacokinetic–pharmacodynamic relationships with respect to anticonvulsant activity and teratogenic potential. However, their hepatotoxic potential could not be evaluated. The metabolism and pharmacokinetics of these derivatives in mice were also studied. VTA lacked anticonvulsant activity, but VTD, DM‐VTD and I‐VTD possessed anticonvulsant activity in the Frings audiogenic seizure susceptible mice (ED50 values of 52, 134 and 126 mg kg−1, respectively). VTA did not have any adverse effect on the reproductive outcome in the Swiss Vancouver/Fnn mice following a single i.p. injection of 600 mg kg−1 on gestational day (GD) 8.5. VTD (600 mg kg−1 at GD 8.5) produced an increase in embryolethality, but unlike valproic acid, it did not induce congenital malformations. DM‐VTD and I‐VTD (600 mg kg−1 at GD 8.5) produced a significant increase in the incidence of gross malformations. The incidence of birth defects increased when the length of the alkyl substituent or the degree of N‐alkylation increased. In mice, N‐alkylated VTDs underwent metabolic N‐dealkylation to VTD. DM‐VTD was first biotransformed to M‐VTD and subsequently to VTD. I‐VTDs fraction metabolized to VTD was 29%. The observed metabolic pathways suggest that active metabolites may contribute to the anticonvulsant activity of the N‐alkylated VTDs and reactive intermediates may be formed during their metabolism. In mice, VTD had five to 10 times lower clearance (CL), and three times longer half‐life than I‐VTD and DM‐VTD, making it a more attractive compound than DM‐VTD and I‐VTD for further development. VTDs extent of brain penetration was only half that observed for the N‐alkylated taurinamides suggesting that it has a higher intrinsic activity that DM‐VTD and I‐VTD. In conclusion, from this series of compounds, although VTD caused embryolethality, this compound emerged as the most promising new antiepileptic drug, having a preclinical spectrum characterized by the highest anticonvulsant potential, lowest potential for teratogenicity and favorable pharmacokinetics.

Metabolism of a new antiepileptic drug, N-methyl-tetramethylcyclopropanecarboxamide, and anticonvulsant activity of its metabolites

N-methyl-tetramethylcyclopropanecarboxamide (MTMCD) is a new antiepileptic drug (AED) structurally related to valproic acid (VPA) that has a broad spectrum of anticonvulsant activity including models of therapy-resistant epilepsy. The purpose of this study was to identify in vivo metabolites of MTMCD that could contribute to its anticonvulsant efficacy. The metabolism of MTMCD was studied in mice, in human liver microsomes (HLM), and in recombinant human CYP isoforms with focus on formation of the hydroxylation product, N-hydroxymethyl-tetramethylcyclopropanecarboxamide (OH-MTMCD) and the N-demethylation product tetramethylcyclopropanecarboxamide (TMCD). The anticonvulsant activity of MTMCDs metabolites was evaluated in the maximal electroshock (MES), subcutaneous metrazole (s.c. Met), and in the 6Hz model in mice. In mice, OH-MTMCD was identified as a phase I metabolite of MTMCD and detected in plasma and brain after administration of MTMCD. In human liver microsomes MTMCD was biotransformed to OH-MTMCD but not to TMCD. Chemical inhibition studies suggested that MTMCD hydroxylation is mainly mediated by CYP 2A6 and CYP 2C19, which was confirmed using cDNA-expressed P450 isozymes. OH-MTMCD was a broad-spectrum anticonvulsant and possessed significant anticonvulsant activity in mouse models of partial and generalized seizures (ED50 values 75-220mg/kg), but was less potent than MTMCD. As OH-MTMCD was also present at lower concentrations than MTMCD in mouse brain, it is likely that MTMCD itself and not one of its metabolites is responsible for its activity in therapy-resistant epilepsy.