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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.
Methods: Optically pure LEV and REV were synthesized, and 20 mg/kg of individual enantiomers was administered intravenously to six dogs. Plasma and urine samples were collected until 24 h, and the concentrations of LEV and REV were determined by an enantioselective assay. The levels of 2-pyrrolidone-N-butyric acid, an acid metabolite of LEV and REV, were determined by high-performance liquid chromatography (HPLC). The data were used for PK analysis of LEV and REV.
Results: LEV and REV had similar mean ± SD values for clearance; 1.5 ± 0.3 ml/min/kg and volume of distribution; 0.5 ± 0.1 L/kg. The half-life (t1/2) and mean residence time (MRT) of REV (t1/2, 4.3 ± 0.8 h, and MRT, 6.0 ± 1.1 h) were, however, significantly longer than those of LEV (t1/2, 3.6 ± 0.8 h, and MRT, 5.0 ± 1.2 h). The renal clearance and fraction excreted unchanged for LEV and REV were significantly different.
Conclusions: In addition to the enantioselective pharmacodynamics, α-ethyl-2-oxo-pyrrolidine acetamide has enantioselective PK. The enantioselectivity was observed in renal clearance. Because REV has more favorable PK in dogs than LEV, the higher antiepileptic potency of LEV is more likely due to intrinsic pharmacodynamic activity rather than to enantioselective PK.
Levetiracetam, (S)-α-ethyl-2-oxo-pyrrolidine acetamide (LEV, ucb LO59), is a new antiepileptic drug (AED) recently approved by the U.S. Food and Drug Administration (FDA) (1). Levetiracetam is an ethyl analogue of piracetam, a nootropic drug used as a cognition enhancer in the elderly and in the treatment of myoclonus (2).
α-Ethyl-2-oxo-pyrrolidine acetamide possesses a single asymmetric carbon atom, and therefore has two enantiomers (Fig. 1): LEV, which is the (S)-enantiomer, and (R)-α-ethyl-2-oxo-pyrrolidine acetamide (REV). Only the (S)-enantiomer has anticonvulsant activity, and consequently, only this enantiomer has been developed and used as a new AED (3).
Figure 1. Chemical structures of levetiracetam (LEV), its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide (REV) and the acid metabolite 2-pyrrolidone-N-butyric acid (PBA).
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LEV expresses significant anticonvulsant activity in numerous animal models of epilepsy. It is active against tonic electroconvulsive seizures and kindling, audiogenic seizures, and chemically induced seizures with median effective dose (ED50) values within 5–30 mg/kg, but it is inactive in the traditional maximal electroshock (MES) and metrazol (scMet) tests (4,5). In humans LEV has proven efficacy in clinical trials of patients with partial and generalized seizures (1). LEV also has been suggested to possess antiepileptogenic activity because it slows the development of scMet kindling in mice and amygdala kindling in rats (6).
LEV has been regarded as a new AED with ideal pharmacokinetics (PK) (7). In humans LEV has a very low potential for drug–drug interactions (7), and it is eliminated mainly by renal excretion with a fraction excreted unchanged (fe) of ∼66%(8,9). LEV has a renal clearance (CLr) of 0.6 ml/min/kg, indicating that the drug is renally excreted by glomerular filtration and tubular reabsorption. In humans, 24% of the LEV dose is biotransformed to 2-pyrrolidone-N-butyric acid (PBA, Fig. 1), which is subsequently excreted in urine (7). The metabolism of LEV in other species has not yet been reported. The volume of distribution of LEV in humans is ∼0.5–0.7 L/kg, a value close to total body water, and the plasma protein binding of LEV in humans is <10%(7).
Several new AEDs [e.g., vigabatrin (VGB), pregabalin, remacemide, and 10-hydroxycarbazepine (MHD)] are chiral compounds (8). Enantioselectivity is, therefore, an important issue in design and development of new AEDs. Enantiomers often exhibit pronounced differences in their PK as well as in their pharmacodynamic properties (10). The pharmacologic effects are determined, on the one hand pharmacodynamically, by the interaction of a drug with a particular enzyme or receptor, and on the other hand, pharmacokinetically by the access of a drug to the site of action (11). Enantioselective PK can lead to enantioselectivity in efficacy, toxicity, and in clinical outcome in general. VGB is marketed as a racemic mixture although only the (S)-enantiomer possesses anticonvulsant activity (12). Whereas pregabalin [(S)-3-isobutyl GABA] is more potent than its (R)-enantiomer in in vitro binding assays, the enantioselectivity is much more pronounced in vivo in rodents, probably due to enantioselective transport or disposition of the compound (13). Oxcarbazepine is a prochiral new AED that undergoes presystemic enantioselective metabolism to its chiral active entity, MHD. In animal (rodent) models, (S)-MHD is slightly more potent as an anticonvulsant than the (R)-enantiomer, but the enantioselective PK of MHD in humans suggest that one of the enantiomers (S-MHD) may be clinically superior (14). Because of the impact of enantioselective PK on drug performance, it is important to investigate the enantioselectivity in the PK of LEV and REV and to evaluate possible enantioselectivity and chiral inversion that might affect the antiepileptic activity of the drug in vivo.
Enantiomerization (chiral inversion) of several chiral drugs, such as thalidomide (15), oxazepam (16), and (R)-ibuprofen (17), has been previously reported. In all of these compounds, the chiral center is in α-position to a carbonyl group (in ibuprofen to carboxylic acid, and in thalidomide and oxazepam, to an amide), which allows tautomerization and consequently chiral inversion. Enantiomerization can be spontaneous (16), or it can proceed by metabolic interconversion (17). In LEV the chiral center is in α-position to the carbonyl of the butyramide, and therefore chiral inversion appears possible.
The evaluation of enantioselective PK is important in assessing the mechanism of action of chiral drugs like LEV. Because the mechanism of action of LEV is currently unknown, the in vitro intrinsic pharmacodynamics of LEV cannot be evaluated. The enantioselectivity in pharmacodynamics can be used as a powerful tool in finding the site of action of LEV, and consequently, it is important to evaluate the possibility of PK-based enantioselective pharmacodynamics.
The purpose of this research was to investigate the PK of LEV and REV enantiomers, to determine their PK parameters after intravenous administration to dogs, and to evaluate the enantioselective disposition and possible in vivo chiral inversion between LEV and REV. This study also evaluated whether the enantioselective pharmacodynamics have a PK basis.
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Mean plasma concentration-versus-time curves for LEV and REV are presented in Fig. 2. Figure 2 also depicts the cumulative amount excreted in urine of both enantiomers and the cumulative amount of PBA excreted in urine as a function of time after administration of LEV or REV. The PK parameters obtained for LEV and REV are presented in Table 1. The total body clearance and volume of distribution of the enantiomers were not significantly different. All other PK parameters were significantly different. The half-life of REV was significantly longer than that of LEV. Consequently, the S/R enantiomeric ratio in plasma concentrations decreased from 1.6 at 5 min after drug administration to 0.8 at 12 h after dosing.
Figure 2. Mean plasma concentrations of levetiracetam (LEV) (A) and its enantiomer (R)-α-ethyl-2-oxo-pyrrolidine acetamide (REV) (B) obtained after individual administration of each enantiomer (20 mg/kg) to six dogs. Cumulative amount excreted in urine of LEV and REV (C) and cumulative amount excreted in urine of the metabolite 2-pyrrolidone-N-butyric acid (PBA) after administration of LEV and REV (D). Data points are presented as an average of six dogs and standard deviation. ▪, administration of LEV; □, administration of REV.
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Table 1. Pharmacokinetic parameters obtained for LEV and REV
| ||S-enantiomer||R-enantiomer||P Valuea|
|CL (ml/min/kg)||1.5 ± 0.3||1.5 ± 0.17||NS|
|Vss (L/kg)||0.45 ± 0.13||0.51 ± 0.11||NS|
|t1/2 (h)||3.6 ± 0.8||4.3 ± 0.8||≤0.05|
|MRT (h)||5.0 ± 1.2||6.0 ± 1.1||≤0.05|
|CLr (ml/min/kg)||0.69 ± 0.16||1.02 ± 0.14||≤0.05|
|fe (%)||50 ± 5||71 ± 10||≤0.05|
|Me/Db (%)||4.5 ± 3.7||2.2 ± 2.1||NS|
The CLr and fraction excreted unchanged of REV were significantly greater than those of LEV. The fraction of the dose excreted as the acid-metabolite PBA was greater after the administration of LEV than after the administration of REV. The fraction excreted unchanged was 50% and 71% for LEV and REV, respectively. The glomerular filtration rate in the dog is 4.0–6.13 ml/min/kg (20,21). In this study the renal clearance was found to be 0.69 ml/min/kg and 1.02 ml/min/kg for LEV and REV, respectively, indicating enantioselective tubular reabsorption. The fraction of the dose excreted as PBA after administration of LEV was higher (4.5%) than that after administration of REV (2.2%), but the difference was not statistically significant. The observed urine concentrations of PBA in dog's urine were in the range of 17–407 mg/L, with most of the samples having concentrations between 200 and 300 mg/L after administration of LEV. After administration of REV, the urine concentrations of PBA were in the range of 15–270 mg/L, with most of the samples with a concentrations of 100–200 mg/L.
After the administration of pure LEV and REV enantiomers separately to dogs, the plasma and urine samples were analyzed by using an enantioselective assay, but none of the second enantiomer could be detected in the samples. This confirmed that no chiral inversion occurs in vivo in the case of LEV or REV.
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Biologic macromolecules are very specific to spatial arrangements, and consequently, enantioselective interactions are frequently encountered. At present, there are no reports in the literature concerning enantioselective PK of LEV, and except our assay (18), there are no published enantioselective methods for LEV and REV analysis needed to explore possible chiral inversion. This study confirmed that the PK of α-ethyl-2-oxo-pyrrolidine acetamide is enantioselective. Enantioselectivity is more likely to be found in organ-specific parameters, such as renal or hepatic clearance, than in whole-body parameters like total body clearance and volume of distribution (22). This study is in agreement with this principle. The total body clearance of the LEV and REV enantiomers was not different, but the CLr was enantioselective. The fact that there was no significant difference in the total-body clearance indicates that in the dog, there may also be enantioselective metabolism that counteracts the enantioselectivity in CLr. This difference in metabolic clearance could not result only from the formation clearance of the acidic metabolite PBA, the major metabolite of LEV in humans, because the fraction of the dose excreted as PBA was rather minor (2–4%) in the dog. Consequently, in the dog, there must be an additional metabolite(s) whose formation clearance is enantioselective. The fraction of LEV metabolized to the corresponding acid is significantly smaller in dogs than in humans (24%). This finding might be of toxicologic importance when extrapolating data from dogs to humans, and the toxicity profile of the acid might need further evaluation. Enantioselective active tubular secretion has been reported for few drugs, such as 10-hydroxynortriptyline (23), ofloxacin (24), salbutamol (25), and pindolol (26), but enantioselective tubular reabsorption is not well known. Both processes can be active, saturable, or carrier-mediated and, consequently, enantioselective. The CLr of LEV and REV is smaller than the glomerular filtration rate, and therefore tubular reabsorption is indicated. Because the renal clearance is enantioselective, it is likely to be due to enantiospecific tubular reabsorption.
The PK of LEV has been studied in humans after oral administration and in rats after i.p. administration (27), but PK in dogs has not been reported in the literature. Because of the lack of data after i.v. administration to any species, the clearance of LEV has not been previously reported. The comprehensive understanding of the pharmacokinetics of an enantiomer requires intravenous administration of both enantiomers. Because REV cannot be administered to humans and a crossover study could not be carried out in rats, the dog was used in this study. As the PK of LEV was found to be linear in rats within a dose range of 20–80 mg/kg (27), and in humans, within a dose range of 1,000–3,000 mg/day (14–42 mg/kg daily), it was assumed that the PK of LEV and REV are linear in the dog. Consequently a single-dose study was found to describe satisfactorily the PK of LEV and REV.
The t1/2 of LEV in rats after i.p. administration was 1.8–3 h (6,27), whereas in humans, the t1/2 is 7–8 h and increases in the elderly (7). The half-life observed in dogs (3.6 h for LEV) was between the values for rats and humans. The volume of distribution of LEV in humans was approximated to 0.5–0.7 L/kg (7), a value similar to the total body water (TBW). The Vss observed in the current study (0.5 l/kg) was slightly smaller than the TBW of a dog. The results are in good agreement with the data reported for humans after oral administration and indicate that LEV distribution in humans and dogs is similar. Like Vss, the CLr of LEV in dogs and humans was quite similar. In humans the CLr was also about one third of the glomerular filtration rate (7), similar to that observed in the dog for LEV. The fe of LEV and the fraction of the dose excreted as the metabolite PBA is very similar in dogs and in children. In children, the fe of LEV is 52%, and the fraction metabolized (fm) to PBA, 9%(7). However, the fraction of the dose excreted as the metabolite PBA in dogs (4.5%) after administration of LEV was much smaller than that observed in adult humans (24%), showing more extensive amide–acid biotransformation in humans than in dogs. This resembles the phenomena observed in dogs and humans after administration of valpromide (VPD), an amide derivative of valproic acid (VPA). In dogs the fm of VPD to VPA was 30–40%, whereas in humans, it was 80–90%(28). LEV might also be regarded as a heterocyclic analogue of VPD that contains the substitution of 2-oxo-pyrrolidine in the β-position of the molecule. Thus, like aliphatic analogues of VPD (29,30), it undergoes metabolic hydrolysis to its corresponding acid PBA. The PK of REV has not been previously reported in any species, and this is also the first time that the PK of both enantiomers, LEV and REV, has been reported after i.v. administration. This study demonstrated that the PK of the enantiomers of (R)- and (S)-α-ethyl-2-oxo-pyrrolidine acetamide in dogs is enantioselective. It also showed that the enantioselectivity in secondary PK parameters is a consequence of enantioselectivity in the organ-specific parameters, such as CLr and metabolic clearance. The inactive enantiomer, REV, has more favorable PK in the dog, as reflected by its longer half-life. The primary PK parameters (CL and Vss) were, however, similar, and therefore the enantiospecificity in the pharmacodynamic activity is likely to be due to a enantioselective mechanism of action or receptor binding rather than a consequence of PK differences between the two enantiomers. This study also demonstrates that LEV and REV do not undergo chiral inversion in vivo in dogs.