Unexpected differences in the pharmacokinetics of N-acetyl-DL-leucine enantiomers after oral dosing and their clinical relevance

Abstract

The enantiomers of many chiral drugs not only exhibit different pharmacological effects in regard to targets that dictate therapeutic and toxic effects, but are also handled differently in the body due to pharmacokinetic effects. We investigated the pharmacokinetics of the enantiomers of N-acetyl-leucine after administration of the racemate (N-acetyl-DL-leucine) or purified, pharmacologically active L-enantiomer (N-acetyl-L-leucine). The results suggest that during chronic administration of the racemate, the D-enantiomer would accumulate, which could have negative effects. Compounds were administered orally to mice. Plasma and tissue samples were collected at predetermined time points (0.25 to 8 h), quantified with liquid chromatography/mass spectrometry, and pharmacokinetic constants were calculated using a noncompartmental model. When administered as the racemate, both the maximum plasma concentration (Cmax) and the area under the plasma drug concentration over time curve (AUC) were much greater for the D-enantiomer relative to the L-enantiomer. When administered as the L-enantiomer, the dose proportionality was greater than unity compared to the racemate, suggesting saturable processes affecting uptake and/or metabolism. Elimination (ke and T1/2) was similar for both enantiomers. These results are most readily explained by inhibition of uptake at an intestinal carrier of the L-enantiomer by the D-enantiomer, and by first-pass metabolism of the L-, but not D-enantiomer, likely by deacetylation. In brain and muscle, N-acetyl-L-leucine levels were lower than N-acetyl-D-leucine, consistent with rapid conversion into L-leucine and utilization by normal leucine metabolism. In summary, the enantiomers of N-acetyl-leucine exhibit large, unexpected differences in pharmacokinetics due to both unique handling and/or inhibition of uptake and metabolism of the L-enantiomer by the D-enantiomer. Taken together, these results have clinical implications supporting the use of N-acetyl-L-leucine instead of the racemate or N-acetyl-D-leucine, and support the research and development of only N-acetyl-L-leucine.

Fig 1. Chemical structure of N-acetyl-leucine.

(a) Stereochemistry of the enantiomers. (b) Amide resonance structures showing similarity to an imine. Extending from the tetrahedral chiral carbon is a solid wedge to indicate a bond projecting above the plane of the paper and a hashed wedge to indicate a bond projecting below the plane of the paper.

Fig 2. Schematic outlining the experimental procedure.

Male mice were orally administered N-acetyl-leucine as either the racemate (50% each enantiomer) or purified L-enantiomer (2.6% D-enantiomer and 97.4% L-enantiomer). At specific times (0.25 to 8 h) after administration, blood was taken, plasma was separated and quantified by chiral liquid chromatography/mass spectrometry. Plots of the plasma concentration of each enantiomer over time were used to visualize pharmacokinetics and a noncompartmental model was used to calculate the pharmacokinetic parameters C_max (maximum peak concentration), T_max (time to reach C_max), k_e (first order elimination rate constant), T_1/2 (half-life) and AUC (area under the curve). Samples of brain and skeletal muscle were also taken at specific times and used to determine compound distribution and to search for metabolites with high-resolution mass spectrometry.

Fig 3. Graphs of plasma concentration of enantiomers versus time after administration of racemic N-acetyl-DL-leucine or purified N-acetyl-L-leucine.

Data are presented as (a,c) linear-linear plots or (b,d) semilog plots. Values are the mean ± standard error of the mean with n = 3 (mice).

Fig 4. Replots of the data to facilitate direct comparison of the plasma concentration of N-acetyl-leucine enantiomers after oral administration of racemic N-acetyl-DL-leucine or purified N-acetyl-L-leucine.

Data are presented as (a,c) linear-linear plots or (b,d) semilog plots. Values are the mean ± standard error of the mean with n = 3 (mice).

Fig 5. Bar charts showing the pharmacokinetic parameters for the enantiomers of N-acetyl-L-leucine after administration of racemic N-acetyl-DL-leucine (denoted as DL) or N-acetyl-L-leucine (denoted as L).

(a-g) Conventional pharmacokinetic parameters calculated from the plasma concentration of drug. (h-j) Parameters derived from the conventional pharmacokinetic parameters to detect and highlight the effects of pharmacokinetic differences between the enantiomers. Values are the mean ± standard error of the mean with n = 3 (mice). Means were statistically analysed by either (a-g) pre-planned t tests; (h and i) a one-sample t test comparing the measured value with the expected value: 1 when administered as DL and 36 when administered as purified L; and (j) a one-sample t test comparing the measured value with the expected value of 2. The means compared are indicated by the horizontal lines on the charts, and exact p values are provided for the comparisons.

Fig 6. Graphs of the concentration of enantiomers in tissue versus time after administration of racemic N-acetyl-DL-leucine or purified N-acetyl-L-leucine.

Data are for (a,b) muscle and (c,d) brain and presented as linear-linear plots. Values are the mean ± standard error of the mean with n = 3 (mice).