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RESEARCH ARTICLE
Unexpected differences in the
pharmacokinetics of N-acetyl-DL-leucine
enantiomers after oral dosing and their
clinical relevance
Grant C. ChurchillID
1
*, Michael StruppID
2
, Antony GalioneID
1
, Frances M. PlattID
1
1Department of Pharmacology, University of Oxford, Oxford, United Kingdom, 2Department of Neurology,
German Center for Vertigo and Balance Disorders, Ludwig Maximilians University Hospital Munich, Munich,
Germany
*grant.churchill@pharm.ox.ac.uk
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 enan-
tiomers 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 (C
max
) 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. Elimi-
nation (k
e
and T
1/2
) 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-enantio-
mer, 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 sum-
mary, the enantiomers of N-acetyl-leucine exhibit large, unexpected differences in pharma-
cokinetics 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.
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 1 / 17
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OPEN ACCESS
Citation: Churchill GC, Strupp M, Galione A, Platt
FM (2020) Unexpected differences in the
pharmacokinetics of N-acetyl-DL-leucine
enantiomers after oral dosing and their clinical
relevance. PLoS ONE 15(2): e0229585. https://doi.
org/10.1371/journal.pone.0229585
Editor: Nicola
´s Pe
´rez-Ferna
´ndez, Clinica
Universidad de Navarra, SPAIN
Received: September 5, 2019
Accepted: February 11, 2020
Published: February 27, 2020
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0229585
Copyright: ©2020 Churchill et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
N-acetyl-leucine has been used as an over-the-counter drug for the treatment of vertigo since
1957 [1]. Although the mechanism of action of N-acetyl-leucine for vertigo is not known, evi-
dence implicates direct action on the central vestibular-related pathways and ocular motor net-
works [2,3], as also demonstrated by [
18
F]fluorodeoxyglucose and Positron Emission
Tomography [4]. At the molecular level, several mechanisms of action have been suggested
including physicochemical partitioning into the phopspholipid bilayer to decrease its fluidity
[1], direct action on glycine recptors and AMPA receptors [1], effects on branched chain ami-
notransferaes affecting glutamate neurotransmission [3], and an increase in glucose metabo-
lism [3]. All of these mechanisms could contrribute to the observed effect of norrmalizing
membrane potential and excitability leading to activation of the vestibulocerebellum and a
deactivation of the posterolateral thalamus [2,3,5].
Recently, N-acetyl-leucine has experienced a renaissance with renewed interest from both
academia and industry as a promising treatment for several disorders with unmet medical
needs including cerebellar ataxia [3,6–8], cognition and mobility in the elderly [9], lysosomal
storage disorders [10,11] migraine [12] and restless legs syndrome [13]. Given the broad thera-
peutic potential of N-acetyl-leucine, its pharmacodynamics and pharmacokinetics warrant fur-
ther exploration.
As N-acetyl-leucine is an analogue of the alpha amino acid leucine, and because it retains
leucine’s stereocentre it exists as a pair of enantiomers (Fig 1). Enantiomers are isomers, com-
pounds with the same molecular formula but which differ in the arrangement of their atoms
in space, having one chiral stereocentre with four different substituents that yields two non-
superimposable mirror image molecules (Fig 1). Often the pharmacological activity of a drug
resides with a single enantiomer because living systems are chiral and formed from chiral con-
stituents [14]. Thus, proteins encoded by mRNA and synthesized on ribosomes from L-amino
acids are chiral and show stereoselective binding of drugs to transporters, receptors and
enzymes [14]. Stereoselective binding can be trivial or profound: S-asparagine is sweet whereas
R-asparagine is bitter; R-thalidomide is a sedative whereas the S-form is teratogenic [15]. The
thalidomide tragedy shifted the importance of drug chirality from inconsequential to crucial
[16] as reflected by the current requirements for developing single enantiomers in drug devel-
opment and regulatory approval [17,18].
The effects of chirality on drug behaviour has shifted from nice to know to effectively need
to know basis for informed dosing and regulatory compliance, safety and efficacy. As N-ace-
tyl-leucine was developed before realization of the importance of drug chirality, it was and
continues to be used and marketed as a racemate (Tanganil
1
, Laboratoires Pierre Fabre)[1].
Subsequent studies in models of vertigo on the individual enantiomers have revealed that the
therapeutic effects of N-acetyl-DL-leucine are due to the L-enantiomer [4,19]. This means
that, as expressed by Ariens [16] for chiral drugs in general, the racemic mixture (N-acetyl-
DL-leucine) is in fact two drugs (the L-enantiomer and the D-enantiomer), each with distinct
properties with one (N-acetyl-D-leucine) at best that does not contribute to the therapeutic
response, and at worst potentially responsible for toxicity. Indeed, inclusion of an inactive
enantiomer provides nothing but an impurity or ‘isomeric ballast, as coined by Ariens [16,20].
The resulting concern was stated by Lees et al. [21]: “An impurity at the level 50% of the active
constituent would never be tolerated by regulatory authorities for any other constituent, which
is neither an active, nor excipient nor solvent.”.
Chirality affects not only the pharmacodynamic properties of potency, efficacy and affinity,
it also affects pharmacokinetic processes of absorption, distribution, metabolism and excretion
[21,22]. Accordingly, both the US Food and Drug Administration’s Guidance for Industry on
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 2 / 17
Funding: This study was financially supported by
IntraBio (https://intrabio.com). The authors (GCC,
MS, AG and FMP) were paid for consultancy work
for IntraBio. Authors, acting in their capacity as
consultants for IntraBio, played roles in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript. FMP is
also a Royal Society Wolfson Research Merit
Award holder and a Wellcome Trust Investigator in
Science.
Competing interests: I have read the journal’s
policy and the authors of this manuscript have the
following competing interests: MS is Joint Chief
Editor of the Journal of Neurology, Editor in Chief
of Frontiers of Neuro-otology and Section Editor of
F1000. He has received speaker’s honoraria from
Abbott, Actelion, Auris Medical, Biogen, Eisai,
Gru¨nenthal, GSK, Henning Pharma, Interacoustics,
Merck, MSD, Otometrics, Pierre-Fabre, TEVA, UCB.
He is a shareholder of IntraBio. He acts as a
consultant for Abbott, Actelion, AurisMedical, Heel,
IntraBio and Sensorion. GCC, AG and FMP are
cofounders, shareholders and consultants to
IntraBio. FMP is a consultant to Actelion. IntraBio
Ltd is the applicant for patents WO2018229738
(Treatment For Migraine), WO2017182802 (Acetyl-
Leucine Or A Pharmaceutically Acceptable Salt
Thereof For Improved Mobility And Cognitive
Function), WO2019078915 and WO2018029658
(Therapeutic Agents For Neurodegenerative
Diseases), WO2018029657 (Pharmaceutical
Compositions And Uses Directed To Lysosomal
Storage Disorders), and WO2019079536
(Therapeutic Agents For Improved Mobility And
Cognitive Function And For Treating
Neurodegenerative Diseases And Lysosomal
Storage Disorders). This does not alter our
adherence to PLOS ONE policies on sharing data
and materials.
the Development of New Stereoisomeric Drugs [17] and the European Medicines Agency [18]
advises that when a single enantiomer has been found to be the pharmacologically active ingre-
dient of a racemic mixture, it is important to not only characterize the pharmacokinetics of the
active enantiomer, but also the effects of the inert member of the stereoisomer pair to deter-
mine if there are potential risks with administering the racemate, or benefits associated with
the use of the single active enantiomer.
With the above as background regarding safety and efficacy of racemic drugs, and the fact
that no data has been published on the pharmacokinetics of the enantiomers of N-acetyl-leu-
cine, we investigated the pharmacokinetics of the racemate (an equal mixture of D and L) as
well as the pharmacologically active L-enantiomer alone. We report significant and unex-
pected differences in the pharmacokinetics of the enantiomers.
Materials and methods
Animal ethics approval
All experimental work in this study was conducted by Admescope Ltd. (Oulu, Finland) and
was prospectively approved by the national Animal Experiment Board of Finland. The study
was conducted under the project licence ESAVI/3047/04.10.07/2016, approved by the national
Animal Experiment Board of Finland under Directive 2010/63/EU of the European Parliament
and of the Council of 22 September 2010 on the protection of animals used for scientific
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.
https://doi.org/10.1371/journal.pone.0229585.g001
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 3 / 17
purposes, with the following national provisions: Act (497/2013) and Decree (564/2013) on
the Protection of Animals Used for Scientific or Educational Purposes (564/2013).
The Animal Welfare Body (AWB) of Admescope Ltd. oversees the animal care and use pro-
gram. The activity of AWB is documented and it is monitored by the Provincial Veterinarian
Officer. The Admescope laboratory Animal Unit is authorized, monitored and regularly
inspected by the Regional State Administrative Agency of Southern Finland (Provincial veteri-
narian officer). The National Animal Experiment Board in Finland is responsible for the pro-
tocol review and authorisation. Approval number: ESAVI/3047/04.10.07/2016.
Details of animal welfare
Animals (5–8 weeks of age during the experiment) were purchased from Scanbur (Denmark)
from Charles River Laboratories (Germany) and were housed in individually ventilated-cages
in groups of six mice. The cages were provided with aspen bedding (4HP and PM90L, Tapvei,
Estonia) and paper strands (Sizzlenest, Datesand, UK) as nesting material, and a paper pulp
cabin and red polycarbonate cylinder (Datesand, UK) as cage enrichment. The temperature
(22 ±2˚C), humidity (55 ±10%) and air exchange rate (75 (times/h) of the individually venti-
lated-cages and 12/12-h light/dark cycle (500 lux lighting on at 6 am, 1.5 lux lighting on at 6
pm) of the animal holding room were automatically controlled and maintained. Animals were
allowed to acclimatize to the site for at least five days prior to the study. Animals had ad libitum
access to food (SDS diets, RM1 (E) 801002, Special Diets Services, UK) and tap water at all
times, and their welfare was assured with daily observations.
To minimize animal suffering and distress, clinical signs and general behaviour of the ani-
mals were recorded when necessary. Noninvasively monitoring enables detection of any signs
that a drug was not well tolerated in this species and strain. Note that no adverse effects were
noted. The animals were not anesthetized during administration of study formulations or
during blood sampling, as the pain inflicted by these procedures is considered as minor. Addi-
tionally, the following internationally acknowledged primary standards, regulations, recom-
mendations and legislation are applied to the institutional animal care and use program to
support animal welfare, acknowledging 3R principles and transparency at Admescope Ltd: 1,
European Convention for the Protection of Vertebrate Animals Used for Experimental and
Other Scientific Purposes, Council of Europe (ETS 123); 2, The Guide for the Care and Use of
Laboratory Animals, NRC, 2011; 3, FELASA Guidelines and Recommendations; 4, Directive
2010/63/EU; and 5, National Centre for the replacement, refinement and reduction of animals
in research recommendations (NC3Rs).
In the case where only plasma and no tissue samples were collected, the animals were killed
humanely by using CO
2
, followed by cervical dislocation as described in the animal ethics
approval held by Admescope. When both plasma and tissue samples were collected, the ani-
mals were killed under isoflurane anaesthesia via cervical dislocation.
We used male BALB/c mice because this inbred strain of mice as it is commonly used for
preclinical pharmacokinetic studies [23]. Mice are commonly used because of their short life-
span, allowing for the growth of a large number of animals in a short period of time and, con-
sequently, the feasibility of many studies to predict pharmacokinetic profiles in humans [24].
We used male mice as this is the sex that is most commonly used; however, we note that sex
differences have been reported [25] and this could be investigated in the future for N-acetyl-
leucine. Pharmacokinetic data obtained from mice can be used to extrapolate to humans
through allometric scaling, but differences between specific drugs require empirical studies in
humans [26].
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 4 / 17
The animals were weighed on the day prior to dosing. The compound was administered to
male BALB/c mice (n = 3 per time point) p.o. (100 mg/kg; 10 mL/kg) by oral gavage. Blood
samples were collected into potassium EDTA tubes by venepuncture from the saphenous vein.
Within 30 min following the sampling, blood was centrifuged for plasma separation (room
temperature; 10 min; 2700 xg). The plasma samples were transferred into plastic tubes, frozen
and stored at –20˚C.
Chemicals and suppliers
HPLC grade methanol and acetonitrile were from Merck (Darmstadt, Germany). HPLC grade
formic acid, acetic acid and ammonium formate were from BDH Laboratory Supplies (Poole,
UK). Other chemicals were from Sigma Aldrich (Helsinki, Finland), and of the highest purity
available. Water was from a Direct-Q3 (Millipore Oy, Espoo, Finland) purification system and
UP grade (ultrapure, 18.2 MW). N-acetyl-DL-leucine was obtained from Molekula
(#73891210) and N-acetyl-L-leucine was obtained from Sigma Aldrich (#441511).
Sample preparation
The plasma samples were prepared for analysis by mixing 50 μL of plasma with 100 μL of ace-
tonitrile and mixed. In addition to plasma, skeletal muscle and brain tissue was also taken at
less frequent time points (0.5, 2, 6, 24 and 48 h) and prepared for analysis by homogenization
of 50 mg of tissue with 100 μL of acetonitrile. The samples were transferred to Waters 96-well
plate and the sample was evaporated under nitrogen gas flow. The sample was reconstituted
into 150 μl of 50% methanol:water and analysed by LC/MS. Standard plasma samples were
prepared by spiking the injection solution with concentrations from 1 to 10 000 ng/mL by
using one volume of spiking solution and nine volumes of injection solution. These samples
were then prepared for analysis in the same way as the samples. Quality control (QC) samples
were prepared both from racemic-N-Acetyl-Leucine and from N-Acetyl-L-Leucine in two
different concentrations. QC samples from racemic-N-Acetyl-Leucine were prepared into
concentrations of 40, 400 and 4000 ng/mL, corresponding to 20, 200 and 200 ng/mL concen-
tration of both D- and L-enantiomers, respectively. QC samples of N-Acetyl-L-Leucine was
then prepared into concentrations of 20, 200 and 2000 ng/mL. QC samples were then prepared
for analysis in the same way as the samples.
Quantification with liquid chromatography-mass spectrometry and chiral-
HPLC
Quantification by HPLC was performed using a Supelco Astec CHIROBIOTIC T chiral HPLC
column (2.1 x 150 mm, 5 μm particle size) with a Waters Acquity UPLC + Thermo Q-Exactive
hybrid Orbitrap MS, using ESI negative polarity, nitrogen auxiliary gas (450˚C), capillary volt-
age was 2000 and 350˚C and controlled with the software Xcalibur 4.1. Samples were injected
as a 4-μL volume and eluted with a gradient of buffer A (20 mM ammonium acetate) and
buffer B (methanol) with a flow rate of 0.3 mL/min and column oven temperature of 30˚C.
The gradient was 80% A at 0 min; 20% A at 3.5 min and 80% A at 4.5 min Parallel Reaction
Monitoring (PRM) and Full-MS-dd-MS2 were measured at the same time. In PRM, quadru-
pole was used as a mass filter and depending whether deuterated or non-deuterated N-Acetyl-
L-Leucine was detected, either m/z 172 or 176 only got through. Ions with aforementioned m/
z was then collided and leucine fragment (m/z 130 or 134) was used in quantitation. In full-
MS-dd-MS2 mode, every ion with intensity over a certain intensity was collided and fragments
analyzed.
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 5 / 17
Metabolite identification with reverse phase ultrahigh performance liquid
chromatography
Metabolite identification was performed using a Waters Acquity UPLC + Thermo Q-Exactive
hybrid Orbitrap MS and a Waters Acquity HSS T3 column (50 x 2.1 mm, 1.8 μm particle size).
MS was as described above over the mass range of 70–1000 using an acquisition time of 7 Hz
for full scan, IT 100 ms for DDI MS/MS, an AGC Target of 1E6, maximum IT of 100 ms and
35 000 (FWHM @ m/z 200) for full scan, 17 500 for MS/MS in DDI mode off for full scan; 20
+40+60 for DDI MS/MS inclusion list for expected metabolites ON; also other unexpected
most abundant metabolites chosen for MS/MS. Samples were injected as a 4-μL volume and
eluted at a flow rate of 0.5 mL/min and a column oven temperature of 35˚C with a gradient
consisting of Buffer A 0.1% formic acid and Buffer B acetonitrile. The gradient was (min, %A):
0, 98; 0.5, 98; 2, 50; 3, 5 and 3.5, 5. Ion chromatograms were extracted from the total ion chro-
matograms using calculated monoisotopic accurate masses with 10 mDa window. The metab-
olites were mined from the data using software-aided data processing (Thermo Compound
Discoverer 2.0 including structure-intelligent dealkylation tool & mass defect filter) with man-
ual confirmation.
Pharmacokinetic calculations
Plasma pharmacokinetic parameters of the N-acetyl-leucine enantiomers were calculated
using Phoenix 64 (Build 6.4.0.768) WinNonlin (version 6.4) software, using non-compartmen-
tal method with sparse sampling. Nominal doses were used for all animals. The terminal phase
half-life (T
1/2
), the time for 50% of the plasma concentration to decrease after some point of
elimination, was calculated by least-squares regression analysis of the terminal linear part of
the log concentration–time curve using the relationship 0.693/k
e
. The area under the plasma
concentration–time curve (AUC), an estimation of plasma drug exposure over time, was
determined with the linear trapezoidal rule for increasing values and log trapezoidal rule for
decreasing values up to the last measurable concentration (AUC
0-last
). The first order elimina-
tion rate constant k
e
was calculated as the slope (minimum 3 points) from the terminal log
plasma concentration time curve. The maximum concentration (C
max
) and the time taken to
achieve the peak concentration (T
max
) after oral dose were obtained directly from the plasma
concentration data without interpolation. The theoretical background and interpretation of
the pharmacokinetic data was based on [23]. Where appropriate, data are expressed as the
mean ±standard error of the mean. Means were statistically analysed by either pre-planned t
tests or a one-sample t test comparing the measured value with the expected value. Graphs
were plotted using Prism 7 (GraphPad Software Inc) and organized and formatted in Illustra-
tor (Adobe Inc).
Results
N-acetyl-D-leucine exhibits larger C
max
and AUC following racemate
administration
To determine whether the enantiomers of N-acetyl-leucine have different pharmacokinetics,
we orally dosed mice with either a racemate or the L-enantiomer (Fig 2 and S1 Fig). Following
an oral dose of N-acetyl-DL-leucine (100 mg/kg and 10 mg/mL), in the plasma, the concentra-
tion of the D-enantiomer was greater than the L-enantiomer at all time points (Fig 3A). Note
that direct comparison of the enantiomers is shown in replots of these data (Fig 4) and will be
discussed below. This asymmetry in the plasma concentrations of the D- and L-enantiomers
can be quantitated by comparing, respectively, C
max
of 86100 ng/mL verses 341 ng/mL (Fig 5A
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 6 / 17
and Table 1) and AUC of 75800 h�ng/mL versus 2560 h�ng/mL (Fig 5D and Table 1). The
elimination rate was similar for both enantiomers, indicated by the linear and parallel curves
on a semilog graph (Fig 3B) using a noncompartmental model giving a k
e
of 2.2 h
-1
for the D-
enantiomer and 2.8
−1
h for L-enantiomer (Fig 5C and Table 1), with corresponding T
1/2
values
of 0.31 h and 0.40 h (Fig 5E and Table 1). The D-enantiomer remained detectable until 8 h
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.
https://doi.org/10.1371/journal.pone.0229585.g002
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).
https://doi.org/10.1371/journal.pone.0229585.g003
Pharmacokinetics of the N-acetyl-leucine enantiomers
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(Fig 5G) with the last concentration of 247 ng/mL (Fig 5F). In contrast, the L-enantiomer
remained detectable until 2 h (Fig 5G) with the last concentration of 623 ng/mL (Fig 5F).
Pharmacokinetics of the enantiomers following N-acetyl-L-leucine
administration
For oral dosing with purified N-acetyl-L-leucine, the commercial source of this was found to
contain 97.4% L-enantiomer and 2.6% of the D-enantiomer (S1 Fig). This trace contamination
enabled us to evaluate the pharmacokinetics of the D-enantiomer at a much lower dose, and
allowed for an internal control and comparator. Following an oral dose of the purified L-enan-
tiomer at 100 mg/kg and 10 mg/mL, the concentration of the L-enantiomer was greater at all
time points (Fig 3C). Quantitatively, for the D- and L-enantiomers, respectively, had a C
max
of
436 ng/mL versus 16900 ng/mL (Fig 5A and Table 1) and an AUC of 573 h�ng/mL and 11400
h�ng/mL (Fig 5D and Table 1). As with administration of the racemate (Fig 3A and 3B), after
dosing with purified L-enantiomer, the elimination rate was similar for both enantiomers, indi-
cated by the linear and parallel curves on a semilog graph (Fig 3D) and was well-fit with a non-
compartmental model giving a k
e
of 1.7 h
-1
for the D-enantiomer and 2.4
−1
for L-enantiomer
(Fig 5C and Table 1), with corresponding T
1/2
values of 0.25 h and 0.29 h (Fig 5E and Table 1).
Both enantiomers remained detectable in the plasma until 8 h and 6 h (Fig 5G) with a last con-
centration of 16 ng/mL and 168 ng/mL (Fig 5F). However, the C
last
and T
last
are somewhat mis-
leading for all measurements, as in looking at the profiles, the main elimination was over for all
enantiomers at around 4 h when these terminal concentrations were reached (Fig 3B and 3D).
Dose proportionality is greater than unity
Dose proportionality refers to the effect of an increase in dose on C
max
and AUC [23]. We can
assess dose proportionality with our data by using the amount of each enantiomer present in
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).
https://doi.org/10.1371/journal.pone.0229585.g004
Pharmacokinetics of the N-acetyl-leucine enantiomers
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the composition administered. The D-enantiomer was dosed as 50% of the administered race-
mate and as 2.6% of the administered purified L-enantiomer, for a difference in dose propor-
tionality of 19-fold. The actual dose proportionality was 197-fold for C
max
(86100/436;
Table 1) and 101 fold for AUC (57800/573; Table 1). The L-enantiomer was dosed as 50% of
the administered racemate and 97.4% of the administered purified L-enantiomer, for a differ-
ence in dose proportionality of 1.9-fold. The actual dose proportionality was 4.9-fold for C
max
(16800/3410; Table 1 and Fig 5J) and 4.6 fold for AUC (11400/2560; Table 1 and Fig 5J).
Direct comparison of enantiomers highlights pharmacokinetic differences
To facilitate comparison of the racemate with the purified L-enantiomer, we re-plotted the
plasma concentration versus time profiles of the two enantiomers on the same graph over the
first two hours (Fig 4). The amount of D-enantiomer in the plasma is significantly higher
when dosed with the racemate compared to the much lower amount present when dosed with
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.
https://doi.org/10.1371/journal.pone.0229585.g005
Pharmacokinetics of the N-acetyl-leucine enantiomers
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purified L-enantiomer, and is consistent with the measured 2.6% D contamination in the puri-
fied L-enantiomer (S1 Fig). The semilog plot nicely shows the equal rates of elimination at all
concentrations and times, demonstrating that the D-enantiomer is not affected by dosing with
either the DL or L form. As would be expected with administering a 97.4% to 2.6% mixture of
N-acetyl-L-leucine to N-acetyl-D-leucine, the L-enantiomer dominated in the plasma (Fig 4A
and 4B). Administration of DL or L alone only affected C
max
and AUC, but did not affect elim-
ination (k
e
o T
1/2
). Plotting the L-enantiomer in the plasma on the same graph to compare dos-
ing with DL with L alone (Fig 4C), graphically shows the dramatic differences in C
max
and
AUC, but show the same rate of elimination (parallel curves when fit to a noncompartmental
model).
Another way to compare administration of the racemate to the purified L-enantiomer on
the pharmacokinetics of the enantiomers was to calculate the ratio of enantiomers in regard to
C
max
and AUC. As we verified the administered compound to be a true racemate (50% each
enantiomer; S1 Fig), deviations from a ratio of 1 reveal significantly different pharmacokinetics
between the D- and L-enantiomers. When administered as the racemate, the ratio of D/L
enantiomer was about 25 for both C
max
(Fig 5H; 26 versus 1, p = 0.014) and AUC (Fig 5I, 25 vs
1, p = 0.015). As the purified L-enantiomer administered contained 97.4% L-enantiomer and
2.6% D-enantiomer (S1 Fig), if the enantiomers had identical pharmacokinetics, the ratio of L/
D would be predicted to be 36 (that is, 97.4/2.6). When administered as the purified L-enantio-
mer, the ratio of L/D was 32 for C
max
(Fig 5H; 31.7 versus 36, p= 0.17) and 20 for AUC (Fig 5I;
19.8 versus 36, p= 0.006).
Enantiomers show differences in distribution and metabolism
To investigate the effect of administering either the racemate or purified L-enantiomer of N-
acetyl-leucine on the distribution of the enantiomers, muscle and brain were analysed. At spe-
cific times after oral dosing, the mice were euthanized and the amount of D- and L-enantiomer
present in the tissues was determined. Following oral dosing with the racemate, muscle con-
tained much more D-enantiomer than L-enantiomer (Fig 6A). In muscle, the D-enantiomer
was only detectable at 30 min and 2 h (Fig 6A). In contrast, following oral dosing of the L-
enantiomer alone, in muscle, the L-enantiomer was not detected at any time point and the D-
Table 1. The calculated pharmacokinetic parameters for N-Acetyl-D-Leucine and N-Acetyl-L-Leucine plasma after oral administration of N-Acetyl-DL-Leucine or
N-Acetyl-L-Leucine at a nominal dose of 100 mg/kg.
Compound administered N-Acetyl-DL-Leucine N-Acetyl-L-Leucine
Compound quantified N-Acetyl-D-Leucine N-Acetyl-L-Leucine N-Acetyl-D-Leucine N-Acetyl-L-Leucine
Parameter Unit Value Value Value Value
r
2
- 0.91 0.93 0.85 0.72
k
e
- 2.2 2.8 1.7 2.4
T
max
h<0.25 <0.25 <0.25 <0.25
C
max
ng/mL 86 100 3410 436 16 800
T
last
h 8.00 2.00 8.00 6.00
C
last
ng/mL 247 623 16.2 168
T
1/2
h 0.31 0.4 0.25 0.29
AUC
0-last
h x ng/mL 57 800 2 560 573 11 400
Ratio C
max
L/D
#
- 0.04 38.5
Ratio AUC L/D
#
- 0.04 19.8
#
The ratio of corresponding value between L and D enantiomers
https://doi.org/10.1371/journal.pone.0229585.t001
Pharmacokinetics of the N-acetyl-leucine enantiomers
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enantiomer was detectable but at a much lower concentration (Fig 6C) than after administra-
tion of the racemate (Fig 6A). Neither the D- nor L-enantiomer was detected in muscle after 2
hours from the time or dosing (Fig 6A and 6C). Following oral dosing with the racemate, the
brain contained detectable D-enantiomer at only the 30 min time point and L-enantiomer was
not detectable at any time point (Fig 6B). Following oral dosing with purified L-enantiomer,
neither of the enantiomers were detected at any time point (Fig 6D).
We investigated the identity of the metabolites of both enantiomers in muscle, but no
metabolites of either N-acetyl-D-leucine or N-acetyl-L-leucine were detected (data not
shown).
Discussion
We investigated the pharmacokinetics of the enantiomers of N-acetyl-leucine after oral admin-
istration of the racemate, which has been marketed under the name Tanganil
1
for the treat-
ment of vertigo in France since 1957 [1], and the purified L-enantiomer, which is the
pharmacologically active enantiomer in models of acute vertigo [4,19]. We report significant
and unexpected differences in the pharmacokinetics of the enantiomers. The major findings of
this study are as follows: First, when administered as the racemate (N-acetyl-DL-leucine), the
D-enantiomer was present at much higher plasma maximal concentration (C
max
) and (area
under the curve; AUC) relative to the L-enantiomer, resulting in greater total exposure. Sec-
ond, when administered as purified N-acetyl-L-leucine, both the C
max
and the AUC for N-ace-
tyl-L-leucine were higher compared to administration as the racemate, even when scaled for
the relative dose. Third, both enantiomers distributed to the tissues monitored, muscle and
brain, but the D-enantiomer was found at much higher concentrations relative to the L-enan-
tiomer in both tissues.
Origin of the differences in C
max
and AUC
The larger AUC for N-acetyl-L-leucine when administered as the purified enantiomer com-
pared to when administered as the racemate, and factoring in the actual amounts of L-enantio-
mer present in each (that is, 97.6% and 50%, respectively), is fully accounted for the by
increase in C
max
because after C
max
and T
max
, the clearance (k
e
and T
1/2
) is the same for both
enantiomers. In other words, after 15 min, the pharmacokinetic parameters are the same for
both enantiomers. Therefore, the large differences in C
max
have to be due to processes occur-
ring in the first 15 min and before the L-enantiomer enters the plasma. Consequently, we can
deduce that the D-enantiomer is interfering with the bioavailability (the amount of drug orally
administered that is systemically available) of the L-enantiomer during the first 15 min follow-
ing oral administration. Differences between enantiomers indicate interaction with protein
targets; therefore, two possible explanations that are not mutually exclusive exist: competition
at a carrier on cells in the intestine and/or differences in first-pass metabolism.
Stereoisomer-mediated pharmacokinetics arising from uptake
The bioavailability of a drug is determined by its ability to penetrate and cross the gastrointes-
tinal epithelial cell membrane, either by passive diffusion or via a carrier. Uptake by passive
diffusion is determined by physicochemical properties, primarily hydrophobicity, which
allows penetration of the membrane’s core [27,28]. The N-acetylation of leucine would be pre-
dicted to greatly increases passive membrane transport, as it eliminates one (NH
3+
) of the two
(NH
3+
and COO
-
) charges present on all amino acids at physiological pH, which can increase
transport rates up to 10
10
-fold [29,30]. However, as this physicochemical effect (loss of charge
and increase in hydrophobicity) is identical for the enantiomers, it cannot underlie the
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 11 / 17
differences observed in the pharmacokinetics of the N-acetyl-leucine enantiomers. In contrast,
uptake by carriers requires molecular recognition at saturable binding sites and would give
rise to interference between the enantiomers. The identity of the carrier for N-acetyl-leucine
on the intestinal brush-border membrane is unknown; however, given that N-acetyl-leucine is
a modified amino acid, the most likely candidates are amino acid transporters, as 52 families
exist that show distinct substrate selectivity[31–33]. These possibilities can be narrowed down
further based on the effect of N-acetylation, which forms an amide bond (Fig 1). An amide
bond would both make N-acetyl-leucine appear more like a dipeptide and, through resonance,
given the C-N bond partial double bond character with a bond order 1.5 [34], making it an
analogue of an imine (Fig 1B). These predict that N-acetyl-leucine would be a substrate for the
low affinity/high capacity a H
+
-coupled di/tripeptide transporter termed PepT1, which is
highly expressed and responsible for 80% of all amino acids are taken up from the small intes-
tine lumen, or an imino acid transporter which has 100-fold greater affinity for N-modified
amino acids and shows only 2-fold stereoselectivity [35].
Stereoisomer-mediated pharmacokinetics arising from first-pass
metabolism
Another likely contributing process accounting for the differences between enantiomers in
C
max
and AUC is first-pass metabolism [21]. As first-pass metabolism is an enzymatic process,
it exhibits molecular recognition at saturable binding sites and would also give rise to interfer-
ence between the enantiomers. Such stereoselective first-pass effects are known to alter oral
drug bioavailability of the enantiomers of propranolol and verapamil [22,36]. Indeed, the 2-
3-fold stereoisomer effect we detected for N-acetyl-leucine is similar to the 2–3 fold greater
oral bioavailability of (–)-verapamil compared to (+)-verapamil caused by first-pass metabo-
lism [21]. Most often first-pass metabolism is mediated by cytochrome P-450 oxidation in the
stomach, intestine or liver [21]; however, N-acetyl-L-leucine is more likely handled like a
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).
https://doi.org/10.1371/journal.pone.0229585.g006
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 12 / 17
nutrient than a xenobiotic, as it is a naturally occurring metabolite of L-leucine and a transace-
tylase has been reported that interconvert N-acetyl-L-leucine and L-leucine, using other L-
amino acids as the substrate or product [37,38]. Therefore, a likely enzyme for first-pass
metabolism of N-acetyl-L-leucine would be the acylase reported in intestinal strips that was
able to remove the acetyl group from most amino acids [39], and showed 40,000-fold selectiv-
ity for L-amino acids over D-amino acids [37–40].
Stereoisomer effects manifested by tissue uptake and metabolism
In regard to the presence of the enantiomers in muscle and brain, the amounts were much
lower than in the plasma (10-fold to undetectable), and the D-enantiomer was present at a
much higher concentration than the L-enantiomer. In general, our results showing that N-ace-
tyl-leucine is blood-brain barrier permeable are consistent with studies in monkeys in which
radioactive racemic N-acetyl-leucine was administered intravenously and radioactivity was
subsequently detected in the brains [41]. However, the
14
C label was in the alpha carbon of leu-
cine and autoradiography was used for quantification, so there is no ability to determine
whether the radioactivity was due to N-acetyl-DL-leucine itself or a metabolite [41]. Therefore,
the data with radioactivity is ambiguous in terms of both the effect of stereoisomerism and
whether N-acetylation promotes uptake and whether it is rapidly metabolized to L-leucine.
In contrast to the situation with uptake from the gut to the plasma in which the D-enantio-
mer was reducing uptake, in muscle and brain, the presence of the D-enantiomer was associ-
ated with increased presence of N-acetyl-L-leucine. Uptake from the plasma into cells and
tissues, as described for the intestinal cells above, occurs through both passive diffusion and
carriers. The explanation of competitive inhibition for a common carrier used for the asymme-
try in uptake between the enantiomers into the plasma of competition cannot explain this
observation. Indeed, such an effect would result in less of the L-enantiomer, not more, when
N-acetyl-D-leucine was also present. A more likely explanation is competitive inhibition of the
enantiomers at an enzyme that metabolizes N-acetyl-L-leucine. A likely explanation is that the
D-enantiomer is inhibiting the deacetylation of N-acetyl-L-leucine. It is also important to note
that the amount of N-acetyl-L-leucine in tissues is a steady state measure of the compound,
and relates not to lack of uptake but rather rapid utilization. By comparison, the D-enantiomer
was present in higher amounts, consistent with it being metabolically inert based on feeding
N-acetyl-D-leucine to rats, where it was excreted in the urine unchanged [38]. The simplest
explanation is that the N-acetyl-L-leucine is rapidly converted to L-leucine and utilized in
metabolism. Rapid utilization and metabolism of L-leucine is consistent with the results of a
study using stable isotope-labelled leucine itself upon oral administration [42]. Moreover, our
inability to detect metabolites is consistent with the disappearance of N-acetyl-L-leucine
though metabolism to L-leucine, which would be undetectable on the background of endoge-
nous L-leucine. Slowing the conversion of N-acetyl-L-leucine to L-leucine, and subsequently
its regulatory effect on muscle protein synthesis and oxidative metabolism [43,44], and possi-
bly impact on its efficacy as a drug. Taken together, these data showing low amounts of N-ace-
tyl-leucine in the brain and muscle suggest that the mechanism of action of N-acetyl-L-leucine
requires metabolism.
Clinical implications of stereoselective pharmacokinetics
The different pharmacokinetics of the enantiomers would conceivably result in disproportion-
ate total exposure (increase in the AUC) to the D-enantiomer when the racemate is dosed, as
the L-enantiomer would be eliminated much faster. Importantly, chronic treatment with mul-
tiple doses over time would cause accumulation in the body of the D-enantiomer of N-acetyl-
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 13 / 17
leucine. Historically, it was presumed that the ‘inactive’ enantiomer was harmless [16], a
notion disabused by the thalidomide tragedy [15]. Although the N-acetyl-D-leucine is not
reported to be toxic, concerns about the toxicity of D-amino acids in general have been raised
as the reason for the original evolutionary selection and biological presence of D-amino acid
oxidase [45,46]. Evidence that the D-leucine is having a biological effect comes from a report
in which low amounts (about 1/10th of endogenous L-form) of D-leucine suppressed endoge-
nous levels of L-leucine by almost half [47].
Conclusions
In conclusion, firstly, the L-enantiomer–which is the pharmacologically active form in models
of acute vertigo–has different pharmacokinetics when administered with the D-enantiomer as
the racemate (N-acetyl-DL-leucine) compared to administration as the purified L-enantiomer.
Secondly, we found evidence for an accumulation of the D-enantiomer, which would be exac-
erbated by chronic dosing of the racemate, with unknown and possibly unwanted deleterious
effects on cell function. Thirdly, the results of this study, taken together with the regulatory
guidelines of the FDA [17] and the EMA [18], strongly supports the research and development
of isolated N-acetyl-L-leucine.
Supporting information
S1 Fig. Chiral high performance liquid chromatography/mass spectrometry analysis show-
ing separation and quantification of the compounds used in these studies. (a) Spectrum of
racemate. (b) Spectrum of purified N-acetyl-L-leucine. Note that the peak areas are not directly
comparable with concentration due to differences in the extent of ionization of the compound
in the mass spectrometers ionization chamber due to relative differences in aqueous and
organic solvent concentrations in the mobile phase at those time points due to a gradient elu-
tion. Therefore, quantification was based on a standard curve specific to each enantiomer. The
result is that the racemate contained 50.2% N-acetyl-D-leucine and 49.8% N-acetyl-L-leucine.
The purified N-acetyl-L-leucine contained 2.6% N-acetyl-D-leucine and 97.4% N-acetyl-L-leu-
cine. The limit of detection and the limit of quantification was, respectively, 10 ng/mL and 25
ng/mL for N-acetyl-D-leucine, and 25 ng/mL and 50 ng/mL for N-acetyl-L-leucine.
(TIF)
S1 Table. Measured concentrations of N-Acetyl-L-Leucine and N-Acetyl-D-Leucine in
mouse plasma and tissues after p.o administration N-Acetyl-DL-Leucine at 100 mg/kg.
(DOCX)
S2 Table. Measured concentrations of N-Acetyl-L-Leucine and N-Acetyl-D-Leucine in
mouse plasma and tissues after p.o administration N-Acetyl-L-Leucine at 100 mg/kg.
(DOCX)
Author Contributions
Conceptualization: Grant C. Churchill, Antony Galione, Frances M. Platt.
Data curation: Grant C. Churchill.
Formal analysis: Grant C. Churchill.
Funding acquisition: Grant C. Churchill, Antony Galione, Frances M. Platt.
Methodology: Grant C. Churchill, Antony Galione, Frances M. Platt.
Pharmacokinetics of the N-acetyl-leucine enantiomers
PLOS ONE | https://doi.org/10.1371/journal.pone.0229585 February 27, 2020 14 / 17
Visualization: Grant C. Churchill.
Writing – original draft: Grant C. Churchill.
Writing – review & editing: Michael Strupp, Antony Galione, Frances M. Platt.
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