Brain kinetics of methylphenidate (Ritalin) enantiomers after oral administration.
ABSTRACT Methylphenidate (MP) (Ritalin) is widely used for the treatment of attention deficit hyperactivity disorder (ADHD). It is a chiral drug, marketed as the racemic mixture of d- and l-threo enantiomers. Our previous studies (PET and microdialysis) in humans, baboons, and rats confirm the notion that pharmacological specificity of MP resides predominantly in the d-isomer. A recent report that intraperitoneally (i.p.) administered l-threo-MP displayed potent, dose-dependent inhibition of cocaine- or apomorphine-induced locomotion in rats, raises the question of whether l-threo-MP has a similar effect when given orally. It has been speculated that l-threo-MP is poorly absorbed in humans when it is given orally because of rapid presystemic metabolism. To investigate whether l-threo-MP or its metabolites can be delivered to the brain when it is given orally, and whether l-threo-MP is pharmacologically active. PET and MicroPET studies were carried out in baboons and rats using orally delivered C-11-labeled d- and l-threo-MP ([methyl-(11)C]d-threo-MP and [methyl-(11)C]l-threo-MP). In addition, we assessed the effects of i.p. l-threo-MP on spontaneous and cocaine-stimulated locomotor activity in mice. There was a higher global uptake of carbon-11 in both baboon and rat brain for oral [(11)C]l-threo-MP than for oral [(11)C]d-threo-MP. Analysis of the chemical form of radioactivity in rat brain after [(11)C]d-threo-MP indicated mainly unchanged tracer, whereas with [(11)C]l-threo-MP, it was mainly a labeled metabolite. The possibility that this labeled metabolite might be [(11)C]methanol or [(11)C]CO(2), derived from demethylation, was excluded by ex vivo studies in rats. When l-threo-MP was given i.p. to mice at a dose of 3 mg/kg, it neither stimulated locomotor activity nor inhibited the increased locomotor activity due to cocaine administration. These results suggest that, in animal models, l-threo-MP or its metabolite(s) is (are) absorbed from the gastrointestinal tract and enters the brain after oral administration, but that l-threo-MP may not be pharmacologically active. These results are pertinent to the question of whether l-threo-MP contributes to the behavioral and side effect profile of MP during treatment of ADHD.
- [Show abstract] [Hide abstract]
ABSTRACT: Methylphenidate (MPD) is one of the most prescribed pharmacological agents, which is also used for cognitive enhancement and recreational purposes. The objective of this study was to investigate the repetitive dose-response effects of MPD on circadian rhythm of locomotor activity pattern of female WKY rats. The hypothesis is that a change in the circadian activity pattern indicates a long-lasting effect of the drug. Four animal groups (saline control, 0.6, 2.5, and 10.0 mg/kg MPD dose groups) were housed in a sound-controlled room at 12:12 light/dark cycle. All received saline injections on experimental day 1 (ED 1). On EDs 2-7, the control group received saline injection; the other groups received 0.6, 2.5, or 10.0 mg/kg MPD, respectively. On ED 8-10, injections were withheld. On ED 11, each group received the same dose as EDs 2-7. Hourly histograms and cosine statistical analyses calculating the acrophase (ϕ), amplitude (A), and MESOR (M) were applied to assess the 24-h circadian activity pattern. The 0.6 and 2.5 mg/kg MPD groups exhibited significant (p < 0.05) change in their circadian activity pattern on ED 11. The 10.0 mg/kg MPD group exhibited tolerance on ED 11 and also a significant change in activity pattern on ED 8 compared to ED 1, consistent with withdrawal behavior (p < 0.007). In conclusion, chronic MPD administration alters circadian locomotor activity of adult female WKY rats and confirms that chronic MPD use elicits long-lasting effects.Journal of Neural Transmission 07/2013; · 3.05 Impact Factor
- Drugs 04/2006; 66(5). · 4.13 Impact Factor
- Radiotherapy and Oncology - RADIOTHER ONCOL. 01/2011; 99.
Brain Kinetics of Methylphenidate
(Ritalin) Enantiomers After Oral
YU-SHIN DING,* S. JOHN GATLEY, PANAYOTIS K. THANOS, COLLEEN SHEA, VICTOR GARZA,
YOUWEN XU, PAULINE CARTER, PAYTON KING, DON WARNER, NICHOLAS B. TAINTOR,
DANIEL J. PARK, BEA PYATT, JOANNA S. FOWLER, AND NORA D. VOLKOW
Chemistry and Medical Departments, Brookhaven National Laboratory, Upton, New York 11973-5000
methylphenidate; Ritalin; ADHD; dopamine transporter; positron
emission tomography; chiral drugs
tion deficit hyperactivity disorder (ADHD). It is a chiral drug, marketed as the racemic
mixture of d- and l-threo enantiomers. Our previous studies (PET and microdialysis) in
predominantly in the d-isomer. A recent report that intraperitoneally (i.p.) administered
l-threo-MP displayed potent, dose-dependent inhibition of cocaine- or apomorphine-induced
locomotion in rats, raises the question of whether l-threo-MP has a similar effect when given
orally. It has been speculated that l-threo-MP is poorly absorbed in humans when it is given
orally because of rapid presystemic metabolism. To investigate whether l-threo-MP or its
metabolites can be delivered to the brain when it is given orally, and whether l-threo-MP is
pharmacologically active. PET and MicroPET studies were carried out in baboons and rats
using orally delivered C-11-labeled d- and l-threo-MP ([methyl-11C]d-threo-MP and [methyl-
11C]l-threo-MP). In addition, we assessed the effects of i.p. l-threo-MP on spontaneous and
in both baboon and rat brain for oral [11C]l-threo-MP than for oral [11C]d-threo-MP. Analysis
of the chemical form of radioactivity in rat brain after [11C]d-threo-MP indicated mainly
unchanged tracer, whereas with [11C]l-threo-MP, it was mainly a labeled metabolite. The
possibility that this labeled metabolite might be [11C]methanol or [11C]CO2, derived from
demethylation, was excluded by ex vivo studies in rats. When l-threo-MP was given i.p. to
locomotor activity due to cocaine administration. These results suggest that, in animal
models, l-threo-MP or its metabolite(s) is (are) absorbed from the gastrointestinal tract and
enters the brain after oral administration, but that l-threo-MP may not be pharmacologi-
cally active. These results are pertinent to the question of whether l-threo-MP contributes to
the behavioral and side effect profile of MP during treatment of ADHD. Synapse 53:
Methylphenidate (MP) (Ritalin) is widely used for the treatment of atten-
© 2004 Wiley-Liss, Inc.
Racemic methylphenidate (MP, dl-threo-methyl-2-
phenyl-2-(2-piperidyl)acetate, Ritalin) is a CNS stimu-
lant that is the most commonly prescribed drug for the
treatment of attention deficit hyperactivity disorder
(ADHD). ADHD has become America’s No. 1 childhood
psychiatric disorder (Barkley, 1977) with an estimated
prevalence of 5–10% of the general population (Swan-
son et al., 1998). The therapeutic effect of MP has been
linked to its blockade of the dopamine transporter,
resulting in enhanced levels of synaptic dopamine
(Volkow et al., 2001). We have labeled both d- and
l-threo-MP with carbon-11 ([11C]d-threo-MP and [11C]l-
threo-MP) and used positron emission tomography
(PET) (1994a,b, 1995, 1997) to show high specific bind-
Contract grant sponsor: U.S. Department of Energy and Office of Biological
Environmental Research; Contract grant number: DE-AC02-98CH10886; Con-
tract grant sponsor: National Institutes of Health, National Institute for Bio-
medical Imaging and Bioengineering; Contract grant number: EB002630; Con-
tract grant sponsor: National Institute on Drug Abuse; Contract grant number:
DA-06278; Contract grant sponsor: Office of National Drug Control Policy.
*Correspondence to: Yu-Shin Ding, Ph.D., Neuroscience and Imaging Group,
Chemistry Department, Brookhaven National Laboratory, Upton, New York
11973-5000. E-mail: email@example.com
Received 14 October 2003; Accepted 26 April 2004
Published onlinein Wiley InterScience (www.interscience.wiley.
SYNAPSE 53:168–175 (2004)
© 2004 WILEY-LISS, INC.
ing of [11C]d-threo-MP as compared to mostly nonspe-
cific binding of [11C]l-threo-MP after i.v. injection into
humans and baboons (Ding et al., 1997). Studies that
employed computer testing of children with ADHD
suggested a drug-induced improvement in sustained
attention that was entirely attributable to the d-enan-
tiomer (Srinivas et al., 1992b). Microdialysis studies in
free-moving rats, used for measuring the changes in
extracellular dopamine concentrations produced by i.p.
injection of individual unlabeled d-threo-MP and
l-threo-MP, further demonstrate that pharmacological
specificity of MP with respect to MP-induced DA in-
creases resides predominantly in the d-threo isomer
and the binding of l-isomer is mostly nonspecific
(Aoyama et al., 1994, 1996).
reospecific presystemic metabolism (Aoyama et al.,
1990; Hubbard et al., 1989; Srinivas et al., 1987) in
both human and animals to form predominantly the
hydrolysis product, ritalinic acid (RA), resulting in a
low absolute oral bioavailability, and an oral bioavail-
ability of d- and l-MP of 22% and 5%, respectively
(Srinivas et al., 1993). Animal studies have suggested
that the primary site of presystemic metabolism is in
the gut and/or intestinal wall and not the liver or lungs
(Aoyama et al., 1990). RA concentrations in the blood
may be 30–60-fold higher than MP concentrations.
Although RA is a polar compound and circulates in
higher concentration than MP, it does not appear to
have CNS activity even when administered intracere-
broventricularly (Patrick et al., 1984; Sheppard et al.,
Studies from our laboratory and other research
groups have shown that injections of racemic MP and
d-threo-MP increase locomotor activity in rodents
(Ding et al., 1997; Gerasimov et al., 2000). A recent
report that i.p. administered l-threo-MP displayed a
dose-dependent inhibitory effect on the locomotor ac-
tivity induced by d-threo-MP, cocaine, or apomor-
phine in intact adult rats (Baldessarini, 2001) raised
the question of whether l-threo-MP has a similar
effect when given orally. This is relevant since
ADHD subjects are treated with oral MP and it is
therefore crucial to assess whether l-threo-MP plays
a role or is inactive in the brain when used thera-
We report here our studies using PET and C-11-
labeled d- and l-threo-MP ([11C]d-threo-MP and [11C]l-
threo-MP) to compare the uptake of these tracers in the
brain after oral administration to individual baboons
and rats. Rats were sacrificed after tracer administra-
tion to investigate the chemical form of C-11 in brain.
To evaluate whether l-threo-MP interfered with co-
caine-induced locomotion, we conducted experiments
with these two drugs in mice.
MATERIALS AND METHODS
Unlabeled dl-threo-MP.HCl was purchased from Re-
search Biochemicals (Natick, MA). The individual un-
labeled enantiomers (d-threo-MP and l-threo-MP) were
separated in our laboratory according to a literature
method (Ding et al., 1994b; Patrick et al., 1987). Co-
caine was obtained from the NIDA.
All studies were conducted in accordance with the
guidelines established by the National Institutes of
Health and were approved by the Institutional Animal
Care and Use Committee of Brookhaven National Lab-
oratory. Adult female baboons (Papio anubis) housed
at BNL for several years were used for PET studies.
Adult male Sprague Dawley rats and Swiss-Webster
mice were purchased from Taconic Farms (German-
town, NY). Rats had a mean weight of 375 ? 12 g when
utilized, and mice had a mean weight of 30 ? 5 g.
Animals were individually housed in a room controlled
for temperature and humidity as well as a 12-h light/
dark cycle. Water (except where indicated) and food
were available ad libitum.
Radiosynthesis of [11C]d-threo-MP and
[11C]d-threo-MP and [11C]l-threo-MP were prepared
from an N-protected d-threo- or l-threo-ritalinic acid
derivative in two steps: O-methylation with [11C]CH3I
followed by hydrolysis of the protective group. The total
synthesis time was 40 min, with an average specific
activity of 1.5 Ci/?mol (EOB), radiochemical purity
? 98%, and enantiomeric purity of 99% (Ding et al.,
PET studies of oral [11C]d-threo-MP and
[11C]l-threo-MP in baboons
Three baboons were used in the PET studies over an
8-month period with at least 3 weeks between studies
to allow recovery from anesthesia and blood sampling.
The baboons were anesthetized and prepared for PET
studies as described previously (Ding et al., 1995).
Briefly, animals were anesthetized with an intramus-
cular dose of ketamine (10 mg/kg) and then intubated
and ventilated with a mixture of isoflurane (Forane,
1–4%), nitrous oxide (1,500 ml/min), and oxygen (800
ml/min). Catheters were inserted in a popliteal artery
and a radial arm vein for arterial sampling and radio-
tracer injection, respectively. An oral gastric tube was
inserted. The baboons were positioned on an individu-
alized padded restraining table to minimize motion
and repositioning errors. An attenuation scan was per-
formed prior to radiotracer administration. During the
study, heart rate, respiration rate, PO2, and tempera-
tureweremeasuredusinga pediatric monitor
KINETICS OF ORAL METHYLPHENIDATE ENANTIOMERS
(SpaceLabs Pediatric Patient Care Monitoring Sys-
tem). For each paired study, tracer doses (4–7 ?g per
injection) of [11C]d-threo-MP and [11C]l-threo-MP were
administered via a gastric tube with a 2–3-h time pe-
riod between the two doses. Each injection contained
3–10 mCi of radioactivity in 10 mL of H2O, and the
gastric tube was then flushed with 30–50 mL water.
This procedure allowed us to compare the kinetics of
each enantiomer in the brain of the same baboon. The
order of administration of the enantiomers was varied
to control possible effects of prolonged anesthesia.
Scanning was performed for 90 min on an HR? high-
resolution PET scanner (63 slices; 4.5 ? 4.5 ? 4.5 mm)
operating in 3D mode. The following scanning se-
quence was used: 10 frames for 1 min each; four frames
for 5 min each, eight frames for 7.5 min each. Gamma
shielding was placed across the baboon body to reduce
effects of scattered radiation on brain images after the
administration via a gastric tube. Arterial blood sam-
pling and determination of unchanged labeled methyl-
phenidate in blood plasma were carried out as reported
previously (Ding et al., 1995). The gastric tube was
taken out at the end of the second study. The residual
radioactivity in the gastric tube was measured and
decay-corrected to the time of the injection to deter-
mine the amount of radioactivity that was delivered to
the stomach. To minimize the movement and distur-
bance of the baboon, the gastric tube was not removed
after the first study.
Image and data analysis
In our previous studies, after the i.v. administration
of [11C]d-threo-MP or [11C]l-threo-MP, regions of inter-
est (ROIs) of the baboon brain were clearly defined and
were drawn directly on the PET scans (Ding et al.,
1997). However, there was much less radioactivity in
the brain after oral administration of [11C]d-threo-MP
or [11C]l-threo-MP, and thus only the uptakes of the
C-11-labeled individual enantiomers in the whole brain
were determined and compared. In one of the studies
that showed the highest uptakes in the brain, radioac-
tivity in the striatum and cerebellum was also com-
pared. For studies performed on the HR? scanners,
time frames were summed to obtain an image on which
a global region comprised 5–7 central slices. The
summed image was then projected onto the dynamic
sequence to obtain time–activity data. Time–activity
curves were compared for [11C]d-threo-MP vs. [11C]l-
threo-MP in the whole brain in the same animal stud-
ied on the same day.
MicroPET studies in rats
Rats were anesthetized i.p. with a mixture of ket-
amine (100 mg/kg) and xylazine (10 mg/kg) and placed
in a stereotaxic head holder in a prone position on the
bed of a microPET R4 scanner (Concorde Microsys-
tems, Knoxville, TN). The methodology of Thanos et al.
(2002) was used. The total acquisition time was 75 min
[(26 frames: 6 (10 sec); 3 (20 sec); 8 (60 sec); 5 (300 sec);
4 (600 sec)] and data was acquired in fully 3D mode
with maximum axial acceptance angle (?28°). Images
were reconstructed using FORE rebinning followed by
2D filtered backprojection with a ramp filter cutoff at
Nyquist. Using the rat stereotaxic atlas (Paxinos and
Watson, 1993) and the harderian glands as a reference
point, the coronal planes of striatum and cerebellum
were identified in the same manner. Specifically, for
each animal the striatum and cerebellum was identi-
fied as 3 and 7 slices, respectively, caudal to the hard-
erian glands (slice thickness was 1.2 mm). It has been
previously shown that the harderian glands (located
just rostral to the brain) because of their uptake of
radioactivity are convenient anatomical markers in ro-
dent PET studies (Thanos et al., 2002).
Animals were injected via the tail vein with [11C]d-
threo-MP (244 ?Ci/cc) or [11C]l-threo-MP (291 ?Ci/cc)
in a volume of 0.2 ml of sterile saline. The same two
rats were used for oral administration studies 3 weeks
after the i.v. administration experiments. Between the
i.v. and oral studies they were trained to drink water
on demand by depriving them of access to water for
24-h periods. This strategy avoided the need for sur-
gery to implant gastric tubes and thus reduced the
animal’s levels of stress. The exact amount of radioac-
tivity consumed by each animal was determined by
measuring the activity in the drinking tube before and
Analysis of the chemical form of
carbon-11 in tissues
Rats were sacrificed at about 100 min after admin-
istration of tracers, when microPET imaging had been
completed. Striatum (ST), cerebellum (CB), and the
rest of the brain (ROB) were dissected out, weighed,
and homogenized in a 2:1 acetonitrile/methanol mix-
ture (volumes ranged from 0.5–1.0 mL depending on
the weight of tissue) using a Tissue Tearor (Biospec
Products, Bartlesville, OK) at full speed for 1 min. The
homogenates were transferred to 2-mL Eppendorf
tubes and centrifuged to produce clear supernatants.
Supernatants and pellets were counted to determine
extraction efficiency. All counting was done on a Mi-
naxi 5000 auto gamma counter (Packard Instruments,
Meriden, CT). Supernatants were spiked with unla-
beled methylphenidate standard and analyzed by
HPLC to determine percent unchanged methylpheni-
date in the tissues (HPLC conditions: Phenomenex
Spherisorb 10 ODS1 250 ? 4.6, 60:40 acetonitrile/
0.15M ammonium phosphate, flow 1.3 mL/min). The
fraction of the labeled MP in each sample was taken as
the amount of radioactivity co-eluting with the unla-
beled MP (identified by monitoring UV activity at 254
nm) relative to the total amount injected into the
Y.-S. DING ET AL.
HPLC. Radioactivity in the entire gastrointestinal
tract was also measured. Tissue radioactivity concen-
trations were expressed as percent injected radioactiv-
ity per gram (%IA/g).
In another study, rats (n ? 2 for each enantiomer)
were sacrificed 30 min after drinking aqueous solutions
of [11C]d-threo-MP or [11C]l-threo-MP and tissue radio-
activity concentrations determined as described above.
In addition, aliquots of the supernatants were also
analyzed to determine the extent to which radioactivity
in the brain could be [11C]methanol or [11C]CO2, de-
rived from demethylation of the labeled MP. For each
brain sample, a paired study was carried out (Fox et
al., 1985). Aliquots of the supernatants from various
brain regions were placed into two tubes containing 3
mL of isopropyl alcohol and 1.0 mL of 0.9 M sodium
bicarbonate. One mL of 6 N HCl was added to one
member of each pair and 1 mL of 0.1 N NaOH was
added to the other. Radioactivity in both samples was
assayed with a gamma-well counter before and after
sonication in a heated ultrasonic bath for 10 min.
Cocaine induced locomotor activity
Outbred male Swiss-Webster mice (25–30 g) were
purchased from Taconic Farms and maintained in our
animal facility for at least 5 days before use on a 12-h
light/dark cycle. Locomotor experiments were per-
formed 1 h into the dark cycle, to maintain relatively
high levels of spontaneous activity. Activity monitors
were obtained from San Diego Instruments (San Diego,
CA). They consisted of 16-inch Plexiglas cubical cham-
bers in which horizontal movements of the mice inter-
rupted infrared beams. Two mice were placed in each of
the four chambers available, in order to increase the
number of beam crossings recorded. The mice were
allowed to explore the chambers for 1 h before admin-
istration of drugs. In a typical experimental session,
one pair of mice was then injected with vehicle, one
pair with l-threo-MP (3 mg/kg) alone, one pair with
cocaine (10 mg/kg) alone, and one pair with l-threo-MP
(3 mg/kg) plus cocaine (10 mg/kg). A total of six ses-
sions were conducted, with chambers matched with
drug group in a randomized manner on each day of
testing. Average beam crossings for the six pairs of
mice in each of the four conditions were graphed. Ad-
ditionally, for each pair of animals, the mean activity
(number of beam breaks) between 65 and 130 min was
divided by the mean activity between 45 and 60 min.
These activity ratios were used to calculate normalized
average values for the four groups—mean ? SE (n ?
6). We routinely placed mice in pairs in the locomotor
activity boxes to increase the number of beam breaks.
In our hands this gives more reproducible results, prob-
ably because animals placed individually in boxes oc-
casionally remain in one place for long periods. This
strategy could be criticized on the grounds that group
differences could reflect drug effects on interactions
between animals, rather than drug effects on locomotor
activity. However, we consider this unlikely in the
PET studies of oral [11C]d-threo-MP and
[11C]l-threo-mp in baboons
In our previous studies, after i.v. injections of [11C]d-
threo-MP or [11C]l-threo-MP the radioactivity reached
a peak uptake (?0.05% injected dose/cc) in baboon
brain in less than 8 min (Ding et al., 1997). As ex-
pected, much slower brain uptake of carbon-11 was
observed when these two tracers were given orally via
a gastric tube. In most cases the uptake in the brain did
not reach peak concentrations until at least 30 min
after injection of [11C]l-threo-MP and 40 min after in-
jection of [11C]d-threo-MP. This slow kinetics was con-
sistent with our previous baboon PET study with orally
administered racemic [11C]MP (Volkow et al., 1998).
Peak uptake values for five studies are summarized in
Table I. Time–activity curves for a representative
paired study are shown in Figure 1. Uptake in the
whole brain for [11C]l-threo-MP was always higher
than for [11C]d-threo-MP in all of the studies, regard-
less of the injected dose or the order of the injections.
However, the ratio of the radioactivity in striatum to
that in cerebellum was higher for oral injection of
[11C]d-threo-MP than that for [11C]l-threo-MP (data not
Measurement of total carbon-11 in baboon plasma
after oral injection of radiotracers indicated a higher
plasma integral for [11C]l-threo-MP than for [11C]d-
threo-MP. However, plasma integrals for parent radio-
tracer (i.e., with the metabolite correction) were similar
after [11C]d-threo-MP and [11C]l-threo-MP injections in
the same baboon, and represented a small fraction of
total radioactivity (Fig. 2).
The decay-corrected radioactivity for the two enanti-
omers remaining in the gastric tube was negligible.
Thus, the measured injected dose was not confounded
by the possibility that part of the activity adhered to
the tube and was not available for biodistribution.
TABLE I. Baboon studies with oral administration of
[11C]d-threo-MP or [11C]l-threo-MP
Tracer BaboonDose (mCi)
Global brain uptakea
aMaximum global brain uptake value based on the time–activity curve.
bThis second study was cancelled due to a technical problem.
KINETICS OF ORAL METHYLPHENIDATE ENANTIOMERS
MicroPET studies of i.v. and oral
[11C]d-threo-MP and [11C]l-threo-MP in rats
A clear and significant difference was observed be-
tween the two isomers with [11C]d-threo-MP, but not
[11C]l-threo-MP, showing high striatal uptake follow-
ing i.v. injection in rats. These results are consistent
with our previous studies in baboon and human (Ding
et al., 1997). However, after oral administration of the
two enantiomers microPET did not give good images of
the brain, possibly due to more rapid metabolism in
rodents than primates, and the insensitivity of micro-
PET to very low radioactivity concentrations.
Radioactivity assay in various tissues after oral
[11C]d-threo-MP and [11C]l-threo-MP in rats
At both 30 and 100 min after oral administration, the
total fraction of radioactivity in the whole brain after
[11C]l-threo-MP was higher than that after [11C]d-
threo-MP (Table II). The radioactivity in the gastroin-
testinal tract was highest for oral d-threo-MP at 30
min, but highest for l-threo-MP at 100 min postoral
injection. HPLC analysis indicated that radioactivity
in the brain regions after oral l-threo-MP administra-
tion did not represent the parent labeled compound
([11C]l-threo-MP); in contrast, most of the radioactivity
in the brain after oral [11C]d-threo-MP) was unmetabo-
Determination of the contributions to total
radioactivity of [11C]methanol or [11C]CO2
The evaporation experiments performed under ei-
ther acidic or basic conditions did not produce any
significant loss of radioactivity before and after sonica-
tion in a heated ultrasonic bath (data not shown).
[11C]l-threo-MP (solid squares) and [11C]d-threo-MP (solid circles) in
the same baboon.
Global uptake in baboon brain after oral administration of
injection of [11C]d-threo-MP (circles) and [11C]l-threo-MP (squares).
Solid symbols represent data that have not been corrected for metab-
olite, open symbols represent data that have been corrected for me-
tabolite. Note that the plasma integrals for both [11C]d-threo-MP and
[11C]l-threo-MP were similar after they were corrected for the pres-
ence of labeled metabolite(s).
Plasma integral of the radioactivity in baboon after oral
TABLE II. Oral [11C]d-threo-MP and [11C]l-threo-MP in rats
Oral [11C]d-threo-MP at 30 min
?50% of the C-11 radioactivity in the brain was MP
Oral [11C]l-threo-MP at 30 min
?5% of the C-11 radioactivity in the brain was MP
Oral [11C]d-threo-MP at 100 min
?30% of the C-11 radioactivity in the brain was MP
Oral [11C]l-threo-MP at 100 min
The C-11 radioactivity in the brain was not MP,11CO2, or11CH3OH
Values are given for the percent injected activity per gram (%IA/g) in the
striatum (ST), cerebellum (CB), the rest of brain (ROB), and the gastrointestinal
tract. Percent unchanged tracer was determined by HPLC.
Y.-S. DING ET AL.
These results suggest that the observed radioactivity
in the brain was not due to either [11C]methanol or
Effect of l-threo-MP on cocaine-induced
Averaged time–activity data for mice treated with
saline, l-threo-MP, cocaine, or cocaine plus l-threo-MP
are shown in Figure 3. The combination of l-threo-MP
and cocaine appeared to slightly enhance the activity
over that seen with cocaine alone. However, when data
for each pair of animals during the 70-min period after
drug treatment was normalized to activity in that pair
in the 15-min period before drug, we obtained the fol-
lowing ratios. Saline, 0.56 ? 0.04; l-threo-MP, 0.60 ?
0.07; cocaine, 1.33 ? 0.17; cocaine plus l-threo-MP,
1.22 ? 0.06. There was no significant difference be-
tween the two groups without cocaine, or between the
two groups with cocaine. However, either group with
cocaine had a significantly higher ratio than either
group without cocaine (all four group differences were
P ? 0.01). Thus, cocaine, as expected, was stimulatory.
However, l-threo-MP neither stimulated locomotion
nor, in contrast to the report of Baldessarini et al.
(2002), did this compound reduce cocaine-stimulated
locomotion. A similar experiment using four pairs of
mice per condition and drug doses of 6 mg/kg
l-threo-MP and 10 mg/kg cocaine also did not find a
reduction of cocaine-stimulated locomotor activity
(data not shown).
Methylphenidate (MP, Ritalin) treatment for ADHD
has been considered one of the great successes in psy-
chiatry. It is a commonly prescribed oral drug for chil-
dren and a growing number of adults with ADHD. It is
a chiral drug and is marketed as the dl-threo racemic
form. There is no evidence of interconversion between
the two enantiomers in vivo. Racemic MP is absorbed
quickly and completely after oral administration in
humans (Faraj et al., 1974), but d-threo-MP is found in
the urine at 10-fold higher levels than l-threo-MP. Con-
versely, urine contains 2–3-fold higher levels of l-threo-
ritalinic acid than d-threo-ritalinic acid after oral ad-
ministration ofMP. In
enantiomeric difference was found for MP or ritalinic
acid in urine after i.v. administration. These findings
have been attributed to enantioselective presystemic
conversion of MP to ritalinic acid rather than enantio-
selective excretion (Srinivas et al., 1992a) and have led
to the speculation that l-threo-MP given orally would
not enter the brain (Srinivas et al., 1992b). It has also
been suggested by some investigators (Srinivas et al.,
1992b), but not others (Jonkman et al., 1998), that
l-threo-MP is not absorbed by the gastrointestinal tract
as evidenced by the failure to detect l-threo-MP in
plasma after oral MP administration.
To our surprise, a higher global uptake of radioactiv-
ity in the brain was observed for [11C]l-threo-MP than
for [11C]d-threo-MP when they were given orally in
both baboons and rats. One of the advantages of our
using MP labeled with C-11 on the methyl ester moiety
is that the demethylated metabolite RA is no longer
radioactive; that is, it does not contribute to the PET
images and does not complicate interpretation of re-
sults. Therefore, brain radioactivity after l-threo-MP is
not in the form of ritalinic acid. Our results in rats also
exclude unchanged [11C]l-threo-MP as well as the de-
methylation products [11C]methanol and [11C]CO2. Al-
though our data are consistent with previous observa-
tions that l-threo-MP cannot be detected in plasma
samples after administration of pharmacological doses
of l-threo-MP (Srinivas et al., 1992a), they indicate
formation of a metabolite bearing the methyl ester
group that is able to cross the blood–brain barrier. At
present, we have not definitively identified the metab-
olite, although it has the same HPLC retention time as
p-hydroxy-MP (Ding et al., unpubl.), of which the hy-
drolyzed form has been previously recognized as a me-
tabolite of racemic MP in human urine (Faraj and
Jenkins, 1973; Srinivas et al., 1991). Other polar me-
tabolites that have been identified in dog and rat urine
after oral administration of14C-labeled MP are methyl
6-oxo-a-phenylpiperidineacetate, the glucuronide of p-
hydroxy-MP and their hydrolyzed forms (oxo-RA and
p-hydroxy-RA) (Egger et al., 1981). An argument
against the assignment of the labeled metabolite of
l-threo-MP as p-hydroxy-l-threo-MP is the reported low
brain penetrability of racemic p-hydroxy-MP (Patrick
et al., 1984). Further studies to elucidate these issues
are necessary. However, our results with oral admin-
istration to monkeys and rats of the enantiomers of
[11C]MP do not contradict the common assumption that
locomotor activity. Y axis indicates movements (i.e., locomotion). Sa-
line (open diamonds); l-threo-MP (triangles); cocaine (10 mg/kg, black
diamonds); and combination of l-threo-MP and cocaine (double
crosses). Drugs were injected i.p. in mice. Each of the four curves was
generated based on the data from 12 mice.
The effect of l-threo-MP (3 mg/kg, i.p.) on cocaine induced
KINETICS OF ORAL METHYLPHENIDATE ENANTIOMERS
the pharmacological specificity of MP resides entirely
in the d-threo isomer. The key observations are that,
after oral administration of [11C]d-threo-MP, most of
the radioactivity in the brain represented the parent
compound and a higher uptake was observed in stria-
tum than that in cerebellum. These findings were con-
sistent with our previous studies when the tracers were
given intravenously (Ding et al., 1997).
In our locomotor activity studies in mice, l-threo-MP
given alone did not affect locomotor activity, which is
consistent with our previous finding in rat brain micro-
dialysis studies that l-threo-MP did not alter extracel-
lular dopamine levels (Ding et al., 1997). These results
are also in agreement with the recent report by Davids
et al. (2002). However, our data did not support the
finding of an inhibitory effect of l-threo-MP on cocaine-
stimulated locomotor activity that was recently re-
ported by Baldessarini et al. (2001). This would be an
important finding, if verified by other workers, because
of its possible implications for the treatment of cocaine
Taken together, our studies indicate for the first time
that there is a compound that is absorbed by the gas-
trointestinal tract and then enters the brain after the
oral administration of l-threo-MP. Although our baboon
and microPET studies were done at tracer doses of the
C-11-labeled MPs and they do not perfectly mimic the
in vivo situation when a person takes a pill, and there
may be differences in the metabolism and kinetics of
MP among different species, these results raise the
question of whether l-threo-MP contributes to the be-
havioral and side-effect profile of MP during treatment
of ADHD. Numerous examples from a range of thera-
peutic areas confirm that single enantiomers can en-
hance clinical efficacy, reduce adverse effects, cause
fewer interactions with other drugs, and minimize re-
sponse variations among patients by offering more pre-
dictable pharmacokinetics and greater selectivity
(Rosenbaum, 2002). In some cases, these advantages
are simply due to the removal of an inactive enantio-
mer; but in other cases, a given dose of a single isomer
offers greater benefits when administered alone than
when administered as the racemic mixture, suggesting
that the opposite enantiomer actually has detracting
effects. This assumes the inactive enantiomer is always
detrimental. On the other hand, one can speculate that
perhaps in some cases the presence of the inactive
enantiomer may contribute to therapeutic efficacy of a
drug by enhancing bioavailability and other factors.
The racemic form of MP has been widely used as the
treatment of ADHD; however, neither the therapeutic
mechanism(s) nor side effects have been well charac-
terized. Our findings strongly suggest the importance
and urgent need to better understand this drug.
In conclusion, these comparative studies of enantio-
merically pure [11C]d-threo-MP and [11C]l-threo-MP in
both baboon and rat brain demonstrate a higher global
uptake of radioactivity for [11C]l-threo-MP than for
[11C]d-threo-MP when they were given orally. How-
ever, although we confirm that [11C]d-threo-MP enters
the brain after oral administration, the chemical form
of radioactivity in brain after administration of [11C]l-
threo-MP is not the administered labeled compound
but a metabolite tentatively identified as p-hydroxy-
MP. These PET and microPET studies, combined with
our previous studies with i.v. injection of [11C]d-threo-
MP and [11C]l-threo-MP, strongly indicate that phar-
macological specificity of MP resides predominantly in
the d-threo isomer. This conclusion is supported by
studies in mice indicating that l-threo-MP may be be-
haviorally inactive. Most important, these results sup-
port the further examination in humans of the compar-
ative absorption, metabolism, and pharmacological
activity of the individual enantiomers of MP and the
nature of their interaction when the drug is given as a
This research was carried out at Brookhaven Na-
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Ferrieri, and D. Schlyer for their assistance in various
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