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Original Articles
Isopropylphenidate: An Ester Homolog of Methylphenidate
with Sustained and Selective Dopaminergic
Activity and Reduced Drug Interaction Liability
John S. Markowitz, PharmD,
1,2
Hao-Jie Zhu, PhD,
3
and Kennerly S. Patrick, PhD
4
Abstract
Objective: The most widely utilized pharmacological treatment of attention-deficit/hyperactivity disorder (ADHD) is the
psychostimulant methylphenidate (MPH). Most MPH formulations consist of the racemic mixture of d-threo-(R, R)-MPH
and l-threo-(S, S)-MPH isomers. MPH is characterized by its low bioavailability and short half-life (2–3 hours). Additionally,
significant inter-individual variability in MPH pharmacokinetics has been consistently documented. Accordingly, efforts
have been directed at developing alternatives to MPH as therapeutic agents. A wide range of MPH analogues (dl-a-[2-
piperidyl]-phenylacetic acid esters) have been synthesized with the dopamine transporter (DAT) and norepinephrine
transporter (NET) as principle neuropharmacological targets. The present study investigated the metabolic profiles and
pharmacological activity of the isopropyl ester derivative of MPH, dl-isopropylphenidate (IPH), both in vitro and in vivo.
Methods: The synthesis, monoaminergic transporter binding, cellular upta ke profiles, and assessment of metabolic hydrolysis
and transesterification in the presence of ethanol are described using MPH as a comparator. Additionally, an in vivo as-
sessment of IPH stimulant effects (vs. saline) in rats was performed with locomotor activity as a pharmacodynamic outcome.
Results: IPH displayed unique pharmacological characteristics including greater DAT than NET binding and cellular uptake
activity, and greater resistance to hydrolysis and transesterification via carboxylesterase 1 relative to MPH. Further, sustained
psychostimulant properties offer the prospect of an enhanced duration of action.
Conclusions: Our findings are consistent with IPH exhibiting attributes distinguishing it from MPH and warranting further
study and development of IPH as a novel psychotherapeutic agent.
Introduction
Attention-deficit/hyperactivity disorder (ADHD) is a
complex neurobehavioral disorder characterized by varying
degrees of inattention, hyperactivity, and impulsivity (Biederman
2005). It is perhaps the single most common chronic health prob-
lems affecting school-age children, with an estimated worldwide
prevalence of 8–12% (Faraone et al. 2003). The most widely uti-
lized pharmacological treatment of ADHD is the psychostimulant
methylphenidate (MPH) consisting of the racemic (50:50) mix-
ture of d-threo-(R,R)-MPH and l-threo-(S,S)-MPH isomers (Fig.
1). MPH is generally an effective and well-tolerated treatment that
hasbeeninclinicalusefor >50 years. MPH is widely available as
an immediate-release (IR) tablet and displays a short plasma half-
life of 2–3 hours as a result of rapid and stereoselective metabo-
lism via de-esterification (Patrick et al. 2005a). This hydrolytic
processismediatedbyhepaticcarboxylesterase1(CES1)inhu-
mans, yielding the major, albeit inactive, metabolite ritalinic acid
(RA) that typically attains blood concentrations 30–60 times those
of the parent compound (Patrick and Markowitz 1997). Use of IR
formulations of MPH result in a relatively brief duration of action
necessitating multiple daily doses to achieve symptom control
throughout the day (Markowitz et al. 2003; Patrick et al. 2005a).
However, administering MPH throughout the day poses problems
in terms of convenience, compliance, security against diversion,
and patient self-esteem, particularly for the school-age child
(Greenhill et al. 2002). Accordingly, common clinical practice in
the United States is to prescribe one of several modified-release
(MR) pharmaceutical formulations (Markowitz et al. 2003) that
vary in duration of action and release profile. Therefore, MR
formulations of MPH have largely supplanted the use of IR for-
mulations as the primary dosage forms employed in the treatment
1
Department of Pharmacotherapy and Translational Research, University of Florida, Gainesville, Florida.
2
Center for Pharmacogenomics, University of Florida, Gainesville, Florida.
3
Department of Clinical, Social, and Administrative Sciences, University of Michigan College of Pharmacy, Ann Arbor, Michigan.
4
Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, South Carolina.
JOURNAL OF CHILD AND ADOLESCENT PSYCHOPHARMACOLOGY
Volume 23, Number 10, 2013
ªMary Ann Liebert, Inc.
Pp. 648–654
DOI: 10.1089/cap.2013.0074
648
of ADHD. Although the half-life parameter of the MPH remains
unchanged upon release from such formulations, this approach
can provide the often necessary all-day symptom ‘‘coverage’’
with a single daily dose. Despite these advances in biopharma-
ceutical delivery of MPH, in many instances these formulations
are not successful for patients because of inter-individual physio-
logical differences in drug absorption as such MR formulations
pass the various regions of the gut (McConnell et al. 2008), or are
prohibitively expensive. Significant inter-individual variability in
response to MPH pharmacotherapy has been consistently docu-
mented in clinical research literature. For example, up to 30% of
patients receiving MPH do not derive adequate therapeutic effects,
or experience treatment-limiting side effects (Patrick et al. 2005a).
The variation in therapeutic outcomes have been partially attributed
to significant variation of CES1 activity and expression associated
with CES1 genetic variants (Zhu et al. 2008) and environmental
factors (i.e., CES1 inhibitors and/or inducers). Therefore, MPH
congeners that are similar in terms of pharmacologic activity, yet
resistant to CES1 hydrolysis, have emerged as an area of active
drug development. Ideally, such compounds will extend the dura-
tion of action and provide more predictable clinical outcomes as a
result of reduced inter-individual variability of CES1-medicated
drug metabolism.
In the present study, the MPH homolog dl-isopropylphenidate
(IPH) (Fig. 1) was synthesized and assessed for its pharmacological
activity and metabolic profiles. Monoamine transporter binding and
cellular uptake experiments were performed on racemic IPH, as
well as on the comparators MPH and the ethyl ester congener
previously characterized by our group, dl-ethylphenidate (EPH)
(Patrick et al. 2005b). Additionally, an assessment of metabolic
hydrolysis of the three compounds by CES1 was performed uti-
lizing CES1 transfected cell lines, human liver microsomes (HLM),
and human intestinal microsomes (HIM). Furthermore, the poten-
tial for a CES1-mediated transesterification reaction between IPH
and ethanol to ethylphenidate was evaluated and compared with
that of MPH in vitro, in view of the metabolic drug interaction
associated with the MPH–ethanol combination (Patrick et al.
2007). Finally, an in vivo assessment of IPH stimulant effects (vs.
saline) on locomotor activity was performed utilizing experimental
methodology previously employed to establish the in vivo loco-
motor activity of the separate d- and l-isomers of MPH in rats
(Patrick et al. 1987). The overarching goal of this series of exper-
iments was to explore the pharmacological characteristics of IPH as
a potential new therapeutic entity.
Materials and Methods
Chemical compounds and animals
MPH was purchased from Sigma-Aldrich (St. Louis, MO). EPH
synthesis was conducted in our laboratory as previously described
(Patrick et al. 2005b). IPH was synthesized in our laboratory using the
following method: Briefly, ( –)-ritalinic acid (2 m M) w as dissolved
in isopropyl alcohol saturated with HCl gas (75 mL) and refluxed for
24 hours under nitrogen. The solution was evaporated to dryness
under reduced pressure and purged with nitrogen, and the white
residue was then dissolved in a minimum volume of warm isopropyl
alcohol. Diethyl ether was then added to turbidity and the flask was
stored for 24 hours at 2C. The resulting white crystalline product
was filtered, washed with diethyl ether, and dried under vacuum. The
purity of the synthetic material was confirmed by gas chromatography-
mass spectrometry. All compounds studied were assessed as their
HCl salts. The remaining reagents and solvents were of the highest
grade commercially available. Animals used in these experiments
were male Sprague–Dawley rats weighing 200–300 g and obtained
from Charles River Laboratories (Wilmington, MA).
Transporter binding and cellular uptake
All described monoamine transporter binding studies as well as
cellular uptake assays were performed in duplicate by CEREP
(Celle l’Evescault, France) and are described subsequently. Further
details of all assays performed may be accessed at the CEREP web
site (www.cerep.com). Cellular-based assays were conducted to
provide complementary in vitro functional measures to comple-
ment the standard transporter assays as well as to provide direct
comparisons of the homologs IPH with MPH and EPH with regard
to dopamine (DA), norepinephrine (NE), and 5-HT uptake in
synaptosomal preparations. The concentration of MPH, EPH, and
IPH utilized in these studies was 10 lM.
Monoamine transporter radioligand binding assays
Dopamine transporter radioligand binding assay. Eval-
uation of the affinity of all three compounds for the human DA
transporter (DAT) in transfected Chinese hamster ovary (CHO) cells
was determined in a validated radioligand binding assay. Cell
membrane homogenates wereincubated for 120 minutes at 4Cwith
0.5 nM [
3
H] GBR 12935 in the absence or presence of each test
compound in a standard buffer solution. Nonspecific binding was
determined in the presence of 10 lM N-(1-[Benzo[b]thien-2-yl-
cyclohexyl]) piperidine (BTCP). Following incubation, the samples
were filtered under vacuum through glass filters and rinsed several
times with ice-cold 50 mM Tris-HCl using a 96 sample cell har-
vester. The filters were then dried and measured for radioactivity
with a scintillation counter (TopCount, Packard) using a liquid
scintillation cocktail (Microscint 0, Packard). The results of DAT
binding experiments as wellas the other transporter assays described
in the following sections were expressed as a percent inhibition of the
control radioligand specific binding. The standard reference com-
pound was BTCP, which was tested in each experiment at several
concentrations in order to obtain a competition curve from which its
inhibitory concentration (IC)
50
was calculated.
Norepinephrine transporter radioligand binding assay. Eval-
uation of the affinity of compounds for the human NE transporter
(NET) in transfected CHO cells was determined in a radioligand
assay analogous to the procedures described for the DAT assay.
Cell membrane homogenates were incubated for 90 minutes at
FIG. 1. Chemical structures of methylphenidate (MPH) and iso-
propylphenidate (IPH).
METABOLISM AND PHARMACOLOGY OF ISOPROPYLPHENIDATE 649
4C with 1 nM [
3
H] nisoxetine in the absence or presence of each
test compound in a standard buffer solution. Nonspecific binding
was determined in the presence of 1 lM of desipramine. Following
incubation, the samples were filtered rapidly under vacuum and
rinsed several times with a buffer solution. The filters were then
dried, and radioactivity counts were obtained. The results are ex-
pressed as percent inhibition of the control radioligand specific
binding. The standard reference compound was the tricyclic anti-
depressant (TCA) protriptyline, which was tested in each experiment
at several concentrations to generate a competition curve from which
its IC
50
was calculated.
Serotonin transporter radioligand binding assay. An
evaluation of the respective compounds was also conducted re-
garding their affinity for the human serotonin 5-HT transporter
(SERT) in transfected CHO cells. Briefly, cell membrane homog-
enates were incubated for 90 minutes at 4C with 2 nM of [
3
H]
imipramine in the absence and presence of each of the assessed
compounds. Nonspecific binding was determined in the presence of
10 lM of imipramine. After incubation, the samples were filtered
rapidly under vacuum and rinsed several times with buffer solution.
The filters were then dried and measured for radioactivity via
scintillation counter. The standard reference compound was the
TCA imipramine, which was tested in each experiment at several
concentrations to generate a competition curve from which its IC
50
was calculated.
Cellular-based monoamine uptake assays
Norepinephrine uptake. The evaluation of the effects of each
compound of interest (MPH, EPH, IPH) on NE uptake utilized
synaptosomes prepared from the rat hypothalamus. These synapto-
somes (100 lg) were incubated for 20 minutes at 37Cwith0.1lCi
[
3
H]norepinephrine in the absence (i.e. control) or presence of the
test compound or the reference compound in a standard buffer so-
lution. Basal control activity was determined by incubating the same
mixture for 20 minutes at 0C in the presence of 10 lM protriptyline
to block the uptake. Following incubation, the samples were filtered,
counted using a scintillation instrument, and the results expressed as
a percent inhibition of the control uptake of [
3
H]norepinephrine. The
standard inhibitory reference compound was the TCA protriptyline,
which was tested in each experiment at several concentrations to
obtain an inhibition curve from which its IC
50
value was calculated.
DA uptake. The evaluation of the effects of the three com-
pounds on DA uptake again utilized synaptosomes, but this time
prepared from the rat striatum. The synaptosomes were incubated for
15 minutes at 37Cwith0.1lCi [
3
H]dopamine in the absence and
presence of the test compound or the reference compound in a buffer
standard buffer solution. Basal control activity was determined by
incubating the same mixture for 15 minutes at 4C in the presence of
1lM GBR12909 to block the uptake. After incubation, the samples
were filtered, counted, and the results expressed as a percent inhi-
bition of the control uptake of [
3
H]dopamine by scintillation count.
The standard inhibitory reference compound was GBR12909, which
was tested in each experiment at several concentrations to obtain an
inhibition curve from which its IC
50
value was calculated.
Serotonin uptake. The assessment of the effects of the three
compounds on 5-HT uptake utilized measures of [
3
H]5-HT incor-
poration into synaptosomes prepared from the rat brain. The syn-
aptosomes were incubated for 15 minutes at 37C with [
3
H]5-HT
(0.2 lCi/mL) in the absence and presence of each of the three as-
sessed compounds or the reference compounds. Following incu-
bation, the samples were filtered, counted using a scintillation
instrument, and the results expressed as a percent inhibition of the
control uptake of [
3
H]5-HT. The standard inhibitory reference
compound was the TCA imipramine, which was tested in each
experiment at several concentrations to obtain an inhibition curve
from which its IC
50
value was calculated.
Determination of relative hydrolytic rates
of MPH, EPH, and IPH
The assessment of relative rates of hydrolysis of the three ester
homologs utilized CES1 transfected cell lines, HLM, and HIM. The
s9 fractions from the HEK293 cells transfected with human
CES1A1 gene were prepared utilizing a method developed in our
laboratory and described previously (Zhu et al. 2008). Hydrolysis
of IPH, EPH, and MPH was assessed by measuring the formation of
the hydrolytic metabolite RA following incubation with cell s9
fractions, HLM, and HIM. Briefly, 50 lL of freshly prepared sub-
strates (racemc IPH, EPH, and MPH) was mixed with 50 lLof
enzymes in 1.5 mL Eppendorf tubes. Both substrates and enzymes
were diluted in Dulbecco’s Phosphate-Buffered Saline (DPBS,
20 mM HEPES, pH 7.4). The final substrate concentration was
1 mM, and the final enzyme concentrations were 0.5 mg/mL,
0.2 mg/mL, and 0.2 mg/mL for the s9 fractions, HLM, and HIM,
respectively. Following incubation at 37C for 60 minutes, the
reaction was terminated by adding 500 lL of methanol. The sam-
ples were then centrifuged at 16,000gat 4C for 5 minutes to
remove precipitated protein. The concentrations of the common
hydrolytic metabolite RA of all three compounds were determined
utilizing a validated high-performance liquid chromatography
(HPLC) method we previously described (Zhu et al. 2008).
CES1 catalyzed transesterification IPH and MPH
in the presence of ethanol
The working solutions of the s9 fractions of CES1 transfected
cells and the substrates (MPH, IPH, and ethanol) were prepared in
DPBS containing 20 mM HEPES (pH 7.4). The reaction was ini-
tiated by mixing 200 lL of the s9 fractions, 100 lL of MPH or IPH,
and 100 lL of ethanol. The final concentrations of the s9 fraction
protein, MPH, IPH, and ethanol were 2 mg/mL, 1mM, 1mM, and
10 mM, respectively. After incubation at 37C for 1h, the reaction
was terminated by adding a fourfold volume of ice-cold methanol.
The samples were then briefly vortexed and centrifuged at 16,000g
for 5 minutes at 4C, and the resulting supernatant was subjected to
HPLC analysis to assess the concentrations of the transesterifica-
tion product EPH from both MPH and IPH, and common metab-
olite RA. The s9 fractions prepared from the vector transfected cells
were included as a negative control for any nonenzymatic hydro-
lysis that might occur. The CES1-mediated metabolism of MPH
and IPH (hydrolysis and transesterification) were estimated by
substracting the amounts of RA and EPH produced in the vector s9
samples from that observed in the CES1 s9 fraction samples.
Locomotor activity measurement in rats
Locomotor-inducing activity of IPH was measured according to
methods previously described for the differential pharmacology of
MPH enantiomers (Patrick et al. 1987). The present study compared
only the IPH homolog versus saline dosing since the other com-
pounds have been extensively investigated in vivo previously
650 MARKOWITZ ET AL.
(Williard et al. 2007). In brief, following an initial 60 minute ha-
bituation period to the activity chamber, racemic IPH (or saline)
was administered intraperitonealy (i.p.) to each animal (n=5 per
active drug group; n=3 per saline group) at a dose of 10 mg/kg to
correspond with 10 mg of racemic MPH (delivering 5 mg of d-
MPH), which was previously determined to produce near maximal
behavioral responses (Patrick et al. 1987). Locomotor activity was
recorded within doughnut-shaped cages with six photocell sensors
equally spaced around a 9 cm runway. Activity counts as assessed
by light beam interruptions were recorded for each animal in in-
crements of 10 minutes over a 2 hour period. Differences in the
cumulative motor activity accounts were assessed by the unpaired,
two tailed Student ttest. The level of significance was set at
p<0.05.
Results
Monoamine transporter binding and cellular
uptake studies
The binding of racemic IPH, MPH, and EPH to the prominent
cellular monoamine transporters DAT, NET, and SERT revealed
that, as a group, binding affinities were greatest for DAT. All
compounds produced significant effects at the DAT, with insig-
nificant differences noted among the three compounds (Table 1).
With regard to NET, as anticipated, MPH exhibited substantial
binding as measured by inhibition of specific control binding,
whereas EPH exhibited approximately half the binding affinity of
MPH, and IPH was the lowest at a value approximately one third
that of MPH at the tested concentration (10 lM). In the case of
SERT, none of the compounds assessed either approached or ex-
ceeded 50% inhibition of control specific binding.
The results of monoamine cellular (i.e., functional) assays are
presented in Table 2. With regard to DA, all three compounds
exhibited significant effects on the uptake of this monoamine, with
little difference observed among the agents. Norepinephrine uptake
studies indicated that MPH and EPH exerted the most effects,
whereas IPH was significantly lower at the concentration of 10 lM.
Finally, 5-HT uptake was not affected to any significant degree by
any of the three assessed compounds.
Hydrolysis of IPH, EPH, and MPH
CES1 is highly expressed in the human liver, and is purported to
account for the majority of hydrolytic activity within the organ,
whereas CES2 is the predominant hydrolase in the human intestine
(Kagan and Hoffman 2008). In the present study, the susceptibility of
MPH, EPH, and IPH to CES1-mediated hydrolysis was investigated
in parallel by incubation of each substrate with the cell s9 fractions of
CES1 transfected cells as well as HLM and HIM preparations. The
results indicated that the catalytic efficiency of CES1-mediated hy-
drolysis upon MPH is approximately 10-fold higher than for IPH
(Fig. 2). When HLM preparations were utilized for incubations, EPH
appeared to be the most vulnerable substrate relative to MPH and
IPH, whereas IPH was the most resistant substrate to HLM (Fig. 2).
Data from the HIM incubation studies where CES2 (but not CES1)
was extensively expressed indicated that EPH was an efficient sub-
strate of CES2. In contrast, catalytic activity of HIM was extremely
low with regard to both MPH and IPH hydrolysis (Fig. 2).
In summary, the present data demonstrate that IPH is a poor
substrate of CES1. Nevertheless, albeit at a markedly lower rate,
IPH was predominantly metabolized (i.e., hydrolyzed) by CES1,
with little to no contribution from CES2, an observation similar to
that which is known to be the case with MPH and established
structure-activity relations for hCES1 and its ester substrates (Ross
and Crow 2007).
Transesterification potential of IPH versus MPH
MPH was efficiently converted to EPH via CES1 catalyzed
transesterification in the presence of ethanol, with a velocity of
423.3 –44.4 pmole/min/mg protein. In contrast, IPH displayed
significant resistance to CES1-mediated transesterification, ex-
hibiting a velocity of 47.5 –3.3 pmole/min/mg protein (Fig. 3).
Furthermore, no EPH formation was observed following the in-
cubation of MPH and IPH with ethanol when CES1 was not
Table 1. Transporter Binding Affinity Study of MPH, EPH, and IPH for the Major Monoamine Transporters
Compound assessed (10lM)
(% inhibition of control specific binding)
Monoamine transporter Ligand
Reference
compound IPH MPH EPH
Dopamine (DAT) (h)[
3
H]GBR-12935 BTCP 97 101 97
Norepinephrine (NET) (h)[
3
H]nisoxetine Protriptyline 27 88 45
Serotonin (SERT) (h)[
3
H]imipramine Imipramine 20 6 29
BTCP, N-[1-(Benzo[b]thien-2-yl-cyclohexyl)]piperidine; [
3
H]GBR-1293, 1-[2-(Diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine; MPH, racemic
(dl) methylphenidate; EPH, racemic ethylphenidate; IPH, racemic isopropylphenidate.
Table 2. Cellular Uptake Studies of MPH, EPH, and IPH
Compound assessed (10lM)
(% inhibition of control uptake)
Cellular uptake
assay substrate Measured parameter IPH MPH EPH
Dopamine uptake [
3
H]DA [
3
H]DA incorporation into synaptosomes 96 90 90
Norepinephrine uptake [
3
H]NE [
3
H]NE incorporation into synaptosomes 62 117 116
Serotonin uptake [
3
H]5-HT [
3
H]5-HT incorporation into synaptosomes 28 35 48
MPH, racemic (dl) methylphenidate; EPH, racemic ethylphenidate; IPH, racemic isopropylphenidate.
METABOLISM AND PHARMACOLOGY OF ISOPROPYLPHENIDATE 651
present. In addition to yielding the novel transesterification me-
tabolite EPH (Markowitz et al. 1999; Patrick et al., 2007), ethanol
also significantly decreased the formation of the hydrolytic product
RA following incubation with MPH.
Rat locomotor activity
The time course of locomotor response over a 120 minute period
following i.p. administration of IPH (10 mg/kg) versus saline is
shown in Figure 4a. As expected, IPH produced robust effects on
locomotor activity in rats when compared with saline injections at
10 minute intervals recorded post-dosing, with a mean of nearly
1200 counts recorded during the initial 10 minute measurement.
Also shown in Figure 4b are the cumulative locomotor activity
counts over the entire 120 minute study period. Racemic IPH ad-
ministration significantly elevated the cumulative locomotor ac-
tivity counts in comparison with saline-injected rats ( p<0.01).
Discussion
The present study provides a systematic assessment of in vitro and
in vivo pharmacological activity, including the catalytic hydrolysis
and ethanolysis rates of CES1-mediated actions using the MPH ester
homolog IPH. Evaluation of the binding affinities of IPH, MPH, and
EPH for DAT, NET, and SERT revealed substantial differences
among these ester congeners. Overall, the transporter binding data
generated for the prototype ‘‘phenidate’’ compound, MPH, was
consistent with the majority of published in vitro reports conducted
using similar methodologies (Markowitz and Patrick 2008). All
tested compounds showed similar and significant binding affinities
for DAT with little interaction with SERT (Table 1). With regard to
NET, IPH exhibited substantially less binding affinity than MPH
which was the highest of the three compounds, and significantly less
than EPH which exhibited modest binding affinity at NET when
these compounds were tested ata single concentration of 10lM. The
results of the complementary cellular functional studies indicated
that all tested agents produce a high degree of DA uptake inhibition
and few differences in these action were noted among the three
(Table 2). Uptake of NE was found to be significantly lower for IPH
relative to both MPH and EPH at the concentration of 10 lM. Fi-
nally, 5-HT uptake was not significantly influenced by any of the
agents, although EPH appeared to exert the greatest effect of the
three compounds under the present experimental conditions.
Taken together, the preliminary pharmacological screening of
monoamine transporter binding and cellular uptake studies suggest
that IPH is primarily a dopaminergic compound with significantly
less noradrenergic activity than MPH or EPH at the concentration
of 10 lM. Although there is substantial evidence of a noradrenergic
component in some cases of ADHD pathophysiology as well as
MPH pharmacotherapy, the prevailing view is that often clinically
significant cardiovascular side effects (increased heart rate and
blood pressures) associated with MPH, as well as amphetamines,
are primarily mediated by the noradrenergic, sympathomimetic
component of these psychostimulant actions. Accordingly, an agent
that appears to have less noradrenergic activity, such as IPH, could
FIG. 2. Hydrolysis of methylphenidate (MPH), ethylphenidate (EPH), and isopropylphenidate (IPH) by carboxylesterase 1 (CES1)
cell s9 fractions (a), human liver microsomes (HLM) (b), and human intestinal microsomes (HIM) (c). The metabolite ritalinic acid
(RA) formed from the hydrolysis of MPH, EPH, and IPH was determined by a validated high-performance liquid chromatography
(HPLC) assay following incubation with the s9 fractions, HLM, and HIM at 37C for 60 minutes. Data are presented as mean –SD of
three to six independent experiments.
FIG. 3. Carboxylesterase 1 (CES1) catalyzed hydrolysis and
transesterification of methylphenidate (MPH) and iso-
propylphenidate (IPH) in the presence of ethanol. The hydrolytic
metabolite ritalinic acid (RA) and the transesterification product
ethylphenidate (EPH) were determined after the incubation of
MPH and IPH with CES1 cell s9 fractions in the presence of
ethanol at 37C for 60 minutes. Data are means from three in-
dependent experiments with error bars representing SD.
652 MARKOWITZ ET AL.
potentially provide an improved safety profile relative to existing
drugs (e.g., MPH) presently employed in ADHD treatment.
The results of enzymatic hydrolysis experiments conducted us-
ing HLM, HIM, and transfected cells overexpressing CES1 also
produced substantially different results for the IPH compound
relative to MPH and EPH. The data generated in the present study
demonstrate that IPH is a relatively poor substrate of CES1, which
is in contrast to what is observed for MPH and EPH (Figure 2). IPH
was predominantly metabolized (i.e., hydrolyzed) by CES1 to RA,
an inactive metabolite common to all three compounds, at a sig-
nificantly lower rate relative to MPH and EPH. There was little to
no contribution to IPH metabolism by CES2.
The pharmacologically active metabolite d-EPH can be formed in
subjects co-ingesting MPH and ethanol, but it is not formed to the
extent that the inactive l-EPH is formed (Patrick et al. 2005b, 2007).
Co-abuse of MPH and alcohol has been well documented, and the
recognized interaction between MPH and ethanol has become an
area of concern in view of the associated elevation of d-MPH by
ethanol (Patrick et al., 2007). Our in vitro incubation studies revealed
that IPH displays great resistance to CES1-mediated transester-
ification, indicating that IPH has significantly reduced interaction
potential when co-ingested with ethanol relative to MPH (Fig. 3).
The mechanism by which IPH is less efficiently hydrolyzed or
transesterified relative to MPH can be speculated as based on the
bulkier isopropyl substituent providing sufficient steric hindrance
in accessing the active site of CES1 relative to MPH. This is con-
sistent with current theory on structural requirements for CES1
substrates, which generally describes molecules esterified by a
small alcohol group and also containing a large acyl group (e.g.,
MPH) (Imai 2006). It is noted, conversely, CES2 tends to show
greater catalytic activity toward structures with larger alcohol
groups and smaller acyl groups (Imai 2006).
FIG. 4. Locomotor activity at 10 minute intervals (a) and during the entire study period (b) in rats intraperitoneally injected with
isopropylphenidate (IPH) (10 mg/kg) or saline. **p<0.01.
METABOLISM AND PHARMACOLOGY OF ISOPROPYLPHENIDATE 653
In the present investigation, i.p. administration of racemic IPH to
rats produced potent locomotor activity effects, as consistent with the
pharmacology of MPH and other DAT-active psychostimulants that
have proven useful in the management of ADHD. These data add to
existing in vitro data suggesting IPH to be a significantly active
central nervous system (CNS) compound at mg/kg doses similar to
those used in assessments of MPH in eliciting classic stimulant-
induced behavioral responses in rodents (Patrick et al. 1987).
Furthermore, when compared with the earlier study by Patrick and
associates (1987) characterizing the pharmacology of MPH isomers,
racemic IPH dosed at 10 mg/kg appeared to produce more potent and
sustained locomotor responses than those of d-MPH dosed at 5 mg/kg.
Conclusions
The present report provides in vitro evidence that IPH displays
pharmacological characteristics of a CNS stimulant with a high
affinity for DAT, as well as potent effects on cellular uptake of
DA. However, unlike MPH, IPH has only minor effects on NE,
which could theoretically provide a more desirable safety/tox-
icity profile. Additionally, a substantially slower rate of enzy-
matic hydrolysis and transesterification via CES1 was noted
relative to MPH, suggesting less potential of drug interaction
with ethanol, a longer duration of action and an extended dosage
interval could be utilized, which is viewed as necessary in the
current treatment of ADHD with MPH. Finally, in vivo studies
demonstrated potent stimulating effects on locomotor activity in
IPH dosed rats similar to that typically observed following MPH
or amphetamine dosing. Forthcoming dose-response and time
course in vivo activity assessments incorporating direct com-
parisons with MPH and expanded in vitro investigation of
monoamine activities (see Patrick et al. 2005b) will further ad-
vance the present preclinical investigations, setting the stage for
potential new drug development.
Clinical Significance
MPH exhibits a relatively short half-life as a result of rapid
hydrolytic metabolism catalyzed by hepatic CES1, which neces-
sitates multiple daily doses, or the use of expensive MR formula-
tions to achieve symptom control throughout the day. Additionally,
significant inter-individual variability in response to MPH therapy
has been consistently documented, which is, at least in part, be-
cause of varied CES1 expression and activity caused by genetic
polymorphisms and environmental factors. The present study re-
vealed that IPH is more resistant to CES1-catalyzed hydrolysis and
transesterification reactions than MPH, indicating that IPH may
offer a longer duration of action and less potential for drug–drug
interactions via CES1, and that it will be the subject of addition
preclinical studies.
Acknowledgment
The authors thank Dr. George R. Breese, University of North
Carolina-Chapel Hill, in whose laboratory the locomotor activity
study was conducted.
Disclosures
Dr. Patrick has been a consultant and expert witness with
Johnson & Johnson, and a consultant for Celgene, Janssen-Ortho,
Novartis, Noven, and UCB. Drs. Markowitz and Zhu have no
conflict of interests to declare.
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Address correspondence to:
John S. Markowitz, PharmD
Department of Pharmacotherapy and Translational Research
University of Florida College of Pharmacy
1600 SW Archer Road, RM PG-23
Gainesville, FL 32610-0486
E-mail: jmarkowitz@cop.ufl.edu
654 MARKOWITZ ET AL.
