Article - Wagner — USA v. Jittaphol (Dkt. 70-7, D. Mass.)

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U.S. District Court for the District of Massachusetts · No. 1:21-cr-10270-MLW · Doc. 70-7 · 2022-04-06 · Docket on CourtListener

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1521-009X/45/7/770–778$25.00
https://doi.org/10.1124/dmd.116.074708
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 45:770–778, July 2017
Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics
Interaction and Transport of Methamphetamine and its Primary
Metabolites by Organic Cation and Multidrug and Toxin
Extrusion Transporters s
David J. Wagner, Jennifer E. Sager, Haichuan Duan, Nina Isoherranen, and Joanne Wang
Department of Pharmaceutics, University of Washington, Seattle, Washington
Received December 17, 2016; accepted April 17, 2017
ABSTRACT
Methamphetamine is one of the most abused illicit drugs with
roughly 1.2 million users in the United States alone. A large portion of
methamphetamine and its metabolites is eliminated by the kidney
with renal clearance larger than glomerular filtration clearance. Yet
the mechanism of active renal secretion is poorly understood.
The goals of this study were to characterize the interaction of
methamphetamine and its major metabolites with organic cation
transporters (OCTs) and multidrug and toxin extrusion (MATE)
transporters and to identify the major transporters involved in the
disposition of methamphetamine and its major metabolites, am-
phetamine and para-hydroxymethamphetamine (p-OHMA). We used
cell lines stably expressing relevant transporters to show that
methamphetamine and its metabolites inhibit human OCTs 1–3
(hOCT1–3) and hMATE1/2-K with the greatest potencies against
hOCT1 and hOCT2. Methamphetamine and amphetamine are sub-
strates of hOCT2, hMATE1, and hMATE2-K, but not hOCT1 and
hOCT3. p-OHMA is transported by hOCT1–3 and hMATE1, but not
hMATE2-K. In contrast, organic anion transporters 1 and 3 do not
interact with or transport these compounds. Methamphetamine and
its metabolites exhibited complex interactions with hOCT1 and
hOCT2, suggesting the existence of multiple binding sites. Our
studies suggest the involvement of the renal OCT2/MATE pathway in
tubular secretion of methamphetamine and its major metabolites
and the potential of drug-drug interactions with substrates or
inhibitors of the OCTs. This information may be considered when
prescribing medications to suspected or known abusers of meth-
amphetamine to mitigate the risk of increased toxicity or reduced
therapeutic efficacy.
Introduction
Methamphetamine is a widely abused illicit drug with approximately
1.2 million reported users in the United States (Volkow, 2013). Also
known as meth, crystal, speed, or ice, methamphetamine is a potent and
highly addictive central nervous stimulant that acts by inhibition and
reversal of neurotransmitter transporters of dopamine, norepinephrine,
and serotonin (Carvalho et al., 2012; Panenka et al., 2013). Illicit
methamphetamine is sold as either a racemic mixture or the d-metham-
phetamine isomer since the dextro isomer is much more psychoactive (de la
Torre et al., 2004). High or repeated doses of methamphetamine can affect
multiple organ systems, leading to profound neurotoxicity, cardiotoxicity,
acute renal failure, and pulmonary toxicity (Volkow et al., 2010; Carvalho
et al., 2012).
Following oral, inhalation, or intranasal administration, methamphet-
amine is well-absorbed into the bloodstream (Harris et al., 2003; Schep
et al., 2010) and is distributed into many organs with the highest uptake
occurring in lungs, liver, brain, and kidneys (Volkow et al., 2010).
Methamphetamine is eliminated by both hepatic metabolism and renal
excretion. In the liver, it is metabolized by the polymorphic enzyme
cytochrome P450 2D6 to the p-hydroxylation metabolite, para-
hydroxymethamphetamine (p-OHMA), and the N-demethylation prod-
uct, amphetamine (Lin et al., 1997; Shima et al., 2008). Both metabolites
have been reported to circulate in plasma of methamphetamine abusers
up to the micromolar range (Shima et al., 2008). Amphetamine is also
highly psychoactive and addictive with a mechanism of action similar to
methamphetamine (Panenka et al., 2013). p-OHMA is not psychoactive
but acts as a cardiovascular agent with hypertensive and adrenergic
effects (Römhild et al., 2003). Concurrent use of CYP2D6 substrates or
inhibitors with methamphetamine and related designer drugs represents
a risk of potential drug interactions leading to toxicity (Wu et al., 1997;
Pritzker et al., 2002; Newton et al., 2005).
Renal excretion is another major elimination pathway for metham-
phetamine and its metabolites. Approximately 37%–54% of metham-
phetamine is recovered unchanged in the urine although more may be
eliminated renally in CYP2D6 poor metabolizers (Kim et al., 2004). The
renal excretion rate of methamphetamine is highly dependent on urinary
pH (Beckett and Rowland, 1965b,c, Cook et al., 1992, 1993). The
fraction unbound (fu) of methamphetamine is about 0.8 (de la Torre et al.,
2004). The reported renal clearance of methamphetamine is highly
variable (e.g., 67–371 ml/min) and much larger than the glomerular
filtration rate in some individuals, suggesting that the drug is actively
This study was supported by the National Institutes of Health National Institute
on Drug Abuse [Grant P01 DA032507] and National Institutes of Health General
Medical Sciences [Grant T32 GM07750].
The content of this paper is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of Health.
https://doi.org/10.1124/dmd.116.074708.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: DDI, drug-drug interaction; HBSS, Hanks’ balanced salt solution; hMATE, human multidrug and toxin extrusion; hOAT, human
organic anion transporter; hOCT, human organic cation transporter; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MATE,
multidrug and toxin extrusion; OCT, organic cation transporter; p-OHMA, para-hydroxymethamphetamine.
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secreted by the kidney (Beckett and Rowland, 1965b,c; Kim et al.,
2004). Positron emission tomography imaging also revealed that metham-
phetamine is highly accumulated in the kidney (Volkow et al., 2010). Both
metabolites (p-OHMA and amphetamine) also undergo urinary excretion
with a possible active secretion component (Shima et al., 2006).
Little is currently known about the involvement of drug transporters in
renal elimination and tissue distribution of methamphetamine and its
metabolites. With a pKa of ;9.9, methamphetamine and its primary
metabolites exist predominantly as protonated cations at physiologic
pH (de la Torre et al., 2004). The reported or calculated log D values
of methamphetamine, amphetamine, and p-OHMA at 7.4 are 20.38,
20.62, and 21.11, respectively (Fowler et al., 2007), suggesting a low
passive membrane diffusion for the protonated species. In rats,
methamphetamine renal clearance was significantly reduced by cimet-
idine, a classic inhibitor of the renal organic cation secretion system
(Kitaichi et al., 2003). In vitro studies have indicated that amphetamine
is an inhibitor of human organic cation transporters (hOCTs) (Amphoux
et al., 2006; Zhu et al., 2010). However, the inhibition potency, substrate
specificity, and transport kinetics of methamphetamine and metabolites
toward renal organic cation uptake and efflux transporters have not been
comprehensively characterized. This information is important for
understanding the mechanisms involved in the disposition and potential
drug-drug interaction (DDI) of methamphetamine. The goals of this
study were to characterize the interaction of methamphetamine and its
major metabolites with hOCT1–3 and human multidrug and toxin
extrusion (hMATE) transporters 1 and 2-K (hMATE1/2-K) and to
identify the major transporters involved in renal secretion of metham-
phetamine, amphetamine, and p-OHMA.
Materials and Methods
Materials. d-Methamphetamine, d-amphetamine, and p-OHMA were pur-
chased from Sigma-Aldrich (St. Louis, MO) and were of analytical grade.
Currently, there is no evidence that organic cation transporters (OCTs) have
stereo-selective interaction with cationic substrates (Yin et al., 2015). We focused
our study on the dextro isoforms of methamphetamine and amphetamine because
they are the psychoactive forms. In all our studies, methamphetamine and
amphetamine refer to the dextro isoforms unless specified otherwise. Metham-
phetamine-D11 and amphetamine-D11 were purchased from Cerilliant Corpora-
tion (Round Rock, TX). [14C]Metformin (98 mCi/mmol) was purchased from
Moravek Biochemicals, Inc. (Brea, CA). [3H]Estrone sulfate (50 Ci/mmol), and
[3H]para-aminohippurate (3 Ci/mmol) were purchased from American Radiola-
beled Chemicals, Inc. (St. Louis, MO). Optima grade acetonitrile, water, and
formic acid were purchased from Fisher Scientific (Waltham, MA). Cell culture
media and reagents were purchased from Invitrogen (Carlsbad, CA). All other
chemicals were commercially available and of analytical grade or higher.
Uptake and Inhibition Assays in HEK293 Cells. Flp-in HEK293 cells
stably expressing hOCT1, hOCT2, hOCT3, hMATE1, hMATE2-K, human
organic anion transporter (hOAT)1, and hOAT3 were previously generated in our
laboratory (Duan and Wang, 2010; Duan et al., 2015; Yin et al., 2015). The cells
were cultured in high glucose Dulbecco’s modified Eagle’s medium with 10%
fetal bovine serum, 1 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml
streptomycin, and 150 mg/ml hygromycin B supplementation at 37C with 5%
CO2 and high humidity. All cell culture plastic surfaces were coated with 0.1%
poly-D-lysine to improve cell attachment. Uptake and inhibition assays were
performed as previously described with modification for analysis of metham-
phetamine and its metabolites by liquid chromatography–tandem mass spectrom-
etry (LC-MS/MS) (Duan and Wang, 2010; Duan et al., 2015; Yin et al., 2015).
Briefly, cells were seeded in 96-well plates at 100,000 cells/well and grown
overnight. Prior to incubation initiation, cells were washed with prewarmed
Hanks’ balanced salt solution (HBSS) and allowed to acclimate for 10 minutes
at 37C or preincubated with HBSS containing 30 mM ammonium chloride for
20 minutes for multidrug and toxin extrusion (MATE) experiments to acidify the
intracellular compartment and drive MATE uptake (Tanihara et al., 2007). Media
were removed and incubation initiated by addition of 100 ml of HBSS at pH 7.4
containing a substrate with or without inhibitor. Uptake was stopped by removal
of media and washing the cells three times with ice cold HBSS. Cells were either
lysed with 100 ml of 1 M NaOH and neutralized with 100 ml of 1 M HCl for
incubations containing a radiolabeled substrate for measurement by liquid
scintillation counting (Tri-Carb B3110TR; PerkinElmer, Waltham, MA) or
permeabilized with 100 ml of methanol containing 100 nM stable labeled internal
standard for analysis by LC-MS/MS. Protein content in the lysate in each well was
measured by the BCA Protein Assay Kit (Pierce Chemical, Rockford, IL) and the
uptake in cells was normalized to their total protein concentrations. The inhibitory
effect of methamphetamine, amphetamine, and p-OHMA on hOCT1, hOCT2,
hOCT3, hMATE1, and hMATE2-K was assessed in transporter-expressing
HEK293 cells using [14C]metformin, a well-established and clinically relevant
probe substrate for these transporters (European Medicines Agency, 2012; Food and
Drug Administration, 2012; Hillgren et al., 2013). The concentration of metformin
in the inhibition experiments (11 mM, 1 mCi/ml) was selected to be much lower than
its Km values (780–1500 mM) for the transporters tested (Koepsell et al., 2007;
Tanihara et al., 2007). Inhibition and kinetic experiments were performed during
the initial rate period using a short incubation time as specified in the legends for
Fig. 1, Fig. 2, Fig. 3, Fig. 4, and Fig. 5. Transport experiments were performed
in triplicate and repeated three times independently. Uptake was performed in
both empty vector- and transporter-transfected cells; and transporter-specific
uptake was calculated by subtracting uptake in vector-transfected cells.
LC-MS/MS Analysis of Methamphetamine and its Metabolites. Meth-
amphetamine, amphetamine, and p-OHMA levels were quantified using an
LC-MS/MS system consisting of an API 4500 triple quadrupole mass spectrom-
eter (AB-Sciex, Foster City, CA) coupled with an LC-20AD ultra-fast liquid
chromatography system (Shimadzu Co., Kyoto, Japan). The Turbo Ion Spray
interface was operated in positive ion mode. Ten microliters of cell lysate was
injected onto an Agilent Eclipse Plus C18 column (1.8 mm; 4.6 
 50 mm)
(Agilent, Santa Clara, CA) running with an isocratic method consisting of
0.28 ml/min 0.2% formic acid in water and 0.12 ml/min acetonitrile. Mass
transitions (m/z) were 150 →119, 136 →91, 166 →135, 161 →97, and 147 →
98 for methamphetamine, amphetamine, p-OHMA, methamphetamine-d11, and
amphetamine-d11, respectively. Data were analyzed using Analyst software
version 1.6.2 (AB Sciex). Assay accuracy and precision were within 15% (20%
for the lower limit of quantification).
Data Analysis. Transport experiments were performed in triplicate and
repeated three times independently. Data representation and replicates with
specific n numbers are detailed in each figure legend. The transport kinetics were
fitted using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA) for
inhibitory interactions and uptake kinetics of hOCT1-3. WinNonLin Phoenix
6.4.0 (Certara, Princeton, NJ) was used for fitting hMATE apparent transport
kinetics. The IC50 values were calculated by fitting the log inhibitor concentration
versus the transporter-specific uptake normalized to the vehicle control using the
following equation:
v ¼ Bottom þ
Top 2 Bottom
1 þ 10ðlogIC50 2 IÞ*H
ð1Þ
where v is the rate of uptake in the presence of the inhibitor; Bottom is the residual
baseline value; Top is the rate of uptake in the absence of inhibitor; I is the
inhibitor concentration; and H is the Hill coefficient. Two-site inhibition data were
fitted using the following equation:
v ¼ Bottom þ ðTop 2 BottomÞ


Fraction1
1 þ 10I 2 logEC50;1 þ 1 2 Fraction1
1 þ 10I 2 logEC50;2

ð2Þ
One- and two-site inhibition equations were compared by an extra sum-of-squares
F test using the data from all three independent experiments modeled
simultaneously. Apparent hMATE1/2-K, Km, and Vmax values were obtained by
simultaneously fitting the data to the Michaelis-Menten equation with a passive
diffusion component in transporter-transfected cells and only the passive diffusion
component in vector-transfected cells (Brouwer et al., 2013):
v ¼ Vmax*S
Km þ S þ Pdiff*S
ð3Þ
where V is the velocity of uptake; Vmax is the maximum velocity of uptake; S is the
substrate concentration; Km is the Michaelis-Menten constant; and Pdif is the
Amphetamine Renal Secretion Involves hOCT2/hMATEs
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nonsaturable passive diffusion rate constant. The sigmoidal saturation kinetics of
p-OHMA hOCT2 transport were obtained by fitting transporter-mediated uptake
to the Michaelis-Menten equation with a Hill slope for the substrate concentration
and half-maximal transport concentration (K1/2 in place of Km) after inspection of
the Eadie-Hofstee plot (Copeland, 2000):
v ¼
Vmax*SH
K1=2H þ SH
ð4Þ
Amphetamine hOCT2 specific uptake kinetics were fit to a biphasic Michaelis-
Menten equation:
v ¼ Vmax1*S
Km1 þ S þ Vmax2*S
Km2 þ S
ð5Þ
Results
Inhibitory Effect of Methamphetamine and its Metabolites on
hOCT1–3 and hMATE1/2-K. The transport activities of hOCT1,
hOCT2, hOCT3, hMATE1, and hMATE2-K in the Flp-in HEK293
expression systems were first confirmed with metformin uptake in the
presence or absence of the prototypical inhibitor cimetidine (Supple-
mental Fig. 1). Methamphetamine, amphetamine, and p-OHMA
inhibited metformin uptake by hOCT1–3 and hMATE1/2-K in a
concentration-dependent manner (Fig. 1; Supplemental Fig. 2).
The IC50 values are summarized in Table 1. Methamphetamine and
amphetamine were 4- to 20-fold more potent for hOCT1 and hOCT2
than for hOCT3, hMATE1, and hMATE2-K, with hOCT2 showing the
greatest sensitivity to both psychostimulants (hOCT2 IC50 values of 15.0 6
6.81 and 20.3 6 16.9 mM, respectively). p-OHMA was a more potent
inhibitor of hOCT1 than other transporters. Addition of the 4-hydroxyl
group to the aromatic phenyl ring (p-OHMA) greatly increased binding
to hOCT3 but decreased its potency toward hOCT2 compared with
methamphetamine. Interestingly, the Hill slope of methamphetamine
and amphetamine inhibition of hOCT1 and hOCT2 was approximately
0.5 (Table 1). Conversely, the Hill slope of p-OHMA against hOCT2
was approximately 1.5. hMATE2-K also had steep Hill slopes ranging
between 1.6 and 1.9 for methamphetamine and its metabolites. These
Hill slopes suggest more complex interactions than simple competitive
inhibition may be occurring with these transporters.
Inspection of methamphetamine and amphetamine dose-dependent
inhibition of hOCT1 and hOCT2 revealed biphasic inhibition charac-
teristics (Supplemental Fig. 3). A two-site inhibition model was
compared with a one-site inhibition model using an extra sum-of-
squares F test and a cutoff significance value of 0.05 by simultaneously
fitting the data from three independent experiments each run in triplicate.
The two-binding site model fit significantly better for methamphetamine
inhibition of hOCT1 and hOCT2 as well as amphetamine inhibition of
hOCT2 (P , 0.0001) but not hOCT1 (Table 2). The high-affinity EC50
values were in the low micromolar range (0.72–5.29 mM), while the
apparent low-affinity interactions appeared to be in the high micromolar
range (58.2–400 mM) for these transporters.
Uptake of Methamphetamine and Metabolites by hOCT1-3,
hMATE1, and hMATE2-K. The substrate potential of methamphet-
amine, amphetamine, and p-OHMA was assessed by measuring the
uptake of these compounds (1 mM) in control cells and transporter-
expressing cells (Fig. 2). After 5-minute incubation, methamphetamine
and amphetamine showed approximately 2-fold greater uptake in cells
expressing hOCT2, hMATE1, and hMATE2-K. p-OHMA accumulated
extensively in hOCT1, hOCT2, and hOCT3, and to a lesser degree in
hMATE1, but did not accumulate at all in hMATE2-K-transfected cells
Fig. 1. Inhibition by methamphetamine, amphetamine, and p-OHMA of hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K. Uptake of [14C]metformin (11 mM) in the
absence and presence of inhibitor was measured in both transporter-expressing and control human embryonic kidney cells. Transporter-specific uptake was obtained by
subtracting the uptake in vector-transfected cells from the uptake in transporter-expressing cells. Incubations were performed at 2, 0.5, 2, 5, and 0.5 minutes for of hOCT1
(A), hOCT2 (B), hOCT3 (C), hMATE1 (D), and hMATE2-K (E), respectively, which are within the linear initial rate of uptake. Activity in the absence of inhibitor (100%)
corresponds to 28.2, 373, 60.2, 52.9, and 100 pmol/min/mg protein for hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K, respectively. Each data point represents the
mean 6 S.D. from one representative experiment in triplicate. Curves from two additional independent repeats are displayed in Supplemental Fig. 2. The IC50 values shown
in Table 1 are mean 6 S.D. of the IC50 values from the three independent experiments.
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when compared with control cells (Fig. 2). These data suggest that renal
secretion of methamphetamine and its primary metabolites may involve
the hOCT2/hMATE pathway.
Interaction of Methamphetamine and Metabolites with Renal
hOAT1 and hOAT3. WhilehOCT2andhOAT1/3mediaterenalsecretion
of organic cations and organic anions, respectively, some substrate and
inhibitor overlap between hOCT and hOATs has been reported (Lai et al.,
2010). We then investigated if methamphetamine and metabolites interact
with hOAT1 and hOAT3 (Fig. 3, A and B). hOAT1- and hOAT3-mediated
para-aminohippurate or estrone sulfate uptake was completely suppressed
by the reference inhibitor probenecid. In contrast, methamphetamine and
amphetamine showed no inhibitory effect on hOAT1 or hOAT3 at 1 mM.
Only p-OHMA showed significant inhibition of hOAT1 and hOAT3 at
1 mM with 47 6 17 and 38 6 28% inhibition, respectively. Uptake studies
showed that none of the compounds were substrates of hOAT1 or hOAT3
(Fig. 3, C–E), suggesting a primary role of the hOCT2/hMATE pathway
in active renal secretion of these compounds.
Methamphetamine and Metabolites Uptake Kinetics by Cation
Transporters. The kinetics of hOCT1, hOCT2, hOCT3, hMATE1, and
hMATE2-K in transporting methamphetamine, amphetamine, and
p-OHMA was assessed by determining concentration-dependent trans-
port rates. The specific uptake was obtained for hOCT1–3 by subtracting
uptake in the control cells and Eadie-Hofstee plots were evaluated to
identify the type of interaction. Due to high passive diffusion at high
concentrations, methamphetamine kinetics can only be accurately
determined at a low concentration range (0–15 mM). Within this range,
methamphetamine displayed saturable kinetics with a Km value of
2.09 6 0.88 mM (Fig. 4A; Supplemental Fig. 4; Table 3), which is very
close to the high-affinity, half-inhibitory concentration (1.21 6 0.19
mM) observed in Supplemental Fig. 2B. For amphetamine, we were able
to cover a wider concentration range (0–600 mM). As shown in Fig. 4B,
biphasic transport kinetics were observed, and the Km values for the
apparent high- and low-affinity binding sites were determined to be
0.830 6 0.55 and 534 6 350 mM, respectively. Interestingly, p-OHMA
displayed sigmoidal kinetics for hOCT2 as clearly revealed by the Eadie-
Hofstee plot (Fig. 4D). The p-OHMA hOCT2 half-maximal transport
concentration (K1/2) is 31.8 6 9.3 mM and the Hill slope is 1.64 6 0.15.
For p-OHMA transport by hOCT1 and hOCT3, no apparent sigmodal
or biphasic pattern was observable in the Eadie-Hofstee plot. Fitting
to a standard Michaelis-Menten equation yielded apparent Km values of
14.5 6 8.7 and 53.3 6 6.2 mM for hOCT1 and hOCT3, respectively
(Fig. 4; Supplemental Fig. 4; Table 4).
hMATE1/2-K transport studies were conducted after intracellular
acidification to provide an outwardly directed proton gradient to drive
substrate uptake because the MATE transporters function as proton/organic
cation exchangers. Under this condition, we observed very high uptake of
methamphetamine and metabolites in vector-transfected cells, likely due to
a pH effect on passive diffusion. The high uptake in vector-transfected cells
makes it difficult to discern transporter-specific uptake at high substrate
concentrations. Therefore, we fitted the concentration-dependent uptake in
transporter-expressing cells to a Michaelis-Menten equation with a non-
saturable passive diffusion component (Fig. 5; Supplemental Fig. 5;
Table 4). This simultaneous fitting of both carrier- and noncarrier-mediated
uptake allowed for an estimate of the apparent Km values for hMATE1/2-K
in the presence of a high-passive permeability component (Table 4).
Discussion
In spite of the major role of renal clearance in methamphetamine
disposition, the molecular mechanisms underlying the tubular secretion
TABLE 1
IC50 values of methamphetamine, amphetamine, and p-OHMA for hOCT1–3 and hMATE1/2-K determined by
one-binding site fitting
Results represent mean 6 S.D. of three independent experiments each run in triplicate.
Transporter
One-Binding Site
Methamphetamine
Amphetamine
p-OHMA
IC50
Hill Slope
IC50
Hill Slope
IC50
Hill Slope
mM
mM
mM
hOCT1
21.1 6 8.8
0.55 6 0.02
96.7 6 37
0.61 6 0.28
12.0 6 3.4
1.17 6 0.14
hOCT2
15.0 6 6.8
0.56 6 0.07
20.3 6 16.9
0.44 6 0.1
83.8 6 22.3
1.55 6 0.32
hOCT3
300 6 139
1.42 6 0.68
363 6 56.4
1.1 6 0.2
44.4 6 25.5
1.1 6 0.26
hMATE1
107 6 38
0.79 6 0.09
94.0 6 25.3
0.89 6 0.2
59.1 6 14.3
0.84 6 0.12
hMATE2-K
84.3 6 12.9
1.63 6 0.14
158 6 48
1.9 6 0.6
234 6 86.8
1.21 6 0.11
TABLE 2
EC50 values of methamphetamine and amphetamine for hOCT1 and hOCT2 determined by two-binding site fitting
The EC50 values were obtained by fitting inhibition data in Fig. 1 and Supplemental Fig. 2 using eq. 2 described in Materials and
Methods with the fit shown in Supplemental Fig. 3. The last column lists the P values obtained by comparing eqs. 1 and 2 with an extra
sum-of-squares F test. Results represent mean 6 S.D. of three independent experiments each run in triplicate.
Inhibitor
Two-Binding Site
Two-Binding versus One-Binding Site
Transporter
EC50 Value
High Affinity
Low Affinity
mM
mM
Methamphetamine
hOCT1
5.29 6 0.66
400 6 229
P , 0.0001
hOCT2
1.21 6 0.19
58.2 6 23.4
P , 0.0001
Amphetamine
hOCT1
NA
NA
P = 0.55
hOCT2
0.72 6 0.29
145 6 104
P , 0.0001
NA, not applicable.
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of methamphetamine and its major metabolites had not been fully
elucidated (Caldwell et al., 1972; Kim et al., 2004; Carvalho et al.,
2012). Here, we showed methamphetamine and its metabolites interact
with hOCT1–3 and hMATE1/2-K at clinically relevant concentrations
(Melega et al., 2007; Shima et al., 2009). We further demonstrated that
methamphetamine and amphetamine are substrates of hOCT2,
hMATE1, and hMATE2-K, but not hOCT1or hOCT3. Interestingly,
p-OHMA was a substrate of hOCT1–3 as well as hMATE1, but not
hMATE2-K. Methamphetamine and its metabolites do not interact with
renal hOAT1 or hOAT3. Methamphetamine and its metabolites
demonstrated complex inhibitory and substrate kinetics with hOCT2.
Our data suggest that the hOCT2/hMATE pathway is involved in renal
secretion of methamphetamine and its metabolites, and that inhibition of
hOCT2 and hMATEs by methamphetamine may lead to potential DDIs
for drugs that are eliminated by the hOCT2/hMATE pathway.
The importance of renal elimination of methamphetamine has long
been known; however, the exact molecular mechanisms of renal
secretion had not been identified (Beckett and Rowland, 1965c;
Caldwell et al., 1972). Here, we identified the hOCT2/hMATE pathway
as being involved in the active renal secretion of methamphetamine and
Fig. 2. Uptake of 1 mM methamphetamine, amphetamine, and p-OHMA by hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K. Uptake was measured after 5-minute
incubation at 37C. Data are illustrated as the mean 6 S.D. from three independent experiments performed in triplicate. Uptake in transporter-expressing cells was compared
with that in control cells (**P , 0.01; ***P , 0.001).
Fig. 3. Interactions of methamphetamine, amphetamine, and p-OHMA with renal hOAT1 and hOAT3. Effect of methamphetamine, amphetamine, and p-OHMA on para-
aminohippurate (PAH) (1 mM) uptake by hOAT1 (A) and estrone sulfate (0.06 mM) uptake by hOAT3 (B) was measured at 1 minute after incubation at 37C. Transporter-
specific uptake was obtained by subtracting the uptake in vector-transfected cells from the uptake in transporter-expressing cells. The classic organic anion transporter
inhibitor probenecid was used as the control. Activity in the absence of an inhibitor (100%) corresponded to 36.6 and 2.1 pmol/min/mg protein for PAH and estrone sulfate
uptake, respectively. Uptake of methamphetamine (C), amphetamine (D), and p-OHMA (E) by hOAT1 and hOAT3 was measured after 5-minute incubation at 37C. Data
are illustrated as the mean 6 S.D. from three independent experiments performed in triplicate. Uptake in transporter-expressing cells was compared with that in control cells
(**P , 0.01; ***P , 0.001).
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amphetamine. Methamphetamine may be a potential victim of DDIs by
inhibitors (e.g., cimetidine, zalcitabine, dolutegravir) of OCT2 and/or
MATE transporters, which could reduce its renal clearance and increase
exposure (Jung et al., 2008; Reese et al., 2013). Located at the apical
membrane of renal proximal tubule cells, the MATE transporters
function as proton/organic cation exchangers, which rely on the
transmembrane proton gradient to drive organic cation secretion into the
urine (Otsuka et al., 2005). The pH dependence of methamphetamine
and amphetamine renal excretion rates has long been known, where
urine acidification increases renal excretion while urine alkalization has
an opposite effect (Beckett and Rowland, 1965a,b,c). The effect of
urinary pH on methamphetamine or amphetamine renal excretion has
been mostly attributed to the pH effect on ionization and membrane
partitioning, which affects tubular reabsorption of these weak bases
Fig. 4. Methamphetamine, amphetamine, and p-OHMA uptake kinetics
by hOCTs. Concentration-dependent uptake of substrate was measured in
both transporter-expressing and control cells at 37C after 1-minute
incubations. Transporter-specific uptake was obtained by subtracting the
uptake in vector-transfected cells from the uptake in transporter-
expressing cells. Panels display saturation curves (v vs. s) and Eadie-
Hofstee transformations (v vs. v/s) for the kinetic data. Based on the
Eadie-Hofstee plots, the kinetics for hOCT2-mediated methamphetamine
transport (A) and hOCT- and hOCT3-mediated p-OHMA transport (C and
E) were fitted with the standard Michaelis-Menten equation. hOCT2-
mediated amphetamine transport (B) was fitted to a biphasic Michaelis-
Menten equation (eq. 5). hOCT2-mediated p-OHMA transport (D) was
fitted to the Michaelis-Menten equation with a Hill slope (eq. 4). Each
data point represents the mean 6 S.D. from one representative experiment
in triplicate. Curves from two additional independent repeats are displayed
in Supplemental Fig. 4. The kinetic parameters in Table 3 are mean 6
S.D. of the values from three independent experiments.
Amphetamine Renal Secretion Involves hOCT2/hMATEs
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(Beckett and Rowland, 1965a,b,c). Here, our data suggest that
renal secretion of methamphetamine and amphetamine involves the
pH-dependent MATE transporters. Therefore, the increased excre-
tion rates observed with acidic urine could be due to a combined
effect of acidic pH in reducing partition-mediated reabsorption
along with increasing MATE-mediated tubular secretion.
The liver is the major site of methamphetamine metabolism. In-
triguingly, methamphetamine and amphetamine were not substrates
of hOCT1, the major OCT isoform responsible for hepatic uptake
of organic cations. Therefore, hepatic uptake of methamphetamine
and amphetamine may be facilitated by other transporters yet to be
identified or be driven by passive diffusion. Interestingly, p-OHMA
was transported by hOCT1, suggesting that the para-hydroxyl group
may be important for OCT1 transport selectivity of substituted
amphetamines. hOCT1 may thus be involved in hepatic transport of
p-OHMA.
In this study, we used metformin as the probe substrate because it
is recommended as an in vitro and in vivo probe substrate for evaluat-
ing hOCT2, hMATE1, and hMATE2-K interaction studies by the
International Transporter Consortium (Hillgren et al., 2013), Food and
Drug Administration (2012), and European Medicines Agency (2012).
Substrate-dependent inhibition has previously been demonstrated for
OCTs with a number of substrates and inhibitors (Moaddel et al., 2005;
Gorbunov et al., 2008; Minuesa et al., 2009; Hacker et al., 2015; Yin
et al., 2016). For example, inhibition potencies of several clinical drugs
toward hOCT2 were reported to be approximately 10-fold more potent
when atenolol was used as the substrate compared with metformin (Yin
et al., 2016). Due to the observed complex interactions, the apparent
inhibition potencies of amphetamines may be highly dependent on the
substrate. As an illicit drug, abusers may use methamphetamine while
taking prescription medications. Testing the inhibition potencies with
the specific hOCT substrate drugs used by methamphetamine abusers
may be warranted to determine the likelihood of clinically relevant
interactions.
Particularly high levels of methamphetamine abuse are reported in
individuals receiving treatment of human immunodeficiency virus and
hepatitis who may be receiving multiple medications for treatment
(Panenka et al., 2013; Volkow, 2013; Bracchi et al., 2015). Importantly,
numerous antiretrovirals (e.g., lamivudine, zalcitabine) interact with
OCTs and rely on these transporters for cellular uptake into human
immunodeficiency virus–infected CD4 cells (Zhou et al., 2006; Jung
et al., 2008). Methamphetamine and its metabolites inhibited the active
transport of the probe substrate metformin by hOCT1–3 and hMA-
TE1/2-K (Table 1) within the concentration range reported in abusers of
methamphetamine (Melega et al., 2007; Shima et al., 2009). The free
plasma concentrations of methamphetamine in some abusers have been
reported to be in the tens of micromolar range and even 130 mM in one
individual, indicating the potential to reach inhibitory concentrations of
hOCT1–3 and hMATE1/2-K in vivo (de la Torre et al., 2004; Shima
et al., 2008). Inhibition of hOCT1 and hOCT2 may reduce intracellular
levels of some antiretrovirals in human immunodeficiency virus–
infected CD4 cells, reducing their effective concentration and efficacy
at the site of action (Minuesa et al., 2008, 2009; Wagner et al., 2016).
These potential distributional DDIs are of particular concern with
drugs of abuse since patients may not be willing to reveal their use of
illicit drugs.
Methamphetamine and its metabolites demonstrated complex inter-
actions with hOCT1 and hOCT2, suggesting they may have multiple
binding sites on these transporters. The structural basis of the complex
kinetic interactions between amphetamines and hOCTs is currently
unclear since the crystal structures of these transporters have not been
obtained. Amphetamine showed biphasic hOCT2 uptake kinetics with
an apparent high affinity (0.830 6 0.55 mM) and low affinity (534 6
350 mM) aligning with the observed high- and low-affinity inhibitory
TABLE 3
Kinetic parameters of methamphetamine and metabolites determined from modeling the data in Fig. 4 and Supplemental
Fig. 4
Models were chosen based on examination of Eadie-Hofstee plots. Methamphetamine was fit to a standard Michaelis-Menten equation.
Amphetamine uptake kinetics was fit to a biphasic Michaelis-Menten equation (eq. 5). p-OHMA hOCT1- and hOCT3-mediated transport
were fit to a standard Michaelis-Menten equation. Sigmoidal kinetics of hOCT2-mediated p-OHMA transport was obtained by fitting
transporter-mediated uptake to the Michaelis-Menten equation with a Hill slope (eq. 5) for the substrate concentration and half-maximal
transport concentration (K1/2 in place of Km). Results represent mean 6 S.D. of three independent experiments each run in triplicate.
Compound
Transporter
Km1
Vmax1
Km2
Vmax2
mM
pmol/mg/min
mM
pmol/mg/min
Methamphetamine
hOCT2
2.09 6 0.88
49.7 6 12.2
ND
ND
Amphetamine
hOCT2
0.830 6 0.55
34.6 6 23.7
534 6 350
853 6 474
p-OHMA
hOCT1
14.5 6 8.7
312 6 163
NA
NA
hOCT2
K1/2: 31.8 6 9.3; H: 1.64 6 0.15
1780 6 718
NA
NA
hOCT3
53.3 6 6.2
1290 6 830
NA
NA
NA, not applicable; ND, not determined (due to high diffusion).
TABLE 4
Apparent kinetic transport parameters for methamphetamine, amphetamine, and p-OHMA for hMATE1 and hMATE2-K
from simultaneously modeling active and passive accumulation
Results represent Mean 6 S.D. of three independent experiments each run in triplicate (Fig. 5; Supplemental Fig. 5).
Transporter
Methamphetamine
Amphetamine
p-OHMA
Km
Vmax
Km
Vmax
Km
Vmax
mM
pmol/mg/min
mM
pmol/mg/min
mM
pmol/mg/min
hMATE1
20.6 6 4.5
86.6 6 54
14.1 6 4.9
238 6 141
49.8 6 26
257 6 190
hMATE2-K
18.1 6 11
97.7 6 25.3
16.4 6 12.2
92.9 6 8.5
NA
NA
NA, not applicable.
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interactions. Conversely, p-OHMA had a relatively steep hOCT2
inhibition Hill slope (1.55 6 0.32) and demonstrated sigmoidal uptake
kinetics (Fig. 4; Table 3). Sigmoidal uptake kinetics may be character-
istic of homotropic activation (Segel, 1976; Atkins, 2005). Both a large
binding pocket in the outward facing cleft allowing for spatially distinct
binding as well as a distal allosteric binding site have been proposed for
OCTs based on kinetics and biochemical analyses (Gorboulev et al.,
1999; Harper and Wright, 2013; Koepsell, 2015). The possible distal
allosteric binding site has demonstrated very high-affinity interactions
with no transport observed in the concentration range (e.g., 6-41 pM for
1-methyl-4-phenylpyridinium) (Moaddel et al., 2005; Gorbunov et al.,
2008; Minuesa et al., 2009; Koepsell, 2015). Binding within the
transportable region is generally a lower-affinity interaction (e.g.,
0.87-12.3 mM for 1-methyl-4-phenylpyridinium) with known sub-
strates (Moaddel et al., 2005; Gorbunov et al., 2008; Minuesa et al.,
2009; Koepsell, 2015). Recent developments have also suggested
the possibility of simultaneous binding of two substrates within the
transport site (Harper and Wright, 2013; Koepsell, 2015). The ap-
parent affinities observed for methamphetamine and metabolites
across their transportable concentration ranges suggest that both
binding sites may reside within the transport region. More studies
are needed to understand the complex kinetic behaviors of the OCTs
and their structure-function relationships.
In summary, our study determined the molecular mechanisms in-
volved in transport and disposition of methamphetamine and its meta-
bolites. Moreover, our studies showed that methamphetamine has the
potential to inhibit hOCT and hMATE transporters at clinically relevant
concentrations. Finally, we identified complex kinetic interactions
between amphetamines and hOCT2. Our findings provide useful
information that may be considered when prescribing medications to
methamphetamine users to mitigate the risk of DDIs that may potentially
compromise therapeutic efficacy and drug safety.
Acknowledgments
The authors thank Dr. Willian Atkins and Dr. Kent Kunze for thoughtful
discussions and insights.
Authorship Contributions
Participated in research design: Wagner, Isoherranen, Wang.
Conducted experiments: Wagner, Sager, Duan.
Contributed new reagents or analytic tools: Isoherranen.
Performed data analysis: Wagner, Wang.
Wrote or contributed to the writing of the manuscript: Wagner, Sager,
Isoherranen, Wang.
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