How Is Crystal Meth Filtered

• Periodical List
• Drug Metab Dispos
• PMC5478906

Drug Metab Dispos. 2017 Jul; 45(7): 770–778.

Interaction and Transport of Methamphetamine and its Primary Metabolites by Organic Cation and Multidrug and Toxin Extrusion Transporters

Received 2016 Dec 17; Accepted 2017 Apr 17.

Abstract

Methamphetamine is one of the most abused illicit drugs with roughly 1.two 1000000 users in the United States lone. A big portion of methamphetamine and its metabolites is eliminated by the kidney with renal clearance larger than glomerular filtration clearance. Yet the machinery of active renal secretion is poorly understood. The goals of this report 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, amphetamine and para-hydroxymethamphetamine (p-OHMA). We used cell lines stably expressing relevant transporters to show that methamphetamine and its metabolites inhibit human being OCTs 1–three (hOCT1–three) and hMATE1/2-K with the greatest potencies confronting hOCT1 and hOCT2. Methamphetamine and amphetamine are substrates of hOCT2, hMATE1, and hMATE2-1000, simply not hOCT1 and hOCT3. p-OHMA is transported by hOCT1–3 and hMATE1, but not hMATE2-Thousand. In contrast, organic anion transporters 1 and iii do non 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 data may be considered when prescribing medications to suspected or known abusers of methamphetamine to mitigate the adventure of increased toxicity or reduced therapeutic efficacy.

Introduction

Methamphetamine is a widely abused illicit drug with approximately one.2 meg reported users in the United States (Volkow, 2013). Also known as meth, crystal, speed, or ice, methamphetamine is a strong 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-methamphetamine isomer since the dextro isomer is much more psychoactive (de la Torre et al., 2004). High or repeated doses of methamphetamine tin can bear upon multiple organ systems, leading to profound neurotoxicity, cardiotoxicity, acute renal failure, and pulmonary toxicity (Volkow et al., 2010; Carvalho et al., 2012).

Post-obit oral, inhalation, or intranasal administration, methamphetamine is well-captivated 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 Due north-demethylation product, 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 as well highly psychoactive and addictive with a mechanism of activity similar to methamphetamine (Panenka et al., 2013). p-OHMA is not psychoactive but acts as a cardiovascular amanuensis with hypertensive and adrenergic effects (Römhild et al., 2003). Concurrent utilise of CYP2D6 substrates or inhibitors with methamphetamine and related designer drugs represents a take a chance 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 methamphetamine and its metabolites. Approximately 37%–54% of methamphetamine is recovered unchanged in the urine although more may be eliminated renally in CYP2D6 poor metabolizers (Kim et al., 2004). The renal excretion charge per unit of methamphetamine is highly dependent on urinary pH (Beckett and Rowland, 1965b,c, Cook et al., 1992, 1993). The fraction unbound (f _u) of methamphetamine is about 0.8 (de la Torre et al., 2004). The reported renal clearance of methamphetamine is highly variable (east.g., 67–371 ml/min) and much larger than the glomerular filtration charge per unit in some individuals, suggesting that the drug is actively secreted by the kidney (Beckett and Rowland, 1965b,c; Kim et al., 2004). Positron emission tomography imaging also revealed that methamphetamine 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 pGa 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 seven.4 are −0.38, −0.62, and −one.11, respectively (Fowler et al., 2007), suggesting a low passive membrane improvidence for the protonated species. In rats, methamphetamine renal clearance was significantly reduced by cimetidine, a archetype 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 authorization, substrate specificity, and transport kinetics of methamphetamine and metabolites toward renal organic cation uptake and efflux transporters accept 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 ane and ii-K (hMATE1/2-Yard) and to place the major transporters involved in renal secretion of methamphetamine, amphetamine, and p-OHMA.

Materials and Methods

Materials.

d-Methamphetamine, d-amphetamine, and p-OHMA were purchased from Sigma-Aldrich (St. Louis, MO) and were of analytical grade. Currently, there is no evidence that organic cation transporters (OCTs) take 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. Methamphetamine-D₁₁ and amphetamine-D₁₁ were purchased from Cerilliant Corporation (Round Rock, TX). [¹⁴C]Metformin (98 mCi/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA). [³H]Estrone sulfate (50 Ci/mmol), and [^threeH]para-aminohippurate (3 Ci/mmol) were purchased from American Radiolabeled 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 bachelor and of belittling 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 loftier glucose Dulbecco'southward modified Eagle's medium with 10% fetal bovine serum, 1 mM 50-glutamine, 100 U/ml penicillin, 100 μm/ml streptomycin, and 150 μg/ml hygromycin B supplementation at 37°C with v% CO₂ and loftier humidity. All cell civilisation plastic surfaces were coated with 0.1% poly-D-lysine to ameliorate cell zipper. Uptake and inhibition assays were performed equally previously described with modification for assay of methamphetamine and its metabolites by liquid chromatography–tandem mass spectrometry (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 common salt solution (HBSS) and allowed to acclimate for 10 minutes at 37°C or preincubated with HBSS containing 30 mM ammonium chloride for xx minutes for multidrug and toxin extrusion (MATE) experiments to acidify the intracellular compartment and bulldoze MATE uptake (Tanihara et al., 2007). Media were removed and incubation initiated by addition of 100 μl of HBSS at pH 7.iv containing a substrate with or without inhibitor. Uptake was stopped by removal of media and washing the cells three times with ice common cold HBSS. Cells were either lysed with 100 μ50 of 1 M NaOH and neutralized with 100 μfifty of 1 K HCl for incubations containing a radiolabeled substrate for measurement by liquid scintillation counting (Tri-Carb B3110TR; PerkinElmer, Waltham, MA) or permeabilized with 100 μ50 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 past the BCA Poly peptide Assay Kit (Pierce Chemical, Rockford, IL) and the uptake in cells was normalized to their full protein concentrations. The inhibitory consequence of methamphetamine, amphetamine, and p-OHMA on hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-G was assessed in transporter-expressing HEK293 cells using [^xivC]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 μM, 1 μCi/ml) was selected to be much lower than its M _m values (780–1500 μM) 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 fourth dimension equally specified in the legends for Fig. 1, Fig. 2, Fig. iii, 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.

Inhibition past methamphetamine, amphetamine, and p-OHMA of hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-M. Uptake of [¹⁴C]metformin (eleven μThou) 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-Thou (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.nine, and 100 pmol/min/mg protein for hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-One thousand, respectively. Each data point represents the hateful ± Southward.D. from i representative experiment in triplicate. Curves from ii boosted independent repeats are displayed in Supplemental Fig. 2. The IC₅₀ values shown in Tabular array i are hateful ± S.D. of the IC_fifty values from the iii independent experiments.

Uptake of 1 μM methamphetamine, amphetamine, and p-OHMA by hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K. Uptake was measured subsequently 5-infinitesimal incubation at 37°C. Data are illustrated as the mean ± South.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).

Interactions of methamphetamine, amphetamine, and p-OHMA with renal hOAT1 and hOAT3. Result of methamphetamine, amphetamine, and p-OHMA on para-aminohippurate (PAH) (i µYard) uptake by hOAT1 (A) and estrone sulfate (0.06 μGrand) uptake by hOAT3 (B) was measured at 1 minute afterwards incubation at 37°C. 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 every bit the control. Activeness in the absence of an inhibitor (100%) corresponded to 36.vi and ii.1 pmol/min/mg protein for PAH and estrone sulfate uptake, respectively. Uptake of methamphetamine (C), amphetamine (D), and p-OHMA (East) by hOAT1 and hOAT3 was measured later on 5-infinitesimal incubation at 37°C. Information are illustrated as the mean ± S.D. from iii independent experiments performed in triplicate. Uptake in transporter-expressing cells was compared with that in command cells (**P < 0.01; ***P < 0.001).

Methamphetamine, amphetamine, and p-OHMA uptake kinetics by hOCTs. Concentration-dependent uptake of substrate was measured in both transporter-expressing and control cells at 37°C 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. 5/south) for the kinetic information. Based on the Eadie-Hofstee plots, the kinetics for hOCT2-mediated methamphetamine ship (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. v). hOCT2-mediated p-OHMA ship (D) was fitted to the Michaelis-Menten equation with a Loma gradient (eq. iv). Each data point represents the mean ± South.D. from 1 representative experiment in triplicate. Curves from two additional independent repeats are displayed in Supplemental Fig. four. The kinetic parameters in Table 3 are mean ± S.D. of the values from three independent experiments.

Methamphetamine and metabolite uptake kinetics by hMATE1 and hMATE2-Thousand. Methamphetamine, amphetamine, and p-OHMA uptake in hMATE1-expresing cells was performed during the initial linear uptake time at 5, ii, and 2 minutes, respectively (A–C). Methamphetamine and amphetamine uptake in hMATE2-expressing cells was performed during initial linear uptake fourth dimension at 5 minutes (D and E). Incubations were performed at 37°C and data were fitted with a Michaelis-Menten equation with a nonsaturable passive diffusion component (eq. three). Each information point represents the mean ± South.D. from i representative experiment in triplicate. Curves from two additional independent repeats are displayed in Supplemental Fig. v. The kinetic parameters in Tabular array 4 are hateful ± Southward.D. of the values from three contained experiments.

LC-MS/MS Assay of Methamphetamine and its Metabolites.

Methamphetamine, amphetamine, and p-OHMA levels were quantified using an LC-MS/MS system consisting of an API 4500 triple quadrupole mass spectrometer (AB-Sciex, Foster Metropolis, CA) coupled with an LC-20AD ultra-fast liquid chromatography system (Shimadzu Co., Kyoto, Nihon). The Turbo Ion Spray interface was operated in positive ion manner. X microliters of cell lysate was injected onto an Agilent Eclipse Plus C18 column (1.8 μm; 4.vi × 50 mm) (Agilent, Santa Clara, CA) running with an isocratic method consisting of 0.28 ml/min 0.2% formic acid in h2o 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-d_eleven, respectively. Information were analyzed using Analyst software version one.6.2 (AB Sciex). Assay accuracy and precision were inside fifteen% (20% for the lower limit of quantification).

Information Analysis.

Transport experiments were performed in triplicate and repeated three times independently. Information representation and replicates with specific north numbers are detailed in each effigy legend. The send kinetics were fitted using GraphPad Prism half dozen.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:

(ane)

where v is the rate of uptake in the presence of the inhibitor; Bottom is the remainder baseline value; Superlative is the rate of uptake in the absence of inhibitor; I is the inhibitor concentration; and H is the Hill coefficient. Two-site inhibition information were fitted using the following equation:

(2)

Ane- and two-site inhibition equations were compared by an extra sum-of-squares F test using the information from all three independent experiments modeled simultaneously. Apparent hMATE1/2-K, K _grand, and V _max values were obtained past simultaneously fitting the data to the Michaelis-Menten equation with a passive diffusion component in transporter-transfected cells and only the passive improvidence component in vector-transfected cells (Brouwer et al., 2013):

(3)

where V is the velocity of uptake; V _max is the maximum velocity of uptake; S is the substrate concentration; K _m is the Michaelis-Menten constant; and P _dif is the nonsaturable passive improvidence 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 one-half-maximal transport concentration (Yard _1/2 in place of Thousand _m) subsequently inspection of the Eadie-Hofstee plot (Copeland, 2000):

(4)

Amphetamine hOCT2 specific uptake kinetics were fit to a biphasic Michaelis-Menten equation:

(5)

Results

Inhibitory Event of Methamphetamine and its Metabolites on hOCT1–iii and hMATE1/2-K.

The ship 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 (Supplemental Fig. i). Methamphetamine, amphetamine, and p-OHMA inhibited metformin uptake by hOCT1–3 and hMATE1/2-K in a concentration-dependent manner (Fig. one; Supplemental Fig. ii). The IC₅₀ values are summarized in Table 1. Methamphetamine and amphetamine were 4- to xx-fold more potent for hOCT1 and hOCT2 than for hOCT3, hMATE1, and hMATE2-K, with hOCT2 showing the greatest sensitivity to both psychostimulants (hOCT2 IC_fifty values of xv.0 ± six.81 and 20.3 ± xvi.9 μM, respectively). p-OHMA was a more than potent inhibitor of hOCT1 than other transporters. Improver of the 4-hydroxyl grouping to the aromatic phenyl band (p-OHMA) greatly increased bounden to hOCT3 but decreased its potency toward hOCT2 compared with methamphetamine. Interestingly, the Loma slope of methamphetamine and amphetamine inhibition of hOCT1 and hOCT2 was approximately 0.v (Tabular array 1). Conversely, the Hill slope of p-OHMA confronting hOCT2 was approximately 1.5. hMATE2-K too had steep Colina slopes ranging betwixt 1.6 and i.9 for methamphetamine and its metabolites. These Colina slopes advise more complex interactions than simple competitive inhibition may exist occurring with these transporters.

TABLE i

IC₅₀ values of methamphetamine, amphetamine, and p-OHMA for hOCT1–3 and hMATE1/2-Grand determined by one-binding site plumbing equipment

Results stand for mean ± Due south.D. of three independent experiments each run in triplicate.

Transporter	One-Bounden Site
	Methamphetamine		Amphetamine		p-OHMA
	IC50	Colina Slope	IC50	Colina Slope	IC50	Loma Slope
	μM		μM		μM
hOCT1	21.1 ± 8.8	0.55 ± 0.02	96.7 ± 37	0.61 ± 0.28	12.0 ± 3.4	i.17 ± 0.14
hOCT2	15.0 ± 6.8	0.56 ± 0.07	twenty.3 ± 16.9	0.44 ± 0.one	83.8 ± 22.3	1.55 ± 0.32
hOCT3	300 ± 139	one.42 ± 0.68	363 ± 56.4	1.1 ± 0.ii	44.4 ± 25.5	ane.i ± 0.26
hMATE1	107 ± 38	0.79 ± 0.09	94.0 ± 25.iii	0.89 ± 0.2	59.1 ± 14.three	0.84 ± 0.12
hMATE2-Grand	84.three ± 12.nine	1.63 ± 0.xiv	158 ± 48	1.9 ± 0.half dozen	234 ± 86.8	1.21 ± 0.11

Inspection of methamphetamine and amphetamine dose-dependent inhibition of hOCT1 and hOCT2 revealed biphasic inhibition characteristics (Supplemental Fig. iii). 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 plumbing equipment the data from three independent experiments each run in triplicate. The two-binding site model fit significantly amend for methamphetamine inhibition of hOCT1 and hOCT2 as well as amphetamine inhibition of hOCT2 (P < 0.0001) but non hOCT1 (Table ii). The high-analogousness EC₅₀ values were in the depression micromolar range (0.72–five.29 μGrand), while the apparent depression-affinity interactions appeared to be in the high micromolar range (58.two–400 μM) for these transporters.

TABLE 2

EC_l values of methamphetamine and amphetamine for hOCT1 and hOCT2 determined by ii-binding site plumbing equipment

The EC₅₀ values were obtained by fitting inhibition data in Fig. i and Supplemental Fig. 2 using eq. 2 described in Materials and Methods with the fit shown in Supplemental Fig. three. The terminal column lists the P values obtained past comparing eqs. 1 and 2 with an extra sum-of-squares F test. Results represent mean ± S.D. of iii independent experiments each run in triplicate.

Inhibitor	Two-Binding Site			Two-Binding versus One-Binding Site
	Transporter	EC50 Value
		High Analogousness	Low Affinity
		μM	μM
Methamphetamine	hOCT1	5.29 ± 0.66	400 ± 229	P < 0.0001
	hOCT2	1.21 ± 0.nineteen	58.2 ± 23.4	P < 0.0001
Amphetamine	hOCT1	NA	NA	P = 0.55
	hOCT2	0.72 ± 0.29	145 ± 104	P < 0.0001

Uptake of Methamphetamine and Metabolites by hOCT1-3, hMATE1, and hMATE2-Thousand.

The substrate potential of methamphetamine, amphetamine, and p-OHMA was assessed past measuring the uptake of these compounds (1 μM) in control cells and transporter-expressing cells (Fig. 2). After 5-minute incubation, methamphetamine and amphetamine showed approximately two-fold greater uptake in cells expressing hOCT2, hMATE1, and hMATE2-1000. p-OHMA accumulated extensively in hOCT1, hOCT2, and hOCT3, and to a lesser caste in hMATE1, merely did non accumulate at all in hMATE2-One thousand-transfected cells when compared with control cells (Fig. two). These data propose 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.

While hOCT2 and hOAT1/3 mediate renal secretion of organic cations and organic anions, respectively, some substrate and inhibitor overlap betwixt 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 event on hOAT1 or hOAT3 at 1 mM. Only p-OHMA showed significant inhibition of hOAT1 and hOAT3 at ane mM with 47 ± 17 and 38 ± 28% inhibition, respectively. Uptake studies showed that none of the compounds were substrates of hOAT1 or hOAT3 (Fig. iii, C–Due east), suggesting a primary office 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 past determining concentration-dependent transport rates. The specific uptake was obtained for hOCT1–three by subtracting uptake in the control cells and Eadie-Hofstee plots were evaluated to identify the blazon of interaction. Due to high passive diffusion at high concentrations, methamphetamine kinetics can merely be accurately adamant at a low concentration range (0–15 μM). Within this range, methamphetamine displayed saturable kinetics with a K _m value of 2.09 ± 0.88 μChiliad (Fig. 4A; Supplemental Fig. 4; Table three), which is very close to the high-analogousness, half-inhibitory concentration (one.21 ± 0.19 μM) observed in Supplemental Fig. 2B. For amphetamine, nosotros were able to cover a wider concentration range (0–600 μM). Equally shown in Fig. 4B, biphasic send kinetics were observed, and the K _g values for the apparent high- and depression-affinity bounden sites were adamant to be 0.830 ± 0.55 and 534 ± 350 μM, respectively. Interestingly, p-OHMA displayed sigmoidal kinetics for hOCT2 every bit clearly revealed by the Eadie-Hofstee plot (Fig. 4D). The p-OHMA hOCT2 one-half-maximal ship concentration (M _1/two) is 31.eight ± 9.iii μThou and the Hill slope is 1.64 ± 0.15. For p-OHMA transport past hOCT1 and hOCT3, no apparent sigmodal or biphasic pattern was observable in the Eadie-Hofstee plot. Fitting to a standard Michaelis-Menten equation yielded credible K _m values of xiv.five ± 8.vii and 53.iii ± half-dozen.2 μM for hOCT1 and hOCT3, respectively (Fig. four; Supplemental Fig. iv; Tabular array iv).

TABLE 3

Kinetic parameters of methamphetamine and metabolites determined from modeling the data in Fig. four and Supplemental Fig. iv

Models were chosen based on exam 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. five). p-OHMA hOCT1- and hOCT3-mediated ship were fit to a standard Michaelis-Menten equation. Sigmoidal kinetics of hOCT2-mediated p-OHMA send was obtained past plumbing equipment transporter-mediated uptake to the Michaelis-Menten equation with a Hill slope (eq. 5) for the substrate concentration and half-maximal ship concentration (Chiliad _1/ii in place of G ₁₀₀₀). Results correspond hateful ± S.D. of 3 independent experiments each run in triplicate.

Compound	Transporter	K m1	V max1	K m2	V max2
		μM	pmol/mg/min	μM	pmol/mg/min
Methamphetamine	hOCT2	2.09 ± 0.88	49.7 ± 12.2	ND	ND
Amphetamine	hOCT2	0.830 ± 0.55	34.vi ± 23.7	534 ± 350	853 ± 474
p-OHMA	hOCT1	14.5 ± 8.7	312 ± 163	NA	NA
	hOCT2	M i/2: 31.8 ± 9.three; H: 1.64 ± 0.15	1780 ± 718	NA	NA
	hOCT3	53.3 ± 6.2	1290 ± 830	NA	NA

TABLE iv

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 ± S.D. of three independent experiments each run in triplicate (Fig. five; Supplemental Fig. 5).

Transporter	Methamphetamine		Amphetamine		p-OHMA
	K thou	Five max	M m	Five max	Grand m	5 max
	μM	pmol/mg/min	μM	pmol/mg/min	μM	pmol/mg/min
hMATE1	xx.6 ± 4.5	86.vi ± 54	fourteen.1 ± four.9	238 ± 141	49.8 ± 26	257 ± 190
hMATE2-K	xviii.one ± 11	97.7 ± 25.3	xvi.4 ± 12.2	92.9 ± 8.five	NA	NA

hMATE1/2-G ship studies were conducted afterward 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 loftier substrate concentrations. Therefore, we fitted the concentration-dependent uptake in transporter-expressing cells to a Michaelis-Menten equation with a nonsaturable passive diffusion component (Fig. 5; Supplemental Fig. 5; Table 4). This simultaneous plumbing equipment of both carrier- and noncarrier-mediated uptake allowed for an gauge of the apparent One thousand ₁₀₀₀ values for hMATE1/2-G in the presence of a high-passive permeability component (Tabular array 4).

Discussion

In spite of the major function of renal clearance in methamphetamine disposition, the molecular mechanisms underlying the tubular secretion of methamphetamine and its major metabolites had non 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/ii-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-Thousand, but non hOCT1or hOCT3. Interestingly, p-OHMA was a substrate of hOCT1–3 likewise as hMATE1, but not hMATE2-K. Methamphetamine and its metabolites do non interact with renal hOAT1 or hOAT3. Methamphetamine and its metabolites demonstrated complex inhibitory and substrate kinetics with hOCT2. Our data propose that the hOCT2/hMATE pathway is involved in renal secretion of methamphetamine and its metabolites, and that inhibition of hOCT2 and hMATEs past methamphetamine may lead to potential DDIs for drugs that are eliminated past the hOCT2/hMATE pathway.

The importance of renal elimination of methamphetamine has long been known; all the same, the exact molecular mechanisms of renal secretion had not been identified (Beckett and Rowland, 1965c; Caldwell et al., 1972). Hither, we identified the hOCT2/hMATE pathway as being involved in the active renal secretion of methamphetamine and 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 upmost 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 (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 excretion 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. Intriguingly, 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 past passive diffusion. Interestingly, p-OHMA was transported by hOCT1, suggesting that the para-hydroxyl group may be of import for OCT1 transport selectivity of substituted amphetamines. hOCT1 may thus be involved in hepatic ship of p-OHMA.

In this study, nosotros used metformin as the probe substrate because it is recommended every bit an in vitro and in vivo probe substrate for evaluating hOCT2, hMATE1, and hMATE2-Yard interaction studies by the International Transporter Consortium (Hillgren et al., 2013), Food and Drug Assistants (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 circuitous interactions, the apparent inhibition potencies of amphetamines may be highly dependent on the substrate. As an illicit drug, abusers may utilise methamphetamine while taking prescription medications. Testing the inhibition potencies with the specific hOCT substrate drugs used by methamphetamine abusers may be warranted to decide the likelihood of clinically relevant interactions.

Particularly high levels of methamphetamine abuse are reported in individuals receiving handling of homo immunodeficiency virus and hepatitis who may exist receiving multiple medications for treatment (Panenka et al., 2013; Volkow, 2013; Bracchi et al., 2015). Importantly, numerous antiretrovirals (eastward.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 agile transport of the probe substrate metformin by hOCT1–iii and hMATE1/2-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 μK in ane 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 corruption since patients may not be willing to reveal their use of illicit drugs.

Methamphetamine and its metabolites demonstrated complex interactions with hOCT1 and hOCT2, suggesting they may take 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 take not been obtained. Amphetamine showed biphasic hOCT2 uptake kinetics with an apparent high affinity (0.830 ± 0.55 μM) and low analogousness (534 ± 350 μChiliad) aligning with the observed high- and low-affinity inhibitory interactions. Conversely, p-OHMA had a relatively steep hOCT2 inhibition Hill slope (1.55 ± 0.32) and demonstrated sigmoidal uptake kinetics (Fig. iv; Table iii). Sigmoidal uptake kinetics may be characteristic of homotropic activation (Segel, 1976; Atkins, 2005). Both a big binding pocket in the outward facing cleft assuasive for spatially distinct binding as well as a distal allosteric bounden site accept 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-analogousness interactions with no transport observed in the concentration range (due east.g., vi-41 pM for one-methyl-4-phenylpyridinium) (Moaddel et al., 2005; Gorbunov et al., 2008; Minuesa et al., 2009; Koepsell, 2015). Binding inside the transportable region is generally a lower-analogousness interaction (eastward.g., 0.87-12.three μYard for 1-methyl-iv-phenylpyridinium) with known substrates (Moaddel et al., 2005; Gorbunov et al., 2008; Minuesa et al., 2009; Koepsell, 2015). Recent developments accept likewise suggested the possibility of simultaneous binding of two substrates within the transport site (Harper and Wright, 2013; Koepsell, 2015). The apparent affinities observed for methamphetamine and metabolites across their transportable concentration ranges advise that both bounden sites may reside inside the ship region. More than studies are needed to understand the complex kinetic behaviors of the OCTs and their structure-office relationships.

In summary, our report determined the molecular mechanisms involved in transport and disposition of methamphetamine and its metabolites. Moreover, our studies showed that methamphetamine has the potential to inhibit hOCT and hMATE transporters at clinically relevant concentrations. Finally, we identified circuitous kinetic interactions between amphetamines and hOCT2. Our findings provide useful information that may exist considered when prescribing medications to methamphetamine users to mitigate the risk of DDIs that may potentially compromise therapeutic efficacy and drug prophylactic.

Acknowledgments

The authors give thanks Dr. Willian Atkins and Dr. Kent Kunze for thoughtful discussions and insights.

Abbreviations

DDI	drug-drug interaction
HBSS	Hanks' counterbalanced common salt solution
hMATE	man multidrug and toxin extrusion
hOAT	man 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

Authorship Contributions

Participated in enquiry 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.

Footnotes

This study was supported past the National Institutes of Health National Plant on Drug Abuse [Grant P01 DA032507] and National Institutes of Health Full general Medical Sciences [Grant T32 GM07750].

The content of this paper is solely the responsibility of the authors and does not necessarily correspond the official views of the National Institutes of Health.

https://doi.org/ten.1124/dmd.116.074708.

This article has supplemental material available at dmd.aspetjournals.org.

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