Interactions of human organic anion transporters 1–4 and human organic cation transporters 1–3 with the stimulant drug methamphetamine and amphetamine

Legal Medicine 44 (2020) 101689

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Interactions of human organic anion transporters 1–4 and human organic cation transporters 1–3 with the stimulant drug methamphetamine and amphetamine

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Shoetsu Chiba , Ayako Ro, Toru Ikawa, Yukino Oide, Toshiji Mukai Department of Legal Medicine, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ward, Kawasaki, Kanagawa 216-8511, Japan

A R T I C LE I N FO

A B S T R A C T

Keywords: Transporter hOAT hOCT Methamphetamine Amphetamine

Drug membrane transport system proteins, namely, drug transporters, are expressed in the kidney and liver and play a crucial role in the excretion process. This study aimed to elucidate the interactions of the drug transporters human organic anion transporters 1, 2, 3, 4 (hOAT1, 2, 3, 4) and human organic cation transporters 1, 2, 3 (hOCT1, 2, 3), which are expressed primarily in human kidney, liver, and brain, with the stimulants methamphetamine (METH) and amphetamine (AMP). The results of an inhibition study using representative substrates of hOATs and hOCTs showed that METH and AMP significantly inhibited (by > 50%) uptake of the hOCT1 and hOCT3 representative substrate 1-methy1-4-phenylpyridinium ion (MPP+) and hOCT2 representative substrate tetraethyl ammonium (TEA). However, METH and AMP did not inhibit uptake of the representative substrates of hOAT1, hOAT2, hOAT3, and hOAT4, (i.e., p-aminohippuric (PAH) acid, prostaglandin F2α (PGF2α), estron sulfate (ES), and ES respectively). Kinetic analyses revealed that METH competitively inhibited hOCT1-mediated MPP+ and hOCT2-mediated TEA uptake (Ki, 16.9 and 78.6 µM, respectively). Similarly, AMP exhibited competitive inhibition, with Ki values of 78.6 and 42.8 µM, respectively. In contrast, hOCT3 exhibited mixed inhibition of representative substrate uptake; hence, calculating Ki values was not possible. Herein, we reveal that hOCTs mediate the inhibition of METH and AMP. The results of this uptake study suggest that METH and AMP bind specifically to hOCT1 and hOCT2 without passing through the cell membrane, with subsequent passage of METH and AMP via hOCT3.

1. Introduction Illicit drug use is a significant health concern and cause of violent crimes worldwide. The use of stimulants is increasing globally, particularly in Japan, and the proportion of stimulant users among all drug offenders is currently exceptionally high (> 80%), with the recidivism rate maintained at approximately 60%. Stimulants act on the central and sympathetic nervous systems, leading to euphoria and stimulation, which promote habitual use [1]. Hence, the use of stimulants has been associated with a variety of criminal activities. Forensic toxicology has long played a central role in stimulant research. Autopsies are performed to investigate the cause of stimulantrelated deaths [2–4]. In forensic medicine, the use of LC–MS/MS has been established for highly sensitive analyses of the quantity of stimulants present in the body. However, the mechanism underlying stimulant-related deaths remains unclear. Therefore, we investigated the yet to be elucidated mechanism of stimulant excretion in the

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pharmacokinetics of the drug transporters human organic anion transporter 1, 2, 3, and 4 (hOAT1, hOAT2, hOAT3, and hOAT4; i.e., hOATs) and human organic cation transporter 1, 2, and 3 (hOCT1, hOCT2, and hOCT3; i.e., hOCTs). These transporters are carrier proteins primarily localized in the membrane of kidney and liver cells. The stimulants used in this study were methamphetamine (METH) and amphetamine (AMP). Because a number of drugs are highly lipophilic, some substances exhibit increased hydrophilicity due to the first-phase reaction, which is a hydroxide reaction, followed by the second-phase reaction, which is a conjugation reaction. Finally, the substrate is eliminated by biliary excretion from the liver via the basolateral membrane of the bile canaliculus or by urinary excretion from the kidney via the basolateral membrane of the renal proximal tubules. However, because hydrophilic compounds cannot penetrate the lipid bilayer of cell membranes, a transporter is required to facilitate the translocation of hydrophilic compounds across the cell membrane. We examined seven different

Corresponding author. E-mail address: [email protected] (S. Chiba).

https://doi.org/10.1016/j.legalmed.2020.101689 Received 22 August 2019; Received in revised form 29 January 2020; Accepted 14 February 2020 Available online 18 February 2020 1344-6223/ © 2020 Published by Elsevier B.V.

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Fig. 1. Comparison of the inhibitory effects of 5 µM–1 mM methamphetamine and 5 µM–1 mM amphetamine on the prototypical organic anion and cation uptake mediated by hOATs and hOCTs. (a) hOAT1, (b) hOAT2, (c) hOAT3, (d) hOAT4, (e) hOCT1, (f) hOCT2, (g) hOCT3, or mock transporter were incubated in a solution containing 5 µM [14C]PAH (hOAT1), 50 nM [3H]PGF2α (hOAT2), 50 nM [3H]ES (hOAT3 and hOAT4), 5 nM [3H]MPP＋(hOCT1), 5 µM [14C]TEA (hOCT2), or 10 nM [3H]MPP+ (pH 8.5 solution) (hOCT3) in the presence of 1 mM methamphetamine or amphetamine at 37 °C for 5 min. Each value represents the mean ± SEM of three determinations: ***P < 0.001 vs. control.

organs, including the placenta, brain, kidney, and liver. Various substrates, known as bi-substrates, have been shown to be transported via hOATs and hOCTs [30]. This type of multi-specificity could cause drug–drug interactions when several medicines are taken concomitantly. In this context, special caution should be applied when stimulants are concomitantly used with other medicines that share corresponding transporters for renal tubular and bile canalicular excretion. This is because concomitant administration of stimulants with other medicines may induce significant increases in the plasma concentrations of the stimulants or other medicines, resulting in a high risk of death due to drug intoxication.

types of transporters that mediate the transport of drugs into cells. cDNAs have been successively cloned that encode the organic anion transporters (OATs) OAT1 [5–9], OAT2 [10–13], OAT3 [14–17], and OAT4 [18–20], and their sites of localization have been elucidated. Whereas hOAT1, hOAT3, and hOAT4 are primarily expressed in the kidney, hOAT2 is primarily expressed in the liver. All four of these hOATs transport organic anions. cDNAs have also been successively cloned that encode various organic cation transporters (OCTs), including OCT1 [21,22], OCT2 [23–26], and OCT3 [27–29]. hOCT1 is primarily expressed in the liver, whereas hOCT2 is expressed predominantly in the kidney. hOCT3 is expressed in a wide range of

2

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(9.25 MBq), [3H]PGF2α (1.85 MBq), [3H]MPP+ (1.85 MBq), and [14C]TEA (1.85 MBq) were purchased from Muromachi Kikai Co., Ltd. (Tokyo, Japan), and [3H]ES (9.25 MBq) was purchased from Perkin Elmer (Waltham, MA, USA). METH was purchased from Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan), and AMP was provided by Dr. Takeshi Saito of Tokai University (Kanagawa, Japan). Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, and geneticin were purchased from Sigma (St. Louis, MO, USA); fetal bovine serum (FBS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and penicillin/streptomycin were purchased from Invitrogen (Carlsbad, CA, USA); and NaHCO3 was purchased from Wako (Osaka, Japan).

Table 1 IC50 and Ki values for METH and AMP in inhibition of hOCT1-3–mediated 3HMPP+ uptake and hOCT2-mediated 14C-TEA uptake. TP

Substance

IC50 (µM)

Ki (µM)

hOCT1

METH AMP METH AMP METH AMP

6.5 ± 0.5 78.3 ± 6.6 19.1 ± 1.4 43.8 ± 8.1 168.9 ± 21.6 500.4 ± 98.0

16.9a 78.6a 17.7a 42.8a n.c.b n.c.b

hOCT2 hOCT3

n.c.: Not calculated. a Competitive inhibition. b Mixed inhibition.

2.2. Cell culture

The objectives of the present study were as follows: (i) use S2hOAT1, 2, 3, and 4 cells and S2-hOCT1, 2, and 3 cells to test whether stimulants (METH and AMP) inhibit representative substrates ([14C]paminohippuric acid [PAH], [3H]prostaglandin F2α [PGF2α], and [3H] estron sulfate [ES] for hOATs; [14C]tetraethyl ammonium bromide [TEA] and [3H]methyl-4-phenylpyridinium iodide [MPP+] for hOCTs); (ii) determine the concentration required to achieve 50% inhibition (IC50 value) and the inhibition constant (Ki value) for stimulants that inhibit representative substrates; (iii) conduct additional experiments to produce Lineweaver–Burk and Eadie–Hofstee plots and establish the inhibition type (i.e., competitive or mixed); and (iv) elucidate the mechanisms of stimulant uptake via hOCTs in a cell uptake study using LC–MS/MS analysis.

S2-hOATs (hOAT1, hOAT2, hOAT3, and hOAT4) and S2-hOCTs (hOCT1, hOCT2, and hOCT3) cells were provided by Dr. Hitoshi Endou (professor emeritus at the University of Kyourin, Tokyo, Japan). The proximal tubule S2 cell line was established by microdissecting cells of the second segment (S2) of proximal tubules obtained from transgenic mice harboring the temperature-sensitive simian virus 40 (SV40) large T antigen [31]. The S2 cells were transfected with pcDNA3.1 vectors containing inserted cDNA sequences encoding the hOATs or hOCTs and pSV2neo (neomycin resistance gene), which were designated hOATand hOCT-expressing cells, respectively. An empty vector lacking the hOAT or hOCT cDNA insert was transfected into S2 cells as a mock [8]. The cells were grown in a humidified incubator at 33 °C with 10% CO2 in DMEM containing 10% FBS, 100 U/mL penicillin/streptomycin, 10 mM HEPES, 540 µg/mL geneticin, 2 mM L-glutamine, and 3.7 mg/ mL NaHCO3, and the pH was adjusted to 7.2 using 1 N HCl. The cells were subcultured using 0.05% trypsin–ethylenediaminetetraacetic acid (EDTA) solution containing 137 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 4 mM NaHCO3, 0.5 mM EDTA, and 5 mM HEPES (pH 7.2).

2. Materials and methods 2.1. Materials Radioactive

materials

(representative

substrates)

[14C]PAH

Fig. 2. Inhibitory effects of various concentrations of methamphetamine and amphetamine on prototypical organic cation uptake mediated by hOCTs. hOCT1 (a, b), hOCT2 (c, d), or hOCT3 (e, f) cells were incubated in a solution containing 5 nM [3H]MPP＋(hOCT1), 5 µM [14C]TEA (hOCT2), or 10 nM [3H]MPP+ (pH 8.5 solution) (hOCT3) in the presence of various concentrations (3–1000 µM) of methamphetamine (a, c, and e) or amphetamine (b, d, and f) at 37 °C for 5 min. Each value represents the mean ± SEM of three determinations. 3

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Fig. 3. Kinetic analyses of the inhibitory effects of methamphetamine and amphetamine on uptake of various concentrations of prototypical organic cation mediated by hOCTs. hOCT1 (a, b), hOCT2 (c, d), or hOCT3 (e, f) cells were incubated in a solution containing various concentrations (10–250 µM) of MPP+ (hOCT1), TEA (hOCT2), or MPP+ (pH 8.5 solution) (hOCT3) in the presence or absence of methamphetamine (a, c, and e) or amphetamine (b, d, and f) of an amount equivalent to the respective IC50 value at 37 °C for 5 min. Each value represents the mean ± SEM of three determinations. Lineweaver–Burk and Eadie–Hofstee plot analyses were performed.

METH and AMP, as described above. Subsequently, the uptake reaction was stopped by adding ice-cold D-PBS, and the cells were washed three times using the same solution. The cells in each well were lysed with 0.5 mL of 0.1 N NaOH, followed by addition of 3 mL of Insta-Gel Plus (Perkin Elmer). Radioactivity was determined using a liquid scintillation counter (LSC-6100; Aloka, Hyogo, Japan). Protein concentration was determined using the Bradford method. First, we confirmed that METH and AMP (5 µM–1 mM) inhibited the uptake of each representative substrate by hOATs and hOCTs by > 50%. Second, to determine the IC50 values (half the maximal inhibitory concentration values), hOCT cells were incubated in a solution containing each representative substrate and/or various concentrations (3, 10, 30, 100, 300, and 1000 µM) of METH and AMP.

2.3. Inhibition study hOATs and hOCTs cells were seeded in 24-well tissue culture plates at a density of 1 × 105 cells/well. After 2 days of culture, the cells reached sub-confluence and were washed three times with Dulbecco’s modified phosphate-buffered saline (D-PBS; 137 mM NaCl, 3 mM KCl, 8 mM NaHPO4, 1 mM KH2PO4, 1 mM CaCl2, and 0.5 mM MgCl2; pH 7.4) supplemented with 5.5 mM glucose. Next, the cells were pre-incubated in the same solution in a water bath at 37 °C for 10 min. To evaluate the inhibitory effects of METH and AMP upon organic anion uptake by hOAT1, hOAT2, hOAT3, and hOAT4 and organic cation uptake by hOCT1, hOCT2, and hOCT3, the cells were incubated in solution containing a representative hOAT or hOCT substrate, namely, 5 µM [14C]PAH for 2 min (for hOAT1), 50 nM [3H]PGF2α for 30 s (for hOAT2), 50 nM [3H]ES for 2 min (for hOAT3 and hOAT4), 5 nM [3H] MPP+ for 5 min (for hOCT1), 5 µM [14C]TEA for 5 min (for hOCT2), or 10 nM [3H]MPP+ for 15 min (for hOCT3), in the absence or presence of

2.4. Kinetic analysis of inhibition hOCT cells were incubated for a fixed duration as described above at 4

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Fig. 4. Time course of the uptake of 10 µM methamphetamine and 10 µM amphetamine via hOCT3. hOCT3 cells and mock control cells were incubated in 10 µM methamphetamine (a), 10 µM amphetamine (c) at 37 °C or on ice (b, d) for 1, 2, 5, 15, 30, and 45 min. Each value represents the mean ± SEM of three determinations.

ZORBAX Eclipse Plus C18 2.1 mm i.d. × 100 mm column (Agilent Technologies) was used. The gradient method was as follows: 5 mM ammonium formate and 0.1% formic acid: acetonitrile (90:10) (v/v) at the beginning and 0:100 (v/v) at 20 min for positive mode. METH and AMP were monitored via electrospray ionization in positive ion mode under the following conditions: (i) cap voltage, 4000 V (positive); (ii) gas temperature, 300 °C; (iii) dry gas flow, 10 L/min; (iv) nebulizer, 50 psi; (v) sheath gas temperature, 375 °C; (vi) sheath gas flow, 10 L/min. Samples (10 µL) were analyzed by LC–MS/MS with the following compound settings: i) METH: 150.1 → 119.2 m/z, fragmentor = 80, collision energy = 5, cell accelerator voltage = 7; ii) AMP: 136.1 → 118.9 m/z, fragmentor = 80, collision energy = 5, cell accelerator voltage = 4.

37 °C in 0.25 mL of D-PBS containing various concentrations (10, 13, 18, 30, 70, and 250 µM) of hOCT representative substrate in the absence or presence of METH or AMP at the corresponding IC50 value. The kinetics of the inhibitory effects were analyzed using Lineweaver–Burk plots. When the inhibition was competitive, inhibition constants (Ki values) were calculated using the following equation: Ki = concentration of inhibitor/([Km of representative substrate with inhibitor]/[Km of representative substrate without inhibitor] – 1). Ki values indicative of mixed inhibition for METH and AMP could not be calculated. 2.5. Uptake study METH and AMP uptake and LC–MS/MS experiments were conducted using cell culture conditions similar to those discussed in Section 2.3. hOCT cells in a 24-well plate were pre-incubated in D-PBS in a water bath at 37 °C or on-ice for 10 min. hOCT cell uptake experiments were conducted with 10 µM METH or 10 µM AMP without radioisotope (RI)-labeled substrate at various time points (1, 2, 5, 15, 30, and 45 min) in a water bath at 37 °C or on ice. Uptake experiments were stopped by adding ice-cold D-PBS, and the cells were washed three times using the same solution. The 24-well plate was sealed and kept cold in a freezer (−20 °C). Following removal from the freezer, extraction solution (400 µL; 1:1:1 MeOH:ACN:H2O) was added to each well, and the cells were scraped into suspension, transferred to a tube (1.5 mL), sonicated (5 min), and centrifuged using a mini-spin centrifuge (5 min at 12,000 × g). The supernatants were then transferred to analytical vials and immediately analyzed via LC–MS/MS using a 6420 type spectrometer (Agilent Technologies; Santa Clara, CA, USA) equipped with a 1260 type HPLC system (Agilent Technologies). A

2.6. Statistical analysis Data are expressed as mean ± SEM. The statistical significance of differences was evaluated using Dunnett’s test. P-values < 0.05 were considered statistically significant. 3. Results 3.1. Inhibitory effects of METH and AMP on representative substrate uptake by hOATs and hOCTs To determine whether hOATs and hOCTs interact with METH and AMP, we examined the impact of these stimulants on hOAT- and hOCTmediated uptake of representative substrates. As illustrated in Fig. 1, METH and AMP inhibited hOCT-mediated representative substrate uptake by > 50%. In contrast, METH and AMP failed to inhibit hOAT5

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inhibition of the equivalent transport by hOATs (Fig. 1). Hence, METH and AMP were found to be candidate substrates for hOCTs. To determine the substrate specificity of hOCTs using kinetic analyses, we performed IC50 and Ki studies. The results revealed that the inhibitory potency of these stimulants for hOCTs differ markedly. The calculated results for the IC50 and Ki values of METH and AMP are indicated in Figs. 2 and 3 and Table 1. METH had the highest affinity for hOCT1 (IC50, 6.5 ± 0.5 µM; Ki, 16.9 µM) and the lowest affinity for hOCT3 (IC50, 168.9 ± 21.6 µM), whereas AMP had the highest affinity for hOCT2 (IC50, 43.8 ± 8.1 µM; Ki, 42.8 µM) and the lowest affinity for hOCT3 (IC50, 500 ± 98.0 µM). Therefore, METH and AMP exhibit potent inhibition against hOCT1 and hOCT2 but not hOCT3, demonstrating the distinct substrate-dependent specificity of hOCTs. To determine the mode of action of inhibition of representative substrate transport by hOCTs, we prepared Lineweaver–Burk plots to distinguish between competitive, noncompetitive, and mixed inhibition types. Competitive inhibition is indicated by the intersection of inhibitor and control plots at the Y-axis and by different slopes of the two lines. In contrast, noncompetitive inhibition is indicated by the intersection of control and inhibitor plots at the X-axis and by different slopes of the two lines. Mixed inhibition is indicated by differing X- and Y-axis intercepts of control and inhibitor plots and the intersection of the two lines in the second quadrant (X < 0 and Y > 0). Therefore, the interactions of METH and AMP with hOCT1 and hOCT2 involved competitive inhibition, whereas their interaction with hOCT3 involved mixed inhibition of uptake. The inhibition elicited by METH and AMP may provide evidence that hOCTs mediate the uptake of METH and AMP into hepatocytes via the basolateral membrane of the bile canaliculus or tubular cells via the basolateral membrane of proximal tubules. Alternatively, METH and AMP could selectively bind to hOCTs to inhibit the uptake of the representative substrate. Particular care must be taken when METH and AMP are concomitantly used with other drugs (such as over-the-counter [OTC] medications) that share transporters with METH and AMP for renal tubular or bile canalicular excretion. In other words, concomitant administration of METH or AMP with other drugs such as OTC medications may induce an increase in the plasma concentrations of METH and AMP or other drugs, including the OTC medications, resulting in significant toxicity. The data from our inhibition study indicate that hOATs are poor transporters of METH and AMP and that hOCTs mediate the inhibition of METH and AMP uptake. To illustrate that an inhibitor is not necessarily equivalent to an hOCT substrate, we utilized LC–MS/MS to quantitatively analyze METH and AMP uptake. The experiment examining uptake at 37 °C resulted in the straightforward prediction that hOCT3 is involved in the cellular uptake of METH and AMP. To confirm these results, an uptake experiment was performed on ice (i.e., under a condition of transporter deactivation). The values were significantly lower on ice as compared with 37 °C, suggesting that the assay values obtained for METH and AMP at 37 °C indicate cellular uptake via hOCT3 specifically rather than non-specific adsorption or simple diffusion into the cells. Moreover, although METH and AMP do not pass through the cell membrane via hOCT1 and hOCT2, due to their selective binding to hOCT1 and hOCT2 (which are expressed on the cellular membrane surface), they are predicted to be potent inhibitors of drugs excreted via hOCT1 and hOCT2. Limited information is available concerning the excretion of METH and AMP via the bile canaliculus and renal tubules. The results of this uptake inhibition study suggest that the pathways for METH and AMP excretion are involved in the transport of various drugs in the liver and kidney. The results of this study indicated that stimulant drugs affect hOCT1 and hOCT2 as powerful inhibitors, hOCT3 as the substrate. hOCT1, hOCT2, and hOCT3 are expressed primarily in the liver, kidney, and both organs, respectively. Thus, for hOCT1 and hOCT2, our results suggest that the stimulant drugs are strongly and selectively connected with the transporters. It is unknown whether stimulant drugs remain

mediated representative substrate uptake by 50% (Fig. 1). Therefore, in the subsequent IC50 study, analyses were performed using only hOCTs to determine the strength of inhibition. The IC50 values of METH and AMP are listed in Table 1 and illustrated in Fig. 2. METH and AMP exhibited higher-affinity interactions with hOCT1 and hOCT2 than with hOCT3. The IC50 values were 6.5 ± 0.5 µM for METH (via hOCT1), 78.3 ± 6.6 µM for AMP (via hOCT1), 19.1 ± 1.4 µM for METH (via hOCT2), 43.8 ± 8.1 µM for AMP (via hOCT2), 168.9 ± 21.6 µM for METH (via hOCT3), and 500.4 ± 98.0 µM for AMP (via hOCT3) (Table 1). 3.2. Kinetic analysis of the inhibitory effects of METH and AMP on representative substrate uptake by hOCTs To further describe the inhibitory kinetics of METH and AMP on hOCT-mediated representative substrate uptake, Lineweaver–Burk plot analyses were performed and Ki values determined for each compound (Table 1). The results are illustrated in Fig. 4. Lineweaver–Burk plots for the interactions of METH and AMP with hOCT1 and hOCT2 indicated competitive inhibition of representative substrate uptake. In contrast, Lineweaver–Burk plots of hOCT3 inhibition by METH and AMP indicated mixed inhibition (Fig. 3). The Ki values were 16.9 µM for METH (via hOCT1), 78.6 µM for AMP (via hOCT1), 17.7 µM for METH (via hOCT2), and 42.8 µM for AMP (via hOCT2) (Table 1); however, the Ki values for hOCT3 could not be calculated because of mixed inhibition. 3.3. Uptake of METH and AMP via hOCTs Cellular uptake of METH and AMP via hOCTs was examined via direct uptake experiments rather than indirect uptake inhibition experiments, with quantitative analysis of samples using LC–MS/MS. The use of LC–MS/MS, that LC–MS/MS has lower sensitivity than that of conventional RI methods, enabled the analysis of METH and AMP using a very small number of cells. These assays indicated uptake of METH and AMP via hOCT3 but not via OCT1 or OCT2. In addition, in experiments comparing METH and AMP uptake via OCT3 at 37 °C and on ice, the values obtained on ice were significantly lower than those obtained at 37 °C (Fig. 4). 4. Discussion Chemical compounds entering the body must be excreted. Many cationic and anionic compounds bind to plasma proteins; for such compounds that do not pass via glomerular filtration in humans, renal tubular or bile canalicular excretion via drug transporters (hOATs and hOCTs) is the primary route of excretion. These transporters have been shown to mainly mediate the transport of small organic anions and cations (molecular weight < 500 Da) through the kidney and liver [32]. hOATs and hOCTs act as xenobiotic transporters that also serve as essential multi-specific organic anion or cation transporters. Immunohistochemical analyses revealed that hOAT1, hOAT2, hOAT3, and hOCT2 are localized on the basolateral side of the proximal tubule [33], whereas hOAT4 is localized on the apical side [34]. In contrast, hOCT1 is localized on the basolateral side of hepatocytes [21,35]. hOCT3 is expressed in a wide range of organs, such as the placenta [27], heart, brain [28], intestines, skeletal muscle, kidney, and liver. Measurement of the uptake of directly radiolabeled test drug (i.e., METH and AMP in this study) via a transporter is the best method for evaluating substrate specificity in pharmacokinetic studies. Radiolabeled METH and AMP (100% import) are not legally available in Japan because their import is prohibited. We investigated the interactions between hOATs or hOCTs and these substances using competitive inhibition assays of radiolabeled representative substrate uptake into S2-hOAT- and S2-hOCToverexpressing cells to determine the entry pathway of METH and AMP. We demonstrated that METH and AMP inhibited > 50% of hOCTmediated representative substrate transport while exhibiting no 6

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there for a long time or require a long time to transport into the cells of the proximal tubules, based on the above data. Therefore, if a person simultaneously ingests cationic drugs, the pharmacokinetics of these drugs can be markedly affected by blocked excretion mediated by stimulant drugs [36]. On the other hand, the passage of stimulant drugs through hOCT3 is proven. Thus, it is presumed the excretion of stimulant drugs is inhibited in cases of simultaneously ingesting drugs that connect specifically with hOCT3 and stimulant drugs [37]. There are individual differences in the activity and expression levels of transporters [38]. Transporters are greatly affected by the pharmacokinetic parameters known as “ADME” – acronyms representing the process of pharmacokinetic until drugs are excreted after being taken: A (absorption), D (distribution), M(metabolism), E (excretion), primarily “A”, “D”, and “E”. In other words, the blood concentration is associated with absorption (A), the organ concentration with distribution (D), and the urinary and bile concentrations with excretion (E) [39,40] Pharmacokinetic parameters can be particularly affected by SNPs in transporter genes. The concentration in the body can differ between individuals in cases in which individuals take a fixed dose of a drug. Even if one orally ingests a lethal dose of a drug, death may not ensue in situations in which absorption transporters are decreased expression in the digestive canal. In contrast, if excretion transporters are decreased expression, resulting in the maintenance of high blood levels of a drug over time, death can occur, even with ingestion of a small, sub-lethal dose of a drug [41–46]. Regarding these issues, kinetic analyses of hOCTs using cells overexpressing hOCT genes should help elucidate the molecular features of the toxicokinetics and pharmacokinetics of METH and AMP, which should aid in the development of drug–drug interaction studies. In autopsies involving suspected intoxication due to interactions between OTC and illicit drugs such as stimulant drugs, the significant connection between absorption, distribution, and excretion in terms of pharmacokinetics should be considered. The concentration of a drug in the body can depend on drug-drug interactions.

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

Acknowledgements

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We would like to express our deepest appreciation to Dr. Hitoshi Endou, Professor Emeritus at the University of Kyorin, who donated stable hOAT- and hOCT-expressing cells, and Dr. Takeshi Saito, Associate Professor at the Tokai University School of Medicine, who donated amphetamine and provided helpful comments and suggestions.

[24]

[25]

Funding source

[26]

St. Marianna University School of Medicine provided financial support for this research. No other financial support from any funding agency in the public, commercial, or not-for-profit sectors was received.

[27]

[28]

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