In Vitro Inhibitory Effects of APINACA on Human Major Cytochrome P450, UDP-Glucuronosyltransferase Enzymes, and Drug Transporters

APINACA (known as AKB48, N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide), an indazole carboxamide synthetic cannabinoid, has been used worldwide as a new psychoactive substance. Drug abusers take various drugs concomitantly, and therefore, it is necessary to characterize the potential of APINACA-induced drug–drug interactions due to the modulation of drug-metabolizing enzymes and transporters. In this study, the inhibitory effects of APINACA on eight major human cytochrome P450s (CYPs) and six uridine 5′-diphospho-glucuronosyltransferases (UGTs) in human liver microsomes, as well as on the transport activities of six solute carrier transporters and two efflux transporters in transporter-overexpressed cells, were investigated. APINACA exhibited time-dependent inhibition of CYP3A4-mediated midazolam 1′-hydroxylation (Ki, 4.5 µM; kinact, 0.04686 min−1) and noncompetitive inhibition of UGT1A9-mediated mycophenolic acid glucuronidation (Ki, 5.9 µM). APINACA did not significantly inhibit the CYPs 1A2, 2A6, 2B6, 2C8/9/19, or 2D6 or the UGTs 1A1, 1A3, 1A4, 1A6, or 2B7 at concentrations up to 100 µM. APINACA did not significantly inhibit the transport activities of organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)1, OCT2, P-glycoprotein, or breast cancer resistance protein at concentrations up to 250 μM. These data suggest that APINACA can cause drug interactions in the clinic via the inhibition of CYP3A4 or UGT1A9 activities.


Introduction
Synthetic cannabinoids (SCs) are a type of new psychoactive substance that mimic ∆9-tetrahydrocannabinol (THC), the active component of cannabis, and typically bind to cannabinoid receptor type 1 or type 2 [1]. The misuse of SCs has increased worldwide, and 169 of these SCs have been monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) through the EU Early Warning System of December 2016.
APINACA (known as AKB48, N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide) is an SC classified as an indazole carboxamide ( Figure 1) [2]. APINACA was identified for the first time in Japanese herbal smoking blends in 2012 and has been categorized in Schedule I of the Controlled Substances Act Substances Act by the US Drug Enforcement Administration since 2013. APINACA is extensively metabolized to 10 metabolites in vitro and in vivo via hydroxylation and oxidation at pentyl and adamantyl moieties by cytochrome P450 (CYP) enzymes, such as CYPs 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4, and via carboxylation on the pentyl group by alcohol dehydrogenase/acetaldehyde dehydrogenase [3][4][5][6][7]. Drugs not only are the substrates for drug-metabolizing enzymes, such as CYP and uridine 5′diphospho-glucuronosyltransferase (UGT), but also may cause drug interactions with coadministered drugs and affect drug metabolism via the inhibition or induction of CYP and UGT enzymes [6,8,9]. Drugs are also the substrate for transporters that play crucial roles in the absorption and disposition of these drugs: therefore, transporter-mediated drug-drug interactions have become an important issue in drug-metabolizing enzyme-mediated drug interactions [10]. According to the guidelines of the US Food and Drug Administration and the International Transporter Consortium, it is necessary to evaluate the effects of new drug candidates on clinically important solute carrier transporters, including organic cation transporter (OCT)1, OCT2, organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, and OATP1B3; and efflux transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to predict transportermediated drug-drug interactions.
The effects of various abused drugs on transporters have been reported to predict transportermediated drug interactions: for example, buprenorphine, norbuprenorphine, ibogaine, methadone, and THC inhibited P-gp in HEK293-MDR1 cells, and buprenorphine, norbuprenorphine, and ibogaine inhibited BCRP in HEK293BCRP cells [25]. Phytocannabinoids (THC, cannabinol, and cannabidiol), JWH-200, and WIN-55,212-2 inhibited BCRP ATPase activity [26,27]. Diclofensine, glaucine, 2,5-dimethoxy-4-iodoamphetamine, N-isopropyl-1,2-diphenylethylamine, and N-(1phenylcyclohexyl)-3-ethoxypropanamine stimulated similar ATPase activity to verapamil and sertraline [28]. Glaucine, JWH-200, mitragynine, and WIN-55,212-2 were shown not to be P-gp substrates, but P-gp inhibitors, in Caco-2 cells [29]. APINACA inhibited dopamine uptake in the human dopamine transporter overexpressed system, with a Ki value of 4.6 µM [30]. Drugs not only are the substrates for drug-metabolizing enzymes, such as CYP and uridine 5 -diphospho-glucuronosyltransferase (UGT), but also may cause drug interactions with coadministered drugs and affect drug metabolism via the inhibition or induction of CYP and UGT enzymes [6,8,9]. Drugs are also the substrate for transporters that play crucial roles in the absorption and disposition of these drugs: therefore, transporter-mediated drug-drug interactions have become an important issue in drug-metabolizing enzyme-mediated drug interactions [10]. According to the guidelines of the US Food and Drug Administration and the International Transporter Consortium, it is necessary to evaluate the effects of new drug candidates on clinically important solute carrier transporters, including organic cation transporter (OCT)1, OCT2, organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, and OATP1B3; and efflux transporters, such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to predict transporter-mediated drug-drug interactions.
However, no report exists concerning the in vitro and in vivo inhibitory effects of APINACA on major human drug-metabolizing enzymes, such as CYPs and UGTs, solute carrier transporters, and efflux transporters.
The purpose of this study was to investigate the in vitro inhibitory effects of APINACA on the activities of eight major human CYPs and six UGTs in ultrapooled human liver microsomes and on the transport activities of six solute carrier transporters and two efflux transporters in transporter-overexpressed cells to predict the potentials for APINACA-induced drug interactions.
Based on an enzyme kinetics study, APINACA noncompetitively inhibited UGT1A9-catalyzed mycophenolic acid glucuronidation with a Ki value of 5.9 µM in human liver microsomes ( Figure 5).   Based on an enzyme kinetics study, APINACA noncompetitively inhibited UGT1A9-catalyzed mycophenolic acid glucuronidation with a K i value of 5.9 µM in human liver microsomes ( Figure 5).

Inhibitory Effect of APINACA on Drug Transporters
The functionality of these transport systems was confirmed by the significantly greater transport rates in the probe substrates in HEK293 cells overexpressing solute carrier transporters or LLC-PK1 cells overexpressing efflux transporters than in mock cells (Table 2), consistent with our previous reports [31 -33]. Using the same system, inhibitory potentials of typical inhibitors of uptake and efflux transporters were evaluated. Cimetidine inhibited OCT1 and OCT2 with IC50 values of 61.8 µM and

Inhibitory Effect of APINACA on Drug Transporters
The functionality of these transport systems was confirmed by the significantly greater transport rates in the probe substrates in HEK293 cells overexpressing solute carrier transporters or LLC-PK1 cells overexpressing efflux transporters than in mock cells (Table 2), consistent with our previous reports [31][32][33]. Using the same system, inhibitory potentials of typical inhibitors of uptake and efflux transporters were evaluated. Cimetidine inhibited OCT1 and OCT2 with IC 50 values of 61. The inhibitory effects of APINACA on eight major transporters were evaluated using mammalian cells overexpressing OCT1, OCT2, OAT1, OAT3, OATP1B1, OATP1B3, P-gp, and BCRP. APINACA did not inhibit significantly the transport activities of OAT1, OAT3, OATP1B1, OATP1B3, OCT1, OCT2, P-gp, or BCRP in the concentration ranges tested ( Figure 6). The data are expressed as the means ± SD from triplicate measurements.
On the other hand, APINACA poorly inhibited solute carrier transporters, such as OAT1, OAT3, OCT1, OCT2, OATP1B1, and OATP1B3; and efflux transporters, such as P-gp and BCRP, even though APINACA was treated at high concentrations (up to 250 µM), suggesting that APINACA has a low potential for drug interactions in association with these transporters. Therefore, APINACA exposure in human blood caused by APINACA abuse might not potentiate the transporter-mediated toxicity or adverse events of APINACA.
For the accurate prediction of APINACA-induced drug interaction potential in the clinic from in vitro data, information regarding APINACA pharmacokinetics in humans, including plasma concentrations, protein binding, tissue distribution, etc., is necessary. However, there has been no report on the absorption, distribution, and excretion of APINACA in humans and animals. In one study, APINACA was determined concomitantly with 5F-APINACA in blood samples of three "driving under the influence of drugs" cases, and its blood concentrations were 0.66-67.1 nM [42], which did not accurately reflect the maximum blood concentrations and the tissue concentrations of APINACA in the liver. Although the inhibition of CYP and UGT activities in vitro does not necessarily translate into drug interactions in clinical situations, it is necessary to evaluate the potential of in vivo pharmacokinetic drug-drug interactions via APINACA-induced inhibition of CYP3A4 and UGT1A9 activities in the clinic.
To evaluate the inhibitory effect of APINACA on CYP2B6-catalyzed bupropion hydroxylation, each incubation mixture in a total volume of 100 µL contained 50 mM potassium phosphate buffer (pH 7.4), 10 mM MgCl 2 , 0.2 mg/mL pooled human liver microsomes, 50 µM bupropion, and various concentrations of APINACA in methanol (final concentrations of 0.1-100 µM), according to our previous report [43]. After 3 min of preincubation at 37 • C, the reaction mixtures were incubated with the addition of NADPH in a shaking water bath for 15 min at 37 • C. The reaction was stopped by adding 100 µL of ice cold d 9 -1 -hydroxybufuralol (internal standard) in methanol. The mixtures were centrifuged at 13,000× g for 8 min at 4 • C. All incubations were performed in triplicate, and the average values were used for subsequent calculations.
To measure the time-dependent inhibition, human liver microsomes were preincubated with the various concentrations of APINACA (final concentrations of 0.1−100 µM) and NADPH for 30 min at 37 • C. Next, the reaction mixtures were incubated with a seven-CYP probe substrate cocktail or bupropion for 15 min at 37 • C. The control reaction was performed by adding methanol instead of the test compounds.
The LC-MS/MS system was comprised of an Agilent 6495 triple quadrupole mass spectrometer coupled with an Agilent 1290 Infinity system (Agilent Technologies, CA, USA). The column and autosampler temperatures were 40 • C and 4 • C, respectively.
The metabolites formed from the eight CYP substrates were simultaneously separated on an Atlantis dC18 (3 µm, 2.1 mm internal diameter × 100 mm; Waters Co., MA, USA) using a gradient elution of 5% methanol in 0.1% formic acid (mobile phase A) and 95% methanol in 0.1% formic acid (mobile phase B) at a flow rate of 0.3 mL/min: 15% mobile phase B for 1.5 min, 15% to 50% mobile phase B for 0.5 min, 50% to 95% mobile phase B for 2 min, 95% mobile phase B for 2 min, 95% to 15% mobile phase B for 0.

Inhibitory Effect of APINACA on Six Major UGT Activities
The inhibitory effect of APINACA and typical UGT inhibitors on UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 were evaluated following our previous method by LC-MS/MS after incubation of ultrapooled human liver microsomes with a cocktail of UGT substrates [44].
To measure the time-dependent inhibition, human liver microsomes were preincubated with the various concentrations of APINACA (final concentrations of 0.1-100 µM) and UDPGA for 30 min at 37 • C. Next, the reaction mixtures were incubated with UGT probe substrate cocktail sets for 60 min at 37 • C. The control reaction was performed by adding methanol instead of the test compounds.
The metabolites formed from the six UGT substrates were simultaneously separated using an Atlantis dC18 system (3 µm, 2.1 mm internal diameter × 100 mm) with a gradient elution of 5% acetonitrile in 0.1% formic acid (mobile phase A) and 95% acetonitrile in 0.1% formic acid (mobile phase B) at a flow rate of 0.3 mL/min: 10% mobile phase B for 1 min, 10% to 60% mobile phase B for 1 min, 60% to 95% mobile phase B for 1 min, 95% mobile phase B for 2 min, 95% to 10% mobile phase B for 0.1 min, and 10% mobile phase B for 2.9 min. The ESI source settings in both the positive and negative ion modes were as follows: gas temperature, 200 • C; gas flow, 14 L/min; nebulizer, 40 psi; sheath gas temperature, 380 • C; sheath gas flow, 11 L/min; capillary voltage, 4500 V; and nozzle voltage, 500 V. Each metabolite was quantified via selected reaction monitoring in the negative ion mode (chenodeoxycholic acid 24-acyl-β-glucuronide, m/z 567.1 → 391.

Time-Dependent Inhibition of CYP3A4 Activity by APINACA in Human Liver Microsomes
The kinetic parameters for the time-dependent inhibition potency of APINACA against human liver microsomal CYP3A4 activity were evaluated. Ultrapooled human liver microsomes (1 mg/mL) were preincubated with various concentrations of APINACA (final concentrations of 1-40 µM) in 50 mM potassium phosphate buffer (pH 7.4) in the presence of NADPH. Aliquots (10 µL) of the preincubated mixtures were withdrawn 5, 10, 20, and 30 min after incubation was commenced and were added to other tubes containing 2 µM midazolam, 1 mM NADPH, 50 mM potassium phosphate buffer (pH 7.4), and 10 mM MgCl 2 in 90-µL reaction mixtures. The second reactions were terminated after 10 min by adding 100 µL of ice cold methanol containing d 9 -1 -hydroxybufuralol. The incubation mixtures were centrifuged at 13,000× g for 8 min at 4 • C, and 50 µL of each supernatant was diluted with 50 µL of water. Aliquots (5 µL) of the diluted supernatants were analyzed by LC-MS/MS.

Enzyme Kinetic Analysis for the Inhibition of UGT1A9 by APINACA
To determine the enzyme kinetic parameters and mode of inhibition of UGT1A9 by APINACA, various concentrations of APINACA (final concentrations of 0-10 µM) and mycophenolic acid (final concentrations of 0.2-1.0 µM) as the UGT1A9 substrate were incubated with human liver microsomes (0.15 mg/mL), 10 mM MgCl 2 , 5 mM UGPGA, and 50 mM Tris buffer (pH 7.4) in a total volume of 100 µL for 60 min at 37 • C. The reaction was stopped by adding 100 µL of ice cold acetonitrile containing propofol glucuronide (internal standard), and the mixtures were centrifuged at 13,000× g for 4 min. Next, 50 µL of the supernatant was diluted with 50 µL of water, and aliquots (5 µL) were analyzed by LC-MS/MS.

Inhibitory Effect of APINACA on the Transport Activities of Efflux Transporters
LLC-PK1-MDR1 and LLC-PK1-mock cells were grown in tissue culture flasks in medium 199 supplemented with 8% FBS, 50 µg/mL of gentamycin, and 50 µg/mL of hygromycin. The cells were seeded onto filter membranes at a density of 2 × 10 5 cells/well for 5 days with TEER values over 450 Ω·cm 2 . The B to A transport of 0.1 µM [3-H]digoxin in LLC-PK1-MDR1 cells was measured by adding 1.5 mL of HBSS containing APINACA (final concentrations of 0-100 µM) on the basal side and by adding 0.5 mL of HBSS without APINACA on the apical side of the insert using protocols identical to those described above. The B to A transport of 0.1 µM [3-H]digoxin in LLC-PK1-mock cells was also measured using the same protocol for comparison.
LLC-PK1-BCRP and -mock cells were grown in tissue culture flasks in DMEM supplemented with 10% FBS, 5 mM nonessential amino acids, and 100 U/mL of penicillin-streptomycin. The cells were seeded onto filter membranes at a density of 2 × 10 5 cells/well for 5 days with TEER values over 300 Ω·cm 2 . The B to A transport of 0.1 µM [3-H]ES was measured by adding 1.5 mL of HBSS containing APINACA (final concentrations of 0-100 µM) on the basal side and by adding 0.5 mL of HBSS without APINACA on the apical side of the insert using protocols identical to those described above. The B to A transport of 0.1 µM [3-H]ES in LLC-PK1-mock cells was also measured using the same protocols for comparison.
Aliquots (100 µL) of transport samples were mixed with 200 µL of Optiphase cocktail solution (Perkin Elmer Inc.; Boston, MA, USA). The radioactivity of the probe substrate in the cells was measured using a liquid scintillation counter.

Inhibitory Effect of APINACA on the Transport Activities of Solute Carrier Transporters
HEK293 cells overexpressing OAT1, OAT3, OATP1B1, OATP1B3, OCT1, and OCT2 transporters and HEK293-mock cells were seeded in poly-d-lysine-coated 96-well plates at a density of 10 5 cells/well and were cultured in DMEM supplemented with 10% FBS, 5 mM nonessential amino acids, and 2 mM sodium butyrate in a humidified atmosphere of 5% CO 2 at 37 • C. For the experiments, the growth medium was discarded after 24 h, and the attached cells were washed with HBSS and preincubated for 10 min in HBSS at 37 • C.
To examine the effects of APINACA and typical inhibitors on transporter activity, the uptake of a probe substrate into HEK293 cells overexpressing the respective solute carrier transporters was measured in the presence of APINACA (final concentrations of 0-250 µM) or typical inhibitors for 5 min. The concentrations and probe substrates were selected as follows: 0.1 µM The uptake of the probe substrate into HEK293-mock cells was also measured using the same protocol for comparison. The typical inhibitors were selected as follows: cimetidine (0-250 µM) for OCT1 and OCT2, probenecid (0-250 µM) for OAT1 and OAT3, rifampin (0-250 µM) for OATP1B1 and OATP1B3, verapamil (0-250 µM) for P-gp, and sulfasalazine (0-250 µM) for BCRP.
The cells were then washed three times with 100 µL of ice cold HBSS immediately after placement of the plates on ice, and the cells were lysed with 50 µL of 10% sodium dodecyl sulfate and mixed with 150 µL of Optiphase cocktail solution (Perkin Elmer Inc.; Boston, MA, USA). The radioactivity of the probe substrates in the cells was measured using a liquid scintillation counter.

Data Analysis
The IC 50 (the concentration of the inhibitor to show half-maximal inhibition) values were calculated using SigmaPlot ver. 12.5 (Systat Software, Inc.; San Jose, CA, USA). K i (the inhibition constant), k inact (the maximal rate of enzyme inactivation), and the mode of inhibition of CYP3A4 and UGT1A9 activities were determined using Enzyme Kinetics ver. 1.1 (Systat Software, Inc.).

Conclusions
The in vitro inhibitory effect of APINACA on eight major clinically important CYP and six UGT enzymes in ultrapooled human liver microsomes and on six solute carrier transporters and two efflux transporters using a transporter expression system was investigated for the first time to predict the drug interaction potential of APINACA via the modulation of drug-metabolizing enzymes and transporters. APINACA showed potent time-dependent inhibition of CYP3A4-mediated midazolam 1 -hydroxylation (K i , 4.5 µM) and noncompetitive inhibition of UGT1A9-mediated mycophenolic acid glucuronidation (K i , 5.9 µM), but did not show inhibition of other tested CYPs, UGTs, solute carrier transporters, and efflux transporters. These results suggest that it is necessary to evaluate the potential of APINACA as an in vivo cause of pharmacokinetic drug interactions via the inhibition of CYP3A4 and UGT1A9 enzymes.