Inhibitory Effect of AB-PINACA, Indazole Carboxamide Synthetic Cannabinoid, on Human Major Drug-Metabolizing Enzymes and Transporters

Indazole carboxamide synthetic cannabinoid, AB-PINACA, has been placed into Schedule I of the Controlled Substances Act by the US Drug Enforcement Administration since 2015. Despite the possibility of AB-PINACA exposure in drug abusers, the interactions between AB-PINACA and drug-metabolizing enzymes and transporters that play crucial roles in the pharmacokinetics and efficacy of various substrate drugs have not been investigated. This study was performed to investigate the inhibitory effects of AB-PINACA on eight clinically important human major cytochrome P450s (CYPs) and six uridine 5′-diphospho-glucuronosyltransferases (UGT) in human liver microsomes and the activities of six solute carrier transporters and two efflux transporters in transporter-overexpressing cells. AB-PINACA reversibly inhibited the metabolic activities of CYP2C8 (Ki, 16.9 µM), CYP2C9 (Ki, 6.7 µM), and CYP2C19 (Ki, 16.1 µM) and the transport activity of OAT3 (Ki, 8.3 µM). It exhibited time-dependent inhibition on CYP3A4 (Ki, 17.6 µM; kinact, 0.04047 min−1). Other metabolizing enzymes and transporters such as CYP1A2, CYP2A6, CYP2B6, CYP2D6, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, OAT1, OATP1B1, OATP1B3, OCT1, OCT2, P-glycoprotein, and BCRP, exhibited only weak interactions with AB-PINACA. These data suggest that AB-PINACA can cause drug-drug interactions with CYP3A4 substrates but that the significance of drug interactions between AB-PINACA and CYP2C8, CYP2C9, CYP2C19, or OAT3 substrates should be interpreted carefully.


Inhibitory
Effects of AB-PINACA on the Transport Activities of OCTs, OATs, OATPs, P-gp, and BCRP HEK293-OCT1, -OCT2, -OAT1, -OAT3, -OATP1B1, and -OATP1B3 cells and HEK293-mock cells were seeded at a density of 10 5 cells/well in poly-d-lysine-coated 96-well plates and cultured in DMEM supplemented with 10% FBS, 5 mM nonessential amino acids, and 2 mM sodium butyrate for 24 h at 37 • C in a humidified atmosphere of 8% CO 2 . Twenty-four hours after seeding of the cells, the growth medium was discarded and the attached cells were washed with prewarmed HBSS and preincubated for 10 min in prewarmed HBSS at 37 • C.
LLC-PK1-MDR1, LLC-PK1-BCRP, and LLC-PK1-mock cells were grown in tissue culture flasks in medium 199 supplemented with 10% FBS, and 50 µg/mL of gentamycin. The cells were seeded at a density of 10 5 cells/well onto the insert membrane of 24 well Transwell plates and cultured for 5 days in medium 199 supplemented with 10% FBS, and 50 µg/mL of gentamycin. After reaching TEER values of the cells over 450 Ω·cm 2 , the B to A transport of digoxin in LLC-PK1-MDR1 and LLC-PK1-mock cells was initiated by adding 0.8 mL of HBSS containing 0.1 µM 3 H-digoxin and AB-PINACA (0-100 µM) to the basal side and by adding 0.4 mL of fresh pre-warmed HBSS to the apical side of the Transwell chamber. Every 15 min for 1 h, an aliquot of 0.3 mL was taken from the apical side of the Transwell chamber and volume loss in the apical side was compensated by addition of 0.3 mL of fresh pre-warmed HBSS. The B to A transport of estrone-3-sulfate in LLC-PK1-BCRP and LLC-PK1-mock cells was also measured using the same protocol in the presence of 0.1 µM [ 3 H]estrone-3-sulfate and AB-PINACA (0-100 µM) on the basal side. Aliquots (100 µL) of transport samples were mixed with 200 µL of Optiphase scintillation cocktail. The radioactivity of the probe substrate in the cocktail mixture was measured using a liquid scintillation counter. The B to A transport of digoxin and ES mediated by MDR1 and BCRP, respectively, was calculated by subtracting the transport of probe substrate in LLC-PK1-mock cells from that in LLC-PK1-MDR1 and LLC-PK1-BCRP cells and the B to A transport rate of probe substrate was calculated from the slope of the B to A transport of probe substrate versus time graph [18,25]. The active sites were determined with reference to the PDB. For docking analysis at the active site, spherical binding sites were formed on CYP2C8, CYP2C9, CYP2C19, and CYP3A4. AB-PINACA was docked at the binding site through the CDOCKER protocol. After docking, the interaction of protein and ligand in the binding site was analyzed using the protein-ligand interaction tool. The number of poses per ligand was set to at least 10, and the lowest CDOCKER interaction energy was used. Other parameters were set at default values.

Data Analysis
To calculate IC 50 values (half-maximal inhibitory concentrations) of AB-PINACA, the inhibition data were fitted to an inhibitory effect model using SigmaPlot (version 12.0; Systat Software Inc., San Jose, CA, USA). The values of the inhibition constants (K i ) of AB-PINACA and the mode of inhibition were calculated from Lineweaver-Burk and Dixon plots [26]. Parameters for time-dependent inhibition such as K i and k inact (maximal rate of enzyme inactivation) were determined using SigmaPlot version 12.0 [18].

Inhibitory Effects of AB-PINACA on CYP and UGT Activities in Human Liver Microsomes
Inhibitory effects of AB-PINACA on eight CYPs were estimated in ultrapooled human liver microsomes. To determine whether the time dependency of CYP inhibition by AB-PINACA is involved, the inhibitory effects of AB-PINACA on the catalytic activities of eight CYPs were measured with or without NADPH preincubation for 30 min ( Figure 1, Table 1). AB-PINACA inhibited CYP2C8-, CYP2C9-, and CYP2C19-catalyzed hydroxylation of amodiaquine, diclofenac, and [S]-mephenytoin with IC 50 values of 32.5 µM, 17.4 µM, and 20.0 µM, respectively, in human liver microsomes, but 30-min preincubation of AB-PINACA with NADPH did not significantly change the inhibitory potential of AB-PINACA toward CYP2C8, CYP2C9, and CYP2C19. However, preincubation of AB-PINACA with NADPH resulted in a 2.7-fold IC 50 shift of CYP3A4-catalyzed midazolam 1 -hydroxylation (66.4 µM without NADPH preincubation vs. 24.4 µM with NADPH preincubation, Figure 1, Table 1), suggesting that AB-PINACA is a time-dependent inhibitor of CYP3A4. However, AB-PINACA did not significantly inhibit the catalytic metabolic activities of CYP1A2, CYP2A6, CYP2B6, and CYP2D6 in ultrapooled human liver microsomes regardless of preincubation of AB-PINACA with NADPH (IC 50 > 100 µM in all cases) ( Figure 1, Table 1).

Inhibitory Effects of AB-PINACA on CYP and UGT Activities in Human Liver Microsomes
Inhibitory effects of AB-PINACA on eight CYPs were estimated in ultrapooled human liver microsomes. To determine whether the time dependency of CYP inhibition by AB-PINACA is involved, the inhibitory effects of AB-PINACA on the catalytic activities of eight CYPs were measured with or without NADPH preincubation for 30 min ( Figure 1, Table 1). AB-PINACA inhibited CYP2C8-, CYP2C9-, and CYP2C19-catalyzed hydroxylation of amodiaquine, diclofenac, and [S]-mephenytoin with IC50 values of 32.5 µM, 17.4 µM, and 20.0 µM, respectively, in human liver microsomes, but 30-min preincubation of AB-PINACA with NADPH did not significantly change the inhibitory potential of AB-PINACA toward CYP2C8, CYP2C9, and CYP2C19. However, preincubation of AB-PINACA with NADPH resulted in a 2.7-fold IC50 shift of CYP3A4-catalyzed midazolam 1′-hydroxylation (66.4 µM without NADPH preincubation vs. 24.4 µM with NADPH preincubation, Figure 1, Table 1), suggesting that AB-PINACA is a time-dependent inhibitor of CYP3A4. However, AB-PINACA did not significantly inhibit the catalytic metabolic activities of CYP1A2, CYP2A6, CYP2B6, and CYP2D6 in ultrapooled human liver microsomes regardless of preincubation of AB-PINACA with NADPH (IC50 > 100 µM in all cases) ( Figure 1, Table 1).  Next, we investigated the inhibitory effects of AB-PINACA on the catalytic activities of CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4 -hydroxylation, and CYP2C19-mediated [S]-mephenytoin 4 -hydroxylation with different substrate concentrations and varying concentrations of AB-PINACA and the results were transformed into Lineweaver-Burk and Dixon plots to determine the inhibition mode of AB-PINACA on the catalytic activities of CYP2C8, CYP2C9, and CYP2C19 along with the K i values (Figure 2). Enzyme kinetic results on the inhibition of CYP2C8 and CYP2C19 activities revealed mixed inhibition with K i values of 16.9 µM and 6.7 µM, respectively (Figure 2A,C, Table 2). AB-PINACA competitively inhibited CYP2C9 activity with a K i value of 16.1 µM ( Figure 2B, Table 2). To characterize the time-dependent inhibition of CYP3A4 by AB-PINACA, the inactivation kinetics for the formation of 1 -hydroxymidazolam from midazolam in the presence of AB-PINACA were measured: K i and k inact values of AB-PINACA were 17.6 µM and 0.04047 min −1 , respectively ( Figure 3 and Table 2).
AB-PINACA had low inhibitory potential for the glucuronidation activities of UGT1A3, UGT1A6, and UGT1A9 with IC 50 values of 84.1 µM, 94.0 µM, and 65.2 µM, respectively, and showed negligible inhibition of the glucuronidation activities of UGT1A1, UGT1A4, and UGT2B7 in human liver microsomes in the concentration range of 0.1-100 µM AB-PINACA ( Figure 4). Data represent the average of three measurements.
Next, we investigated the inhibitory effects of AB-PINACA on the catalytic activities of CYP2C8catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, and CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation with different substrate concentrations and varying concentrations of AB-PINACA and the results were transformed into Lineweaver-Burk and Dixon plots to determine the inhibition mode of AB-PINACA on the catalytic activities of CYP2C8, CYP2C9, and CYP2C19 along with the Ki values ( Figure 2). Enzyme kinetic results on the inhibition of CYP2C8 and CYP2C19 activities revealed mixed inhibition with Ki values of 16.9 µM and 6.7 µM, respectively (Figure 2A,C, Table 2). AB-PINACA competitively inhibited CYP2C9 activity with a Ki value of 16.1 µM ( Figure 2B, Table 2).  To characterize the time-dependent inhibition of CYP3A4 by AB-PINACA, the inactivation kinetics for the formation of 1′-hydroxymidazolam from midazolam in the presence of AB-PINACA were measured: Ki and kinact values of AB-PINACA were 17.6 µM and 0.04047 min −1 , respectively ( Figure 3 and Table 2).   Table 2).  AB-PINACA had low inhibitory potential for the glucuronidation activities of UGT1A3, UGT1A6, and UGT1A9 with IC50 values of 84.1 µM, 94.0 µM, and 65.2 µM, respectively, and showed negligible inhibition of the glucuronidation activities of UGT1A1, UGT1A4, and UGT2B7 in human liver microsomes in the concentration range of 0.1-100 µM AB-PINACA (Figure 4).
Among the eight transporters tested, OCT1 and OAT3 transporters that were inhibited by AB-PINACA were further subjected to enzyme kinetic studies to determine the mode of inhibition and K i values. Lineweaver-Burk and Dixon plots of the inhibitory effect of AB-PINACA on the OCT1-mediated methyl-4-phenylpyridinium uptake revealed mixed inhibition with a K i value of 145.7 µM ( Figure 6A and Table 2). AB-PINACA competitively inhibited OAT3-mediated ES uptake with a K i value of 8.3 µM ( Figure 6B and Table 2).  Among the eight transporters tested, OCT1 and OAT3 transporters that were inhibited by AB-PINACA were further subjected to enzyme kinetic studies to determine the mode of inhibition and Ki values. Lineweaver-Burk and Dixon plots of the inhibitory effect of AB-PINACA on the OCT1mediated methyl-4-phenylpyridinium uptake revealed mixed inhibition with a Ki value of 145.7 µM ( Figure 6A and Table 2). AB-PINACA competitively inhibited OAT3-mediated ES uptake with a Ki value of 8.3 µM ( Figure 6B and Table 2).
However, AB-PINACA, an indazole carboxamide synthetic cannabinoid, inhibited CYP2C8-catalyzed amodiaquine N-deethylation (K i , 16.9 µM), and CYP2C9-catalyzed diclofenac 4 -hydroxylation (K i , 6.7 µM) with a mixed mode of inhibition (Table 2). It also competitively inhibited CYP2C19-mediated [S]-mephenytoin 4 -hydroxylation with a K i value of 16.1 µM ( Table 2). As a result that the reported plasma concentration of AB-PINACA is in the range of 1.8-127 nM, although the dose regimen was not specified [10], and was much lower than the K i values of AB-PINACA, the inhibition of CYP2C8, CYP2C9, and CYP2C19 by the presence of AB-PINACA will not cause clinically significant drug-drug interactions.
Previous studies have reported that the active site cavity of CYP2C8 is located on either side of the helix B-C loop and is bound by helix I, the helix F-G region, portions of β-sheet 1, the turn in β-sheet 4, and the loop between helix K and β-sheet 1 [27]; therefore, it has sufficient space to capture AB-PINACA. However, unlike with CYP2C8-9-cis-retinoic acid crystal complex, in which 9-cis-retinoic acid formed hydrogen bonds with Arg241, the carboxamide group of AB-PINACA formed hydrogen bonds with heme (2.95 Å) of CYP2C8 ( Figure 7A,B). Importantly, AB-PINACA shared the same amino acids, Ile113, Phe205, Leu208, Val296, and Val336 with 9-cis-retinoic acid to form hydrophobic interactions with CYP2C8, which formed additional interaction with Ile106 and Ile476 ( Figure 7A,B and Table 3). As a result that CYP2C8 forms a homodimer that is connected by two molecules of palmitic acid and contains two 9-cis-retinoic acids in the active pocket in its crystal structure, it is not clear whether or not the inhibition of AB-PINACA against CYP2C8 requires two cis-retinoic acids.
As a result that human CYP2C9 and CYP2C19 have roughly 91% amino acid identity, it is predicted that the 3D structures and the substrate selectivity may be redundant. However, although published structures of these two enzymes do not show marked differences in the active site cavity, it is surprising that they exhibit distinct substrate selectivity [29,33,34]. This suggests that conformational changes underlie differences in the substrate selectivity [28]. Our study yielded docking scores of AB-PINACA against CYP2C9 and CYP2C19 of −26.99 and −33.15 kcal/mol, respectively ( Table 3). The difference in binding energy may be due to the interactions of hydrogen bonds between AB-PINACA and Arg108 of CYP2C9 or Asp239/Heme of CYP2C19 ( Figure 7C). Structural analyses between AB-PINACA and CYP2C9 or CYP2C19 revealed that the distance between AB-PINACA and Arg108 was 4.47 Å, whereas CYP2C19 had distances of 2.95 and 2.62 Å between AB-PINACA and Asp239 and Heme, respectively ( Figure 7C-F). Moreover, our results revealed that AB-PINACA efficiently inhibited CYP2C19 activity with a lower K i value than CYP2C9 (Table 2). Additionally, CYP3A4 had diverse hydrogen bonds to Arg105, Arg372, Glu374, and Heme with 2.26-2.89 Å and energy score of −32.34 kcal/mol ( Figure 7G,H and Table 3). Since interactions of CYP3A4 with tert-butyl{6-oxo-6-[(pyridin-3-ylmethyl)amino]hexyl}carbamate or midazolam, a CYP3A4 substrate, are known to occur through Arg106, Ala370, and Glu374, or Arg105 and Ile369, respectively [30], it is possible that the interaction between CYP3A4 and AB-PINACA is through the region making up the active site of CYP3A4 containing Arg105/106 and Ilu369-Glu374. These results suggest that AB-PINACA may preferentially interact with CYP2C19 at the active site under physiological conditions. However, the observation that the distances between AB-PINACA and amino acids of CYP3A4 are short for hydrogen bonds ( Figure 7G,H and Table 3) means that crystallization of AB-PINACA and CYP3A4 may be necessary to resolve this issue.
AB-PINACA inhibited OCT1-mediated methyl-4-phenylpyridinium uptake with a mixed mode of inhibition and also competitively inhibited OAT3-mediated estrone-3-sulfate uptake with K i values of 145.7 µM and 8.3 µM, respectively ( Figure 6 and Table 2). Although the clinical data regarding the pharmacokinetics of AB-PINACA for prediction of AB-PINACA-induced drug interaction are limited, plasma concentration of AB-PINACA (1.8-127 nM) in humans much lower than the in vitro K i values of AB-PINACA for OCT1 and OAT3 is suspected for AB-PINACA intoxication [10]. Thus suggesting a low possibility of clinically significant drug-drug interaction between AB-PINACA and concomitantly administered OCT1 or OAT3 substrate drugs (i.e., metformin, cimetidine, pravastatin, diuretics, and non-steroidal anti-inflammatory drugs) [45]. However, other transporters, such as OCT2, OAT1, OATP1B1, OATP1B3, P-gp, and BCRP, interact poorly with AB-PINACA, even at high AB-PINACA concentrations (up to 250 µM). This suggests that AB-PINACA shows remote drug interaction likelihood for OCT2, OAT1, OATP1B1, OATP1B3, P-gp, and BCRP transporters.

Conclusions
The CYP, UGT, and transporter-mediated drug interaction potentials of AB-PINACA were evaluated based on in vitro inhibitory effects of AB-PINACA on major CYP and UGT enzyme activities and the transport activities of eight drug transporters. Clinically significant drug interaction potentials between AB-PINACA and CYP2C8, CYP2C9, CYP2C19, CYP3A4, or OAT3 substrates should be evaluated carefully, although the plasma concentrations of AB-PINACA reported in drug abusers were much lower than the K i values.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/11/1036/s1, Figure S1. SRM chromatograms of CYP metabolites formed from human liver microsomal incubation of eight CYP cocktail substrates with NADPH and two internal standards (IS), Figure S2. SRM chromatograms of six UGT metabolites formed from human liver microsomal incubation of six UGT cocktail substrates with UDPGA and two IS, Table S1. SRM parameters of eight CYP metabolites, six UGT metabolites, and their internal standards (IS).