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Inhibition of methotrexate uptake by glucuronides of nonsteroidal anti-inflammatory drugs via organic anion transporters OAT1 and OAT3
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
Quantification of relative contributions of individual cytochrome P450 (CYP) isoforms to overall metabolic clearance using reaction phenotyping studies is essential when predicting potential drug-drug interactions. A method that is commonly utilized for such studies is relative activity factor (RAF) using recombinant human CYP isoforms and human liver microsomes. In this approach, the intrinsic clearance of a CYP-selective probe reaction is assessed in both systems to establish a RAF. The RAF acquired is subsequently applied to the intrinsic clearance for a potential drug candidate to assess the relative contribution of each CYP isoform. However, multiple binding sites with diverse substrate selectivity have been eminent for several CYP isoforms, namely CYP2C9 and CYP3A4 which are important in drug metabolism. Based on this rather unique property of CYP, it is conceivable that the drug candidate of interest may not always interact with the same binding site as the probe substrate, from which the RAF was derived. In the present study, CYP-selective substrates were used to establish RAF values for CYP3A4 (i.e., midazolam, testosterone and nifedipine) and CYP2C9 (i.e., S-warfarin, diclofenac and tolbutamide) using recombinant human CYP isoforms and human liver microsomes. The RAF values were further exploited to assess the relative contributions of CYP3A4 or CYP2C9 towards metabolism of these very same probe substrates in a crossover manner. In addition, these RAF values were applied to several CYP2C9 and CYP3A4 model drugs to determine the contributions of CYP isoforms to their overall metabolism. The results demonstrated substantial differences in the relative contributions of the CYP isoforms depending on which probe substrates were used for RAF on a given CYP isoform, further emphasizing the potential impact toward overall metabolic clearance and drug-drug interaction. In conclusion, multiple probe substrates in CYP reaction phenotyping studies using the RAF approach should be evaluated when attempting to predict potential drug-drug interactions in CYP2C9 and CYP3A4. P86 IN VITRO CHARACTERIZATION OF DRUG METABOLIZING ENZYMES AND TRANSPORTERS TO ENABLE A MECHANISTIC DRUG-DRUG INTERACTION ASSESSMENT FOR VENETOCLAX Ryota Kikuchi, Mohamad Shebley, Daniel A.J. Bow, Robert A. Carr, Marjoleen Nijsen, Sonia M. de Morais. AbbVie Inc., North Chicago, IL, USA
In vitro tools were used to characterize the drug-drug interaction (DDI) potential for venetoclax (ABT-199) and its human major metabolite M27 as victims or perpetrators of Cytochrome P450 (CYP), Uridine 5'-diphosphoglucuronosyltransferase (UGT) enzymes and drug transporters. Venetoclax and M27 were predominantly metabolized by CYP3A4, while UGTs were not involved in the metabolism. Both venetoclax and M27 were identified as substrates for the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) but not for hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3 or organic cation transporter (OCT) 1. The nonclinical predictions for venetoclax were consistent with the clinical findings observed with ketoconazole (CYP3A4/ P-gp inhibitor) and rifampin (CYP3A4/P-gp inducer). Venetoclax PBPK model simulations were consistent with results from the clinical ketoconazole and rifampin studies: predicted vs observed ratios for venetoclax Cmax and AUC∞ were within 0.8-1.25 fold. At a 400 mg dose, venetoclax and M27 are not predicted to inhibit CYPs but venetoclax may weakly inhibit UGT1A1 (predicted AUCR 1.38) based on the mechanistic static model. Venetoclax and M27 are P-gp and BCRP inhibitors with IC50 values of 0.79 and 0.13 mM (venetoclax) and 0.83 and 1.48 mM (M27), respectively, and at clinically relevant exposures may inhibit these transporters ([I]2/ IC50 >10 and [I]1/IC50 >0.1). Venetoclax is an OATP1B1 inhibitor (IC50 10.1 mM in the presence of 4% bovine serum albumin) and an R-total value of 1.33 (calculated using total plasma exposure) indicates that venetoclax may interact with drugs that are substrates for this transporter. No interaction with other transporters (OATP1B3, OCT1, OCT2, organic anion transporter (OAT) 1, OAT3, multidrug and toxin extrusion protein (MATE) 1 or MATE2K) was predicted. Overall, an in vitro characterization of drug metabolizing enzyme and transporters enabled a mechanistic assessment of DDI potential for venetoclax. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was
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provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. P87 INHIBITION OF METHOTREXATE UPTAKE BY GLUCURONIDES OF NONSTEROIDAL ANTI-INFLAMMATORY DRUGS VIA ORGANIC ANION TRANSPORTERS OAT1 AND OAT3 Masahiro Iwaki 1, Yuri Irino 2, Manami Take 2, Sachiko Egashira 2. 1 Department of Pharmacy, Faculty of Pharmacy, Kindai University, Higashi-Osaka, Japan; 2 Kindai University, Higashi-Osaka, Japan Introduction: Combined administration of methotrexate (MTX) and nonsteroidal anti-inflammatory drugs (NSAIDs) can result in an elevation of plasma concentration of MTX. This drug interaction has been thought to involve not only the inhibition of basolateral organic anion transporters (OATs) but not apical multidrug resistance proteins (MRPs) by NSAIDs. Most recently, we have shown that the glucuronide conjugates of several NSAIDs exhibit stereoselective and concentration-dependent inhibitory effects on MTX transport via MRP2 and MRP4, with a tendency of the glucuronides to have stronger potencies compared to their parent drugs [1]. As the glucuronides of some NSAIDs are detected in plasma, inhibition of OATs-mediated MTX uptake by the glucuronides may also not be ignored. The purpose of this study was to investigate the inhibitory effects of the glucuronides of several NSAIDs (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen and Rand S-naproxen) on OAT1- and OAT3-mediated MTX transport. Methods: b-1-O-glucuronides of NSAIDs were prepared biosynthetically in vitro from respective NSAIDs using rat liver microsomes. Uptake experiments were performed using human OAT1- and OAT3-expressing HEK293 cells to assess the inhibitory effects of the glucuronides against hOATs-mediated MTX transport in the presence or absence of the glucuronides. The amounts of [3H]MTX taken into the cells were determined using a liquid scintillation counter. The 50% inhibitory concentrations (IC50) of the glucuronides were obtained from curve-fitting of the resulting concentration-inhibition curves by nonlinear regression analysis. Results and Discussion: All examined glucuronides of NSAIDs exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3 with different potencies. Diclofenac glucuronide had most potent inhibitory effect against OAT3 (IC50¼3.2 mM) among the glucuronides tested, and this value was extremely lower than that against OAT1 (IC50¼ 265 mM). Similarly, the IC50 values against OAT3 in all glucuronides of the enantiomers were 5-15 times smaller than those against OAT1, showing that the glucuronides potently inhibit OAT3-mediated MTX transport. There was no difference between both enantiomers of each NSAID in the inhibitory potencies unlike the MRPs-mediated MTX transport previously reported [1]. The present results suggest that inhibition of OAT3 by plasma glucuronides of NSAIDs may be the fourth competitive site underlying complex drug interaction between MTX and NSAIDs in addition to inhibition of basolateral OATs by parent NSAIDs and of apical MRPs by NSAIDs and their glucuronide. Conclusion: The glucuronides of NSAIDs, as well as the parent NSAIDs, can inhibit OAT1and OAT3-mediated MTX transport. Stereoselectivity was not observed in their inhibitory effect on both OAT1 and OAT3. 1. Kawase A., Yamamoto T., Egashira S., and Iwaki M., Stereoselective inhibition of methotrexate excretion by glucuronides of nonsteroidal antiinflammatory drugs via multidrug resistance proteins 2 and 4, J Pharmacol. Exp. Ther., n press. P88 INTERACTION BETWEEN LOBEGLITAZONE AND ATORVASTATIN IN RATS: INVOLVMENT OF TRANSPORTER-MEDIATED INTERACTION IN THE HEPATIC UPTAKE Chang-Soon Yim, Yoo-Seong Jeong, Song-Yi Lee, Heon-Min Ryu, Wooin Lee, Dae-Duk Kim, Suk-Jae Chung. College of Pharmacy, Seoul National University, Seoul, South Korea In our previous study, we demonstrated that lobeglitazone (LB), a new drug currently marketed in Korea, was interacted with hOATP1B1. Since statins are known substrates to hOATP1B1 and likely to be concomitantly
Quantification of relative contributions of individual cytochrome P450 (CYP) isoforms to overall metabolic clearance using reaction phenotyping studies is essential when predicting potential drug-drug interactions. A method that is commonly utilized for such studies is relative activity factor (RAF) using recombinant human CYP isoforms and human liver microsomes. In this approach, the intrinsic clearance of a CYP-selective probe reaction is assessed in both systems to establish a RAF. The RAF acquired is subsequently applied to the intrinsic clearance for a potential drug candidate to assess the relative contribution of each CYP isoform. However, multiple binding sites with diverse substrate selectivity have been eminent for several CYP isoforms, namely CYP2C9 and CYP3A4 which are important in drug metabolism. Based on this rather unique property of CYP, it is conceivable that the drug candidate of interest may not always interact with the same binding site as the probe substrate, from which the RAF was derived. In the present study, CYP-selective substrates were used to establish RAF values for CYP3A4 (i.e., midazolam, testosterone and nifedipine) and CYP2C9 (i.e., S-warfarin, diclofenac and tolbutamide) using recombinant human CYP isoforms and human liver microsomes. The RAF values were further exploited to assess the relative contributions of CYP3A4 or CYP2C9 towards metabolism of these very same probe substrates in a crossover manner. In addition, these RAF values were applied to several CYP2C9 and CYP3A4 model drugs to determine the contributions of CYP isoforms to their overall metabolism. The results demonstrated substantial differences in the relative contributions of the CYP isoforms depending on which probe substrates were used for RAF on a given CYP isoform, further emphasizing the potential impact toward overall metabolic clearance and drug-drug interaction. In conclusion, multiple probe substrates in CYP reaction phenotyping studies using the RAF approach should be evaluated when attempting to predict potential drug-drug interactions in CYP2C9 and CYP3A4. P86 IN VITRO CHARACTERIZATION OF DRUG METABOLIZING ENZYMES AND TRANSPORTERS TO ENABLE A MECHANISTIC DRUG-DRUG INTERACTION ASSESSMENT FOR VENETOCLAX Ryota Kikuchi, Mohamad Shebley, Daniel A.J. Bow, Robert A. Carr, Marjoleen Nijsen, Sonia M. de Morais. AbbVie Inc., North Chicago, IL, USA
In vitro tools were used to characterize the drug-drug interaction (DDI) potential for venetoclax (ABT-199) and its human major metabolite M27 as victims or perpetrators of Cytochrome P450 (CYP), Uridine 5'-diphosphoglucuronosyltransferase (UGT) enzymes and drug transporters. Venetoclax and M27 were predominantly metabolized by CYP3A4, while UGTs were not involved in the metabolism. Both venetoclax and M27 were identified as substrates for the efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) but not for hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1, OATP1B3 or organic cation transporter (OCT) 1. The nonclinical predictions for venetoclax were consistent with the clinical findings observed with ketoconazole (CYP3A4/ P-gp inhibitor) and rifampin (CYP3A4/P-gp inducer). Venetoclax PBPK model simulations were consistent with results from the clinical ketoconazole and rifampin studies: predicted vs observed ratios for venetoclax Cmax and AUC∞ were within 0.8-1.25 fold. At a 400 mg dose, venetoclax and M27 are not predicted to inhibit CYPs but venetoclax may weakly inhibit UGT1A1 (predicted AUCR 1.38) based on the mechanistic static model. Venetoclax and M27 are P-gp and BCRP inhibitors with IC50 values of 0.79 and 0.13 mM (venetoclax) and 0.83 and 1.48 mM (M27), respectively, and at clinically relevant exposures may inhibit these transporters ([I]2/ IC50 >10 and [I]1/IC50 >0.1). Venetoclax is an OATP1B1 inhibitor (IC50 10.1 mM in the presence of 4% bovine serum albumin) and an R-total value of 1.33 (calculated using total plasma exposure) indicates that venetoclax may interact with drugs that are substrates for this transporter. No interaction with other transporters (OATP1B3, OCT1, OCT2, organic anion transporter (OAT) 1, OAT3, multidrug and toxin extrusion protein (MATE) 1 or MATE2K) was predicted. Overall, an in vitro characterization of drug metabolizing enzyme and transporters enabled a mechanistic assessment of DDI potential for venetoclax. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research was
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provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. P87 INHIBITION OF METHOTREXATE UPTAKE BY GLUCURONIDES OF NONSTEROIDAL ANTI-INFLAMMATORY DRUGS VIA ORGANIC ANION TRANSPORTERS OAT1 AND OAT3 Masahiro Iwaki 1, Yuri Irino 2, Manami Take 2, Sachiko Egashira 2. 1 Department of Pharmacy, Faculty of Pharmacy, Kindai University, Higashi-Osaka, Japan; 2 Kindai University, Higashi-Osaka, Japan Introduction: Combined administration of methotrexate (MTX) and nonsteroidal anti-inflammatory drugs (NSAIDs) can result in an elevation of plasma concentration of MTX. This drug interaction has been thought to involve not only the inhibition of basolateral organic anion transporters (OATs) but not apical multidrug resistance proteins (MRPs) by NSAIDs. Most recently, we have shown that the glucuronide conjugates of several NSAIDs exhibit stereoselective and concentration-dependent inhibitory effects on MTX transport via MRP2 and MRP4, with a tendency of the glucuronides to have stronger potencies compared to their parent drugs [1]. As the glucuronides of some NSAIDs are detected in plasma, inhibition of OATs-mediated MTX uptake by the glucuronides may also not be ignored. The purpose of this study was to investigate the inhibitory effects of the glucuronides of several NSAIDs (diclofenac, R- and S-ibuprofen, R- and S-flurbiprofen and Rand S-naproxen) on OAT1- and OAT3-mediated MTX transport. Methods: b-1-O-glucuronides of NSAIDs were prepared biosynthetically in vitro from respective NSAIDs using rat liver microsomes. Uptake experiments were performed using human OAT1- and OAT3-expressing HEK293 cells to assess the inhibitory effects of the glucuronides against hOATs-mediated MTX transport in the presence or absence of the glucuronides. The amounts of [3H]MTX taken into the cells were determined using a liquid scintillation counter. The 50% inhibitory concentrations (IC50) of the glucuronides were obtained from curve-fitting of the resulting concentration-inhibition curves by nonlinear regression analysis. Results and Discussion: All examined glucuronides of NSAIDs exhibited concentration-dependent inhibitory effects on MTX uptake via OAT1 and OAT3 with different potencies. Diclofenac glucuronide had most potent inhibitory effect against OAT3 (IC50¼3.2 mM) among the glucuronides tested, and this value was extremely lower than that against OAT1 (IC50¼ 265 mM). Similarly, the IC50 values against OAT3 in all glucuronides of the enantiomers were 5-15 times smaller than those against OAT1, showing that the glucuronides potently inhibit OAT3-mediated MTX transport. There was no difference between both enantiomers of each NSAID in the inhibitory potencies unlike the MRPs-mediated MTX transport previously reported [1]. The present results suggest that inhibition of OAT3 by plasma glucuronides of NSAIDs may be the fourth competitive site underlying complex drug interaction between MTX and NSAIDs in addition to inhibition of basolateral OATs by parent NSAIDs and of apical MRPs by NSAIDs and their glucuronide. Conclusion: The glucuronides of NSAIDs, as well as the parent NSAIDs, can inhibit OAT1and OAT3-mediated MTX transport. Stereoselectivity was not observed in their inhibitory effect on both OAT1 and OAT3. 1. Kawase A., Yamamoto T., Egashira S., and Iwaki M., Stereoselective inhibition of methotrexate excretion by glucuronides of nonsteroidal antiinflammatory drugs via multidrug resistance proteins 2 and 4, J Pharmacol. Exp. Ther., n press. P88 INTERACTION BETWEEN LOBEGLITAZONE AND ATORVASTATIN IN RATS: INVOLVMENT OF TRANSPORTER-MEDIATED INTERACTION IN THE HEPATIC UPTAKE Chang-Soon Yim, Yoo-Seong Jeong, Song-Yi Lee, Heon-Min Ryu, Wooin Lee, Dae-Duk Kim, Suk-Jae Chung. College of Pharmacy, Seoul National University, Seoul, South Korea In our previous study, we demonstrated that lobeglitazone (LB), a new drug currently marketed in Korea, was interacted with hOATP1B1. Since statins are known substrates to hOATP1B1 and likely to be concomitantly