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Unbound tissue exposure (Kpuu) of oatps substrates in the cynomolgus monkey
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
decrease of intracellular EK in the presence of their respective inhibitors. However, the cellular accumulation of EK was strongly reduced by 100 mM adenosine, cytidine, guanosine, uridine and in Na+ free medium, which indicated that EK uptake in BeWo cells were transported by nucleoside transporters. Furthermore, 100 mM but not 1 mM NBMPR resulted in slight inhibition of the EK uptake, which implied EK might be a weak substrate of ENT2 but not ENT1. RT-qPCR results proved that the mRNA of hCNT3, hENT1 and hENT2 was expressed in BeWo cells. Therefore, the accumulation of EK in BeWo cells was predominately transported by hCNT3. To further characterize the transport of EK by a nucleoside transporter, we measured the uptake of EK by MDCK cells that heterologously expressed hCNT2/3. The results demonstrated and found that EK was a substrate of hCNT3 but not of hCNT2. The accumulation of EK in BeWo cells was increased by verapamil, MK571 and GF120918, which indicated that the EK was a substrate of P-gP, MRP and BCRP. Finally, human primary trophoblast cells were used to confirm the contribution of hCNT3 in the uptake of EK. The above results indicated that EK could across human placenta and hCNT3 plays a predominant role in its placental transfer. Our data benefit the safe use of EK in the pregnant women. P273 UNBOUND TISSUE EXPOSURE (KPUU) OF OATPS SUBSTRATES IN THE CYNOMOLGUS MONKEY Larry Tremaine, Manthena V. Varma, Rachel E. Kosa, Keith Riccardi, Emi Kimoto, Sweta Modi. Pharmacokinetics, Pharmacodynamics, and Metabolism, Pfizer, Inc., Groton, CT, USA Purpose: Solute carrier (SLC) substrates subjected to active uptake may show higher intracellular unbound tissue concentrations relative to those in systemic circulation. Prediction of both systemic pharmacokinetics and tissue unbound concentration in relation to unbound drug in plasma (Kpuu) is important in establishing reliable PK/PD modeling for drug efficacy. However, obtaining tissue concentration is limited in humans; and cynomolgus monkey could serve as a preclinical model for these purposes due to the high similarity in transporter protein homologies. This study examined the time course of Kpuu in liver, pancreas, muscle, kidney and brain for three organic anion transporting polypeptides (OATPs) substrates, valsartan (V), rosuvastatin (R) and pravastatin (P). Method: Nine male cynomolgus monkeys were dosed intravenously with a cocktail of V, R and P, each at 1 mg/kg. Three animals were euthanized at 0.25, 0.75 and 3 hr post-dose. Blood for plasma and approximately 1 gram of each tissue (liver, kidney cortex, brain cortex, gastrocnemius muscle and pancreas) were collected. Plasma and tissues were diluted with acetate buffer or 6:4 Isopropanol: water, respectively, the tissues were homogenized, and both were extracted with acetonitrile prior to quantitation by LC-MS-MS. Unbound concentrations of each drug in diluted, homogenized tissue and plasma were determined by equilibrium dialysis and LC-MS-MS analysis. Results: The mean (N¼3) liver-to-plasma Kpuu values based on the AUC (0-3 hr) are 26.7, 6.3 and 0.64 for V, R and P, respectively. Whilst, mean kidney-to-plasma Kpuu values are 27.8, 21.6 and 13.9 for V, R and P, respectively. All three drugs showed the highest Kpuu in liver and kidney at 3 hr. On the other hand, Kpuu in muscle and brain is lower than unity for all the 3 compounds. Conclusions: Based on Kpuu, all three compounds are highly accumulated in kidney. V and R also accumulate in liver, whereas only V accumulates in pancreas, suggestive of net active uptake by these tissues. Observed higher free concentrations in tissues can be attributed to the OATPs-mediated uptake by hepatocytes and OAT3mediated uptake by kidney proximal tubule cells. Restriction from muscle and brain may be explained by the low passive permeability and efflux liability for these compounds. Overall, cynomolgus monkey can serve as a preclinical model to assess tissue Kpuu for transporter substrates. P274 UREMIC TOXINS LIKELY BOOST THE PLASMA EXPOSURE OF MORINIDAZOLE CONJUGATED METABOLITES IN THE RENAL FAILURE PATIENTS Fandi Kong, Xiaoyan Pang, Kan Zhong, Xiuli Li, Zitao Guo, Dafang Zhong, Xiaoyan Chen. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
S105
Morinidazole, R,S-1-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-morpholinopropan-2-ol, is a 5-nitroimidazole antimicrobial drug approved for the treatment of amoebiasis, trichomoniasis and anaerobic bacterial infections in China. The elimination of morinidazole is mainly via the biotransformation to the sulfate conjugate, M7, and glucuronide conjugates, M8-1 and M8-2 excreted through the kidney in humans. In the severe renal failure patients, the exposures of M7, M8-1 and M8-2 increased to 29 %, 75 % and 380 % of the parent drug, whereas the percentage in the healthy subjects was 2.8, 5.4 and 32 %, respectively. In the previous study, we found that M7 was the substrate of OAT1 and OAT3, and M8-1 and M8-2 were the substrates of OAT3 [1]. We speculated that the dramatic elevation in the plasma exposures may be mediated by the altered activity of OAT1 and OAT3. The rats with 5/6 nephrectomy were employed as the model to mimic the chronic kidney failure (CKF) in humans. Similar to those in humans, the plasma exposures of M7, M8-1 and M8-2 in CKF rats also increased by 13, 11 and 14 times, respectively. The uptakes of the conjugates to the fresh kidney slices and the mRNA levels of OAT1 and OAT3 were measured. The data revealed that the conjugates accumulations had no significant difference between normal and CKF kidney slices although the transcription of OAT1 and OAT3 in CKF rats was both reduced by 50 %. In addition, we discovered that uremic toxins, indoxyl sufate (IS) and hippuric acid (HA) were both elevated by around 5 times in CKF rats. In vitro, the transport of M7, M8-1 and M8-2 in kidney slices were decreased to 41%, 33% and 57% at 500 mM of HA and IS mixture. Upon further validation, 200 mM IS, 100 mM HA, 300 mM 3-carboxy-4-methyl-5-propyl-2furanpropionate, and 500 mM indoleacetate inhibited M7 uptake by 55, 55, 66 and 72% in the OAT1 overexpressed HEK293 cells, respectively, and by 33, 47, 93 and 54 % in the OAT3 overexpressed HEK293 cells, respectively. M8-1 uptake was decreased by 61, 65, 95 and 62 %, respectively, and M8-2 uptake by 47, 58, 94 and 57 %, respectively, in the OAT3 overexpressed HEK293 cells. Taking together, our data indicate that the inhibition of uremic toxins on the uptakes to the renal epithelial cells may be responsible for the boosted plasma exposure of the morinidazole conjugates in severe renal impairment patients instead of the altered expression of transporters. 1. Zhong K. et al. Effects of renal impairment on the pharmacokinetics of morinidazole: uptake transporter-mediated renal clearance of the conjugated metabolites. Antimicrob Agents Chemother. 2014, 58(7):4153-4161. P275 WARFARIN IS A SUBSTRATE OF BREAST CANCER RESISTANCE PROTEIN, AN EFFLUX DRUG TRANSPORTER Meng-Syuan Yang 1, Chung-Ping Yu 1, Shiuan-Pey Lin 1, Pei-Dawn Lee Chao 1, Yu-Chi Hou 1, 2. 1 School of Pharmacy, China Medical University, Taichung, Taiwan; 2 Department of Pharmacy, China Medical University Hospital, Taichung, Taiwan Warfarin, a racemic mixture of R- and S- forms, is one of the clinically important oral anticoagulants. However, the therapeutic window of warfarin is narrow, and the anticoagulation effect of warfarin is routinely monitored by the use of International Normalized Ratio (INR) in clinical practice. Nowadays, numerous unexpected interactions of warfarin with drugs, herbs, foods and dietary supplements are still emerging in clinical practice, and the interactions remained not fully explainable by known mechanism. We speculate that there are some unrecognized mechanisms involved in these interactions. In regard to the physiochemical property, warfarin is acidic (pKa ¼ 4.94) and exists mainly in anionic form under physiological pH. We thus hypothesized that breast cancer resistance protein (BCRP, ABCG2), an efflux drug transporter, might mediate the transmembrane efflux of warfarin. Therefore, this study aimed to clarify whether warfarin was a substrate of BCRP by using BCRP-overexpressing cells and Bcrp-/- mice. The concentrations of R-warfarin and S-warfarin in cell and plasma were simultaneously analyzed by LC-MS using Astec Chirobiotic V column. The anticoagulation effect was evaluated by measuring INR by using CoaguChek® XS System. The result of cell study showed that the intracellular concentrations of R-warfarin and S-warfarin in BCRP-overexpressing cells were significantly lower than those in wildtype cells, indicating that both R-warfarin and S-warfarin were extruded by BCRP. Animal study revealed that the plasma concentrations of Rwarfarin and S-warfarin in Bcrp-/- mice at 6 h after warfarin dosing were
decrease of intracellular EK in the presence of their respective inhibitors. However, the cellular accumulation of EK was strongly reduced by 100 mM adenosine, cytidine, guanosine, uridine and in Na+ free medium, which indicated that EK uptake in BeWo cells were transported by nucleoside transporters. Furthermore, 100 mM but not 1 mM NBMPR resulted in slight inhibition of the EK uptake, which implied EK might be a weak substrate of ENT2 but not ENT1. RT-qPCR results proved that the mRNA of hCNT3, hENT1 and hENT2 was expressed in BeWo cells. Therefore, the accumulation of EK in BeWo cells was predominately transported by hCNT3. To further characterize the transport of EK by a nucleoside transporter, we measured the uptake of EK by MDCK cells that heterologously expressed hCNT2/3. The results demonstrated and found that EK was a substrate of hCNT3 but not of hCNT2. The accumulation of EK in BeWo cells was increased by verapamil, MK571 and GF120918, which indicated that the EK was a substrate of P-gP, MRP and BCRP. Finally, human primary trophoblast cells were used to confirm the contribution of hCNT3 in the uptake of EK. The above results indicated that EK could across human placenta and hCNT3 plays a predominant role in its placental transfer. Our data benefit the safe use of EK in the pregnant women. P273 UNBOUND TISSUE EXPOSURE (KPUU) OF OATPS SUBSTRATES IN THE CYNOMOLGUS MONKEY Larry Tremaine, Manthena V. Varma, Rachel E. Kosa, Keith Riccardi, Emi Kimoto, Sweta Modi. Pharmacokinetics, Pharmacodynamics, and Metabolism, Pfizer, Inc., Groton, CT, USA Purpose: Solute carrier (SLC) substrates subjected to active uptake may show higher intracellular unbound tissue concentrations relative to those in systemic circulation. Prediction of both systemic pharmacokinetics and tissue unbound concentration in relation to unbound drug in plasma (Kpuu) is important in establishing reliable PK/PD modeling for drug efficacy. However, obtaining tissue concentration is limited in humans; and cynomolgus monkey could serve as a preclinical model for these purposes due to the high similarity in transporter protein homologies. This study examined the time course of Kpuu in liver, pancreas, muscle, kidney and brain for three organic anion transporting polypeptides (OATPs) substrates, valsartan (V), rosuvastatin (R) and pravastatin (P). Method: Nine male cynomolgus monkeys were dosed intravenously with a cocktail of V, R and P, each at 1 mg/kg. Three animals were euthanized at 0.25, 0.75 and 3 hr post-dose. Blood for plasma and approximately 1 gram of each tissue (liver, kidney cortex, brain cortex, gastrocnemius muscle and pancreas) were collected. Plasma and tissues were diluted with acetate buffer or 6:4 Isopropanol: water, respectively, the tissues were homogenized, and both were extracted with acetonitrile prior to quantitation by LC-MS-MS. Unbound concentrations of each drug in diluted, homogenized tissue and plasma were determined by equilibrium dialysis and LC-MS-MS analysis. Results: The mean (N¼3) liver-to-plasma Kpuu values based on the AUC (0-3 hr) are 26.7, 6.3 and 0.64 for V, R and P, respectively. Whilst, mean kidney-to-plasma Kpuu values are 27.8, 21.6 and 13.9 for V, R and P, respectively. All three drugs showed the highest Kpuu in liver and kidney at 3 hr. On the other hand, Kpuu in muscle and brain is lower than unity for all the 3 compounds. Conclusions: Based on Kpuu, all three compounds are highly accumulated in kidney. V and R also accumulate in liver, whereas only V accumulates in pancreas, suggestive of net active uptake by these tissues. Observed higher free concentrations in tissues can be attributed to the OATPs-mediated uptake by hepatocytes and OAT3mediated uptake by kidney proximal tubule cells. Restriction from muscle and brain may be explained by the low passive permeability and efflux liability for these compounds. Overall, cynomolgus monkey can serve as a preclinical model to assess tissue Kpuu for transporter substrates. P274 UREMIC TOXINS LIKELY BOOST THE PLASMA EXPOSURE OF MORINIDAZOLE CONJUGATED METABOLITES IN THE RENAL FAILURE PATIENTS Fandi Kong, Xiaoyan Pang, Kan Zhong, Xiuli Li, Zitao Guo, Dafang Zhong, Xiaoyan Chen. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
S105
Morinidazole, R,S-1-(2-methyl-5-nitro-1H-imidazol-1-yl)-3-morpholinopropan-2-ol, is a 5-nitroimidazole antimicrobial drug approved for the treatment of amoebiasis, trichomoniasis and anaerobic bacterial infections in China. The elimination of morinidazole is mainly via the biotransformation to the sulfate conjugate, M7, and glucuronide conjugates, M8-1 and M8-2 excreted through the kidney in humans. In the severe renal failure patients, the exposures of M7, M8-1 and M8-2 increased to 29 %, 75 % and 380 % of the parent drug, whereas the percentage in the healthy subjects was 2.8, 5.4 and 32 %, respectively. In the previous study, we found that M7 was the substrate of OAT1 and OAT3, and M8-1 and M8-2 were the substrates of OAT3 [1]. We speculated that the dramatic elevation in the plasma exposures may be mediated by the altered activity of OAT1 and OAT3. The rats with 5/6 nephrectomy were employed as the model to mimic the chronic kidney failure (CKF) in humans. Similar to those in humans, the plasma exposures of M7, M8-1 and M8-2 in CKF rats also increased by 13, 11 and 14 times, respectively. The uptakes of the conjugates to the fresh kidney slices and the mRNA levels of OAT1 and OAT3 were measured. The data revealed that the conjugates accumulations had no significant difference between normal and CKF kidney slices although the transcription of OAT1 and OAT3 in CKF rats was both reduced by 50 %. In addition, we discovered that uremic toxins, indoxyl sufate (IS) and hippuric acid (HA) were both elevated by around 5 times in CKF rats. In vitro, the transport of M7, M8-1 and M8-2 in kidney slices were decreased to 41%, 33% and 57% at 500 mM of HA and IS mixture. Upon further validation, 200 mM IS, 100 mM HA, 300 mM 3-carboxy-4-methyl-5-propyl-2furanpropionate, and 500 mM indoleacetate inhibited M7 uptake by 55, 55, 66 and 72% in the OAT1 overexpressed HEK293 cells, respectively, and by 33, 47, 93 and 54 % in the OAT3 overexpressed HEK293 cells, respectively. M8-1 uptake was decreased by 61, 65, 95 and 62 %, respectively, and M8-2 uptake by 47, 58, 94 and 57 %, respectively, in the OAT3 overexpressed HEK293 cells. Taking together, our data indicate that the inhibition of uremic toxins on the uptakes to the renal epithelial cells may be responsible for the boosted plasma exposure of the morinidazole conjugates in severe renal impairment patients instead of the altered expression of transporters. 1. Zhong K. et al. Effects of renal impairment on the pharmacokinetics of morinidazole: uptake transporter-mediated renal clearance of the conjugated metabolites. Antimicrob Agents Chemother. 2014, 58(7):4153-4161. P275 WARFARIN IS A SUBSTRATE OF BREAST CANCER RESISTANCE PROTEIN, AN EFFLUX DRUG TRANSPORTER Meng-Syuan Yang 1, Chung-Ping Yu 1, Shiuan-Pey Lin 1, Pei-Dawn Lee Chao 1, Yu-Chi Hou 1, 2. 1 School of Pharmacy, China Medical University, Taichung, Taiwan; 2 Department of Pharmacy, China Medical University Hospital, Taichung, Taiwan Warfarin, a racemic mixture of R- and S- forms, is one of the clinically important oral anticoagulants. However, the therapeutic window of warfarin is narrow, and the anticoagulation effect of warfarin is routinely monitored by the use of International Normalized Ratio (INR) in clinical practice. Nowadays, numerous unexpected interactions of warfarin with drugs, herbs, foods and dietary supplements are still emerging in clinical practice, and the interactions remained not fully explainable by known mechanism. We speculate that there are some unrecognized mechanisms involved in these interactions. In regard to the physiochemical property, warfarin is acidic (pKa ¼ 4.94) and exists mainly in anionic form under physiological pH. We thus hypothesized that breast cancer resistance protein (BCRP, ABCG2), an efflux drug transporter, might mediate the transmembrane efflux of warfarin. Therefore, this study aimed to clarify whether warfarin was a substrate of BCRP by using BCRP-overexpressing cells and Bcrp-/- mice. The concentrations of R-warfarin and S-warfarin in cell and plasma were simultaneously analyzed by LC-MS using Astec Chirobiotic V column. The anticoagulation effect was evaluated by measuring INR by using CoaguChek® XS System. The result of cell study showed that the intracellular concentrations of R-warfarin and S-warfarin in BCRP-overexpressing cells were significantly lower than those in wildtype cells, indicating that both R-warfarin and S-warfarin were extruded by BCRP. Animal study revealed that the plasma concentrations of Rwarfarin and S-warfarin in Bcrp-/- mice at 6 h after warfarin dosing were