The predominant role of human concentrative nucleoside transporter 3 in the placental transfer of EK, an antiviral drug

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structure-activity relationships of ABCC2 modulators using a screening approach. Bioorganic & Medicinal Chemistry. 23:3513-3525. P269 SULFASALAZINE DISPOSITION IN SUBJECT WITH 376C>T (NONSENSE MUTATION) VARIANT IN ABCG2 GENE Takeshi Hirota, Keisuke Gotanda, Tomoko Tokumoto, Masato Fukae, Ichiro Ieiri. Department of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan Background: Breast cancer resistance protein (BCRP), the ABCG2 gene product, is a half-molecule ATP-binding cassette transporter that acts as an efflux transporter. Sulfasalazine (SASP) is a candidate in vivo probe for BCRP function. Some single-nucleotide polymorphisms (SNPs) of the ABCG2 gene have been associated with changes in the pharmacokinetics and pharmacodynamics of clinically important drugs. A non-synonymous 421C>A (rs2231142) is a well-known SNP causing the increased oral bioavailability of BCRP substrates, such as SASP. In this study, we analyzed the association between SNPs of the ABCG2 gene and the pharmacokinetics of SASP. Methods: A clinical study was conducted with an open-label and singlearm design involving 34 healthy participants, who were selected from our study panel based on their genotyping for ABCG2 421C>A polymorphism (wild-type homozygotes (C/C) ¼ 13, heterozygotes (C/A) ¼ 12, mutant homozygotes (A/A) ¼ 8). A single dose of sulfasalazine (2,000 mg) was administered orally after a 12-hours overnight fast. The concentrations of sulfasalazine in plasma were measured by high-performance liquid chromatography. Results and Discussion: The mean AUC0e48 were significantly 3-fold higher in 421A/A than in 421C/C wild-type homozygotes. Our results are consistent with our previous study. However, the SASP concentrations in one apparently heterozygous subject deviated from other 421C/A heterozygotes. The subject showed higher AUC0-48 (779 vs. 292 ± 97 mghr/mL, respectively) than those in other 421C/A heterozygotes (n ¼12). We identified that the subject was heterozygous for 376C>T (rs72552713, 126Q>Stop). Haplotype analysis revealed that the subject was a compound heterozygote (376C wild type and 421A mutant type were located on the same allele). The relative expression level of the BCRP protein in red blood cell membrane in this subject was lower than that in other genotypes. Conclusion: In this study, the subject with 376C>T and 421C>A alleles showed higher SASP concentrations. This is the first study to assess the impact of 376C>T on the pharmacokinetics of BCRP substrate drugs. P270 THE INFLUENCE OF BOVINE SERUM ALBUMIN ON MRP2 AND BCRP IN THE VESICULAR TRANSPORT ASSAY € stedt, Heidi Kidron. University of Helsinki, Helsinki, Feng Deng, Noora Sjo Finland Multidrug resistance associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) are ABC transporters expressed in many tissues important for distribution and excretion of xenobiotics. They interacts with several drugs affecting their pharmacokinetics, which is why interactions with these proteins are studied in vitro in drug development to predict in vivo outcomes. The addition of albumin is known to affect the kinetic parameters of metabolic enzymes measured in vitro in human liver microsomes or membrane preparations of recombinant enzymes. Because similar membrane preparations are used to study transport by MRP2 and BCRP using the vesicular transport assay, we wanted to investigate whether bovine serum albumin can affect the kinetic parameters of transport. We studied the effect of bovine serum albumin (BSA) on transport of 5(6)-carboxy-2’,7’-dichlorofluorescein (CDCF) and estradiol17-b-D-glucuronide (E217bG) by MRP2 and Lucifer yellow (LY) by BCRP. The maximal uptake of CDCF into the MRP2 vesicles in the presence of 0.1 % BSA was 2-fold higher than in the control (absence of BSA) and E217bG uptake was 1.5-fold higher. Maximal LY uptake in BCRP vesicles was only 1.3-fold compared to control. Km values tended to increase, but the change was not statistically significant. Oleic acid, the most abundant fatty acid in Sf9 insect cells inhibited both MRP2 and BCRP at low micromolar concentrations in the assay but the extent of inhibition was diminished when 0.1% BSA was added. This supports the hypothesis from metabolic studies

that albumin increases transport by removing inhibitory fatty acids released during membrane extraction. Altogether the results point to a minor effect of albumin on MRP2 and BCRP, but it should be kept in mind if albumin is used for example as a solubilizer, since effects may be transporter or preparation specific. P271 THE N-TERMINAL REGION OF ORGANIC ANION TRANSPORTING POLYPEPTIDE 1B3 (OATP1B3) PLAYS AN ESSENTIAL ROLE IN REGULATING ITS PLASMA MEMBRANE TRAFFICKING Se-Eun Chun 1, Nilay Thakkar 2, Ji Eun Park 1, Songhee Han 1, Hyunggu Hahn 1, Sang Hyun Maeng 1, Young-Ran Lim 3, Byung Woo Han 1, Wooin Lee 1. 1 Seoul National University, Seoul, South Korea; 2 University of Kentucky, Lexington, KY, USA; 3 Konkuk University, Seoul, South Korea Organic anion transporting polypeptide 1B3 (OATP1B3) mediates the hepatic uptake of various endogenous and xenobiotic compounds and it is shown to influence in vivo disposition and toxicity profiles of clinically important drugs including statins and paclitaxel. Recently, we and others identified the cancer-type OATP1B3 variant which arises from the use of an alternative transcription initiation site and lacks the N-terminal 28 amino acids compared to liver-type OATP1B3. In contrast with liver-type OATP1B3, cancer-type OATP1B3 is mainly located in cytoplasm, implying a potential role of the N-terminal sequence of OATP1B3 in regulating its membrane trafficking. Here, we set out to verify the importance of the N-terminal region of OATP1B3 in regulating its membrane trafficking and to identify responsible sequence motif(s) in that region. We prepared a number of expression plasmids harboring truncation and point mutants of OATP1B3 fused with a C-terminal myc tag via site-directed mutagenesis. These constructs were transiently transfected in HEK293T, HCT-8 or MDCKII cells and compared for their cellular localization by immunoblotting analyses of fractionated samples. Our results indicated that the fusion construct containing the N-terminal 50 amino acid sequence of OATP1B3 is effectively localized to the plasma membrane. Point mutations to alanine on several candidate positions of the N-terminal region (positively or negatively charged amino acid residues, potential phosphorylation sites) however did not alter the extent of membrane trafficking of OATP1B3. Similar to our findings with OATP1B3, the closely related OATP1B1 also showed the importance of its N-terminal 50 amino acid sequence in regulating membrane trafficking. Further investigations are ongoing to determine the potential involvement of secondary structures (e.g. beta-turn, alpha-helix) in regulating the membrane trafficking within the N-terminal 50 amino acids of OATP1B3. We are also investigating the importance of the N-terminal region in regulating the murine oatp1b2 (orthologous to OATP1B3 and OATP1B1). Our current findings may provide important mechanistic insights into the regulation and trafficking of OATP1B3 and its related transporters under physiological and pathological conditions. P272 THE PREDOMINANT ROLE OF HUMAN CONCENTRATIVE NUCLEOSIDE TRANSPORTER 3 IN THE PLACENTAL TRANSFER OF EK, AN ANTIVIRAL DRUG Zhiyuan Ma 1, Xi Yang 2, Mengru Bai 2, Wei Wang 1, Dongli Sun 3, Caihong Zheng 3, Su Zeng 2, Huidi Jiang 2. 1 Zhejiang University, Hangzhou, China; 2 College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China; 3 Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, China EK, a nucleoside analogue with a high barrier to drug resistance, was recommended for the first-line therapy of chronic hepatitis B. It is not known if EK crosses the human placenta, whereas the reported studies revealed EK could across the rat placenta, meanwhile our previous study also indicated the placental transfer of EK in pregnant mice. Therefore, the present study aimed to clarify the role of the transporters in the transfer of EK and predict whether EK could permeate human placenta. The BeWo cells, with the expression of human placenta uptake and efflux transporters were used for the interaction of EK with these transporters. The results showed that the cellular uptake of EK was independent with MCT and OATP2B1 and slight influenced by PEPT2 and OCTN2 based on the

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

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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