Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes
Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes
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DOI：
10.1016/j.ejps.2011.05.002
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发表时间：
2011-07-17
影响因子：
4.6
通讯作者：
Annaert, Pieter P.
中科院分区：
文献类型：
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作者：
De Bruyn, Tom;Ye, Zhi-Wei;Annaert, Pieter P.
虽然已经建立了冻存人肝细胞悬液（CHHS）用于体外药物代谢试验的实用性，但关于冻存对人肝细胞转运蛋白活性的影响知之甚少。在本研究中，在14个单独批次和4个合并批次的CHHS中评估了NTCP（牛磺胆酸钠共转运多肽; SLC 10A 1）以及肝脏OATP（有机阴离子转运多肽; SLCO基因家族）和OCT（有机阳离子转运蛋白; SLC 22 A）亚型的活性。在CHHS中，NTCP底物牛磺胆酸盐（1 μ M）的平均累积清除率为27.5（+/-15.0）μ l/mm/百万个细胞，并且当细胞外钠被胆碱替代时降低了10倍。地高辛和OATP底物雌酮-3-硫酸酯和雌二醇-17 β-D-葡糖苷酸（E（2）-17 β-G; 1 μ M）的蓄积清除率分别为9.5（+/- 4.9）、99（+/- 67）和5.2（+/- 2.6）μ l/min/百万个细胞。已知OATP抑制剂利福平（25 μ M）的存在显着（P < 0.01）降低了雌酮-3-硫酸酯和E（2）-17 β-G的积累，分别降至对照值的48%和70%，而对地高辛积累没有观察到显着影响。OCT底物1-甲基-4-苯基吡啶鎓的平均累积清除率为19.8（+/-10.9）μ l/min/百万个细胞。与OCT 1抑制剂哌唑嗪（3 μ M）和OCT 3抑制剂皮质酮（1 μ M）共孵育导致显著（p < 0.01）降低，分别为对照条件下蓄积量的72%和85%。合并CHHS中的实验通常显示蓄积值与单个批次的平均值相当。在5批CHHS中测定的雌酮-3-硫酸酯蓄积值和OATP 1B 3 mRNA水平之间观察到良好的相关性（R（2）= 0.93）。与体外培养的人肝细胞中测定的底物蓄积相比，CHHS中的蓄积值相当（牛磺胆酸盐和地高辛），略高（雌酮-3-硫酸盐）。我们的数据表明，冻存的人肝细胞悬液是一个可靠的体外模型，研究转运蛋白介导的底物摄取的肝脏。多批CHHS转运蛋白活性的系统表征支持针对特定应用合理选择人肝细胞。(C)2011 Elsevier B. V.保留所有权利。
While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes.In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 mu M) was 27.5 (+/- 15.0) mu l/mm/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17 beta-D-glucuronide (E(2)-17 beta-G; 1 mu M) amounted to 9.5 (+/- 4.9), 99 (+/- 67) and 5.2 (+/- 2.6) mu l/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 mu M) significantly (P < 0.01) decreased the accumulation of estrone-3-sulfate and E(2)-17 beta-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (+/- 10.9) mu l/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 mu M) and the OCT3 inhibitor corticosterone (1 mu M) resulted in a significant (p < 0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R(2) = 0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications. (C) 2011 Elsevier B.V. All rights reserved.