Regulation of drug transporter expression by oncostatin M in human hepatocytes

The cytokine oncostatin M (OSM) is a member of the interleukin (IL)-6 family, known to down-regulate expression of drug metabolizing cytochromes P-450 in human hepatocytes. The present study was designed to determine whether OSM may also impair expression of sinusoidal and canalicular drug transporters, which constitute important determinants of drug hepatic clearance. Exposure of primary human hepatocytes to OSM down-regulated mRNA levels of major sinusoidal solute carrier (SLC) influx transporters, including sodium-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter 1 and organic anion transporter 2. OSM also repressed mRNA expressions of ATP binding cassette (ABC) efflux transporters such as multidrug resistance protein (MRP) 2/ABCC2 and breast cancer resistance protein/ABCG2, without however impairing those of multidrug resistance gene 1/P-glycoprotein/ABCB1, MRP3/ABCC3, MRP4/ABCC4 and bile salt export pump/ABCB11. The cytokine concomitantly reduced NTCP, OATP1B1, OATP2B1 and ABCG2 protein expression and NTCP and OATP transport activities. OSM effects towards transporters were found to be dose-dependent and highly correlated with those of IL-6, but not with those of other inflammatory cytokines such as tumor necrosis factor-α or interferon-γ. In addition, OSM-mediated repression of some transporters such as NTCP, OATP1B1 and OATP2B1, was counteracted by knocking-down expression of the type II OSM receptor subunits through siRNA transfection. This OSM-mediated down-regulation of drug SLC transporters and ABCG2 in human hepatocytes may contribute to alterations of pharmacokinetics in patients suffering from diseases associated with increased production of OSM.     

Contribution of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to hepatic uptake of nateglinide, and the prediction of drug-drug interactions via these transporters

Objectives We have investigated the contributions of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to the hepatic uptake of nateglinide, and the possibility of drug–drug interactions via these transporters. Methods Uptake studies using transporter‐expressing HEK293 cells and cryopreserved human hepatocytes were performed to examine the contributions of each transporter. Inhibition studies using cryopreserved human hepatocytes were performed to examine the possibility of drug–drug interactions. Key findings The rate of saturable hepatic uptake of nateglinide using human hepatocytes was 47.6%. A certain increase in uptake was observed in the examination using transporter‐expressing HEK293 cells, indicating contributions of OATP1B1 and OATP1B3 to hepatic nateglinide uptake. The 50% inhibitory concentration (IC50) values of nateglinide using cryopreserved human hepatocytes for uptake of estrone 3‐sulfate (substrate of OATP1B1), and cholecystokinin octapeptide (substrate of OATP1B3) were 168 and 17.4 µmol/l, respectively. Moreover, ciclosporin inhibited saturable hepatic uptake of nateglinide with an IC50 value of 6.05 µmol/l. The calculated 1 + I_in,max,u/IC50 values for inhibition of OATP1B1 and OATP1B3 by nateglinide, and the inhibition of saturable uptake of nateglinide by ciclosporin, were all close to 1, indicating a low clinical risk of drug–drug interaction with nateglinide taken up via OATP1B1 and OATP1B3. Conclusions OATP1B1 and OATP1B3 may have contributed to the hepatic uptake of nateglinide, but the possibility of drug–drug interactions appeared to be low.     

Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells

The HepaRG cell line is a well-differentiated human hepatoma cell line proposed as a surrogate for human hepatocytes, especially for hepatic detoxification studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, which represents a key hallmark of hepato-biliary drug transport, remains however incompletely documented in HepaRG cells. The present study was therefore designed to analyze transporter location in HepaRG cells, which exhibit mRNA expressions of most of hepatic transporters. HepaRG cells were demonstrated, through immunofluorescence staining, to express several drug transporters at their sinusoidal pole, especially the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of HepaRG cells. Moreover, saturable uptake of reference substrates for the sinusoidal transporters sodium-taurocholate cotransporting polypeptide, OATPs and OCT1 and canalicular secretion of reference substrates for the efflux transporters bile salt export pump and MRP2 were observed. This polarized and functional expression of various sinusoidal and canalicular transporters in HepaRG cells highlights the interest of using these hepatoma cells in xenobiotic transport studies.     

Investigation of the role of transporters on the hepatic elimination of an LAT1 selective inhibitor JPH203

JPH203 has been developed as an anticancer drug that inhibits L‐type amino acid transporter 1‐mediated essential amino acid uptake into tumor cells. This study sought to elucidate which drug transporters may be involved in JPH203 hepatic elimination, and to estimate human hepatic clearance. In Sprague–Dawley rats, JPH203 total body clearance approached blood flow rate. JPH203 biotransformation via phase II metabolism produces N‐acetyl‐JPH203 (NAc‐JPH203). NAc‐JPH203 accumulates in the bile, and NAc‐JPH203 canalicular efflux was significantly decreased in Mrp2‐deficient mutant rats (Eisai hyperbilirubinemic rats). JPH203 and NAc‐JPH203 are organic anion transporters [organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, and OAT3] substrates. In human cryopreserved hepatocytes, JPH203 uptake was saturable and inhibited by rifampicin, a prototypical OATP inhibitor. JPH203 metabolic clearance was larger than influx clearance and eventually passive clearance; JPH203 uptake appears to be the rate‐determining process in overall hepatic elimination. Furthermore, unlike rats, the human hepatic clearance was predicted to be intrinsic clearance rate limited. These results suggest that the hepatic uptake transporters are determinant factors to determine JPH203 systemic exposure.     

Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney

The role of carrier-mediated transport in determining the pharmacokinetics of drugs has become increasingly evident with the discovery of genetic variants that affect expression and/or function of a given drug transporter. Drug transporters are expressed at numerous epithelial barriers, such as intestinal epithelial cells, hepatocytes, renal tubular cells and at the blood-brain barrier. Several recent studies have associated alterations in substrate uptake with the presence of SNPs. Here, we summarize the current knowledge on the functional and phenotypic consequences of genetic variation in intestinally, hepatically and renally expressed members of the organic anion-transporting polypeptide family (OATPs; SLC21/SLCO family), the organic anion and organic cation transporters (OATs/OCTs; SLC22 family) and the peptide transporter-1 (PEPT1; SLC15 family).     

Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line.

Functional expression of both sinusoidal and canalicular hepatic drug transporters was investigated in the highly differentiated human hepatoma HepaRG cell line and also, for comparison, in primary human hepatocytes and in the hepatoma HepG2 cell line. Using RT-qPCR assays, differentiated HepaRG cells were found to display a pattern of transporter expression close to that found in primary human hepatocytes, i.e. they exhibit substantial mRNA levels of the influx transporters OCT1, OATP-B, OATP-C and NTCP, and of the secretion transporters MRP2, MRP3, BSEP and P-glycoprotein. By contrast, expression of influx transporters was not present or very weak in HepG2 cells. Drug transport assays allowed to detect functional activities of OCT1, OATPs/OAT2, NTCP, MRPs and P-glycoprotein in differentiated HepaRG cells as in primary human hepatocytes whereas HepG2 cells only showed notable MRP and P-glycoprotein activities. In addition, expression of canalicular transporters in HepaRG cells was found to be up-regulated by known inducers of transporters such as rifampicin, phenobarbital and chenodeoxycholate acting on P-glycoprotein, MRP2 and BSEP, respectively. HepaRG cells thus exhibit functional expression of both sinusoidal and canalicular drug transporters and have retained regulatory pathways controlling transporter levels. These data, associated with the known high expression of drug metabolizing enzymes in HepaRG cells, highlight the interest of such hepatoma cells for analysing hepatic drug detoxification pathways.     

异鼠李素和阿魏酸在大鼠原代肝细胞的肝摄取性质

目的建立大鼠原代肝细胞摄取模型,评价异鼠李素和阿魏酸的肝细胞摄取性质。方法应用RT-qPCR技术测定0,0.5,2和4h悬浮培养原代大鼠肝细胞上摄取转运体有机阴离子转运多肽(Oatp)1和Oatp2,有机阴离子转运体(Oat)2和有机阳离子转运体(Oct)1的mRNA表达。应用已知底物非索非那定、普伐他汀、甲氨蝶呤和阿昔洛韦评价转运体的功能。将已知底物或受试药物在4℃和37℃分别与肝细胞孵育不同时间,LC-MS/MS定量测定细胞摄取量,考察温度和时间对底物摄取的影响。由浓度依赖的摄取实验,计算得到Km、Vmax和主动摄取率等参数。在肝细胞模型上,将转运体的阳性抑制剂与异鼠李素10μmol·L-1和阿魏酸50μmol·L-1共孵育2min,观察抑制剂对受试药转运的影响。结果悬浮培养大鼠原代肝细胞上表达有转运体Oatp1,Oatp2,Oat2和Oct1,并能介导已知底物的主动摄取。转运体的mRNA表达水平随着时间的延长而快速下降,4h的表达水平约为0h的10%。转运体对已知底物摄取活性也随时间呈下降趋势,但程度显著低于表达水平,4h的活性约为0h的28.7%～71.4%。在经验证的肝细胞模型上,异鼠李素的4℃和37℃摄取无显著差异,主动转运率为14.6%。阿魏酸在37℃的摄取量显著高于4℃,主动转运率为84.1%;Oct阳性抑制剂奎尼丁100μmol·L-1能显著抑制阿魏酸的肝摄取,抑制率为64.9%。结论成功建立了大鼠原代肝细胞摄取模型,并用底物进行了验证。异鼠李素通过被动扩散进入肝细胞,阿魏酸的肝细胞摄取则以主动转运为主。     

Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells

Human embryonic and induced pluripotent stem cell-derived hepatocytes (hESC-Hep and hiPSC-Hep) have the potential to provide relevant human in vitro model systems for toxicity testing and drug discovery studies. In this study, the expression and function of important drug metabolizing cytochrome P450 (CYP) enzymes and transporter proteins in hESC-Hep and hiPSC-Hep were compared to cryopreserved human primary hepatocytes (hphep) and HepG2 cells. Overall, CYP activities in hESC-Hep and hiPSC-Hep were much lower than in hphep cultured for 4 h, but CYP1A and 3A activities were comparable to levels in hphep cultured for 48 h (CYP1A: 35% and 26% of 48 h hphep, respectively; CYP3A: 80% and 440% of 48 h hphep, respectively). Importantly, in hESC-Hep and hiPSC-Hep, CYP activities were stable or increasing for at least one week in culture which was in contrast to the rapid loss of CYP activities in cultured hphep between 4 and 48 h after plating. With regard to transporters, in hESC-Hep and hiPSC-Hep, pronounced NTCP activity (17% and 29% of 4 h hphep, respectively) and moderate BSEP activity (6% and 8% of 4 h hphep, respectively) were observed. Analyses of mRNA expression and immunocytochemistry supported the observed CYP and transporter activities and showed expression of additional CYPs and transporters. In conclusion, the stable expression and function of CYPs and transporters in hESC-Hep and hiPSC-Hep for at least one week opens up the possibility to reproducibly perform long term and extensive studies, e.g. chronic toxicity testing, in a stem cell-derived hepatic system.     

Predominant contribution of OATP1B3 to the hepatic uptake of telmisartan, an angiotensin II receptor antagonist, in humans.

Telmisartan, a nonpeptide angiotensin II receptor antagonist, is selectively distributed to liver. In the present study, we have characterized the contribution of organic anion transporting polypeptide (OATP) isoforms to the hepatic uptake of telmisartan by isolated rat hepatocytes, human cryopreserved hepatocytes, and human transporter-expressing cells. Because it is difficult to evaluate the transport activity of telmisartan because of its extensive adsorption to cells and culture materials, we performed the uptake study in the presence of human serum albumin. The saturable uptake of telmisartan into isolated rat hepatocytes took place in a Na(+)-independent manner and was inhibited by pravastatin, taurocholate, and digoxin, which are Oatp substrates and inhibitors, but not by organic cation, tetraethylammonium, indicating the involvement of Oatp isoforms in its uptake into rat hepatocytes. To identify which human OATP transporters are important for the hepatic uptake of telmisartan, the uptake assay was carried out using OATP1B1- and OATP1B3-expressing human embryonic kidney 293 cells and cryopreserved human hepatocytes. The uptake of telmisartan by OATP1B3-expressing cells was saturable (K(m) = 0.81 microM) and significantly higher than that by vector-transfected cells. In contrast, no significant uptake was observed in OATP1B1-expressing cells. We also observed the saturable uptake of telmisartan by human hepatocytes. Thirty micromolar estrone-3-sulfate, which can selectively inhibit OATP1B1-mediated uptake compared with OATP1B3, did not inhibit the uptake of telmisartan in human hepatocytes, whereas it could inhibit the uptake of estradiol 17beta-d-glucuronide mediated by OATP1B1. These results suggest that OATP1B3 is predominantly involved in the hepatic uptake of telmisartan in humans.     

Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin II AT1-receptor, in humans.

Valsartan is a highly selective angiotensin II AT1-receptor antagonist for the treatment of hypertension. Valsartan is mainly excreted into the bile in unchanged form. Because valsartan has an anionic carboxyl group, we hypothesized that a series of organic anion transporters could be involved in its hepatic clearance. In this study, to identify transporters that mediate the hepatic uptake and biliary excretion of valsartan and estimate the contribution of each transporter to the overall hepatic uptake and efflux, we characterized its transport using transporter-expressing systems, human cryopreserved hepatocytes, and Mrp2-deficient Eisai hyperbilirubinemic rats (EHBRs). Valsartan was significantly taken up into organic anion-transporting polypeptide (OATP) 1B1 (OATP2/OATP-C)- and OATP1B3 (OATP8)-expressing HEK293 cells. We also observed saturable uptake into human hepatocytes. Based on our estimation, the relative contribution of OATP1B1 to the uptake of valsartan in human hepatocytes depends on the batch, ranging from 20 to 70%. Regarding efflux transporters, the ratio of basal-to-apical transcellular transport of valsartan to that in the opposite direction in OATP1B1/MRP2 (multidrug resistance-associated protein 2) double transfected cells was the highest among the three kinds of double transfectants, OATP1B1/MRP2, OATP1B1/multi-drug resistance 1, and OATP1B1/breast cancer resistance protein-expressing MDCKII cells. We observed saturable ATP-dependent transport into membrane vesicles expressing human MRP2. We also found that the elimination of intravenously administered valsartan from plasma was markedly delayed, and the biliary excretion was severely impaired in EHBR compared with normal Sprague-Dawley rats. These results suggest that OATP1B1 and OATP1B3 as the uptake transporters and MRP2 as the efflux transporter are responsible for the efficient hepatobiliary transport of valsartan.     

Comparative study of culture conditions for maintaining CYP3A4 and ATP-binding cassette transporters activity in primary cultured human hepatocytes

The aim of this study was to determine suitable culture conditions for maintaining the activity of cytochrome p450 (CYP) 3A4 and drug transporters in primary cultured human hepatocytes. Human hepatocytes were isolated using the two-step collagenase perfusion technique and were cultured with four different media, serum-free William's E medium (serum-free WEM), WEM containing fetal calf serum (FCS-WEM), WEM with human serum (HS-WEM), and Lanford's medium. The albumin levels were maintained for 7 days in hepatocytes. Although CYP3A4 mRNA levels gradually decreased from 3 days, CYP3A4 and hepatocyte nuclear factor-4α alpha protein levels and activities were maintained for 7 days in hepatocytes cultured with serum-free WEM and Lanford's but not in those with FCS-WEM and HS-WEM. Furthermore, CYP3A4 protein levels were significantly increased by the addition of rifampicin and dexamethasone to the culture media, indicating that the induction potential was maintained. The protein levels of P-glycoprotein, multi-drug-resistance-2, and breast cancer-resistance protein were maintained for 7 days in all media. Serum-free WEM and Lanford's also maintained protein levels of CYP2C19, CYP2D6, and organic anion transporter polypeptide in the hepatocytes. Serum-free WEM and Lanford's may be appropriate culture media for maintaining CYP3A4 and drug transporter protein levels in primary cultured hepatocytes.     

Comparison of uptake transporter functions in hepatocytes in different species to determine the optimal model for evaluating drug transporter activities in humans

1. A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data.

2. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared.

3. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis–Menten constant (K_m,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17β-glucuronide and metformin. The K_m,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PS_inf,act) while no difference in %PS_inf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug–drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin.

4. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.

     

Human organic anion transporter 2 is an entecavir,but not tenofovir,transporter

Entecavir (ETV) and tenofovir (TFV) are essential nucleoside analogues in current hepatitis B virus (HBV) treatments. Since these drugs target the HBV polymerase that is localized within human hepatocytes, determining of their cellular uptake process is an important step in fully understanding their pharmacological actions. However, the human hepatic transporters responsible for their uptake have remained unidentified. Therefore, this study aimed at identifying the primary ETV and TFV uptake transporter(s) in human hepatocytes. In transport assays, temperature-sensitive ETV and TFV uptake by human hepatocytes were observed, and their uptake were strongly inhibited by bromosulfophthalein, which is an inhibitor of organic anion transporters/organic anion transporting polypeptides (OATs/OATPs). Given these results, ETV and TFV uptake activities in several human OAT/OATP expression systems were examined. The results showed that, among the transporters tested, only OAT2 possessed ETV transport activity. On the other hand, none of the transporters showed any TFV uptake activity. To summarize, our results identify that human OAT2 is an ETV transporter, thereby suggesting that it plays an important part in the mechanisms underlying ETV antiviral activity. Furthermore, although the hepatic TFV transporters remain unknown, our results have, at least, clarified that these two anti-HBV drugs have different hepatocyte entry routes.     

Enzyme induction in cryopreserved human hepatocyte cultures

Freshly isolated human hepatocytes are considered as the gold standard for in vitro testing of drug candidates. Meanwhile also cryopreserved human hepatocyte suspensions are available. However, a drawback of these cells is the incalculability of attachment to the culture dish. Therefore, we established a technique freezing hepatocytes cultured on a collagen gel. After thawing damaged cells were removed to a certain extent by gentle washing with culture medium prior to adding an upper gel layer. The morphology of the resulting hepatocyte cultures could not be distinguished from that of non-frozen cells. However, basal activities of cytochrome P450 isoforms decreased in cryopreserved compared to non-frozen hepatocytes, as evidenced by analysis of testosterone hydroxylation (OHT) in positions 6β, 16, 2β and 6. Nevertheless, enzyme induction factors caused by 24 h incubation with 50 μM rifampicin were similar in cryopreserved and non-frozen hepatocytes. In cryopreserved hepatocytes rifampicin caused an increase in mean values of 6β-OHT formation from 57.2 to 157.7 pmol/well/min (2.8-fold), compared to an increase from 115.8 to 269.1 pmol/well/min (2.3-fold) in non-frozen cells. Similarly, 16- and 2β-OHT showed induction factors of 2.4- and 2.3-fold in cryopreserved compared to 1.6- and 2.4-fold in non-frozen hepatocytes, respectively. In conclusion, human hepatocytes cryopreserved on collagen gels show a clear induction of CYP3A4 by rifampicin, although the basal activities are reduced compared to non-frozen cells.     

Mechanisms of renal anionic drug transport

By utilizing filtration, active secretion and reabsorption processes, the kidney can conserve essential nutrients, and eliminate drugs and potentially toxic compounds. Active uptake of organic anions and cations across the basolateral membrane, and their extrusion into the urine across the brush border membrane mainly takes place in the renal proximal tubule cells, and is facilitated via a range of substrate-specific tubular transporters. Many drugs and their phase II conjugates are anionic compounds, and therefore renal organic anion transporters are important determinants of their distribution and elimination. Competition for renal excretory transporters may cause drugs to accumulate in the body leading to toxicity, which is a potential hazard of concomitant drug administration. Here, we present a brief update on the most prominent human proximal tubule organic anion transporters, which either belong to the ATP-binding cassette (ABC) or the solute carrier transporter (SLC) families. We focus on the participation of the individual transporters in renal anionic drug elimination, in an attempt to understand their overall biological and pharmacological significance, hoping to inspire further studies in the renal transporters field.     

Renal Transport of Adefovir,Cidofovir, and Tenofovir by SLC22A Family Members (hOAT1, hOAT3, and hOCT2)

Purpose The nephrotoxicity of the nucleotide antivirals adefovir, cidofovir and tenofovir is considered to depend on the renal tubular transport of them. Although it is known that the antivirals are substrates of the human renal organic anion transporter hOAT1 (SLC22A6), there is no information available on other organic ion transporters. The aim of the present study was to investigate whether the other renal organic anion transporter hOAT3 (SLC22A8) and organic cation transporter hOCT2 (SLC22A2) transport the antivirals. Materials and Methods Uptake experiments were performed using HEK293 cells transfected with cDNA of the organic ion transporters. Results The uptake of adefovir, cidofovir and tenofovir in monolayers stably expressing hOAT3 increased time-dependently, compared with control. Probenecid, a typical inhibitor of organic anion transporters, completely inhibited their transport. The amounts of the antivirals taken up by hOAT3 were much lower than those by hOAT1. The transient expression of hOCT2 did not increase uptake of the antivirals. Conclusion These results indicate that adefovir, cidofovir and tenofovir are substrates of hOAT3 as well as hOAT1, but that quantitatively hOAT1 is the major renal transporter for these drugs.