Organic anion transporting polypeptide 1a/1b–knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs (original) (raw)

We have generated and characterized what we believe is a novel mouse model lacking all Slco1a/1b genes. Slco1a/1b–/– mice were viable, fertile, and did not show clear pathological abnormalities, but displayed marked conjugated hyperbilirubinemia and associated jaundice. The conjugated hyperbilirubinemia was unexpected and, as discussed below, it could change our perspective on the functioning of the normal liver. We also found that Oatp1a/1b proteins are involved in the hepatic uptake of unconjugated bile acids. Furthermore, using this model we could demonstrate an important role of Oatp1a/1b proteins in the disposition of various drugs, mainly by their pronounced effect on the hepatic uptake of drugs.

Total bilirubin plasma levels in Slco1a/1b–/– mice were more than 40-fold increased compared with WTs. Remarkably, 95% of this increase could be ascribed to increased levels of conjugated bilirubin. Hepatic glucuronidation by Ugt1a1 is likely the most important detoxification pathway for bilirubin in mice, although it has to be noted that in humans gastrointestinal glucuronidation of bilirubin might also play a (minor) role (17). Bilirubin glucuronides formed within the liver can be transported into bile by Abcc2, Abcg2, and some other, as yet unidentified, canalicular transporter(s) (18, 19) or, under pathological conditions (cholestasis), back into the circulation by Abcc3 (15, 20). Our results lead us to hypothesize the unexpected existence of a substantial and, at first sight, seemingly futile cycling of bilirubin glucuronides in normal, healthy liver, in which bilirubin conjugated in the liver is substantially secreted across the sinusoidal membrane by Abcc3. We propose that the subsequent effective reuptake into the liver is mediated by Oatp1a/1b transporters. Interestingly, in the absence of Oatp1a/1b transporters, hepatobiliary elimination of bilirubin glucuronide was practically halved (Figure 2C), suggesting that under normal conditions at least half of the bilirubin glucuronide formed in the liver undergoes sinusoidal secretion and reuptake.

These findings may alter our perspective on the detoxifying functioning of the normal liver. Rather than a mostly unidirectional process, bilirubin detoxification seems to be dynamic, with the flexibility to reroute glucuronidated bilirubin (and presumably many other conjugated compounds as well) formed in 1 hepatocyte to downstream hepatocytes by secretion via ABCC3 (21) and subsequent OATP1A/1B-mediated reuptake for final biliary excretion. We hypothesize that this “hepatocyte hopping” afforded by the tandem activity of ABCC3 and OATP1A/1B transporters can help to prevent saturation of the canalicular excretion of many conjugated compounds generated in the periportal hepatocytes, which are the first and most highly exposed to xenobiotics and endotoxins. Thus, sinusoidal back-transport of hepatic-conjugated compounds toward the blood by ABCC3 (and ABCC4) would not only be important during cholestasis to afford alternative renal excretion (22), but also during the normal functioning of the liver by allowing the distribution of the excretion load over the entire liver lobule.

An alternative explanation for the observed conjugated hyperbilirubinemia in Slco1a/1b–/– mice might be altered enterohepatic circulation of bilirubin-glucuronide, which, after reaching the portal blood, would be dependent on Oatp1a/1b transporters for the reuptake into the hepatocytes. However, although enterohepatic circulation of UCB has been demonstrated under some specific conditions (e.g., increased intestinal bile acid levels for example during bile acid malabsorption) in rodents (23–25) and humans (26), enterohepatic circulation of conjugated bilirubin has never been demonstrated. We therefore consider this alternative hypothesis less plausible.

Another hypothesis would be that conjugated hyperbilirubinemia in Slco1a/1b–/– mice is somehow caused by the 2-fold decreased canalicular excretion of bilirubin-glucuronide into bile that we observed. Indeed, Abcc2-deficient mice also display a 2-fold reduced output of bilirubin-glucuronide, associated with mildly increased plasma levels of bilirubin-glucuronide (to about 4 μM) (18). Abcc2 is the main canalicular bilirubin-glucuronide exporter in mice, with an ancillary role of Abcg2 (19). However, we found that levels of Abcc2 and Abcg2 proteins are normal in the Slco1a/1b–/– mice, and it is not obvious why the activity of these transporters would be reduced: hepatic accumulation of potentially inhibiting compounds is more likely to be decreased than increased in the Slco1a/1b–/– mice, and there are also no indications for profound changes in hepatic expression of transporters (Supplemental Table 1). Also, biliary excretion of GSH, which is primarily mediated by Abcc2 (18, 19), is unchanged. This strongly supports normal activity of Abcc2 in the Slco1a/1b–/– mice. Finally, the plasma bilirubin-glucuronide level is much higher in the Slco1a/1b–/– mice (>45 μM) than in the Abcc2–/– mice (4 μM), despite similar levels of biliary bilirubin-glucuronide output. Reduced biliary excretion of bilirubin-glucuronide as a primary cause of the conjugated hyperbilirubinemia in Slco1a/1b–/– mice thus seems both mechanistically unlikely and insufficient to explain the data.

Our bilirubin data are in line with in vitro studies demonstrating that Oatp1a1 (27), OATP1B1, and OATP1B3 (28, 29) can transport bilirubin glucuronides with high affinity (Km values < 0.5 μM). Furthermore, in humans the SLCO1B1*1b (Val174Ala), -*5 (Asp130Asn), and -*15 (Asp130Asn + Val174Ala) haplotypes have also been associated with significantly (albeit modestly) increased plasma levels of both conjugated bilirubin and UCB (30–33). Interestingly, results from the study by Sanna et al. suggested that SNPs in SLCO1B1 were mostly associated with conjugated bilirubin, whereas an intronic polymorphism in SLCO1B3 was mainly associated with mild unconjugated hyperbilirubinemia (31). In general, the fact that genetic variation in SLCO1A/1B genes is associated with increased levels of UCB (in addition to conjugated bilirubin) might point to possible species differences in hepatic uptake of UCB; we note that our data do not indicate an essential role of murine Oatp1a/1b transporters in the liver uptake of UCB, as most bilirubin was still effectively conjugated in Slco1a/1b–/– mice and therefore must have entered the hepatocytes. Also, UCB plasma levels were only slightly increased in Slco1a/1b–/– mice, which might be a consequence of some in vivo deconjugation of the circulating bilirubin-glucuronide. However, we cannot exclude that Oatp1a/1b proteins can make a nonessential contribution to UCB uptake into the liver. Whether one or more other sinusoidal uptake transporters, or passive diffusion, are primarily responsible for the UCB uptake into hepatocytes remains an open question.

Bile acids play an important role in several physiological processes and they are therefore subject to strict homeostasis (34). Bile acids are synthesized from cholesterol in the liver and undergo extensive enterohepatic circulation, a process in which Na+ taurocholate cotransporting polypeptide (NTCP), bile acid export pump (BSEP/ABCB11), apical sodium bile acid transporter (ASBT), and organic solute transporter (OSTα/OSTβ) play important roles (reviewed in refs. 35, 36). Although it is widely accepted that NTCP/Ntcp functions as the main hepatic bile acid uptake transporter, Slco1a/1b–/– mice showed markedly increased levels of total bile acids in plasma, and this was almost exclusively due to increased unconjugated bile acid levels (13-fold increased; Figure 2B and Supplemental Figure 1). These results suggest that in vivo, conjugated bile acids are primarily taken up by Ntcp and unconjugated bile acids by Oatp1a/1b (note that expression of Ntcp was not changed in Slco1a/1b–/– mice; Supplemental Table 1). Indeed, in vitro studies with rat hepatocytes showed that Na+-dependent transport dominated the uptake of the conjugated bile acid taurocholate (>80%), whereas unconjugated bile acids were more efficiently taken up by (one or more) Na+-independent transport systems, which could include Oatps (36, 37). We note that unconjugated bile acids only account for a small fraction of the overall bile acid pool in the body, which might explain the unchanged biliary excretion of total bile acids in Slco1a/1b–/– mice that we observed. Together, our data demonstrate that Oatp1a/1b transporters make a substantial contribution to hepatic (re-)uptake of unconjugated but not conjugated bile acids. Partly in line with these results, Xiang et al. recently showed that individuals with the SLCO1B1*5 haplotype have increased plasma levels of unconjugated and conjugated bile acids (33).

The present study demonstrates that Oatp1a/1b transporters play an essential role in the pharmacokinetics of MTX and FEX by mediating most of the hepatic uptake of these compounds. Interestingly, the effect of Oatp1a/1b transporters was virtually instantaneous, and only 3.5 minutes after i.v. injection of MTX and FEX, we observed profoundly increased plasma levels in Slco1a/1b–/– mice and profoundly decreased liver levels of MTX and FEX. Moreover, WT mice showed high liver-to-plasma ratios of MTX and FEX 15 minutes after administration (ratios of 43 and 32, respectively; Tables 1 and 2), whereas in Slco1a/1b–/– mice, these values were below 4. This suggests not only that Oatp1a/1b transporters have a high capacity to transport these drugs, but also that they may actively concentrate these drugs in the liver. In vitro studies have shown that MTX is a substrate for Oatp1a4, OATP1B1, OATP1B3, and OATP1A2 (12–14). FEX is transported in vitro by Oatp1a1, -1a4, -1a5 and -1b2, and OATP1A2 (38, 39), but in vitro studies of OATP1B1 and -1B3-mediated uptake of FEX are quite inconsistent (39–41). In the present study, by using Slco1a/1b–/– mice, we proved that Oatp1a/1b transporters effect a profound impact on MTX and FEX pharmacokinetics by acting as high-capacity hepatic uptake transporters that determine the overall elimination rate of these drugs. Note that the plasma levels of the drugs obtained in these mouse studies were well within the therapeutic range in humans.

Surprisingly, shortly after oral dosing of MTX and FEX, no first-pass effect of hepatic Oatp1a/1b transporters was observed, since at those time points plasma and liver concentrations of the drugs did not differ between WT and Slco1a/1b–/– mice. This is in contrast to the rapid and profound effect of hepatic Oatp1a/1b transporters after i.v. administration of MTX and FEX. A possible explanation might be that because of the effectively approximately 100-fold lower plasma concentrations of the drugs upon intestinal uptake compared with shortly after bolus i.v. injection (Figures 3 and 5), there is much more extensive plasma protein binding of the drugs, reducing the drug fraction immediately available for hepatic uptake. Another possibility might be that shortly after oral dosing, concentrations of the drugs in the portal vein are low compared with the Km values of Oatp1a/1b, allowing other hepatic uptake transporters with higher affinities but lower capacities to mediate most hepatic drug uptake. Indeed, for the transport of FEX by hepatic Oatp1a1 and Oatp1a4, the Km values are 32 and 6 μM (38), respectively, and portal vein concentrations of FEX are far below these Km values (~0.03–0.09 μM; Figure 5).

Slco1a/1b–/– mice did not display reduced intestinal absorption of MTX and FEX upon oral dosing, despite the fact that we could demonstrate Slco1a4 and some Slco1a6 mRNA in the small intestine. In vitro, Oatp1a4 can transport both MTX and FEX (14, 39). An explanation for this might be the intestinal expression of Oatp2b1, which possibly compensates for the lack of Oatp1a/1b transporters or the presence of other intestinal uptake transporters. In addition, MTX is transported by the reduced folate carrier (RFC1) which is also present at the apical membrane of mouse small intestinal enterocytes (but not in hepatocytes) (42, 43). Intestinal absorption of MTX in mice might therefore be primarily mediated by RFC1 rather than Oatp1a/1b transporters. Nevertheless, Oatp1a/1b function does have a profound impact on overall oral availability of MTX and FEX (Figures 3 and 5), most likely by its effect on the hepatic elimination of these drugs.

We could demonstrate extensive in vivo inhibition of Oatp1a/1b by rifampicin. Previous in vitro studies have shown that rifampicin is an inhibitor of Oatp1a4, Oatp1b2, OATP1B1, and OATP1B3 (44–46). Rifampicin, an antibiotic mainly used in the treatment of tuberculosis, has been shown to reduce the elimination of BSP and to increase serum-conjugated bilirubin and UCB levels (30, 47). Importantly, our results indicate that rifampicin is quite Oatp1a/1b specific in vivo (at least in the context of MTX pharmacokinetics), since rifampicin treatment did not affect liver or plasma levels of MTX in Slco1a/1b–/– mice. These data demonstrate that Slco1a/1b–/– mice present an ideal tool to study the efficacy and specificity of (novel) Oatp1a/1b inhibitors.

The results of this study and the mouse model generated for it might have important implications for drug development and drug therapy. Although Slco1b2–/– mice have already shown the impact of hepatic Oatp1b2 on liver uptake of some toxins, statins, and antibiotics (2, 9, 10), in this study we demonstrated that Oatp1a/1b effects an even more profound impact on drug pharmacokinetics by mediating (for some drugs, virtually all) hepatic uptake of drugs. This might have sweeping consequences for drug pharmacokinetics in patients, since many SNPs have been identified in the SLCO1B1, SLCO1B3, and SLCO1A2 genes, some of which are responsible for markedly reduced transport capacities (reviewed in ref. 6). For example, individuals carrying SLCO1B1*15 (388A>G and 521T>C) have markedly increased plasma levels of statins and are at increased risk of developing statin-induced myopathy (6, 7). SLCO1B1*15 has also been associated with life-threatening toxicities in patients treated with irinotecan (8). Moreover, 2 intronic SNPs in SLCO1B1, which are linked to each other and to 521T>C, were recently associated with increased plasma clearance and gastrointestinal toxicity of MTX (mucositis grade 3 or 4) (48). This could imply that individualized therapy, based on SLCO genotype, might partly overcome interindividual variation. This latter phenomenon is causing problems in the clinic, since many drugs (and especially anticancer drugs) have a narrow therapeutic window.

Our results indicate that coadministration of a specific OATP inhibitor could be used to increase systemic exposure and hence oral availability of drugs that otherwise have high OATP1A/1B-mediated hepatic uptake. Furthermore, inhibition of OATP1A/1B transporters might be used to limit hepatic toxicity of some drugs. However, adverse drug reactions elsewhere in the body might occur when drugs reach toxic plasma levels upon (unintentional) coadministration with an OATP inhibitor. The Slco1a/1b–/– mice will be invaluable tools in studying the consequences of SLCO1B deficiencies and in testing OATP1A/1B modulation strategies.

In conclusion, we have shown that the Oatp1a/1b knockout mouse model presents a powerful tool in studying the role of Oatp1a/1b transporters in liver physiology and drug pharmacokinetics. We expect that this mouse model will be of great value during drug development and optimization of drug therapy, especially when complemented with transgenic expression of individual human OATP1A/1B transporters to create humanized mouse models.