Uptake of ursodeoxycholate and its conjugates by human hepatocytes: role of Na(+)-taurocholate cotransporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1 (OATP-C), and oatp1B3 (OATP8)

Ursodeoxycholate (UDCA) is widely used for the treatment of cholestatic liver disease. After oral administration, UDCA is absorbed, taken up efficiently by hepatocytes, and conjugated mainly with glycine to form glycoursodeoxycholate (GUDC) or partly with taurine to form tauroursodeoxycholate (TUDC), which undergo enterohepatic circulation. In this study, to check whether three basolateral transporters--Na(+)-taurocholate cotransporting polypeptide (NTCP, SLC10A1), organic anion transporting polypeptide (OATP) 1B1 (OATP-C), and OATP1B3 (OATP8)-mediate uptake of UDCA, GUDC, and TUDC by human hepatocytes, we investigated their transport properties using transporter-expressing HEK293 cells and human cryopreserved hepatocytes. TUDC and GUDC could be taken up via human NTCP, OATP1B1, and OATP1B3, whereas UDCA could be transported significantly by NTCP, but not OATP1B1 and OATP1B3 in our expression systems. We observed a time-dependent and saturable uptake of UDCA and its conjugates by human cryopreserved hepatocytes, and more than half of the overall uptake involved a saturable component. Kinetic analyses revealed that the contribution of Na(+)-dependent and -independent pathways to the uptake of UDCA or TUDC was very similar, while the Na(+)-independent uptake of GUDC was predominant. These results suggest that UDCA and its conjugates are taken up by both multiple saturable transport systems and nonsaturable transport in human liver with different contributions. These results provide an explanation for the efficient hepatic clearance of UDCA and its conjugates in patients receiving UDCA therapy.     

Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (K_m and V_max) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.     

Genetic polymorphisms in Na+-taurocholate co-transporting polypeptide (NTCP) and ileal apical sodium-dependent bile acid transporter (ASBT) and ethnic comparisons of functional variants of NTCP among Asian populations

Genetic variants of Na(+)-taurocholate co-transporting polypeptide (NTCP; SLC10A1) and ileal apical sodium-dependent bile acid transporter (ASBT; SLC10A2), which greatly contribute to bile acid homeostasis, were extensively explored in the Korean population and functional variants of NTCP were compared among Asian populations. From direct DNA sequencing, six SNPs were identified in the SLC10A1 gene and 14 SNPs in the SLC10A2 gene. Three of seven coding variants were non-synonymous SNPs: two variants from SLC10A1 (A64T, S267F) and one from SLC10A2 (A171S). No linkage was analysed in the SLC10A1 gene because of low frequencies of genetic variants, and the SLC10A2 gene was composed of two separated linkage disequilibrium blocks contrary to the white population. The stably transfected NTCP-A64T variant showed significantly decreased uptakes of taurocholate and rosuvastatin compared with wild-type NTCP. The decreased taurocholate uptake and increased rosuvastatin uptake were shown in the NTCP-S267F variant. The allele frequencies of these functional variants were 1.0% and 3.1%, respectively, in a Korean population. However, NTCP-A64T was not found in Chinese and Vietnamese subjects. The frequency distribution of NTCP-S267F in Koreans was significantly lower than those in Chinese and Vietnamese populations. Our data suggest that NTCP-A64T and -S267F variants cause substrate-dependent functional change in vitro, and show ethnic difference in their allelic frequencies among Asian populations although the clinical relevance of these variants is remained to be evaluated.     

pH-sensitive interaction of HMG-CoA reductase inhibitors (statins) with organic anion transporting polypeptide 2B1

The human organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is ubiquitously expressed and may play an important role in the disposition of xenobiotics. The present study aimed to examine the role of OATP2B1 in the intestinal absorption and tissue uptake of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors (statins). We first investigated the functional affinity of statins to the transporter as a function of extracellular pH, using OATP2B1-transfeced HEK293 cells. The results indicate that OATP2B1-mediated transport is significant for rosuvastatin, fluvastatin and atorvastatin, at neutral pH. However, OATP2B1 showed broader substrate specificity as well as enhanced transporter activity at acidic pH. Furthermore, uptake at acidic pH was diminished in the presence of proton ionophore, suggesting proton gradient as the driving force for OATP2B1 activity. Notably, passive transport rates are predominant or comparable to active transport rates for statins, except for rosuvastatin and fluvastatin. Second, we studied the effect of OATP modulators on statin uptake. At pH 6.0, OATP2B1-mediated transport of atorvastatin and cerivastatin was not inhibitable, while rosuvastatin transport was inhibited by E-3-S, rifamycin SV and cyclosporine with IC(50) values of 19.7 ± 3.3 μM, 0.53 ± 0.2 μM and 2.2 ± 0.4 μM, respectively. Rifamycin SV inhibited OATP2B1-mediated transport of E-3-S and rosuvastatin with similar IC(50) values at pH 6.0 and 7.4, suggesting that the inhibitor affinity is not pH-dependent. Finally, we noted that OATP2B1-mediated transport of E-3-S, but not rosuvastatin, is pH sensitive in intestinal epithelial (Caco-2) cells. However, uptake of E-3-S and rosuvastatin by Caco-2 cells was diminished in the presence of proton ionophore. The present results indicate that OATP2B1 may be involved in the tissue uptake of rosuvastatin and fluvastatin, while OATP2B1 may play a significant role in the intestinal absorption of several statins due to their transporter affinity at acidic pH.     

Substrate- and pH-specific antifolate transport mediated by organic anion-transporting polypeptide 2B1 (OATP2B1-SLCO2B1)

Human organic anion-transporting polypeptide (OATP) 2B1 (OATP-B; SLCO2B1) is expressed in the apical membrane of the small intestine and the hepatocyte basolateral membrane and transports structurally diverse organic anions with a wide spectrum of pH sensitivities. This article describes highly pH-dependent OATP2B1-mediated antifolate transport and compares this property with that of sulfobromophthalein (BSP), a preferred OATP2B1 substrate. At pH 5.5 and low substrate concentrations (~2.5 μM), only [(3)H]pemetrexed influx [in contrast to methotrexate (MTX), folic acid, and reduced folates] could be detected in OATP2B1-transfected HeLa R1-11 cells that lack endogenous folate-specific transporters. Influx was optimal at pH 4.5 to 5.5, falling precipitously with an increase in pH >6.0; BSP influx was independent of pH. Influx of both substrates at low pH was markedly inhibited by the proton ionophore 4-(trifluoromethoxy)phenylhydrazone; BSP influx was also suppressed at pH 7.4. At 300 μM MTX, influx was one-third that of pemetrexed; influx of folic acid, (6S)5-methyltetrahydrofolate, or (6S)5-formyltetrahydrofolate was not detected. There were similar findings in OATP2B1-expressing Xenopus laevis oocytes. The pemetrexed influx K(m) was ~300 μM; the raltitrexed influx K(i) was ~70 μM at pH 5.5. Stable expression of OAPT2B1 in HeLa R1-11 cells resulted in substantial raltitrexed, but modest pemetrexed, growth inhibition consistent with their affinities for this carrier. Hence, OATP2B1 represents a low-affinity transport route for antifolates (relative affinities: raltitrexed > pemetrexed > MTX) at low pH. In contrast, the high affinity of this transporter for BSP relative to antifolates seems to be intrinsic to its binding site and independent of the proton concentration.     

Contribution of OATP (organic anion-transporting polypeptide) family transporters to the hepatic uptake of fexofenadine in humans.

Fexofenadine hydrochloride (FEX), a second generation H(1)-receptor antagonist, is mainly eliminated from the liver into bile in unchanged form. Recent studies have shown that FEX can be accepted by human MDR1 (P-glycoprotein), OATP1A2 [organic anion-transporting polypeptide (OATP)-A, and OATP2B1 (OATP-B)] expression systems. However, other transporters responsible for the hepatic uptake of FEX have not yet been identified. In the present study, we evaluated the contribution of OATP family transporters, namely OATP1B1 (OATP2/OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), to FEX uptake using transporter-expressing HEK293 (human embryonic kidney) cells. The uptake of FEX in OATP1B3-expressing cells was significantly greater than that in vector-transfected cells. On the other hand, OATP1B1- or OATP2B1-mediated uptake of FEX was not statistically significant. OATP1B3-mediated transport could be explained by a one-saturable component with a Michaelis constant (K(m)) of 108 +/- 11 microM. The inhibitory effect of FEX on the uptake of estrone-3-sulfate (E(1)S), cholecystokinin octapeptide (CCK-8), and 17beta-estradiol-17beta-d-glucuronide (E(2)17betaG) was also examined. Both OATP1B1- and OATP1B3-mediated E(2)17betaG uptake was inhibited by FEX. The K(i) values were 148 +/- 61 and 205 +/- 72 microM for OATP1B1 and OATP1B3, respectively. FEX also inhibited OATP1B3-mediated CCK-8 uptake and OATP1B1-mediated E(1)S uptake with a K(i) value of 83.3 +/- 15.3 and 257 +/- 84 microM, respectively, suggesting that FEX could not be used as a specific inhibitor for OATP1B1 and OATP1B3, although FEX was preferentially accepted by OATP1B3. In conclusion, this is, to our knowledge, the first demonstration that OATP1B3 is thought to be a major transporter involved in hepatic uptake of FEX in humans.     

Substrate-dependent drug-drug interactions between gemfibrozil, fluvastatin and other organic anion-transporting peptide (OATP) substrates on OATP1B1, OATP2B1, and OATP1B3.

Hepatic uptake carriers of the organic anion-transporting peptide (OATP) family of solute carriers are more and more recognized as being involved in hepatic elimination of many drugs and potentially associated drug-drug interactions. The gemfibrozil-statin interaction was studied at the level of active hepatic uptake as a model for such drug-drug interactions. Active, temperature-dependent uptake of fluvastatin into primary human hepatocytes was shown. Multiple transporters are involved in this uptake as Chinese hamster ovary or HEK293 cells expressing either OATP1B1 (K(m) = 1.4-3.5 microM), OATP2B1 (K(m) = 0.7-0.8 microM), or OATP1B3 showed significant fluvastatin uptake relative to control cells. For OATP1B1 the inhibition by gemfibrozil was substrate-dependent as the transport of fluvastatin (IC(50) of 63 microM), pravastatin, simvastatin, and taurocholate was inhibited by gemfibrozil, whereas the transport of estrone-3-sulfate and troglitazone sulfate (both used at 3 microM) was not affected. The OATP1B1- but not OATP2B1-mediated transport of estrone-3-sulfate displayed biphasic saturation kinetics, with two distinct affinity components for estrone-3-sulfate (0.23 and 45 microM). Only the high-affinity component was inhibited by gemfibrozil. Recombinant OATP1B1-, OATP2B1-, and OATP1B3-mediated fluvastatin transport was inhibited to 97, 70, and 62% by gemfibrozil (200 microM), respectively, whereas only a small inhibitory effect by gemfibrozil (200 microM) on fluvastatin uptake into primary human hepatocytes was observed (27% inhibition). The results indicate that the in vitro engineered systems can not always predict the behavior in more complex systems such as freshly isolated primary hepatocytes. Therefore, selection of substrate, substrate concentration, and in vitro transport system are critical for the conduct of in vitro interaction studies involving individual liver OATP carriers.     

Interactions of crizotinib and gefitinib with organic anion-transporting polypeptides (OATP)1B1, OATP1B3 and OATP2B1: gefitinib shows contradictory interaction with OATP1B3

1.?The drug–drug interaction (DDI) mediated by organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and OATP2B1 has a major impact on the hepatic clearance of drugs. The effects of tyrosine kinase inhibitors (TKIs) on OATPs have not been well studied. In the present study, we evaluated the contribution of OATPs to the hepatic uptake of crizotinib and gefitinib and the interaction of those TKIs with OATPs to estimate DDIs.

2.?To clarify whether crizotinib and gefitinib were substrates for OATPs, we performed uptake studies. We examined the effects of the TKIs on uptake of typical substrates and fluvastatin via OATPs. IC₅₀ and EC₅₀ values of the TKIs were calculated.

3.?OATP1B3- and OATP2B1-mediated crizotinib uptake and OATP2B1-mediated gefitinib uptake were observed. Gefitinib accelerated OATP1B3-mediated [³H]TCA uptake and inhibited OATP2B1-mediated [³H]E₃S uptake. On the other hand, gefitinib inhibited OATP1B1- and OATP2B1-mediated fluvastatin uptake.

4.?We provided basic information to estimate the DDI on OATPs caused by TKIs. The DDI on OATPs caused by gefitinib could occur in a normal clinical situation. And the uptake of crizotinib into the intrahepatocellular environment via OATPs may induce DDI and liver damage. We therefore emphasize the necessity of careful use of TKIs.     

Further characterization of the electrogenicity and pH sensitivity of the human organic anion-transporting polypeptides OATP1B1 and OATP1B3

Organic anion-transporting polypeptides (OATPs) are involved in the liver uptake of many endogenous and xenobiotic compounds, such as bile acids and drugs, respectively. Using Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells expressing rat Oatp1a1, human OATP1B1, or OATP1B3, the sensitivity of these transporters to extracellular/intracellular pH (pHo/pHi) and changes in plasma membrane potential (ΔΨ) was investigated. In X. laevis oocytes, nonspecific plasma membrane permeability increased only at pHo below 4.5. Above this value, both using oocytes and CHO cells, extracellular acidification affected differently the specific transport of taurocholic acid (TCA) and estradiol 17β-d-glucuronide (E(2)17βG) by Oatp1a1 (stimulation), OATP1B1 (inhibition), and OATP1B3 (stimulation). Changes in substrate uptake in the presence of valinomycin (K(+)-ionophore), carbonyl cyanide 3-chlorophenylhydrazone and nigericin (protonophores), and amiloride (Na(+)/H(+)-inhibitor) and cation replacement in the medium were studied with fluorescent probes for measuring substrate uptake (cholylglycyl amidofluorescein) and changes in pHi (SNARF-4F) and ΔΨ [DilC(1)(5)]. The results suggest that activity of these three carriers is sodium/potassium-independent and affected differently by changes in pHo and ΔΨ: Oatp1a1 was confirmed to be an electroneutral anion exchanger, whereas the function of both OATP1B1 and OATP1B3 was markedly affected by the magnitude of ΔΨ. Moreover, electrophysiological measurements revealed the existence of a net anion influx associated to OATP1B1/OATP1B3-mediated transport of TCA, E(2)17βG, and estrone-3-sulfate. Furthermore, a leakage of Na(+) through OATP1B1 and OATP1B3, which is not coupled to substrate transport, was found. In conclusion, these results suggest that OATP1B1 and OATP1B3 are electrogenic transporters whose activity may be strongly affected under circumstances of displacement of local pH.     

Characterization of ursodeoxycholic and norursodeoxycholic acid as substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and NTCP

Ursodeoxycholic acid (UDCA) is the only approved treatment for primary biliary cirrhosis, and norursodeoxycholic acid (norUDCA) is currently tested in clinical trials for future treatment of primary sclerosing cholangitis because of beneficial effects in cholestatic Mdr2 knock‐out mice. Uptake of UDCA and norUDCA into hepatocytes is believed to be a prerequisite for subsequent metabolism and therapeutic action. However, the molecular determinants of hepatocellular uptake of UDCA and norUDCA are poorly understood. We therefore investigated whether UDCA and norUDCA are substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and Na⁺‐taurocholate co‐transporting polypeptide (NTCP), which are localized in the basolateral membrane of hepatocytes. Uptake of [³H]UDCA and [¹⁴C]norUDCA into Human embryonic kidney (HEK) cells stably expressing OATP1B1, OATP1B3, OATP2B1 or NTCP was investigated and compared with uptake into vector control cells. Uptake ratios were calculated by dividing uptake into transporter‐transfected cells by uptake into respective control cells. Uptake ratios of OATP1B1‐, OATP1B3‐ and OATP2B1‐mediated UDCA and norUDCA uptake were at maximum 1.23 and 1.49, respectively. Uptake of UDCA was significantly higher into HEK‐NTCP cells only at the lowest tested concentration (1 μM, p < 0.001) compared with the control cells with an uptake ratio of 1.34‐fold. NorUDCA was not significantly transported by NTCP. The low uptake rates suggest that OATP1B1, OATP1B3, OATP2B1 and NTCP are not relevant for hepatocellular uptake and effects of UDCA and norUDCA in human beings.     

Effect of genetic polymorphism of OATP-C (SLCO1B1) on lipid-lowering response to HMG-CoA reductase inhibitors

The effect of genetic polymorphism of human organic anion transporting polypeptide C (OATP-C) on the lipid-lowering response to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors was assessed. A retrospective study was conducted on 66 patients who underwent treatment of hyperlipidemia with HMG-CoA reductase inhibitors in a municipal hospital in a community-based cohort of Ehime prefecture in the southern part of Japan. Plasma lipid concentrations before and after administration were analyzed in patients in relation to the 521T/C (Val-174-->Ala) polymorphism in the OATP-C gene (TT: n=44 (66.7%), TC: n=20 (30.3%), CC: n=0 (0.0%), undetermined: n=2 (3.0%)). Total cholesterol level was significantly lowered after treatment with HMG-CoA reductase inhibitors in all patients (p<0.001); moreover, subjects with the 521C allele showed an attenuated total-cholesterol-lowering effect compared with those homozygous for the 521T allele (-22.3+/-8.7% vs. -16.5+/-10.5%, p<0.05). These data suggest that the 521T/C polymorphism of the OATP-C gene modulates the lipid-lowering efficacy of HMG-CoA reductase inhibitors.     

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.     

Potential role of organic anion transporting polypeptide 1B1 (OATP1B1) in the selective hepatic uptake of hematoporphyrin monomethyl ether isomers

Aim:

Hematoporphyrin monomethyl ether (HMME), which consists of equal amounts of isomers HMME-1 and HMME-2, is a novel porphyrin-related drug for photodynamic therapy. This study was aimed to investigate the uptake transporter-mediated selective uptake of HMME into the liver and to identify the major uptake transporter isoforms involved.

Methods:

Adult SD rats were intravenously injected with a single dose of HMME (5 mg/kg) with or without rifampicin (an inhibitor of organic anion transporting polypeptides OATP1B1 and OATP1B3, 25 mg/kg). Blood samples were collected, and HMME concentrations were measured using LC-MS/MS. Rat hepatocytes, human hepatocytes and HEK293 cells expressing OATP1B1, OATP1B3, or OATP2B1 were used to investigate the uptake of HMME or individual isomers in vitro.

Results:

Co-administration of rifampicin significantly increased the exposure of HMME isomers, and decreased the AUC ratio of HMME-1 to HMME-2 from 1.98 to 1.56. The uptake of HMME-2 into human hepatocytes and the HEK293 cells expressing OATP1B1 or OATP2B1 in vitro was 2–7 times greater than that of HMME-1, whereas OATP1B3 mediated a higher HMME-1 uptake. OATP1B1 exhibited a higher affinity for HMME-2 than for HMME-1 (the Km values were 0.63 and 5.61 μmol/L, respectively), which were similar to those in human hepatocytes. By using telmisartan (a non-specific OATP inhibitor) and rifampicin, OATP2B1 was demonstrated to account for <20% of hepatic HMME uptake.

Conclusion:

OATP1B1 is the major transporter involved in the rapid hepatic uptake of HMME, and the greater uptake of HMME-2 by OATP1B1 may lead to a lower exposure of HMME-2 than HMME-1 in humans.     

Functional consequences of genetic variations in the human organic anion transporting polypeptide 1B3 (OATP1B3) in the Korean population

The objectives of this study were to investigate the allele frequencies and linkage disequilibrium (LD) in the organic anion transporting polypeptide 1B3 (OATP1B3) in the Korean population and to examine the functional consequences. Using samples from 48 Koreans, direct sequencing was carried out to determine the allele frequencies and LD of OATP1B3 in a representative Korean population. Thirty-six genetic variations in the transporter were found in Koreans; among them, five undocumented variations (i.e.,-6436G>C in the 5'-upstream region, 26A>C and 586A>G in the protein coding region, and IVS6-72A>T and IVS12-80A>T in intron regions) were identified. In the upstream region, -5035G>A was found to have lowered gene expression, as determined by a reporter gene assay, suggesting that this variation reduces the expression of OATP1B3 in humans. The functional relevance of the genetic variations in the protein coding region was determined by an uptake study involving representative substrates in human embryonic kidney 293 cells expressing wild type or variant forms. Variations involving 699G>A showed a reduced uptake activity for testosterone, but not for estradiol 17β-d-glucuronide or methotrexate, indicating that the functional impact of the variations is substrate specific. Considering the kinetic relevance of OATP1B3, the functionally affected variations may be therapeutically important.     

Hepatic uptake in the dog: comparison of uptake in hepatocytes and human embryonic kidney cells expressing dog organic anion-transporting polypeptide 1B4

Although the dog is frequently used in pharmacological, pharmacokinetic, and drug safety studies, little is known about canine drug transporters. Dog organic anion-transporting polypeptide (Oatp1b4) has recently been cloned (Comp Biochem Physiol C Toxicol Pharmacol 151:393-399, 2010), but the contribution of Oatp1b4 to hepatic uptake has yet to be clarified. This study compares the transport characteristics of dog Oatp1b4 with those of human OATP1B1/1B3 and demonstrates the importance of Oatp1b4 in the uptake of anionic compounds in dog hepatocytes. Oatp1b4 is the predominant Oatp in dog liver with expression levels double and 30 times those of Oatp2b1 and Oatp1a2, respectively. Uptake of a range of typical OATP substrates by Oatp1b4-expressing HEK293 cells was compared with that in fresh dog hepatocytes. All compounds tested were transported by Oatp1b4 and uptake intrinsic clearance (CL(int, uptake)) in dog hepatocytes in sodium-free buffer was correlated significantly with CL(int, uptake) in Oatp1b4-expressing cells. Dog in vivo clearance for five substrates was predicted more accurately from CL(int, uptake) than from metabolic intrinsic clearance (CL(int, met)), indicating that uptake governs the overall in vivo hepatic clearance of these anionic compounds in dog. The substrate specificities of dog Oatp1b4 appear to be similar to those of human OATP1B1/OATP1B3, whereas the relative uptake clearance of substrates for Oatp1b4 correlate better with OATP1B3 than with the more abundant hepatic analog OATP1B1.     

The influence of macrolide antibiotics on the uptake of organic anions and drugs mediated by OATP1B1 and OATP1B3.

Macrolides may cause severe drug interactions due to the inhibition of metabolizing enzymes. Transporter-mediated uptake of drugs into cells [e.g., by members of the human organic anion transporting polypeptide (OATP) family] is a determinant of drug disposition and a prerequisite for subsequent metabolism. However whether macrolides are also inhibitors of uptake transporters, thereby providing an additional mechanism of drug interactions, has not been systematically studied. The human OATP family members OATP1B1 and OATP1B3 mediate the uptake of endogenous substances and drugs such as antibiotics and HMG-CoA reductase inhibitors (statins) into hepatocytes. In this study we investigated the potential role of these uptake transporters on macrolide-induced drug interactions. By using sulfobromophthalein (BSP) and the HMG-CoA reductase inhibitor pravastatin as substrates, the effects of the macrolides azithromycin, clarithromycin, erythromycin, and roxithromycin and of the ketolide telithromycin on the OATP1B1- and OATP1B3-mediated uptake were analyzed. These experiments demonstrated that the OATP1B1- and OATP1B3-mediated uptake of BSP and pravastatin can be inhibited by increasing concentrations of all macrolides except azithromycin. The IC50 values for the inhibition of OATP1B3-mediated BSP uptake were 11 microM for telithromycin, 32 microM for clarithromycin, 34 microM for erythromycin, and 37 microM for roxithromycin. These IC50 values were lower than the IC50 values for inhibition of OATP1B1-mediated BSP uptake (96-217 microM). These macrolides also inhibited in a concentration-dependent manner the OATP1B1- and OATP1B3-mediated uptake of pravastatin. In summary, these results indicate that alterations of uptake transporter function by certain macrolides/ketolides have to be considered as a potential additional mechanism underlying drug-drug interactions.     

Differential interaction of 3-hydroxy-3-methylglutaryl-coa reductase inhibitors with ABCB1, ABCC2, and OATP1B1.

The present study examined the interaction of four 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (atorvastatin, lovastatin, and simvastatin in acid and lactone forms, and pravastatin in acid form only) with multidrug resistance gene 1 (MDR1, ABCB1) P-glycoprotein, multidrug resistance-associated protein 2 (MRP2, ABCC2), and organic anion-transporting polypeptide 1B1 (OATP1B1, SLCO21A6). P-glycoprotein substrate assays were performed using Madin-Darby canine kidney (MDCK) cells expressing MDR1, and the efflux ratios [the ratio of the ratio of basolateral-to-apical apparent permeability and apical-to-basolateral permeability between MDR1 and MDCK] were 1.87, 2.32/4.46, 2.17/3.17, and 0.93/2.00 for pravastatin, atorvastatin (lactone/acid), lovastatin (lactone/acid), and simvastatin (lactone/acid), respectively, indicating that these compounds are weak or moderate substrates of P-glycoprotein. In the inhibition assays (MDR1, MRP2, Mrp2, and OATP1B1), the IC50 values for efflux transporters (MDR1, MRP2, and Mrp2) were >100 microM for all statins in acid form except lovastatin acid (>33 microM), and the IC50 values were up to 10-fold lower for the corresponding lactone forms. In contrast, the IC50 values for the uptake transporter OATP1B1 were 3- to 7-fold lower for statins in the acid form compared with the corresponding lactone form. These data demonstrate that lactone and acid forms of statins exhibit differential substrate and inhibitor activities toward efflux and uptake transporters. The interconversion between the lactone and acid forms of most statins exists in the body and will potentially influence drug-transporter interactions, and may ultimately contribute to the differences in pharmacokinetic profiles observed between statins.     

Interaction of deoxyschizandrin and schizandrin B with liver uptake transporters OATP1B1 and OATP1B3

1. Deoxyschizandrin and schizandrin B have diverse pharmacological effects, including hepatoprotective activity. We aim to study their hepatic uptake and their effects on the hepatic uptake of other clinical drugs mediated by OATP1B1 and OATP1B3.

2. Deoxyschizandrin exhibited a high affinity for OATP1B1 with K_m of 17.61?±?0.43?μM but a low affinity for OATP1B3. Similarly, schizandrin B also showed a strong affinity for OATP1B1 with K_m of 18.45?±?1.23?μM but a weak affinity for OATP1B3.

3. Atorvastatin and rifampicin could inhibit the uptake of deoxyschizandrin and schizandrin B mediated by OATP1B1.

4. Intriguingly, both deoxyschizandrin and schizandrin B significantly promoted the uptake of atorvastatin (with EC₅₀ of 50.58?±?8.08 and 24.70?±?5.82 µM, respectively) and rosuvastatin (with EC₅₀ of 13.46?±?2.70 and 8.99?±?4.73 µM, respectively) mediated by OATP1B1. Deoxyschizandrin could markedly promote the uptake of fluvastatin but inhibit the uptake of sodium taurocholate (TCNa) mediated by OATP1B1.

5. The promotion on hepatic uptake of statins mediated by OATP1B1 might lead to enhanced efficacy of cholesterol lowering and reduced risk of myopathy for hyperlipidemia patients when given statins together with deoxyschizandrin or schizandrin B.     

Inhibition of the organic anion-transporting polypeptide 1B1 by quercetin: an in vitro and in vivo assessment

AIM

To investigate the effect of quercetin on organic anion transporting polypeptide 1B1 (OATP1B1) activities in vitro and on the pharmacokinetics of pravastatin, a typical substrate for OATP1B1 in healthy Chinese-Han male subjects.

METHODS

Using human embryonic kidney 293 (HEK293) cells stably expressing OATP1B1, we observed the effect of quercetin on OATP1B1-mediated uptake of estrone-3-sulphate (E3S) and pravastatin. The influence of quercetin on the pharmacokinetics of pravastatin was measured in 16 healthy Chinese-Han male volunteers receiving a single dose of pravastatin (40 mg orally) after co-administration of placebo or 500 mg quercetin capsules (once daily orally for 14 days).

RESULTS

Quercetin competitively inhibited OATP1B1-mediated E3S uptake with a K_i value of 17.9 ± 4.6 µm and also inhibited OATP1B1-mediated pravastatin uptake in a concentration dependent manner (IC₅₀, 15.9 ± 1.4 µm). In healthy Chinese-Han male subjects, quercetin increased the pravastatin area under the plasma concentration – time curve (AUC(0,10 h) and the peak plasma drug concentration (C_max) to 24% (95% CI 15, 32%, P < 0.001) and 31% (95% CI 20, 42%, P < 0.001), respectively. After administration of quercetin, the elimination half-life (t_1/2) of pravastatin was prolonged by 14% (95% CI 4, 24%, P = 0.027), with no change in the time to reach C_max (t_max). Moreover, quercetin decreased the apparent clearance (CL/F) of pravastatin by 18% (95% CI 75, 89%, P < 0.001).

CONCLUSIONS

These findings suggest that quercetin inhibits the OATP1B1-mediated transport of E3S and pravastatin in vitro and also has a modest inhibitory influence on the pharmacokinetics of pravastatin in healthy Chinese-Han male volunteers. The effects of quercetin on other OATP1B1 substrate drugs deserve further investigation.