Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8)

OBJECTIVE: Hepatocellular uptake transporters are involved in the hepatobiliary elimination of endogenous and xenobiotic substances. Mutations in genes encoding these uptake transporters may be key determinants of interindividual variability in hepatobiliary elimination and drug disposition. Our aim was to investigate the functional consequences of mutations in the SLCO1B3 gene encoding the hepatic uptake transporter for organic anions OATP1B3, formerly termed OATP8. METHODS: Mutations occurring in Caucasian Europeans and observed in databases were introduced into the SLCO1B3 cDNA and the consequences were analyzed in stably transfected canine MDCKII cells and human HEK293 cells. The functional consequences were examined for two frequent polymorphisms SLCO1B3-334T>G, encoding OATP1B3-S112A (allelic frequency of 74%) and SLCO1B3-699G>A, encoding OATP1B3-M233I (allelic frequency of 71%) and one rare polymorphism SLCO1B3-1564G>T, encoding OATP1B3-G522C (allelic frequency of 1.9%) and one artificial mutation SLCO1B3-1748G>A, encoding OATP1B3-G583E. RESULTS: OATP1B3-S112A, OATP1B3-M233I, and the OATP1B3 protein corresponding to the reference sequence (accession NM_019844), showed a comparable lateral localization in stably transfected MDCKII cells, whereas OATP1B3-G522C and OATP1B3-G583E proteins were retained intracellularly. Both latter amino acid substitutions abolished the transport of bile acids mediated by OATP1B3, whereas other substrates, like bromosulfophthalein, were transported by all polymorphic variants of the protein. CONCLUSIONS: The functional consequences of three polymorphisms and one artificial mutation include differences in the localization and in transport characteristics of several OATP1B3 proteins. This study demonstrates the importance of the analysis of genetic variations in genes encoding transport proteins for the understanding of individual variations in the hepatobiliary elimination of substances.     

Functional analysis of single nucleotide polymorphisms of hepatic organic anion transporter OATP1B1 (OATP-C)

OBJECTIVE: Two kinds of single nucleotide polymorphism (SNP; Asn130Asp and Val174Ala) are frequently observed in the liver specific transporter, organic anion transporting polypeptide 1B1 (OATP1B1/OATP-C) gene. Although these two SNPs occur independently in European-Americans, Val174Ala is mostly associated with Asn130Asp in Japanese. Our previous in-vivo studies in Japanese subjects indicated that the non-renal clearance of pravastatin was decreased to 13% of that in wild-type subjects (Nishizato et al. Clin Pharmacol Ther 2003;73(6):554-564). The purpose of the present study is to characterize the function of SNPs variants of OATP1B1 in cDNA transfected cells. METHODS: The localization and transport activity were analyzed in HEK293 cells stably expressing wild-type OATP1B1 (OATP1B1*1a), OATP1B1*1b (Asn130Asp), OATP1B1*5 (Val174Ala) and OATP1B1*15 (Asn130Asp and Val174Ala). To characterize the intrinsic Vmax, observed Vmax in uptake study were normalized by the expression level estimated from Western blotting. RESULTS: All SNP variants are predominantly located on the cell surface. No significant alteration was observed in Km values for the transport of 17beta-estradiol 17beta-d-glucuronide (E217betaG), a typical substrate of OATP1B1, among these SNP variants. However, the normalized Vmax value for OATP1B1*15 was drastically decreased to less than 30% compared with OATP1B1*1a. In contrast, the transport activity of OATP1B1*1b (Asn130Asp) and OATP1B1*5 (Val 174Ala) was similar to that of OATP1B1*1a. CONCLUSIONS: These results are consistent with the results of our previous clinical studies. It is thus suggested that in-vivo disposition may be predicted from in-vitro results using recombinant transporters.     

Modulation of transporter activity of OATP1B1 and OATP1B3 by the major active components of Radix Ophiopogonis

Abstract

1. Radix Ophiopogonis is often an integral part of many traditional Chinese formulas, such as Shenmai injection used to treat cardio-cerebrovascular diseases. This study aimed to investigate the influence of the four active components of Radix Ophiopogonis on the transport activity of OATP1B1 and OATP1B3.

2. The uptake of rosuvastatin in OATP1B1-HEK293T cells were stimulated by methylophiopogonanone A (MA) and ophiopogonin D′ (OPD′) with EC₅₀ calculated to be 11.33?±?2.78 and 4.62?±?0.64?μM, respectively. However, there were no remarkable influences on rosuvastatin uptake in the presence of methylophiopogonanone B (MB) or ophiopogonin D (OPD). The uptake of atorvastatin in OATP1B1-HEK293T cells can be increased by MA, MB, OPD and OPD′ with EC₅₀ calculated to be 6.00?±?1.60, 13.64?±?4.07, 10.41?±?1.28 and 3.68?±?0.85?μM, respectively.

3. The uptake of rosuvastatin in OATP1B3-HEK293T cells was scarcely influenced by MA, MB and OPD, but was considerably increased by OPD′ with an EC₅₀ of 14.95?±?1.62?μM. However, the uptake of telmisartan in OATP1B3-HEK293T cells was notably reduced by OPD′ with an IC₅₀ of 4.44?±?1.10?μM, and barely affected by MA, MB and OPD.

4. The four active components of Radix Ophiopogonis affect the transporting activitives of OATP1B1 and OATP1B3 in a substrate-dependent manner.

     

Rapid genotyping of the OATP1B1 polymorphisms A388G and T521C with real-time PCR FRET assays

The polymorphisms (OATP)1B1 A388G and T521C of the solute carrier organic anion-transporter family member 1B1 gene (SLCO1B1), previously known as OATP-C, have potential impacts on drug metabolism. In order to establish a fast and consistent assay for these polymorphisms, rapid speed polymerase chain reaction (PCR) fluorescence resonance energy transfer (FRET) assays on the LightCycler were developed for both OATP1B1 polymorphisms. A locked nucleic acid (LNA) on the polymorphic location within the sensor probe was necessary to discriminate both alleles of the OATP1B1 T521C polymorphism. To confirm the reliability of both real-time PCR FRET assays, these new methods were validated by genotyping 120 samples using a PCR restriction fragment length polymorphism (RFLP) assay and an allele-specific PCR. The results of the real-time PCR FRET assays were completely in line with conventional PCR methods, indicating that the real-time PCR FRET assays are appropriate for clinical settings.     

Interaction of three regiospecific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity

The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds, including bile acids, steroid conjugates, hormones, and drugs, including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide hormone cholecystokinin-8 (CCK-8) is a high affinity substrate for OATP1B3 but not OATP1B1. We utilized homologous recombination of linear DNA by E. coli to generate a library of cDNA containing monomer size chimeric OATP1B1-1B3 and OATP1B3-1B1 transporters with randomly distributed chimeric junctions to identify three discrete regions of the transporter involved in conferring CCK-8 transport activity. Site-directed mutagenesis of three key residues in OATP1B1 transmembrane helices 1 and 10, and extracellular loop 6, to the corresponding residues in OATP1B3, resulted in a gain of CCK-8 transport by OATP1B1. The residues appear specific to CCK-8, as the mutations did not affect transport of the shared OATP1B substrate atorvastatin or the OATP1B1-specific substrate estrone sulfate. Regions involved in gain of CCK-8 transport by OATP1B1, when mapped to the crystal structures of bacterial transporters from the major facilitator superfamily, are positioned to suggest these regions could readily interact with drug substrates. Accordingly, our data provide new insight into the molecular determinants of the substrate specificity of these hepatic uptake transporters with relevance to targeted drug design and prediction of drug-drug interactions.     

Clinical importance of OATP1B1 and OATP1B3 in drug-drug interactions

OATP1B1 and OATP1B3 are transporters that are expressed on the sinusoidal membrane of hepatocytes; they accept a number of therapeutic reagents as their substrates. In vitro and in vivo studies have shown that some drugs inhibit these transporters and cause clinically relevant drug-drug interactions (DDIs). Among these drugs, cyclosporin A markedly increases the plasma concentrations of OATP1B1 substrates. In such cases, the area under the plasma concentration-time curve and the maximum concentration of the affected drugs are increased to a similar degree. Even for OATP1B1 substrates that are metabolized in the liver, the hepatic uptake rate is a determinant of overall hepatic clearance, and the DDIs are partly caused by the inhibition of OATP1B1. Gemfibrozil displays DDIs with some OATP1B1 substrates, although their extent is small. Rifampicin and some HIV protease inhibitors are also OATP1B1 inhibitors. Rifampicin is also an inducer of metabolic enzymes, and although its single coadministration produces an increase in the plasma concentration of the affected drugs, multiple coadministrations may result in reductions in the plasma concentrations of OATP1B1 and CYP3A4 bisubstrates. As a large number of therapeutic reagents are substrates and/or inhibitors of OATP1B1 and OATP1B3, we should be aware of DDIs caused by the inhibition of these transporters.     

Role of the liver-specific transporters OATP1B1 and OATP1B3 in governing drug elimination

Members of the organic anion transporting polypeptide (OATP) family are responsible for the cellular uptake of a broad range of endogenous compounds and xenobiotics in multiple tissues. This review focuses on OATP1B1 and -1B3, which are specifically expressed in the liver and considered to be of particular importance for hepatic drug elimination and drug pharmacokinetics. Recent literature has indicated that inhibition of these transporters may result in drug-drug interactions. Furthermore, genetic polymorphisms in the genes encoding OATP1B1 and -1B3 have been described that increase or decrease transport in vitro and in vivo. Alteration of transporter function by either of these mechanisms may contribute to interindividual variability in drug disposition and response. In this review an update of this rapidly emerging field is provided.     

Polymorphisms and linkage disequilibrium of the OATP8 (OATP1B3) gene in Japanese subjects

OATP8, a member of the organic anion-transporting polypeptide family, is expressed on the sinusoidal membrane of hepatocytes, and transports endogenous organic anions, such as 17beta-glucuronosyl estradiol, and xenobiotic substances, such as digoxin. The objective of this study is to search for polymorphisms of the OATP8 gene and to assess the allele frequency of the polymorphisms in the Japanese population. Analysis of the OATP8 gene in 79 subjects revealed complete linkage of two deletion polymorphisms in the 5' regulatory region, deletion from position -28 to -11 and deletion from position -7 to -4, with an allele frequency of 0.196 for the deletion allele. The polymorphisms T334G (Ser112Ala) and G699A (Met233Ile) were also shown to be in complete linkage disequilibrium, with an allele frequency of 0.728 for the variant (112Ala/233Ile) allele. Interestingly, linkage disequilibrium was identified between the ins/del polymorphism and SNPs at 112 and 233. The predicted major haplotype was the insert-variant type with a haplotype frequency of 0.60.     

Short-lasting inhibition of hepatic uptake transporter OATP1B1 by tyrosine kinase inhibitor pazopanib

Risk assessment of organic anion transporting polypeptide 1B1 (OATP1B1)-mediated drug-drug interactions (DDIs) is an integral part of drug development, but the difficult aspects in DDI prediction include complex mechanism of OATP1B1 inhibition. Pazopanib, an orally available tyrosine kinase inhibitor, exhibits OATP1B1 inhibition and clinically interacts with some OATP1B1 substrates, although quantitative analysis of DDI potential has not yet been performed. The purpose of the present study was to characterize the inhibitory effect of pazopanib on OATP1B1-mediated transport. Inhibition by pazopanib of OATP1B1-mediated uptake of two typical substrates, [³H]estrone-3-sulfate (E₁S) and [³H]estradiol-17β-glucuronide, assessed in HEK293/OATP1B1 cells, was more obvious after preincubation with pazopanib compared with no preincubation. The reduction in IC₅₀ values was 3–7 times greater and was comparable with the preincubation effect of another long-lasting inhibitor, cyclosporine A (CsA). Preincubation with pazopanib and CsA tended to similarly reduce V_max and increase K_m values of E₁S. However, the reduced OATP1B1 activity by preincubation with pazopanib was more rapidly recovered than CsA. In addition, R value, which predicts the maximum increase in the AUC ratio of victim drugs, was calculated to be 1.09. These results suggest that pazopanib is preincubation-dependent but a short-lasting inhibitor against OATP1B1 with low potential of OATP1B1-mediated DDIs.     

Disposition of ezetimibe is influenced by polymorphisms of the hepatic uptake carrier OATP1B1

OBJECTIVES: Genetic variability in hepatic uptake was recently shown to influence the disposition and cholesterol-lowering effects of statins. Ezetimibe, an inhibitor of the intestinal cholesterol uptake protein Niemann-Pick C 1 like 1, is another drug for which genetic polymorphisms of hepatic organic anion transporting polypeptides (OATPs) are expected to be of clinical relevance because ezetimibe undergoes intensive enterohepatic circulation for which hepatic uptake transporters may be rate-limiting determinants. METHODS: Using OATP1B3-, OATP2B1-, and OATP1B1-transfected HEK cells, including the OATP1B1 variants OATP1B1*1b and OATP1B1*5, we measured the uptake of ezetimibe and its glucuronide and we analyzed the competition with the common OATP-substrate bromosulfophthalein. Disposition and sterol-lowering effects of 20-mg ezetimibe were measured in 35 healthy participants genotyped for OATP1B1, ABCB1, ABCC2, and UGT1A1. RESULTS: Ezetimibe glucuronide inhibited bromosulfophthalein uptake in all OATP-transfected cells (50% inhibitory concentration (IC50): 0.14-0.26 mumol/l) whereas ezetimibe was 30-100 times less potent. Only the glucuronide was accumulated significantly in cells expressing OATP1B1 and OATP2B1. Its uptake in cells expressing OATP1B1*1b and *5 was reduced. In-vivo studies showed there was a gene-dose-dependent decrease in the area under the curve of ezetimibe in participants with the OATP1B1*1b protein (*1a/*1a, N=12, 112+/-66 ngxh/ml vs. *1a/*1b, N=8, 88+/-39 ngxh/ml vs. *1b/*1b, N=5, 55+/-18 ngxh/ml; Jonkheere-Terpstra, P=0.041) and a tendency for increased glucuronide exposure (704+/-296 vs. 878+/-369 vs. 1059+/-363 ngxh/ml; P=0.092). Fecal ezetimibe excretion was significantly decreased whereas renal glucuronide excretion was increased in carriers of *1b/*1b. Fecal excretion was also diminished in carriers of OATP1B1*5 and *15. The sterol-lowering effect of ezetimibe was not influenced by OATP1B1 polymorphisms. CONCLUSION: Pharmacokinetics of ezetimibe is influenced by OATP1B1 polymorphisms in healthy participants after single dose administration.     

Interaction of Sulfonylureas with Liver Uptake Transporters OATP1B1 and OATP1B3

Sulfonylureas (SUs) such as glibenclamide, gliclazide, glimepiride, glipizide and gliquidone are one of the first oral medicines available for the treatment of type 2 diabetes and are widely used for the treatment of hyperglycaemia. The hepatic transporters, organic anion transporting polypeptide 1B1 (OATP1B1) and organic anion transporting polypeptide 1B3 (OATP1B3), play an important role in the disposition of a variety of drugs by mediating their uptake from blood into hepatocytes. Drug–drug interactions mediated by OATP1B1/1B3 may result in the hepatic transporting change for drug substrates. The inhibitory effects of glibenclamide and glimepiride on sulfobromophthalein (BSP) uptake have been previously studied, and glibenclamide has been reported as the substrate of OATP1B3, but it remains unclear whether other SUs such as gliclazide, glipizide and gliquidone are substrates of OATP1B1 and OATP1B3. Here, we investigated the relationship between the five most commonly applied SUs (glibenclamide, gliclazide, glimepiride, glipizide, gliquidone) and OATP1B1 and OATP1B3. We performed uptake and inhibition assays in HEK293T cells stably expressing OATP1B1 or OATP1B3, respectively, and established a liquid chromatography–mass spectrometry (LC‐MS) method for the simultaneous measurement of five SUs. We demonstrated that gliclazide and glimepiride are substrates of OATP1B1 and glibenclamide and glipizide are substrates of OATP1B3. We also confirmed the interaction between these SUs and rosuvastatin. No transporting was observed for gliquidone, suggesting that it is not a substrate of either transporter.     

Post-transcriptional regulation of OATP2B1 transporter by a microRNA,miR-24

Human OATP2B1, a member of organic anion transporting polypeptide family, is expressed in several tissues, including small intestine and liver, and contributes to cellular uptake of endogenous compounds and various drugs. Altered expression of OATP2B1 affects pharmacokinetics of substrate drugs; however, limited information is available on the regulation of OATP2B1 expression. This study aimed to explore microRNAs (miRNAs) that regulate OATP2B1 expression using HEK293 cells transfected with an expression plasmid of OATP2B1 including 3′-UTR (HEK/OATP2B1) and Caco-2 as a model of human intestine. Computational analysis predicted that three miRNAs, miR-143, miR-125b and miR-24, may bind to the 3′-UTR of OATP2B1 mRNA. A luciferase assay using a plasmid containing the 3′-UTR of OATP2B1 gene demonstrated that only miR-24 significantly reduced its expression. The overexpression of miR-24 decreased the expression of OATP2B1 mRNA and protein in HEK/OATP2B1 and Caco-2 cells and uptake of [³H]estrone-3-sulfate by HEK/OATP2B1 cells. However, a statistically significant increase of endogenous OATP2B1 expression was not observed by miR-24 inhibitor in Caco-2 cells. In conclusion, it was found that miR-24 negatively regulates OATP2B1 expression, resulting in suppression of OATP2B1 activity, while its contribution to regulation of apparent expression of OATP2B1 is considered to depend on tissues and cell types.     

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.     

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.     

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.     

有机阴离子转运多肽1B1（OATP1B1）基因多态性对他汀类药物影响的研究进展

有机阴离子转运多肽1B1(OATP1B1)是一种负责转运多种内外源性物质进入肝细胞发挥作用的摄入型转运蛋白。他汀类药物又称3-羟基-3-甲基戊二酸单酰辅酶A(HMG-CoA)还原酶抑制剂,临床上广泛应用于调脂及心脑血管疾病的预防,其疗效及不良反应有显著的个体化差异。研究表明OATP1B1基因多态性是导致他汀类药物个体化差异的重要因素。对OATP1B1基因多态性对他汀类药物影响的研究进展进行综述,为他汀类药物的个体化、安全化应用提供可能的理论指导。     

Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3

Okadaic acid is known as a diarrheal shellfish poison. It is thought that there is no specific target organ for okadaic acid after it has been absorbed into the body. However, the details of its pharmacokinetics are still unknown.In this study, we demonstrated that okadaic acid was more toxic to the hepatocyte-specific uptake transporter OATP1B1- or OATP1B3-expressing cells than control vector-transfected cells. In addition, PP2A activity, which is a target molecule of okadaic acid, was more potently inhibited by okadaic acid in OATP1B1- or OATP1B3-expressing cells compared with control vector-transfected cells. The cytotoxicity of okadaic acid in OATP1B1- or OATP1B3-expressing cells was attenuated by known substrates of OATP1B1- and OATP1B3, but not in control vector-transfected cells. Furthermore, after uptake inhibition study using OATP1B3-expressing cells, Dixon plot showed that okadaic acid inhibited the uptake of hepatotoxin microcystin-LR, which is a substrate for OATP1B1 and OATP1B3, in a competitive manner. These results strongly suggested that okadaic acid is a substrate for OATP1B3 and probably for OATP1B1, and could be involved in unknown caused liver failure and liver cancer. Since okadaic acid possesses cytotoxicity and cell proliferative activity by virtue of its known phosphatase inhibition activity.     

Nostocyclopeptide-M1: a potent, nontoxic inhibitor of the hepatocyte drug transporters OATP1B3 and OATP1B1

We have isolated a novel cyanobacterial cyclic peptide (nostocyclopeptide M1; Ncp-M1) that blocks the hepatotoxic action of microcystin (MC) and nodularin (Nod). We show here that Ncp-M1 is nontoxic to primary hepatocytes in long-term culture. Ncp-M1 does not affect any known intracellular targets or pathways involved in MC action, like protein phosphatases, CaM-KII, or ROS-dependent cell death effectors. In support of this conclusion Ncp-M1 had no protective effect when microinjected into cells. Rather, the antitoxin effect was solely due to blocked hepatocyte uptake of MC and Nod. The hepatic uptake of MC and Nod is mainly via the closely related organic anion transporters OATP1B1 and OATP1B3, which also mediate hepatic transport of endogenous metabolites and hormones as well as drugs. OATP1B3 is also expressed in some aggressive cancers, where it confers apoptosis resistance. We show that Ncp-M1 inhibits transport through OATP1B3 and OATP1B1 expressed in HEK293 cells. The Ncp-M1 molecule has several nonproteinogenic amino acids and an imino bond, which hamper its synthesis. Moreover, a cyclic all L-amino acid heptapeptide analogue of Ncp-M1 also inhibits the OATP1B1/1B3 transporters, and with higher OATP1B3 preference than Ncp-M1 itself. The nontoxic Ncp-M1 and its synthetic cyclic peptide analogues thus provide new tools to probe the role of OATB1B1/1B3 mediated drug and metabolite transport in liver and cancer cells. They can also serve as scaffolds to design new, exopeptidase resistant OATP1B3-specific modulators.     

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.