OATP1B1 Transport Activity Research Articles

Metabolism, Disposition, Excretion, and Potential Transporter Inhibition of 7-16, an Improving 5-HT2A Receptor Antagonist and Inverse Agonist for Parkinson's Disease.

Compound 7-16 was designed and synthesized in our previous study and was identified as a more potential selective 5-HT2A receptor antagonist and inverse agonist for treating Parkinson's disease psychosis (PDP). Then, the metabolism, disposition, and excretion properties of 7-16 and its potential inhibition on transporters were investigated in this study to highlight advancements in the understanding of its therapeutic mechanisms. The results indicate that a total of 10 metabolites of 7-16/[14C]7-16 were identified and determined in five species of liver microsomes and in rats using UPLC-Q Exactive high-resolution mass spectrometry combined with radioanalysis. Metabolites formed in human liver microsomes could be covered by animal species. 7-16 is mainly metabolized through mono-oxidation (M470-2) and N-demethylation (M440), and the CYP3A4 isozyme was responsible for both metabolic reactions. Based on the excretion data in bile and urine, the absorption rate of 7-16 was at least 74.7%. 7-16 had weak inhibition on P-glycoprotein and no effect on the transport activity of OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 transporters. The comprehensive pharmacokinetic properties indicate that 7-16 deserves further development as a new treatment drug for PDP.

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

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

Further Studies to Support the Use of Coproporphyrin I and III as Novel Clinical Biomarkers for Evaluating the Potential for Organic Anion Transporting Polypeptide 1B1 and OATP1B3 Inhibition.

In a recent study, limited to South Asian Indian subjects (n = 12), coproporphyrin (CP) I and CPIII demonstrated properties appropriate for an organic anion-transporting polypeptide (OATP) 1B endogenous probe. The current studies were conducted in healthy volunteers of mixed ethnicities, including black, white, and Hispanic subjects, to better understand the utility of these biomarkers in broader populations. After oral administration with 600 mg rifampin, AUC(0-24h) values were 2.8-, 3.7-, and 3.6-fold higher than predose levels for CPI and 2.6-, 3.1-, and 2.4-fold higher for CPIII, for the three populations, respectively. These changes in response to rifampin were consistent with previous results. The sensitivity toward OATP1B inhibition was also investigated by evaluating changes of plasma CP levels in the presence of diltiazem and itraconazole [administered as part of an unrelated drug-drug interaction (DDI) investigation], two compounds that were predicted to have minimal inhibitory effect on OATP1B. Administration of diltiazem and itraconazole did not increase plasma CPI and CPIII concentrations relative to prestudy levels, in agreement with predictions from in vitro parameters. Additionally, the basal CP concentrations in subjects with SLCO1B1 c.521TT genotype were comparable to those with SLCO1B1 c.521TC genotype, similar to studies with probe substrates. However, subjects with SLCO1B1 c.388AG and c.388GG genotypes (i.e., increased OATP1B1 transport activity for certain substrates) had lower concentrations of CPI than those with SLCO1B1 c.388AA. Collectively, these findings provide further evidence supporting the translational value of CPI and CPIII as suitable endogenous clinical probes to gauge OATP1B activity and potential for OATP1B-mediated DDIs.

Oligomerization Study of Human Organic Anion Transporting Polypeptide 1B1.

Organic anion-transporting polypeptides play important roles in the uptake of various endogenous and exogenous compounds. It has been proposed that OATP family members, as membrane proteins, may form oligomers. However, oligomerization status of OATPs is still largely unclear. In the present study, HEK293 cells stably expressing OATP1B1 were generated to investigate the oligomerization status of the transporter. Chemical cross-linking and coimmunoprecipitation experiments revealed that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. When wild-type OATP1B1 was coexpressed with a loss-of-function mutant W258A, cells showed reduced uptake of prototypic substrate estrone-3-sulfate (ES). Interestingly, such a coexpression did not affect OATP1B1 transport activity of high concentrations ES, implicating that oligomerization status may affect only the high affinity component of ES. OATP1B1 possesses three GXXXG motifs that have been associated with protein dimerization in other membrane proteins. When glycine residues were replaced with alanine, G219A and G393A showed drastically reduced uptake function. Further studies revealed that G219A has a similar association capability to that of the wild-type, while mutation at Gly393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced Vmax for ES uptake. Km of G219A was increased while that of G393A exhibited a decreased value for high affinity component of ES binding. Our studies demonstrated that OATP1B1 may function as oligomers in the high affinity site of ES while acting as monomers for the low affinity binding component of the substrate.

Downregulation of Organic Anion Transporting Polypeptide (OATP) 1B1 Transport Function by Lysosomotropic Drug Chloroquine: Implication in OATP-Mediated Drug-Drug Interactions.

Organic anion transporting polypeptide (OATP) 1B1 mediates the hepatic uptake of many drugs including lipid-lowering statins. Decreased OATP1B1 transport activity is often associated with increased systemic exposure of statins and statin-induced myopathy. Antimalarial drug chloroquine (CQ) is also used for long-term treatment of rheumatoid arthritis and systemic lupus erythematosus. CQ is lysosomotropic and inhibits protein degradation in lysosomes. The current studies were designed to determine the effects of CQ on OATP1B1 protein degradation, OATP1B1-mediated transport in OATP1B1-overexpressing cell line, and statin uptake in human sandwich-cultured hepatocytes (SCH). Treatment with lysosome inhibitor CQ increased OATP1B1 total protein levels in HEK293-OATP1B1 cells and in human SCH as determined by OATP1B1 immunoblot. In HEK293-FLAG-tagged OATP1B1 stable cell line, co-immunofluorescence staining indicated that intracellular FLAG-OATP1B1 is colocalized with lysosomal associated membrane glycoprotein (LAMP)-2, a marker protein of late endosome/lysosome. Enlarged LAMP-2-positive vacuoles with FLAG-OATP1B1 protein retained inside were readily detected in CQ-treated cells, consistent with blocking lysosomal degradation of OATP1B1 by CQ. In HEK293-OATP1B1 cells, without pre-incubation, CQ concentrations up to 100 μM did not affect OATP1B1-mediated [(3)H]E217G accumulation. However, pre-incubation with CQ at clinically relevant concentration(s) significantly decreased [(3)H]E217G and [(3)H]pitavastatin accumulation in HEK293-OATP1B1 cells and [(3)H]pitavastatin accumulation in human SCH. CQ pretreatment (25 μM, 2 h) resulted in ∼1.9-fold decrease in Vmax without affecting Km of OATP1B1-mediated [(3)H]E217G transport in HEK293-OATP1B1 cells. Pretreatment with monensin and bafilomycin A1, which also have lysosome inhibition activity, significantly decreased OATP1B1-mediated transport in HEK293-OATP1B1 cells. Pharmacoepidemiologic studies using data from the U.S. Food and Drug Administration Adverse Event Reporting System indicated that CQ plus pitavastatin, rosuvastatin, and pravastatin, which are minimally metabolized by the cytochrome P450 enzymes, led to higher myopathy risk than these statins alone. In summary, the present studies report novel findings that lysosome is involved in degradation of OATP1B1 protein and that pre-incubation with lysosomotropic drug CQ downregulates OATP1B1 transport activity. Our in vitro data in combination with pharmacoepidemiologic studies support that CQ has potential to cause OATP-mediated drug-drug interactions.

Effects of natural products on the function of human organic anion transporting polypeptide 1B1

In this study, the effects of 136 naturally occurring products, which have been reported to play important roles in modification of Cytochrome P450 (CYP450) activities, on the uptake of estrone-3-sulfate (E3S), a typical OATP1B1 substrate, were evaluated using human embryonic kidney 293 cells stably expressing OATP1B1.At a concentration of 100 μM, 42 natural products inhibited OATP1B1-mediated [3H]E3S uptake by more than 50%, and five of them significantly inhibited OATP1B1-mediated [3H]E3S by more than 80% with the following rank order of potency: quercetin > astragaloside IV > icariin > glycyrrhizic acid > ginsenoside Rc. Inhibitory effects of these natural products on OATP1B1 activity were in a concentration-dependent manner. 11 natural compounds were found exhibiting greater than 50% inhibition at 30 μM with IC50 values ranging from 14.6 ± 3.3 to 28.5 ± 3.0 μM.In conclusion, our data suggest that modification of OATP1B1 transport activity by these natural occurring products may be a mechanism for natural product–drug interactions in humans.

SLCO1B1 Polymorphism and Oral Antidiabetic Drugs

Organic anion-transporting polypeptide 1B1 (OATP1B1; gene: SLCO1B1) is an influx transporter expressed on the sinusoidal membrane of human hepatocytes, where it mediates the uptake of its substrates from blood into liver. In vitro, the SLCO1B1 c.521T>C (p.Val174Ala) single-nucleotide polymorphism (SNP) has been associated with reduced and the c.388A>G (p.Asn130Asp) SNP with both enhanced and reduced transport activity of OATP1B1. In vivo in humans, the c.521C allele (present in SLCO1B1*5 and *15 haplotypes) is associated with decreased hepatic uptake and increased plasma concentrations of several OATP1B1 substrates. The SLCO1B1*1B (c.388G-c.521T) haplotype is associated with enhanced hepatic uptake and decreased plasma concentrations of some OATP1B1 substrates. The SLCO1B1 c.521CC genotype has been associated with an about 60-190% increased, and the SLCO1B1*1B/*1B genotype with an about 30% decreased area under the plasma concentration-time curve of repaglinide. Moreover, SLCO1B1 polymorphism can affect the extent of interaction between OATP1B1 inhibitors and repaglinide. Accordingly, SLCO1B1 genotyping may help in choosing the optimal starting dose of repaglinide. In Chinese individuals, the SLCO1B1 c.521C allele has been associated with increased plasma concentrations of nateglinide, but the association could not be replicated in Caucasians. SLCO1B1 genotype has had no effect on the pharmacokinetics of rosiglitazone, pioglitazone or their metabolites. The hepatic uptake of metformin is mediated by organic cation transporters 1 and 3, and the liver is not important for the elimination or action of the dipeptidylpeptidase 4 inhibitors sitagliptin, vildagliptin and saxagliptin. Therefore, SLCO1B1 polymorphism unlikely affects the response to these antidiabetics. Possible effects of SLCO1B1 polymorphism on sulfonylureas remain to be investigated.

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

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. 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. 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. 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.

Inhibition of human organic anion transporting polypeptide OATP 1B1 as a mechanism of drug-induced hyperbilirubinemia

OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug–drug and drug–endogenous substance interactions.