Human Organic Anion-transporting Polypeptides Research Articles

Functional knockout of the Oatp1d1 membrane transporter affects toxicity of diclofenac in zebrafish embryos

Organic anion transporting polypeptides (OATPs) facilitate the cellular uptake of a large number of compounds. Zebrafish Oatp1d1 matches the functional capabilities of human OATP orthologs, particularly in hormone and drug transport. It is highly expressed in the liver and later stages of embryonic development, indicating its critical role in zebrafish physiology and development. Data from previous in vitro analyses have shown a high affinity of zebrafish Oatp1d1 for pharmaceuticals and xenobiotics, providing the basis for further in vivo studies on its defence and developmental functions. Using CRISPR-Cas9 technology, we have generated an Oatp1d1 zebrafish mutant that has highly reduced Oatp1d1 expression in embryos and adult tissues compared to wild type (WT). The absence of Oatp1d1 was confirmed using custom-made antibodies. To evaluate its ecotoxicological relevance, mutant and WT embryos were exposed to increasing concentrations of diclofenac, an NSAID known for its wide and frequent use, environmental pseudo-persistence and ecological implications. WT embryos showed developmental delays and malformations such as spinal curvature, cardiac edema and blood pooling at higher diclofenac concentrations, whereas the Oatp1d1 mutant embryos showed marked resilience, with milder developmental defects and delayed toxic effects. These observations suggest that the absence of Oatp1d1 impedes the efficient entry of diclofenac into hepatocytes, thereby slowing its biotransformation into potentially more toxic metabolites. In addition, the changes in transcript expression of other uptake transporters revealed a highly probable and complex network of compensatory mechanisms. Therefore, the results of this study point to the importance of Oatp1d1-mediated transport of diclofenac, as demonstrated for the first time in vivo using an Oatp1 deficient zebrafish line. Finally, our data indicates that the compensatory role of other transporters with overlapping substrate preferences needs to be considered for a reliable understanding of the physiological and/or defensive role(s) of membrane transporters.

Synthetic augmentation of bilirubin metabolism in human pluripotent stem cell-derived liver organoids

UGT1A1 (UDP glucuronosyltransferase family 1 member A1)is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic aniontransporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development.

SLCO family of organic anion transporting polypeptides in GtoPdb v.2023.1

The SLCO superfamily is comprised of the organic anion transporting polypeptides (OATPs). The 11 human OATPs are divided into 6 families and ten subfamilies based on amino acid identity. These proteins are located on the plasma membrane of cells throughout the body. They have 12 TM domains and intracellular termini, with multiple putative glycosylation sites. OATPs mediate the sodium-independent uptake of a wide range of amphiphilic substrates, including many drugs and toxins. Due to the multispecificity of these proteins, this guide lists classes of substrates and inhibitors for each family member. More comprehensive lists of substrates, inhibitors, and their relative affinities may be found in the review articles listed below.

Plasma Protein Binding Refinement of the Extended Clearance Classification System: Subclasses for Predicting Hepatic Uptake or Renal Clearance for Classes 1B and 3B.

The Extended Clearance Classification System (ECCS) was established to facilitate the timely anticipation of clearance rate determination according to the physicochemical characteristics of a given compound and invitro passive membrane permeability. Unfortunately, distinguishing between renal and hepatic uptake clearance mechanisms using ECCS class 3B is not possible. We determined the effects of plasma protein binding (PPB) on major hepatic organic anion transporting polypeptide (OATP) and renal organic aniontransporter (OAT) substrates. A modified ECCS could predict when renal or hepatic uptake mechanisms were the main clearance rate determinants (accounting for ≥ 70% of total clearance). A dataset of 66 human OATP and 41 OAT substrates was analyzed to determine the effect of PPB. A total of 63 acidic and zwitterionic, and high-molecular-weight (MW > 400Da) compounds, including 50 drugs in ECCS classes 1B and 3B, were reanalyzed considering their PPB. Statistical analyses revealed that hepatic uptake transporter (OATP1B1 and OATP1B3) substrates possess a high PPB rate of ≥ 90%, whereas OAT1 and/or OAT3 substrates possess low PPB rates of < 90%. By analyzing the 63 drugs on the basis of their PPB, the active hepatic uptakes of acids and zwitterions were determined to be the main clearance mechanisms, with PPB ≥ 90%, whereas renally eliminated drugs exhibited limited PPB (< 90%). Therefore, PPB is an effective parameter for defining clearance rate determination for acidic and zwitterionic drugs with high MWs. Using PPB as an additional parameter in ECCS, clearance mechanisms for class 1B and 3B compounds can be predicted, and OATP and OAT substrates may be readily distinguished.

Structural dissection of 13-epiestrones based on the interaction with human Organic anion-transporting polypeptide, OATP2B1

Human OATP2B1 encoded by the SLCO2B1 gene is a multispecific transporter mediating the cellular uptake of large, organic molecules, including hormones, prostaglandins and bile acids. OATP2B1 is ubiquitously expressed in the human body, with highest expression levels in pharmacologically relevant barriers, like enterocytes, hepatocytes and endothelial cells of the blood-brain-barrier. In addition to its endogenous substrates, OATP2B1 also recognizes clinically applied drugs, such as statins, antivirals, antihistamines and chemotherapeutic agents and influences their pharmacokinetics. On the other hand, OATP2B1 is also overexpressed in various tumors. Considering that elevated hormone uptake by OATP2B1 results in increased cell proliferation of hormone dependent tumors (e.g. breast or prostate), inhibition of OATP2B1 can be a good strategy to inhibit the growth of these tumors.13-epiestrones represent a potential novel strategy in the treatment of hormone dependent cancers by the suppression of local estrogen production due to the inhibition of the key enzyme of estrone metabolism, 17ß-hydroxysteroid-dehydrogenase type 1 (HSD17ß1). Recently, we have demonstrated that various phosphonated 13-epiestrones are dual inhibitors also suppressing OATP2B1 function. In order to gain better insights into the molecular determinants of OATP2B1 13-epiestrone interaction we investigated the effect of C-2 and C-4 halogen or phenylalkynyl modified epiestrones on OATP2B1 transport function. Potent inhibitors (with EC50 values in the low micromolar range) as well as non-inhibitors of OATP2B1 function were identified. Based on the structure-activity relationship (SAR) of the various 13-epiestrone derivatives we could define structural elements important for OATP2B1 inhibition. Our results may help to understand the drug/inhibitor interaction profile of OATP2B1, and also may be a useful strategy to block steroid hormone entry into tumors.

Organic anion transporting polypeptide1A2 mediates fentanyl uptake in cultured cells.

Individual differences in the response to fentanyl, which may be caused by different concentrations of the drug in the central nervous system, can complicate analgesic treatment. It has been reported that the organic anion transporting polypeptide(OATP) at the blood‑brain barrier(BBB) in Sprague‑Dawley rats may serve an important role in the transport of fentanyl across the BBB. However, whether human OATP can transport fentanyl has thus far not been reported. The present study aimed to establish a 293cell line stably overexpressing OATP1A2, and to determine whether OATP1A2 is able to transport fentanyl across the plasma membrane. Initially, 293cells were transfected with an OATP1A2‑expressing plasmid (referred to as 293‑OATP1A2 cells), and single colonies were selected and characterized following geneticin treatment. Subsequently, reverse transcription‑quantitative polymerase chain reaction and western blot analyses were conducted to verify the transfection efficiency. Furthermore, treatment of 293‑OATP1A2 cells with different concentrations of fexofenadine (FEX) and fentanyl was performed to investigate the transport function of OATP1A2 in 293cells. FEX and fentanyl uptake experiments were also performed with naringenin, an inhibitor of OATP1A2. The results indicated that FEX and fentanyl uptake was significantly increased in 293‑OATP1A2 cells compared with that in the control‑transfected cells. The 293‑OATP1A2‑mediated uptake of FEX at concentration of 100nM FEX was ~10‑fold higher than that of 293‑VCcells. The 293‑OATP1A2‑mediated uptake of fentanyl (100nM) was 5.1‑fold higher compared with that in 293‑VCcells. In 293‑OATP1A2 cells, the uptake of FEX without OATP1A2 inhibitor naringenin (100µg/ml) was 2.8‑fold higher compared with that in the presence of naringenin, and the uptake of fentanyl without naringenin was 7.3‑fold higher compared with that in the presence of naringenin (100µg/ml). In conclusion, 293cells that overexpressed OATP1A2 were successfully constructed, and OATP1A2 was revealed to mediate fentanyl uptake in the cultured cells.

Development of a Human Photoacoustic Imaging Reporter Gene Using the Clinical Dye Indocyanine Green.

To develop a photoacoustic imaging (PAI) reporter gene that has high translational potential. Previous research has shown that human organic anion-transporting polypeptide 1b3 (OATP1B3) promotes the uptake of the near-infrared fluorescent dye indocyanine green (ICG). In this study, the authors have established OATP1B3 and ICG as a reporter gene-probe pair for in vivo PAI. Human breast cancer cells were engineered to express OATP1B3. Control cells (not expressing OATP1B3) or OATP1B3-expressing cells were incubated with or without ICG, placed in a breast-mimicking phantom, and imaged with PAI. Control (n = 6) or OATP1B3-expressing (n = 5) cells were then implanted orthotopically into female mice. Full-spectrum PAI was performed before and 24 hours after ICG administration. One-way analysis of variance was performed, followed by Tukey posthoc multiple comparisons, to assess statistical significance. OATP1B3-expressing cells incubated with ICG exhibited a 2.7-fold increase in contrast-to-noise ratio relative to all other controls in vitro (P < .05). In mice, PAI signals after ICG administration were increased 2.3-fold in OATP1B3 tumors relative to those in controls (P < .05). OATP1B3 operates as an in vivo PAI reporter gene based on its ability to promote the cellular uptake of ICG. Benefits include the human derivation of OATP1B3, combined with the use of wavelengths in the near-infrared region, high extinction coefficient, low quantum yield, and clinical approval of ICG. The authors posit that this system will be useful for localized monitoring of emerging gene- and cell-based therapies in clinical applications.© RSNA, 2019Keywords: Animal Studies, Molecular Imaging, Molecular Imaging-Clinical Translation, Molecular Imaging-Reporter Gene Imaging, Optical ImagingSupplemental material is available for this article.

Organic Anion-Transporting Polypeptide Genes Are Not Induced by the Pregnane X Receptor Activator Rifampin: Studies in Hepatocytes In Vitro and in Monkeys In Vivo.

Induction potentials of the pregnane X receptor (PXR) activator rifampin (RIF) on transporter genes [e.g., organic anion-transporting polypeptides (OATPs)] are still in its infancy or remain controversial in the field. The present investigations characterized changes in transporter gene expression by RIF in sandwich-cultured hepatocytes from multiple donors of human and cynomolgus monkey using real-time quantitative reverse transcription polymerase chain reaction method. Three-day treatment of RIF significantly induced CYP3A4 (∼60-fold induction), but not CYP1A2 and CYP2D6 genes. SLC51B was the most highly induced uptake transporter gene (>10-fold) in both human and monkey hepatocytes. A greater induction of CYP2C9 was observed in monkey hepatocytes than that in humans. ATP-binding cassette (ABC)B1 and ABCC2 were induced slightly above 2-fold in human and monkey hepatocytes and appeared to be dose-dependent. The induction of OATP and other transporter genes was generally less than 2-fold and considered not clinically relevant. SLCO2B1 was not detectable in monkey hepatocytes. To investigate in vivo OATP induction, RIF (18 mg/kg per day) was orally dosed to cynomolgus monkeys for 7 days. Pitavastatin and antipyrine were intravenously dosed before and after RIF treatment as exogenous probes of OATP and CYP activities, respectively. Plasma coproporphyrin-I (CP-I) and coproporphyrin-III (CP-III) were measured as OATP endogenous biomarkers. Although a significant increase of antipyrine clearance (CL) was observed after RIF treatment, the plasma exposures of pitavastatin, CP-I, and CP-III remained unchanged, suggesting that OATP function was not significantly altered. The results suggested that OATP transporters were not significantly induced by PXR ligand RIF. The data are consistent with current regulatory guidances that the in vitro characterization of transporter induction during drug development is not required. SIGNIFICANCE STATEMENT: Organic anion-transporting polypeptide (OATP) genes were not induced by rifampin in sandwich-cultured human and monkey hepatocytes OATP functions measured by OATP probe pitavastatin and endogenous marker coproporphyrins were not altered in monkeys in vivo by 7-day rifampin treatment. The data suggested that OATP transporters are unlikely induced by the pregnane X receptor ligand rifampin, which are consistent with current regulatory guidances that the in vitro characterization of OATP1B induction during drug development is not required.

SLCO family of organic anion transporting polypeptides (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The SLCO superfamily is comprised of the organic anion transporting polypeptides (OATPs). The 11 human OATPs are divided into 6 families and ten subfamilies based on amino acid identity. These proteins are located on the plasma membrane of cells throughout the body. They have 12 TM domains and intracellular termini, with multiple putative glycosylation sites. OATPs mediate the sodium-independent uptake of a wide range of amphiphilic substrates, including many drugs and toxins. Due to the multispecificity of these proteins, this guide lists classes of substrates and inhibitors for each family member. More comprehensive lists of substrates, inhibitors, and their relative affinities may be found in the review articles listed below.

Involvement of organic anion-transporting polypeptides and organic cation transporter in the hepatic uptake of jatrorrhizine

Jatrorrhizine possesses a wide spectrum of pharmacological activities. However, the mechanism underlying hepatic uptake of jatrorrhizine remains unclear.Rat liver slices, isolated rat hepatocytes and human embryonic kidney 293 (HEK293) cells stably expressing human organic anion-transporting polypeptide (OATP) and organic cation transporter (OCT) were used to evaluate the hepatic uptake of jatrorrhizine in this study.Uptake of jatrorrhizine in rat liver slices and isolated rat hepatocytes was significantly inhibited by glycyrrhizic acid (Oatp1b2 inhibitor) and prazosin (Oct1 inhibitor), but not by ibuprofen (Oatp1a1 inhibitor) or digoxin (Oatp1a4 inhibitor). Uptake of jatrorrhizine in OATP1B3 and OCT1-HEK293 cells indicated a saturable process with the Km of 8.20 ± 1.28 and 4.94 ± 0.55 μM, respectively. However, the transcellular transport of jatrorrhizine in OATP1B1-HEK293 cells was not observed. Rifampicin (OATP inhibitor) for OATP1B3-HEK293 cells and prazosin for OCT1-HEK293 cells could inhibit the uptake of jatrorrhizine with the IC50 of 5.49 ± 1.05 and 2.77 ± 0.72 μM, respectively.The above data indicate that hepatic uptake of jatrorrhizine is involved in both OATP and OCT, which may have important roles in jatrorrhizine liver disposition and potential drug–drug interactions.

Role of OATP2B1 in Drug Absorption and Drug‐Drug Interactions

BackgroundThe human organic anion‐transporting polypeptide 2B1 (OATP2B1; SLC21A9) is localized to the basolateral membrane of hepatocytes and the brush‐border membrane of small‐intestinal enterocytes. Based on its distribution pattern and functional similarity to OATP1B‐type transporters, it has been hypothesized that OATP2B1 might influence the absorption and disposition properties of a wide range of xenobiotic agents. Although several prescription drugs, including statins such as fluvastatin, have been identified as OATP2B1 substrates in vitro, the lack of a useful in vivo model system has limited our understanding of the pharmacological role of this transporter.Experimental designOATP2B1‐overexpressing cells were generated by transfecting HEK293 cells with an empty vector (VC) or a construct containing human OATP2B1 or mouse OATP2B1 cDNA. Transporter function was evaluated in vitro by measuring the comparative cellular uptake of [3H]estrone‐3‐sulfate (ES) in VC cells and OATP2B1‐overexpressing cells. Candidate substrates and inhibitors were identified based on the difference in intracellular accumulation of radioactivity. OATP2B1‐knockout (KO) mice were obtained from the Slco2b1tm1a(KOMP)Wtsi clone, and derived chimera and subsequent progeny were mated to C57BL/6N for germline production and colony expansion. Mouse genotypes were confirmed by PCR, and OATP2B1 gene deletion was verified by qPCR on major organs. The pharmacokinetic profiles of fluvastatin, a known OATP2B1 substrate, and sorafenib, a known OATP2B1 inhibitor, were determined through serial blood collection and subsequent LC‐MS/MS analysis. Noncompartmental pharmacokinetic parameters were calculated using WinNolin 6.2 software (Pharsight).ResultsAn approximately four‐fold increase in intracellular accumulation of radiolabeled ES (2.5 μM) was observed in the OATP2B1 overexpressing cells compared to VC cells (34.8 ± 2.45 pmol/mg vs 8.38 ± 0.341 pmol/mg, respectively). Subsequently, we identified several drugs as candidate OATP2B1 substrates, including multiple tyrosine kinase inhibitors (TKIs). Knockout of OATP2B1 in organs from KO mice was confirmed at the gene level (P&lt;0.01), and consistent with the intestinal localization of OATP2B1, we found that the absorption of fluvastatin was affected by OATP2B1 deficiency (Tmax: 1 h vs 0.5 h; Cmax: 1.05 μg/mL vs 2.19 μg/mL). No gender differences in mRNA expression levels were detected. Among 24 different agents tested, sorafenib was identified as one of the most potent OATP2B1 inhibitors in the class of TKIs. However, the pharmacokinetic properties of sorafenib and its main metabolites were not significantly affected by OATP2B1 deficiency.ConclusionOur studies have identified novel potent inhibitors of OATP2B1 among TKIs, a rapidly expanding class of drugs used in various therapeutic areas. The availability of a viable, well‐characterized OATP2B1‐deficient mouse model provides a previously unavailable opportunity to unequivocally determine the contribution of this transporter to the absorption and drug‐drug interaction potential of drugs.Support or Funding InformationEli Lilly Fellowship in PharmaceuticsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chimeric mouse model for MRI contrast agent evaluation.

While rodents are the primary animal models for contrast agent evaluation, rodents can potentially misrepresent human organ clearance of newly developed contrast agents. For example, gadolinium (Gd)-BOPTA has ~50% hepatic clearance in rodents, but ~5% in humans. This study demonstrates the benefit of chimeric mice expressing human hepatic OATPs (organic anion-transporting polypeptides) to improve evaluation of novel contrast agents for clinical use. FVB (wild-type) and OATP1B1/1B3 knock-in mice were injected with hepatospecific MRI contrast agents (Gd-EOB-DTPA, Gd-BOPTA) and nonspecific Gd-DTPA. T1 -weighted dynamic contrast-enhanced MRI was performed on mice injected intravenously. Hepatic MRI signal enhancement was calculated per time point. Mass of gadolinium cleared per time point and percentage elimination by means of feces and urine were also measured. Following intravenous injection of Gd-BOPTA in chimeric OATP1B1/1B3 knock-in mice, hepatic MRI signal enhancement and elimination by liver was more reflective of human hepatic clearance than that measured in wild-type mice. Gd-BOPTA hepatic MRI signal enhancement was reduced to 22% relative to wild-type mice. Gd-BOPTA elimination in wild-type mice was 83% fecal compared with 32% fecal in chimeric mice. Hepatic MRI signal enhancement and elimination for Gd-EOB-DTPA and Gd-DTPA were similar between wild-type and chimeric cohorts. Hepatic MRI signal enhancement and elimination of Gd-EOB-DTPA, Gd-BOPTA, and Gd-DTPA in chimeric OATP1B1/1B3 knock-in mice closely mimics that seen in humans. This study provides evidence that the chimeric knock-in mouse is a more useful screening tool for novel MRI contrast agents destined for clinical use as compared to the traditionally used wild-type models.

Multiple circulating saponins from intravenous ShenMai inhibit OATP1Bs in vitro: potential joint precipitants of drug interactions

ShenMai, an intravenous injection prepared from steamed Panax ginseng roots (Hongshen) and Ophiopogon japonicus roots (Maidong), is used as an add-on therapy for coronary artery disease and cancer; saponins are its bioactive constituents. Since many saponins inhibit human organic anion-transporting polypeptides (OATP)1B, this investigation determined the inhibition potencies of circulating ShenMai saponins on the transporters and the joint potential of these compounds for ShenMai-drug interaction. Circulating saponins and their pharmacokinetics were characterized in rats receiving a 30-min infusion of ShenMai at 10 mL/kg. Inhibition of human OATP1B1/1B3 and rat Oatp1b2 by the individual saponins was investigated in vitro; the compounds' joint inhibition was also assessed in vitro and the data was processed using the Chou-Talalay method. Plasma protein binding was assessed by equilibrium dialysis. Altogether, 49 saponins in ShenMai were characterized and graded into: 10-100 μmol/day (compound doses from ShenMai; 7 compounds), 1-10 μmol/day (17 compounds), and <1 μmol/day (25 compounds, including Maidong ophiopogonins). After dosing, circulating saponins were protopanaxadiol-type ginsenosides Rb1, Rb2, Rc, Rd, Ra1, Rg3, Ra2, and Ra3, protopanaxatriol-type ginsenosides Rg1, Re, Rg2, and Rf, and ginsenoside Ro. The protopanaxadiol-type ginsenosides exhibited maximum plasma concentrations of 2.1-46.6 μmol/L, plasma unbound fractions of 0.4-1.0% and terminal half-lives of 15.6-28.5 h (ginsenoside Rg3, 1.9 h), while the other ginsenosides exhibited 0.1-7.7 μmol/L, 20.8-99.2%, and 0.2-0.5 h, respectively. The protopanaxadiol-type ginsenosides, ginsenosides without any sugar attachment at C-20 (except ginsenoside Rf), and ginsenoside Ro inhibited OATP1B3 more potently (IC50, 0.2-3.5 µmol/L) than the other ginsenosides (≥22.6 µmol/L). Inhibition of OATP1B1 by ginsenosides was less potent than OATP1B3 inhibition. Ginsenosides Rb1, Rb2, Rc, Rd, Ro, Ra1, Re, and Rg2 likely contribute the major part of OATP1B3-mediated ShenMai-drug interaction potential, in an additive and time-related manner.

Endogenous probes for human liver organic anion-transporting polypeptides: the intersection of bioanalytical and ADME science.

Endogenous probes for human liver organic anion-transporting polypeptides: the intersection of bioanalytical and ADME science.

Characterization of bile acid sulfate conjugates as substrates of human organic anion-transporting polypeptides

Characterization of bile acid sulfate conjugates as substrates of human organic anion-transporting polypeptides

Influence of OATPs on Hepatic Disposition of Erlotinib Measured With Positron Emission Tomography

To assess the hepatic disposition of erlotinib, we performed positron emission tomography (PET) scans with [11C]erlotinib in healthy volunteers without and with oral pretreatment with a therapeutic erlotinib dose (300 mg). Erlotinib pretreatment significantly decreased the liver exposure to [11C]erlotinib with a concomitant increase in blood exposure, pointing to the involvement of a carrier‐mediated hepatic uptake mechanism. Using cell lines overexpressing human organic anion‐transporting polypeptides (OATPs) 1B1, 1B3, or 2B1, we show that [11C]erlotinib is selectively transported by OATP2B1. Our data suggest that at PET microdoses hepatic uptake of [11C]erlotinib is mediated by OATP2B1, whereas at therapeutic doses OATP2B1 transport is saturated and hepatic uptake occurs mainly by passive diffusion. We propose that [11C]erlotinib may be used as a hepatic OATP2B1 probe substrate and erlotinib as an OATP2B1 inhibitor in clinical drug–drug interaction studies, allowing the contribution of OATP2B1 to the hepatic uptake of drugs to be revealed.

Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3-O-Sulfate Conjugates as Potential Novel Biomarkers for Organic Anion-Transporting Polypeptides.

In the search for novel bile acid (BA) biomarkers of liver organic anion-transporting polypeptides (OATPs), cynomolgus monkeys received oral rifampicin (RIF) at four dose levels (1, 3, 10, and 30 mg/kg) that generated plasma-free Cmax values (0.06, 0.66, 2.57, and 7.79 µM, respectively) spanning the reported in vitro IC50 values for OATP1B1 and OATP1B3 (≤1.7 μM). As expected, the area under the plasma concentration-time curve (AUC) of an OATP probe drug (i.v. 2H4-pitavastatin, 0.2 mg/kg) was increased 1.2-, 2.4-, 3.8-, and 4.5-fold, respectively. Plasma of RIF-dosed cynomolgus monkeys was subjected to a liquid chromatography-tandem mass spectrometry method that supported the analysis of 30 different BAs. Monkey urine was profiled, and we also determined that the impact of RIF on BA renal clearance was minimal. Although sulfated BAs comprised only 1% of the plasma BA pool, a robust RIF dose response (maximal ≥50-fold increase in plasma AUC) was observed for the sulfates of five BAs [glycodeoxycholate (GDCA-S), glycochenodeoxycholate (GCDCA-S), taurochenodeoxycholate, deoxycholate (DCA-S), and taurodeoxycholate (TDCA-S)]. In vitro, RIF (≤100 μM) did not inhibit cynomolgus monkey liver cytosol-catalyzed BA sulfation and cynomolgus monkey hepatocyte-mediated uptake of representative sulfated BAs (GDCA-S, GCDCA-S, DCA-S, and TDCA-S) was sodium-independent and inhibited (≥70%) by RIF (5 μM); uptake of taurocholic acid was sensitive to sodium removal (74% decrease) and relatively refractory to RIF (≤21% inhibition). We concluded that sulfated BAs may serve as sensitive biomarkers of cynomolgus monkey OATPs and that exploration of their utility as circulating human OATP biomarkers is warranted.

Preference of Conjugated Bile Acids over Unconjugated Bile Acids as Substrates for OATP1B1 and OATP1B3.

Bile acids, the metabolites of cholesterol, are signaling molecules that play critical role in many physiological functions. They undergo enterohepatic circulation through various transporters expressed in intestine and liver. Human organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 contribute to hepatic uptake of bile acids such as taurocholic acid. However, the transport properties of individual bile acids are not well understood. Therefore, we selected HEK293 cells overexpressing OATP1B1 and OATP1B3 to evaluate the transport of five major human bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid) together withtheir glycine and taurine conjugates via OATP1B1 and OATP1B3. The bile acids were quantified by liquid chromatography-tandem mass spectrometry. The present study revealed that cholic acid, chenodeoxyxcholic acid, and deoxycholic acid were transported by OATP1B1 and OATP1B3, while ursodeoxycholic acid and lithocholic acid were not significantly transported by OATPs. However, all the conjugated bile acids were taken up rapidly by OATP1B1 and OATP1B3. Kinetic analyses revealed the involvement of saturable OATP1B1- and OATP1B3-mediated transport of bile acids. The apparent Km values for OATP1B1 and OATP1B3 of the conjugated bile acids were similar (0.74–14.7 μM for OATP1B1 and 0.47–15.3 μM for OATP1B3). They exhibited higher affinity than cholic acid (47.1 μM for OATP1B1 and 42.2 μM for OATP1B3). Our results suggest that conjugated bile acids (glycine and taurine) are preferred to unconjugated bile acids as substrates for OATP1B1 and OATP1B3.

Recent advance in the pharmacogenomics of human Solute Carrier Transporters (SLCs) in drug disposition

Drug pharmacokinetics is influenced by the function of metabolising enzymes and influx/efflux transporters. Genetic variability of these genes is known to impact on clinical therapies. Solute Carrier Transporters (SLCs) are the primary influx transporters responsible for the cellular uptake of drug molecules, which consequently, impact on drug efficacy and toxicity. The Organic Anion Transporting Polypeptides (OATPs), Organic Anion Transporters (OATs) and Organic Cation Transporters (OCTs/OCTNs) are the most important SLCs involved in drug disposition. The information regarding the influence of SLC polymorphisms on drug pharmacokinetics is limited and remains a hot topic of pharmaceutical research. This review summarises the recent advance in the pharmacogenomics of SLCs with an emphasis on human OATPs, OATs and OCTs/OCTNs. Our current appreciation of the degree of variability in these transporters may contribute to better understanding the inter-patient variation of therapies and thus, guide the optimisation of clinical treatments.

Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking.

Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.