ABSTRACT
Iron is the most abundant transition metal essential for numerous cellular processes. Although most mammalian cells acquire iron through transferrin receptors, molecular players of iron utilization under iron restriction are incompletely understood. To address this, we performed metabolism-focused CRISPRa gain-of-function screens, which revealed metabolic limitations under stress conditions. Iron restriction screens identified not only expected members of iron utilization pathways but also SLCO2B1, a poorly characterized membrane carrier. SLCO2B1 expression is sufficient to increase intracellular iron, bypass the essentiality of the transferrin receptor, and enable proliferation under iron restriction. Mechanistically, SLCO2B1 mediates heme analog import in cellular assays. Heme uptake by SLCO2B1 provides sufficient iron for proliferation through heme oxygenases. Notably, SLCO2B1 is predominantly expressed in microglia in the brain, and primary Slco2b1-/- mouse microglia exhibit strong defects in heme analog import. Altogether, our work identifies SLCO2B1 as a microglia-enriched plasma membrane heme importer and provides a genetic platform to identify metabolic limitations under stress conditions.
Subject(s)
Heme , Iron , Organic Anion Transporters/metabolism , Animals , Biological Transport , Heme/genetics , Heme/metabolism , Iron/metabolism , Mammals/metabolism , Membrane Transport Proteins/metabolism , Mice , Transcriptional ActivationABSTRACT
Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.
Subject(s)
Amino Acids/metabolism , Mitochondria/metabolism , Stress, Physiological/physiology , Adaptation, Physiological , Amino Acids/toxicity , Carrier Proteins/metabolism , Homeostasis , Membrane Transport Proteins/metabolism , Mitochondria/physiology , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/metabolism , Multivesicular Bodies/metabolism , Organic Anion Transporters/metabolism , Protein Transport , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Vacuoles/metabolismABSTRACT
Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.
Subject(s)
Dicarboxylic Acid Transporters , Humans , Kinetics , Dicarboxylic Acid Transporters/metabolism , Dicarboxylic Acid Transporters/genetics , Mitochondria/metabolism , Mitochondria/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Protein Multimerization , Amino Acid Transport Systems, Acidic/metabolism , Amino Acid Transport Systems, Acidic/genetics , Anion Transport Proteins/metabolism , Anion Transport Proteins/genetics , Anion Transport Proteins/chemistry , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Antiporters/metabolism , Antiporters/genetics , Antiporters/chemistry , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondrial ADP, ATP Translocases/genetics , Biological Transport , Organic Anion Transporters/metabolism , Organic Anion Transporters/genetics , Organic Anion Transporters/chemistry , Adenosine Triphosphate/metabolism , Carrier Proteins , Membrane Transport ProteinsABSTRACT
The coordinated movement of organic anions (e.g., drugs, metabolites, signaling molecules, nutrients, antioxidants, gut microbiome products) between tissues and body fluids depends, in large part, on organic anion transporters (OATs) [solute carrier 22 (SLC22)], organic anion transporting polypeptides (OATPs) [solute carrier organic (SLCO)], and multidrug resistance proteins (MRPs) [ATP-binding cassette, subfamily C (ABCC)]. Depending on the range of substrates, transporters in these families can be considered multispecific, oligospecific, or (relatively) monospecific. Systems biology analyses of these transporters in the context of expression patterns reveal they are hubs in networks involved in interorgan and interorganismal communication. The remote sensing and signaling theory explains how the coordinated functions of drug transporters, drug-metabolizing enzymes, and regulatory proteins play a role in optimizing systemic and local levels of important endogenous small molecules. We focus on the role of OATs, OATPs, and MRPs in endogenous metabolism and how their substrates (e.g., bile acids, short chain fatty acids, urate, uremic toxins) mediate interorgan and interorganismal communication and help maintain and restore homeostasis in healthy and disease states.
Subject(s)
Avena , Organic Anion Transporters , Humans , Avena/metabolism , Remote Sensing Technology , Membrane Transport Proteins/metabolism , Organic Anion Transporters/metabolism , Adenosine TriphosphateABSTRACT
The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, -3, and -4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.
Subject(s)
Aedes , Ecdysone , Insect Proteins , Organic Anion Transporters , Aedes/growth & development , Aedes/physiology , Animals , Drosophila/metabolism , Drosophila melanogaster/metabolism , Ecdysone/metabolism , Female , Insect Proteins/metabolism , Organic Anion Transporters/metabolism , VitellogenesisABSTRACT
Solute carrier organic anion transporter family member 2A1 (SLCO2A1) is a prostaglandin (PG) transporter and serves as the osmosensitive ATP-permeable maxi-anion channel (Maxi-Cl). Since a heterotetrameric complex of annexin A2 (ANXA2) and S100A10 is obligatory for the channel activity, the present study aimed to determine if they regulate SLCO2A1-mediated PG transport. This study examined PGE2 uptake and ATP release in Anxa2 and/or S100a10 knockout (KO) murine breast C127 cells. Deletion of Slco2a1 decreased PGE2-d4 uptake by wild-type (WT) cells in an isotonic medium (290 mosmol/kgH2O). Decreased osmolarity (135 mosmol/kgH2O) stimulated ATP release but did not affect PGE2 uptake kinetics, showing Km (1,280 nM) and Vmax (10.38 pmol/15 s/mg protein) similar to those in isotonic medium (1,227 nM and 10.65 pmol/15 s/mg protein), respectively, in WT cells. Deletion of Anxa2 associated with loss of S100a10 diminished SLCO2A1-mediated ATP release and uncompetitively inhibited PGE2 uptake with lowered Km (376 nM) and Vmax (2.59 pmol/15 s/mg protein). Moreover, the immunoprecipitation assay confirmed the physical interaction of ANXA2 with SLCO2A1 in WT cells. Enforcement of ANXA2 expression to Anxa2 KO cells partially restored PGE2 uptake and increased Km (744.3 nM) and Vmax (9.07 pmol/15 s/mg protein), whereas the uptake clearance (Vmax/Km) did not change much regardless of ANXA2 expression. These results suggest that an ANXA2/S100A10 complex modulates PG transport activity but osmolality has little effect on it; therefore, the bound form of SLCO2A1, which functions as a PG transporter and Maxi-Cl, may exist regardless of changes in the cell volume.NEW & NOTEWORTHY A previous study indicated that the ANXA2/S100A10 complex represents the regulatory component of SLCO2A1-mediated Maxi-Cl channel activity. The present study showed that apparent PGE2 uptake by C127 cells was osmoinsensitive and uncompetitively inhibited by loss of ANXA2 expression, demonstrating that ANXA2 is a regulatory factor of SLCO2A1-mediated PG transport activity.
Subject(s)
Annexin A2 , Organic Anion Transporters , Prostaglandins , S100 Proteins , Animals , Mice , Adenosine Triphosphate/metabolism , Annexin A2/metabolism , Biological Transport , Dinoprostone/metabolism , Organic Anion Transporters/metabolism , Prostaglandins/metabolism , S100 Proteins/metabolismABSTRACT
The organic anion transporting polypeptide (OATP)2B1 [(gene: solute carrier organic anion transporter family member 2B1 (SLCO2B1)] is an uptake transporter that facilitates cellular accumulation of its substrates. Comparison of SLCO2B1+/+ knockin and rSlco2b1-/- knockout rats showed a higher expression of rCYP3A1 in the humanized animals. We hypothesize that humanization of OATP2B1 not only affects cellular uptake but also metabolic activity. To further investigate this hypothesis, we used SLCO2B1+/+ and rSlco2b1-/ - rats and the OATP2B1 and rCYP3A1 substrate erlotinib, which is metabolized to OSI-420, for in vivo and ex vivo experiments. One hour after administration of a single dose of erlotinib, the knockin rats exhibited significantly lower erlotinib serum levels, but no change was observed in metabolite concentration or the OSI-420/erlotinib ratio. Similar results were obtained for liver tissue levels comparing SLCO2B1+/+ and rSlco2b1-/- rats. Liver microsomes isolated from the erlotinib-treated animals were characterized ex vivo for rCYP3A activity using testosterone, showing higher activity in the knockin rats. The contrary was observed when microsomes isolated from treatment-naïve animals were assessed for the metabolism of erlotinib to OSI-420. The latter is in contrast to the higher rCYP3A1 protein amount observed by western blot analysis in rat liver lysates and liver microsomes isolated from untreated rats. In summary, rats humanized for OATP2B1 showed higher expression of rCYP3A1 in liver and reduced serum levels of erlotinib but no change in the OSI-420/erlotinib ratio despite a lower OSI-420 formation in isolated liver microsomes. Studies with CYP3A-specific substrates are warranted to evaluate whether humanization affects not only rCYP3A1 expression but also metabolic activity in vivo. SIGNIFICANCE STATEMENT: Humanization of rats for the organic anion transporting polypeptide (OATP)2B1 increases rCYP3A1 expression and activity in liver. Using the OATP2B1/CYP3A-substrate erlotinib to assess the resulting phenotype, we observed lower erlotinib serum and liver concentrations but no impact on the liver/serum ratio. Moreover, there was no difference in the OSI-420/erlotinib ratio comparing humanized and knockout rats, suggesting that OSI-420 is not applicable to monitor differences in rCYP3A1 expression as supported by data from ex vivo experiments with rat liver microsomes.
Subject(s)
Cytochrome P-450 CYP3A , Organic Anion Transporters , Rats , Animals , Erlotinib Hydrochloride/pharmacology , Cytochrome P-450 CYP3A/metabolism , Quinazolines/pharmacology , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolismABSTRACT
Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively) compared with 19 nM for the wild type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. SIGNIFICANCE STATEMENT: Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting organic ion transporters (OATs). This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 is found to mediate hydrogen bonding.
Subject(s)
Organic Anion Transporters , Uric Acid , Uricosuric Agents , Animals , Organic Anion Transporters/metabolism , Organic Anion Transporters/antagonists & inhibitors , Organic Anion Transporters/genetics , Uric Acid/metabolism , Uric Acid/pharmacology , Binding Sites , Humans , Uricosuric Agents/pharmacology , Organic Cation Transport Proteins/metabolism , Organic Cation Transport Proteins/genetics , Organic Cation Transport Proteins/antagonists & inhibitors , Xenopus laevis , Kidney/metabolism , Kidney/drug effects , Oocytes/metabolism , Oocytes/drug effects , Benzothiazoles/pharmacology , Molecular Docking SimulationABSTRACT
Extrapolating in vivo hepatic clearance from in vitro uptake data is a known challenge, especially for organic anion-transporting polypeptide transporter (OATP) substrates, and the well-stirred model (WSM) commonly yields systematic underpredictions for those anionic drugs, hypothetically due to "albumin-mediated hepatic drug uptake". In the present study, we demonstrate that the WSM incorporating the dynamic free fraction (f D), a measure of drug protein binding affinity, performs reasonably well in predicting hepatic clearance of OATP substrates. For a selection of anionic drugs, including atorvastatin, fluvastatin, pravastatin, rosuvastatin, pitavastatin, cerivastatin, and repaglinide, this dynamic well-stirred model (dWSM) correctly predicts hepatic plasma clearance within 2-fold error for six out of seven OATP substrates examined. The geometric mean of clearance ratios between the predicted and the observed values falls in the range of 1.21-1.38. As expected, the WSM with unbound fraction (f u) systematically underpredicts hepatic clearance with greater than 2-fold error for five out of seven drugs, and the geometric mean of clearance ratios between the predicted and the observed values is in the range of 0.20-0.29. The results suggest that, despite its simplicity, the dWSM operates well for transporter-mediated uptake clearance, and that clearance under-prediction of OATP substrates may not necessarily be associated with the chemical class of the anionic drugs, nor is it a result of albumin-mediated hepatic drug uptake as currently hypothesized. Instead, the superior prediction power of the dWSM confirms the utility of the dynamic free fraction in clearance prediction and the importance of drug plasma binding kinetics in hepatic uptake clearance. SIGNIFICANCE STATEMENT: The traditional well-stirred model (WSM) consistently underpredicts organin anion-transporting polypeptide transporter (OATP)-mediated hepatic uptake clearance, hypothetically due to the albumin-mediated hepatic drug uptake. In this manuscript, we apply the dynamic WSM to extrapolate hepatic clearance of the OATP substrates, and our results show significant improvements in clearance prediction without assuming albumin-mediated hepatic drug uptake.
Subject(s)
Liver , Models, Biological , Organic Anion Transporters , Organic Anion Transporters/metabolism , Liver/metabolism , Humans , Albumins/metabolism , Biological Transport/physiology , Metabolic Clearance Rate , Protein Binding , Pharmaceutical Preparations/metabolism , AnimalsABSTRACT
The organic anion uptake and efflux transporters [organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and multidrug resistance-associated protein (MRP)2 and MRP3] that mediate the transport of the hepatobiliary-specific contrast agent gadoxetate (Gd-EOB-DTPA) are direct or indirect targets of the farnesoid X receptor (FXR), a key regulator of bile acid and lipid homeostasis. In benign liver tumors, FXR expression and activation is not yet characterized. We investigated the expression and activation of FXR and its targets in hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) and their correlation with Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI). Gd-EOB-DTPA MRI patterns were assessed by an expert radiologist. The intensity of the lesions on the hepatobiliary phase was correlated to mRNA expression levels of OATP1B1, OATP1B3, MRP2, MRP3, FXR, and small heterodimer partner (SHP) in fresh surgical specimens of patients with FNH or HCA subtypes. Normal and tumor sample pairs of 43 HCA and 14 FNH were included. All FNH (14/14) were hyperintense. Of the 34 HCA with available Gd-EOB-DTPA-enhanced MRI, 6 were hyperintense and 28 HCA were hypointense. OATP1B3 was downregulated in the hypointense tumors compared with normal surrounding liver tissue (2.77±3.59 vs. 12.9±15.6, P < 0.001). A significant positive correlation between FXR expression and activation and OATP1B3 expression level was found in the HCA cohort. SHP showed a trend toward downregulation in hypointense HCA. In conclusion, this study suggests that the MRI relative signal in HCA may reflect expression level and/or activity of SHP and FXR. Moreover, our data confirms the pivotal role of OATP1B3 in Gd-EOB-DTPA uptake in HCA. SIGNIFICANCE STATEMENT: FXR represents a valuable target for the treatment of liver disease and metabolic syndrome. Currently, two molecules, ursodeoxycholate and obeticholate, are approved for the treatment of primary biliary cirrhosis and cholestasis, with several compounds in clinical trials for the treatment of metabolic dysfunction-associated fatty liver disease. Because FXR expression and activation is associated with gadoxetate accumulation in HCA, an atypical gadoxetate-enhanced MRI pattern might arise in patients under FXR-targeted therapy, thereby complicating the differential diagnosis.
Subject(s)
Adenoma, Liver Cell , Focal Nodular Hyperplasia , Liver Neoplasms , Organic Anion Transporters , Humans , Organic Anion Transporters/metabolism , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/metabolism , Contrast Media/metabolism , Focal Nodular Hyperplasia/diagnosis , Focal Nodular Hyperplasia/metabolism , Focal Nodular Hyperplasia/pathology , Magnetic Resonance Imaging/methods , Multidrug Resistance-Associated Protein 2 , Anions/metabolism , Retrospective StudiesABSTRACT
Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 (collectively, OATP1B) transporters encoded by the solute carrier organic anion transporter (SLCO) genes mediate uptake of multiple pharmaceutical compounds. Nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease (NAFLD), decreases OATP1B abundance. This research characterized the pathologic and pharmacokinetics effects of three diet- and one chemical-induced NAFLD model in male and female humanized OATP1B mice, which comprises knock-out of rodent Oatp orthologs and insertion of human SLCO1B1 and SLCO1B3. Histopathology scoring demonstrated elevated steatosis and inflammation scores for all NAFLD-treatment groups. Female mice had minor changes in SLCO1B1 expression in two of the four NAFLD treatment groups, and pitavastatin (PIT) area under the concentration-time curve (AUC) increased in female mice in only one of the diet-induced models. OATP1B3 expression decreased in male and female mice in the chemical-induced NAFLD model, with a coinciding increase in PIT AUC, indicating the chemical-induced model may better replicate changes in OATP1B3 expression and OATP substrate disposition observed in NASH patients. This research also tested a reported multifactorial pharmacokinetic interaction between NAFLD and silymarin, an extract from milk thistle seeds with notable OATP-inhibitory effects. Males showed no change in PIT AUC, whereas female PIT AUC increased 1.55-fold from the diet alone and the 1.88-fold from the combination of diet with silymarin, suggesting that female mice are more sensitive to pharmacokinetic changes than male mice. Overall, the humanized OATP1B model should be used with caution for modeling NAFLD and multifactorial pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Advanced stages of NAFLD cause decreased hepatic OATP1B abundance and increase systemic exposure to OATP substrates in human patients. The humanized OATP1B mouse strain may provide a clinically relevant model to recapitulate these observations and predict pharmacokinetic interactions in NAFLD. This research characterized three diet-induced and one drug-induced NAFLD model in a humanized OATP1B mouse model. Additionally, a multifactorial pharmacokinetic interaction was observed between silymarin and NAFLD.
Subject(s)
Non-alcoholic Fatty Liver Disease , Organic Anion Transporters , Silymarin , Humans , Male , Female , Mice , Animals , Non-alcoholic Fatty Liver Disease/metabolism , Mice, Transgenic , Solute Carrier Organic Anion Transporter Family Member 1B3/metabolism , Liver-Specific Organic Anion Transporter 1/metabolism , Liver/metabolism , Organic Anion Transporters/metabolism , Membrane Transport Proteins/metabolism , Silymarin/metabolism , Drug InteractionsABSTRACT
The organic anion transporting polypeptide (OATP) 2B1 is considered an emerging drug transporter that is found expressed in pharmacokinetically relevant organs such as the liver, small intestine, and kidney. Despite its interaction with various substrate drugs, the understanding of its in vivo relevance is still limited. In this study, we first validated the interaction of atorvastatin with rat OATP2B1 using transiently transfected HeLa cells. Moreover, we characterized our rSlco2b1-knockout and SLCO2B1-knockin rats for mRNA, protein expression, and localization of OATP2B1 in the liver, small intestine, and kidney. The transporter showed the highest expression in the liver followed by the small intestine. In humanized rats, human OATP2B1 is localized on the sinusoidal membrane of hepatocytes. In enterocytes of wild-type and humanized rats, the transporter was detected in the luminal membrane with the vast majority being localized subapical. Subsequently, we assessed atorvastatin pharmacokinetics in male wild-type, rSlco2b1-knockout, and SLCO2B1-knockin rats after a single-dose administration (orally and intravenously). Investigating the contribution of rat OATP2B1 or human OATP2B1 to oral atorvastatin pharmacokinetics revealed no differences in concentration-time profiles or pharmacokinetic parameters. However, when comparing the pharmacokinetics of atorvastatin after intravenous administration in SLCO2B1-humanized rats and knockout animals, notable differences were observed. In particular, the systemic exposure (area under the curve) decreased by approximately 40% in humanized animals, whereas the clearance was 57% higher in animals expressing human OATP2B1. These findings indicate that human OATP2B1 influences pharmacokinetics of atorvastatin after intravenous administration, most likely by contributing to the hepatic uptake. SIGNIFICANCE STATEMENT: Wild-type, rSlco2b1-knockout, and SLCO2B1-humanized Wistar rats were characterized for the expression of rat and human SLCO2B1/OATP2B1. Pharmacokinetic studies of atorvastatin over 24 hours were conducted in male wild-type, rSlco2b1-knockout, and SLCO2B1-humanized rats. After a single-dose intravenous administration, a lower systemic exposure and an increase in clearance were observed in SLCO2B1-humanized rats compared with knockout animals indicating a contribution of OATP2B1 to the hepatic clearance.
Subject(s)
Atorvastatin , Liver , Organic Anion Transporters , Animals , Atorvastatin/pharmacokinetics , Atorvastatin/administration & dosage , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolism , Humans , Male , Rats , Liver/metabolism , HeLa Cells , Rats, Transgenic , Intestine, Small/metabolism , Gene Knockout Techniques/methods , Kidney/metabolism , Gene Knock-In Techniques/methods , Administration, Oral , Administration, Intravenous , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacokinetics , Hydroxymethylglutaryl-CoA Reductase Inhibitors/administration & dosage , Hepatocytes/metabolism , Tissue DistributionABSTRACT
Morphine is a widely used opioid for the treatment of pain. Differences in drug transporter expression and activity may contribute to variability in morphine pharmacokinetics and response. Using appropriate mouse models, we investigated the impact of the efflux transporters ABCB1 and ABCG2 and the OATP uptake transporters on the pharmacokinetics of morphine, morphine-3-glucuronide (M3G), and M6G. Upon subcutaneous administration of morphine, its plasma exposure in Abcb1a/1b-/-;Abcg2-/--, Abcb1a/1b-/-;Abcg2-/-;Oatp1a/1b-/-;Oatp2b1-/- (Bab12), and Oatp1a/1b-/-;Oatp2b1-/- mice was similar to that found in wild-type mice. Forty minutes after dosing, morphine brain accumulation increased by 2-fold when mouse (m)Abcb1 and mAbcg2 were ablated. Relative recovery of morphine in small intestinal content was significantly reduced in all the knockout strains. In the absence of mOatp1a/1b and mOatp2b1, plasma levels of M3G were markedly increased, suggesting a lower elimination rate. Moreover, Oatp-deficient mice displayed reduced hepatic and intestinal M3G accumulation. Mouse Oatps similarly affected plasma and tissue disposition of subcutaneously administered M6G. Human OATP1B1/1B3 transporters modestly contribute to the liver accumulation of M6G. In summary, mAbcb1, in combination with mAbcg2, limits morphine brain penetration and its net intestinal absorption. Variation in ABCB1 activity due to genetic polymorphisms/mutations and/or environmental factors might, therefore, partially affect morphine tissue exposure in patients. The ablation of mOatp1a/1b increases plasma exposure and decreases the liver and small intestinal disposition of M3G and M6G. Since the contribution of human OATP1B1/1B3 to M6G liver uptake was quite modest, the risks of undesirable drug interactions or interindividual variation related to OATP activity are likely negligible.
Subject(s)
Mice, Knockout , Morphine Derivatives , Morphine , Animals , Morphine/pharmacokinetics , Morphine/metabolism , Morphine Derivatives/metabolism , Morphine Derivatives/blood , Mice , Tissue Distribution , Male , Brain/metabolism , Analgesics, Opioid/pharmacokinetics , Analgesics, Opioid/metabolism , Analgesics, Opioid/blood , Mice, Inbred C57BL , Organic Anion Transporters/metabolism , Organic Anion Transporters/genetics , Liver/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/geneticsABSTRACT
Organic anion transporting polypeptides OATP1A2, OATP1B1, OATP1B3 and OATP2B1 are Na+ - and ATP-independent exchangers of large, organic compounds, encompassing structurally diverse xenobiotics, including various drugs. These OATPs influence intestinal absorption (OATP2B1), hepatic clearance (OATP1B1/3) and blood to brain penetration (OATP1A2, OATP2B1) of their drug substrates. Consequently, OATP-mediated drug or food interactions may lead to altered pharmacokinetics and toxicity. During drug development, investigation of hepatic OATP1B1 and OATP1B3 is recommended by international regulatory agencies. Most frequently, OATP-drug interactions are investigated in an indirect assay, i.e., by examining uptake inhibition of a radioactive or fluorescent probe. However, indirect assays do not distinguish between transported substrates and non-transported OATP inhibitors. To fill this hiatus, a novel assay, termed competitive counterflow (CCF) has been developed and has since been applied for several OATPs to differentiate between substrates and non-transported inhibitors. However, previous OATP CCF assays, with the exception of that for OATP1B1, used radioactive probes. In the current study, we demonstrate that sulforhodamine 101 or pyranine can be used as fluorescent probes in a CCF assay to identify transported substrates of OATP1A2, or OATPs 1B1, 1B3 and 2B1, respectively. With the help of the newly developed fluorescence-based CCF method, we identify the FDA-approved anti-protozoal drug, pentamidine as a unique substrate of OATP1A2. Furthermore, we confirm the selective, OATP1A2-mediated uptake of pentamidine in a cytotoxicity assay. Based on our results, OATP1A2 may be an important determinant of pentamidine transport through the blood-brain barrier.
Subject(s)
Organic Anion Transporters , Organic Anion Transporters/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Pentamidine , Liver-Specific Organic Anion Transporter 1/metabolism , Fluorescence , Biological Transport , PeptidesABSTRACT
Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.
Subject(s)
Estradiol/analogs & derivatives , Organic Anion Transporters, Sodium-Independent , Organic Anion Transporters , Organic Anion Transporters, Sodium-Independent/metabolism , Clotrimazole/pharmacology , Solute Carrier Organic Anion Transporter Family Member 1B3/metabolism , Liver-Specific Organic Anion Transporter 1/metabolism , Molecular Docking Simulation , Organic Anion Transporters/metabolism , Biological TransportABSTRACT
Amyotrophic lateral sclerosis (ALS) is characterized by death and dysfunction of motor neurons that result in a rapidly progressing loss of motor function. While there are some data on alterations at the blood-brain barrier (BBB) in ALS and their potential impact on CNS trafficking of drugs, little is reported on the impact of this disease on the expression of drug-handling proteins in the small intestine and liver. This may impact the dosing of the many medicines that individuals with ALS are prescribed. In the present study, a proteomic evaluation was performed on small intestine and liver samples from postnatal day 120 SOD1G93A mice (a model of familial ALS that harbors a human mutant form of superoxide dismutase 1) and wild-type (WT) littermates (n = 7/genotype/sex). Untargeted, quantitative proteomics was undertaken using either label-based [tandem mass tag (TMT)] or label-free [data-independent acquisition (DIA)] acquisition strategies on high-resolution mass spectrometric instrumentation. Copper chaperone for superoxide dismutase (CCS) was significantly higher in SOD1G93A samples compared to the WT samples for both sexes and tissues, therefore representing a potential biomarker for ALS in this mouse model. Relative to WT mice, male SOD1G93A mice had significantly different proteins (Padj < 0.05, |fold-change|>1.2) in the small intestine (male 22, female 1) and liver (male 140, female 3). This included an up-regulation of intestinal transporters for dietary glucose [solute carrier (SLC) SLC5A1] and cholesterol (Niemann-Pick c1-like 1), as well as for several drugs (e.g., SLC15A1), in the male SOD1G93A mice. There was both an up-regulation (e.g., SLCO2A1) and down-regulation (ammonium transporter rh type b) of transporters in the male SOD1G93A liver. In addition, there was both an up-regulation (e.g., phosphoenolpyruvate carboxykinase) and down-regulation (e.g., carboxylesterase 1) of metabolizing enzymes in the male SOD1G93A liver. This proteomic data set identified male-specific changes to key small intestinal and hepatic transporters and metabolizing enzymes that may have important implications for the bioavailability of nutrients and drugs in individuals with ALS.
Subject(s)
Amyotrophic Lateral Sclerosis , Organic Anion Transporters , Animals , Female , Humans , Male , Mice , Amyotrophic Lateral Sclerosis/genetics , Disease Models, Animal , Liver/metabolism , Mice, Transgenic , Organic Anion Transporters/metabolism , Proteomics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolismABSTRACT
Intravenously administered chemotherapeutic cabazitaxel is used for palliative treatment of prostate cancer. An oral formulation would be more patient-friendly and reduce the need for hospitalization. We therefore study determinants of the oral pharmacokinetics of cabazitaxel in a ritonavir-boosted setting, which reduces the CYP3A-mediated first-pass metabolism of cabazitaxel. We here assessed the role of organic anion-transporting polypeptides (OATPs) in the disposition of orally boosted cabazitaxel and its active metabolites, using the Oatp1a/b-knockout and the OATP1B1/1B3-transgenic mice. These transporters may substantially affect plasma clearance and hepatic and intestinal drug disposition. The pharmacokinetics of cabazitaxel and DM2 were not significantly affected by Oatp1a/b and OATP1B1/1B3 activity. In contrast, the plasma AUC0-120 min of DM1 in Oatp1a/b-/- was 1.9-fold (p < 0.05) higher than that in wild-type mice, and that of docetaxel was 2.4-fold (p < 0.05) higher. We further observed impaired hepatic uptake and intestinal disposition for DM1 and docetaxel in the Oatp-ablated strains. None of these parameters showed rescue by the OATP1B1 or -1B3 transporters in the humanized mouse strains, suggesting a minimal role of OATP1B1/1B3. Ritonavir itself was also a potent substrate for mOatp1a/b, showing a 2.9-fold (p < 0.0001) increased plasma AUC0-120 min and 3.5-fold (p < 0.0001) decreased liver-to-plasma ratio in Oatp1a/b-/- compared to those in wild-type mice. Furthermore, we observed the tight binding of cabazitaxel and its active metabolites, including docetaxel, to plasma carboxylesterase (Ces1c) in mice, which may complicate the interpretation of pharmacokinetic and pharmacodynamic mouse studies. Collectively, these results will help to further optimize (pre)clinical research into the safety and efficacy of orally applied cabazitaxel.
Subject(s)
Organic Anion Transporters, Sodium-Independent , Organic Anion Transporters , Taxoids , Animals , Humans , Male , Mice , Carboxylesterase/metabolism , Docetaxel , Liver/metabolism , Liver-Specific Organic Anion Transporter 1/metabolism , Mice, Transgenic , Organic Anion Transporters/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , Ritonavir , Solute Carrier Organic Anion Transporter Family Member 1B3/metabolismABSTRACT
Organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3, encoded by the SLCO gene family of the solute carrier superfamily, are involved in the disposition of many exogenous and endogenous compounds. Preclinical rodent models help assess risks of pharmacokinetic interactions, but interspecies differences in transporter orthologs and expression limit direct clinical translation. An OATP1B transgenic mouse model comprising a rodent Slco1a/1b gene cluster knockout and human SLCO1B1 and SLCO1B3 gene insertions provides a potential physiologically relevant preclinical tool to predict pharmacokinetic interactions. Pharmacokinetics of exogenous probe substrates, pitavastatin and pravastatin, and endogenous OATP1B biomarkers, coproporphyrin-I and coproporphyrin-III, were determined in the presence and absence of known OATP/Oatp inhibitors, rifampin or silymarin (an extract of milk thistle [Silybum marianum]), in wild-type FVB mice and humanized OATP1B mice. Rifampin increased exposure of pitavastatin (4.6- and 2.8-fold), pravastatin (3.6- and 2.2-fold), and coproporphyrin-III (1.6- and 2.1-fold) in FVB and OATP1B mice, respectively, but increased coproporphyrin-I AUC0-24h only (1.8-fold) in the OATP1B mice. Silymarin did not significantly affect substrate AUC, likely because the silymarin flavonolignan concentrations were at or below their reported IC50 values for the relevant OATPs/Oatps. Silymarin increased the Cmax of pitavastatin 2.7-fold and pravastatin 1.9-fold in the OATP1B mice. The data of the OATP1B mice were similar to those of the pitavastatin and pravastatin clinical data; however, the FVB mice data more closely recapitulated pitavastatin clinical data than the data of the OATP1B mice, suggesting that the OATP1B mice are a reasonable, though costly, preclinical strain for predicting pharmacokinetic interactions when doses are optimized to achieve clinically relevant plasma concentrations.
Subject(s)
Drug Interactions , Liver-Specific Organic Anion Transporter 1 , Mice, Transgenic , Pravastatin , Rifampin , Silymarin , Solute Carrier Organic Anion Transporter Family Member 1B3 , Animals , Rifampin/pharmacokinetics , Mice , Liver-Specific Organic Anion Transporter 1/genetics , Liver-Specific Organic Anion Transporter 1/metabolism , Humans , Silymarin/pharmacokinetics , Pravastatin/pharmacokinetics , Pravastatin/administration & dosage , Solute Carrier Organic Anion Transporter Family Member 1B3/genetics , Solute Carrier Organic Anion Transporter Family Member 1B3/metabolism , Quinolines/pharmacokinetics , Coproporphyrins/metabolism , Male , Organic Anion Transporters/genetics , Organic Anion Transporters/metabolismABSTRACT
Changes in the pharmacokinetic and resulting pharmacodynamic properties of drugs are common in many chronic liver diseases, leading to adverse effects, drug interactions and increased risk of over- or underdosing of medications. Structural and functional hepatic impairment can have major effects on drug metabolism and transport. This review summarizes research on the functional changes in phase I and II metabolic enzymes and in transport proteins in patients with metabolic diseases such as type 2 diabetes, metabolic dysfunction-associated steatotic liver disease, metabolic dysfunction-associated steatohepatitis and cirrhosis, providing a clinical perspective on how these changes affect drug uptake and metabolism. Generally, a decrease in expression and/or activity of many enzymes of the cytochrome P450 family (e.g. CYP2E1 and CYP3A4), and of influx and efflux transporters (e.g. organic anion-transporting polypeptide [OATP]1B1, OATP2B1, OAT2 and bile salt export pump), has been recently documented in patients with liver disease. Decreased enzyme levels often correlate with increased severity of chronic liver disease. In subjects with hepatic impairment, there is potential for strong alterations of drug pharmacokinetics due to reduced absorption, increased volume of distribution, metabolism and extraction. Due to the altered pharmacokinetics, specific drug-drug interactions are also a potential issue to consider in patients with liver disease. Given the huge burden of liver disease in western societies, there is a need to improve awareness among all healthcare professionals and patients with liver disease to ensure appropriate drug prescriptions.
Subject(s)
Diabetes Mellitus, Type 2 , Liver Diseases , Organic Anion Transporters , Humans , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Liver/metabolism , Metabolic Clearance Rate , Drug Interactions , Membrane Transport Proteins/metabolism , Organic Anion Transporters/metabolism , Organic Anion Transporters/pharmacology , Liver Diseases/metabolismABSTRACT
PURPOSE: Olmesartan medoxomil (olmesartan-MX), an ester-type prodrug of the angiotensin II receptor blocker (ARB) olmesartan, is predominantly anionic at intestinal pH. Human organic anion transporting polypeptide 2B1 (OATP2B1) is expressed in the small intestine and is involved in the absorption of various acidic drugs. This study was designed to test the hypothesis that OATP2B1-mediated uptake contributes to the enhanced intestinal absorption of olmesartan-MX, even though olmesartan itself is not a substrate of OATP2B1. METHODS: Tetracycline-inducible human OATP2B1- and rat Oatp2b1-overexpressing HEK 293 cell lines (hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293, respectively) were established to characterize OATP2B1-mediated uptake. Rat jejunal permeability was measured using Ussing chambers. ARBs were quantified by liquid chromatography-tandem mass spectrometry. RESULTS: Significant olmesartan-MX uptake was observed in hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293 cells, whereas olmesartan uptake was undetectable or much lower than olmesartan-MX uptake, respectively. Furthermore, olmesartan-MX exhibited several-fold higher uptake in Caco-2 cells and greater permeability in rat jejunum compared to olmesartan. Olmesartan-MX uptake in hOATP2B1/T-REx-293 cells and in Caco-2 cells was significantly decreased by OATP2B1 substrates/inhibitors such as 1 mM estrone-3-sulfate, 100 µM rifamycin SV, and 100 µM fluvastatin. Rat Oatp2b1-mediated uptake and rat jejunal permeability of olmesartan-MX were significantly decreased by 50 µM naringin, an OATP2B1 inhibitor. Oral administration of olmesartan-MX with 50 µM naringin to rats significantly reduced the area under the plasma concentration-time curve of olmesartan to 76.9%. CONCLUSION: Olmesartan-MX is a substrate for OATP2B1, and the naringin-sensitive transport system contributes to the improved intestinal absorption of olmesartan-MX compared with its parent drug, olmesartan.