Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

BACKGROUND: In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, a phenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. METHODS: The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). RESULTS: This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. CONCLUSIONS: We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Interplay of Ritonavir-Boosted Oral Cabazitaxel with the Organic Anion-Transporting Polypeptide (OATP) Uptake Transporters and Carboxylesterase 1 in Mice.

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.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

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.

Pharmacokinetic interactions of niclosamide in rats: Involvement of organic anion transporters 1 and 3 and organic cation transporter 2.

Niclosamide is an anthelmintic drug with a long history of use and is generally safe and well tolerated in humans. As the conventional dose of niclosamide results in a low but certain level in systemic circulation, drug interactions with concomitant drugs should be considered. We aimed to investigate the interaction between niclosamide and drug transporters, as such information is currently limited. Niclosamide inhibited the transport activity of OATP1B1, OATP1B3, OAT1, OAT3, and OCT2 in vitro. Among them, the inhibitory effects on OAT1, OAT3, and OCT2 were strong, with IC50 values of less than 1 µM. When 3 mg/kg of niclosamide was co-administered to rats, systemic exposure to furosemide (a substrate of OAT1/3) and metformin (a substrate of OCT2) increased, and the renal clearance (CLr) of the drugs significantly decreased. These results suggest that niclosamide inhibits renal transporters, OAT1/3 and OCT2, not only in vitro but also in vivo, resulting in increased systemic exposure to the substrates of the transporters by strongly blocking the urinary elimination pathway in rats. The findings of this study will support a meticulous understanding of the transporter-mediated drug interactions of niclosamide and consequently aid in effective and safe use of niclosamide.

Examining the Association of Rare Allelic Variants in Urate Transporters SLC22A11, SLC22A13, and SLC17A1 with Hyperuricemia and Gout.

Genetic variations in urate transporters play a significant role in determining human urate levels and have been implicated in developing hyperuricemia or gout. Polymorphism in the key urate transporters, such as ABCG2, URAT1, or GLUT9 was well-documented in the literature. Therefore in this study, our objective was to determine the frequency and effect of rare nonsynonymous allelic variants of SLC22A11, SLC22A13, and SLC17A1 on urate transport. In a cohort of 150 Czech patients with primary hyperuricemia and gout, we examined all coding regions and exon-intron boundaries of SLC22A11, SLC22A13, and SLC17A1 using PCR amplification and Sanger sequencing. For comparison, we used a control group consisting of 115 normouricemic subjects. To examine the effects of the rare allelic nonsynonymous variants on the expression, intracellular processing, and urate transporter protein function, we performed a functional characterization using the HEK293A cell line, immunoblotting, fluorescent microscopy, and site directed mutagenesis for preparing variants in vitro. Variants p.V202M (rs201209258), p.R343L (rs75933978), and p.P519L (rs144573306) were identified in the SLC22A11 gene (OAT4 transporter); variants p.R16H (rs72542450), and p.R102H (rs113229654) in the SLC22A13 gene (OAT10 transporter); and the p.W75C variant in the SLC17A1 gene (NPT1 transporter). All variants minimally affected protein levels and cytoplasmic/plasma membrane localization. The functional in vitro assay revealed that contrary to the native proteins, variants p.P519L in OAT4 (p ≤ 0.05), p.R16H in OAT10 (p ≤ 0.05), and p.W75C in the NPT1 transporter (p ≤ 0.01) significantly limited urate transport activity. Our findings contribute to a better understanding of (1) the risk of urate transporter-related hyperuricemia/gout and (2) uric acid handling in the kidneys.

Microcystin-LR-induced epithelial-mesenchymal transition-like cells acquire resistance to multi-toxins.

The protein phosphatase inhibitor microcystin-LR (MC-LR), a hepatocyte-selective cyanotoxin, induces phenotypic changes in HEK293 OATP1B3-expressing (HEK293-OATP1B3) cells, which include cytoskeletal reorganization (HEK293-OATP1B3-AD) and anoikis resistance (HEK293-OATP1B3-FL) transformed cells, respectively. These cells acquire resistance to MC-LR and partial epithelial-mesenchymal transition (EMT) characteristics. In cancer cells, EMT is generally involved in multi-drug resistance. Here, we focused on the multi-drug resistance of HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. The MTT assay and immunoblotting were conducted to examine the responses of HEK293-OATP1B3, HEK293-OATP1B3-AD, and HEK293-OATP1B3-FL cells to multiple toxins and drugs that function as substrates for OATP1B3, including MC-LR, nodularin (Nod), okadaic acid (OA), and cisplatin (CDDP). HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells were more resistant to MC-LR, Nod, and OA than HEK293-OATP1B3 cells. Conversely, the three cell types were equivalently sensitive to CDDP. By using protein phosphatase assay, the reduction of the inhibitory effect of MC-LR and Nod on phosphatase activity might be one reason for the resistance to MC-LR and Nod in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Furthermore, the parental HEK293-OATP1B3 cells showed enhanced p53 phosphorylation and stabilization after MC-LR exposure, while p53 phosphorylation was attenuated in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells. Moreover, in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells, AKT phosphorylation was higher than that of the parental HEK293-OATP1B3 cell line. These results suggest that the multi-toxin resistance observed in HEK293-OATP1B3-AD and HEK293-OATP1B3-FL cells is associated with AKT activation and p53 inactivation.

Renal Organic Anion Transporters 1 and 3 In Vitro: Gone but Not Forgotten.

Organic anion transporters 1 and 3 (OAT1 and OAT3) play a crucial role in kidney function by regulating the secretion of multiple renally cleared small molecules and toxic metabolic by-products. Assessing the activity of these transporters is essential for drug development purposes as they can significantly impact drug disposition and safety. OAT1 and OAT3 are amongst the most abundant drug transporters expressed in human renal proximal tubules. However, their expression is lost when cells are isolated and cultured in vitro, which is a persistent issue across all human and animal renal proximal tubule cell models, including primary cells and cell lines. Although it is well known that the overall expression of drug transporters is affected in vitro, the underlying reasons for the loss of OAT1 and OAT3 are still not fully understood. Nonetheless, research into the regulatory mechanisms of these transporters has provided insights into the molecular pathways underlying their expression and activity. In this review, we explore the regulatory mechanisms that govern the expression and activity of OAT1 and OAT3 and investigate the physiological changes that proximal tubule cells undergo and that potentially result in the loss of these transporters. A better understanding of the regulation of these transporters could aid in the development of strategies, such as introducing microfluidic conditions or epigenetic modification inhibitors, to improve their expression and activity in vitro and to create more physiologically relevant models. Consequently, this will enable more accurate assessment for drug development and safety applications.

SLC22A11 Inserts the Uremic Toxins Indoxyl Sulfate and P-Cresol Sulfate into the Plasma Membrane.

Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.

Effect of probenecid on blood levels and renal elimination of furosemide and endogenous compounds in rats: Discovery of putative organic anion transporter biomarkers.

Transporter-mediated drug-drug interactions (DDIs) are assessed using probe drugs and in vitro and in vivo models during drug development. The utility of endogenous metabolites as transporter biomarkers is emerging for prediction of DDIs during early phases of clinical trials. Endogenous metabolites such as pyridoxic acid and kynurenic acid have shown potential to predict DDIs mediated by organic anion transporters (OAT1 and OAT3). However, these metabolites have not been assessed in rats as potential transporter biomarkers. We carried out a rat pharmacokinetic DDI study using probenecid and furosemide as OAT inhibitor and substrate, respectively. Probenecid administration led to a 3.8-fold increase in the blood concentrations and a 3-fold decrease in renal clearance of furosemide. High inter-individual and intra-day variability in pyridoxic acid and kynurenic acid, and no or moderate effect of probenecid administration on these metabolites suggest their limited utility for prediction of Oat-mediated DDI in rats. Therefore, rat blood and urine samples were further analysed using untargeted metabolomics. Twenty-one m/z features (out of >8000 detected features) were identified as putative biomarkers of rat Oat1 and Oat3 using a robust biomarker qualification approach. These m/z features belong to metabolic pathways such as fatty acid analogues, peptides, prostaglandin analogues, bile acid derivatives, flavonoids, phytoconstituents, and steroids, and can be used as a panel to decrease variability caused by processes other than Oats. When validated, these putative biomarkers will be useful in predicting DDIs caused by Oats in rats.

Hyperoside ameliorates cisplatin-induced acute kidney injury by regulating the expression and function of Oat1.

Cisplatin is a widely used chemotherapeutic agent to treat solid tumours in clinics. However, cisplatin-induced acute kidney injury (AKI) limits its clinical application. This study investigated the effect of hyperoside (a flavonol glycoside compound) on regulating AKI.The model of cisplatin-induced AKI was established, and hyperoside was preadministered to investigate its effect on improving kidney injury.Hyperoside ameliorated renal pathological damage, reduced the accumulation of SCr, BUN, Kim-1 and indoxyl sulphate in vivo, increased the excretion of indoxyl sulphate into the urine, and upregulated the expression of renal organic anion transporter 1 (Oat1). Moreover, evaluation of rat kidney slices demonstrated that hyperoside promoted the uptake of PAH (p-aminohippurate, the Oat1 substrate), which was confirmed by transient over-expression of OAT1 in HEK-293T cells. Additionally, hyperoside upregulated the mRNA expression of Oat1 upstream regulators hepatocyte nuclear factor-1α (HNF-1α) and pregnane X receptor (PXR).These findings indicated hyperoside could protect against cisplatin-induced AKI by promoting indoxyl sulphate excretion through regulating the expression and function of Oat1, suggesting hyperoside may offer a potential tactic for cisplatin-induced AKI treatment.

The fluorescence-based competitive counterflow assay developed for organic anion transporting polypeptides 1A2, 1B1, 1B3 and 2B1 identifies pentamidine as a selective OATP1A2 substrate.

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.

Relevance of the organic anion transporting polypeptide 1B3 (OATP1B3) in the personalized pharmacological treatment of hepatocellular carcinoma.

Although pharmacological treatment is the best option for most patients with advanced hepatocellular carcinoma (HCC), its success is very limited, partly due to reduced uptake and enhanced efflux of antitumor drugs. Here we have explored the usefulness of vectorizing drugs towards the organic anion transporting polypeptide 1B3 (OATP1B3) to enhance their efficacy against HCC cells. In silico studies (RNA-Seq data, 11 cohorts) and immunohistochemistry analyses revealed a marked interindividual variability, together with general downregulation but still expression of OATP1B3 in the plasma membrane of HCC cells. The measurement of mRNA variants in 20 HCC samples showed the almost absence of the cancer-type variant (Ct-OATP1B3) together with marked predominance of the liver-type variant (Lt-OATP1B3). In Lt-OATP1B3-expressing cells, the screening of 37 chemotherapeutical drugs and 17 tyrosine kinase receptors inhibitors (TKIs) revealed that 10 classical anticancer drugs and 12 TKIs were able to inhibit Lt-OATP1B3-mediated transport. Lt-OATP1B3-expressing cells were more sensitive than Mock parental cells (transduced with empty lentiviral vectors) to some Lt-OATP1B3 substrates (paclitaxel and the bile acid-cisplatin derivative Bamet-UD2), but not to cisplatin, which is not transported by Lt-OATP1B3. This enhanced response was abolished by competition with taurocholic acid, a known Lt-OATP1B3 substrate. Tumors subcutaneously generated in immunodeficient mice by Lt-OATP1B3-expressing HCC cells were more sensitive to Bamet-UD2 than those derived from Mock cells. In conclusion, Lt-OATP1B3 expression should be screened before deciding the use of anticancer drugs substrates of this carrier in the personalized treatment of HCC. Moreover, Lt-OATP1B3-mediated uptake must be considered when designing novel anti-HCC targeted drugs.

The regulation of human organic anion transporter 4 by insulin-like growth factor 1 and protein kinase B signaling.

Human organic anion transporter 4 (hOAT4), mainly expressed in the kidney and placenta, is essential for the disposition of numerous drugs, toxins, and endogenous substances. Insulin-like growth factor 1 (IGF-1) is a hormone generated in the liver and plays important roles in systemic growth, development, and metabolism. In the current study, we explored the regulatory effects of IGF-1 and downstream signaling on the transport activity, protein expression, and SUMOylation of hOAT4. We showed that IGF-1 significantly increased the transport activity, expression, and maximal transport velocity Vmax of hOAT4 in kidney-derived cells. This stimulatory effect of IGF-1 on hOAT4 activity was also confirmed in cells derived from the human placenta. The increased activity and expression were correlated well with the reduced degradation rate of hOAT4 at the cell surface. Furthermore, IGF-1 significantly increased hOAT4 SUMOylation, and protein kinase B (PKB)-specific inhibitors blocked the IGF-1-induced regulations on hOAT4. In conclusion, our study demonstrates that the hepatic hormone IGF-1 regulates hOAT4 expressed in the kidney and placenta through the PKB signaling pathway. Our results support the remote sensing and signaling theory, where OATs play a central role in the remote communications among distal tissues.

Recent Advances in Synthetic Drugs and Natural Actives Interacting with OAT3.

Organic anion transporter 3 (OAT3) is predominantly expressed in the kidney and plays a vital role in drug clearance. Consequently, co-ingestion of two OAT3 substrates may alter the pharmacokinetics of the substrate. This review summarizes drug-drug interactions (DDIs) and herbal-drug interactions (HDIs) mediated by OAT3, and inhibitors of OAT3 in natural active compounds in the past decade. This provides a valuable reference for the combined use of substrate drugs/herbs for OAT3 in clinical practice in the future and for the screening of OAT3 inhibitors to avoid harmful interactions.

Cellular uptake of rose bengal is mediated by OATP1B1/1B3 transporters.

Due to its fluorescent properties and high yield of singlet oxygen, rose bengal (RB) is one of the most promising photosensitizers for cancer treatment. However, the negative charge of RB molecule may significantly hamper its intracellular delivery by passive diffusion through the cell membrane. Thus, specific membrane protein transporters may be needed. The organic anion transporting polypeptides (OATPs) are a well-characterized group of membrane protein transporters, responsible for cellular uptake of a number of drugs. To our knowledge, this is the first study that evaluates cellular transport of RB mediated by the OATP transporter family. First, electrified liquid-liquid interface, together with biophysical analysis and molecular dynamics simulations were used to characterize the interaction of RB with several models of a cellular membranes. These experiments proved that RB interacts only with the membrane's surface, without spontaneously crossing the lipid bilayer. Evaluation of intracellular uptake of RB by flow cytometry and confocal microscopy showed significant differences in uptake between liver and intestinal cell line models differing in expression of OATP transporters. The use of specific pharmacological inhibitors of OATPs, together with Western blotting and in silico analysis, indicated that OATPs are crucial for cellular uptake of RB.

Characterization of Bile Acid Sulfate Conjugates as Substrates of Human Organic Anion Transporting Polypeptides.

Drug interactions involving the inhibition of hepatic organic anion transporting polypeptides (OATPs) 1B1 and OATP1B3 are considered important. Therefore, we sought to study various sulfated bile acids (BA-S) as potential clinical OATP1B1/3 biomarkers. It was determined that BA-S [e.g., glycochenodeoxycholic acid 3-O-sulfate (GCDCA-S) and glycodeoxycholic acid 3-O-sulfate (GDCA-S)] are substrates of OATP1B1, OATP1B3, and sodium-dependent taurocholic acid cotransporting polypeptide (NTCP) transfected into human embryonic kidney 293 cells, with minimal uptake evident for other solute carriers (SLCs) like OATP2B1, organic anion transporter 2, and organic cation transporter 1. It was also shown that BA-S uptake by plated human hepatocytes (PHH) was inhibited (≥96%) by a pan-SLC inhibitor (rifamycin SV), and there was greater inhibition (≥77% versus ≤12%) with rifampicin (OATP1B1/3-selective inhibitor) than a hepatitis B virus myristoylated-preS1 peptide (NTCP-selective inhibitor). Estrone 3-sulfate was also used as an OATP1B1-selective inhibitor. In this instance, greater inhibition was observed with GDCA-S (76%) than GCDCA-S (52%). The study was expanded to encompass the measurement of GCDCA-S and GDCA-S in plasma of SLCO1B1 genotyped subjects. The geometric mean GDCA-S concentration was 2.6-fold (90% confidence interval 1.6, 4.3; P = 2.1 × 10-4) and 1.3-fold (1.1, 1.7; P = 0.001) higher in individuals homozygous and heterozygous for the SLCO1B1 c.521T > C loss-of-function allele, respectively. For GCDCA-S, no significant difference was noted [1.2-fold (0.8, 1.7; P = 0.384) and 0.9-fold (0.8, 1.1; P = 0.190), respectively]. This supported the in vitro data indicating that GDCA-S is a more OATP1B1-selective substrate (versus GCDCA-S). It is concluded that GCDCA-S and GDCA-S are viable plasma-based OATP1B1/3 biomarkers, but they are both less OATP1B1-selective when compared to their corresponding 3-O-glucuronides (GCDCA-3G and GDCA-3G). Additional studies are needed to determine their utility versus more established biomarkers, such as coproporphyrin I, for assessing inhibitors with different OATP1B1 (versus OATP1B3) inhibition signatures.

Development of a Suite of Gadolinium-Free OATP1-Targeted Paramagnetic Probes for Liver MRI.

Five amphiphilic, anionic Mn(II) complexes were synthesized as contrast agents targeted to organic anion transporting polypeptide transporters (OATP) for liver magnetic resonance imaging (MRI). The Mn(II) complexes are synthesized in three steps, each from the commercially available trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) chelator, with T1-relaxivity of complexes ranging between 2.3 and 3.0 mM-1 s-1 in phosphate buffered saline at an applied field strength of 3.0 T. Pharmacokinetics were assessed in female BALB/c mice by acquiring T1-weighted images dynamically for 70 min after agent administration and determining contrast enhancement and washout in various organs. Uptake of Mn(II) complexes in human OATPs was investigated through in vitro assays using MDA-MB-231 cells engineered to express either OATP1B1 or OATP1B3 isoforms. Our study introduces a new class of Mn-based OATP-targeted contrast that can be broadly tuned via simple synthetic protocols.

Potential Involvement of Organic Anion Transporters in Drug Interactions with Shuganning Injection, a Traditional Chinese Patent Medicine.

Traditional Chinese medicine injections have been widely used in China for the treatment of various diseases. Transporter-mediated drug-drug interactions are a major contributor to adverse drug reactions. However, the research on transporter-mediated Traditional Chinese medicine injection-drug interactions is limited. Shuganning injection is a widely used Traditional Chinese medicine injection for treating various liver diseases. In this study, we investigated the inhibitory effect of Shuganning injection and its four main ingredients (baicalin, geniposide, chlorogenic acid, and oroxylin A) on 9 drug transporters. Shuganning injection strongly inhibited organic anion transporter 1 and organic anion transporter 3 with IC50 values < 0.1% (v/v), and moderately inhibited organic anion transporter 2, organic anion transporting-polypeptide 1B1, and organic anion transporting-polypeptide 1B3 with IC50 values < 1.0%. Baicalin, the most abundant bioactive ingredient in the Shuganning injection, was identified as both an inhibitor and substrate of organic anion transporter 1, organic anion transporter 3, and organic anion transporting-polypeptide 1B3. Oroxylin A had the potential to act as both an inhibitor and substrate of organic anion transporting-polypeptide 1B1 and organic anion transporting-polypeptide 1B3. In contrast, geniposide and chlorogenic acid had no significant inhibitory effect on drug transporters. Notably, Shuganning injection markedly altered the pharmacokinetics of furosemide and atorvastatin in rats. Using Shuganning injection as an example, our findings support the implementation of transporter-mediated Traditional Chinese medicine injection-drug interactions in the development of Traditional Chinese medicine injection standards.

Characterization of Elimination Pathways and the Feasibility of Endogenous Metabolites as Biomarkers of Organic Anion Transporter 1/3 Inhibition in Cynomolgus Monkeys.

Organic anion transporters 1 and 3 (OAT1/3) occupy a key role in mediating renal elimination. Kynurenic acid (KYNA) was previously discovered as an effective endogenous biomarker to assess drug-drug interaction (DDI) for OAT inhibitors. Here, further in vitro and in vivo investigation was performed to characterize the elimination routes and feasibility of KYNA, along with other reported endogenous metabolites, as biomarkers of Oat1/3 inhibition in bile duct-cannulated (BDC) cynomolgus monkeys. Our results suggested that KYNA is a substrate of OAT1/3 and OAT2, but not OCT2, MATE1/2K, or NTCP, and that it shares comparable affinities between OAT1 and OAT3. Renal and biliary excretions and plasma concentration-time profiles of KYNA, pyridoxic acid (PDA), homovanillic acid (HVA), and coproporphyrin I (CP-I) were assessed in BDC monkeys dosed with either probenecid (PROB) at 100 mg/kg or the control vehicle. Renal excretion of KYNA, PDA, and HVA was determined to be the major elimination route. The maximum concentration and the area under the plasma concentration-time curve (Cmax and AUC0-24h) of KYNA were about 11.6- and 3.7-fold higher in the PROB group than in the vehicle group. Renal clearance of KYNA decreased by 3.2-fold, but biliary clearance (CLbile) was not altered after PROB administration. A similar trend was observed for PDA and HVA. Interestingly, an elevation of plasma concentration and reduction of CP-I CLbile were observed after PROB treatment, which suggested inhibition of the CP-I Oatp-Mrp2 transport axis by PROB. Overall, our results indicated that KYNA could potentially facilitate early and reliable assessment of DDI liabilities of Oat inhibition in monkeys. SIGNIFICANCE STATEMENT: This work reported renal excretion as the major elimination pathway for kynurenic acid, pyridoxic acid, and homovanillic acid. Administration of probenecid reduced renal clearance and increased plasma exposure of these biomarkers in monkeys, consistent with the observation in humans. These endogenous biomarkers discovered in monkeys could be potentially used to evaluate the clinical drug-drug interactions in the early phase of drug development.