Structure and inhibition of the human lysosomal transporter Sialin.

Sialin, a member of the solute carrier 17 (SLC17) transporter family, is unique in its ability to transport not only sialic acid using a pH-driven mechanism, but also transport mono and diacidic neurotransmitters, such as glutamate and N-acetylaspartylglutamate (NAAG), into synaptic vesicles via a membrane potential-driven mechanism. While most transporters utilize one of these mechanisms, the structural basis of how Sialin transports substrates using both remains unclear. Here, we present the cryogenic electron-microscopy structures of human Sialin: apo cytosol-open, apo lumen-open, NAAG-bound, and inhibitor-bound. Our structures show that a positively charged cytosol-open vestibule accommodates either NAAG or the Sialin inhibitor Fmoc-Leu-OH, while its luminal cavity potentially binds sialic acid. Moreover, functional analyses along with molecular dynamics simulations identify key residues in binding sialic acid and NAAG. Thus, our findings uncover the essential conformational states in NAAG and sialic acid transport, demonstrating a working model of SLC17 transporters.

Evaluation of the Clinical Drug-Drug Interaction Potential of Pritelivir on Transporters and CYP450 Enzymes Using a Cocktail Approach.

Pritelivir is a novel viral helicase-primase inhibitor active against herpes simplex virus. In vitro drug-drug interaction studies indicated that pritelivir has the potential for clinically relevant interactions on the cytochrome P450 (CYP) enzymes 2C8, 2C9, 3A4, and 2B6, and intestinal uptake transporter organic anion transporting polypeptide (OATP) 2B1 and efflux transporter breast cancer resistance protein (BCRP). This was evaluated in 2 clinical trials. In 1 trial the substrates flurbiprofen (CYP2C9), bupropion (CYP2B6), and midazolam (CYP3A4) were administered simultaneously as part of the Geneva cocktail, while the substrate celiprolol (OAPT2B1) was administered separately. In another trial, the substrates repaglinide (CYP2C8) and rosuvastatin (BCRP) were administered separately. Exposure parameters of the substrates and their metabolites (flurbiprofen and bupropion only) were compared after administration with or without pritelivir under therapeutic concentrations. The results of these trials indicated that pritelivir has no clinically relevant effect on the exposure of substrates for the intestinal uptake transporter OATP2B1 and the CYP enzymes 3A4, 2B6, 2C9, and 2C8, and has a weak inhibitory effect on the intestinal efflux transporter BCRP. In summary, the results suggest that pritelivir has a low drug-drug interaction potential.

Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1.

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.

Inhibition of human drug transporter activities by succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

Design, synthesis, and biological evaluation of 3-phenyl substituted pyridine derivatives as potential dual inhibitors of XOR and URAT1.

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are two most widely studied targets involved in production and reabsorption of uric acid, respectively. Marketed drugs almost target XOR or URAT1, but sometimes, single agents might not achieve aim of lowering uric acid to ideal value in clinic. Thus, therapeutic strategies of combining XOR inhibitors with uricosuric drugs were proposed and implemented. Based on our initial work of virtual screening, A and B were potential hits for dual-targeted inhibitors on XOR/URAT1. By docking A/B with XOR/URAT1 respectively, compounds I1-7 were designed to get different degree of inhibition effect on XOR and URAT1, and I7 showed the best inhibitory effect on XOR (IC50 = 0.037 ± 0.001 µM) and URAT1 (IC50 = 546.70 ± 32.60 µM). Further docking research on I7 with XOR/URAT1 led to the design of compounds II with the significantly improved inhibitory activity on XOR and URAT1, such as II11 and II15. Especially, for II15, the IC50 of XOR is 0.006 ± 0.000 µM, superior to that of febuxostat (IC50 = 0.008 ± 0.000 µM), IC50 of URAT1 is 12.90 ± 2.30 µM, superior to that of benzbromarone (IC50 = 27.04 ± 2.55 µM). In acute hyperuricemia mouse model, II15 showed significant uric acid lowering effect. The results suggest that II15 had good inhibitory effect on XOR/URAT1, with the possibility for further investigation in in-vivo models of hyperuricemia.

Discovery of digallic acid as XOD/URAT1 dual target inhibitor for the treatment of hyperuricemia.

The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC50 of 5.34 ± 0.65 µM, which is less potent than benzbromarone (2.01 ± 0.36 µM) but more potent than lesinurad (10.36 ± 1.23 µM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC50 of 1.04 ± 0.23 µM. Its metabolic product, gallic acid, inhibited XOD with an IC50 of 0.91 ± 0.14 µM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug.

Conversion of Olmesartan to Olmesartan Medoxomil, A Prodrug that Improves Intestinal Absorption, Confers Substrate Recognition by OATP2B1.

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.

Effects of rifampicin on the pharmacokinetics and safety of carotegrast methyl in healthy subjects: A randomized 2 × 2 crossover study.

AIMS: To evaluate the effect of the combination of carotegrast methyl with rifampicin, a potent inhibitor of organic anion transporter polypeptide, on the pharmacokinetics (PKs), safety and tolerability of carotegrast methyl. METHODS: In this 2 × 2 crossover study in 20 healthy Japanese adults, 10 subjects received carotegrast methyl 960 mg and rifampicin 600 mg on day 1 and received carotegrast methyl 960 mg on day 8. The subjects in the other sequence received the same treatments but in the opposite order. The 90% confidence interval (CI) of the geometric mean ratio of the Cmax and AUC0-t for carotegrast, the main active metabolite of carotegrast methyl, with/without rifampicin was calculated. If the 90% CI fell within the range of 0.80-1.25, this indicated the absence of any drug-drug interaction. Adverse events (AEs) were monitored. RESULTS: The geometric mean ratios (90% CI) of the Cmax and AUC0-t for carotegrast with/without rifampicin were 4.78 (3.64-6.29) and 5.59 (4.60-6.79), respectively, indicating that carotegrast has a PK interaction with rifampicin. The combination with rifampicin increased the exposure of carotegrast and also that of its metabolites. The incidence of any AEs with/without rifampicin was five (25.0%) and one (5.0%), respectively. CONCLUSIONS: Coadministration of carotegrast methyl with rifampicin significantly increased the exposure of carotegrast compared with carotegrast methyl administration alone. In this single dose study, the incidence of AEs of carotegrast methyl with rifampicin increased compared with carotegrast methyl alone, but the incidence of adverse drug reactions did not increase with combination administration.

Prognostic Biomarker SLCO4A1 Is Correlated with Tumor Immune Infiltration in Colon Adenocarcinoma.

Background: Solute carrier organic anion transporter family member 4A1 (SLCO4A1), a member of solute carrier organic anion family, is a key gene regulating bile metabolism, organic anion transport, and ABC transport. However, the association of SLCO4A1 with prognosis and tumor immune infiltration in colon adenocarcinoma (COAD) remains indistinct. Methods: Firstly, we explored the expression level of SLCO4A1 in COAD via GEPIA, Oncomine, and UALCAN databases. Secondly, we used the Kaplan-Meier plotter and PrognoScan databases to investigate the effect of SLCO4A1 on prognosis in COAD patients. In addition, the correlation between SLCO4A1 and tumor immune infiltration was studied by using TIMER and TISIDB databases. Results: Our results showed that SLCO4A1 was overexpressed in COAD tissues. At the same time, our study showed that high expression of SLCO4A1 was associated with poor overall survival, disease-free survival, and disease-specific survival in COAD patients. The expression level of SLCO4A1 was negatively linked to the infiltrating levels of B cells, CD8+ T cells, and dendritic cells in COAD. Moreover, the expression of SLCO4A1 was significantly correlated with numerous immune markers in COAD. Conclusions: These results indicated that SLCO4A1 could be associated with the prognosis of COAD patients and the levels of tumor immune infiltration. Our study suggested that SLCO4A1 could be a valuable biomarker for evaluating prognosis and tumor immune infiltration in COAD patients.

Blockade of Organic Anion Transport in Humans After Treatment With the Drug Probenecid Leads to Major Metabolic Alterations in Plasma and Urine.

Probenecid is used to treat gout and hyperuricemia as well as increase plasma levels of antiviral drugs and antibiotics. In vivo, probenecid mainly inhibits the renal SLC22 organic anion transporters OAT1 (SLC22A6), OAT3 (SLC22A8), and URAT1 (SLC22A12). To understand the endogenous role of these transporters in humans, we administered probenecid to 20 healthy participants and metabolically profiled the plasma and urine before and after dosage. Hundreds of metabolites were significantly altered, indicating numerous drug-metabolite interactions. We focused on potential OAT1 substrates by identifying 97 metabolites that were significantly elevated in the plasma and decreased in the urine, indicating OAT-mediated clearance. These included signaling molecules, antioxidants, and gut microbiome products. In contrast, urate was the only metabolite significantly decreased in the plasma and elevated in the urine, consistent with an effect on renal reuptake by URAT1. Additional support comes from metabolomics analyses of our Oat1 and Oat3 knockout mice, where over 50% of the metabolites that were likely OAT substrates in humans were elevated in the serum of the mice. Fifteen of these compounds were elevated in both knockout mice, whereas six were exclusive to the Oat1 knockout and 4 to the Oat3 knockout. These may be endogenous biomarkers of OAT function. We also propose a probenecid stress test to evaluate kidney proximal tubule organic anion transport function in kidney disease. Consistent with the Remote Sensing and Signaling Theory, the profound changes in metabolite levels following probenecid treatment support the view that SLC22 transporters are hubs in the regulation of systemic human metabolism.

Discovery of novel verinurad analogs as dual inhibitors of URAT1 and GLUT9 with improved Druggability for the treatment of hyperuricemia.

Verinurad (RDEA3170) is a selective URAT1 inhibitor under investigation for the treatment of gout and hyperuricemia. In an effort to further improve the pharmacodynamics/pharmacokinetics of verinurad and to increase the structural diversity, we designed novel verinurad analogs by introducing a linker (e.g. aminomethyl, amino or oxygen) between the naphthalene and the pyridine ring to increase the flexibility. These compounds were synthesized and tested for their in vitro URAT1-inhibitory activity. Most compounds exhibited potent inhibitory activities against URAT1 with IC50 values ranging from 0.24 µM to 16.35 µM. Among them, compound KPH2f exhibited the highest URAT1-inhibitory activity with IC50 of 0.24 µM, comparable to that of verinurad (IC50 = 0.17 µM). KPH2f also inhibited GLUT9 with an IC50 value of 9.37 ± 7.10 µM, indicating the dual URAT1/GLUT9 targeting capability. In addition, KPH2f showed little effects on OAT1 and ABCG2, and thus was unlikely to cause OAT1/ABCG2-mediated drug-drug interactions and/or to neutralize the uricosuric effects of URAT1/GLUT9 inhibitors. Importantly, KPH2f (10 mg/kg) was equally effective in reducing serum uric acid levels and exhibited higher uricosuric effects in a mice hyperuricemia model, as compared to verinurad (10 mg/kg). Furthermore, KPH2f demonstrated favorable pharmacokinetic properties with an oral bioavailability of 30.13%, clearly better than that of verinurad (21.47%). Moreover, KPH2f presented benign safety profiles without causing hERG toxicity, cytotoxicity in vitro (lower than verinurad), and renal damage in vivo. Collectively, these results suggest that KPH2f represents a novel, safe and effective dual URAT1/GLUT9 inhibitor with improved druggabilities and is worthy of further investigation as an anti-hyperuricemic drug candidate.

Pharmacologic and genetic inhibition of cholesterol esterification enzymes reduces tumour burden: A systematic review and meta-analysis of preclinical models.

Cholesterol esterification proteins Sterol-O acyltransferases (SOAT) 1 and 2 are emerging prognostic markers in many cancers. These enzymes utilise fatty acids conjugated to coenzyme A to esterify cholesterol. Cholesterol esterification is tightly regulated and enables formation of lipid droplets that act as storage organelles for lipid soluble vitamins and minerals, and as cholesterol reservoirs. In cancer, this provides rapid access to cholesterol to maintain continual synthesis of the plasma membrane. In this systematic review and meta-analysis, we summarise the current depth of understanding of the role of this metabolic pathway in pan-cancer development. A systematic search of PubMed, Scopus, Web of Science, and Cochrane Library for preclinical studies identified eight studies where cholesteryl ester concentrations were compared between tumour and adjacent-normal tissue, and 24 studies where cholesterol esterification was blocked by pharmacological or genetic approaches. Tumour tissue had a significantly greater concentration of cholesteryl esters than non-tumour tissue (p < 0.0001). Pharmacological or genetic inhibition of SOAT was associated with significantly smaller tumours of all types (p ≤ 0.002). SOAT inhibition increased tumour apoptosis (p = 0.007), CD8 + lymphocyte infiltration and cytotoxicity (p ≤ 0.05), and reduced proliferation (p = 0.0003) and metastasis (p < 0.0001). Significant risk of publication bias was found and may have contributed to a 32% overestimation of the meta-analysed effect size. Avasimibe, the most frequently used SOAT inhibitor, was effective at doses equivalent to those previously reported to be safe and tolerable in humans. This work indicates that SOAT inhibition should be explored in clinical trials as an adjunct to existing anti-neoplastic agents.

CDER167, a dual inhibitor of URAT1 and GLUT9, is a novel and potent uricosuric candidate for the treatment of hyperuricemia.

Urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) are important targets for the development of uric acid-lowering drugs. We previously showed that the flexible linkers of URAT1 inhibitors could enhance their potency. In this study we designed and synthesized CDER167, a novel RDEA3710 analogue, by introducing a linker (methylene) between the naphthalene and pyridine rings to increase flexibility, and characterized its pharmacological and pharmacokinetics properties in vitro and in vivo. We showed that CDER167 exerted dual-target inhibitory effects on both URAT1 and GLUT9: CDER167 concentration-dependently inhibited the uptake of [14C]-uric acid in URAT1-expressing HEK293 cells with an IC50 value of 2.08 ± 0.31 µM, which was similar to that of RDEA3170 (its IC50 value was 1.47 ± 0.23 µM). Using site-directed mutagenesis, we demonstrated that CDER167 might interact with URAT1 at S35 and F365. In GLUT9-expressing HEK293T cells, CDER167 concentration-dependently inhibited GLUT9 with an IC50 value of 91.55 ± 15.28 µM, whereas RDEA3170 at 100 µM had no effect on GLUT9. In potassium oxonate-induced hyperuricemic mice, oral administration of CDER167 (10 mg·kg-1 · d-1) for 7 days was more effective in lowering uric acid in blood and significantly promoted uric acid excretion in urine as compared with RDEA3170 (20 mg·kg-1 · d-1) administered. The animal experiment proved the safety of CDER167. In addition, CDER167 displayed better bioavailability than RDEA3170, better metabolic stability and no hERG toxicity at 100 µM. These results suggest that CDER167 deserves further investigation as a candidate antihyperuricemic drug targeting URAT1 and GLUT9.

Novel natural scaffold as hURAT1 inhibitor identified by 3D-shape-based, docking-based virtual screening approach and biological evaluation.

As a promising therapeutic target for gout, hURAT1 has attracted increasing attention. In this work, we identified a novel scaffold of hURAT1 inhibitors from a personal natural product database of verified herb-treated gout. First, we constructed more than 800 natural compounds from Chinese medicine that were verified to treat gout. Following the application of both shape-based and docking-based virtual screening (VS) methods, taking into account the shape similarity and flexibility of the target, we identified isopentenyl dihydroflavones that might inhibit hURAT1. Specifically, 9 compounds with commercial availability were tested with biochemical assays for the inhibition of 14C-uric acid uptake in high-expression hURAT1 cells (HEK293-hURAT1), and their structure-activity relationship was evaluated. As a result, 8-isopentenyl dihydroflavone was identified as a novel scaffold of hURAT1 inhibitors since isobavachin (DHF3) inhibited hURAT1 with an IC50 value of 0.39 ± 0.17 µM, which was comparable to verinurad with an IC50 value of 0.32 ± 0.23 µM. Remarkably, isobavachin also displayed an eminent effect in the decline of serum uric acid in vivo experiments. Taken together, isobavachin is a promising candidate for the treatment of hyperuricemia and gout.

Identification of the organic anion transporting polypeptides responsible for the hepatic uptake of the major metabolite of epyrifenacil, S-3100-CA, in mice.

Epyrifenacil is a novel herbicide that acts as an inhibitor of protoporphyrinogen oxidase (PPO) and produces hepatotoxicity in rodents by inhibiting PPO. Our previous research revealed that the causal substance of hepatotoxicity is S-3100-CA, a major metabolite of epyrifenacil, and that human hepatocyte uptake of S-3100-CA was significantly lower than rodent one, suggesting less relevant to hepatotoxicity in humans. To clarify the species difference in the uptake of S-3100-CA, we focused on organic anion transporting polypeptides (OATPs) and carried out an uptake assay using human, rat, and mouse OATP hepatic isoforms-expressing 293FT cells. As a result, all the examined OATPs were found to contribute to the S-3100-CA uptake, suggesting that the species difference was not due to the differences in selectivity toward OATP isoforms. When [14 C]epyrifenacil was administered to mice, the liver concentration of S-3100-CA was higher in males than in females. Furthermore, when [14 C]epyrifenacil was administered with OATP inhibitors, the liver/plasma ratio of S-3100-CA was significantly decreased by rifampicin, an Oatp1a1/Oatp1a4 inhibitor in mice, but not by digoxin, an Oatp1a4-specific inhibitor. This result indicates that Oatp1a1, the predominant transporter in male mice, is the main contributor to the hepatic transport of S-3100-CA, and consequently to the gender difference. Moreover, we conclude that the species difference in the hepatic uptake of S-3100-CA observed in our previous research is not due to differences in the selectivity toward OATP isoforms but rather to the significantly higher expression of OATPs which mediate uptake of S-3100-CA in rodents than in humans.

Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1).

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.

Impact of fedratinib on the pharmacokinetics of transporter probe substrates using a cocktail approach.

INTRODUCTION: Fedratinib, an oral, selective Janus kinase 2 inhibitor, has been shown to inhibit P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter (OCT) 2, and multidrug and toxin extrusion (MATE) 1 and MATE2-K in vitro. The objective of this study was to evaluate the influence of fedratinib on the pharmacokinetics (PK) of digoxin (P-gp substrate), rosuvastatin (OATP1B1/1B3 and BCRP substrate), and metformin (OCT2 and MATE1/2-K substrate). METHODS: In this nonrandomized, fixed-sequence, open-label study, 24 healthy adult participants received single oral doses of digoxin 0.25 mg, rosuvastatin 10 mg, and metformin 1000 mg administered as a drug cocktail (day 1, period 1). After a 6-day washout, participants received oral fedratinib 600 mg 1 h before the cocktail on day 7 (period 2). An oral glucose tolerance test (OGTT) was performed to determine possible influences of fedratinib on the antihyperglycemic effect of metformin. RESULTS: Plasma exposure to the three probe drugs was generally comparable in the presence or absence of fedratinib. Reduced metformin renal clearance by 36% and slightly higher plasma glucose levels after OGTT were observed in the presence of fedratinib. Single oral doses of the cocktail ± fedratinib were generally well tolerated. CONCLUSIONS: These results suggest that fedratinib has minimal impact on the exposure of P-gp, BCRP, OATP1B1/1B3, OCT2, and MATE1/2-K substrates. Since renal clearance of metformin was decreased in the presence of fedratinib, caution should be exercised in using coadministered drugs that are renally excreted via OCT2 and MATEs. TRIAL REGISTRATION: Clinicaltrials.gov NCT04231435 on January 18, 2020.

Effect of Berberine on Hyperuricemia and Kidney Injury: A Network Pharmacology Analysis and Experimental Validation in a Mouse Model.

PURPOSE: Berberine (BBR) is an active component of Phellodendri Cortex (PC), which is a traditional Chinese medicine that has been prescribed clinically for hyperuricemia (HUA) for hundreds of years. Many studies reported the anti-inflammatory and nephroprotective properties of BBR and PC; however, the therapeutic effects of BBR on HUA have not been explored. This study aims to investigate the efficacy and mechanism of BBR for treating HUA. METHODS: The mechanism of BBR in the treatment of HUA were predicted by network pharmacology. A mouse model of HUA established by potassium oxonate and hypoxanthine was used to verify the prediction. The levels of serum uric acid (UA), urea nitrogen (BUN) and creatinine (CRE) were determined by biochemical test kits. Hematoxylin and eosin staining of kidney tissues was used to observe the kidney damage. ELISA kits were applied to detect the levels of interleukin (IL)-1ß and IL-18 in serum and kidney tissues. Quantitative real-time PCR and Western blotting were adopted to analyze the expression of NLRP3, ASC, Caspase1, IL-1ß and URAT1. The expressions of URAT1 in the kidney tubules were visualized by immunohistochemical staining. Molecular docking was used to assess the interaction between URAT1 and BBR. RESULTS: The network pharmacology screened out 82 genes and several inflammation-related signaling pathways related to the anti-hyperuricemia effect of BBR. In the in vivo experiment, BBR substantially decreased the level of UA, BUN and CRE, and alleviated the kidney damage in mice with HUA. BBR reduced IL-1ß and IL-18, and downregulated expressions of NLRP3, ASC, Caspase1 and IL-1ß. BBR also inhibited expression of URAT1 and exhibited strong affinity with this target in silico docking. CONCLUSION: BBR exerts anti-HUA and nephroprotective effects via inhibiting activation of NLRP3 inflammasome and correcting the aberrant expression of URAT1 in kidney. BBR might be a novel therapeutic agent for treating HUA.

ABCB1 and ABCG2, but not CYP3A4 limit oral availability and brain accumulation of the RET inhibitor pralsetinib.

BACKGROUND AND PURPOSE: Pralsetinib is an FDA-approved oral small-molecule inhibitor for treatment of rearranged during transfection (RET) proto-oncogene fusion-positive non-small cell lung cancer. We investigated how the efflux transporters ABCB1 and ABCG2, the SLCO1A/1B uptake transporters and the drug-metabolizing enzyme CYP3A influence pralsetinib pharmacokinetics. EXPERIMENTAL APPROACH: In vitro, transepithelial pralsetinib transport was assessed. In vivo, pralsetinib (10 mg/kg) was administered orally to relevant genetically modified mouse models. Pralsetinib concentrations in cell medium, plasma samples and organ homogenates were measured using liquid chromatography-tandem mass spectrometry. KEY RESULTS: Pralsetinib was efficiently transported by human (h)ABCB1 and mouse (m)Abcg2, but not hACBG2. In vivo, mAbcb1a/1b markedly and mAbcg2 slightly limited pralsetinib brain penetration (6.3-and 1.8-fold, respectively). Testis distribution showed similar results. Abcb1a/1b;Abcg2-/- mice showed 1.5-fold higher plasma exposure, 23-fold increased brain penetration, and 4-fold reduced recovery of pralsetinib in the small intestinal content. mSlco1a/1b deficiency did not affect pralsetinib oral availability or tissue exposure. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted pralsetinib plasma exposure (1.3-fold) and brain penetration (19.6-fold) in wild-type mice. Additionally, pralsetinib was a modest substrate of mCYP3A, but not of hCYP3A4, which did not noticeably restrict the oral availability or tissue distribution of pralsetinib. CONCLUSIONS AND IMPLICATIONS: SLCO1A/1B and CYP3A4 are unlikely to affect the pharmacokinetics of pralsetinib, but ABCG2 and especially ABCB1 markedly limit its brain and testis penetration, as well as oral availability. These effects are mostly reversed by oral coadministration of the ABCB1/ABCG2 inhibitor elacridar. These insights may be useful in the further clinical development of pralsetinib.

Physiologically Based Pharmacokinetic Modeling of Transporter-Mediated Hepatic Disposition of Imaging Biomarker Gadoxetate in Rats.

Physiologically based pharmacokinetic (PBPK) models are increasingly used in drug development to simulate changes in both systemic and tissue exposures that arise as a result of changes in enzyme and/or transporter activity. Verification of these model-based simulations of tissue exposure is challenging in the case of transporter-mediated drug-drug interactions (tDDI), in particular as these may lead to differential effects on substrate exposure in plasma and tissues/organs of interest. Gadoxetate, a promising magnetic resonance imaging (MRI) contrast agent, is a substrate of organic-anion-transporting polypeptide 1B1 (OATP1B1) and multidrug resistance-associated protein 2 (MRP2). In this study, we developed a gadoxetate PBPK model and explored the use of liver-imaging data to achieve and refine in vitro-in vivo extrapolation (IVIVE) of gadoxetate hepatic transporter kinetic data. In addition, PBPK modeling was used to investigate gadoxetate hepatic tDDI with rifampicin i.v. 10 mg/kg. In vivo dynamic contrast-enhanced (DCE) MRI data of gadoxetate in rat blood, spleen, and liver were used in this analysis. Gadoxetate in vitro uptake kinetic data were generated in plated rat hepatocytes. Mean (%CV) in vitro hepatocyte uptake unbound Michaelis-Menten constant (Km,u) of gadoxetate was 106 µM (17%) (n = 4 rats), and active saturable uptake accounted for 94% of total uptake into hepatocytes. PBPK-IVIVE of these data (bottom-up approach) captured reasonably systemic exposure, but underestimated the in vivo gadoxetate DCE-MRI profiles and elimination from the liver. Therefore, in vivo rat DCE-MRI liver data were subsequently used to refine gadoxetate transporter kinetic parameters in the PBPK model (top-down approach). Active uptake into the hepatocytes refined by the liver-imaging data was one order of magnitude higher than the one predicted by the IVIVE approach. Finally, the PBPK model was fitted to the gadoxetate DCE-MRI data (blood, spleen, and liver) obtained with and without coadministered rifampicin. Rifampicin was estimated to inhibit active uptake transport of gadoxetate into the liver by 96%. The current analysis highlighted the importance of gadoxetate liver data for PBPK model refinement, which was not feasible when using the blood data alone, as is common in PBPK modeling applications. The results of our study demonstrate the utility of organ-imaging data in evaluating and refining PBPK transporter IVIVE to support the subsequent model use for quantitative evaluation of hepatic tDDI.