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.

Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.

N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.

Integrated metabolomics and network pharmacology to reveal the mechanism of areca nut addiction.

As a chewing hobby, areca nut (Areca catechu L.) has become the most common psychoactive substance in the world, besides tobacco, alcohol and caffeinated beverages. Moreover, as a first-class carcinogen designated by International Agency for Research on Cancer, long-term chewing areca nut can result in oral mucosal diseases and even oral cancer. To clarify the potential mechanism of areca nut addiction, an integrated strategy of metabolomics and network pharmacology was adopted in this study. Network pharmacology study indicated that all the key targets related to areca nut addiction could be regulated by arecoline and pointed out the importance of G-protein coupled receptor signalling pathway. Analysis results of mice plasma metabolome and faeces metabolome intervened by arecoline suggested that the component may affect the dopamine system and 5-HT system by regulating phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, primary bile acid biosynthesis, glycerophospholipid metabolism and intestinal flora structure. Moreover, the potential importance of bile acids in formation of addictive behaviour of chewing areca nut was investigated by integrative analysis of the relationships between metabolites and intestinal flora. The study can provide scientific basis for the addiction intervention and treatment of areca nut chewers.

Trehalose metabolism targeting as a novel strategy to modulate acid tolerance of yeasts and its application in food industry.

Some spoilage yeasts are able to develop resistance to commonly used weak-acid preservatives. We studied the trehalose metabolism and its regulation in Saccharomyces cerevisiae in response to propionic acid stress. We show interruption of trehalose synthetic pathway caused the mutant hypersensitive to the acid stress, while its overexpression conferred acid-tolerance to yeast. Interestingly, this acid-tolerance phenotype was largely independent of trehalose but relied on the trehalose synthetic pathway. We demonstrate trehalose metabolism played a vital role in regulation of glycolysis flux and Pi/ATP homeostasis in yeast during acid-adaptation, and the PKA and TOR signaling pathways were involved in regulating trehalose synthesis at transcriptional level. This work confirmed the regulatory function of trehalose metabolism and improved our understanding of molecular mechanism of acid-adaptation of yeast. By exemplifying trehalose metabolism interruption limited the growth of S. cerevisiae exposed to weak acids, and trehalose pathway overexpression conferring acid-resistance to Yarrowia lipolytica enhanced citric acid production, this work provides new insights into the development of efficient preservation strategies and robust organic acid producers.

Up Front Unfolded Protein Response Combined with Early Protein Secretion Pathway Engineering in Yarrowia lipolytica to Attenuate ER Stress Caused by Enzyme Overproduction.

Engineering the yeast Yarrowia lipolytica as an efficient host to produce recombinant proteins remains a longstanding goal for applied biocatalysis. During the protein overproduction, the accumulation of unfolded and misfolded proteins causes ER stress and cell dysfunction in Y. lipolytica. In this study, we evaluated the effects of several potential ER chaperones and translocation components on relieving ER stress by debottlenecking the protein synthetic machinery during the production of the endogenous lipase 2 and the E. coli ß-galactosidase. Our results showed that improving the activities of the non-dominant translocation pathway (SRP-independent) boosted the production of the two proteins. While the impact of ER chaperones is protein dependent, the nucleotide exchange factor Sls1p for protein folding catalyst Kar2p is recognized as a common contributor enhancing the secretion of the two enzymes. With the identified protein translocation components and ER chaperones, we then exemplified how these components can act synergistically with Hac1p to enhance recombinant protein production and relieve the ER stress on cell growth. Specifically, the yeast overexpressing Sls1p and cytosolic heat shock protein Ssa8p and Ssb1p yielded a two-fold increase in Lip2p secretion compared with the control, while co-overexpressing Ssa6p, Ssb1p, Sls1p and Hac1p resulted in a 90% increase in extracellular ß-galp activity. More importantly, the cells sustained a maximum specific growth rate (µmax) of 0.38 h-1 and a biomass yield of 0.95 g-DCW/g-glucose, only slightly lower than that was obtained by the wild type strain. This work demonstrated engineering ER chaperones and translocation as useful strategies to facilitate the development of Y. lipolytica as an efficient protein-manufacturing platform.

Mechanistic Study on the Species Differences in Excretion Pathway of HR011303 in Humans and Rats.

Excretion of [14C]HR011303-derived radioactivity showed significant species difference. Urine (81.50% of dose) was the main excretion route in healthy male subjects, whereas feces (87.16% of dose) was the main excretion route in rats. To further elucidate the underlying cause for excretion species differences of HR011303, studies were conducted to uncover its metabolism and excretion mechanism. M5, a glucuronide metabolite of HR011303, is the main metabolite in humans and rats. Results of a rat microsome incubation study suggested that HR011303 was metabolized to M5 in the rat liver. According to previous studies, M5 is produced in both human liver and kidney microsomes. We found that M5 in the human liver can be transported to the blood by multidrug resistance-associated protein (MRP) 3, and then the majority of M5 can be hydrolyzed to HR011303. HR011303 enters the human kidney or liver through passive diffusion, whereas M5 is taken up through organic anion transporter (OAT) 3, organic anion-transporting polypeptide (OATP) 1B1, and OATP1B3. When HR011303 alone is present, it can be metabolized to M5 in both sandwich-cultured rat hepatocytes (SCRH) and sandwich-cultured human hepatocytes (SCHH) and excreted into bile as M5 in SCRH. Using transporter inhibitors in sandwich-cultured model and membrane vesicles expressing MRP2 or Mrp2, we found that M5 was a substance of MRP2/Mrp2, and the bile efflux of M5 was mainly mediated by MRP2/Mrp2. Considering the significant role of MRP3/Mrp3 and MRP2/Mrp2 in the excretion of glucuronides, the competition between them for M5 was possibly the determinant for the different excretion routes in humans and rats. SIGNIFICANCE STATEMENT: Animal experiments are necessary to predict dosage and safety of candidate drugs prior to clinical trials. However, extrapolation results often differ from the actual situation. For HR011303, excretory pathways exhibited a complete reversal, through urine in humans and feces in rats. Such phenomena have been observed in several drugs, but no in-depth studies have been conducted to date. In the present study, the excretion species differences of HR011303 can be explained by the competition for M5 between MRP2/Mrp2 and MRP3/Mrp3.

Mechanistic Study on the Effect of Renal Impairment on the Pharmacokinetics of Vildagliptin and its Carboxylic Acid Metabolite.

PURPOSE: To clarify the mechanism of renal impairment leading to different degrees of increased plasma exposure to dipeptidyl peptidase 4 inhibitor vildagliptin and its major metabolite, M20.7. METHODS: The 5/6 nephrectomized (5/6 Nx) rat model, to simulate chronic renal failure (CRF) patients, combined with kidney slices and transporter studies in vitro were used to assess this pharmacokinetic differences. RESULTS: After intragastric administration to 5/6 Nx rats, vildagliptin showed increased plasma levels by 45.8%, and M20.7 by 7.51 times, which was similar to patients with severe renal impairment. The recovery rate of M20.7 in urine and feces increased by less than 20%, showing limited effect of renal impairment on vildagliptin metabolism. In vitro studies found M20.7 to be the substrate for organic anion transporter 3 (OAT3). However, the active uptake of M20.7 in renal slices showed no difference between the 5/6 Nx and normal rats. In OAT3 overexpressed cells, the protein-bound uremic toxins, 3-carboxy-4-methyl-5propyl-2-furanpropionate (CMPF), hippuric acid (HA) and indoxyl sulfate (IS), which accumulate in CRF patients, inhibited M20.7 uptake with IC50 values of 5.75, 29.0 and 69.5 µM respectively, far lower than plasma concentrations in CRF patients, and showed a mixed inhibition type. CONCLUSIONS: The large increase in plasma exposure of M20.7 could be attributed to the accumulation of uremic toxins in CRF patients, which inhibited OAT3 activity and blocked renal excretion of M20.7, while vildagliptin, with high permeability, showed a slight increase in plasma exposure due to reduced glomerular filtration.

Itraconazole and rifampicin, as CYP3A modulators but not P-gp modulators, affect the pharmacokinetics of almonertinib and active metabolite HAS-719 in healthy volunteers.

Almonertinib is a novel third-generation EGFR tyrosine kinase inhibitor. It is mainly metabolized by CYP3A in vitro, and N-desmethylated almonertinib (HAS-719) is the major active metabolite in human plasma. In this study, we investigated the effects of CYP3A inhibitor itraconazole and CYP3A inducer rifampicin on the pharmacokinetics of almonertinib and HAS-719 in 64 healthy volunteers. We found that when co-administered with itraconazole, the maximal plasma concentration (Cmax) and the plasma exposure (AUC0-t) of almonertinib were increased by 56.3% and 2.38-fold, respectively, whereas the Cmax and AUC0-t of HAS-719 were reduced by 86.8% and 71.8%, respectively. Co-administration with rifampicin reduced the Cmax and AUC0-t of almonertinib by 79.3% and 92.6%, but the AUC0-t of HAS-719 was unexpectedly decreased by 72.5%. In vitro assays showed that both almonertinib and HAS-719 were substrates of CYP3A and P-gp. Co-administration of rifampicin in Beagle dogs reduced the fecal recovery of almonertinib and HAS-719, and markedly increased the levels of metabolites derived from further metabolism of HAS-719, which was consistent with human plasma data, suggesting that although rifampicin was also a potent inducer of P-gp, the pharmacokinetic alternation of HAS-719 was mainly due to its further metabolism but not excretion changes. Moreover, we revealed that almonertinib was a moderately sensitive substrate of CYP3A in vivo. Special attention should be paid to the interaction between almonertinib and drugs or food affecting CYP3A activity in the clinical application of almonertinib.

Furmonertinib (Alflutinib, AST2818) is a potential positive control drug comparable to rifampin for evaluation of CYP3A4 induction in sandwich-cultured primary human hepatocytes.

Furmonertinib (Alflutinib, AST2818), as a third-generation epidermal growth factor receptor inhibitor with an advanced efficacy and a relatively wide safety window, has been commercially launched in China recently. However, previous clinical studies demonstrated its time- and dose-dependent clearance in a multiple-dose regimen. In vitro drug metabolism and pharmacokinetic studies have suggested that furmonertinib is mainly metabolized by cytochrome P450 3A4 (CYP3A4) and can induce these enzymes via an increased mRNA expression. This study investigated two important evaluation criteria of CYP3A4 induction by furmonertinib through quantitative proteomics and probe metabolite formation: simultaneous (1) protein expression and (2) enzyme activity with sandwich-cultured primary human hepatocytes in the same well of cell culture plates. Results confirmed that furmonertinib was a potent CYP3A4 inducer comparable with rifampin and could be used as a positive model drug in in vitro studies to evaluate the induction potential of other drug candidates in preclinical studies. In addition, inconsistencies were observed between the protein expression and enzyme activities of CYP3A4 in cells induced by rifampin but not in groups treated with furmonertinib. As such, furmonertinib could be an ideal positive control in the evaluation of CYP3A4 induction. The cells treated with 10 µM rifampin expressed 20.16 ± 5.78 pmol/mg total protein, whereas the cells induced with 0.5 µM furmonertinib expressed 4.8 ± 0.66 pmol/mg protein compared with the vehicle (0.1% dimethyl sulfoxide), which contained 0.65 ± 0.45 pmol/mg protein. The fold change in the CYP3A4 enzyme activity in the cells treated with rifampin was 5.22 ± 1.13, which was similar to that of 0.5 µM furmonertinib (3.79 ± 0.52).

Recombinant Human Arresten and Canstatin Inhibit Angiogenic Behaviors of HUVECs via Inhibiting the PI3K/Akt Signaling Pathway.

Angiogenetic inhibitors are crucial in tumor therapy, and endogenous angiogenesis inhibitors have attracted considerable attention due to their effectiveness, safety, and multi-targeting ability. Arresten and canstatin, which have anti-angiogenesis effects, are the c-terminal fragments of the α1 and α2 chains of type IV collagen, respectively. In this study, human arresten and canstatin were recombinantly expressed in Escherichia coli (E. coli), and their effects on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated. Regarding the cell cycle distribution test and 5-ethynyl-2'-deoxyuridine (EdU) assays, arresten and canstatin could repress the proliferation of HUVECs at a range of concentrations. Transwell assay indicated that the migration of HUVECs was significantly decreased in the presence of arresten and canstatin, while tube formation assays suggested that the total tube length and junction number of HUVECs were significantly inhibited by these two proteins; moreover, they could also reduce the expression of vascular endothelial growth factor (VEGF) and the phosphorylation levels of PI3K and Akt, which indicated that the activation of the 3-kinase/serine/threonine-kinase (PI3K/Akt) signaling pathway was inhibited. These findings may have important implications for the soluble recombinant expression of human arresten and canstatin, and for the related therapy of cancer.

Preparation and Characterization of an Ancient Aminopeptidase Obtained from Ancestral Sequence Reconstruction for L-Carnosine Synthesis.

As a biologically active peptide, L-carnosine has been widely used in the pharmaceutical, cosmetic and health care industries due to its various physiological properties. However, relatively little research is available regarding L-carnosine's enzymatic synthesis function. In this study, a potential enzyme sequence with the function of carnosine synthesizing was screened out using the ancestral sequence reconstruction (ASR) technique. Identified with L-carnosine synthesis activity, this enzyme was further confirmed using autoproteolytic phenomenon via Western blot and N-terminal sequencing. After purification, the enzymatic properties of LUCA-DmpA were characterized. The melting temperature (Tm) and denaturation enthalpy (ΔH) of LUCA-DmpA were 60.27 ± 1.24 °C and 1306.00 ± 26.73 kJ·mol-1, respectively. Circular dichroism (CD) spectroscopy results showed that this ancestral enzyme was composed of α-helix (35.23 ± 0.06%), ß-sheet (11.06 ± 0.06%), ß-turn (23.67 ± 0.06%) and random coil (32.03 ± 0.06%). The enzyme was characterized with the optimal temperature and pH of 45 °C and 9.0, respectively. Notably, LUCA-DmpA was also characterized with remarkable pH tolerance based on the observation of more than 85% remaining enzymatic activity after incubation at different pH buffers (pH = 6-11) for 12 h. Additionally, rather than being improved or inhibited by metal ions, its enzymatic activity was found to be promoted by introducing organic solvent with a larger log P value. Based on these homology modeling results, the screened LUCA-DmpA is suggested to have further optimization potential, and thereafter to be offered as a promising candidate for real industrial applications.

Dual Action of Acidic Microenvironment on the Enrichment of the Active Metabolite of Disulfiram in Tumor Tissues.

Disulfiram, an antialcoholism drug, could potentially be repurposed as an anticancer drug because of the formation of copper(II) diethyldithiocarbamate (CuET) from dithiocarb (DTC, a reduced metabolite of disulfiram) and Cu2+ CuET exhibited preferential distribution to tumor tissues. This study investigated the mechanism of CuET accumulation in tumor tissues by employing MDA-MB-231 human breast cancer cells. The concentration of CuET in cells treated with DTC and Cu2+ in acidic culture medium (pH 6.8) was significantly higher than that of the control group (pH 7.4). Subsequently, the effects of pH on the uptake of DTC, Cu2+, and CuET were investigated separately. The acidic environment significantly increased the uptake rate of DTC and Cu2+ but had no effect on CuET. MDA-MB-231 cells overexpressing copper transporter hCTR1 were constructed to evaluate its intermediate role in CuET accumulation. After treatment with CuCl2 followed by DTC for 15 minutes, the levels of CuET and Cu2+ in hCTR1-overexpressed cells were 2.5 times as much as those of vector group. In the tumors of cancer xenograft models constructed by hCTR1-MDA-MB-231 cells, the concentrations of CuET and Cu were also significantly higher than those of control group. In conclusion, the acidic microenvironment of tumors can promote the enrichment of CuET in tumors through dual action. On the one hand, it can promote transmembrane transport of DTC by converting ionic DTC into molecular state. On the other hand, it enhances Cu2+ uptake by activating hCTR1, which ultimately leads to the enrichment of CuET. SIGNIFICANCE STATEMENT: Increasing evidence suggests that the antitumor activity of disulfiram is related to the formation of a copper(II) diethyldithiocarbamate (CuET) of its reducing metabolite dithiocarb with copper(II) ion, which is preferentially distributed in tumor tissues. We showed that the acidic microenvironment, a common feature of many solid tumor tissues, could promote intracellular CuET accumulation through dual action without changing CuET uptake. This result is helpful for the formulation of clinical dosage regimens of disulfiram in cancer treatment.

Drug interaction of ningetinib and gefitinib involving CYP1A1 and efflux transporters in non-small cell lung cancer patients.

AIMS: Ningetinib is a tyrosine kinase inhibitor for the treatment of non-small cell lung cancer (NSCLC). The present study aims to investigate the drug interaction of ningetinib and gefitinib and the mechanism of high plasma exposure of N-demethylated ningetinib (M1) in NSCLC patients. METHODS: Patients with NSCLC were recruited. Metabolism and transport assays were performed using in vitro models. Deuterated M1 was used to study the effects of ningetinib and gefitinib on M1 efflux in Institute of Cancer Research (ICR) mice. RESULTS: Upon co-administration of ningetinib with gefitinib, the plasma exposure of M1 was reduced by 80%, whereas that of ningetinib was not affected. In vitro experiments indicated that CYP1A1 was primarily responsible for M1 formation. Gefitinib was demonstrated to be a strong inhibitor of CYP1A1 with Ki value of 0.095 µM. M1 was identified as a substrate of efflux transporters P-gp and BCRP, while ningetinib and gefitinib were demonstrated to be their inhibitors, which was consistent with the results in mice. However, the inhibitory effect of gefitinib on efflux in vivo was negligible in the presence of ningetinib. CONCLUSION: The high plasma exposure of M1 in patients was attributed to the inhibition of M1 efflux by ningetinib and its low tissue affinity. When co-administered, gefitinib inhibited the formation of M1, but due to the low metabolic yield of M1 in vivo, the pharmacokinetics of ningetinib was not influenced. Inhibition of CYP1A1 may increase the concentration of ningetinib in target tissues, and the long-term safety and efficacy of ningetinib combined with gefitinib should be evaluated.

Nimesulide increases the aldehyde oxidase activity of humans and rats.

An increasing number of drugs are metabolized by aldehyde oxidase (AOX), but AOX-mediated drug interactions are seldom reported due to the lack of appropriate inhibitors and inducers. A recent study reported that nimesulide (NIM) could increase the liver injury risk of methotrexate. The latter was mainly metabolized by AOX to form hepatotoxic 7-hydroxymethotrexate (7-OH MTX). Thus, we speculated that NIM could induce AOX. In this study, we investigated the potential induction of AOX activity by NIM using methotrexate as the probe substrate. Treatment of primary human and rat hepatocytes with NIM (20 µM) for 24 h caused a 2.0- and 3.1-fold, respectively, increase in 7-OH MTX formation. Oral administration of NIM (100 mg·kg-1·d-1, for 5 days) to rats significantly increased the systematic exposure (6.5-fold), liver distribution (2.5-fold), and excretion (5.2-fold for urinary excretion and 2.1-fold for fecal excretion) of 7-OH MTX. The 7-OH MTX formation in liver cytosol from rats pretreated with 20, 50, and 100 mg·kg-1·d-1 NIM for 5 days increased by 1.9-, 3.2-, and 3.7-fold, respectively, compared with that of rats pretreated with the vehicle. We revealed that the elevation of AOX activity was accompanied by an increase in AOX1 protein levels but not the corresponding mRNA levels. Collectively, our results demonstrate for the first time that NIM can increase the AOX activity of humans and rats, and may raise concerns regarding the risk of drug interactions between NIM and AOX substrates in clinical practice.

Alflutinib (AST2818), primarily metabolized by CYP3A4, is a potent CYP3A4 inducer.

Alflutinib (AST2818) is a third-generation epidermal growth factor receptor (EGFR) inhibitor that inhibits both EGFR-sensitive mutations and T790M mutations. Previous study has shown that after multiple dosages, alflutinib exhibits nonlinear pharmacokinetics and displays a time- and dose-dependent increase in the apparent clearance, probably due to its self-induction of cytochrome P450 (CYP) enzyme. In this study, we investigated the CYP isozymes involved in the metabolism of alflutinib and evaluated the enzyme inhibition and induction potential of alflutinib and its metabolites. The data showed that alflutinib in human liver microsomes (HLMs) was metabolized mainly by CYP3A4, which could catalyze the formation of AST5902. Alflutinib did not inhibit CYP isozymes in HLMs but could induce CYP3A4 in human hepatocytes. Rifampin is a known strong CYP3A4 inducer and is recommended by the FDA as a positive control in the CYP3A4 induction assay. We found that the induction potential of alflutinib was comparable to that of rifampin. The Emax of CYP3A4 induction by alflutinib in three lots of human hepatocytes were 9.24-, 11.2-, and 10.4-fold, while the fold-induction of rifampin (10 µM) were 7.22-, 19.4- and 9.46-fold, respectively. The EC50 of alflutinib-induced CYP3A4 mRNA expression was 0.25 µM, which was similar to that of rifampin. In addition, AST5902 exhibited much weak CYP3A4 induction potential compared to alflutinib. Given the plasma exposure of alflutinib and AST5902, both are likely to affect the pharmacokinetics of CYP3A4 substrates. Considering that alflutinib is a CYP3A4 substrate and a potent CYP3A4 inducer, drug-drug interactions are expected during alflutinib treatment.

Carboxylesterase 2 and Intestine Transporters Contribute to the Low Bioavailability of Allisartan, a Prodrug of Exp3174 for Hypertension Treatment in Humans.

Exp3174 is an active metabolite of losartan for the treatment of hypertension. Allisartan (ALS3) is a marketed ester prodrug of Exp3174 to reduce bioavailability variation of losartan in China. However, ALS3 exhibited a lower oral absorption than losartan in humans. In this study, the enzymes and transporters involved in ALS3 and Exp3174 disposition were investigated to clarify the mechanisms. ALS3 underwent extensive hydrolysis to Exp3174 in S9 of Caco-2 cells, human intestine microsomes (HIM), recombinant carboxylesterase (rCES) 1, and rCES2. ALS3 exhibited similar affinity in HIM and rCES2 with K m values of 6.92 and 6.77 µM, respectively, indicating that ALS3 is mainly hydrolyzed to Exp3174 in human intestine by CES2. Transport assays of ALS3 and Exp3174 suggested that ALS3 and Exp3174 are substrates of P-glycoprotein, breast cancer resistance protein, and multidrug resistance protein 2 with poor permeability. Organic anion-transporting polypeptide 2B1 showed higher affinity and clearance toward ALS3 (K m 0.75 µM and intrinsic clearance 215 µl/min/mg) than those of Exp3174 (K m 7.85 µM and intrinsic clearance 16.1 µl/min/mg), indicating that ALS3 is preferred to be uptaken into intestinal epithelia. Hydrolysis of ALS3 was increased from approximately 30% to 55% in CES2-transfected human embryonic kidney 293-OATP2B1 cells, indicating the possible interplay between OATP2B1 and CES2. The influx and efflux of ALS3 across Caco-2 cells increased the potential of ALS3 hydrolysis to Exp3174, and the produced Exp3174 was rapidly pumped out, which led to undetectable ALS3 and Exp3174 in basolateral (receiver) side in Caco-2 cells. Overall, our study provided supportive evidences that the interplay between CES2 and transporters in intestine contributes to the low bioavailability of ALS3 in humans.

Increased Plasma Exposures of Conjugated Metabolites of Morinidazole in Renal Failure Patients: A Critical Role of Uremic Toxins.

Morinidazole is a 5-nitroimidazole drug. Its sulfate conjugate M7 was a sensitive substrate of organic anion transporter 1 (OAT1) and OAT3, whereas N+-glucuronides M8-1 and M8-2 were only OAT3 substrates. In chronic renal failure (CRF) patients, plasma exposures of the three conjugates increased by 15-fold, which were also found in 5/6 nephrectomized (5/6 Nx) rats in this study. Although the transcriptions of Oat1 and Oat3 in 5/6 Nx rat kidneys decreased by 50%, no difference was observed on the three conjugate uptakes between control and 5/6 Nx rat kidney slices. Thus, the highly elevated endogenous uremic toxins in 5/6 Nx rats and humans, namely, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippuric acid (HA), and indoxyl sulfate (IS), were considered as influential factors. In rat kidney slices, the uptake of M7, M8-1, and M8-2 was dose dependently reduced by HA and IS, whose plasma concentrations were elevated 5 times in 5/6 Nx rats. In OAT3-overexpressed cells, the three conjugate uptakes were inhibited by CMPF, HA, and IS with IC50 values of 19.2, 87.4, and 222 µM (M7); 8.53, 39.4, and 161 µM (M8-1); and 6.75, 24.1, and 78.3 µM (M8-2), respectively. In OAT1-overexpressed cells, CMPF, HA, and IS showed weak inhibition on M7 uptake with IC50 values of 187, 162, and 200 µM, correspondingly. Results suggest that the reduced mRNA expression of renal transporters in CRF patients may not influence the activities of these transporters. However, accumulated uremic toxins may inhibit the transporters, particularly OAT3, leading to plasma exposure changes of relevant substrates.

Impact of curcumin on the pharmacokinetics of rosuvastatin in rats and dogs based on the conjugated metabolites.

1. Plasma concentrations of curcumin-O-glucuronide (COG) and curcumin-O-sulfate (COS) significantly increased after Sprague-Dawley rats dealt with the Oatp inhibitor rifampicin, with the Cmax ascending 2.9 and 6.7 times, and the AUC0-∞ ascending 4.4 and 10.8 times, respectively. When pretreated with the Oat inhibitor probenecid, the Cmax increased 4.4 and 20 times, and the AUC0-∞ increased 3.2 and 13.9 times, respectively. The results suggested that COG and COS may be the substrates of Oatp and Oat. 2. The accumulation of curcumin significantly increased in organic anion transporting polypeptide (OATP)- and organic anion transporter (OAT)-transfected human embryonic kidney (HEK) 293 systems, which suggested that curcumin was a substrate of OATP1B1, OATP1B3, OATP2B1, OAT1, and OAT3; and COG was a substrate of OATP1B1, OATP1B3, and OAT3. 3. Inhibition study using rosuvastatin as the substrate in OATP1B1- and OATP1B3-transfected cells indicated that curcumin was an OATP1B1 and 1B3 inhibitor, with IC50 at 5.19 ± 0.05 and 3.68 ± 0.05 µM, respectively; the data for COG were 1.04 ± 0.01 and 1.08 ± 0.02 µM, respectively. COS was speculated to be an inhibitor of hepatic OATP1B1 as calculated using the ADMET Predictor. 4. COG and COS are substrates and inhibitors of OATP/Oatp. Co-administration of curcumin significantly increased rosuvastatin concentration in rat and dog plasma.

Fasiglifam (TAK-875) Inhibits Hepatobiliary Transporters: A Possible Factor Contributing to Fasiglifam-Induced Liver Injury.

Fasiglifam (TAK-875), a selective G-protein-coupled receptor 40 agonist, was developed for the treatment of type 2 diabetes mellitus; however, its development was terminated in phase III clinical trials because of liver safety concerns. Our preliminary study indicated that intravenous administration of 100 mg/kg of TAK-875 increased the serum total bile acid concentration by 3 to 4 times and total bilirubin levels by 1.5 to 2.6 times in rats. In the present study, we examined the inhibitory effects of TAK-875 on hepatobiliary transporters to explore the mechanisms underlying its hepatotoxicity. TAK-875 decreased the biliary excretion index and the in vitro biliary clearance of d8-taurocholic acid in sandwich-cultured rat hepatocytes, suggesting that TAK-875 impaired biliary excretion of bile acids, possibly by inhibiting bile salt export pump (Bsep). TAK-875 inhibited the efflux transporter multidrug resistance-associated protein 2 (Mrp2) in rat hepatocytes using 5 (and 6)-carboxy-2',7'-dichlorofluorescein as a substrate. Inhibition of MRP2 was further confirmed by reduced transport of vinblastine in Madin-Darby canine kidney cells overexpressing MRP2 with IC50 values of 2.41 µM. TAK-875 also inhibited the major bile acid uptake transporter Na(+)/taurocholate cotransporting polypeptide (Ntcp), which transports d8-taurocholic acid into rat hepatocytes, with an IC50 value of 10.9 µM. TAK-875 significantly inhibited atorvastatin uptake in organic anion transporter protein (OATP) 1B1 and OATP1B3 cells with IC50 values of 2.28 and 3.98 µM, respectively. These results indicate that TAK-875 inhibited the efflux transporter MRP2/Mrp2 and uptake transporters Ntcp and OATP/Oatp, which may affect bile acid and bilirubin homeostasis, resulting in hyperbilirubinemia and cholestatic hepatotoxicity.

Potential role of organic anion transporting polypeptide 1B1 (OATP1B1) in the selective hepatic uptake of hematoporphyrin monomethyl ether isomers.

AIM: Hematoporphyrin monomethyl ether (HMME), which consists of equal amounts of isomers HMME-1 and HMME-2, is a novel porphyrin-related drug for photodynamic therapy. This study was aimed to investigate the uptake transporter-mediated selective uptake of HMME into the liver and to identify the major uptake transporter isoforms involved. METHODS: Adult SD rats were intravenously injected with a single dose of HMME (5 mg/kg) with or without rifampicin (an inhibitor of organic anion transporting polypeptides OATP1B1 and OATP1B3, 25 mg/kg). Blood samples were collected, and HMME concentrations were measured using LC-MS/MS. Rat hepatocytes, human hepatocytes and HEK293 cells expressing OATP1B1, OATP1B3, or OATP2B1 were used to investigate the uptake of HMME or individual isomers in vitro. RESULTS: Co-administration of rifampicin significantly increased the exposure of HMME isomers, and decreased the AUC ratio of HMME-1 to HMME-2 from 1.98 to 1.56. The uptake of HMME-2 into human hepatocytes and the HEK293 cells expressing OATP1B1 or OATP2B1 in vitro was 2-7 times greater than that of HMME-1, whereas OATP1B3 mediated a higher HMME-1 uptake. OATP1B1 exhibited a higher affinity for HMME-2 than for HMME-1 (the Km values were 0.63 and 5.61 µmol/L, respectively), which were similar to those in human hepatocytes. By using telmisartan (a non-specific OATP inhibitor) and rifampicin, OATP2B1 was demonstrated to account for <20% of hepatic HMME uptake. CONCLUSION: OATP1B1 is the major transporter involved in the rapid hepatic uptake of HMME, and the greater uptake of HMME-2 by OATP1B1 may lead to a lower exposure of HMME-2 than HMME-1 in humans.