bs_bs_banner Research Paper Journal of Pharmacy And Pharmacology Glucuronidation of the broad-spectrum antiviral drug arbidol by UGT isoforms Jin-Hui Songa,*, Zhong-Ze Fangb,e,*, Liang-Liang Zhud, Yun-Feng Caoc, Cui-Min Hue, Guang-Bo Ged and De-Wei Zhaoa aOrthopedics Department, Affiliated Zhongshan Hospital of Dalian University, Dalian, bLiaoning Medical University, Jinzhou, cKey Laboratory of Contraceptives and Devices Research (NPFPC), Shanghai Engineer and Technology Research Center of Reproductive Health Drug and Devices, Shanghai Institute of Planned Parenthood Research, Shanghai, dLaboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China and eLaboratory of Metabolism, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Keywords Abstract arbidol; glucuronidation; UDP-glucuronosyltransferases (UGTs) Objectives The aim of this work was to identify the uridine glucuronosyltrans- ferase (UGT) isoforms involved in the metabolism of the broad-spectrum antivi- Correspondence ral drug arbidol. De-Wei Zhao, Orthopedics Department, Methods A human liver microsome (HLM) incubation system was employed to Affiliated Zhongshan Hospital of Dalian catalyse the formation of arbidol glucuronide. The glucuronidation activity of University, Dalian 116 001, China. E-mail: [email protected] commercially recombinant UGT isoforms towards arbidol was screened. A combi- nation of kinetic analysis and chemical inhibition study was used to determine the Received June 21, 2012 UGT isoforms involved in arbidol’s glucuronidation. Accepted November 14, 2012 Key findings The arbidol glucuronide was detected when arbidol was incubated with HLMs in the presence of UDP-glucuronic acid. The Eadie–Hofstee plot doi: 10.1111/jphp.12014 showed that glucuronidation of arbidol was best fit to the Michaelis–Menten Ϯ *These two authors equally contributed to kinetic model, and Km and apparent Vmax were calculated to be 8.0 0.7 mm and Ϯ this work. 2.03 0.05 nmol/min/mg protein, respectively. Assessment of a panel of recom- binant UGT isoforms revealed that UGT1A1, UGT1A3 and UGT1A9 could cata- lyse the glucuronidation of arbidol. Kinetic analysis and chemical inhibition study demonstrated that UGT1A9 was the predominant UGT isoform involved in arbidol glucuronidation in HLMs. Conclusions The major contribution of UGT1A9 towards arbidol glucuronida- tion was demonstrated in this study. Introduction Arbidol, ethyl-6-bromo-4-[(dimethylamino)-methyl]-5- dose of arbidol, the time to reach maximal plasma concen- hydroxy-1-methyl-2-[(phenylthio) methyl]-indole-3- trations (Tmax) was determined to be 1.2 h. The half-life 1 carboxylate (Figure 1), is an antiviral drug developed by the (t /2) of the drug in the body is 17–21 h and about 40% of Chemical Drug Center of All Russian Research Institute of drug is excreted unchanged.[7] It is metabolized in the liver Pharmaceutical Chemistry together with the Institute of and arbidol glucuronide is a major metabolite in human Medical Radiology.[1] It has been demonstrated to exhibit urine.[8] inhibitory activity towards various types of viruses, such as The human uridine glucuronosyltransferases (UGTs) are influenza virus, respiratory syncytial virus, adenovirus, rhi- membrane proteins of the endoplasmic reticulum and can novirus, parainfluenza virus, hepatitis B virus, hepatitis C conjugate various endogenous substances and exogenous virus and coxsackie virus.[2–6] Therefore, arbidol is regarded compounds.[9] Glucuronidation reactions catalysed by as a broad-spectrum antiviral compound. Recently, arbidol UGTs account for >35% of all phase II drug metabolism.[10] has been approved for the treatment of influenza in Russia, UGT-mediated metabolism is normally regarded as a China and some European countries. detoxifying process, and glucuronides have greater polarity The pharmacokinetic behaviour of arbidol has been than the parent drugs. Nevertheless, some glucuronides reported in a previous publication.[7] After a single 50-mg have been reported to show pharmacological and © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society 2013 Journal of Pharmacy and Pharmacology, 65, pp. 521–527 521 UGT isoforms for arbidol glucuronidation Jin-Hui Song et al. CH3 donors signed informed consents approved by the local ethical committee. Information on the medication history N of the sample donors was not gained. A panel of human O CH3 H3C liver microsomes (HLMs) was prepared from 12 liver O samples obtained from male and female patients by differ- ential ultracentrifugation as described previously.[13,14] HO Microsomal protein concentrations were determined by the Lowry method with bovine serum albumin as standard.[15] A panel of recombinant human UGT supersomes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, N S Br UGT2B4, UGT2B7, UGT2B15 and UGT2B17) expressed in baculovirus-infected insect cells were purchased from BD CH3 Gentest Corp. (Woburn, MA, USA). Figure 1 The structure of arbidol. Arbidol glucuronidation assay [11] toxicological effects. The human UGT superfamily is A typical incubation mixture (200 ml total volume) was classified into two families (UGT1 and UGT2) and three composed of 50 mm Tris-HCl buffer (pH = 7.4), 5 mm subfamilies (UGT1A, UGT2A and UGT2B) based on their MgCl2,5mm UDPGA, 25 mg/ml alamethicin, 10 mm [12] sequence identity. The following UGT enzymes are d-saccharic acid 1,4-lactone, 100 mm arbidol and 0.5 mg/ml expressed in the human liver: UGT1A1, UGT1A3, UGT1A4, HLMs. Arbidol was dissolved in methanol and the final UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, concentration of methanol in the incubation system was UGT2B11, UGT2B15, UGT2B17 and UGT2B28. 1% (v/v). After pre-incubation at 37°C for 5 min, the reac- Identification of drug-metabolizing enzymes (DMEs) is tion was initiated by addition of UDPGA. The reaction very important to understand the variation in drug effect mixture was incubated at 37°C for 1 h and terminated by and metabolism. Moreover, identification of DMEs could addition of 100 ml ice-cold acetonitrile. The mixture was provide pivotal information for potential drug–drug inter- centrifuged at 20 000g for 10 min and a portion of the actions, and the information provided can be used to guide supernatant fraction was transferred to a 0.3-ml auto- clinical practice and reduce side effects in clinical treatment. injector vial for HPLC analysis. Control incubations Therefore, the objective of this experiment was to investi- (without UDPGA, without substrate or without micro- gate the glucuronidation of arbidol using an in-vitro somes) were performed to ensure that the formation of human liver microsomal system. Human UGT isoforms metabolites was microsome- and UDPGA-dependent. The responsible for the formation of metabolites were identified HPLC system (Shimadzu, Kyoto, Japan) contained an SCL- by screening with recombinant expressed UGT isoforms 10A system controller, two LC-10AT pumps, a SIL-10A and chemical inhibition study. To our knowledge, this is the auto-injector, an SPD-10AVP UV detector and a C18 column first study to investigate the UGT isoforms involved in the (4.6 ¥ 150 mm, 5 mm, Kromasil, Sigma-Aldrich) was used glucuronidation of arbidol. to separate arbidol and its metabolites. The mobile phase consisted of CH3CN (A) and H2O containing 0.5% (v/v) Materials and Methods formic acid (B). The following gradient condition was used: 0–15 min, 98–10% B; 15–20 min, 10% B; 20–25 min, 5% B; Materials 25–35 min, 98% B. The flow rate of the mobile phase was Arbidol hydrochloride (purity > 98%) was obtained from set at 1 ml/min. The injection volume was 30 ml and the the National Institute for the Control of Pharmaceutical scan wavelength was set at 316 nm. Due to the absence of and Biological Products (Beijing, People’s Republic of authentic standards for arbidol glucuronide, quantification China). Alamethicin, magnesium chloride (MgCl2), of the glucuronide in the incubation mixtures was per- d-saccharic acid 1,4-lactone, UDP-glucuronic acid triso- formed using a standard curve of arbidol. The calculation dium salt (UDPGA), androsterone, mefenamic acid and curve was generated by peak area of arbidol over the con- erlotinib were purchased from Sigma-Aldrich (St Louis, centration range 0.1–100 mm. The curve was linear over this MO, USA). All other reagents were of HPLC grade or of the concentration range (r2 > 0.99). The limits of detection and highest grade commercially available. quantification were determined at signal-to-noise ratios of Human liver samples were obtained from Dalian Medical 3 and 10, respectively. The accuracy and precision of the University (Dalian, Liaoning province, China) with the back-calculated values for each concentration were less approval of the local ethics committee at the university. All than 5%. © 2012 The Authors. JPP © 2012 522 Royal Pharmaceutical Society 2013 Journal of Pharmacy and Pharmacology, 65, pp. 521–527 Jin-Hui Song et al. UGT isoforms for arbidol glucuronidation Identification of arbidol glucuronide by for 20 min. For recombinant human UGT1A1, UGT1A3 mass/mass analysis and UGT1A9, 0.025 mg/ml of protein and various concen- trations of arbidol were used to determine the kinetic The arbidol glucuronide was separated and collected for parameters. Metabolite formation was linear under assay mass/mass (MS/MS) analysis. MS/MS analysis was carried conditions. The kinetic constants were estimated by nonlin- out on an ABI 4000 Q-Trap hybrid triple quadrupole linear ear regression analysis using the Michaelis–Menten equa- ion-trap mass spectrometer (Applied Biosystems, Foster tion (Equation 1): City, CA, USA). MS conditions used were as follows: ion- spray voltage, 5000; temperature, 550; collision energy, 35; vV=∗(max [ S ]) ( K m + [ S ]) (1) collision energy spread, 15. A collision-induced dissociation (CID) MS/MS spectrum was obtained to further elucidate where v is the rate of reaction, Vmax is the maximum veloc- the structure of metabolite. Data were processed using ity, Km is the Michaelis constant (substrate concentration at Analyst 4.1 software (Applied Biosystems). 0.5 Vmax) and [S] is the substrate concentration.