Evaluation of the Inhibitory Effects of Antihypertensive Drugs on Human Carboxylesterase in Vitro

Drug Metab. Pharmacokinet. 28 (6): 468–474 (2013). Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX) Regular Article Evaluation of the Inhibitory Effects of Antihypertensive Drugs on Human Carboxylesterase In Vitro

† † Xu YANJIAO ,ZhangCHENGLIANG ,LiXIPING,WuTAO,RenXIUHUA and Liu DONG* Department of Pharmacy, Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, PR China

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: Human carboxylesterase (CES) 1A and CES2, two major forms of human CES, dominate the pharmacokinetics of most prodrugs such as imidapril and irinotecan (CPT-11). Antihypertensive drugs are often prescribed for clinical therapy concurrently with others. Moreover, two or more antihypertensive drugs are ubiquitously combined. The inﬂuences of antihypertensive drugs on the activity of CES remain undeﬁned. In the present study, the inhibitory effects of 17 antihypertensive drugs on the CES1A1 and CES2 activities were evaluated. Imidapril and CPT-11 were used as substrates and cultured with liver microsomes in vitro. The imidapril hydrolase activities by recombinant CES1A1 and human liver micro-

somes (HLM) were intensely inhibited by telmisartan and nitrendipine (Ki =0.49« 0.09 and 1.12 « 0.39 µM for CES1A1, 1.69 « 0.17 µM and 1.24 « 0.27 µM for HLM, respectively). However, other drugs did not exert strong inhibition. The enzyme hydrolase activity of recombinant CES2 was substantially inhibited by

diltiazem and verapamil (Ki =0.25« 0.02 and 3.84 « 0.99 µM, respectively). Hence, diltiazem, verapamil, nitrendipine and telmisartan may attenuate the drug efﬁcacy of catalyzed prodrugs by changing the activities of CES1A1 and CES2.

Keywords: carboxylesterase; antihypertensive drugs; enzyme kinetics; inhibition; microsomes; toxicology; prodrugs; drug interaction

mainly catalyzes the hydrolysis of a wide variety of substrates, Introduction especially those with acyl moieties sterically larger than alcohol Human carboxylesterase (CES), a member of the serine groups, such as imidapril and oseltamivir.5,6) In contrast, CES2 can hydrolase superfamily, metabolizes a variety of ester, carbamate, hydrolyze the substrates with small acyl groups, such as CPT-11 thioester and amide drug compounds, environmental toxicants and and heroin.7,8) carcinogens. In humans, the CES1A and CES2 families are the two In multiple drug therapy, drug interactions are important issues major isoforms of CES and they play important roles in drug that must be taken into consideration. Probably, co-administering metabolism. The two isoforms of human CES1A, CES1A1 and several drugs can change the efficacy of each drug per se by CES1A2, share high homology at the mRNA level (99.3%).1) inhibiting drug-metabolizing enzymes. Human CES1A and CES2 Since mature proteins produced from both CES1A1 and CES1A2 govern the pharmacokinetic behaviors of most prodrugs, the mRNA are identical, Hosokawa et al. revealed that in the liver, the activities of which are influenced by the direct interactions. Goel levels of CES1A1 mRNA transcribed from the CES1A1 gene were et al. conducted a clinical trial to identify potential drug-drug substantially higher than those of CES1A2 mRNA transcribed interactions between capecitabine and irinotecan. They exhibited from the CES1A2 gene.2) Therefore, it is plausible that the level in vitro synergistic anti-cancer activity and both are substrates of of CES1A1 mRNA rather than that of CES1A2 mRNA affects CES. The outcome revealed that capecitabine significantly delayed the level of mature protein and enzyme activity.3) CES1A is the conversion of irinotecan to SN-38, suggesting the drug-drug predominantly expressed in the liver and the lung, whereas CES2 interaction at the level of CES.9) Another clinical trial reported that is expressed in the gastrointestinal tract and the liver.4) CES1A a severe mix mania episode occurred in a patient treated with

Received December 11, 2012; Accepted April 22, 2013 J-STAGE Advance Published Date: May 7, 2013, doi:10.2133/dmpk.DMPK-12-RG-143 *To whom correspondence should be addressed: Dong LIU, Ph.D., Department of Pharmacy, Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology, 1095 Jiefang Avenue Wuhan, Hubei 430030, PR China. Tel. ©86-27-8366-3643, Fax. ©86-27-8366-3643, E-mail: [email protected] †These authors contributed equally to this work. This project was supported by a grant from Hubei Natural Science Foundation of China (No. 2011CDB550) and the National Natural Science Foundation of China (No. 81301953).

468 CESs Inhibition by Antihypertensive Drugs In Vitro 469 the combined aripiprazole and dl-threo-methylphenidate (MPH), incubation mixture was determined. The variability and the relative and MPH was mainly metabolized by the human CES1 enzyme. recovery rate were suitable for inhibitory study. However, the underlying mechanism was unclear.10) Zhu et al. SN-38 formation: SN-38 formation from CPT-11 was revealed the inhibitory effect of antipsychotics on the metabolism determined by slightly modifying the methods described pre- of MPH in vitro, and found that aripiprazole, thioridazine and viously.14,15) A typical incubation mixture (total volume of 200 µl) fluoxetine were the most potent inhibitors of CES1.11) Moreover, contained the microsomes, 100 mM phosphate buffer (pH 7.4) and Fukami et al. examined the inhibitory effects of various anti- inhibitors. After 2 min of preincubation at 37°C, the reaction was diabetic and antihyperlipidemic drugs on human CES enzyme initiated by the addition of CPT-11 (5 µM), and then the mixture activity, and found simvastatin (Ki, 0.11 µM), troglitazone (Ki, was incubated at 37°C for 10 min. The reaction was terminated by 0.62 µM) and fenofibrate (Ki, 0.04 µM) inhibited CES1A1 and adding 200 µl of ice-cold methanol. Camptothecine was added as CES2 enzyme activities.12) It is well known that patients suffering the internal standard. After 10 min of centrifugation at 15,000©g, from hypertension disease may be prone to heart failure, hyper- the supernatant (10 µl) was determined by high performance liquid lipidemia or diabetes, and such patients are concurrently treated chromatography. CPT-11 and SN-38 were separated on a Hypersil with multiple medications including antihypertensive, antihyper- ODS C18 (4.6 mm © 250 mm, 5 µm) column utilizing the mobile lipidemic or antidiabetic drugs. Furthermore, two or more kinds phase of 0.05 mol0L¹1 phosphate-methanol (50:50, v/v) contain- of antihypertensive drugs are frequently co-prescribed in clinic ing 0.25‰ triethylamine (adjusted to pH 3.0 with phosphate),16) practice, among which angiotensin-converting enzyme (ACE) and the chromatograms were recorded by being monitored at inhibitors that are mostly hydrolyzed by CES1A dominate the the excitation and emission wavelengths of 380 nm and 550 nm, combined drug regimen. Inhibiting the CES enzyme activity by respectively. The linearity of the standard curve of SN-38 was coadministered drugs may impair the effectiveness of pharmaco- confirmed (r = 0.9992), within which the concentration in the therapy or even give rise to toxicity. Nevertheless, there have been incubation were determined. The variability and relative recovery hardly any systematic studies assessing the inhibitory potential for rate were suitable for inhibitory study. CES enzyme activity by antihypertensive drugs in humans per- p-Nitrophenyl acetate hydrolase activity: The p-nitrophenyl formed or published. Therefore, we herein investigate the inhibi- acetate hydrolase activity was determined according to the methods tory effects of different kinds of antihypertensive drugs on the described previously.9,17) activities of CES1A and CES2 in vitro. Inhibition analysis of CES enzyme activities: The inhibitory effects of 17 drugs on the imidapril and CPT-11 hydrolase activities Materials and Methods were investigated. Hydrochlorothiazide, losartan, telmisartan, Materials: Imidapril and imidaprilat were purchased from candesartan, nifedipine, felodipine, nicardipine, lacidipine and Sigma (St. Louis, MO). CPT-11, SN-38, p-nitrophenyl acetate and cilnidipine were dissolved in DMSO. Propranolol, atenolol, meto- p-nitrophenol were purchased from Toronto Research Chemicals prolol, timolol, amlodipine, nitrendipine, diltiazem and verapamil Inc. (North York, ON, Canada). Cetirizine (purity >99.7%) and were dissolved in distilled water. These drugs were added to the camptothecine were purchased from National Institute for the incubation mixtures described above to investigate their inhibitory Control of Pharmaceutical and Biological Product (Beijing, China). effects on the imidaprilat and SN-38 formations. The final con- Hydrochlorothiazide, losartan, telmisartan, candesartan, nifedipine, centration of DMSO in the incubation mixture was <0.1%. All amlodipine, felodipine, nitrendipine, nicardipine, diltiazem, vera- data were analyzed using the mean « SD. pamil, lacidipine, cilnidipine, propranolol, atenolol, metoprolol To screen of the inhibitory effects, the enzyme activities at and timolol were purchased from Sigma-Aldrich (St. Louis, MO). 100 µM imidapril and 5 µM CPT-11 were examined in the presence 18) Pooled human liver microsomes (HLM) were purchased from of the 17 drugs (200 µM). For determination of the Ki (inhibi- BD Gentest (Woburn, MA). Pooled human jejunum microsomes tion constant) values of hydrolase activity, the concentrations of (HJM) were purchased from Tissue Transformation Technologies imidapril ranged between 25 and 200 µM. The concentrations of (Edison, NJ). Human CES1A1 and human CES2 expressed in the inhibitors for the imidapril hydrolase activity test ranged as Baculovirus-insect cells were obtained from BD Gentest. All other follows: telmisartan, 0.2 to 2 µM and 1 to 3 µM for recombinant chemicals and solvents were of analytical or HPLC grade. CES1A1 and HLM, respectively; nitrendipine, 0.5 to 5 µM and Imidaprilat formation: Imidaprilat formation from imidapril 0.5 to 10 µM, respectively. The protein concentrations of HLM was determined according to the method described previously with and CES1A1 were 0.15 and 0.42 mg/ml, respectively. 13) a slight modification. A typical incubation mixture (total volume For determination of the Ki values, the concentrations of CPT-11 of 200 µl) contained the microsomes, 100 mM Tris-HCl buffer ranged from 2 to 20 µM for recombinant CES2 and 5 to 50 (pH 7.4) and inhibitors. After 2 min of preincubation at 37°C, the µM for HLM and HJM, respectively. The concentrations of the reaction was initiated by adding imidapril (100 µM), and then the inhibitors for the CPT-11 hydrolase activity test ranged as follows: mixture was incubated at 37°C for 15 min. The reaction was termi- diltiazem, 0.2 to 2 µM for recombinant CES2, 2 to 15 µM for HLM nated by adding 200 µl of ice-cold ethyl acetate and the mixture was and 1 to 10 µM for HJM; and verapamil, 0.5 to 10 µM for recom- next dried by N2. Cetirizine was added as the internal standard. binant CES2, 0.5 to 20 µM for HLM and 5 to 30 µM for HJM. After centrifuging the solution at 15,000©g for 5 min, imidaprilat in The protein concentrations of recombinant CES2, HLM and HJM the supernatant (10 µl) was determined by a liquid chromatography- were 0.38, 0.06 and 0.14 mg/ml, respectively. tandem mass spectrometry system. A Diamonsil C18 column (5 µm, To compare the inhibitory effects, the enzyme activities at 150 © 2.1 mm) was used as the analytical column with acetonitrile- 150 µM p-nitrophenyl acetate were examined in the presence of 0.1% (v/v) formic acid (1:2, v/v) as the mobile phase at the flow the 17 drugs (200 µM).12) To determine the inhibitor concentra- rate of 0.3 ml/min. The standard curve of imidaprilat was verified tion that caused 50% inhibition (IC50), the p-nitrophenyl acetate to be linear (r = 0.9987), within which the concentration in the hydrolase activities by recombinant CES1A1 and CES2 at 150 µM

Copyright © 2013 by the Japanese Society for the Study of Xenobiotics (JSSX) 470 Xu YANJIAO, et al. were examined in the presence of the inhibitors. The concen- (0.1%) inhibited the imidapril hydrolase activity by CES1A1 by trations of the inhibitors ranged as follows: telmisartan, 0.2–5 µM, 1.2%. The inhibition rates of the drugs dissolved in DMSO were nitrendipine, 0.5–30 µM, diltiazem, 0.5–20 µM, and verapamil, calculated as the percentages of the tested activity to the control 1–50 µM. one after eliminating the impact of 0.1% DMSO. The imidaprilat The Ki,Km and Vmax values and inhibition types were deter- formation catalyzed by CES1A1 was strongly inhibited by mined by ﬁtting the kinetic data to a competitive, noncompetitive, telmisartan (percentage of the control: 0.8%), nitrendipine uncompetitive or mixed inhibition model by nonlinear regression (1.3%), diltiazem (12%) and felodipine (16%). The activity of analysis using GraphPad Prism 5 (GraphPad Software Inc., recombinant CES1A1 was moderately inhibited by losartan and San Diego, CA). The Ki,Km and Vmax values were expressed propranolol (20–50% of the control). The rest inhibited less than as the mean « SD. IC50 values were determined by plotting the 50% of the imidapril hydrolysis activity. percentage of experimental activity to the control one versus log Inhibitory effects of 17 drugs on CPT-11 hydrolase activities concentration, and ﬁtting the data to a sigmoidal dose-response of recombinant CES2: To investigate the inhibitory effects of equation. the drugs on human CES2 enzyme activity, the CPT-11 hydrolase activity that was suppressed by 0.1% DMSO by 0.8% was Results evaluated (Fig. 1B). In case of being dissolved in DMSO, the Inhibitory effects of 17 drugs on imidapril hydrolase inhibition rates of the drugs were calculated as the percentages of activities of recombinant human CES1A1: The inhibitory the experimental activity to the control one. The CPT-11 hydrolase effects of the 17 drugs on the imidapril hydrolase activity of activity of recombinant CES2 was dramatically inhibited by diltia- human recombinant CES1A1 were investigated (Fig. 1A). DMSO zem (percentage of control: 4.2%) and verapamil (7%), and moderately inhibited by timolol and cilnidipine (20–50% of the control). The others exhibited weak inhibition (>50% of the control). Inhibition constant and inhibition patterns of imidapril hydrolase activities of recombinant human CES1A1 and HLM: The Ki values and inhibition patterns of telmisartan and nitrendipine that tremendously inhibited the imidapril hydrolase activities of recombinant CES1A1 and HLM were determined, and the representative Lineweaver-Burk plots are illustrated in Figure 2. The Ki values of telmisartan and nitrendipine for recombinant CES1A1 were 0.49 « 0.09 and 1.12 « 0.39 µM, respectively, with competitive- and mixed-type inhibition. In contrast, the Ki values of telmisartan and nitrendipine for HLM were 1.69 « 0.17 and 1.24 « 0.27 µM with noncompetitive- and competitive-type inhibition, respectively. Inhibition constant and inhibition patterns of CPT-11 hydrolase activities of recombinant human CES2, HLM, and HJM: The Ki values and inhibition patterns of diltiazem and verapamil evidently inhibiting the CPT-11 hydrolase activities by recombinant CES2, HLM and HJM were determined, and the representative Lineweaver-Burk plots are shown in Figure 3. The Ki values of diltiazem and verapamil for recombinant CES2 were 0.25 « 0.02 and 3.84 « 0.99 µM with noncompetitive- and competitive-type inhibition, respectively. The Ki values of diltiazem and verapamil for HLM were 2.89 « 0.39 and 11.54 « 1.20 µM with noncompetitive-type inhibition. The Ki values of diltiazem and verapamil for HJM were 4.67 « 2.12 and 15.75 « 2.63 µM with mixed- and noncompetitive-type inhibition, respectively. Inhibitory effects of 17 drugs on p-nitrophenyl acetate hydrolase activities of recombinant human CES1A1 and CES2: To compare the inhibitory effects of the drugs on human CES1A1 and CES2 enzyme activities, the activities of the p- nitrophenyl acetate hydrolase catalyzed by both CES1A1 and CES2 were assessed (Fig. 4). DMSO (0.1%) inhibited the p- nitrophenyl acetate hydrolase activity by 0.5%. If the drugs were dissolved in DMSO, the inhibition rates were calculated as the Fig. 1. Inhibitory effects of 17 antihypertensive drugs on imidapril percentage of the tested activity to the control one. Telmisartan hydrolase activity of recombinant CES1A1 (A) and on CPT-11 hydrolase and nitrendipine remarkably inhibited (percentage of control: 1.8% activity of recombinant CES2 (B) and 3%, respectively) the p-nitrophenyl acetate hydrolase activity The concentrations of imidapril and CPT-11 were 100 µM and 5 µM, and the concentrations of the 17 drugs were 200 µM. Each column represents the of CES1A1, but mildly inhibited that of CES2 (higher than 50% mean « SD. The control activity was 1.45 nmol/min/mg and 12.5 pmol/min/mg of the control). Diltiazem substantially suppressed (21% and 1.6% for CES1A1 and CES2, respectively. of the control, respectively) the activities of CES1A1 and CES2.

Fig. 2. Inhibitory effects of telmisartan (A and C) and nitrendipine (B and D) on imidapril hydrolase activities of recombinant CES1A1 (A and B) and HLM (C and D) Each data point represents the mean « SD. The Ki value represents the mean « SE.

However, verapamil inhibited their activities differently (5% and determine the drug efficacy eventually. Although antihypertensive 74% of the control, respectively). In addition, nifedipine and drugs and other drugs such as antihyperlipidemic and antidiabetic felodipine led to modest inhibitions, but the rest performed weakly drugs have been extensively co-prescribed for clinical therapy, the and diversely. profiles of the drug-drug interaction between antihypertensive IC50 value of p-nitrophenyl acetate hydrolase activities of drugs and other combined therapeutic drugs have not been fully recombinant human CES1A1 and CES2: Telmisartan and established. In particular, the influences of the hypertension nitrendipine intensely inhibited the p-nitrophenyl acetate hydrolase therapy medications on human CES remain essentially unexplored. activity of recombinant CES1A1 (Fig. 4A), while diltiazem In the present study, we investigated the inhibitory effects of and verapamil apparently inhibited that of recombinant CES2 antihypertensive drugs on both CES1A1 and CES2 utilizing (Fig. 4B). To further compare the inhibitory effects of telmisartan, imidapril and CPT-11 as the representative substrates. nitrendipine, diltiazem and verapamil on the CES1A1 and CES2 In the imidaprilat formation from imidapril catalyzed by enzyme activities, the IC50 values of p-nitrophenyl acetate hydro- CES1, telmisartan and nitrendipine strongly inhibited both HLM lase activity were determined (Fig. 5) based on 150 µM substrate. and recombinant CES1A1, and the Ki value of telmisartan for The IC50 values of telmisartan and nitrendipine for recombinant recombinant CES1A1 (0.49 « 0.09 µM) was lower than that of CES1A1 were 0.89 and 3.7 µM, respectively, while those of nitrendipine (1.12 « 0.39 µM). Furthermore, telmisartan compet- diltiazem and verapamil were 18.8 and 20.1 µM. Besides, the IC50 itively inhibited recombinant CES1A1, suggesting that telmisartan value of telmisartan and nitrendipine for recombinant CES2 were may bind to the active site of CES1A1. The results may partially be >5 µM and >30 µM, respectively. The IC50 values of diltiazem attributed to the CES expression pattern and the plastic nature of and verapamil for recombinant CES2 were 3.98 and 7.94 µM, the active site that accommodates variously structured substrates.19) respectively. Thus, the activities of CES1A1 and CES2 were SN-38 formation from CPT-11 was inhibited evidently by differently inhibited by these drugs. diltiazem and verapamil, which are calcium channel blocks (CCBs) prescribed simultaneously with ACE inhibitors for hypertension Discussion therapy. In this study, we investigated the inhibitory effects Drug interactions may lead to undesirable side effects. of diltiazem and verapamil on HLM, HJM and recombinant Alterations in drug-metabolizing enzymes in particular inhibitions CES2. The Ki values of diltiazem for HLM, HJM and recombinant are recognized as a prevalent factor, so it is necessary to understand CES2 were all lower than those of verapamil, and the Ki value and clarify the inhibitory effects. Human CES1A and CES2, which of verapamil for recombinant CES2 was 15-fold higher than account for the biotransformation of both endogenous and exoge- that of diltiazem. Chemically, CCBs are classified into three nous compounds into polar products to be facilely eliminated, classes, benzothiazepines (e.g., diltiazem), dihydropyridines (e.g.,

Fig. 3. Inhibitory effects of diltiazem (A, C and E) and verapamil (B, D and F) on imidapril hydrolase activities of recombinant CES2 (A and B), HLM (C and D) and HJM (E and F) Each data point represents the mean « SD. The Ki value represents the mean « SE.

Fig. 4. Inhibitory effects of 17 antihypertensive drugs on p-nitrophenyl acetate hydrolase activities of recombinant CES1A1 (A) and CES2 (B) The activities were determined at 150 µM p-nitrophenyl acetate, and the concentrations of the 17 drugs were 200 µM. Each data point represents the mean « SD. The control activities by recombinant CES1A1 and CES2 were 3.25 nmol/min/mg and 38.9 pmol/min/mg, respectively.

Fig. 5. IC50 values of telmisartan (A), nitrendipine (B), diltiazem (C) and verapamil (D) on p-nitrophenyl acetate hydrolase activities of recombinant CES1A1 and CES2 The activities were determined at 150 µM p-nitrophenyl acetate. Each data point represents the mean « SD. The control activities by recombinant CES1A1 and CES2 were 3.88 nmol/min/mg and 42.4 pmol/min/mg, respectively.

nifedipine) and phenylalkylamines (e.g., verapamil).20) Metabolic In this study, we found that telmisartan, nitrendipine, diltiazem intermediate (MI) complexing agents, such as diltiazem, com- and verapamil exhibited potent inhibitory effect on human CES monly contain an amine functional group and allow N-deal- in vitro. However, the corresponding results in vivo are still kylation. However, the formation mechanisms concerning such unrevealed because the drugs undergo sophisticated metabolism complexes on the basis of benzothiazepines and phenylalkylamines and may be susceptible to many physiological factors. Fukami have never been reported hitherto.21,22) The differently structured et al. reported that simvastatin and lovastatin strongly inhibited the diltiazem and verapamil functioned distinctly. In the meantime, CES1A1 enzyme activity in vitro.12) However, in contrast to that the Ki values of diltiazem and verapamil for HLM, HJM and in vitro study, simvastatin and lovastatin may not affect CES1A1 recombinant CES2 also differed. Firstly, more CES1 mRNAs hydrolase activity in vivo in humans. Thus, the inhibitory effects of are expressed in human liver than those in the small intestine.4) drugs on human CES in vivo as well as the relevant mechanisms In contrast, human CES2 is abundant in the small intestine.23) still need to be evaluated comprehensively. Secondly, recombinant CES2 is subject to post-translational In addition to its major role in the hydrolysis of numerous modifications specifically in insect cells, which somewhat affects therapeutically active compounds, CES1 has been reported to be the interactions between the protein and substrates/inhibitors. As one of the key enzymes responsible for the biotransformation of a result, the differences between CES2 expressions in human liver a number of pyrethroid organophosphate and carbamate insecti- and intestine and recombinant factor may contribute to distinct cides.24,25) CES1 activity is crucial to the detoxification process of inhibition behaviors against HLM, HJM and recombinant CES2. these increasingly used insecticides. Hence, patients administered To compare the inhibitory effects of telmisartan, nitrendipine, with CES1 inhibitors may be prone to suffering from toxic reac- diltiazem and verapamil on human CES1A1 and CES2 enzyme tions after being exposed to those insecticides. activities, the IC50 values of the p-nitrophenyl acetate hydrolase In conclusion, we evaluated the inhibitory effect of antihyper- activity were evaluated (Fig. 5). Telmisartan and nitrendipine tensive agents on human CES, and found that telmisartan and inhibited CES1A1 more effectively than CES2. In contrast, diltia- nitrendipine dramatically inhibited both HLM and recombinant zem and verapamil inhibited CES2 more potently than CES1. CES1A1. Besides, the CES2 enzyme activity was strongly inhib- Moreover, telmisartan and diltiazem showed strongest inhibition ited by CCBs such as diltiazem and verapamil belonging to on CES1A1 (IC50 = 0.89 µM) and CES2 (IC50 = 3.98 µM), respec- the benzothiazepine and phenylalkylamine class, respectively. tively. Collectively, the different inhibitions may be ascribed to the This study provides theoretical insights for predicting drug-drug structural differences between drugs and enzymes. interactions.

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