Epicatechin gallate and epigallocatechin gallate are potent inhibitors of human arylacetamide deacetylase

Kaori Yasuda a, *, Kazuki Watanabe b, Tatsuki Fukami c, d, Shimon Nakashima c, Shin-ichi Ikushiro b, Miki Nakajima c, d, Toshiyuki Sakaki a
a Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
b Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan
c Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University Kakuma-machi, Kanazawa 920-1192, Japan
d WPI Nano Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan


Recently, in addition to carboxylesterases (CESs), we found that arylacetamide deacetylase (AADAC) plays an important role in the metabolism of some clinical drugs. In this study, we screened for food-related natural compounds that could specifically inhibit human AADAC, CES1, or CES2. AADAC, CES1, and CES2 activities in human liver microsomes were measured using phenacetin, fenofibrate, and procaine as specific substrates, respectively. In total, 43 natural compounds were screened for their inhibitory effects on each of these enzymes. Curcumin and quercetin showed strong inhibitory effects against all three enzymes, whereas epicatechin, epicatechin gallate (ECg), and epigallocatechin gallate (EGCg) specifically inhibited AADAC. In particular, ECg and EGCg showed strong inhibitory effects on AADAC (IC50 values: 3.0 ± 0.5 and 2.2 ± 0.2 mM, respectively). ECg and EGCg also strongly inhibited AADAC-mediated rifampicin hydrolase activity in human liver microsomes with IC50 values of 2.2 ± 1.4 and 1.7 ± 0.4 mM, respectively, whereas it weakly inhibited p-nitrophenyl acetate hydrolase activity, which is catalyzed by AADAC, CES1, and CES2. Our results indicate that ECg and EGCg are potent inhibitors of AADAC.

Arylacetamide deacetylase Carboxylesterase Catechins
Drug hydrolysis Epigallocatechin gallate Flavonoids

1. Introduction

Drug-metabolizing enzymes play essential roles in the detoxi- fication, elimination, and pharmacological activation of drugs. Hy- drolases contribute to the metabolism of 10% of current clinical drugs that contain ester, amide, or thioester bonds [1], among which carboxylesterases (CES) are well-known enzymes involved in drug metabolism. In humans, CES enzymes are classified into five subfamilies, CES1, CES2, CES3, CES4A, and CES5A, with CES1 and CES2 being widely studied since they are largely responsible for the metabolism of drugs in the liver [1]. Recently, we found that ary- lacetamide deacetylase (AADAC), which is also expressed in the liver, also plays an important role in the hydrolysis of some clinical drugs such as ketoconazole, phenacetin, and rifamycins [2e5].
It is important to understand drug-drug or drug-food in- teractions (DIs) as they can cause unexpected side effects by increasing or decreasing drug levels, thereby affecting their phar- macodynamic or toxicological effects. One of the most well-known examples of a compound responsible for a drug-food interaction is bergamottin, which is found in grapefruit juice and can cause serious side effects for some drugs due to its ability to inhibit CYP3A4 [6,7]. DIs for CES1 and CES2 have been widely studied in vitro. Nonetheless, no known CES-mediated DIs have been clin- ically documented [8], although several interactions with ethanol were reported [9,10]. The formation of reactive metabolites as a result of drug metabolism is also known to cause side effects, with P450s receiving great attention. Recently, we demonstrated that drug metabolism by CES1, CES2, and AADAC can cause side effects as a result of the production of reactive metabolites [5,11e14]. For example, the metabolic reactions catalyzed by CES1 and/or CES2 are involved in prilocaine- and lidocaine-induced methemoglobi- nemia [12], whereas the hydrolysis of phenacetin and ketoconazole by AADAC has been linked with methemoglobinemia and hepato- toxicity, respectively [5,14]. If compounds specifically inhibit CES1, CES2, or AADAC, they could possibly suppress the production of toxic reactive metabolites and reduce the side effects related to drugs.
CES1 plays important roles in triacylglycerol and cholesteryl ester metabolism. In particular, CES1 levels were found to be elevated in obese individuals and in patients with type 2 diabetes [15,16]. Moreover, CES1 inhibitors were shown to have beneficial effects on lipid and glucose metabolism; therefore, CES1 inhibitors are being developed as treatments for hypertriglyceridemia, obesity, and type 2 diabetes [15]. In turn, CES2 inhibitors are being developed to treat irinotecan-induced diarrhea, which is caused by a hydrolytic metabolite of irinotecan [17,18]. Furthermore, both CES1 and CES2 inhibitors are being developed for the purpose of modulating the oral bioavailability of drugs and for alleviating drug toxicity. Previously, we screened 542 chemicals for their ability to inhibit CESs and AADAC [19]. We found that digitonin and telmi- sartan specifically inhibit CES1 and CES2 activity, respectively, whereas vinblastine inhibits both CES2 and AADAC activities. However, to date, no specific inhibitor for AADAC has been identified.
As mentioned, specific inhibitors for AADAC, CES1, or CES2 would be promising agents for suppressing the production of toxic reactive metabolites and reducing drug side effects. In this study, we screened for natural compounds that could specifically inhibit each of these enzymes.

2. Materials and methods

2.1. Materials

Human liver microsomes (HLM, 150 donor-pool) were pur- chased from Corning (Corning, NY, USA). Phenacetin, p-nitro- phenol, and p-aminobenzoic acid were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Fenofibric acid, rifampicin, and 25-desacetylrifampicin were purchased from Toronto Research Chemicals (Toronto, Canada). Fenofibrate, procaine hydrochloride, p-nitrophenyl acetate (PNPA), and p-phenetidine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The suppliers of the 43 compounds tested as inhibitors are shown in Supplemental Table 1. All other chemicals were of analytical grade or the highest quality commercially available.

2.2. Phenacetin, fenofibrate, and procaine hydrolase activities in HLM

Phenacetin, fenofibrate, and procaine were used as specific substrates for AADAC, CES1, and CES2, respectively [3,20]. The hy- drolase activity of each enzyme was measured as described previ- ously with some modifications [3,19,20]. The substrate concentration for assay was determined based on the Km values for hydrolase activities of phenacetin, fenofibrate, and procaine in HLM, 3.3 mM, 4.1 mM, and 0.8 mM, respectively [3,20]. Briefly, to measure the phenacetin hydrolase activity, the reaction mixture containing 100 mM phosphate buffer (pH 7.4), 0.4 mg/mL HLM, and 1 mM phenacetin was incubated for 20 min at 37 ◦C. The reaction was stopped by adding 3% perchloric acid (final concentration). To measure the fenofibrate hydrolase activity, the reaction mixture containing 100 mM phosphate buffer (pH 7.4), 0.025 mg/mL HLM, and 10 mM fenofibrate was incubated for 30 s at 37 ◦C. The reaction was stopped by adding an equal volume of acetonitrile to the re- action mixture. To measure the procaine hydrolase activity, the reaction mixture containing 100 mM phosphate buffer (pH 7.4), 0.8 mg/mL HLM, and 1 mM procaine was incubated for 30 min at 37 ◦C. The reaction was stopped by adding an equal volume of methanol. After the termination of each reaction, each solution was centrifuged at 20,000×g for 10 min, and the supernatant was analyzed by high performance liquid chromatography (HPLC) as described below.

2.3. HPLC analysis of metabolites

HPLC was performed on a CAPCELL PACK C18UG120 (5 mm particle size, 4.6 mm i.d 250 mm; Nippon Soda Co., Tokyo, Japan). The mobile phase was 30% acetonitrile containing 25 mM potas- sium dihydrogen phosphate, 60% acetonitrile containing 20 mM citric acid (pH 3.9), and 20% methanol containing 20 mM ammo- nium acetate (pH 4.0), for measuring phenacetin, fenofibrate, and procaine hydrolase activities, respectively. The flow rate was 1.0 mL/min, and the column temperature was maintained at 40 ◦C. The elution was detected by measuring absorption at 232, 287, and 280 nm for detection of p-phenetidine, fenofibric acid, and p- aminobenzoic acid, respectively, which are the hydrolyzed me- tabolites of phenacetin, fenofibrate, and procaine.

2.4. Inhibition studies of each hydrolase activity

For screening the compounds inhibiting either AADAC, CES1, or CES2, each of the 43 natural compounds were added to the above described reaction mixture at a final concentration of 100 mM in 1% of DMSO. As control sample, DMSO was added to the reaction mixture at 1% of final concentration.
For the determination of IC50 values of EC, ECg, and EGCg toward phenacetin hydrolase activities, the reactions were performed in the same conditions as described above using the compounds at concentrations ranging from 1 to 100 mM. IC50 values were calcu- lated using the following equation: IC50 10[log(A/B) (50 C)/ (D C) log(B)] where A and B are the higher and lower con- centrations near 50% inhibition, respectively, and C and D are the inhibition percentages at B and A concentrations.
For the determination of Ki values of EC, ECg, and EGCg toward the phenacetin hydrolase activity, phenacetin was added in con- centrations ranging from 0.5 to 4 mM, and the inhibitors were added in concentrations ranging from 25 to 100 mM for EC, and from 1 to 25 mM for ECg and EGCg. Ki values were calculated by the Dixon plot. The goodness-of-fit to each of the four inhibition equations, competitive, noncompetitive, uncompetitive or mixed- inhibiton, was determined by comparison of Akaike Information Criterion values using SigmaPlot software (Systat Software, Inc. CA, USA).

2.5. Inhibitory effect of EGCg on PNPA and rifampicin hydrolase activities in HLM

The PNPA hydrolase activity was measured according to a method reported previously [2] with slight modifications (the concentration of HLM: 0.1 mg/mL; substrate concentration: 100 mM; incubation time: 1 min). Rifampicin hydrolase activity at substrate concentrations of 100 mM was measured according to a method reported previously [4]. Concentration of substrate for assay was determined based on the Km values for hydrolase activ- ities of PNPA and rifampicin in HLM, 195 and 76 mM, respectively [4]. IC50 values of ECg and EGCg in HLM were determined as described above.

3. Results

3.1. Screening for potential inhibitors of AADAC, CES1, or CES2

The inhibitory effects of 43 types of natural compounds on the hydrolysis of phenacetin, fenofibrate, and procaine (catalyzed by AADAC, CES1, and CES2, respectively) in HLM were evaluated. Fig. 1 shows the remaining activities in the presence of 100 mM of the various natural products tested. Each compound showed different degrees of inhibition for each hydrolase. Among all natural prod- ucts assessed, quercetin and curcumin potently inhibited all three enzymes. The activities of AADAC, CES1, and CES2 were reduced to 29%, 22%, and 30% of the control levels, respectively, in the presence of 100 mM quercetin, and to 26%, 33%, and 13% of the control levels in the presence of 100 mM curcumin. The IC50 values for quercetin inhibition of CES1 activity and for curcumin inhibition of CES2 ac- tivity were 34.0 ± 5.2 and 7.6 ± 0.3 mM, respectively (values are the mean ± standard deviation of triplicate determinations; data not shown), suggesting that they were less potent inhibitors when compared to previously reported flavonoids. Hesperetin inhibited both AADAC and CES2 activity to 20% and 16% of the control levels, whereas it barely inhibited CES1 (Fig. 1). It should be noted that the epicatechin analogs ECg and EGCg potently and specifically inhibited AADAC to 18% and 16% of the control levels, respectively, whereas EC moderately and specifically inhibited AADAC to 41% of the control level. Interestingly, catechin and EGC did not inhibit any of the three hydrolases including AADAC (Fig. 1). Supplemental Fig. 1 shows the chemical structures of catechin and its analogs used in this study. Our results suggest that a cis configuration at the C2 and C3 positions and a galloyl group at the C3 position might be critical for the inhibitory activity.

3.2. Inhibitory effects of EC, ECg, and EGCg on AADAC, CES1, or CES2

The inhibitory effects of EC, ECg, and EGCg on AADAC, CES1, or CES2 were further evaluated to clarify their specificity for AADAC inhibition. All three compounds failed to inhibit CES1 or CES2 at any of the concentrations tested, whereas they strongly inhibited AADAC (Fig. 2). The IC50 values of EC, ECg, and EGCg for phenacetin hydrolase activities were 65.6 ± 3.3, 3.0 ± 0.5, and 2.2 ± 0.2 mM, respectively, further demonstrating that ECg and EGCg strongly and specifically inhibit AADAC. The respective Ki values of EC, ECg, and EGCg were 57 ± 12, 5.4 ± 0.7, and 3.5 ± 0.7 mM, exhibiting competitive inhibition (Fig. 3).

3.3. Inhibitory effects of ECg and EGCg on PNPA and rifampicin hydrolase activities in HLM

The inhibitory effects of ECg and EGCg on AADAC activity in HLM were further evaluated using PNPA and rifampicin. PNPA is hy- drolyzed by all three hydrolases [2], whereas rifampicin is specif- ically hydrolyzed by AADAC [4]. PNPA hydrolase activity in HLM was weakly inhibited by ECg and EGCg (Fig. 4). This is likely because PNPA is not only hydrolyzed by AADAC but also by CES1 and CES2. The more potent inhibition by ECg rather than EGCg at high con- centrations of catechins might be due to the difference in their inhibition potencies toward CES2 (Fig. 2), which shows efficient PNPA hydrolase activity [21]. The IC50 value of ECg and EGCg for rifampicin hydrolase activity in HLM was 2.2 ± 1.4 and 1.7 ± 0.4 mM, respectively, demonstrating that ECg and EGCg potently inhibit AADAC regardless of the type of substrate used.

4. Discussion

This study aimed to search for natural compounds that specif- ically inhibit the drug-metabolizing hydrolases AADAC, CES1, and CES2. CES1 and CES2 inhibitors, such as flavonoids, have been extensively explored, but there are few studies describing AADAC inhibitors. We have previously searched for inhibitors of AADAC, CES1, or CES2, but we were unable to identify specific inhibitors of AADAC. In the current study, several experiments confirmed that ECg and EGCg potently and specifically inhibit AADAC, whereas CES1 and CES2 specific inhibitors were not identified. To our knowledge, this is the first report identifying compounds that can specifically inhibit human AADAC.
The inhibition of drug-metabolizing enzymes is often undesir- able as it can result in unexpected side effects by increasing the concentration of drugs in the blood to levels greater than needed for efficacy. However, in recent years, the inhibition of drug- metabolizing enzymes has been explored as an alternative way to improve the kinetics of poorly bioavailable drugs [22]. The inhibi- tion of AADAC, CES1, or CES2 by natural compounds could also help reduce potential side effects caused by their reactive metabolites.
Herein, all 43 natural compounds investigated showed various degrees of inhibition against AADAC, CES1, and CES2. Of note, curcumin and quercetin inhibited all three hydrolases (Fig. 1). Previous studies reported that some flavonoids have strong inhibitory effects on CES1 and CES2. In particular, the Ki values of bavachinin and corylin for CES1 activity are 0.5 and 0.7 mM, respectively, and that of corylifol A, which is found in Fructus Psoraleae, for CES2 activity is 0.62 mM [23,24]. Compared to those of these two compounds, the inhibitory effects of curcumin and quercetin towards CES1 and CES2 in the current study were not as strong. Furthermore, it is known that both curcumin and quer- cetin are potent inhibitors of drug-metabolizing P450. Specifically, it was shown that curcumin inhibits CYP2C9 and the CYP3A4- dependent metabolism of 7-benzyloxymethyloxy-3- cyanocoumarin to 3-cyano-7-hydroxycoumarin with IC50 values of 12 and 15 mM, respectively [25]. Moreover, quercetin can inhibit CYP1A2-mediated phenacetin O-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated S-mephenytoin 40- hydroxylation, and CYP3A4-mediated midazolam 10-hydroxyl- ation with IC50 values of 0.9, 1.7, 1.8, and 4.3 mM, respectively [26].
It is also known that the plasma concentration of a caffeine- derived CYP1A2 metabolite, 1,7-dimethylxanthine, is reduced by 10% when humans take a 100 mg caffeine capsule after 13 days of taking a 500 mg quercetin capsule once per day [27]. Because both curcumin and quercetin had higher IC50 values for AADAC, CES1, and CES2 than for P450s, it is unlikely that these compounds affect the pharmacokinetics of substrates of AADAC, CES1, CES2 in humans.
In the present study, EC, ECg, and EGCg all showed specific in- hibition of AADAC among the three hydrolases tested. EC, ECg, and EGCg are found in green tea, with four cups of green tea containing 88, 180, and 440 mg of EC, ECg, and EGCg, respectively [28]. These compounds are known to inhibit various P450s [29] but with IC50 values that are higher than those for AADAC (Table 1). This suggests that EC, ECg, and EGCg all inhibit AADAC more potently than P450s. A previous in vivo study demonstrated that there was a 20% in- crease in the area under the curve for CYP3A4 metabotropic drugs after human intake of EGCg at a dose of 800 mg/day for 4 weeks [30]. It is known that the bioavailability of EGCg is low, and thus, the inhibitory effects toward CYP3A4 in the small intestine might be large. AADAC is highly expressed in both the liver and small in- testine [2], similar to CYP3A4. Thus, EGCg could also inhibit AADAC metabolism in vivo, although further in vivo studies will be required.
In this study, we also compared the inhibitory effects of catechin-related analogs. Catechin and epicatechin are stereoiso- mers with two chiral centers at C2 and C3, of which catechin is the trans configuration isomer, whereas epicatechin is the cis configu- ration isomer. ECg and EGCg have a galloyl group at the C3 position of EC and EGC, respectively. It is interesting to note that compounds with the galloyl group were stronger inhibitors of AADAC than epicatechin and its analogs. Unlike that for CES1 and CES2, the crystal structure of AADAC has not been determined yet, but once available, it will provide very important information. Furthermore, more specific and potent AADAC inhibitors than ECg and EGCg could be expected to be produced using docking models. None- theless, it is clear that the present results demonstrate that ECg and EGCG can specifically and strongly inhibit AADAC, representing useful tools for in vitro metabolic studies for drug development [31].


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