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  • Screening of specific inhibitors for human carboxylesterases or arylacetamide deacetylase.

Screening of specific inhibitors for human carboxylesterases or arylacetamide deacetylase.

Drug metabolism and disposition: the biological fate of chemicals (2014-04-23)
Mai Shimizu, Tatsuki Fukami, Miki Nakajima, Tsuyoshi Yokoi
ABSTRACT

Esterases catalyze the hydrolysis of therapeutic drugs containing esters or amides in their structures. Human carboxylesterase (CES) and arylacetamide deacetylase (AADAC) are the major enzymes that catalyze the hydrolysis of drugs in the liver. Characterization of the enzyme(s) responsible for drug metabolism is required in drug development and to realize optimal drug therapy. Because multiple enzymes may show a metabolic potency for a given compound, inhibition studies using chemical inhibitors are useful tools to determine the contribution of each enzyme in human tissue preparations. The purpose of this study was to find specific inhibitors for human CES1, CES2, and AADAC. We screened 542 chemicals for the inhibition potency toward hydrolase activities of p-nitrophenyl acetate by recombinant CES1, CES2, and AADAC. We found that digitonin and telmisartan specifically inhibited CES1 and CES2 enzyme activity, respectively. Vinblastine potently inhibited both AADAC and CES2, but no specific inhibitor of AADAC was found. The inhibitory potency and specificity of these compounds were also evaluated by monitoring the effects on hydrolase activity of probe compounds of each enzyme (CES1: lidocaine, CES2: CPT-11, AADAC: phenacetin) in human liver microsomes. Telmisartan and vinblastine strongly inhibited the hydrolysis of CPT-11 and/or phenacetin, but digitonin did not strongly inhibit the hydrolysis of lidocaine, indicating that the inhibitory potency of digitonin was different between recombinant CES1 and liver microsomes. Although we could not find a specific inhibitor of AADAC, the combined use of telmisartan and vinblastine could predict the responsibility of human AADAC to drug hydrolysis.

MATERIALS
Product Number
Brand
Product Description

Supelco
Phenacetin, Pharmaceutical Secondary Standard; Certified Reference Material
Lidocaine, European Pharmacopoeia (EP) Reference Standard
Supelco
Lidocaine, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Acetaminophen Related Compound F, Pharmaceutical Secondary Standard; Certified Reference Material
Bupivacaine impurity F, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
4-Nitrophenol solution, 10 mM
Supelco
Phenacetin melting point standard, Pharmaceutical Secondary Standard; Certified Reference Material
USP
Lidocaine, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
4-Nitrophenyl acetate, esterase substrate
Sigma-Aldrich
4-Nitrophenol, spectrophotometric grade
Sigma-Aldrich
Irinotecan hydrochloride, topoisomerase inhibitor
Sigma-Aldrich
Lidocaine, analytical standard
Sigma-Aldrich
Lidocaine, powder
Supelco
4-Nitrophenol, PESTANAL®, analytical standard
Sigma-Aldrich
Phenacetin, ≥98.0% (HPLC)
Sigma-Aldrich
4-Nitrophenol, ReagentPlus®, ≥99%
Sigma-Aldrich
4-Ethoxyaniline, 98%
USP
Phenacetin Melting Point Standard, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
7-Ethyl-10-hydroxycamptothecin, ≥98% (HPLC), powder
Supelco
2,6-Dimethylaniline, analytical standard
Supelco
2,6-Dimethylaniline, PESTANAL®, analytical standard
Sigma-Aldrich
2,6-Dimethylaniline, 99%