A trapping method for semi-quantitative assessment of reactive metabolite formation using 35S cysteine and 14C cyanide

Kazuko Inoue, Yoshihiro Shibata, Hiroyuki Takahashi, Tomoyuki Ohe, Masato Chiba, Yasuyuki Ishii

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

A trapping approach for semi-quantitative assessment of bioactivation potential has been established for new chemical entities by using [35S]cysteine and [14C]sodium cyanide as trapping reagents. Reactive metabolites were trapped as radioactive adducts with the trapping reagents to be analyzed by radio-LC(/MS). As a reference, hepatotoxic drugs (clozapine, diclofenac, R-(+)-pulegone and troglitazone) were tested in the [35S]cysteine trapping assay and the proposed structures of the cysteine adducts were consistent with glutathione adducts previously reported. The accuracy of this methodology to predict bioactivation potential of structurally diverse non-radiolabeled test compounds was evaluated by comparing the radiochromatographic peak area obtained in this assays with the extent of covalent binding to protein assessed by the conventional method using radiolabeled test compounds. The value obtained from the [35S]cysteine trapping assay in human liver microsomes predicted potential for covalent binding of the test compounds to proteins with reasonable accuracy. A combination of trapping reagents ([35S]cysteine and [14C]cyanide) improved the accuracy for prediction of bioactivation potential by simultaneously trapping both types of electrophilic reactive metabolites. This method is expected to be a useful to prioritize compounds for further development based on the bioactivation liability, especially at the lead optimization stage.

Original languageEnglish
Pages (from-to)245-254
Number of pages10
JournalDrug Metabolism and Pharmacokinetics
Volume24
Issue number3
DOIs
Publication statusPublished - 2009
Externally publishedYes

Fingerprint

Cyanides
Cysteine
troglitazone
Sodium Cyanide
Diclofenac
Clozapine
Liver Microsomes
Radio
Glutathione
Carrier Proteins
Pharmaceutical Preparations
Proteins

Keywords

  • Analytical method
  • Bioactivation
  • Covalent binding
  • Reactive metabolites
  • Trapping assay

ASJC Scopus subject areas

  • Pharmacology (medical)
  • Pharmacology
  • Pharmaceutical Science

Cite this

A trapping method for semi-quantitative assessment of reactive metabolite formation using 35S cysteine and 14C cyanide. / Inoue, Kazuko; Shibata, Yoshihiro; Takahashi, Hiroyuki; Ohe, Tomoyuki; Chiba, Masato; Ishii, Yasuyuki.

In: Drug Metabolism and Pharmacokinetics, Vol. 24, No. 3, 2009, p. 245-254.

Research output: Contribution to journalArticle

Inoue, Kazuko ; Shibata, Yoshihiro ; Takahashi, Hiroyuki ; Ohe, Tomoyuki ; Chiba, Masato ; Ishii, Yasuyuki. / A trapping method for semi-quantitative assessment of reactive metabolite formation using 35S cysteine and 14C cyanide. In: Drug Metabolism and Pharmacokinetics. 2009 ; Vol. 24, No. 3. pp. 245-254.
@article{dfca902c871e408aaf214cffd986d106,
title = "A trapping method for semi-quantitative assessment of reactive metabolite formation using 35S cysteine and 14C cyanide",
abstract = "A trapping approach for semi-quantitative assessment of bioactivation potential has been established for new chemical entities by using [35S]cysteine and [14C]sodium cyanide as trapping reagents. Reactive metabolites were trapped as radioactive adducts with the trapping reagents to be analyzed by radio-LC(/MS). As a reference, hepatotoxic drugs (clozapine, diclofenac, R-(+)-pulegone and troglitazone) were tested in the [35S]cysteine trapping assay and the proposed structures of the cysteine adducts were consistent with glutathione adducts previously reported. The accuracy of this methodology to predict bioactivation potential of structurally diverse non-radiolabeled test compounds was evaluated by comparing the radiochromatographic peak area obtained in this assays with the extent of covalent binding to protein assessed by the conventional method using radiolabeled test compounds. The value obtained from the [35S]cysteine trapping assay in human liver microsomes predicted potential for covalent binding of the test compounds to proteins with reasonable accuracy. A combination of trapping reagents ([35S]cysteine and [14C]cyanide) improved the accuracy for prediction of bioactivation potential by simultaneously trapping both types of electrophilic reactive metabolites. This method is expected to be a useful to prioritize compounds for further development based on the bioactivation liability, especially at the lead optimization stage.",
keywords = "Analytical method, Bioactivation, Covalent binding, Reactive metabolites, Trapping assay",
author = "Kazuko Inoue and Yoshihiro Shibata and Hiroyuki Takahashi and Tomoyuki Ohe and Masato Chiba and Yasuyuki Ishii",
year = "2009",
doi = "10.2133/dmpk.24.245",
language = "English",
volume = "24",
pages = "245--254",
journal = "Drug Metabolism and Pharmacokinetics",
issn = "1347-4367",
publisher = "Japanese Society for the Study of Xenobiotics",
number = "3",

}

TY - JOUR

T1 - A trapping method for semi-quantitative assessment of reactive metabolite formation using 35S cysteine and 14C cyanide

AU - Inoue, Kazuko

AU - Shibata, Yoshihiro

AU - Takahashi, Hiroyuki

AU - Ohe, Tomoyuki

AU - Chiba, Masato

AU - Ishii, Yasuyuki

PY - 2009

Y1 - 2009

N2 - A trapping approach for semi-quantitative assessment of bioactivation potential has been established for new chemical entities by using [35S]cysteine and [14C]sodium cyanide as trapping reagents. Reactive metabolites were trapped as radioactive adducts with the trapping reagents to be analyzed by radio-LC(/MS). As a reference, hepatotoxic drugs (clozapine, diclofenac, R-(+)-pulegone and troglitazone) were tested in the [35S]cysteine trapping assay and the proposed structures of the cysteine adducts were consistent with glutathione adducts previously reported. The accuracy of this methodology to predict bioactivation potential of structurally diverse non-radiolabeled test compounds was evaluated by comparing the radiochromatographic peak area obtained in this assays with the extent of covalent binding to protein assessed by the conventional method using radiolabeled test compounds. The value obtained from the [35S]cysteine trapping assay in human liver microsomes predicted potential for covalent binding of the test compounds to proteins with reasonable accuracy. A combination of trapping reagents ([35S]cysteine and [14C]cyanide) improved the accuracy for prediction of bioactivation potential by simultaneously trapping both types of electrophilic reactive metabolites. This method is expected to be a useful to prioritize compounds for further development based on the bioactivation liability, especially at the lead optimization stage.

AB - A trapping approach for semi-quantitative assessment of bioactivation potential has been established for new chemical entities by using [35S]cysteine and [14C]sodium cyanide as trapping reagents. Reactive metabolites were trapped as radioactive adducts with the trapping reagents to be analyzed by radio-LC(/MS). As a reference, hepatotoxic drugs (clozapine, diclofenac, R-(+)-pulegone and troglitazone) were tested in the [35S]cysteine trapping assay and the proposed structures of the cysteine adducts were consistent with glutathione adducts previously reported. The accuracy of this methodology to predict bioactivation potential of structurally diverse non-radiolabeled test compounds was evaluated by comparing the radiochromatographic peak area obtained in this assays with the extent of covalent binding to protein assessed by the conventional method using radiolabeled test compounds. The value obtained from the [35S]cysteine trapping assay in human liver microsomes predicted potential for covalent binding of the test compounds to proteins with reasonable accuracy. A combination of trapping reagents ([35S]cysteine and [14C]cyanide) improved the accuracy for prediction of bioactivation potential by simultaneously trapping both types of electrophilic reactive metabolites. This method is expected to be a useful to prioritize compounds for further development based on the bioactivation liability, especially at the lead optimization stage.

KW - Analytical method

KW - Bioactivation

KW - Covalent binding

KW - Reactive metabolites

KW - Trapping assay

UR - http://www.scopus.com/inward/record.url?scp=69949185975&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=69949185975&partnerID=8YFLogxK

U2 - 10.2133/dmpk.24.245

DO - 10.2133/dmpk.24.245

M3 - Article

VL - 24

SP - 245

EP - 254

JO - Drug Metabolism and Pharmacokinetics

JF - Drug Metabolism and Pharmacokinetics

SN - 1347-4367

IS - 3

ER -