Carvacrol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Data source

Reference

Reference Type:

publication

Title:

Identification of CYP isoforms involved in the metabolism of thymol and carvacrol in human liver microsomes (HLMs)

Author:

Dong, Fang, Zhu, Ge, Cao, Li, Hu, Yang, Liu.

Year:

2012

Bibliographic source:

Pharmazie 67: 1002–1006 (2012)

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of the study was to investigate the role of cytochrome P450s (CYPs) in the metabolism of thymol and carvacrol. After incubation with human liver microsomes (HLMs) in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. A combination of chemical inhibition studies and assays with recombinant CYP isoforms was used to investigate what aret the predominant drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Carvacrol

EC Number:

207-889-6

EC Name:

Carvacrol

Cas Number:

499-75-2

Molecular formula:

C10H14O

IUPAC Name:

2-methyl-5-(propan-2-yl)phenol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Carvacrol; Aladdin Corp. (Shanghai, China)
- Purity: 99%

- Source of test material: Thymol; Aladdin Corp. (Shanghai, China0
- Purity: 99%

Radiolabelling:

no

Results and discussion

Main ADME results

Type:

metabolism

Results:

CYP2A6 was demonstrated to be the major drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.

Metabolite characterisation studies

Metabolites identified:

no

Details on metabolites:

After incubation with HLMs in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. These metabolies were not identified.

Any other information on results incl. tables

Detection of thymol and carvacrol metabolites in HLM

After incubation of thymol with HLMs and NADPH-generating system, a new peak was eluted at 14.5 min (M) for thymol (Fig. 2A). This new peak was not detected in the negative controls without NADPH, without substrate, and without microsomes. For the metabolism of carvacrol, two new peaks were eluted at 14.7 min (M-1) and 15.3 min (M-2) (Fig. 2B). These two new peaks were not detected in the negative controls without NADPH, without substrate, and without microsomes.

Kinetic study

Under the experimental conditions used, the metabolism of thymol in HLMs obeyed typical Michaelis-Menten kinetics, as evidenced by Eadie-Hofstee plots (Fig. 3A). The kinetic parameters (apparent Vmax and Km) were calculated to be 0.58±0.02 nmol/min/mg pro and 19.8±2.2µM. The metabolism of carvacrol in HLMs obeyed the typical Michaelis-Menten kinetics, as evidenced by Eadie-Hofstee plots (Fig. 3B and Fig. 3C).The kinetic parameters (Km and Vmax) were 9.8µM and 0.78 nmol/min/mg pro for M-1, and 9.3 µM and 0.37 nmol/min/mg pro for M-2.

Chemical inhibition study

The effect of various chemical inhibitors on the metabolism of thymol was investigated in pooled HLMs (Fig. 4A). ABT, the broad CYP inhibitor, strongly inhibited the formation of thymol metabolite, suggesting that CYPs were the major drug-metabolizing enzymes involved in the metabolism of thymol. Among the selective inhibitors of nine CYP iso- forms, 8-methoxypsolaren (the selective inhibitor of CYP2A6) almost completely inhibited the formation of thymol metabolite. As shown in Fig. 4B, ABT, the broad CYP inhibitor, strongly inhibited the formation of carvacrol metabolites (M-1, M-2), suggesting that CYPs were the major drug metabolizing enzymes involved in the metabolism of carvacrol. 8-methoxypsolaren (the selective inhibitor of CYP2A6) almost completely inhibited the formation of M-1 and M-2.

Assay with human recombinant CYP isoforms

Nine recombinant CYP isoforms were employed to identify the CYP isoforms involved in the metabolism of thymol and carvacrol. The results (Fig. 5A) showed that CYP1A2, CYP2A6 and CYP2B6 could catalyze the formation of thymol metabolite. The levels of involvement of other CYP isoforms in the metabolism of thymol were negligible. For the metabolism of carvacrol, the results (Fig. 5B) showed that CYP1A2, and CYP2A6 could catalyze the formation of M-1, and CYP1A2, CYP2A6 and CYP2B6 could catalyze the formation of M-2. The levels of involvement of other CYP isoforms in the metabolism of carvacrol were negligible.

Applicant's summary and conclusion

Conclusions:

After incubation with human liver microsomes (HLMs) in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. These metabolites were not identified. A combination of chemical inhibition studies and assays with recombinant CYP isoforms demonstrated that CYP2A6 was the predominant drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.

Executive summary:

In an vitro enzyme assay (Dong et al., 2012), the role of cytochrome P450s (CYPs) in the metabolism of thymol and carvacrol was investigated.

After incubation with human liver microsomes (HLMs) in the presence of NADPH, a new metabolite and two metabolites were detected for thymol and carvacrol, respectively. These metabolites were not identified. A combination of chemical inhibition studies and assays with recombinant CYP isoforms demonstrated that CYP2A6 was the predominant drug-metabolizing enzyme involved in the metabolism of thymol and carvacrol.