p-menth-1-en-4-ol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Determination of cytochrome P450 enzymes involved in the metabolism of (−)-terpinen-4-ol by human liver microsomes

Author:

Miyazawa M, Haigou R

Year:

2011

Bibliographic source:

Xenobiotica, 2011; 41(12): 1056–1062

Report date:

2011

Materials and methods

Objective of study:

metabolism

toxicokinetics

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study, the oxidation of the (-) enantiomer of the test substance by cytochrome P450 enzymes in human liver microsokmes was examined.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

p-menth-1-en-4-ol

EC Number:

209-235-5

EC Name:

p-menth-1-en-4-ol

Cas Number:

562-74-3

Molecular formula:

C10H18O

IUPAC Name:

4-methyl-1-(propan-2-yl)cyclohex-3-en-1-ol

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- The (-) enantiomer of the test substance was used in this study
- Source: Tokyo Chemical Industry Co., Ltd., Tokyo, Japan

Radiolabelling:

no

Test animals

Species:

other: human

Details on test animals or test system and environmental conditions:

TEST SYSTEM
- Type: Human liver microsomes available with complete catalytic properties
- Source: Gentest Corporation (Woburn, MA)
- Microsomes: HG-03, HG-06, HG-74, HG-88, HH-13, HH-18, HH-47, HH-74, HK-37, HH-741

Administration / exposure

Vehicle:

other: methanol

Doses / concentrations

Dose / conc.:

200 other: µM

Details on study design:

IN VITRO ASSAY WITH HUMAN LIVER MICROSOMES
- Concentration: 200 µM (at <1% v/V in final solvent concentration)
- Incubation: 30 min at 37°C

IN VITRO ASSAY WITH cDNA-EXPRESSED P450 ENZYMES
- Concentration: 200 µM
- Incubation: 30 min at 37°C

INHIBITION EXPERIMENTS:
- Investigatiing selective inhibitory effects of known P450 enzymes
- Inhibitors: alpha-naphthoflavone (CYP1A2), menthofuran (CYP2A6), ketoconazole (CYP3A4)
- Substrate concentration: 50 mM
- Incubation: 30 min at 37°C

KINETIK ANALYSIS
- Vmax and Km were estimated using a computer program for nonlinear regression analysis
- Concentrations used for the assay: 10, 30, 50, 100, 200, and 400 µM

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

(−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol and (−)-(1R,2S,4R)-1,2-epoxy-p-menthan-4-ol. CYP1A2, CYP2A6 and CYP3A4 catalysed oxidations of (−)-terpinen-4-ol to (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol. (−)-(1R,2S,4R)-1,2-epoxy-p-menthan-4-ol activity was found to be catalysed by CYP3A4. Other P450 enzymes had very low activities or activities below the limit of detection.

(+)-Menthofuran inhibited oxidation of the test substance to >50% to (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol. Ketoconazole inhibited the oxidation of the test substance to >50% to (−)-(1R,2S,4R)-1,2-epoxy-p-menthan-4-ol.

Any other information on results incl. tables

Table 1: Kinetic analysis of the oxidation of the test substance by human liver microsomes

Oxidation of (−)-terpinen-4-ol
(−)-(1S,2R,4R)-1,2-Epoxy-p-menthan-4-ol			(−)-(1R,2S,4R)-1,2-Epoxy-p-menthan-4-ol
Enzymes source	Km(μM)	Vmax	Vmax/Km	Km(μM)	Vmax	Vmax/Km
HH-18	205 ± 53	0.51 ± 0.2a	2.49c	60 ± 6	0.04 ± 0.01	10.7
CYP1A2	378 ± 46	5.16 ± 1.3b	13.7d
CYP2A6	91 ± 5	13.9 ± 0.6	152.7
CYP3A4	267 ± 77	3.5 ± 0.7	13.1	143 ± 20	1.44 ± 0.50	10.1

a=Vmax expressed in nmol/min/mg protein for human liver microsomes.

b0Vmax expressed in nmol/min/nmol P450 for recombinant human P450 enzymes.

c=Intrinsic clearance (Vmax/Km) expressed in μL/min/mg protein for human liver microsomes.

d=Intrinsic clearance (Vmax/Km) expressed in μL/min/nmol P450 for recombinant human P450 enzymes.

Applicant's summary and conclusion

Conclusions:

In conclusion, the results demosntrated that the (-) enantiomer of the test substance was converted to (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol by CYP1A2, CYP2A6, and CYP3A4, whereas (−)-(1R,2S,4R)-1,2-epoxy-p-menthan-4-ol was only converted by CYP3A4 in human liver microsomes. CYP2A6 was suggested to be a major enzyme in human liver microsomes.

Executive summary:

The metabolism of the (-) enantiomer of the test substance was investigated in an in vitro assay using human liver microsomes and recombinant enzymes. The biotransformation was analyzed by gas chromatography-mass spectrometry. Two metabolic products were found, namely (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol and (−)-(1R,2S,4R)-1,2-epoxy-p-menthan-4-ol transformed by CYP1A2, CAP2A6, CYP3A4 and CYP3A4 exclusively, respectively. CYP2A6 showed the highest activity for oxidation of the test substance and the oxidation was inhibited by (+)-menthofuran.

Kinetic analysis showed that the Vmax/Km values for (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol. Human recombinant CYP2A6 catalysed (−)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with Vmax values of 13.9 nmol/ min/nmol P450 and apparent Km values of 91 μM.