Cannabis L. (Cannabis sativa)
Identification of cytochrome P450 enzymes responsible for metabolism of cannabidiol by human liver microsomes CSV JSON

AIMS:

Cannabidiol (CBD), one of the major constituents in marijuana, has been shown to be extensively metabolized by experimental animals and humans. However, human hepatic enzymes responsible for the CBD metabolism remain to be elucidated. In this study, we examined in vitro metabolism of CBD with human liver microsomes (HLMs) to clarify cytochrome P450 (CYP) isoforms involved in the CBD oxidations.

MAIN METHODS:

Oxidations of CBD in HLMs and recombinant human CYP enzymes were analyzed by gas chromatography/mass spectrometry.

KEY FINDINGS:

CBD was metabolized by pooled HLMs to eight monohydroxylated metabolites (6α-OH-, 6β-OH-, 7-OH-, 1″-OH-, 2″-OH-, 3″-OH-, 4″-OH-, and 5″-OH-CBDs). Among these metabolites, 6α-OH-, 6β-OH-, 7-OH-, and 4″-OH-CBDs were the major ones as estimated from the relative abundance of m/z 478, which was a predominant fragment ion of trimethylsilyl derivatives of the metabolites. Seven of 14 recombinant human CYP enzymes examined (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) were capable of metabolizing CBD. The correlations between CYP isoform-specific activities and CBD oxidative activities in 16 individual HLMs indicated that 6β-OH- and 4″-OH-CBDs were mainly formed by CYP3A4, which was supported by inhibition studies using ketoconazole and an anti-CYP3A4 antibody. The correlation and inhibition studies also showed that CBD 6α-hydroxylation was mainly catalyzed by CYP3A4 and CYP2C19, whereas CBD 7-hydroxylation was predominantly catalyzed by CYP2C19.

SIGNIFICANCE:

This study indicated that CBD was extensively metabolized by HLMs. These results suggest that CYP3A4 and CYP2C19 may be major isoforms responsible for 6α-, 6β-, 7-, and/or 4″-hydroxylations of CBD in HLMs.

1 . 6-alpha-OH-CBD-ketoconazole (id=NPDI-otngFw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — Not specified

cannabidiol (CBD)

ketoconazole

6α-OH-cannabidiol

  • CYP3A4 4306811

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 Not available

Results

Table 4 (100-(residual activity (% of control))

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

In-house preparation

0.5 mL

20 min (60 min for recombinant CYP isoforms)

MgCl2
NADPH regenerating system

6.4 μM

1 μM

2 . 6-alpha-OH-CBD-omeprazole (id=NPDI-jJ11Ig)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — Not specified

cannabidiol (CBD)

omeprazole

6α-OH-cannabidiol

  • CYP2C19 4311137

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 Not available

Results

Table 4 (100-(residual activity (% of control))

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

In-house preparation

0.5 mL

20 min (60 min for recombinant CYP isoforms)

MgCl2
NADPH regenerating system

6.4 μM

5 μM

3 . 6-alpha-OH-CBD-quinidine (id=NPDI-RPqAvQ)

In Vitro Enzyme Inhibition Experiment

Negligible Inhibition was detected.  Cutoff used — Not specified

cannabidiol (CBD)

quinidine

6α-OH-cannabidiol

  • CYP2D6 4173631

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 Not available

Results

Table 4 (100-(residual activity (% of control))

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

In-house preparation

0.5 mL

20 min (60 min for recombinant CYP isoforms)

MgCl2
NADPH regenerating system

6.4 μM

1 μM

4 . 6-alpha-OH-CBD-sulfaphenazole (id=NPDI-tjxX2A)

In Vitro Enzyme Inhibition Experiment

Negligible Inhibition was detected.  Cutoff used — Not specified

cannabidiol (CBD)

sulfaphenazole

6α-OH-cannabidiol

  • CYP2C9 4309227

Recombinant expression system Baculovirus-insect cells
Cytochrome B5 Not available

Cytochrome b5 purchased but no mention of how used in the study

Results

Table 4 (100-residual activity (% of control))

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

In-house preparation

0.5 mL

20 min (60 min for recombinant CYP isoforms)

MgCl2
NADPH regenerating system

6.4 μM

5 μM