Cannabis (Cannabis sativa)
Comparison in the in vitro inhibitory effects of major phytocannabinoids and polycyclic aromatic hydrocarbons contained in marijuana smoke on cytochrome P450 2C9 activity.
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Inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, and polycyclic aromatic hydrocarbons (PAHs) contained in marijuana smoke on catalytic activity of human cytochrome P450 (CYP) 2C9 were investigated. These phytocannabinoids concentration-dependently inhibited S-warfarin 7-hydroxylase and diclofenac 4'-hydroxylase activities of human liver microsomes (HLMs) and recombinant CYP2C9 (rCYP2C9). In contrast, none of the twelve PAHs including benz[a]anthracene and benzo[a]pyrene exerted substantial inhibition (IC₅₀ > 10 µM). The inhibitory potentials of Δ⁹-THC (Ki = 0.937-1.50 µM) and CBN (Ki = 0.882-1.29 µM) were almost equivalent regardless of the enzyme sources used, whereas the inhibitory potency of CBD (Ki > = 0.954-9.88 µM) varied depending on the enzyme sources and substrates used. Δ⁹-THC inhibited both S-warfarin 7-hydroxylase and diclofenac 4'-hydroxylase activities of HLMs and rCYP2C9 in a mixed manner. CBD and CBN competitively inhibited the activities of HLMs and rCYP2C9, with the only notable difference being that CBD and CBN exhibited mixed-type inhibitions against diclofenac 4'-hydroxylation and S-warfarin 7-hydroxylation, respectively, by rCYP2C9. None of Δ⁹-THC, CBD, and CBN exerted metabolism-dependent inhibition. These results indicated that the three major phytocannabinoids but not PAHs contained in marijuana smoke potently inhibited CYP2C9 activity and that these cannabinoidscan be characterized as direct inhibitors for CYP2C9.
1 . rCYP2C9: CBD-warfarin (id=NPDI-P2rdYg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
IC50 preincubation time recorded at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
6 pmol
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
2 . pHLM: CBD-warfarin (id=NPDI-XouN0Q)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Pooled human liver microsomes -7999662
Results
Tables 2, 3, and 4.
IC50 pre-incubation recorded at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
20 μg
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
3 . iHLMs: CBD-warfarin (id=NPDI-dvyC1w)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Individual human liver microsomes
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
50 μg
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
4 . pHLM: CBN-warfarin (id=NPDI-mGu2ew)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Pooled human liver microsomes -7999662
Results
Tables 2, 3, and 4.
IC50 pre-incubation recorded at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Commercially available
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
5 . iHLM: CBN-warfarin (id=NPDI-ZNPhvw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Individual human liver microsomes
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
50 μg
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
6 . rCYP2C9: CBN-warfarin (id=NPDI-wt2yDg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
IC50 preincubation time recorded at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
6 pmol
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
7 . iHLM: THC-warfarin (id=NPDI-vUechg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Individual human liver microsomes
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
50 μg
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
8 . rCYP2C9: THC-warfarin (id=NPDI-59dZ4A)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
IC50 preincubation time recorded at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
6 pmol
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
9 . pHLM: THC-warfarin (id=NPDI-jZO6oQ)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Pooled human liver microsomes -7999662
Results
Tables 2, 3, and 4.
IC50 pre-incubation recorded at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
20 μg
Commercially available
200 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
10 . iHLM: CBD-diclofenac (id=NPDI-Fqa3JA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Individual human liver microsomes
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
50 μg
Commercially available
250 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
11 . rCYP2C9: CBD-diclofenac (id=NPDI-LKS7Ow)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
2 pmol
Commercially available
250 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
12 . rCYP2C9: CBN-diclofenac (id=NPDI-KqafWg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
2 pmol
Commercially available
250 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
13 . iHLM: CBN-diclofenac (id=NPDI-V1SemA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Individual human liver microsomes
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
50 μg
Commercially available
250 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
14 . rCYP2C9: THC-diclofenac (id=NPDI-bM6NOQ)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
2 pmol
Commercially available
250 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant
15 . iHLM: THC-diclofenac (id=NPDI-Jb4JFA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP2C9 4309227
Cell fraction Individual human liver microsomes
Results
Tables 2 and 3
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
50 μg
Commercially available
250 μL
20 min
MgCl2
NADPH regenerating system
5 min
NADPH with precipitant