Cannabis (Cannabis sativa)
Characterization of major phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent inhibitors of human CYP1 enzymes
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Inhibitory effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K(i) value (0.155microM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 (K(i)=0.0790 and 0.148microM, respectively) compared with CYP1A1 (K(i)=0.541microM). Delta(9)-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition (K(i)=2.47-7.54microM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Delta(9)-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k(inact)/K(I) value (540l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1.
1 . 1A1: CBD (id=NPDI-jYDUsw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A1 4173297
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2, 3, and 4.
IC50 at 20 min.
Fig 4. % inhibition estimated with 0.625 μM
Fig 5. With NADPH at 9 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
10 fmol
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
2 . 1A2: CBD (id=NPDI-FompNA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A2 4312402
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2, 3, and 4
IC50 at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
60 fmol
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
3 . HLMs: CBD (id=NPDI-bxWjqg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A1 4173297
Cell fraction Pooled human liver microsomes -7999662
Results
Tables 2 and 3.
IC50 at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
5 μg
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
4 . 1A1: CBN (id=NPDI-v65nxg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A1 4173297
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2, 3, and 4
IC50 at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
10 fmol
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
5 . 1A2: CBN (id=NPDI-RC8Kxw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A2 4312402
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2 and 3
IC50 at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
60 fmol
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
6 . HLMs: CBN (id=NPDI-XKoD2A)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A1 4173297
Cell fraction Pooled human liver microsomes -7999662
Results
Tables 2 and 3.
IC50 at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
5 μg
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
7 . 1A1: THC (id=NPDI-4tAywg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A1 4173297
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2, 3, and 4
IC50 at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
10 fmol
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
8 . 1A2: THC (id=NPDI-F-0XWg)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A2 4312402
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Tables 2 and 3
IC50 at 20 min
Fig 4. %inhibition estimated for 8 μM
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
60 fmol
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM
9 . HLMs: THC (id=NPDI-DQGnJw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- CYP1A1 4173297
Cell fraction Pooled human liver microsomes -7999662
Results
Tables 2 and 3.
IC50 at 20 min
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
96-well
5 μg
Commercially available
200 μL
30 min
NADPH
150 nM
5 min
No NADPH with precipitant
0.025-25 μM