Cannabis (Cannabis sativa)
Involvement of UDP-Glucuronosyltransferases UGT1A9 and UGT2B7 in Ethanol Glucuronidation, and Interactions with Common Drugs of Abuse
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Ethyl glucuronide (EtG) determination is increasingly used in clinical and forensic toxicology to document ethanol consumption. The enzymes involved in EtG production, as well as potential interactions with common drugs of abuse, have not been extensively studied. Activities of human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as of 12 major human recombinant UDP-glucuronosyltransferases (UGTs), toward ethanol (50 and 500 mM) were evaluated in vitro using liquid chromatography-tandem mass spectrometry. Enzyme kinetic parameters were determined for pooled microsomes and recombinant UGTs with significant activity. Individual contributions of UGTs were estimated using the relative activity factor approach, proposed for scaling activities obtained with cDNA-expressed enzymes to HLM. Interaction of morphine, codeine, lorazepam, oxazepam, nicotine, cotinine, cannabinol, and cannabidiol (5, 10, 15 mg/l) with ethanol (1.15, 4.6, 11.5 g/l; i.e., 25, 100, 250 mM) glucuronidation was assessed using pooled HLM. Ethanol glucuronidation intrinsic clearance (Cl(int)) was 4 and 12.7 times higher for HLM than for HKM and HIM, respectively. All recombinant UGTs, except UGT1A1, 1A6, and 1A10, produced EtG in detectable amounts. UGT1A9 and 2B7 were the most active enzymes, each accounting for 17 and 33% of HLM Cl(int), respectively. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation. Cannabinol increased ethanol glucuronidation in a concentration-dependent manner, whereas cannabidiol significantly inhibited EtG formation in a noncompetitive manner (IC(50) = 1.17 mg/l; inhibition constant (K(i)) = 3.1 mg/l). UGT1A9 and 2B7 are the main enzymes involved in ethanol glucuronidation. In addition, our results suggest that cannabinol and cannabidiol could significantly alter ethanol glucuronidation.
1 . CBD-UGT2B7 Inhibition (id=NPDI-DpYeow)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- UGT2B7
Cell system Human cryopreserved hepatocytes
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
In-house preparation
100 μL
45 min
MgCl2
UDPGA
25 mM
5 min
No NADPH with no precipitant
0, 5, 10,15 mg/L
2 . CBD on ethanol glucuronidation (id=NPDI-ILsKWw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- UGT
Cell system Human cryopreserved hepatocytes
Results
%inhibition estimated from Fig 4 at 15 mg/L.
Ki (3.1 mg/L) from Fig 5.
IC50 (1.17 mg/L) and Ki converted from mg/L to µg/mL
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
In-house preparation
100 μL
45 min
MgCl2
UDPGA
25 mM
5 min
No NADPH with no precipitant
0, 5, 10,15 mg/L
3 . CBD-UGT1A9 Inhibition (id=NPDI-dh6TjQ)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- UGT1A9
Cell system Human cryopreserved hepatocytes
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
In-house preparation
100 μL
45 min
MgCl2
UDPGA
25 mM
5 min
No NADPH with no precipitant
0, 5, 10,15 mg/L
4 . CBN-UGT1A9 Inhibition (id=NPDI-jwN2Vw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — Not specified
- UGT1A9
Cell system Human cryopreserved hepatocytes
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
In-house preparation
100 μL
45 min
MgCl2
UDPGA
25 mM
5 min
No NADPH with no precipitant
0, 5, 10,15 mg/L
1 . CBN-UGT2B7 Induction (id=NPDI-JvqWDQ)
In Vitro Enzyme Induction Experiment
Induction was detected. Cutoff used — Not specified
- UGT2B7
Cell system Human cryopreserved hepatocytes
Results
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Enzyme activity
44
0, 5, 10, 15 mg/L
45 m
2 . CBN on ethanol glucuronidation (id=NPDI-7hkXUg)
In Vitro Enzyme Induction Experiment
Induction was detected. Cutoff used — Not specified
- UGT
Cell system Human cryopreserved hepatocytes
Results
Estimated from Fig 4 at 15 mg/L.
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Enzyme activity
44
0, 5, 10, 15 mg/L
45 m