Kratom (Mitragyna speciosa)
Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth) on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5’- diphospho-glucuronosyltransferase isoforms
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Background: Glucuronidation catalyzed by uridine 5’- diphospho-glucuronosyltransferase (UGT) is a major phase II drug metabolism reaction which facilitates drug elimination. Inhibition of UGT activity can cause drug-drug interaction. Therefore, it is important to determine the inhibitory potentials of drugs on glucuronidation.
Objective: The objective was to evaluate the inhibitory potentials of mitragynine, 7-hydroxymitragynine, ketamine and buprenorphine, respectively on 4-methylumbelliferone (4-MU) glucuronidation in rat liver microsomes, human liver microsomes and recombinant human UGT1A1 and UGT2B7 isoforms.
Materials and Methods: The effects of the above four compounds on the formation of 4-MU glucuronide from 4-MU by rat liver microsomes, human liver microsomes, recombinant human UGT1A1 and UGT2B7 isoforms were determined using high-performance liquid chromatography with ultraviolet detection.
Results: For rat liver microsomes, ketamine strongly inhibited 4-MU glucuronidation with an IC50 value of 6.21 ± 1.51 μM followed by buprenorphine with an IC50 value of 73.22 ± 1.63 μM. For human liver microsomes, buprenorphine strongly inhibited 4-MU glucuronidation with an IC50 value of 6.32 ± 1.39 μM. For human UGT1A1 isoform, 7-hydroxymitragynine strongly inhibited 4-MU glucuronidation with an IC50 value of 7.13 ± 1.16 μM. For human UGT2B7 isoform, buprenorphine strongly inhibited 4-MU glucuronidation followed by 7-hydroxymitragynine and ketamine with respective IC50 values of 5.14 ± 1.30, 26.44 ± 1.31, and 27.28 ± 1.18 μM.
Conclusions: These data indicate the possibility of drug-drug interaction if 7-hydroxymitragynine, ketamine, and buprenorphine are co-administered with drugs that are UGT2B7 substrates since these three compounds showed significant inhibition on UGT2B7 activity. In addition, if 7-hydroxymitragynine is to be taken with other drugs that are highly metabolized by UGT1A1, there is a possibility of drug-drug interaction to occur.
1 . Inhibition of UGT1A1 by 7-hydroxymitragynine (id=NPDI-4do0FA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected.
Cutoff used — IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT1A1 40782950
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
Concentration of precipitant and % inhibition of UGT1A1 (values estimated from figure 4)
0.01 μM: 6 ± 1%
0.1 μM: 17 ± 1% (p < 0.05)
1 μM: 26 ± 2% (p < 0.05)
10 μM:49 ± 3% (p < 0.05)
100 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Tested concentrations of precipitant only up to 100 μM due to limited solubility
Control=no inhibitor
200 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-100 μM
2 . Inhibition of UGT in human liver microsomes by buprenorphine (id=NPDI-pMOhyQ)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT
Cell fraction Pooled human liver microsomes -7999662
Results
Concentration of precipitant and % inhibition of pooled human liver microsomes (values estimated from figure 3) (p<0.05 for all values)
0.01 μM: 27 ± 2%
0.1 μM: 39 ± 1%
1 μM: 43 ± 3%
10 μM: 55 ± 1%
100 μM: 61 ± 2%
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Pooled human liver microsomes were purchased from Sigma-Aldrich; The enzyme activity assay mixture (250 μL) contained Tris-HCl (100 mM) (pH 7.4), MgCl (5 mM), microsomal protein (0.1 mg/mL), Triton X-100 (0.005%), UDPGA (3 mM), and 4-MU (100 μM).
Control=no inhibitor
0.1 mg/mL
Commercially available
250 μL
30 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-1000 μM
3 . Inhibition of UGT2B7 by buprenorphine (id=NPDI-ZTY8Qw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected.
Cutoff used — IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT2B7
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
Concentration of precipitant and % inhibition of UGT2B7 (values estimated from figure 5)
0.01 μM: -8 ± 4%
0.1 μM: 19 ± 3% (p < 0.05)
1 μM: 31 ± 7% (p < 0.05)
10 μM: 61 ± 3% (p < 0.05)
100 μM: 81± 3% (p < 0.05)
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Control=no inhibitor
150 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
350 μM
0.01-1000 μM
4 . Inhibition of UGT1A1 by diclofenac (id=NPDI-4_m2Gw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected.
Cutoff used — IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT1A1 40782950
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
Concentration of precipitant and % inhibition of UGT1A1 (values estimated from figure 4) (p < 0.05 for all values)
0.01 μM: 17 ± 4%
0.1 μM: 32 ± 2%
1 μM: 47 ± 2%
10 μM:55 ± 3%
100 μM: 69 ± 2%
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Control=no inhibitor
200 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-1000 μM
5 . No inhibition of UGT in human liver microsome activity by 7-hydroxymitragynine (id=NPDI-Q5Hs5A)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT
Cell fraction Pooled human liver microsomes -7999662
Results
The IC50 values were all greater than the highest concentrations used (IC50 > 100 μM) since inhibition at more than 50% did not occur at the highest concentration.
Concentration of precipitant and % inhibition of pooled human liver microsomes (intermediate values estimated from figure 3)
0.01 μM: 5 ± 1%
0.1 μM: 9 ± 5%
1 μM: 10 ± 2% (P < 0.05)
10 μM:10 ± 3% (P < 0.05)
100 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Pooled human liver microsomes were purchased from Sigma-Aldrich; The enzyme activity assay mixture (250 μL) contained Tris-HCl (100 mM) (pH 7.4), MgCl (5 mM), microsomal protein (0.1 mg/mL), Triton X-100 (0.005%), UDPGA (3 mM), and 4-MU (100 μM).
Tested concentrations of precipitant only up to 100 μM due to limited solubility
Control=no inhibitor
0.1 mg/mL
Commercially available
250 μL
30 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-100 μM
6 . Negligible inhibition of UGT2B7 by 7-hydroxymitragynine (id=NPDI-3C6gmg)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT2B7
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
Concentration of precipitant and % inhibition of UGT2B7 (values estimated from figure 5)
0.01 μM: 3 ± 5%
0.1 μM: 13 ± 2% (p < 0.05)
1 μM: 26 ± 5% (p < 0.05)
10 μM:33 ± 5% (p < 0.05)
100 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Tested concentrations of precipitant only up to 100 μM due to limited solubility
Control=no inhibitor
150 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
350 μM
0.01-100 μM
7 . Negligible inhibition of UGT1A1 by buprenorphine (id=NPDI-3eovVg)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT1A1 40782950
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
The IC50 values were all greater than the highest concentrations used (IC50 > 1000 μM) since inhibition at more than 50% did not occur at the highest concentration.
Concentration of precipitant and % inhibition of UGT1A1 (intermediate values estimated from figure 4)
0.01 μM: 12 ± 4%
0.1 μM: 24 ± 2% (p < 0.05)
1 μM: 25 ± 3% (p < 0.05)
10 μM:32 ± 2% (p < 0.05)
100 μM: 35 ± 3% (p < 0.05)
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Control=no inhibitor
200 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-1000 μM
8 . Negligible inhibition of UGT in human liver microsomes by diclofenac (id=NPDI-u96XKg)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT
Cell fraction Pooled human liver microsomes -7999662
Results
Concentration of precipitant and % inhibition of pooled human liver microsomes (values estimated from figure 3) (p<0.05 for all values)
0.01 μM: 8 ± 3%
0.1 μM: 9 ± 2%
1 μM: 20 ± 1%
10 μM: 21 ± 1%
100 μM: 41 ± 1%
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Pooled human liver microsomes were purchased from Sigma-Aldrich; The enzyme activity assay mixture (250 μL) contained Tris-HCl (100 mM) (pH 7.4), MgCl (5 mM), microsomal protein (0.1 mg/mL), Triton X-100 (0.005%), UDPGA (3 mM), and 4-MU (100 μM).
Control=no inhibitor
0.1 mg/mL
Commercially available
250 μL
30 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-1000 μM
9 . Negligible inhibition of UGT2B7 by diclofenac (id=NPDI-NZQ3aw)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT2B7
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
Concentration of precipitant and % inhibition of UGT2B7 (values estimated from figure 5)
0.01 μM: -1 ± 3%
0.1 μM: 3 ± 5%
1 μM: 8 ± 4%
10 μM: 38 ± 2% (p < 0.05)
100 μM: 84± 1% (p < 0.05)
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Control=no inhibitor
150 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
350 μM
0.01-1000 μM
10 . No inhibition of UGT in human liver microsomes by ketamine (id=NPDI-ipamgg)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT
Cell fraction Pooled human liver microsomes -7999662
Results
The IC50 values were all greater than the highest concentrations used (IC50 > 1000 μM) since inhibition at more than 50% did not occur at the highest concentration.
Concentration of precipitant and % inhibition of pooled human liver microsomes (intermediate values estimated from figure 3)
0.01 μM: 8 ± 3%
0.1 μM: 16 ± 1% (p<0.05)
1 μM: 18 ± 2% (p<0.05)
10 μM: 21 ± 4% (p<0.05)
100 μM: 21 ± 2% (p<0.05)
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Pooled human liver microsomes were purchased from Sigma-Aldrich; The enzyme activity assay mixture (250 μL) contained Tris-HCl (100 mM) (pH 7.4), MgCl (5 mM), microsomal protein (0.1 mg/mL), Triton X-100 (0.005%), UDPGA (3 mM), and 4-MU (100 μM).
Control=no inhibitor
0.1 mg/mL
Commercially available
250 μL
30 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-1000 μM
11 . No inhibition of UGT1A1 by ketamine (id=NPDI-Ve-SYA)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT1A1 40782950
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
The IC50 values were all greater than the highest concentrations used (IC50 > 1000 μM) since inhibition at more than 50% did not occur at the highest concentration.
Concentration of precipitant and % inhibition of UGT1A1 (intermediate values estimated from figure 4)
0.01 μM: 9 ± 1%
0.1 μM: 14 ± 2% (p < 0.05)
1 μM: 17 ± 5% (p < 0.05)
10 μM:18 ± 3% (p < 0.05)
100 μM: 20 ± 1% (p < 0.05)
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Control=no inhibitor
200 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-1000 μM
12 . No inhibition of UGT2B7 by ketamine (id=NPDI-bX8XPA)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT2B7
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
Concentration of precipitant and % inhibition of UGT2B7 (values estimated from figure 5)
0.01 μM: -5 ± 6%
0.1 μM: 4 ± 5%
1 μM: 11 ± 5%
10 μM: 34 ± 3% (p < 0.05)
100 μM: 70 ± 1% (p < 0.05)
1000 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Control=no inhibitor
150 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
350 μM
0.01-1000 μM
13 . Negligible inhibition of UGT2B7 by mitragynine (id=NPDI-9spGvg)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT2B7
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
The IC50 values were all greater than the highest concentrations used (IC50 > 100 μM) since inhibition at more than 50% did not occur at the highest concentration.
Concentration of precipitant and % inhibition of UGT2B7 (intermediate values estimated from figure 5)
0.01 μM: 8 ± 4%
0.1 μM: 11 ± 2%
1 μM: 23 ± 0% (p < 0.05)
10 μM:36 ± 5% (p < 0.05)
100 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Tested concentrations of precipitant only up to 100 μM due to limited solubility
Control=no inhibitor
150 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
350 μM
0.01-100 μM
14 . Negligible inhibition of UGT in human liver microsomes by mitragynine (id=NPDI-Nl2Cww)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT
Cell fraction Pooled human liver microsomes -7999662
Results
The IC50 values were all greater than the highest concentrations used (IC50 > 100 μM) since inhibition at more than 50% did not occur at the highest concentration.
Concentration of precipitant and % inhibition of pooled human liver microsomes (intermediate values estimated from figure 3) (p < 0.05 for all values)
0.01 μM: 16 ± 3%
0.1 μM: 26 ± 3%
1 μM: 29 ± 3%
10 μM:30 ± 2%
100 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
Pooled human liver microsomes were purchased from Sigma-Aldrich; The enzyme activity assay mixture (250 μL) contained Tris-HCl (100 mM) (pH 7.4), MgCl (5 mM), microsomal protein (0.1 mg/mL), Triton X-100 (0.005%), UDPGA (3 mM), and 4-MU (100 μM).
Tested concentrations of precipitant only up to 100 μM due to limited solubility
Control=no inhibitor
0.1 mg/mL
Commercially available
250 μL
30 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-100 μM
15 . Negligible inhibition of UGT1A1 by mitragynine (id=NPDI-5VhYlA)
In Vitro Enzyme Inhibition Experiment
Negligible Inhibition was detected. Cutoff used —
IC50 > 10 μM are low potential inhibitors.
1 μM < IC50 < 10 μM are moderate potential inhibitors.
IC50 < 1 μM are high potential inhibitors.
- UGT1A1 40782950
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Not available
Results
The IC50 values were all greater than the highest concentrations used (IC50 > 100 μM) since inhibition at more than 50% did not occur at the highest concentration.
Concentration of precipitant and % inhibition of UGT1A1 (some values estimated from figure 4)
0.01 μM: -4 ± 0%
0.1 μM: 8 ± 6%
1 μM: 15 ± 4% (p < 0.05)
10 μM:19 ± 0% (p < 0.05)
100 μM: See above data
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
The amount of protein, the incubation time and the concentration of 4-MU used in the measurement of 4-MU glucuronidation were described in the method by Uchaipichat et al.
Microsomes expressing recombinant human UGT1A1 and UGT2B7 were obtained from BD Biosciences (Woburn, MA, United States);incubations (total volume 250 μL) contained phosphate buffer (100 mM) (pH 7.4), MgCl 2 (5 mM), cell lysate (83.3 μg/mL for UGT1A1 or 62.5 μg/mL for UGT2B7), UDPGA (5 mM), and 4-MU (substrate) (100 μM for UGT1A1 assay or 350 μM for UGT2B7 assay).
Tested concentrations of precipitant only up to 100 μM due to limited solubility
Control=no inhibitor
200 μg/incubation
Commercially available
250 μL
120 min
MgCl2
UDPGA
Not available
Not available
100 μM
0.01-100 μM