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 CSV JSON

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.

Kratom (Mitragyna speciosa)

26692748

2015485354

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.

4-methylumbelliferone

7-hydroxymitragynine

4-methylumbelliferone glucuronide

  • 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 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.

4-methylumbelliferone

buprenorphine

4-methylumbelliferone glucuronide

  • 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

3 . 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.

4-methylumbelliferone

buprenorphine

4-methylumbelliferone glucuronide

  • 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

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.

4-methylumbelliferone

diclofenac 1124300

4-methylumbelliferone glucuronide

  • 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 . 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.

4-methylumbelliferone

7-hydroxymitragynine

4-methylumbelliferone glucuronide

  • 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

6 . 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.

4-methylumbelliferone

7-hydroxymitragynine

4-methylumbelliferone glucuronide

  • 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

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.

4-methylumbelliferone

buprenorphine

4-methylumbelliferone glucuronide

  • 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 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.

4-methylumbelliferone

diclofenac 1124300

4-methylumbelliferone glucuronide

  • 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

9 . 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.

4-methylumbelliferone

diclofenac 1124300

4-methylumbelliferone glucuronide

  • 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

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.

4-methylumbelliferone

ketamine

4-methylumbelliferone glucuronide

  • 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.

4-methylumbelliferone

ketamine

4-methylumbelliferone glucuronide

  • 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.

4-methylumbelliferone

ketamine

4-methylumbelliferone glucuronide

  • 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 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.

4-methylumbelliferone

mitragynine

4-methylumbelliferone glucuronide

  • 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

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.

4-methylumbelliferone

mitragynine

4-methylumbelliferone glucuronide

  • 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 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.

4-methylumbelliferone

mitragynine

4-methylumbelliferone glucuronide

  • 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