Kratom (Mitragyna speciosa)
Refined Prediction of Pharmacokinetic Kratom-Drug Interactions: Time-Dependent Inhibition Considerations CSV JSON

  • All kratom extracts inhibited CYP2C9, CYP2D6 and CYP3A (hepatic and intestinal) activities by similar extents, indicating minimal product to product variability with respect to CYP inhibition. Mitragynine inhibited CYP2C9, CYP2D6, and CYP3A activity by greater than 50%, warranting further investigation as a reversible or a time dependent inhibitor of the CYP activities.
  • The 7-fold shift observed with mitragynine against intestinal and hepatic CYP3A activity suggests mitragynine is a time dependent inhibitor. IC50values were within the concentration range reported in post-mortem human plasma and tissues. Although no leftward shift was observed for CYP2D6, the IC50was also within the concentration range reported in post-mortem human plasma and tissues.
  • Mitragynine was shown to be a strong competitive inhibitor of CYP2D6 activity. The robust Ki(~1 µM) will be applied to mechanistic and PBPK models to predict drug interaction potential.
  • Mitragynine is a time dependent inhibitor of both intestinal and hepatic CYP3A activity. The efficiency of inactivation (kinact/KI) was similar to that of the clinically relevant time dependent inhibitor verapamil. The kinetic parameters, KIand kinactwill be applied to mechanistic and PBPK models to predict drug interaction potential.

1 . Screening of kratom extracts as inhibitors of CYP2D6 activity in HLM (id=NPDI-o6FZsw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

dextromethorphan 1119510

dextrorphan 4349487

  • CYP2D6 4173631

Cell fraction Pooled human liver microsomes -7999662

Three methanolic Kratom extracts were tested, K-50, K-51 and K-52.

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP activity in HLM. Mitragynine at 10 µM inhibited CYP2C9, CYP2D6, and CYP3A activity by 42%, 87%, and 50%, respectively. Kratom extracts at 20 µg/mL inhibited these activities by 81-88%, 87-93%, and 86-89%, respectively.  

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1010191

400 µL

10 min

NADPH

Available

Available

4 µM

2, 10, and 20 µg/ml

2 . Screening of mitragynine as inhibitors of CYP2D6 activity in HLM (id=NPDI-sfeVpQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

dextromethorphan 1119510

mitragynine

dextrorphan 4349487

  • CYP2D6 4173631

Cell fraction Pooled human liver microsomes -7999662

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP activity in HLM. Mitragynine at 10 µM inhibited CYP2C9, CYP2D6, and CYP3A activity by 42%, 87%, and 50%, respectively. Kratom extracts at 20 µg/mL inhibited these activities by 81-88%, 87-93%, and 86-89%, respectively.  

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1010191

400 µL

10 min

NADPH

Available

Available

4 µM

1, 10, and 100 µM

3 . Inhibition kinetics of CYP2D6 activity by mitragynine in HLM (id=NPDI-ksOM2w)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

N/A

dextromethorphan 1119510

mitragynine

dextrorphan 4349487

  • CYP2D6 4173631

Cell fraction Pooled human liver microsomes -7999662

Results

Mitragynine was shown to be a strong competitive inhibitor of CYP2D6 activity, with a Kiof 0.97  ± 0.07 µM, 1.25  ± 0.09 µM, and 0.95 ±  0.11 µM, respectively, for three individual experiments. [Ki(Mean ± SEM) values were determined using nonlinear least-squares regression and competitive inhibition model.]

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Quinidine (2 µM) served as positive control inhibitors of CYP2D6 activity.

NA

0.05 MG/ML

Commercially available

1010191

200 µL

10 min

NADPH

Available

Available

0.12 - 10 µM

4 . IC50 shift determination for mitragynine towards CYP2D6 activity in HLM without NADPH (id=NPDI--jKvig)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

dextromethorphan 1119510

mitragynine

dextrorphan 4349487

  • CYP2D6 4173631

Cell fraction Pooled human liver microsomes -7999662

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1010191

200 µL

10 min

NADPH

Available

Available

4 µM

250 µL

30 min

NADPH with no precipitant

200 µL

10 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP2D6 activity in HLM with NADPH)

5 . IC50 shift determination for mitragynine towards CYP2D6 activity in HLM with NADPH (id=NPDI-XFPmtA)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

dextromethorphan 1119510

mitragynine

dextrorphan 4349487

  • CYP2D6 4173631

Cell fraction Pooled human liver microsomes -7999662

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1010191

NADPH

Available

Available

4 µM

250 µL

30 min

NADPH with precipitant

200 µL

10 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP2D6 activity in HLM without NADPH)

6 . Screening of kratom extracts as inhibitors of CYP2C9 activity in HLM (id=NPDI-71pBaQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

diclofenac 1124300

4'-hydroxydiclofenac

  • CYP2C9 4309227

Cell fraction Pooled human liver microsomes -7999662

Three methanolic Kratom extracts were tested, K-50, K-51 and K-52.

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP activity in HLM. Mitragynine at 10 µM inhibited CYP2C9, CYP2D6, and CYP3A activity by 42%, 87%, and 50%, respectively. Kratom extracts at 20 µg/mL inhibited these activities by 81-88%, 87-93%, and 86-89%, respectively.  

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1010191

400 µL

10 min

NADPH

Available

Available

4 µM

2, 10, and 20 µg/ml

7 . Screening of mitragynine as inhibitors of CYP2C9 activity in HLM (id=NPDI-bOmVTQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

diclofenac 1124300

mitragynine

4'-hydroxydiclofenac

  • CYP2C9 4309227

Cell fraction Pooled human liver microsomes -7999662

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP activity in HLM. Mitragynine at 10 µM inhibited CYP2C9, CYP2D6, and CYP3A activity by 42%, 87%, and 50%, respectively. Kratom extracts at 20 µg/mL inhibited these activities by 81-88%, 87-93%, and 86-89%, respectively.  

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1010191

400 µL

10 min

NADPH

Available

Available

4 µM

1, 10, and 100 µM

8 . IC50 shift determination for mitragynine towards CYP2C9 activity in HLM with NADPH (id=NPDI-Xrn4xQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

diclofenac 1124300

mitragynine

4'-hydroxydiclofenac

  • CYP2C9 4309227

Cell fraction Pooled human liver microsomes -7999662

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1010191

NADPH

Available

Available

4 µM

250 µL

30 min

NADPH with precipitant

200 µL

10 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP2C9 activity in HLM without NADPH)

9 . IC50 shift determination for mitragynine towards CYP2C9 activity in HLM without NADPH (id=NPDI-xK3lmw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

diclofenac 1124300

mitragynine

4'-hydroxydiclofenac

  • CYP2C9 4309227

Cell fraction Pooled human liver microsomes -7999662

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1010191

NADPH

Available

Available

4 µM

250 µL

30 min

NADPH with no precipitant

200 µL

10 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP2C9 activity in HLM with NADPH)

10 . Screening of kratom extracts as inhibitors of CYP3A activity in HLM (id=NPDI-7-Gg-g)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

midazolam 708298

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Three methanolic Kratom extracts were tested, K-50, K-51 and K-52.

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP activity in HLM. Mitragynine at 10 µM inhibited CYP2C9, CYP2D6, and CYP3A activity by 42%, 87%, and 50%, respectively. Kratom extracts at 20 µg/mL inhibited these activities by 81-88%, 87-93%, and 86-89%, respectively.  

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1010191

400 µL

2 min

NADPH

Available

Available

2 µM

2, 10, and 20 µg/ml

11 . IC50 shift determination for mitragynine towards CYP3A activity in HLM with NADPH (id=NPDI-4_UdSg)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

 

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

A 7-fold shift in IC50was observed towards CYP3A activity in HLM, 18.9 ±1.8 vs. 2.6 ±0.3 µM, in absence and presence of NADPH, respectively. [IC50values (Mean ± SEM) were determined using nonlinear least-squares regression]

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1010191

NADPH

Available

Available

2 µM

250 µL

30 min

NADPH with precipitant

200 µL

2 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP3A activity in HLM without NADPH)

12 . Inhibition kinetics of CYP3A activity by mitragynine in HIM (id=NPDI-Tr3Eyg)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

N/A

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human intestinal microsomes -7999663

Results

The KIand kinactvalues for mitragynine towards CYP3A activity in HIM were 4.5  ± 1.1 µM and 0.072  ± 0.006 min-1,  6.0  ± 0.5 µM and 0.074  ± 0.002 min-1, and  7.3  ± 2.5 µM and 0.17  ± 0.02 min-1, respectively, for three individual experiments.[KIand kinact(Mean ± SEM) values were determined using nonlinear least-squares regression.]

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. 6',7'-Dihydroxybergamottin (1 and 2 µM) served as a positive control inhibitor of CYP3A activity.

NA

0.05 mg/mL

Commercially available

1610314

NADPH

Available

Available

20 µM

1100 µL

0, 2, 5, 10, and 20 min

NADPH with precipitant

200 µL

5 min

No dilution

0.56 - 30 µM

13 . Screening of mitragynine as inhibitors of CYP3A activity in HLM (id=NPDI-ov597g)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP activity in HLM. Mitragynine at 10 µM inhibited CYP2C9, CYP2D6, and CYP3A activity by 42%, 87%, and 50%, respectively. Kratom extracts at 20 µg/mL inhibited these activities by 81-88%, 87-93%, and 86-89%, respectively.  

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1010191

400 µL

2 min

NADPH

Available

Available

2 µM

1, 10, and 100 µM

14 . Inhibition kinetics of CYP3A activity by mitragynine in HLM (id=NPDI-515DHw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

N/A

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

The KIand kinactvalues for mitragynine towards CYP3A activity in HLM were 4.1  ± 0.7 µM and 0.051  ± 0.002 min-1,  4.0  ± 0.5 µM and 0.057  ± 0.002 min-1, and  5.9  ± 0.9 µM and 0.17  ± 0.01 min-1, respectively, for three individual experiments.
[KIand kinact(Mean ± SEM) values were determined using nonlinear least-squares regression.]

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. 6',7'-Dihydroxybergamottin (1 and 2 µM) served as a positive control inhibitor of CYP3A activity.

NA

0.05 mg/mL

Commercially available

1010191

NADPH

Available

Available

20 µM

1100 µL

0, 2, 5, 10, and 20 min

NADPH with precipitant

200 µL

5 min

No dilution

0.56 - 30 µM

15 . IC50 shift determination for mitragynine towards CYP3A activity in HLM without NADPH (id=NPDI-EjMg0g)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

A 7-fold shift in IC50was observed towards CYP3A activity in HLM, 18.9 ±1.8 vs. 2.6 ±0.3 µM, in presence and absence of NADPH, respectively. [IC50values (Mean ± SEM) were determined using nonlinear least-squares regression]

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1010191

NADPH

Available

Available

2 µM

250 µL

30 min

NADPH with no precipitant

200 µL

2 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP3A activity in HLM with NADPH)

16 . Screening of kratom extracts as inhibitors CYP3A activity in HIM (id=NPDI-QbMPuw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human intestinal microsomes -7999663

Three methanolic Kratom extracts were tested, K-50, K-51 and K-52.

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP3A activity in HIM. Mitragynine at 10 µM inhibited CYP3A activity by 44%. Kratom extracts at 20 µg/mL inhibited CYP3A activity by 82-90%. 

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1610314

400 µL

10 min

NADPH

Available

Available

2 µM

2, 10, 20 µg/mL

17 . Screening of mitragynine as inhibitors CYP3A activity in HIM (id=NPDI-vr5BAA)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

50% inhibition at the highest tested concentration

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human intestinal microsomes -7999663

Results

Mitragynine and kratom extracts showed concentration-dependent inhibition of CYP3A activity in HIM. Mitragynine at 10 µM inhibited CYP3A activity by 44%. Kratom extracts at 20 µg/mL inhibited CYP3A activity by 82-90%. 

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Methanol (0.8 % v/v) served as solvent control. Sulphaphenazole (1 µM), quinidine (2 µM), and ketoconazole (0.01 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A, respectively.

NA

0.05 mg/mL

Commercially available

1610314

400 µL

10 min

NADPH

Available

Available

2 µM

1, 10, and 100 µM

18 . IC50 shift determination for mitragynine towards CYP3A activity in HIM with NADPH (id=NPDI-A66pwQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human intestinal microsomes -7999663

Results

A 7-fold shift in IC50was observed towards CYP3A activity in HIM, 21.9 ±2.7 vs. 3.2 ±0.3 µM, in absence and presence of NADPH, respectively. [IC50values (Mean ± SEM) were determined using nonlinear least-squares regression]

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1610314

NADPH

Available

Available

2 µM

250 µL

30 min

NADPH with precipitant

200 µL

10 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP3A activity in HIM without NADPH)

19 . IC50 shift determination for mitragynine towards CYP3A activity in HIM without NADPH (id=NPDI-o8WmjA)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used —

IC50<10 µM for reversible inhibition and IC50shift 1.5 fold for time dependent inhibition

midazolam 708298

mitragynine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human intestinal microsomes -7999663

Results

A 7-fold shift in IC50was observed towards CYP3A activity in HIM, 21.9 ±2.7 vs. 3.2 ±0.3 µM, in presence and absence of NADPH, respectively. [IC50values (Mean ± SEM) were determined using nonlinear least-squares regression]

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

A cocktail of probe substrates for CYP2C9 (diclofenac), CYP2D6 (dextromethorphan), and CYP3A (midazolam) was used for the IC50shift determination.
Methanol (0.8 % v/v) served as solvent control. Tienilic acid (0.4 and 0.8 µM), paroxetine (0.25 and 0.5 µM), and 6',7'-dihydroxybergamottin (1 and 2 µM) served as positive control inhibitors of CYP2C9, CYP2D6, and CYP3A activities, respectively.

NA

0.05 mg/mL

Commercially available

1610314

NADPH

Available

Available

4 µM

250 µL

30 min

NADPH with no precipitant

200 µL

10 min

No dilution

0.015 - 100 µM

IC50 Shift Related Experiment Information (IC50 shift determination for mitragynine towards CYP3A activity in HIM with NADPH)