Kratom (Mitragyna speciosa)
The inhibitory effects of mitragynine on P-glycoprotein in vitro CSV JSON

Mitragynine is a major component isolated from Mitragyna speciosa Korth or kratom, a medicinal plant known for its opiate-like and euphoric properties. Multiple toxicity and fatal cases involving mitragynine or kratom have been reported but the underlying causes remain unclear. P-glycoprotein (P-gp) is a multidrug transporter which modulates the pharmacokinetics of xenobiotics and plays a key role in mediating drug-drug interactions. This study investigated the effects of mitragynine on P-gp transport activity, mRNA, and protein expression in Caco-2 cells using molecular docking, bidirectional assay, RT-qPCR, Western blot analysis, and immunocytochemistry techniques, respectively. Molecular docking simulation revealed that mitragynine interacts with important residues at the nucleotide binding domain (NBD) site of the P-gp structure but not with the residues from the substrate binding site. This was consistent with subsequent experimental work as mitragynine exhibited low permeability across the cell monolayer but inhibited digoxin transport at 10 μM, similar to quinidine. The reduction of P-gp activity in vitro was further contributed by the downregulation of mRNA and protein expression of P-gp. In summary, mitragynine is likely a P-gp inhibitor in vitro but not a substrate. Hence, concurrent administration of mitragynine-containing kratom products with psychoactive drugs which are P-gp substrates may lead to clinically significant toxicity. Further clinical study to prove this point is needed.

Kratom (Mitragyna speciosa)

30604191

625786142

1 . Permeability of mitragynine across Caco-2 monolayer cells (id=NPDI-Ue8YDg)

In Vitro Transporter Kinetics Experiment

No Transport Activity was detected.  Cutoff used —

(not provided)

mitragynine

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Figure 2a for Papp ratio

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Cell Culture Conditions

100000 cells / cm^2 per insert

Transwell® polycarbonate membrane cell culture insert

21

Viability and Function

350 - 600 Ωcm^2

Assay Conditions

72 h

0.1, 1, 10 µM

Control Experiment Information

1 . Induction of P-gp protein expression by 0.001 µM rifampicin (id=NPDI-GETspg)

In Vitro Transporter Induction Experiment

Induction was detected.  Cutoff used —

p < 0.05 compared with control

rifampicin

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Protein expression

0.001 µM

72 h

Western blot

2 . Induction of P-gp protein expression by 0.01 µM rifampicin (id=NPDI-U0t5zQ)

In Vitro Transporter Induction Experiment

Induction was detected.  Cutoff used —

p < 0.05 compared with control

rifampicin

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Protein expression

0.01 µM

72 h

Western blot

3 . Induction of P-gp protein expression by 0.1 µM rifampicin (id=NPDI-gdI_mg)

In Vitro Transporter Induction Experiment

Induction was detected.  Cutoff used —

p < 0.05 compared with control

rifampicin

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Protein expression

0.1 µM

72 h

Western blot

4 . Induction of P-gp protein expression by 1 µM rifampicin (id=NPDI-4k7d3A)

In Vitro Transporter Induction Experiment

Induction was detected.  Cutoff used —

p < 0.05 compared with control

rifampicin

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Protein expression

1 µM

72 h

Western blot

5 . Induction of P-gp protein expression by 10 µM rifampicin (id=NPDI-8GmUTQ)

In Vitro Transporter Induction Experiment

Induction was detected.  Cutoff used —

p < 0.05 compared with control

rifampicin

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

Protein expression

10 µM

72 h

Western blot

6 . Down regulation of P-gp mRNA expression by 1 µM mitragynine (id=NPDI-nWIYpA)

In Vitro Transporter Induction Experiment

Down Regulation was detected.  Cutoff used —

p < 0.05 compared with control

mitragynine

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Estimate from Fig 3a

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

mRNA expression

1 µM

72 h

Reverse transcription-quantitative real-time PCR

7 . Down regulation of P-gp mRNA expression by 10 µM mitragynine (id=NPDI-DEOq7g)

In Vitro Transporter Induction Experiment

Down Regulation was detected.  Cutoff used —

p < 0.05 compared with control

mitragynine

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

Mean taken from text. SD estimated from Figure 3a.

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

mRNA expression

10 µM

72 h

Reverse transcription-quantitative real-time PCR

8 . Down regulation of P-gp mRNA expression by 1 µM quinidine (id=NPDI-ye4dPw)

In Vitro Transporter Induction Experiment

Down Regulation was detected.  Cutoff used —

p < 0.05 compared with control

quinidine

  • P-gp (ABCB1)

Cell system Caco-2 cells

Results

% Inhibition mean taken from text. SD estimated from Figure 3b.

Sample Compound measured Value Measurement Study sequence Additional information N replicates

Experimental Conditions

mRNA expression

1 µM

72 h

Reverse transcription-quantitative real-time PCR