Kratom (Mitragyna speciosa)
Evaluation of In Vitro Absorption, Distribution, Metabolism, and Excretion (ADME) Properties of Mitragynine, 7-Hydroxymitragynine, and Mitraphylline
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Mitragyna speciosa (kratom) is a popular herb in Southeast Asia, which is traditionally used to treat withdrawal symptoms associated with opiate ad- diction. Mitragynine, 7-hydroxymitragynine, and mitraphylline are reported to be the central nervous system active alkaloids which bind to the opiate receptors. Mitraphylline is also present in the bark of Uncaria tomentosa (catʼs claw). Several therapeutic properties have been reported for these compounds but limited information is available on the absorption and distribution properties. This study focuses on evaluating the absorption, distribution, metabolism, and excretion (ADME) properties of these compounds and their effect on major efflux transporter P-glycoprotein, using in vitro methods. Quantitative analysis was performed by the Q‐TOF LC‐MS system. Mitragynine was unstable in simulated gastric fluid with 26% degradation but stable in simulated intestinal fluid. 7-Hydroxymitragynine degraded up to 27% in simulated gastric fluid, which could account for its conversion to mitragynine (23%), while only 6% degradation was seen in simulated intestinal fluid. Mitraphylline was stable in simulated gastric fluid but unstable in simulated intes- tinal fluid (13.6% degradation). Mitragynine and 7-hydroxymitragynine showed moderate permeability across Caco-2 and MDR-MDCK monolayers with no significant efflux. However, mitraphylline was subjected to efflux mediated by P-glycoprotein in both Caco-2 and MDR-MDCK monolayers. Mitragynine was found to be metabolically stable in both human liver microsomes and S9 fractions. In contrast, both 7-hydroxymitragynine and mitraphylline were metabolized by human liver microsomes with half-lives of 24 and 50min, re-
spectively. All three compounds exhibited high plasma protein binding (>90%) determined by equilibrium dialysis. Mitragynine and 7-hydroxymitragynine inhibited P-glycoprotein with EC50 values of 18.2 ± 3.6 μM and 32.4 ± 1.9 μM, respectively, determined by the calcein-AM fluorescent assay, while no inhibition was seen with mitra- phylline. These data indicate the possibility of a drug interaction if mitragynine and 7-hydroxy- mitragynine are coadministered with drugs that are P-glycoprotein substrates.
1 . 7-Hydroxymitragynine P-gp Inhibition (id=NPDI-_5ld1Q)
In Vitro Transporter Inhibition Experiment
Inhibition was detected.
Cutoff used —
EC50 positive control verapamil (22.3± 1.4 μM)
EC50 positive control verapamil (22.3± 1.4 μM)
- P-gp (ABCB1)
Transfected / siRNA knock-out / injected cells MDCK transfected cells
Results
positive control verapamil (22.3± 1.4 μM)
Fig 1S (with exact values in text pg 574)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
70 000 cells/well
200 μL
Transwell
2
30-42
>1100 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5 uM
5 uM
ChromaDex
2 . Inhibition of P-gp by Mitragynine (id=NPDI-mJ7zbw)
In Vitro Transporter Inhibition Experiment
Inhibition was detected.
Cutoff used —
positive control verapamil (22.3± 1.4 μM)
positive control verapamil (22.3± 1.4 μM)
- P-gp (ABCB1)
Transfected / siRNA knock-out / injected cells MDCK transfected cells
Results
positive control verapamil (22.3± 1.4 μM)
Fig 1S (with exact values in text pg 574)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
70 000 cells/well
200 μL
Transwell
2
30-42
>1100 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5 uM
5 uM
ChromaDex
1 . Mitraphylline P-gp transport kinetics (id=NPDI-ruor3g)
In Vitro Transporter Kinetics Experiment
Transport Activity was detected. Cutoff used — ER of 2
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
ER for 10 uM is 3.4
p<0.001
(Table 3)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
70 000 cells/well
200 μL
Transwell
2
30–42
>400 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5,10 uM
2 . Mitraphylline p-gp transport kinetics MDCK (id=NPDI-2HCqSQ)
In Vitro Transporter Kinetics Experiment
Transport Activity was detected. Cutoff used — ER of 2
- P-gp (ABCB1)
Transfected / siRNA knock-out / injected cells MDCK transfected cells
Results
p<0.001
ER for 10 uM was 5.9 also significant
(Table 4)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
70 000 cells/well
200 μL
Transwell
2
30–42
>1100 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5,10 uM
3 . 7-hyrdoxymitragynine P-gp Transport kinetics (id=NPDI-TjrSew)
In Vitro Transporter Kinetics Experiment
No Transport Activity was detected. Cutoff used — ER of 2
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
ER for 10 uM also 1.2
(Table 3)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
70 000 cells/well
200 μL
Transwell
2
30–42
>400 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5,10 uM
4 . 7-Hydroxymitragynine p-gp transport kinetics MDCK (id=NPDI-iGAFkQ)
In Vitro Transporter Kinetics Experiment
No Transport Activity was detected. Cutoff used — 2
- P-gp (ABCB1)
Transfected / siRNA knock-out / injected cells MDCK transfected cells
Results
ER for 10 uM also 1.2
(Table 4)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
7 x 10^4 cells/well
200 μL
Transwell
2
30–42
>1100 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5,10 uM
5 . Mitragynine P-gp transport activity (id=NPDI-2VLnZA)
In Vitro Transporter Kinetics Experiment
No Transport Activity was detected. Cutoff used — ER of 2
- P-gp (ABCB1)
Cell system Caco-2 cells
Results
Results above for 5 uM
ER for 10 uM values reported: 1.1 N=3
(Table 3)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
70 000 cells/well
200 μL
Transwell
2
30–42
>400 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5,10 uM
6 . Mitragynine p-gp transport kinetics MDCK (id=NPDI-yG9O9A)
In Vitro Transporter Kinetics Experiment
No Transport Activity was detected. Cutoff used — ER of 2
- P-gp (ABCB1)
Transfected / siRNA knock-out / injected cells MDCK transfected cells
Results
ER for 10 uM also 1.1
(Table 4)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
70 000 cells/well
200 μL
Transwell
2
30–42
>1100 ohm/cm2
TEER measurements and Ly permeability.
120 min
37 deg C
7.4
5,10 uM