Oxidative Metabolism as a Modulator of Kratom's Biological Actions

Abstract

The leaves of Mitragyna speciosa (kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitro and in vivo pharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom's major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.

Figure 1.

(A) Kratom alkaloids; major alkaloid natural product (B) metabolites of mitragynine in mice.

Figure 2.

Kratom, mitragynine, and its metabolites show MOR-dependent antinociception with reduced constipation except for 7OH but retain reinforcing behavior except for MP in C57BL/6J mice. Oral tail flick dose–response curves (A,B): groups of mice (n = 8) per dose were given different doses of po kratom (A) or po mitragynine (B) by gavage and tested for the antinociceptive response at 20 min. ED₅₀ (and 95% CI) was 17.1 (11.87–23.92) mg/kg for po kratom and 6.25 (4.49–8.68) mg/kg for po mitragynine. Subcutaneous tail flick dose–response curves (C,D): Groups of mice (n = 8) per dose were given different doses of sc 7OH and sc morphine (C) and sc MP and sc morphine (D) and tested for antinociceptive response at 20 min for 7OH and MP and 30 min for morphine. ED₅₀ (and 95% CI) was 0.3 (0.19–0.48) mg/kg for sc 7OH, 0.26 (0.15–0.43) mg/kg for sc MP, and 2.48(1.57–3.87) mg/kg for sc morphine. The means of each point in each determination were calculated as percentage maximal possible effect (%MPE) [(observed latency − baseline latency)/(maximal latency − baseline latency)] × 100. Kratom alkaloid fraction, mitragynine showed antinociception given orally while the putative metabolites 7OH and MP showed antinociception given sc and about 10 fold more potent over sc morphine. Antinociception in KO mice (E–H): An ~ED₈₀ antinociceptive dose of alkaloid extract (45 mg/kg, po), mitragynine (45 mg/kg, po), 7OH (1 mg/kg, sc), and MP (3 mg/kg, sc) was evaluated in group of (n = 8) of wildtype (WT), MOR KO, KOR KO, and DOR KO mice. Antinociceptive effects of all these drugs were found attenuated in MOR KO, whereas the effect was found intact in KOR KO and DOR KO mice (****p < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison comparisons test). Effects on GI transit in C57BL/6J mice (I–L): a group of C57BL/CJ mice (n = 8) were administered with either saline, morphine (10 mg/kg, sc), alkaloid extract (85.5 mg/kg, po), mitragynine (20 mg/kg, po) 7OH (1.5 mg/kg, sc), or MP (0.76, 1.5, 4 mg/kg, sc) and then were fed a charcoal meal. After 3 h, morphine significantly reduced the distance traveled by the charcoal through the intestines, consistent with the action of a MOR agonist (5.07 ± 0.57 cm), compared to 33 ± 0.68 cm for saline-treated mice (****p < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison test). In contrast, the alkaloid extract (24.6 ± 2.54 cm) showed constipation compared to saline (*p < 0.0019, one-way ANOVA followed by Dunnett’s multiple comparison test) but less than morphine, while mitragynine (34.7 ± 0.53 cm) showed no significant effect (I,J). 7OH (1.5 mg/kg, sc) showed significant reduction in the distance traveled by the charcoal (11.6 ± 1.8 cm) through the intestines (****p < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparison test) (K). MP showed constipation at all doses (*p = 0.016, ***p = 0.001, **p = 0.005, one way ANOVA followed by Dunnett’s multiple comparison test) compared to saline but less than morphine. Conditioned place preference or aversion in C57BL/CJ mice (CPP/CPA) (M–P): A group of C57BL/CJ mice (n = 18–24) were habituated to freely explore both sides of a two-compartment apparatus for 3 h each for 2 days prior testing. The baseline preferences of 20 min were determined as the pre-condition test prior to the administration of a drug on the conditioning day. Mice were conditioned for 20 min on each session with either saline, morphine (10 mg/kg/d, sc), U50, 488H (30 mg/kg/d, sc), alkaloid extract (100 mg/kg/d), mitragynine (30 mg/kg/d) 7OH (1 mg/kg/d, sc), and MP (3.2 mg/kg/d, sc) after being habituated for 1 h. On the testing day, animals were placed in the side paired with saline and are allowed to freely explore both compartments for 20 min. The difference score was calculated to determine CPP/CPA. MP did not display significant CPP or CPA, whereas kratom alkaloid fraction, mitragynine, and 7OH displayed CPP (M–P); (p < 0.05) as determined by ANOVA followed by Tukey’s multiple-comparison test. Note: Figure 2F,G; data reprinted (adapted or reprinted in part) with permission from [Kruegel, A. C.; Uprety, R.; Grinnell, S. G.; Langreck, C.; Pekarskaya, E. A.; Le Rouzic, V.; Ansonoff, M.; Gassaway, M. M.; Pintar, J. E.; Pasternak, G. W.; Javitch, J. A.; Majumdar, S.; Sames, D. 7-Hydroxymitragynine Is an Active Metabolite of Mitragynine and a Key Mediator of Its Analgesic Effects. ACS Cent. Sci. 2019, 5 (6), 992–1001.] Copyright [2019/American Chemical Society]. Figure 2L,P data reprinted (adapted or reprinted in part) with permission from [Váradi, A.; Marrone, G. F.; Palmer, T. C.; Narayan, A.; Szabó, M. R.; Le Rouzic, V.; Grinnell, S. G.; Subrath, J. J.; Warner, E.; Kalra, S.; Hunkele, A.; Pagirsky, J.; Eans, S. O.; Medina, J. M.; Xu, J.; Pan, Y. X.; Borics, A.; Pasternak, G. W.; McLaughlin, J. P.; Majumdar, S. Mitragynine/Corynantheidine Pseudoindoxyls As Opioid Analgesics with Mu Agonism and Delta Antagonism, Which Do Not Recruit β-Arrestin-2. J. Med. Chem. 2016, 59 (18), 8381–8397.] Copyright [2016/American Chemical Society].

Figure 3.

Concentration time profile for mitragynine to its metabolites was confirmed in vivo in C57BL/6 mice using LC–MS/MS. (A) Mitragynine was detected in both the plasma and brains of mice treated with mitragynine (30 mg/kg, po). n = 3 per time point for plasma; n = 3 per time point for brain. At the same time gradient, metabolites formed from mitragynine, namely, 7-OH (B), MP (C), 3DM (D), 9OH (E), and 9G-mitragynine (F) were also detected in the plasma and brains of the same animals, but at lower concentrations. 7OH was the major metabolite followed by MP and 3DM as other important metabolites.

Figure 4.

Metabolism of mitragynine to 7OH, MP, and 3DM in liver microsomes: mitragynine (1 μM) was incubated in the potassium phosphate buffer (0.1 M; pH 7.4) at 37 °C in the presence of liver microsomes of mice (129Sv6 and C57BL/6J) or human (HLM) for 60 min. Each point in the curve represents three independent determinations. Level of intact mitragynine decreases over time due to microsomal disintegration. The half-lives of mitragynine were 7.5 ± 0.82, 5.9 ± 0.3, and 13.5 ± 1.1 min in HLM, 129Sv6, and C57BL/6J mice liver microsomes. (A). Usage of a CYP3A blocker ketoconazole (1 μM). The half-lives of mitragynine in the presence of ketoconazole were 37.4 ± 7.5, 24.4 ± 4.9, and 35.8 ± 4 min in HLM, 129Sv6, and C57BL/6J mice liver microsomes (B). Liver microsomes from a Cyp3a KO male mice also inhibit the depletion of mitragynine. The half-life of mitragynine was 25.2 ± 2.05 min in Cyp3a KO mice microsomes (C). These liver microsomal assays indicated the formation of three major metabolites 7OH, MP, and 3DM (D–F). A decrease in the formation metabolites from mitragynine including 7OH and MP was observed in the presence of ketoconazole at the peak effect compared to control, where ketoconazole was not included in microsomal incubations (p < 0.05 as determined by unpaired t-test) (G–H). The formation of 3DM was found to be independent of CYP3A (I).

Figure 5.

Metabolism of mitragynine to 7OH, MP, and 3DM in HLM and S9 fractions in the presence and absence of NADPH: mitragynine (1 μM) was incubated in the potassium phosphate buffer (0.1 M; pH 7.4) at 37 °C in the presence of HLM for 60 min. Level of intact mitragynine decreases over time due to microsomal disintegration. (A). In the presence of NADPH in HLM, concentration of mitragynine decreases with formation of 7OH, MP; 7OH being the major metabolite. (B) In the absence of NADPH in HLM, the depletion of mitragynine was inhibited with minimal formation of 7OH and MP, while formation of 3DM is less effected. (C) In the presence of NADPH in S9, the fraction concentration of mitragynine decreases with formation of 7OH, MP, and 3DM; 7OH being the major metabolite. (D) A decrease in the formation metabolites from mitragynine including 7OH and MP was observed in S9 fraction in the absence of NADPH, while 3DM levels were less effected.

Figure 6.

GPRome screening of 7OH and 3DM: 7OH and 3DM were screened against 318 non-olfactory GPCRs for agonism in the β-arrestin2 recruitment assay PRESTO-Tango. Each point shows change in the luminescence signal as fold-of-basal at a given GPCR with (A) 10 μM 7OH and (B) 10 μM 3DM. For clarity, GPCRs are listed alphabetically along the X-axis, and every 10th receptor is labeled. A full list of receptors is reported previously (Kroeze et al. PRESTO-Tango Nat. Struc. Mol. Bio. 2015). Both 7OH and 3DM induce increases in signal >3-fold of basal at opioid receptors. Non-opioid receptor screening hits (C) BB3, (D) CXCR7, and (E) CCR3 were tested against a concentration–response of 3DM, 7OH, and reference (if available) using PRESTO-Tango. Experiments were performed in triplicate and independently replicated three times.

Figure 7.

Detection of metabolic product 3-dehydromitragynine in brain and plasma in mice and its toxicity. Groups of mice (n = 6 per dose) were given different doses of sc 3DM to 129Sv6 mice (A) or CD1 mice (B) and tested for lethality. Similarly, C57BL/6J mice (n = 4) at 50 mg/kg, sc and MOR KO mice (n = 4) (C) at 50 mg/kg, sc and C57BL/6J mice pretreated with norBNI (10 mg/kg, sc; n = 15) for 24 h and NTI (20 mg/kg, sc; n = 12) for 15 min were tested for lethality. While 3-dehydromitragynine was not an antinociceptive agent, it was found to be toxic with a LD₅₀ of 48.4 (20.85–129.8) mg/kg in 129Sv6 (A) and 74 (48.08–120.5) mg/kg in CD1 mice (B). It caused 100% lethality at 50 mg/kg, sc in C57BL/6J mice (C) and at 50 mg/kg, sc in MOR KO mice, suggesting that the toxicity is not MOR-mediated. Similarly, in the presence of KOR antagonist (nor-BNI) and DOR antagonist (NTI), the lethality was still observed. Results suggest lethality is opioid receptor-independent. The metabolite 3DM was detected at 0.035 ± 0.01 and 0.09 ± 0.025 μM in the brain of 129Sv6 and C57BL/6J mice when mice were dosed at ED₈₀ po antinociceptive doses of mitragynine (D). Similarly, about 0.0275 ± 0.01 and 0.037 ± 0.01 μM were seen in plasma of 129Sv6 and C57BL/6J mice (D).

Scheme 1.

Synthesis of Mitragynine Metabolites and Mitragynine N-Oxide