(M1230-02-12) Metabolite Identification of a Kratom Alkaloid Speciogynine in Human Liver Microsomes and Hepatocytes

Purpose: Mitragyna speciosa Korth, widely known as kratom, is a medicinal plant indigenous to Southeast Asia. It has been used traditionally for the treatment of pain and opioid use disorder^1,2,3. Speciogynine, a diastereomer of mitragynine, is an indole-based alkaloid and is the third major alkaloid (5-10%) present in the plant⁴. Speciogynine was found to be a weak antagonist of µ (MOR) opioid receptors and high affinity toward serotonin receptors, in contrast with its major diastereomer, mitragynine, and exhibited antinociceptive properties in rats via an opioid receptor-independent mechanism2. The purpose of this study was to evaluate the metabolism of speciogynine in human liver microsomes and hepatocytes for its further development as a non-opioid treatment of pain.

Methods: Speciogynine (30 µM) was incubated in mixed-gender pooled human liver microsomes (1 mg/mL) in 50 mM phosphate buffer (pH 7.4) containing 3 mM of GSH and 1 mM NADPH. For both phase I and II metabolite identification, speciogynine (30 µM) was incubated in mixed-gender pooled human hepatocytes (1 million cells/mL) in Invitrogro KHB buffer (pH 7.4). Aliquots (50 µL) were withdrawn at 0 and 60 min and protein precipitation was performed by the addition of an equal volume of ice-cold acetonitrile containing 0.1% v/v formic acid to stop the reaction and filtered through 0.45 µM filter plates. Filtrates were then analyzed on the Agilent 6540 instrument to obtain the LC-DAD-Q-TOF data.

Results: A total of 31 putative metabolites of speciogynine were identified. 9-O-demethyl speciogynine and 7-hydroxyspeciogynine were found as major metabolites based on relative UV intensity and by confirming with reference standards. Glutathione conjugation, O-demethylation of either at 9-methoxyindole or the β-methoxyacrylate position, and the hydrolysis of the methyl acrylate ester of speciogynine is the other major metabolic pathway. Multiple minor metabolites including monooxidative, di-oxidative, mono or di-dehydrogenated, monooxidative products of demethylated or hydrolyzed metabolites were identified in liver microsomes and hepatocytes.

Conclusion: Speciogynine underwent extensive metabolism, primarily through monooxidation and O-demethylation metabolic pathways in liver microsomes and hepatocytes. A major metabolite of speciogynine, 9-O-demethyl speciogynine has also shown activity on serotonin receptors, and further studies are required to understand the contribution of active metabolite in the overall therapeutic activity of speciogynine.

References: 1. Kamble, S. H., Sharma, A., King, T. I., León, F., McCurdy, C. R., & Avery, B. A. (2019). Metabolite profiling and identification of enzymes responsible for the metabolism of mitragynine, the major alkaloid of Mitragyna speciosa (kratom). Xenobiotica, 49(11), 1279–1288. https://doi.org/10.1080/00498254.2018.1552819
2. León F, Obeng S, Mottinelli M, Chen Y, King TI, Berthold EC, Kamble SH, Restrepo LF, Patel A, Gamez-Jimenez LR, Lopera-Londoño C, Hiranita T, Sharma A, Hampson AJ, Canal CE, McMahon LR, McCurdy CR. Activity of Mitragyna speciosa ("Kratom") Alkaloids at Serotonin Receptors. J Med Chem. 2021 Sep 23;64(18):13510-13523. doi: 10.1021/acs.jmedchem.1c00726. PMID: 34467758.
3. Kamble SH, Berthold EC, Kanumuri SRR, King TI, Kuntz MA, León F, Mottinelli M, McMahon LR, McCurdy CR, Sharma A. Metabolism of Speciociliatine, an Overlooked Kratom Alkaloid for its Potential Pharmacological Effects. AAPS J. 2022 Jul 19;24(5):86. doi: 10.1208/s12248-022-00736-8. PMID: 35854066.
4. Philipp AA, Wissenbach DK, Weber AA, Zapp J, Maurer HH. Phase I and II metabolites of speciogynine, a diastereomer of the main Kratom alkaloid mitragynine, identified in rat and human urine by liquid chromatography coupled to low- and high-resolution linear ion trap mass spectrometry. J Mass Spectrom. 2010 Nov;45(11):1344-57. doi: 10.1002/jms.1848. PMID: 20967737.