Physicochemical Characterization of Mitragyna speciosa Alkaloid Extract and Mitragynine using In Vitro High Throughput Assays.

AIM AND OBJECTIVE: Mitragynine, a major active alkaloid of Mitragyna speciosa, acts as an agonist on µ-opioid receptors, producing effects similar to morphine and other opioids. It has been traditionally utilized to alleviate opiate withdrawal symptoms. Besides consideration about potency and selectivity, a good drug must possess a suitable pharmacokinetic profile, with suitable absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) profile, in order to have a high chance of success in clinical trials. MATERIAL AND METHOD: The purity of mitragynine in a Mitragyna speciosa alkaloid extract (MSAE) was determined using Ultra-Fast Liquid Chromatography (UFLC). In vitro high throughput ADMETox studies such as aqueous solubility, plasma protein binding, metabolic stability, permeability and cytotoxicity tests were carried out to analyze the physicochemical properties of MSAE and mitragynine. The UFLC quantification revealed that the purity of mitragynine in the MSAE was 40.9%. RESULTS: MSAE and mitragynine are highly soluble in aqueous solution at pH 4.0 but less soluble at pH 7.4. A parallel artificial membrane permeability assay demonstrated that it is extensively absorbed through the semi-permeable membrane at pH 7.4 but very poorly at pH 4.0. Both are relatively highly bound to plasma proteins (> 85 % bound) and are metabolically stable to liver microsomes (> 84 % remained unchanged). In comparison to MSAE, mitragynine showed higher cytotoxicity against WRL 68, HepG2 and Clone 9 hepatocytes after 72 h treatment. CONCLUSION: The obtained ADME and cytotoxicity data demonstrated that both MSAE and mitragynine have poor bioavailability and have the potential to be significantly cytotoxic.

Evaluation of pharmacokinetics and blood-brain barrier permeability of mitragynine using in vivo microdialysis technique.

A microdialysis system coupled with a sensitive ultra-fast liquid chromatography-mass spectrometry (UFLC-MS) method was developed for the pharmacokinetic analysis of mitragynine in rat blood and striatum. Mitragynine is an active alkaloid of Mitragyna speciosa and has been proposed to be used for opioid withdrawal therapy. In this study, chromatographic separation was performed in a gradient elution mode with 0.1% formic acid and acetonitrile on a Zorbax Eclipse C18 column. The mass spectrometric (MS) analysis was carried out in a positive electrospray mode and mitragynine ion (m/z 399.2) was monitored in extracted ion chromatography. A good linearity range was obtained from 10-1000ng/mL with acceptable accuracy and precision parameters. The microdialysate was collected simultaneously from the striatum and the right jugular vein using microdialysis probes. After a single intravenous administration of 10mg/kg mitragynine, mitragynine showed a two-compartmental drug elimination pattern with half-life (T1/2) of approximately 13h. The percent of AUCbrain/AUCplasma of mitragynine was calculated and shown to be 65.8±4.5%. The results indicated that mitragynine could be a suitable molecule to develop into an opioid replacement drug based on its ideal pharmacokinetic properties, namely, small molecular size, lipophilic in nature and with excellent blood-brain barrier (BBB) permeability.

Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay.

The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.