Human Liver Microsomes-200 Donors

Determination of Contribution of CYP Enzymes in Mitragynine Metabolism Using rCYPs and HLM

Mitragynine is the major indole-based alkaloid of Mitragyna speciosa (kratom). Kratom has become increasingly utilized for mood elevation, pain treatment, and as a means of self-treating opioid addiction. There have been reports of deaths or toxic events associated with the use of kratom. However, information on the metabolic pathways and related enzymes of mitragynine is limited. Kamble et al. aimed to investigate the metabolic characteristics of mitragynine in human liver microsomes (HLM) (200 donors: 100 male and 100 female).

Kamble et al. first analyzed the metabolite profile of mitragynine in HLM using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Subsequently, they performed cytochrome P450 (CYP) reaction phenotyping using recombinant CYP enzymes (rCYPs) and HLM. When incubated with various CYP enzymes, mitragynine was significantly metabolized by rCYP3A4, with minimal metabolism by other CYPs (Fig. 1). Similarly, ketoconazole (a potent CYP3A4 and CYP3AS inhibitor) and CYP3cide (a selective CYP3A4 inhibitor) substantially inhibited mitragynine metabolism in HLM incubations (Fig. 2), indicating that CYP3A4 plays predominant role in the metabolism of mitragynine. In addition to studying mitragynine metabolism, they investigated how various CYP enzymes contribute to the formation of specific primary metabolites (Met2, Met4, Met5, Met6, Met7, Met8, and Met11) in both rCYPs and HLM. Ketoconazole and CYP3cide significantly inhibited the formation of Met2, Met4, Met5, Met6, and Met11 by 80%, indicating a major role for CYP3A4 in their formation (Fig. 1 and 2). Conversely, quinidine, a CYP2D6 inhibitor, significantly hindered the formation of Met7 and Met8, which aligns with rCYP2D6 data, suggesting CYP2D6's predominant role. The CYP2B6 inhibitor (PPP) also inhibited Met7 and Met8 formation due to cross-reactivity with CYP2D6.

Fig. 1. The first order disappearance of mitragynine and metabolite formation (%) of mitragynine in the presence of various recombinant cytochrome P450 (rCYPs) (Kamble, S. H., Sharma, A., et al., 2019).

Fig. 2. Mitragynine metabolism and metabolite formation (%) of mitragynine in the presence of selective chemical inhibitors of CYPs in human liver microsomes (Kamble, S. H., Sharma, A., et al., 2019).

Identification of Metabolites of EPT, 4-OH-EPT, and 5-MeO-EPT after Incubation with pHLM

N-Ethyl-N-propyltryptamine (EPT), 4-Hydroxy-N-ethyl-N-propyltryptamine (4-OH-EPT), and 5-Methoxy-N-ethyl-N-propyltryptamine (5-MeO-EPT) are new psychoactive substances classified as tryptamines. EPT is known to form metabolites by indole ring hydroxylation among others, but the metabolism of 4-OH-EPT and 5-MeO-EPT has not been reported. Many tryptamines metabolize rapidly, and identifying the appropriate metabolites to reveal intake is essential. Therefore, Bergh et al. aimed to identify the major metabolites of EPT, 4-OH-EPT, and 5-MeO-EPT by incubating them with pooled human liver microsomes (pHLM).

The six to seven most abundant metabolites of EPT, 4-OH-EPT, and 5-MeO-EPT formed after 60 min, but also detectable after 240 min incubation with pHLM, were characterized to potentially serve as metabolite markers in forensic case work. Possible metabolite structures were elucidated by comparing the fragmentation patterns of the parent compounds with those of the metabolites. Incubation of EPT with pHLM resulted in the formation of the seven most abundant metabolites, and the proposed structure is shown in Figure 3A. The metabolite structure formed by incubation of 5-MeO-EPT with pHLM is shown in Figure 3B. The metabolite structure formed by incubation of 4-OH-EPT with pHLM is shown in Figure 3C.

Fig. 3. Main metabolites of PET (A), 5-MeO-EPT (B) and 4-OH-EPT (C) identified after incubation with pHLM (Bergh M S, Bogen I L, et al., 2024).