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 a class of psychoactive chemicals known as tryptamines. EPT has been reported to form metabolites via indole ring hydroxylation and other methods, but it is unknown how 4-OH-EPT and 5-MeO-EPT are converted. Most tryptamines are quick to break down, so it's important to pinpoint which metabolites will reveal intake. 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).

These six or seven most prolific metabolites (EPT, 4-OH-EPT, and 5-MeO-EPT formed 60 min after incubation but could be detected at 240 min under pHLM) were identified as potentially useful metabolite markers for forensic analysis. Possible metabolite structures were explained by comparing the fragmentation of parent compounds with the metabolites. The seven most abundant metabolites were formed when EPT was incubated with pHLM, and the suggested structure is represented in Figure 3A. Figure 3B shows the metabolite profile from 5-MeO-EPT incubation with pHLM. Figure 3C shows the metabolite complex obtained by heating 4-OH-EPT with pHLM.

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).