In silico investigation of mitragynine and 7-hydroxymitragynine metabolism

Objective Mitragynine is the main active compound of Mitragyna speciose (Kratom in Thai). The understanding of mitragynine derivative metabolism in human body is required to develop effective detection techniques in case of drug abuse or establish an appropriate dosage in case of medicinal uses. This in silico study is based upon in vivo results in rat and human by Philipp et al. (J Mass Spectrom 44:1249–1261, 2009). Results Gas-phase structures of mitragynine, 7-hydroxymitragynine and their metabolites were obtained by quantum chemical method at B3LYP/6-311++G(d,p) level. Results in terms of standard Gibbs energies of reaction for all metabolic pathways are reported with solvation energy from SMD model. We found that 7-hydroxy substitution leads to changes in reactivity in comparison to mitragynine: position 17 is more reactive towards demethylation and conjugation with glucuronic acid and position 9 is less reactive towards conjugation with glucuronic acid. Despite the changes, position 9 is the most reactive for demethylation and position 17 is the most reactive for conjugation with glucuronic acid for both mitragynine and 7-hydroxymitragynine. Our results suggest that 7-hydroxy substitution could lead to different metabolic pathways and raise an important question for further experimental studies of this more potent derivative. Electronic supplementary material The online version of this article (10.1186/s13104-019-4461-3) contains supplementary material, which is available to authorized users.


Introduction
Mitragynine is the alkaloid derived from Kratom (Mitragyna speciose), a plant commonly found in Thailand and throughout the South East Asia region. (1,2) Like many other opioid plants, there are claims of medical uses (3,4) but these plants are also potentially illegal drugs of abuse. (5,6) In additional to natural sources, mitragynine and its derivative may be obtained by total syntheses reported by researchers in Japan(7) and the US. • O-demethylation of the 9-methoxy group, • O-demethylation of the 17-methoxy group, which may proceed via an aldehyde intermediate and result in corresponding carboxylic acid or alcohol by oxidation or reduction respectively, • subsequent conjugation to glucuronides at one of the three position above, • subsequent conjugation to sulfates only at the 9-methoxy group, and • combination of the steps above. Figure 1 is the basis of our study and our aim is to complement the experimental ndings with results from DFT calculation. For comparison purpose, 7-hydroxymitragynine, a representative of naturally occurring and more potent derivatives of mitragynine is also included in our in silico investigation.

Main Text
All quantum chemical calculations were performed using the Q-Chem 5.1 program package (18). Gasphase structures were obtained at B3LYP/6-311++G(d,p) level and were con rmed to be minima on the electronic potential energy surface by frequency calculations. Solvation energy in water from SMD model (19) was obtained from gas-phase geometries. The energies were corrected to standard state in solution condition of 1 M, 1 atm at 298.15 K with an exception of water in which case 55.34 M was used.
(Shell script, spreadsheet template and Mathematica (20) notebook are modi ed from our previous work.
(21) All output les and other associated codes to obtain the standard Gibbs energies of reaction are provided in the electronic supporting information.) For reporting purpose, metabolites are coded by molecular formula and abbreviated names as shown in Figure 1 and Table 1. To compute reaction energy from Figure 1, relevant additional reagents (water, sulfate ion, hydronium ion, protonated nicotinamide (22), gluconic acid, oxygen) and products (water, methanol, reduced nicotinamide) were also added to the scheme to complete the thermodynamic calculation. These compounds are commonly found in biological systems and are likely to be reasonable energy reference point for these demethylation, oxidation/reduction of aldehyde and conjugation to a glucuronide/sulfate reactions.

FIGURE 2
Relative standard Gibbs energies of metabolites in Figure 1 for mitragynine (left) and 7hydroxymitragynine (right). The parent compounds, M_mmm and H_mmm are the reference points for energy comparison.
All calculations were completed with no imaginary frequency. The lowest energy structures of mitragynine and 7-hydroxymitragynine are shown in Figure 1S. The detailed result for all reactions is listed in Table 1S (Supplementary Information) by type of reaction, number of steps from the parent compound and by position of reaction.
Gas-phase Gibbs energies of reaction show similar trend to aqueous-phase energies. The differences between gas-phase and aqueous-phase energies for conjugation to sulfates and reduction reactions are considerably large due to the presence of charged species on the reactant and/or product sides.
The major determining factor for the energy of reaction is the type of reaction. Conjugation to sulfates, oxidation and reductions are highly exergonic and should occur easily. However, demethylation and conjugation to glucuronides have mixed results. The positions therefore play an important roles in these cases to determine which pathway is more energetically favorable.
The average standard Gibbs energies of reaction for each reaction-position pair is shown in Figure 2S (Supplementary Information). As far as the three positions are concerned, position 9 is the most reactive for demethylation and position 17 is the most reactive for conjugation to glucuronide.
There are no conclusive trends from the number of steps from the parent compound.
The effect of 7-hydroxy substitution can be seen in Figure 2S (decreases in d17, g17 and an increase in g9 average reaction energies) and Figure 2 (lower position of H_mmd, H_dmd and H_mmG, signi cantly lower position of H_dmr and slightly higher position of H_gmr). Despite the decreases and increase, the general conclusion above that position 9 is the most reactive for demethylation and position 17 is the most reactive for conjugation to a glucuronide are still true for both mitragynine and 7hydroxymitragynine.
Based on existing experimental evidence (11,16), gas-phase structures of mitragynine, 7hydroxymitragynine and their metabolites (in total 36 compounds) were obtained by quantum chemical method at B3LYP/6-311++G(d,p). Standard Gibbs energies of reaction in solution-phase was calculated by SMD solvation model. Four factors, type of reaction, number of steps from the parent compound, position of reaction and 7-hydroxy substitution effect were studied in this work. When compared to mitragynine, the 7-hydroxy substitution makes demethylation and conjugation to a glucuronide at position 17 more favorable and makes conjugation to a glucuronide at position 9 less favorable. Our results can be a basis for further experimental investigation of physiological effects of the compounds and/or detection of Kratom use.

Limitations
During the preparation of this paper, relevant work (23)(24)(25) emerged in the literature. Readers may be interested to see a recent in vivo pharmacokinetics study of mitragynine in rats (23) and in vitro conversion of mitragynine to 7-hydroxymitragynine. (24,25) The importance of 7-hydroxymitragynine was raised as there are clear evidences that it can be formed by in vivo metabolism. This is different to the basic premise in this manuscript that 7-hydroxymitragynine is obtained from Kratom as a minor alkaloid component. However, this fact does not materially change our conclusion in the current study.

Declarations
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Availability of data and material: All data generated or analysed during this study are included in this published article and its supplementary information les. All output les and other associated codes to obtain the standard Gibbs energies of reaction are provided in supplementary information les.   Table 1 for abbreviated name convention. Compounds in brackets are not found experimentally but are assumed to be intermediates. Left, right and down arrows are for reactions at positions 17, 16 and 9 respectively. Metabolites found in rat or human are indicated by R or H respectively.)

Figure 2
Relative standard Gibbs energies of metabolites in Figure 1 for mitragynine (left) and 7hydroxymitragynine (right). The parent compounds, M_mmm and H_mmm are the reference points for energy comparison.

Supplementary Files
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