Effects of Itraconazole on Pharmacokinetics of Mitragynine and 7-Hydroxymitragynine in Healthy Volunteers

Abstract

CYP3A4-mediated metabolic conversion of mitragynine to 7-hydroxymitragynine (7OH) has been demonstrated in human liver microsomes, and in rodents. Pharmacokinetics (PK) of mitragynine and 7OH in humans is still limited. We aimed to examine the pharmacokinetics of mitragynine and the formation of 7OH in healthy volunteers. To elucidate involvement of CYP3A4 in 7OH formation, inhibition by itraconazole was implemented. Two study periods with PK study of mitragynine alone in period 1, followed by period 2 including itraconazole pretreatment was conducted. Freshly prepared kratom tea consisting of 23.6 mg of mitragynine was given to participants in both study periods. Serial blood samplings were performed for 72 hours, and analyzed using a validated LCMS in multiple reaction monitoring mode. The median Cmax for mitragynine of 159.12 ± 8.68 ng/mL was attained in 0.84 h. While median Cmax for 7OH of 12.81 ± 3.39 ng/mL was observed at 1.77 h. In period 1, Cmax and AUC 0-inf of 7OH accounted for 9% and 20 %, respectively, of those parameters for mitragynine. The geometric mean ratio of AUC0-72 for 7OH/mitragynine (metabolic ratio, MR) was 13.25 ± 1.07. Co-administration of itraconazole 200 mg per day orally for 4 days (period 2) decreased 7OH exposure by 56% for Cmax and 43% for AUC0-72 after a single oral dose of kratom tea. While the Cmax of mitragynine increased by 1.5 folds without a significant change in Tmax. The geometric mean metabolic ratio was 3.30 ± 1.23 (period 2), indicating the attenuation for the formation of 7OH by the pretreatment with itraconazole. This suggested the CYP3A4-mediated formation of 7OH from mitragynine in healthy volunteers. This study provides the first evidence of metabolic conversion of mitragynine to 7OH in humans.

Mitragynine and 7-hydroxymitragynine (7OH) are well-characterized alkaloids found in the leaves of Mitragyna speciosa Korth. (kratom), comprising approximately 66 and 2% of the total alkaloid content, respectively.^1,2 Mitragynine acts as a partial agonist at μ-opioid receptors, and a competitive antagonist at both κ and δ receptors.^3,4 Similarly, 7OH has been shown to bind and activate with higher affinity for the μ-opioid receptor subtype.⁵⁻⁸ Based on studies of their interaction with opioid receptors, 7OH is 46 times more potent than mitragynine at the mu opioid receptor and 13 times more potent than morphine.^9,10 Preliminary studies in mice have demonstrated that analgesic properties of mitragynine and 7OH occurred via opioid receptor-mediated pathways, with 7OH showing more potency than mitragynine.⁵

In a survey of kratom users, a majority of respondents indicated using kratom for pain relief (91.3%), anxiety (67.2%), and depression (64.5%).¹¹ In another online survey, 48% of kratom users reported using it for pain relief.¹² While the analgesic properties of mitragynine were initially proposed to be due to opioid-like effects, it has also been suggested that mitragynine may activate alpha-2 adrenergic postsynaptic receptors, which modulate “descending” pain pathways.^13,14 In addition, the inhibition of cyclooxygenase-2 and prostaglandin E2 can provide the indirect antinociceptive properties of mitragynine.^14,15 Moreover, due to reduced arrestin-2 recruitment rendered by mitragynine and 7OH, some traditional opioid adverse effects, e.g., respiratory depression, sedation, and constipation, could be reduced, compared to morphine.^14,16

Recent in vitro studies have shown that mitragynine is converted to 7OH primarily by cytochrome P450 (CYP)3A4 and, to a lesser extent, by CYP2C19 and CYP2D6.^8,17,18 In a tail flick study in mice, 7OH was found to be ∼5 folds more potent than mitragynine when administered orally.¹⁷ However, the antinociceptive effects of mitragynine were not observed from subcutaneous administration. Therefore, first-pass metabolism-mediated pharmacological effects and the involvement of an active metabolite were strongly postulated for the analgesic effect of mitragynine. The evidence showing that 7OH is formed to explain the antinociceptive effects of mitragynine was fully elaborated in animals.^8,17 In vitro data have suggested that 7OH is formed more efficiently from mitragynine in human liver microsomes than in mouse liver microsomes.¹⁷ Although a trial in humans recently demonstrated that kratom significantly increased pain tolerance compared to placebo,¹⁹ differences in conversion of mitragynine to 7OH in mouse liver microsome and in vivo have been reported.¹⁷ Thus, the formation of 7OH in humans to exert analgesic effects still requires further investigation.

Pharmacokinetics (PK) of mitragynine following the oral administration of kratom in humans is limited. A recent PK study in Asian populations has shown linear PK of mitragynine, with an apparent volume of distribution (V_d) of 38.0 ± 24.3 L/kg, terminal half-life (T_1/2) of 23.2 ± 16.1 h, and renal excretion of unchanged mitragynine of 0.14%.²⁰ The data, however, did not describe related parameters for any metabolite of mitragynine, especially 7OH. Additionally, the study was conducted in chronic kratom users, with several doses given. Since the metabolism of mitragynine in chronic users can be affected by several factors, e.g., existing metabolites of mitragynine and other alkaloids. Pharmacokinetics of mitragynine in this population might not be applicable to nonusers. Therefore, we aimed to examine the PK of mitragynine and the formation of 7OH in healthy volunteers. To elucidate the metabolic conversion by CYP3A4, itraconazole was used as a CYP3A4 inhibitor.

Results and Discussion

LCMS and Chromatographic Conditions

The operating parameters for the HPLC were optimized to obtain the best UV absorption spectrometric performance for mitragynine, 7OH, and ajmalicine (IS). The UV absorption was observed at a wavelength of 245 nm (Figure S1). Linearity was determined in the range of 0.50–20.0 μg/mL for mitragynine and 0.50–10 μg/mL for 7OH. The peak areas of mitragynine and 7OH vs the nominal concentrations of mitragynine and 7OH showed a good linear relationship over the mentioned concentration ranges (Table S1).

Development of the LCMS method began with optimization of both the analytes and IS mass spectra ionization and fragmentation. Full scan mass spectra of mitragynine, 7OH, and IS (Figure 1) were acquired by autoinjection of individual standard solutions of mitragynine and IS (1 μg/mL each) and 7OH (500 ng/mL) on a 6490 triple quadrupole mass spectrometer coupled with an ESI source under positive ionization mode. Quantitation was performed using the MRM mode of transitions precursor ion > product ion at a m/z of 399 > 174 for mitragynine, 415 > 190 for 7OH, and 353 > 144 for IS at optimal values of collision energies at 28, 38, and 32 eV, respectively (Figure 1). While other parameters were adopted per the suggested default values for the instrument (Table S2). The retention times of 7OH, IS, and mitragynine were at 5.35, 6.47, and 7.12 min (Figure S2), respectively. Liquid–liquid extraction method (LLE) for analyte separation from the plasma was adopted due to its ease, high efficiency, good recovery, and cost saving.

Figure 1.

LCMS spectra and multiple reaction monitoring transitions of mitragynine (A), 7-hydroxymitragynine (B), and ajmalicine (IS, C).

Chromatographic conditions were optimized to attain adequate separation with acceptable retention times. Mobile phase composition, a mixture of 0.1% formic acid in acetonitrile and 0.1% formic acid in milli-Q water in a gradient mode was optimized according to Todd et al.⁷ and Manwill et al.²¹ with some modification. The gradient elution starting at 30–90% of the organic solvent over 9 min led to the highest sensitivity, ionization efficiency, accuracy, and good peak shape for the analytes of interest. After optimization, the MRM chromatograms of mitragynine, 7OH, and IS from human plasma samples showed acceptable peak shapes, high density, and good symmetry (Figure S2). The total run time of each sample was 15 min, including washing and equilibration.

Method Validation

Selectivity of the bioanalytical method employed was adequate, as indicated by the absence of any endogenous interference at the peaks for mitragynine, 7OH, and IS, as observed from chromatograms of blank plasma compared to plasma spiked with those compounds. Linearity of the calibration curve over the concentration ranges of 5.0, 10, 25, 50, 100, 200, 300, and 400 ng/mL of mitragynine; and 2.5, 5.0, 10, 25, 50, and 100 ng/mL of 7OH, were demonstrated by the coefficient of correlation (r²) > 0.996. The regression equations for mitragynine: y = 0.0160 x + 0.0582 (r² = 0.9990); for 7OH, y = 0.0026 x + 0.0019 (r² = 0.9967). The limits of detection (LOD) for mitragynine and 7OH were 0.5377 ng/mL and 0.0045 ng/mL , respectively. The lower limit of quantification (LLOQ) for mitragynine and 7OH in plasma was 1.6296 ng/ml (%CV = 25.8%), and 0.137 ng/mL (%CV = 16.7%), respectively.

The accuracy and precision of the analysis were evaluated using quality control (QC) samples prepared at 20.0 ng/mL (LQC), and 120 ng/mL (HQC) for mitragynine and 5.0 ng/mL (LQC) and 30.0 ng/mL (HQC) for 7OH. The interday accuracy and precision were determined by repeated analysis of the QC samples on 5 different days. The results revealed that the intraday accuracy and precision for mitragynine were 93.1 ± 6.63% (% CV = 7.12%), and 95.1 ± 6.01% (%CV = 6.32%) for LQC and HQC, respectively. Inter-day accuracy and precision for mitragynine were 80.2 ± 14.5% (%CV = 11.5%) and 84.1 ± 7.38% (%CV = 4.87%) for LQC and HQC, respectively. Intra-day accuracy and precision for 7OH were 108.8 ± 4.04% (%CV = 3.71%), and 89.4 ± 5.02% (% CV = 5.62%) for LQC and HQC, respectively. Inter-day accuracy and precision for 7OH were 89.3 ± 28.7% (%CV = 14.7%) and 74.1 ± 11.1% (% CV = 11.5%) for LQC and HQC, respectively. Both precision and accuracy of mitragynine and 7OH were within the acceptable limits (≤15%) as specified by FDA guidelines for bioanalytical method validation.

The conditions employed in this study yielded good selectivity with a total run time of 15 min. The LOD for mitragynine was 0.54 ng/mL, slightly higher than LOD reported²² and the LLOQ for mitragynine was 1.63 ng/mL. While, LOD and LLOQ for 7OH were determined to be 0.004 and 0.137 ng/mL, respectively. The recoveries for extraction were ≥90% for mitragynine and ≥77.8% for 7OH. Coefficient of variation (CV) of the extraction recoveries of 7OH at 1.0 ng/mL was greater than 20%. Extraction by ethyl acetate produced acceptable recoveries for both mitragynine and 7OH. Large deviation for the recovery values can be due to solvent volatility and the high polarity of 7OH. In summary, a simple LCMS method was developed and validated as per FDA guidelines. The method used requires a small sample volume (200 μL) and a simple liquid–liquid extraction. The method has a dynamic range of 5–400 ng/mL and a short run time (15 min) that allows for efficient analysis. The validated method was applied for the simultaneous determination of mitragynine and 7OH in human plasma obtained following the pharmacokinetic study of mitragynine and 7OH in healthy volunteers.

In our study, mitragynine was found to be 89.4% of the original amount at 6 h at room temperature. While percentage remained of 7OH at 12 h was 45.2 and 76.3% without and with the presence of PMSF, respectively (Figure 2). Mitragynine was reported to be stable in plasma at room temperature for 6 h with less than 11% deviation.²³ It was confirmed later for the stability over a 12 h period at same condition.²⁴ Kamble et al.²⁵ has shown the lability of 7OH in human plasma (∼40% 7OH remained at 120 min) with a half-life (T_1/2) of 98.7 min. A protease inhibitor cocktail was shown to improve the stability of 7OH efficiently.²⁵ In our study, a serine protease inhibitor, PMSF, reduced the rate of 7OH degradation in plasma by 31.1% compared to untreated human plasma (Figure 2). The compound was then used to prevent degradation of 7OH in human plasma during sample preparation.

Figure 2.

Degradation profile of mitragynine and 7-hydroxymitragynine (7OH) in human plasma.

Pharmacokinetic Study

Participant Demographic Characteristics

Sixteen healthy participants were enrolled and completed the study. Demographic characteristics of the population enrolled in the study are listed in Table S3. Of the 16 participants, 5 were male and 11 were female. The ethnicity and race of the volunteers were of Thai descent. The median (range) age of the participants was 23 (20–44) years, and the median (range) body mass index was 22.0 (16.5–25.2) kg/m².

Effect of Itraconazole on Mitragynine and 7OH Pharmacokinetic

In this study, each participant received 25 ± 1.0 mL of kratom tea (0.8146–0.9364 mg/mL mitragynine), which was 23.6 mg of mitragynine. In period 1, median C_max for mitragynine of 159.12 ± 8.68 ng/mL was attained in 1.0 h (Figure 3 and Table 1), indicating fast absorption of mitragynine from kratom tea. The T_max observed in period 1 was similar to the earlier pharmacokinetic studies (0.75–1.5 h).^22,26 The elimination half-life (T_1/2) of mitragynine was 8.72 h, which was consistent with a recent pharmacokinetic study in healthy subjects,²² but much less than that reported at 23 h in chronic kratom users.²⁰ The initial 7OH was observed as early as 0.25 h in most participants, with a median C_max of 12.81 ± 3.39 ng/mL, attained at 1.77 h (Figure 4 and Table 1). The C_max and AUC_0–∞ for 7OH were accounted for 9 and 20%, respectively, of the corresponding parameters for mitragynine. 7-hydroxymitragynine appeared to have a similar T_1/2 (7.41 h), compared to that for mitragynine (8.72 h, Table 1). The median ratio of AUC₇₂ of 7OH to AUC₇₂ of mitragynine in our study (13.25 ± 1.07, Table 1) reflected an efficient conversion of mitragynine to 7OH in humans, compared to those recently reported in rats (1.4 ± 0.2).²⁷

Figure 3.

Plasma concentration time profile of mitragynine and 7-hydroxymitragynine (7OH) from healthy participants after oral administration of kratom tea in period 1 (N = 15).

Table 1. Pharmacokinetic Parameters of Mitragynine and 7-Hydroxymitragynine (7OH) Following Oral Administration of Kratom Tea without (Period 1) or with (Period 2) Itraconazole Treatment in Human Volunteers (N = 15)^a,b.

parameters	period 1		period 2
	mitragynine	7OH	mitragynine	7OH
Cmax (ng/mL)	159.12 (134.06, 217.44)	12.81 (9.47, 22.23)	244.86 (206.34, 322.420	4.32 (4.49, 13.50)
AUC0–72 (ng h/mL)	809.82 (586.29, 1471.50)	107.28 (88.78, 168.06)	1031.94 (825.49, 1518.74)	34.06 (22.24, 90.24)
AUC0–∞ (ng h/mL)	940.42 (588.17, 2016.73)	171.63 (121.74, 414.16)	1075.54 (854.14, 1617.98)	42.00 (29.44,112.65)
AUC ratio [7OH/mitragynine]*	NA	13.25 (11.47, 16.17)	NA	3.30 (2.67, 5.77)
Tmax (h)	0.84 (0.61, 1.39)	1.77 (1.44, 2.83)c	1.14 (0.98, 1.49)	2.56 (2.08, 3.86)
T1/2 (h)	8.72 (7.26, 17.34)	7.41 (5.69, 15.45)c	3.47 (2.54, 11.62)	3.03 (2.09, 6.30)
Ke (h–1)	0.05 (0.01, 0.18)	0.04 (0.04, 0.17)d	0.20 (0.19, 0.54)	0.18 (0.13, 0.50)
Vd/F (L)	521.1 (394.7, 858.1)f	23.01 (−885.11, 2518.79)	109.81 (83.81, 242.42)	13.06 (6.88, 32.41)
CL/F (L/h)	24.90 (16.17, 52.65)	0.84 (−5.61, 19.07)e	21.94 (17.64, 32.66)	2.38 (1.59, 6.61)
MRT (h)	17.2 (13.56, 40.75)c	29.1 (6.95, 194.14)c	5.95 (3.81, 14.13)	7.56 (−5.69,41.93)

^aData presented as geometric mean (95% CI).

^bC_max, maximum plasma concentration; AUC_0–72, area under the plasma-concentration time curve from time zero to 72 h; AUC_0–∞, area under the plasma-concentration time curve from time zero to time infinity; AUC ratio, 7-hydroxymitragynine to mitragynine AUC 0–72 h ratio; T_max, time to reach C_max; T_1/2, terminal half-life; K_e, elimination rate constant; V_d/F, apparent volume of distribution during the terminal phase; CL/F, oral clearance; MRT, mean residence time; NA: not applicable.

^cStatistically significant difference compared to period 2 according to Wilcoxon signed rank test p < 0.02.

^dp < 0.03.

^ep < 0.05.

^fp < 0.003.

Figure 4.

Plasma concentration time profile of mitragynine and 7-hydroxymitragynine (7OH) from healthy participants after oral administration of kratom tea in period 2 (N = 15).

The rapid absorption of mitragynine was first postulated to be passive. Oral bioavailability of mitragynine in humans was reported as 3–17%,¹⁰ and 30%.²⁶ Mitragynine is lipophilic (log P = 1.73), BCS class II, dissolution-limited absorption.³ It was initially controversial about the efflux by P-glycoprotein (P-gp) due to the lack of polarization in the bidirectional transport study at high-tested mitragynine concentrations. However, P-gp efflux of mitragynine was evident at low mitragynine concentrations (0.3 μM).²⁸ Though in vivo data is still needed, these in vitro data can serve as important data indicating potential P-gp-mediated efflux of mitragynine, leading to low oral bioavailability of mitragynine.

Large volume of distribution of mitragynine in this study (V_d/F = 521.1 L) is quite consistent with previous study of 38.04 ± 24.3 L/kg²⁰ and 37–90 L/kg.⁴ Plasma concentration profiles of mitragynine displayed a biphasic distribution characteristic (Figure 3), which agreed well with the two compartment model pharmacokinetic proposed.^20,26 During study period 2, peak exposure of mitragynine has increased 1.5-fold with nonsignificant changes of T_max compared to those in period 1 (Table 1). However, the geometric mean ratio of AUC_0–72 and AUC_0–∞ of mitragynine, in period 2 vs those of period 1, were inequivalent according to the 80–125% for 90% CI (Table S4). Maximum 7OH concentration was attained almost 1.0 h later than that of mitragynine (1.77 vs 0.84 h). In period 2, T_max of 7OH was extended, while C_max and AUC_0–72 of 7OH decreased significantly by 56 and 43%, respectively (Table 1). This can simply suggest the slower and decreased formation of 7OH in period 2.

Approximately 4-fold differences in metabolic ratio (MR) between period 1 and period 2 was obtained, while quite large variation among participants were observed (Figures 5 and S3 and Table 1). Although the other minor metabolic pathways (e.g., CYP2D6 and UGTs) could contribute to the overall metabolism of mitragynine. The influence of gender on itraconazole PK, i.e., AUC and C_max, was recently presented.²⁹ In females, there was significant reduction of C_max and AUC, and increment of V_d of itraconazole and hydroxy-itraconazole, compared with males.²⁹ Given unequal numbers of males and females in our study, i.e., 5 males and 11 females, less exposure to itraconazole in females than in males could occur. This might contribute to the variation for the degree of CYP3A4 inhibition following itraconazole pretreatment. Although the MR in period 1, estimated from all participants, was about 4 folds of the MR in period 2 (Table 1). Subgroup calculation has shown MR differences in periods 1 and 2 for approximately 6.79 (males) and 3.1 folds (females). Moreover, the compliance of the participants for itraconazole administration can be confirmed by measuring itraconazole and its metabolite (hydroxy-itraconazole) in plasma.²⁹

Figure 5.

Metabolic ratio for 7-hydroxymitragynine (7OH) to mitagynine from period 1 and 2 (N = 15).

Larger volume of distribution (V_d/F) of mitragynine than 7OH (521.1 L vs 23.01 L) in this study should be due to the difference in lipophilicity of the compounds. Manda et al. have reported the permeability in epithelial cell for mitragynine, which was 1.45–1.50 times higher than that of 7OH.³⁰ Less permeability rendered by 7OH should be related to the presence of the hydroxy group in the molecule. Moreover, greater permeability into brain for mitragynine than 7OH was associated with their lipophilicity (log P_Octanol = 4.11 vs 1.67, mitragynine, and 7OH, respectively).²⁸ Although both compounds were shown for the extensive efflux in rats, mitragynine revealed about 18-fold higher brain tissue uptake than 7OH.²⁸ Moreover, it has recently shown extensive tissue distribution with a slower redistribution to the systemic circulation of mitragynine.²⁶ Excessive brain tissue binding of mitragynine in rats, compared to 7OH, established by Yusof et al., was associated with an unbound fraction in the brain of 0.0265 and 0.26, respectively, for mitragynine and 7OH.²⁸

The observed smaller V_d/F of mitragynine during period 2 could be ascribed to altered tissue binding or altered oral bioavailability (F). Although itraconazole binds to plasma albumin for 99%, it can distribute widely to several tissues, i.e., skin, gynecological tissues, and alveolar cells, with reported a V_ss of 11.0 L/kg.³¹ The extensive and sustained tissue binding of itraconazole was shown for different organs of the female genital tract.³¹ Such strong tissue binding of itraconazole might interfere with or lessen the tissue binding capacity of mitragynine, leading to the elevated unbound fraction of mitragynine in tissues. This might result in a decrease in distribution volume of mitragynine. Alternatively, considering the reported bioavailability of 17.0–31% for mitragynine in rats³² and 3–17% in human.⁴ Although the enzyme expression in humans can be different from that in rats, potential first-pass metabolism of mitragynine should be reduced by itraconazole, and the systemic exposure of mitragynine increased. Although we cannot indicate conclusively whether a decrease in distribution or an increase in F is leading to smaller V_d/F of mitragynine during period 2 in this study. The effect of mitragynine in reducing intestinal transit times and gastrointestinal motility can influence plasma mitragynine and 7OH. During absorption, variation in GI motility and gastric emptying after a single oral dose of mitragynine could result in inconsistent bioavailability.

Half-lives of mitragynine and 7OH reported earlier were ≈3.5 and ≈2.5 h, respectively,³³ with peak effects occurring 2–4 h after ingestion.² The observed effects of kratom last for 5–7 h.³³ The T_1/2 of mitragynine in this study (8.74 h) was consistent with earlier reported values,⁴² and pharmacokinetic study in healthy subjects,²² but dissimilar to that reported by Trakulsrichai et al.²⁰ Healthy volunteers unexposed to kratom employed in the study, with a single oral administration of standardized kratom tea, were the major similar points between our study and that by Buasri.²² While study in participants with kratom uses history, with the varying doses of mitragynine, and 24 h sample collection, which might not yield the suitable pharmacokinetic profile.²⁰ Definite comparison of our pharmacokinetic parameters to those in that study was quite disincentive. The T_1/2 observed in this study can explain some undetectable mitragynine levels after 36 h, which were more than 3 times of the elimination half-life of mitragynine. During period 2, shorter half-lives were observed for both 7OH and mitragynine. Though there are insignificant changes, decreased V_d/F without changes in CL/F can cause the reduced T_1/2 of mitragynine. Significant reduction in T_1/2 of 7OH can be attributable to the increase in CL/F, with unaltered V_d/F.

Itraconazole has replaced ketoconazole in drug–drug interaction studies due to safety concerns. Itraconazole acts as a strong CYP3A inhibitor, with a well-described and acceptable safety profile.³⁴ With an absolute bioavailability of about 55%, the best absorption of itraconazole capsules occurs when taken after a full meal. Itraconazole and hydroxy-itraconazole are CYP3A4 substrates and inhibitors, affecting their own metabolic clearance and those of the other drugs.³⁵ The T_1/2 of itraconazole, and hydroxy- itraconazole are 20–24, and 12 h, respectively.³⁶ The dose and duration of itraconazole pretreatment in this study has been based upon the extent of CYP3A inhibition and safety profiles. Itraconazole 100–200 mg/day with a 3-day lead-in have demonstrated adequate CYP3A inhibition in a number of drug–drug interaction studies.^37,38 With several days of oral itraconazole dosing, the expected maximal CYP3A inhibition was achieved early in the postdosing period.³⁹

Design of our study, performing PK study of mitragynine alone in period 1 (where the general pharmacokinetic parameters were characterized), followed by period 2 PK study with itraconazole pretreatment, is considered acceptable. The 2-period study design reduces intersubject variability by dosing the substrate with and without itraconazole in the same subjects.⁴⁰ The washout period of 14 days between 2 study periods was adequate with regard to the elimination half-life of mitragynine as well as the undetectable predose concentration of mitragynine and 7OH in period 2.

Safety

Total of 15 adverse effects were recorded during period 1: drowsiness (56.2%), vomiting (31.2%), dizziness (31.2%), headache (18.7%), fatigue (18.7%), and nausea (12.5%). Others observed adverse effects including diarrhea, fever, skin itchy/irritation, constipation, anorexia, heartburn, dry mouth, and muscle pain. In period 2, the only adverse effect observed was vomiting (6.25%). All events were resolved on the same day without any treatment and did not lead to any drop out.

Increase in blood pressure and pulse rate at the eighth hour and tongue numbness after administration of kratom tea was reported in chronic kratom users,²⁰ which were not observed in our study. Most commonly reported adverse effects among kratom users (without other drugs of abuse) were agitation/irritability (23%), tachycardia (21.4%), nausea (14.6%), drowsiness/lethargy (14.3%), vomiting (13.2%), confusion (10.6%), and hypertension (10.1%).⁴¹ In this study, drowsiness, dizziness, and vomiting were main adverse effects, while the other typical adverse effects, including diarrhea, fever, skin itchy, irritation, constipation, anorexia, and heartburn, were not observed. Drowsiness and fatigue, mostly found in period 1, can be related to opioid-like effects of 7OH. Nausea and vomiting, mostly occurring during the first 2 h after tea administration, was commonly reported for opioids. These were found in a greater number of subjects in period 1, compared to those in period 2. Association of the events with 7OH concentration was then speculated, since about 43% decreased C_max of 7OH in period 2 was observed.

This study provided evidence for the formation of 7OH in humans following the administration of kratom tea. However, there were some limitations in our study. First, a small number of participants participated in this study. For a better characterization of interindividual variability of the PK of mitragynine and other related alkaloids, a larger sample size should be included. Second, our study assessed the PK after a single oral dose of kratom tea, containing quite low amount of mitragynine (23.6 mg mitragynine). Extrapolation of our results to multiple dosing or higher dose should be cautiously performed. Third, the analgesic properties have not been examined with and without itraconazole. A number of major oxidative metabolites of mitragynine was recently demonstrated in mice receiving oral mitragynine, and it was reported that the antinociception of oral mitragynine, resulted from its bioactivation to potent 7OH (major metabolites) and minor metabolites, especially mitragynine pseudoindoxyl.⁸ Further studies on PK/PD can clarify the roles of 7OH in analgesic effects in humans. Moreover, numerous metabolites of mitragynine have been found in human urine.³³ PK study considering urinary excretion of the parent mitragynine and metabolites in period 1 and period 2, can help in elaboration of elimination pathway of mitragynine.

Conclusions

Our study has provided descriptive pharmacokinetic parameters of mitragynine and 7OH in healthy adult participants after a single dose of a well-defined kratom tea. Moreover, the formation of 7OH from mitragynine metabolism was addressed since the 7OH exposure was reduced by treatment with a selective CYP3A4 inhibitor, itraconazole. Mild adverse effects were reported and related to opioid-like effects of 7OH. Due to its major role in first-pass metabolism, CYP3A4 conversion was found necessary for the analgesic effects of mitragynine in healthy volunteers.

Materials and Methods

Study Chemicals and Drugs

Mitragynine and 7OH were provided by Professor Dr. Dalibor Sames, Columbia University, USA. Itraconazole was purchased from Charoen Bhaesaj Lab Co.Ltd. (Spornar), Bangkok, Thailand. Ajmalicine (IS) was purchased from Sigma-Aldrich (Spruce St., St. Louis, USA), and phenylmethylsulfonyl fluoride (PMSF) was from Acros Organics (Fisher Scientific, USA). Ethyl alcohol, methanol, and acetonitrile of HPLC grade were purchased from RCI Labscan, Bangkok. Formic acid (Optima LC/MS grade 99.5%) was obtained from Fisher Chemical, UK.

Preparation of Kratom Tea

Standardized kratom tea was prepared from the fresh, mature red-veined kratom leaves collected from Nam Phu subdistrict, Ban Na San, Sura Thani province, Thailand. The voucher specimen (Wungsintaweekul, J. N5/001) was identified by Associate Professor Dr. Juraithip Wungsintaweekul and deposited at the Herbarium of Faculty of Sciences, Prince of Songkla University.⁴³ Initially, the fresh leaves were washed with tap water and dried at 50 °C for 20 h. The dried leaves were crushed into coarse powder, and 1.5 kg of dried coarse powder was boiled in 4.5 L of water for 0.5–1.0 h. The resulting liquid was filtered through a clean muslin sheet, and the liquid was concentrated by cooking at low heat. Amount of mitragynine in kratom was determined by the HPLC method. Two mitragynine concentrations (0.936 and 0.814 mg/mL) of tea were prepared. The tea was kept in sterile 60 mL amber-colored bottles at −20 °C and further analyzed for stability on days 0, 57, and 72 days.

Analysis of Kratom Tea

A 1 mL sample of kratom tea was centrifuged at 7,000g for 10 min at 25 °C. A 50 μL aliquot of supernatant was diluted 400x fold with 25% acetonitrile. The analysis was performed on HPLC, and the conditions included 65% of mobile phase A (10 mM ammonium acetate, pH 5 adjusted with 2% acetic acid) and 35% mobile phase B (acetonitrile) in an isocratic elution method. Column used was C₈ column (ACE 5-C8, 150 mm × 4.6 mm) at column temperature 30 ± 5 °C. Flow rate was 1 mL/min, and the wavelength was at 245 nm. The mitragynine % remaining on day 57 and 72 was 96.7% and 84.1%, respectively. The standardized kratom tea with a total mitragynine concentration of 23.6 mg was filled in a sterile glass amber color bottles and kept at −20 °C until further use.

Stability

Prior to collecting blood from participants, the stability of mitragynine and 7OH in human plasma was examined in the presence of PMSF. The study involved incubating 100 nM mitragynine, 96.5 nM 7OH, and 1.42 μM IS in human plasma at 37 °C with 0.2 mM PMSF, which was compared to untreated plasma and analyzed using LCMS.

Human Subject Protection

This was an open-label, two-period study conducted at the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand, in accordance with Good Clinical Practice guidelines. The study protocol was approved by the Ethics Review Board of the Human Research Ethics Committee Health Sciences at Prince of Songkla University (Grant No. HSc-HREC-63–033–1–1). Participants were informed about the risks and benefits of the study and provided with detailed treatment information. Written informed consent was obtained from all volunteers before any study procedures were performed.

Study Participants

The study recruited healthy individuals aged 18–45 years with a normal body mass index (BMI). Participant’s health was evaluated through vital signs, medical history, physical exams, and laboratory tests, including HIV testing. Exclusively applied to those with abnormal blood pressure or high pulse rates, smokers, those with a history of substance abuse or allergies to mitragynine and its metabolites, individuals on medications, or those who have participated in recent clinical studies. Additionally, individuals could not have used certain drugs of abuse (methamphetamine and opioids), over-the-counter drugs, or hormonal contraceptives within specified time frames and were asked to ovoid alcohol for at least a week before and during the study period.

Sample Size Calculation

Sample size calculation was determined by the equation for the studies where data was quantitative. This was obtained by measurement of the total drug exposure across time (Area under the curve, AUC_0–∞).⁴⁴

where,

n = sample size

Z_α/2 = the value when a = 0.05% will get Z_α/2 = 1.96

σ = standard deviation

E = tolerance of the mean (10%)

So, the reported human pharmacokinetic value, AUC_0–∞ was 277.63 ± 109.1 ng/mL/h.²² Therefore, to calculate the sample size

Study Design and Treatment

Two periods of pharmacokinetic study, separated by 14 days of washing out, were conducted (Figure 6). The washout period was estimated in accordance with the reported T_1/2 of mitragynine in humans 23.2 ± 16.1 h.²⁰ In the first period, the PK of mitragynine after oral administration of kratom tea was examined over 72 h. Participants were asked to fast overnight (at least 10 h) prior to the study (Figure 6). On the morning of study day, kratom tea (25 mL) consisting of 23.6 mg of mitragynine was given to each participant. Water was allowed afterward, with limited volume of not more than 100 mL. Serial blood samples (5 mL) were collected on 17 occasions, into BD Heparin vacutainer collection tubes via an indwelling venous catheter at the following collection time points: predose (time 0), 0.25, 0.50, 1.0, 1.50, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10, 12, 24, 36, 48, and 72 h. Lunch was provided after the 4.0 h of blood collection. The IV catheter was removed from the forearm after 12 h, and a single blood sample was drawn at 24, 36, 48, and 72 h after kratom tea administration. After a 14-day washout, the participants received itraconazole 200 mg/day for 4 consecutive days. On the fifth day, the same pattern as those carried out in period 1 was performed, with kratom tea administration in the morning. Vital signs were monitored periodically during the entire study period. Additional abnormal signs and symptoms were carefully observed throughout the study.

Figure 6.

Schematic design for period 2 pharmacokinetic studies. With the preceding study for pharmacokinetics of mitragynine and 7OH following kratom tea administration in period 1, the second period started after a 14-day washout. Period 2 involved oral itraconazole pretreatment for 4 consecutive days prior to kratom tea administration on Day 5.

All blood samples were immediately centrifuged at 3,000 rpm for 10 min at 25 °C. After separation and transfer to a new tube, the plasma samples were then treated with 0.2 mM PMSF, followed by vortex mixing and stored at −80 °C until LCMS analysis for mitragynine and 7OH concentration.

Mitragynine, 7OH, and IS were extracted from plasma using liquid–liquid extraction. To 0.2 mL of plasma in an eppendorf tube was added 10 μL of 50 ng/mL IS and alkalinized with 0.5 M Na₂HPO₄ (pH 10), followed by 1.0 mL of ethyl acetate. The extraction was performed by horizontal shaking for 10 min. The organic layer was separated and dried under a stream of nitrogen gas, and the residue was reconstituted with 200 μL of the mobile phase. A 10 μL aliquot was injected into the LCMS analysis. Method validation was conducted according to FDA guidelines for bioanalytical methods.

Safety Assessment

Blood pressure, body temperature, and pulse rate were recorded prior to tea administration and every 2 h after tea administration. Additionally, participants were periodically interviewed to detect any unusual symptoms.

LCMS Analysis of Mitragynine, 7OH, and IS

The plasma concentrations of mitragynine, 7OH, and IS were analyzed using an Agilent LC1290 series (Agilent Technologies, Germany) coupled with a 6490 triple quadrupole mass spectrometer (Agilent Technologies, USA) operating in an electrospray ionization source in positive and multiple reaction monitoring (MRM) modes. Chromatographic separation was achieved on a Zorbax Eclipse XDB-C18 column (4.6 mm × 150 mm; 5 μm; Agilent Technologies, USA) at 35 ± 0.5 °C under gradient conditions at a flow rate of 0.35 mL/min. The initial mobile phase was 70% of 0.1% formic acid in water (mobile phase A) and 30% of 0.1% formic acid in acetonitrile (mobile phase B) for 1 min, followed by a 10% (A) and 90% (B) over 8 min, maintained at 2% (A) and 98% (B) for 2 min, and 70% (A) and 30% (B) for next 2 min. The total run time was 15 min. The mass transitions for MRM were m/z 399 → 174 for mitragynine, 415 → 190 for 7OH, and 353 → 144 for IS. The operational parameters for MS analysis were followed according to the suggested value in the instruments by default (Table S1). The calibration standard responses were linear in the range of 20.0–5000 ng/mL using a weighted (L/concentration) linear least-squares regression. The between-day assay accuracy, expressed as the percentage of relative error for QC concentrations, ranged from 9.0 to 9.5% for the low (20.0 ng/mL), medium 1 (60.0 ng/mL), medium 2 (400.0 ng/mL), and high (4000.0 ng/mL) QC samples. Assay precision, expressed as the between-day percent coefficient of variation of the mean estimated concentrations of the QC samples, was ≤5.9%.

Pharmacokinetic Parameters and Statistical Analysis

The pharmacokinetic parameters were analyzed by the noncompartmental method using the PK solver analyzer.⁴⁵ The area under the plasma concentration–time curve (AUC_0–t) was calculated using the trapezoidal rule, and the total AUC (AUC_0–∞) was estimated from the sum of AUC_0–t and AUC_t–∞., where AUC_t–∞ = C_last/k. Elimination half-life (T_1/2) was estimated as T_1/2 = ln2/k, where k was the elimination rate constant obtained through linear regression of plasma concentrations in the terminal phase. C_max and T_max were observed directly from the plasma concentration–time profiles. Mean residence time (MRT), which represents the average time a drug molecule spends in the body before elimination, was calculated from MRT = AUMC_0–∞ /AUC_0–∞. The metabolite-to-parent ratio for AUC (AUC₇₂ 7OH/AUC₇₂ mitragynine) was calculated to demonstrate the relative availability of 7OH to mitragynine in the circulation. Data are reported as medians with ranges. Wilcoxon signed rank test was performed on data generated from PK solver comparing periods 1 and 2, and their differences were considered statistically significant at p < 0.05. The Kolmogorov-Smimov test was used to assess normal distribution. Statistical analysis was performed using SPSS (version 25.0; SPSS Inc., Chicago, IL, USA).

Bioequivalence of mitragynine in periods 1 and 2 was determined by Phoenix WinNonlin 8.3 (Certara, Princeton, NJ, USA), through All research Co. Ltd., Thailand. The two products were considered bioequivalent if the 90% confidence interval (CI) of the test-to-reference geometric mean ratios for AUC_0–72, AUC_0–∞, and C_max fell within the range of 80.0–125.0%. In our study, “Test” is represented by those parameters for period 2, and “Reference” is represented by the parameters for period 1. Statistical analysis for sources of variation that can have an effect on the response variable, i.e., sequence, subject within sequence, period, and formulation, in the ANOVA model. The differences between groups were determined by two one-sided tests. The probability value less than 0.05 was considered statistically significant.

Data Availability Statement

The raw data that support the findings of this study are not available to the public due to a lack of volunteer consent for data sharing.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00335.

• Details about the HPLC chromatogram and linear regression parameters, optimized LCMS parameters, demographic characteristics of the enrolled participants, bioequivalence analysis of mitragynine, total ion chromatogram of blood plasma, and metabolic ratio for 7-hydroxymitragynine (7OH) to mitagynine from period 1 and 2 (PDF)

Author Contributions

K.W.: conceptualization, supervision, and project administration. K.W. and A.J. designed experiments. P.M. and A.J. performed experiments and analyzed the data. T.I. and K.W. did the human volunteer medical screening and safety assessment. J.W. and A.J. provided the drug and reagents. P.M. and K.W. wrote the first draft of the manuscript. K.W. and A.J. edited and revised the manuscript. All authors edited and reviewed the manuscript.

This research was funded by the Research and Development Office, Prince of Songkla University (Grant number PHA6402090S).

The authors declare no competing financial interest.