Activity of Mitragyna speciosa ("Kratom") Alkaloids at Serotonin Receptors

Abstract

Kratom alkaloids have mostly been evaluated for their opioid activity but less at other targets that could contribute to their physiological effects. Here, we investigated the in vitro and in vivo activity of kratom alkaloids at serotonin receptors (5-HTRs). Paynantheine and speciogynine exhibited high affinity for 5-HT_1ARs and 5-HT_2BRs, unlike the principal kratom alkaloid mitragynine. Both alkaloids produced antinociceptive properties in rats via an opioid receptor-independent mechanism, and neither activated 5-HT_2BRs in vitro. Paynantheine, speciogynine, and mitragynine induced lower lip retraction and antinociception in rats, effects blocked by a selective 5-HT_1AR antagonist. In vitro functional assays revealed that the in vivo 5-HT_1AR agonistic effects may be due to the metabolites 9-O-desmethylspeciogynine and 9-O-desmethylpaynantheine and not the parent compounds. Both metabolites did not activate 5-HT_2BR, suggesting low inherent risk of causing valvulopathy. The 5-HT_1AR agonism by kratom alkaloids may contribute to the mood-enhancing effects associated with kratom use.

Figure 1.

Chemical structures of the selected isolated kratom alkaloids and metabolites.

Figure 2.

Radioligand competition binding at human 5-HT_1ARs and 5-HT_2BRs. Ordinates: percent specific binding of the radioligand to receptors expressed in cell membranes (described in the Experimental Section). Abscissae: concentrations of test ligands (log scale). The left panel shows percent specific binding of [³H]8-OH-DPAT at 5-HT_1ARs. The right panel shows percent specific binding of [¹²⁵I]DOI at 5-HT_2BRs. These experiments were conducted in duplicate and repeated twice (N = 2), and the data shown were obtained by Eurofins. We conducted additional affinity tests to validate these results, and the binding curves are shown in the Supporting Information (Figure S21).

Figure 3.

Three-dimensional structure comparison of mitragynine and speciogynine (most stable conformation, Chemdraw3D).

Figure 4.

Two- and three-dimensional chemical structures for speciogynine and geissoschizine methyl ether.

Figure 5.

Effects of i.p. administration of various compounds alone and in combination with the 5-HT_1AR antagonist WAY 100635 on expression of lower lip retraction in rats. Abscissae: vehicle and cumulative compound dose in mg/kg (i.p., log scale). Ordinates: percentage of subjects showing lower lip retraction. Each point represents the mean ± SEM (N = 8, four subjects per sex). No lower lip retraction was observed during each baseline measurement. Following the baseline measurement, a vehicle was administered i.p. 60 min prior to the next measurement (open circles). Following the second measurement, each lowest dose of test compounds was administered. Cumulative doses of each test compound were administered every 15 min prior to each measurement. Note that ipsapirone, paynantheine, speciogynine, and mitragynine produced 100% lower lip retraction. Each gray symbol indicates a significant difference from the respective vehicle. Using separate groups of rats, sensitivity of respective expression of lower lip retraction to WAY 100635 (0.01 mg/kg) was assessed. Following the baseline measurement, WAY 100635 (0.01 mg/kg) and then one of the highest doses of test compounds were administered i.p. 60 and 15 min, respectively, prior to measurement (squares). Behaviorally toxic effects were observed in a pilot observational study of higher doses. WAY 100635 significantly antagonized lower lip retraction produced by each test compound. Each square with cross hatch indicates a significant difference from the respective highest dose of test compounds. Details for statistical analyses are shown in Table 3 and Tables S2 and S3.

Figure 6.

In vitro function of speciogynine and paynantheine at human 5-HT_1ARs (A) and 5-HT_2BRs (B). Abscissae: concentrations of test compounds (molar, log scale). Ordinates: (A) serotonin and ipsapirone activated 5-HT_1ARs as measured by inhibition of forskolin-stimulated production of cAMP, whereas speciogynine and paynantheine showed no significant functional effects. (B) Serotonin activated 5-HT_2BRs as measured by increased production of inositol phosphate 1 (IP1), whereas SB 206553 inactivated 5-HT_2BRs; speciogynine and paynantheine showed no significant functional effects. Each symbol represents the mean ± SEM from at least three independent determinations.

Figure 7.

In vitro binding and function at human 5-HT_1ARs (A, C) and 5-HT_2BRs (B, D). Abscissae: concentrations of test compounds (molar, log scale). Ordinates: (A) 5-HT_1AR competition binding with [³H]8-OH-DPAT. (B) 5-HT_2BR competition binding with [³H]LSD. Note that serotonin data from both 5-HT_1AR [³H]8-OH-DPAT and 5-HT_2BR [³H]LSD competition binding fit best to a two-site model, relative to a one-site model (P values: <0.05). K_i values for serotonin at 5-HT_1ARs were 0.41 nM (high affinity) and 6.7 nM (low affinity), and K_i values for serotonin at 5-HT_2BRs were 5.0 nM (high affinity) and 230 nM (low affinity); R² values were 0.726 and 0.924, respectively. The K_i results for serotonin at 5-HT_1A and 5-HT_2BRs reported in Table 2 of the manuscript are from one-site analyses, wherein R² values were 0.715 and 0.895, respectively. (C) Serotonin, 9-O-desmethylpaynantheine, and 9-O-desmethylspeciogynine activated 5-HT_1ARs as measured by inhibition of forskolin-stimulated production of cAMP. (D) Serotonin activated 5-HT_2BRs as measured by increased production of IP1, whereas SB 206553, 9-O-desmethylpaynantheine, and 9-O-desmethylspeciogynine inactivated 5-HT_2BRs. Table 4 shows the pIC/EC₅₀ and E/I_max values. Each data point represents the mean (±SEM) of at least three independent experiments. In each experiment, serotonin and SB 206553 were tested in duplicate while the kratom alkaloids were tested in triplicate.

Figure 8.

Effects of i.v. injection of ipsapirone, paynantheine, and speciogynine on hotplate latency in catheterized rats. The catheterized rats differ from rats in Figure 5. Abscissae: vehicle, cumulative compound dose in mg/kg (i.v., log scale). Ordinates: percentage of maximum possible effects. The hotplate was maintained at 52 ± 0.1 °C. Each symbol represents the mean ± SEM (N = 6, three subjects per sex). In baseline values across six groups (ipsapirone alone and in combination with WAY 100635, paynantheine alone and in combination with WAY 100635, and speciogynine alone and in combination with WAY 100635), there was no significant difference of the treatment group (F_5,24 = 1.44; P = 0.245), sex (F_1,24 = 3.16; P = 0.088), and interaction of the two (F_5,24 = 1.54; P = 0.215). Following the baseline measurement, the vehicle was administered i.p. 60 min prior to the next measurement (open squares). Following the second measurement, cumulative doses were sequentially administered. Cumulative doses of each test compound were administered every 5 min prior to each measurement. Note that ipsapirone, paynantheine, and speciogynine significantly increased the maximum possible effect. Each gray symbol indicates a significant difference from the respective vehicle. Using separate groups of rats, sensitivity of respective antinociceptive effects to WAY 100635 (0.01 mg/kg) was assessed. Following the baseline measurement, WAY 100635 (0.01 mg/kg) was administered i.p. 60 min prior to measurement (squares). WAY 100635 shifted to the right dose-effect functions of each test compound. Paynantheine (32 mg/kg) in the presence of WAY 100635 caused lethality in all rats tested. Details for statistical analyses are shown in Table 3 and Table S4.

Figure 9.

Correlations between in vitro 5-HT_1AR affinity (K_i values) and potency to produce lower lip retraction and antinociception. Ordinates: (left panel) ED₅₀ value in μmol/kg (i.p.) to produce lower lip retraction; (right panel) ED₅₀ value in μmol/kg (i.v.) to produce antinociception. Abscissae: K_i values at 5-HT_1ARs in nM. (Left panel) The R² value was 0.807 (P = 0.0508). (Lower panels) The R² values were 0.9025 (P = 0.142).

Figure 10.

Mean plasma concentration-time profile of (left) i.v. paynantheine and (right) i.v. speciogynine in male rats. Each symbol represents mean ± SEM (n = 4 paynantheine, n = 3 speciogynine).