Isolation and Pharmacological Characterization of Six Opioidergic Picralima nitida Alkaloids

Abstract

The seeds of the akuamma tree (Picralima nitida) have been used as a traditional treatment for pain and fever. Previous studies have attributed these effects to a series of indole alkaloids found within the seed extracts; however, these pharmacological studies were significantly limited in scope. Herein, an isolation protocol employing pH-zone-refining countercurrent chromatography was developed to provide six of the akuamma alkaloids in high purity and quantities sufficient for more extensive biological evaluation. Five of these alkaloids, akuammine (1), pseudo-akuammigine (3), akuammicine (4), akuammiline (5), and picraline (6), were evaluated against a panel of >40 central nervous system receptors to identify that their primary targets are the opioid receptors. Detailed in vitro investigations revealed 4 to be a potent kappa opioid receptor agonist, and three alkaloids (1-3) were shown to have micromolar activity at the mu opioid receptor. The mu opioid receptor agonists were further evaluated for analgesic properties but demonstrated limited efficacy in assays of thermal nociception. These findings contradict previous reports of the antinociceptive properties of the P. nitida alkaloids and the traditional use of akuamma seeds as analgesics. Nevertheless, their opioid-preferring activity does suggest the akuamma alkaloids provide distinct scaffolds from which novel opioids with unique pharmacologic properties and therapeutic utility can be developed.

Figure 1.

pH-zone-refining countercurrent chromatography chromatogram of akuamma alkaloid DCM extract. Elution of the alkaloids akuammiline (5) and picraline (6) occurred between high pH 3 to low-mid pH 4 range. Elution of pseudo-akuammigine (3) occurred between mid pH 4 to low pH 5. Elution of akuammicine (4) occurred between low to high pH 5.

Figure 2.

Receptor binding profiles of akuamma alkaloids. The akuamma alkaloids akuammine (1), pseudo-akuammigine (3), akuammicine (4), akuammiline (5), and picraline (6) were assessed at 10 μM for their ability to displace radiolabeled ligands from membranes expressing individual receptors. The heatmap represents mean displacement of radioligand from four replicates.

Figure 3.

Pharmacological characterization of akuamma alkaloids at μOR, κOR, and δORs. The akuamma alkaloids akuammine (1), akuammidine (2), pseudo-akuammigine (3), akuammicine (4), akuammiline (5), and picraline (6) were characterized for binding affinity using [³H]DAMGO, [³H]U69,593 and [³H]DPDPE (A, B, C), inhibition of forskolin-induced cAMP in a Glo-sensor assay in transfected HEK-293 cells (D, E, F) and the ability of the alkaloids to recruit β-arrestin 2 in a PathHunter assay. (G, H, I) at μOR (A, D, G), κOR (B, E, H), and δOR (C, F, I). All curves are representative of the averaged values from a minimum of 3 independent assays.

Figure 4.

Effects of pseudo-akuammigine in mouse models of thermal nociception. Antinociception by pseudo-akuammigine (3) was tested at doses of 5 mg/kg (p.o., n=16) (A-B) and 5 and 10 mg/kg (s.c., n=8) (C-D) in C57BL/6 mice via the tail flick assay (A and C) and the hot plate assay (B and D) at various time points. Morphine (6 mg/kg, s.c., n=8) served as a positive control (A-B). All data is expressed as maximum possible effect (%MPE) normalized to a saline baseline (treatment – saline baseline). For the 5 mg/kg doses, ^P < 0.05 vs. vehicle and ^^P < 0.01 vs vehicle. For morphine, ***P < 0.001 vs. vehicle. Morphine and 10 mg/kg alkaloid 3 data was analyzed with a paired t-test. Data for 5 mg/kg alkaloid 3 (p.o. and s.c.) was analyzed with one-way, repeated measures ANOVA followed by Sidak’s multiple comparisons post-test.

Figure 5.

Effects of akuammine and akuammidine in mouse models of thermal nociception. Antinociception by akuammine (1) (3, 10, 30, and 60 mg/kg (s.c.)), A-B) and akuammidine (2) (3, 10, and 30 mg/kg (s.c.)), C-D) was assessed in C57BL/6 mice (n=8, per alkaloid) via the tail flick assay (A and C) and the hot plate assay (B and D) at various time points. All data is expressed as maximum possible effect (%MPE) normalized to a saline baseline (treatment – saline baseline). For the 3 mg/kg doses, ^P < 0.05 vs vehicle and ^^P < 0.01 vs vehicle. For the 10 mg/kg doses, statistical significance is indicated as ^∂P < 0.05 vs vehicle. For the 30 mg/kg doses, ^#P < 0.05 vs vehicle. For the 60 mg/kg doses, *P < 0.05 vs vehicle and **P < 0.01 vs vehicle. Data was analyzed with one-way, repeated measures ANOVA followed by Sidak’s multiple comparisons post-test.