Betulinic acid analogs inhibit N- and T-type voltage-gated calcium channels to attenuate nerve-injury associated neuropathic and formalin models of pain

Abstract

Over the past three decades, there has been a significant growth in the use of natural products, with approximately 80% of individuals using them for some aspect of primary healthcare. Our laboratories have identified and studied natural compounds with analgesic effects from dry land plants or their associated fungus during the past ten years. Here, we isolated and characterized thirteen betulin analogs and fifteen betulinic acid analogs for their capacity to prevent calcium influx brought on by depolarization in sensory neurons. The in vitro inhibition of voltage-gated calcium channels by the top drugs was then assessed using whole cell patch clamp electrophysiology. In vivo experiments, conducted at two sites, evaluated the best compound in acute and tonic, neuropathic, inflammatory, post-operative and visceral models of pain. We found that the betulinic acid analog 8 inhibited calcium influx in rat dorsal root ganglion neurons by inhibiting N- (CaV2.2) and T- (CaV3) type voltage-gated calcium channels. Moreover, intrathecal delivery of analog 8 had analgesic activity in both spared nerve injury model of neuropathic pain and acute and tonic pain induced by formalin. The results presented herein highlight the potential antinociceptive properties of betulinic acid analog 8 and set the stage for the development of novel non-opioid pain therapeutics based on the triterpenoid scaffold of betulinic acid.

Keywords: Analgesic; BA, Betulinic acid; Betulin analogs; Betulinic acid analogs; CaV2.2, N-type voltage-gated calcium channel; CaV3, T-type voltage-gated calcium channel; DRG, dorsal root ganglia; Formalin model; HVA, high voltage-gated; LVA, low voltage-gated; Pain; SNI, spared nerve injury; Spared nerve injury mode; VGCCs, Voltage-gated calcium channels; Voltage-gated calcium channels.

Graphical abstract

Fig. 1

Effect of betulinic acid and betulin analogs on depolarization-evoked calcium influx in rat DRG sensory neurons. Peak calcium responses of female DRG sensory neurons incubated overnight with 0.1 % DMSO (vehicle) or 10 µM of BA analogs in response to 40-mM KCl. Betulinic acid (1) was used as a control (n = 33–365 neurons). The underlined compounds demonstrated more than 50 % inhibition in comparison to vehicle (0.1 % DMSO)- treated controls. Average responses were normalized to that of the vehicle and are shown as mean ± SEM. One-way ANOVA with Dunnett’s post-hoc test (see Table S1 for full statistics).

Fig. 2

Total calcium currents are inhibited by betulinic acid analog 8 in isolated dorsal root ganglia. (A) Representative family of total calcium currents recorded from small- to medium-sized DRG neurons from female rats. (B) Current density–voltage relationship of control (0.1 % DMSO), analogs 8 and 18 evoked from −70 to +60 mV for 200-milliseconds. (C) Summary showing peak calcium current density (DMSO −70.4 ± 6.3, 8–31.7 ± 3.2, 18–58.3 ± 11.9). (D) Boltzmann fits for voltage dependence activation and inactivation. Table 1 summarizes the half-maximal activation potential of activation and inactivation (V_1/2) and slop values (k) for voltage-dependence activation and inactivation. n = 12–13 cells: error bars indicate mean ± SEM; p values as indicated, one-way ANOVA with Tukey’s post hoc test (see Table S1 for full statistics).

Fig. 3

Analog 8 inhibits N-type (Cav2.2) calcium currents in dorsal root ganglia neurons. (A) Representative family of N-type calcium currents evoked by voltage steps from −70 to +60 mV for 200-milliseconds. (B) Current-voltage relationship of control (0.1 %-DMSO), compound 8, evoked from −70 to +60 mV for 200-milliseconds. (C) Peak calcium current density summarized as scatter plots (DMSO −44.0 ± 7.52 (n = 11), 8 –18.8 ± 2.7 (n = 10)). (D) Steady-state inactivation and activation curves. To evaluate steady-state inactivation, currents were elicited by a 20 ms pulse at 10 mV after a 1500 ms pre-pulses ranging from −100 mV to 30 mV in 10 mV increments from a holding potential of −60 mV. Table 2 summarizes the half-maximal activation potential of activation and inactivation (V_1/2) and slop values (k) for voltage-dependence activation and inactivation. n = 10–11 cells; error bars indicate mean ± SEM; p values as indicated (see Table S1 for full statistics).

Fig. 4

T-type calcium currents are reduced by compound 8. (A) Representative T-type Ca²⁺ current traces evoked by voltage steps from −70 to 0 mV in increments of 5 mV. (B) Average current density–voltage plot of control (0.1 % DMSO) and compound 8 (20 µM). Currents were measured from a holding potential of −90 mV with voltage steps of 200 ms of duration applied at 0.5 s intervals in 5 mV increments (C) Peak current density summarized as scatter plot (0.1 %-DMSO, −60.1 ± 5.0, compound 8–35.3 ± 5.7). (D) Boltzmann fits for and steady-state inactivation curves. After a 1.5 s pre-pulses ranging from −100 mV to −10 mV in 10 mV increments from a holding potential of −90 mV. Half-maximal activation potential of activation, inactivation (V_1/2) and slope values (k) for activation and inactivation are presented in Table 3. n = 9–13 cells; error bars indicate mean ± SEM; p values as indicated (see Table S1 for full statistics).

Fig. 5

Compound 8 reduces calcium current through CaV2.2 and CaV3.2 channels expressed in HEK cells. Current density–voltage relationships from HEK cells transiently transfected with plasmid constructs encoding CaV1.2 and CaV1.3 channels (A left), CaV2.2 channels (B left); CaV3.1, CaV3.2, and CaV3.3 channels (C, left). The corresponding peak current densities are shown on the right of each I-V plot. Black symbols indicate overnight incubation with 0.1 % DMSO (control condition). Pink symbols indicate cells treated overnight with 20 µM of compound 8. p values as indicated, Student’s t-test.

Fig. 6

Betulinic acid analog 8 is antinociceptive 3 h after intrathecal delivery. Time course (A) and quantification (B) of paw withdrawal threshold (PWT) of male rats with neuropathic pain induced by spared nerve injury (SNI). At 14 days following SNI, analog 8 was intrathecally administered (2 µg in 5 µl). Compound 8 produced antinocieption 3 h following injection (vehicle 18.4 ± 8.2 (n = 6), analog 8 1.9 ± 0.7 (n = 5)). p values as indicated, two-way ANOVA with Mann-Whitney post hoc test (see Table S1 for full statistics). The behavioral experiments were performed by an experimenter who was blinded to the experimental groups and treatments.