Analgesic effect of highly reversible ω-conotoxin FVIA on N type Ca2+ channels

Abstract

Background: N-type Ca2+ channels (Ca(v)2.2) play an important role in the transmission of pain signals to the central nervous system. ω-Conotoxin (CTx)-MVIIA, also called ziconotide (Prialt®), effectively alleviates pain, without causing addiction, by blocking the pores of these channels. Unfortunately, CTx-MVIIA has a narrow therapeutic window and produces serious side effects due to the poor reversibility of its binding to the channel. It would thus be desirable to identify new analgesic blockers with binding characteristics that lead to fewer adverse side effects.

Results: Here we identify a new CTx, FVIA, from the Korean Conus Fulmen and describe its effects on pain responses and blood pressure. The inhibitory effect of CTx-FVIA on N-type Ca2+ channel currents was dose-dependent and similar to that of CTx-MVIIA. However, the two conopeptides exhibited markedly different degrees of reversibility after block. CTx-FVIA effectively and dose-dependently reduced nociceptive behavior in the formalin test and in neuropathic pain models, and reduced mechanical and thermal allodynia in the tail nerve injury rat model. CTx-FVIA (10 ng) also showed significant analgesic effects on writhing in mouse neurotransmitter- and cytokine-induced pain models, though it had no effect on acute thermal pain and interferon-γ induced pain. Interestingly, although both CTx-FVIA and CTx-MVIIA depressed arterial blood pressure immediately after administration, pressure recovered faster and to a greater degree after CTx-FVIA administration.

Conclusions: The analgesic potency of CTx-FVIA and its greater reversibility could represent advantages over CTx-MVIIA for the treatment of refractory pain and contribute to the design of an analgesic with high potency and low side effects.

Figure 1

CTx-FVIA cloned from Conus Fulmen. (A) Conus Fulmen lives in an area of subtropical sea south of Jeju island, South Korea. This piscivorous snail captures its prey mainly by paralyzing it with a mixture of toxins. (B) Nucleotide sequence and deduced amino acid sequence of CTx-FVIA. Δindicates the position at which the carboxy terminal of Arg is cleaved by conotoxin precursor proteases, and ▲ indicates the C-terminal cysteine that is thought to be amidated by monooxygenase. The mature sequence of CTx-FVIA is underlined. (C) Conotoxins homologous with CTx-FVIA are listed. Black box indicates similarity of all the peptides; dark and light gray boxes indicate similarity between five and four peptides, respectively. Six cysteines form three disulfide bonds (1st-4th, 2nd-5th, 3rd-6th) and four intercysteine loops.

Figure 2

Electrophysiological characteristics of CTx-FVIA and -MVIIA. (A) Representative traces of N-type Ca²⁺ currents (control) in C2D7 cells showing blockade by 100 nM CTx-FVIA. (B) Concentration-response curves for CTx-FVIA and -MVIIA (n = 3). (C) Time-course of the block induced by 100 nM CTx-FVIA or -MVIIV and its washout. Cells were held at -80 mV and depolarized to 0 mV. 100 nM CTx-FVIA (Δ) or CTx-MVIIA (○) were applied until a complete block was achieved and then washed out. Currents were measured every 15 s. (D) Potency of CTx-FVIA and -MVIIA and percent recovery after washout. (E) Averaged time constants τ_on for CTx-FVIA and -MVIIA determined from time-course graphs fitted with a three-parameter single exponential function.

Figure 3

Effects of intrathecal CTx-FVIA and -MVIIA on pain responses induced by intraplantar (left hindpaw) formalin injection. Mice were subcutaneously injected with formalin solution (5%, 10 μl) 5 min after intrathecal administration of CTx-FVIA and -MVIIA. (A) Effect of intrathecal CTx-FVIA and -MVIIA (32 ng) on the cumulative time spent licking and biting the injected paw. Measurements were made during the first phase (0-5 min) and second phase (20-40 min) of the formalin response. There were 8-10 animals in each group. *P < 0.05, **P < 0.01, ***P < 0.005 vs. control. (B) Dose-response curves for the indicated toxins during the indicated period.

Figure 4

Effects of intrathecal CTx-FVIA and -MVIIA on acute and inflammatory pains. (A) Effects of CTx-FVIA on acute pain responses. CTx-FVIA (10 ng) was administered intrathecally 5 min before the indicated tests. There were 7-10 animals in each group. (B-D) Effects of CTx-FVIA and -MVIIA on pain responses elicited by acetic acid (intraperitoneal), substance P, glutamate, TNF-α, IL-1β and IFN-γ (intrathecal). These agents were applied 5 min after 10 ng CTx-FVIA or -MVIIA was administered intrathecally. Pain responses, including writhing, licking, biting and scratching, were measured for 30 min after injection. (B) Acetic acid-induced writhing counts. (C) Cumulative duration of neurotransmitter-evoked pain behavior. (D) Cumulative duration of inflammatory cytokine-evoked pain behavior. The data are presented as means ± SEM. There were 9-13 animals in each group. Con (control) refers to the saline-treated group. *P < 0.05, **P < 0.01, ***P < 0.005 vs. control.

Figure 5

Analgesic effects of intrathecal CTx-FVIA on neuropathic pain. (A-C) Time-course of antinociceptive effects of intrathecal CTx-FVIA (6.5, 20, 65 and 200 ng/kg) on allodynia in an TNI rat model: (A) mechanical allodynia; (B) cold allodynia; and (C) warm allodynia. The data are presented as means ± SEM. There were 5-8 animals in each group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the saline group (two-way ANOVA followed by Bonferroni's tests).

Figure 6

Effects of intravenous CTx-FVIA and -MVIIA on blood pressure. (A) Effects of intravenous CTx-FVIA and -MVIIA (100 μg/kg) on mean arterial pressure measured in the femoral artery of anaesthetized rats and (B) on mean blood pressure measured using the tail-cuff method. The data are presented as means ± SEM. *P < 0.01 vs. the pre-dosing value (repeated measures ANOVA followed by Dunnett's test). #P < 0.05 CTx-FVIA vs. CTx-MVIIA (10 min to 60 min, repeated measures ANOVA).