The potency with which CTx-FVIA blocks N-type Ca
2+ channels and its homology with CTx-MVIIA suggests CTx-FVIA should be effective against pain
in vivo. To test this hypothesis, we used several rodent pain models. Pain is classified as acute pain, which is essential for survival of the organism, and persistent pain, which is pathological and often accompanied by a lowered pain threshold. The formalin test is useful to investigate drug effects on both acute and persistent pain because it produces two phases of nociceptive behavior. First phase pain is caused by direct effects on nociceptors leading to activation of primary afferent fibers, but second phase pain is caused by tonic inflammatory nociceptive responses. The effects of CTx-FVIA and -MVIIA were more pronounced during the second phase than during the first, indicating that CTxs reduce pathological pain more effectively than acute pain (Figure
3). When an effective dose (10 ng) of CTx-FVIA was intrathecally administered in acute thermal pain models (hot plate or tail-flick), the latency times were little affected or perhaps even reduced (plantar test) (Figure
4A). By contrast, CTx-FVIA effectively reduced thermal allodynia caused by peripheral nerve injury (Figure
5B and
5C). These findings are similar to results previously reported for CTx-MVIIA in tail-flick or formalin tests [
6,
8,
42] and suggest that N-type Ca
2+ channel blockers are more effective against chronic pain than acute pain. This may be due in part the fact that expression of N-type Ca
2+ channels is up-regulated by chronic pain, reflecting the plasticity of the affected synapses [
37,
38,
43,
44].
Chronic pain is further divided into inflammatory pain and neuropathic pain, based on the underlying mechanism [
43]. Inflammatory pain is caused by the actions of various inflammatory mediators, including cytokines, growth factors, neurotransmitters and protons, while neuropathic pain is caused by nervous system dysfunction and is well characterized in rodents with respect to the location and form of the nerve injury involved [
45,
46]. In the present study, intraperitoneal injection of acetic acid or intrathecal injection of glutamate, substance P, TNF-α, IL-1β or IFN-γ were used to simulate inflammatory pain [
47]. Intraperitoneal injection of acetic acid is considered to be a model of visceral inflammatory pain, while neurotransmitters and pro-inflammatory cytokines play important roles in the development of inflammatory pain in the spinal cord, where they produce pain signals by directly activating nociceptors or by sensitizing the peripheral nervous system [
43]. At effective doses (e.g., 10 ng), CTx-FVIA and -MVIIA exerted analgesic effects in all of these inflammatory pain models, with the exception of IFN-γ-induced pain (Figure
3 and
4), which suggests the involvement of N-type Ca
2+ channels in the pain pathway activated by these inflammatory mediators. The ways in which inflammatory mediators increase pain sensitization through ion channel modulation have been described in other cases. For example, second phase pain in the formalin test is related to an inflammatory reaction in peripheral tissues [
30] and requires nociceptive neurotransmitter release and activation of postsynaptic N-methyl-D-aspartate (NMDA) receptors [
48]. Intrathecal neurotransmitters and pro-inflammatory cytokines also induce inflammatory pain by modulating ion channels [
49‐
51]. In particular, TNF-α and IL-1β increase tetrodotoxin-resistant Na
+ channel currents and sensitize N-type Ca
2+ channels [
52,
53]. It is noteworthy that CTx-FVIA and -MVIIA do not reduce IFN-γ induced pain, although they effectively alleviate pain induced by acetic acid, substance P, glutamate, TNF-α or IL-1β (Figure
4B, C and
4D). It may be that different nociceptive stimuli use different pain pathways or modulate different channels [
43,
54]. It is not well understood how substance P, glutamate and IL-1β affect N-type Ca
2+ channels, but our results suggest they at least evoke pain signals via N-type Ca
2+ channels, probably situated postsynaptically. In two neuropathic pain models, SNL (data not shown) and TNI, CTx-FVIA also exerted dose- and time-dependent anti-allodynic effects (Figure
5). These effects of N-type Ca
2+ channel blockers have been observed in several neuropathic pain models [
6,
55,
56], and our results showing that CTx-FVIA is effective against neuropathic pain developed by S1/S2 nerve injury are similar. Taken together, our results indicate that the CTx-FVIA is as effective an analgesic as CTx-MVIIA in inflammatory and neuropathic pain models, and establishes that the residues essential for blocking N-type Ca
2+ channels are effective on a scaffold of CTx-FVIA (Figure
1C) [
24,
57‐
59].