The word pain derives from the Latin poena, which means “penalty” [1], and treating pain has always been a challenge to mankind since ancient times. Nowadays, the International Association for the Study of Pain describes it as “an unpleasant sensory and emotional experience associated with real or potential tissue damage”. Further understanding on the mechanisms by which pain signals are interpreted, transmitted and maintained will improve pain treatment strategies.

A number of novel approaches using animal toxins to relieve pain have been investigated [2,3,4,5,6,7]. Indeed, peptides derived from animal venoms, including scorpions, spiders, amphibians, snakes and marine organisms, have been explored as antinociceptive agents. Many of them selectively inhibit voltage-activated Ca²⁺ and Na⁺ channels, acid-sensitive ion channels or glutamate ionotropic receptors [8,9,10,11,12] (for a review please refer to [3,5,6,7,13,14]). A successful example is the drug (Prialt^®) derived from ω-conotoxin MVIIA, a peptide from Conus magus snail venom, known to have a pronounced analgesic effect resulting from the inhibition of voltage-activated Ca²⁺ channels [15,16].

It is also known that analgesia can be achieved by modulation of opioid and cannabinoid pathways [17,18]. Some peptides that have their mechanism of action based on this modulation have been characterized. One example is Crotalphine, an antinociceptive peptide based on the natural analgesic factor, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus [19], that has its analgesic effect mediated by the release of peripheral dynorphin A, an endogenous agonist of κ-opioid receptors, and this release is dependent on cannabinoid receptor CB₂ activation [20]. Some scorpion toxins, such as alpha-anatoxin Amm VIII, a weak modulator of Na_v1.2 channel, and the depressant insect-selective beta-toxin LqqIT2, caused antinociception when injected in mammals, being the activation of endogenous opioid system suggested to be implicated [21]. Additionally, very recently, our group showed that a synthetic peptide called PnPP-19, derived from another toxin of the spider Phoneutria nigriventer, exhibits antinociception in rats via opioid and cannabinoid systems [22].

In the present work, we focused on δ-CNTX-Pn1a, a peptide isolated from the venom of the “armed” spider Phoneutria nigriventer. This spider is responsible for most of the envenomation cases in Brazil. Its venom is mainly composed of peptides, some proteins/enzymes, salts, amino acids and biogenic amines (for a review, please refer to [5,7,23,24]). Different peptides from this venom have been shown to target ion channels, as Na_v channels [25,26]; Ca_v channels [27,28,29], and K_v channels [30]. Another interesting group of peptides isolated from the venom of P. nigriventer comprises the insect toxins [31,32,33,34,35], which include PnTx4(6-1) or δ-CNTX-Pn1a, the toxin studied in this work, previously characterized to target insect sodium channels [9]. Herein we renamed this toxin, previously named PnTx4(6-1), taking into account the nomenclature proposed by King and co-authors [36], and we also highlight this new nomenclature for the other P. nigriventer peptides. δ-CNTX-Pn1a is a 48-amino-acid polypeptide, with 5 disulfide bridges (MW 5244.6 Da) [31]. The cDNA encoding this toxin was isolated and the toxin probably has a signal peptide followed by a propeptide [37]. This toxin binds to site 3 of sodium channels in cockroach nerve cord synaptosomes (Periplaneta americana) [9]. Indeed, although δ-CNTX-Pn1a slows down the inactivation of sodium currents in cockroach-isolated axons, it does not affect the currents in rat skeletal muscle (rNa_v1.4/rSKM1) or brain (rNa_v1.2/rBIIA) [9]. Moreover, at a high concentration (30 μg) it does not cause apparent toxicity to mice, when intracerebroventricularly (i.c.v) injected [31]. However, an action of this toxin in other sub-types of Na_vs from mammals cannot be discarded, as we recently demonstrated for the recombinant PnTx4(5-5) (rΓ-ctenitoxin-Pn1a), another insect toxin from P. nigriventer venom, which shows 63% of identity with δ-CNTX-Pn1a [35]. rΓ-ctenitoxin-Pn1a was able to inhibit sodium currents on all mammalian Na_v channels tested (Na_v1.2 to Na_v1.6), with the highest current inhibition on Na_v1.3 (38.43% ± 8.04%, IC₅₀ = 1.5 μM). Despite its apparent lack of toxicity to mammals, we previously showed that Γ-ctenitoxin-Pn1a native reversibly inhibited the current of N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors in rat hippocampal neurons [32]. In addition, we demonstrated that this toxin causes antinociception when peripherally injected in rats [38]. Additionally, it was recently shown that Γ-ctenitoxin-Pn1a promotes neuroprotection under insults of high levels of glutamate, in primary-cultured corticostriatal neurons from wild type mice, as well as from a mouse model of Huntington's disease [39]. It is well known that NMDA receptors are involved in nociception and that NMDA antagonists can produce antinociception [40,41].

Taking into account that δ-CNTX-Pn1a shows significant similarity with PnTx4(5-5) (Γ-ctenitoxin-Pn1a), which antagonized NMDA-receptor current in rat neurons and induced antinociception when peripherally tested in rats [38], this work aims at looking for the putative antinociceptive effect of δ-CNTX-Pn1a in different experimental pain models in rats: inflammatory, neuropathic and nociceptive. After confirming the antinociceptive effect of δ-CNTX-Pn1a in all these pain models, we proceeded to investigate the mechanisms underlying its analgesic properties, revealing the involvement of endogenous opioid and cannabinoid systems.

The antinociceptive potential of animal toxins has been subject of several investigations that demonstrate the antinociceptive effect of venoms and many of their derivative peptides [5,6,12,43,44,45,46]. Our results show the analgesic effect of δ-CNTX-Pn1a in inflammatory, neuropathic and nociceptive in vivo pain models. Moreover, we reveal that the antinociceptive effect of δ-CNTX-Pn1a in the nociceptive pain model involves both opioid and cannabinoid endogenous systems.

Intraplantar administration of δ-CNTX-Pn1a inhibited inflammatory pain only when administered 2 h and 30 min after carrageenan injection. This effect may be related to the kinetics of mediators release in carrageenan-induced inflammation. In fact, many mediators are involved in carrageenan inflammation, including histamine, serotonin, kinins and prostaglandins [47]. The mediators involved in this nociceptive effect are most likely prostaglandins, which are known to decrease the threshold of nociceptor activation two hours after carrageenan administration [48].

The pharmacological treatment of neuropathic pain remains a challenge. Indeed, the current pharmacological clinical management of neuropathic pain achieves clinically significant pain relief in less than 50% of patients [49]. Here, we tested δ-CNTX-Pn1a in the neuropathic pain model and found that this peptide reversed the hyperalgesia caused by sciatic constriction. The effect of δ-CNTX-Pn1a was significant up to 10 min after injection, decreasing progressively over time, and losing effect after 20 min.

According to Dalmolin and co-authors [46] and Oliveira and co-authors [50], two peptides isolated from the venom of Phoneutria nigriventer induce marked antinociceptive effect in an experimental model of neuropathic pain. Both peptides are able to modulate voltage-sensitive calcium channels: Tx3-3 (ω-ctenitoxin-Pn2a) blocks P/Q and R types, while Tx3-5 (U₇-ctenitoxin-Pn1a) is a selective and potent blocker of L type. In low doses (3–300 fmol/site), Tx3-5 (U₇-ctenitoxin-Pn1a) was able to produce antinociception in postoperative, neuropathic and cancer-related pain models. The analgesic potential of these toxins isolated from Phoneutria nigriventer venom seems evident. However, future studies using different concentrations of δ-CNTX-Pn1a may reveal better toxin-induced reversal of the hyperalgesia in our neuropathic model.

It is known that loss of spinal opioid receptors and increased activity of physiological opioid antagonist systems occur in neuropathic pain. In contrast, no biologically relevant decrease in the number of CB₁ receptors was evident after dorsal injury [49]. Although we did not investigate the mechanism of action of δ-CNTX-Pn1a in the neuropathic pain model, the effect of the peptide on CB₁ receptors, observed for the nociceptive pain model discussed below, may encourage future investigations regarding a possible relationship between these receptors and the peptide analgesia observed in our neuropathic pain model.

When intrathecally administered, δ-CNTX-Pn1a showed a dose-dependent analgesic effect in PGE₂-induced hyperalgesia. This effect was comparable to the antinociceptive effect of ω-conotoxin MVIIA, a peptide purified from the venom of the snail Conus magus. However, these two peptides apparently have distinct mechanisms of action. Indeed, the synthetic version of ω-conotoxin MVIIA is a powerful analgesic drug (Prialt^®, Dublin, Ireland) that has a unique mechanism of action involving potent and selective blockage of N-type calcium channels [16]. On the other hand, the mechanism underlying the analgesic effect of δ-CNTX-Pn1a involves both cannabinoid and opioid receptors. It is well known that these receptors are involved in antinociceptive pathways. However, there are few studies regarding the effect of animal toxins on these receptors. Here, we show that only AM251, a selective CB₁ receptor antagonist, when intrathecally administered, antagonizes the antinociceptive effect of δ-CNTX-Pn1a in PGE₂-induced hyperalgesia (Figure 5a). Using naloxone, a nonselective opioid receptor antagonist, the antinociceptive effect of δ-CNTX-Pn1a was reverted (Figure 6a). In addition, we show that μ and δ opioid, but not κ receptors, are involved in δ-CNTX-Pn1a antinociception (Figure 6b,c). Pnpp19, a peptide synthetized by our group, in addition to potentiating erectile function in rats, showed antinocieptive activity against PGE₂ hyperalgesia through activation of µ and δ opioid, and CB₁ cannabinoid receptors. This peptide also seems to be able to indirectly induce antinociception through inhibition of a neuronal endopeptidase responsible for the cleavage of the endogenous opioid peptide encephalin [22]. Different from these findings, Crotalphine peptide reduced PGE₂-induced hyperalgesia through an increased activation of both κ-opioid and CB₂ cannabinoid receptors, being this effect mediated by dynorphin A [20]. This result reinforces the interaction between cannabinoid and opioid systems, as observed in our work and highlights the complexity of the nociceoptitive pathways.

Finally, we recently showed that rPnTx4(5-5) (rΓ-ctenitoxin-Pn1a), another insect toxin from P. nigriventer showing 63% of similarity to δ-CNTX-Pn1a, obtained by heterologous expression in E. coli, besides strongly slowing down the inactivation of Bg Na_v (a sodium channel from Blatella germanica cockroach) also inhibited sodium currents on all tested Na_vs from mammals (Na_v1.2 to Na_v1.6). It is worth noting that rΓ-ctenitoxin-Pn1a, as observed for δ-CNTX-Pn1a, did not show any apparent toxicity to mice, when i.c.v. injected (up to 30 μg). rΓ-ctenitoxin-Pn1a caused the highest current inhibition on Na_v1.3 (38.43% ± 8.04%, IC₅₀ = 1.5 μM) [35]. In addition to Na_v1.3, some subtypes of Na_vs, especially Na_vs 1.7, l.8 and 1.9, have been described to play a significant role in pain processes. However, in order to investigate if the antinociceptive effect of these insect-toxins involves the inhibition of sodium channels, other assays are required, including the test of its effects on Na_vs 1.7, 1.8 and 1.9, which are critically involved in pain mechanisms. Another possible target of these toxins, as observed for rΓ-ctenitoxin-Pn1a [38,39] is the NMDA receptor, which could also be involved in the observed antinociceptive effects. Other studies are in progress to try to clarify this point.

In the present study, we reveal that δ-CNTX-Pn1a, a peptide isolated from the venom of Phoneutria nigriventer spider and previously characterized as an insect toxin, shows a clear analgesic effect in three different in vivo pain models. Moreover, we show, for the first time, the involvement of CB₁ cannabinoid receptor and μ and δ opioid receptors in the antinociceptive effect of δ-CNTX-Pn1a. Taken together, our results may contribute for the development of novel therapeutic agents of a wide spectrum to manage pain, although studies are still necessary to better clarify the mechanisms involved in the effects of δ-CNTX-Pn1a.

This study received financial support from INCTTox (INCTTOX 573790/2008-6), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig), Capes-Toxinologia (Capes 063/10-23038.006280/2011-07), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We would like to acknowledge Dr. Marie France Martin Eauclaire for reviewing this manuscript.

The following abbreviations are used in this manuscript:

Maria Elena de Lima proposed the project and, together with Igor Dimitri G. Duarte, supervised the research and revised this paper. Marta N. Cordeiro, Márcia Helena Borges and Suely G. Figueiredo isolated the toxin and performed its biochemical characterization. Renata C.M. Ferreira and Bruna Luiza Emerich performed the nociceptive experiments and data analyses, besides writing the paper. Adriano M.C. Pimenta reviewed the manuscript and participated in discussions during the work. All authors contributed extensively to the work presented in this paper.

The authors declare no conflicts of interest.