Potential Uses of Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review.

Neuropathic pain is a subset of chronic pain that is caused by neurons that are damaged or firing aberrantly in the peripheral or central nervous systems. The treatment guidelines for neuropathic pain include antidepressants, calcium channel α2 delta ligands, topical therapy, and opioids as a second-line option. Pharmacotherapy has not been effective in the treatment of neuropathic pain except in the treatment of trigeminal neuralgia with carbamazepine. The inability to properly treat neuropathic pain causes frustration in both the patients and their treating physicians. Venoms, which are classically believed to be causes of pain and death, have peptide components that have been implicated in pain relief. Although some venoms are efficacious and have shown benefits in patients, their side-effect profile precludes their more widespread use. This review identifies and explores the use of venoms in neuropathic pain relief. This treatment can open doors to potential therapeutic targets. We believe that further research into the mechanisms of action of these receptors as well as their functions in nature will provide alternative therapies as well as a window into how they affect neuropathic pain.

Omega-conotoxins as experimental tools and therapeutics in pain management.

Neuropathic pain afflicts a large percentage of the global population. This form of chronic, intractable pain arises when the peripheral or central nervous systems are damaged, either directly by lesion or indirectly through disease. The comorbidity of neuropathic pain with other diseases, including diabetes, cancer, and AIDS, contributes to a complex pathogenesis and symptom profile. Because most patients present with neuropathic pain refractory to current first-line therapeutics, pharmaceuticals with greater efficacy in pain management are highly desired. In this review we discuss the growing application of ω-conotoxins, small peptides isolated from Conus species, in the management of neuropathic pain. These toxins are synthesized by predatory cone snails as a component of paralytic venoms. The potency and selectivity with which ω-conotoxins inhibit their molecular targets, voltage-gated Ca2+ channels, is advantageous in the treatment of neuropathic pain states, in which Ca2+ channel activity is characteristically aberrant. Although ω-conotoxins demonstrate analgesic efficacy in animal models of neuropathic pain and in human clinical trials, there remains a critical need to improve the convenience of peptide drug delivery methods, and reduce the number and severity of adverse effects associated with ω-conotoxin-based therapies.

An evaluation of the antinociceptive effects of Phα1ß, a neurotoxin from the spider Phoneutria nigriventer, and ω-conotoxin MVIIA, a cone snail Conus magus toxin, in rat model of inflammatory and neuropathic pain.

Voltage-sensitive calcium channels (VSCCs) underlie cell excitability and are involved in the mechanisms that generate and maintain neuropathic and inflammatory pain. We evaluated in rats the effects of two VSCC blockers, ω-conotoxin MVIIA and Phα1ß, in models of inflammatory and neuropathic pain induced with complete Freund's adjuvant (CFA) and chronic constrictive injury (CCI), respectively. We also evaluated the effects of the toxins on capsaicin-induced Ca(2+) influx in dorsal root ganglion (DRG) neurons obtained from rats exposed to both models of pain. A single intrathecal injection of Phα1ß reversibly inhibits CFA and CCI-induced mechanical hyperalgesia longer than a single injection of ω-conotoxin MVIIA. Phα1ß and MVIIA also inhibited capsaicin-induced Ca(2+) influx in DRG neurons. The inhibitory effect of Phα1ß on capsaicin-induced calcium transients in DRG neurons was greater in the CFA model of pain, while the inhibitory effect of ω-conotoxin MVIIA was greater in the CCI model. The management of chronic inflammatory and neuropathic pain is still a major challenge for clinicians. Phα1ß, a reversible inhibitor of VSCCs with a preference for N-type Ca(2+) channels, has potential as a novel therapeutic agent for inflammatory and neuropathic pain. Clinical studies are necessary to establish the role of Phα1ß in the treatment of chronic pain.

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

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.

Divergent M- and O-superfamily peptides from venom of fish-hunting Conus parius.

Six novel peptides from the piscivorous cone snail, Conus parius were purified by reverse-phase HPLC fractionation of crude venom. With the use of matrix-assisted laser desorption ionization mass spectrometry and standard Edman sequencing methods, the peptides were characterized. Two peptides were identified as members of the m-2 and m-4 branches of the M-superfamily and were designated as pr3a and pr3b, while four peptides were identified as members of the O-superfamily and were designated as pr6a, pr6b, pr6c and pr6d. Peptide pr3a differs from the majority of the M-superfamily peptides in the presence of two prolines, which are not modified to 4-trans-hydroxyproline. In peptide pr3b, five amino acids out of the 16 non-cysteine residues are identical with those of mu-GIIIA and mu-PIIIA, suggesting that pr3b may be a divergent mu-conotoxin. Peptide pr6a is notable because of its extreme hydrophobicity. Peptide pr6c has three prolines that are unhydroxylated. Peptides pr6b and pr6d differ from the previously characterized O-superfamily peptides in the presence of an extended N-terminus consisting of six amino acids. Peptides pr3a, pr3b, pr6a and pr6b were demonstrated to be biologically active when injected intraperitoneally in fish. The identification and characterization of these peptides in venom of a fish-hunting species establish the divergence of gene products and their patterns of post-translational modification within superfamilies in a single Conus species.

Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.

Neuronal (N)-type Ca(2+) channel-selective omega-conotoxins have emerged as potential new drugs for the treatment of chronic pain. In this study, two new omega-conotoxins, CVIE and CVIF, were discovered from a Conus catus cDNA library. Both conopeptides potently displaced (125)I-GVIA binding to rat brain membranes. In Xenopus laevis oocytes, CVIE and CVIF potently and selectively inhibited depolarization-activated Ba(2+) currents through recombinant N-type (alpha1(B-b)/alpha(2)delta1/beta(3)) Ca(2+) channels. Recovery from block increased with membrane hyperpolarization, indicating that CVIE and CVIF have a higher affinity for channels in the inactivated state. The link between inactivation and the reversibility of omega-conotoxin action was investigated by creating molecular diversity in beta subunits: N-type channels with beta(2a) subunits almost completely recovered from CVIE or CVIF block, whereas those with beta(3) subunits exhibited weak recovery, suggesting that reversibility of the omega-conotoxin block may depend on the type of beta-subunit isoform. In rat dorsal root ganglion sensory neurons, neither peptide had an effect on low-voltage-activated T-type channels but potently and selectively inhibited high voltage-activated N-type Ca(2+) channels in a voltage-dependent manner. In rat spinal cord slices, both peptides reversibly inhibited excitatory monosynaptic transmission between primary afferents and dorsal horn superficial lamina neurons. Homology models of CVIE and CVIF suggest that omega-conotoxin/voltage-gated Ca(2+) channel interaction is dominated by ionic/electrostatic interactions. In the rat partial sciatic nerve ligation model of neuropathic pain, CVIE and CVIF (1 nM) significantly reduced allodynic behavior. These N-type Ca(2+) channel-selective omega-conotoxins are therefore useful as neurophysiological tools and as potential therapeutic agents to inhibit nociceptive pain pathways.

Recombinant omega-conotoxin MVIIA possesses strong analgesic activity.

BACKGROUND: omega-Conotoxin (CTX) MVIIA is a specific antagonist of N-type voltage-sensitive calcium channels. A synthetic peptide version of CTX MVIIA (ziconotide) has been approved by the US FDA for severe and chronic pain. Given the high cost and complexity of the synthetic process of the disulfide-rich peptide, the genetic recombinant approach may simplify the development of this potent therapeutic agent. AIM: In this study, we report a new method for production of the recombinant CTX MVIIA. METHOD: A novel DNA fragment encoding CTX MVIIA was designed using Escherichia coli-preferred codons, and the fragment was cloned into the expression vector pGEX(2T). The fusion protein, CTX MVIIA and glutathione-S-transferase (GST) [GST-CTX MVIIA], was expressed in E. coli and purified by affinity chromatography on a glutathione-agarose column. After digestion with thrombin, the CTX MVIIA fragment was purified on a Sephacryl S-100 HR column and identified by mass spectrometry. The bioactivity of the peptide was evaluated by the hot tail-flick assay, in which the CTX MVIIA was intracerebroventricularly administered into Sprague-Dawley rats and its antinociceptive effect measured. RESULTS: The analgesic activity of the conotoxin was about 800 times stronger than that of morphine. CONCLUSION: The recombinant CTX MVIIA expressed in E. coli has shown marked analgesic activity, which may have potential in clinical application.

Development of antibody-based assays for omega-conotoxin MVIIA.

Omega-conotoxin MVIIA (CTX MVIIA) is a specific peptide blocker of the N-type voltage-sensitive calcium channel in neurons. The synthetic version of CTX MVIIA, Ziconotide, has been recently approved by FDA for management of severe and chronic pains. Currently, the chemical synthetic CTX MVIIA has been analyzed by RP-HPLC, and there are no chemical or immunological assays available for determination of the peptide. In this article, we report a novel method for preparation of polyclonal antibody against CTX MVIIA, and the antibody-based assays for the analysis of CTX MVIIA. The DNA sequences encoding the conotoxin were chemically synthesized and then cloned into the expression vector pGEX-2T. The GST fusion protein of CTX MVIIA was expressed in E. coli BL21 (DE3) with induction of IPTG. The purified fusion protein was used to immunize the male rabbits with standard protocols. The produced antiserum was purified through anion-exchange chromatography. Another thioredoxin (Trx) fusion protein of CTX MVIIA was employed to cross-examine the antibody against the conotoxin. Our Western blot and ELISA results show that the polyclonal antibody was capable of binding the conotoxin parts of both GST and Trx fusion proteins, and the antibody titer is 1:8192. Thus, the assays based on this antibody are useful for the conotoxin analysis.

A new omega-conotoxin that targets N-type voltage-sensitive calcium channels with unusual specificity.

A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named omega-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 degrees C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of omega-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated omega-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a K(d) of 36.3 pM. Its binding is competitive with omega-Ctx MVIIA at low concentration (K(i) = 2 pM). Moreover, omega-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated omega-Ctx GVIA and omega-Ctx MVIIC. Although omega-Ctx CNVIIA clearly blocked N-type Ca(2+) current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to omega-Ctx GVIA. omega-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca(2+) channels in the animal kingdom.