Exploring the structural and functional aspects of the phospholipase A2 from Naja spp.

Naja spp. venom is a natural source of active compounds with therapeutic application potential. Phospholipase A2 (PLA2) is abundant in the venom of Naja spp. and can perform neurotoxicity, cytotoxicity, cardiotoxicity, and hematological disorders. The PLA2s from Naja spp. venoms are Asp 49 isoenzymes with the exception of PLA2 Cys 49 from Naja sagittifera. When looking at the functional aspects, the neurotoxicity occurs by PLA2 called ß-toxins that have affinity for phosphatidylcholine in nerve endings and synaptosomes membranes, and by α-toxins that block the nicotinic acetylcholine receptors in the neuromuscular junctions. In addition, these neurotoxins may inhibit K+ and Ca++ channels or even interfere with the Na+/K+/ATPase enzyme. The disturbance in the membrane fluidity also results in inhibition of the release of acetylcholine. The PLA2 can act as anticoagulants or procoagulant. The cytotoxicity exerted by PLA2s result from changes in the cardiomyocyte membranes, triggering cardiac failure and hemolysis. The antibacterial activity, however, is the result of alterations that decrease the stability of the lipid bilayer. Thus, the understanding of the structural and functional aspects of PLA2s can contribute to studies on the toxic and therapeutic mechanisms involved in the envenomation by Naja spp. and in the treatment of pathologies.

Cobra neurotoxin produces central analgesic and hyperalgesic actions via adenosine A1 and A2A receptors.

Cobra neurotoxin, a short-chain peptide isolated from snake venom of Naja naja atra, showed both a central analgesic effect and a hyperalgesic effect in mice tests. In order to explore mechanisms, a hypothesis is put forward that cobra neurotoxin takes effect through adenosine receptor pathway. The central effects of cobra neurotoxin were evaluated using the hot plate test (a model of acute pain) and the spinal cord injury (a model of central pain) in mice and using A1 receptor antagonist (DPCPX) and A2A receptor antagonist (ZM241385); behaviors were scored and signal molecules such as reactive oxygen species and adenosine triphosphate levels and mitogen-activated protein kinases/extracellular signal-regulated protein kinase expression were measured. Low dose of cobra neurotoxin (25 µg/kg) had analgesic effects which were inhibited by DPCPX, while high dose of cobra neurotoxin (100 µg/kg) had hyperalgesic effects which were blocked by ZM241385. Cobra neurotoxin reduced reactive oxygen species and increased adenosine triphosphate in brain tissues, and extracellular signal-regulated protein kinase expression was markedly inhibited by cobra neurotoxin. Cobra neurotoxin may take effect through mitogen-activated protein kinases/extracellular signal-regulated protein kinase pathway inhibition by activating adenosine A1Rs and cause changes of reactive oxygen species and adenosine triphosphate through feedback mechanisms. Overdose cobra neurotoxin further activates the adenosine A2ARs to generate pain sensitization. This research proposes a new central analgesic mechanism of cobra neurotoxin and discloses dual regulation of pain.

Dimerization of resveratrol induced by red light and its synergistic analgesic effects with cobra neurotoxin.

Resveratrol polymer has better effects than monomer in some aspects as reported, but most of synthetic methods acquire severe conditions and no analgesic effects are investigated. A novel method is found to synthesize resveratrol polymer by excitation of photosensitizer pheophorbide at red light of 630 nm. The polymer was analyzed by fluorescence spectra and HPLC, further isolated by preparative liquid chromatography and identified as a resveratrol dimer by MS and NMR. Analgesic effects were measured by acetic acid writhing and hot-plate test in mice. The resveratrol dimer has the stronger analgesic effects than monomer, and drug combination of the dimer and cobra neurotoxin enhances and prolongs analgesic effects, suggesting the synergistic action. Simulation of molecular interaction reveals that the dimer spontaneously binds to cobra neurotoxin and makes a complex substance. The dimer can interact with cyclooxygenase-2, µ receptor and nicotine receptor, the synergistic analgesic effects of the complex are attributed to its multiple targets role. The combination of resveratrol dimer and cobra neurotoxin may make up for their deficiencies in analgesic effects, and has prospects in clinical use.

Involvement of cholinergic system in suppression of formalin-induced inflammatory pain by cobratoxin.

AIM: To investigate the analgesic effect of cobratoxin (CTX), a long-chain α-neurotoxin from Thailand cobra venom, in a rat model of formalin-induced inflammatory pain. METHODS: Inflammatory pain was induced in SD rats via injecting 5% formalin (50 µL) into the plantar surface of their right hind paw. CTX and other agents were ip administered before formalin injection. The time that the animals spent for licking the injected paw was counted every 5 min for 1 h. RESULTS: CTX (25, 34, and 45 µg/kg) exhibited a dose-dependent analgesic effect during the phase 1 (0-15 min) and phase 2 (20-60 min) response induced by formalin. Pretreatment with naloxone (0.5 or 2.5 mg/kg) did not block the analgesic effect of CTX. Pretreatment with atropine at 5 mg/kg, but not at 2.5 mg/kg, antagonized the analgesic effect of CTX. Treatment with the nonselective nAChR antagonist mecamylamine (3 mg/kg) inhibited the analgesic effects of CTX in Phase 1 and Phase 2 responses, while with the selective α7-nAChR antagonist methyllycaconitine (3 mg/kg) antagonized the effect of CTX only in the Phase 1 response. Treatment with the α7-nAChR agonist PNU282987 (3 mg/kg) significantly reduced the formalin-induced phase 2 pain response, but only slightly reduced the Phase 1 pain response. CONCLUSION: The results suggest that CTX exerts an antinociceptive effect in formalin-induced inflammatory pain, which appears to be mediated by mAChR and α7-nAChR.

Inhibition of non-neuronal alpha7-nicotinic receptor reduces tumorigenicity in A549 NSCLC xenografts.

Nicotinic acetylcholine receptors (nAChR) are expressed on bronchial epithelial and non-small cell lung cancer cells and are involved in cell growth regulation. Nicotine (classical nAChR agonist) induced cell proliferation, whereas nAChR antagonists, d- tubocurarine or alpha-cobratoxin (alpha-CbT), induced cell death. In the current study, we further explored the antitumor potential mechanisms and activities of alpha-CbT. NOD/SCID mice were grafted intraperitoneally or orthotopically and treated with alpha-CbT. alpha-CbT treatment [0.04 ng/kg or 0.12 ng/kg] induced a strong reduction in tumor size ( approximately 90%) in comparison with mice treated with the vehicle alone. Tumor inhibition was related to severe induction of apoptosis. Moreover, neoangiogenesis was strongly inhibited (reduction of cells positive to vascular endothelial growth factor and CD31). Biochemical analyses of the cells, isolated by the primary lung tumor in alpha-CbT-treated mice, showed apoptosis features characterized by: (i) inhibition of BAD phosphorylation at Ser(112) and Ser(136); (ii) BAD dissociation from 14-3-3; (iii) BAD association with BCL-XL; and (iv) cleavage of caspase-9. Moreover, these cells were unable to grow in soft agar and develop tumor, when reinjected into mice. The small interfering RNA-mediated silencing of the alpha7-nAChR gene confirmed that alpha-CbT specifically inhibited the alpha7-nAChR-mediated survival pathway in A549 cells. Furthermore, the specificity of alpha-CbT is reinforced by the lack of effect of short chain toxin (Erabutoxin-a). Once more, the no effect of the low-affinity R33E-modified alpha-CbT strengthened the specificity of this inhibition. Although alpha7-nAChR antagonists, such as alpha-CbT, are unlikely to be a primary therapy, it may provide lead compounds for the design of clinically useful drugs.

Inhibition of nonneuronal alpha7-nicotinic receptor for lung cancer treatment.

RATIONALE: Studies strongly suggest that the nicotinic acetylcholine receptors for nicotine (nAChRs) play a significant role in lung cancer predisposition and natural history. The nAChR alpha7 subunit has been found to be pivotal in the control of nicotine-induced lung cancer development and in growth signal transduction induced by nicotine binding to nAChRs. OBJECTIVES: To investigate the anticancer effects of alpha7-nAChR antagonists. METHODS: (1) To check the correlation between alpha7-nAChR presence and alpha-cobratoxin (alpha-CbT) sensitivity, binding experiments were performed in various normal human cells, lung cancer cell lines, and primary tumoral cells; (2) to demonstrate that alpha-CbT might be an efficient adjuvant therapy for non-small cell lung cancer (NSCLC) we expanded our previous observations to a panel of NSCLCs of various subtypes orthotopically grafted on nonobese diabetic/severe combined immunodeficient mice; (3) to gain insight into the mechanism of alpha-CbT-induced tumor reduction, the cells obtained after enzymatic digestion of tumors were analyzed for procaspase-9, Bax, Bad, and Bcl-X(L) protein; and (4) Snail/E-cadherin expression was evaluated to acquire information about the chemoresistance of cancer cells to alpha-CbT. MEASUREMENTS AND MAIN RESULTS: We report herein the results of an experimental strategy aimed at investigating the antitumor effects of a powerful alpha7-nAChR antagonist, alpha-CbT, in an in vivo setting set to mimic the clinical setting of lung cancer; in addition, a possible explanation for alpha-CbT selectivity toward cancer cells is presented. CONCLUSIONS: We report the prolonged survival of alpha-CbT-treated animals in our mouse model of NSCLC, which is most likely the result of multiple mechanisms, including various antiproliferative and antiangiogenic effects.

Suppression of complete Freund's adjuvant-induced adjuvant arthritis by cobratoxin.

AIM: Cobratoxin (CTX), the long-chain alpha-neurotoxin from Thailand cobra venom, has been demonstrated to have analgesic action in rodent pain models. The present study evaluated the anti-inflammatory and anti-nociceptive effects of CTX on adjuvant arthritis (AA) in rats. METHODS: Arthritis was induced by injection of complete Freund's adjuvant (CFA) in rats. Paw swelling and hyperalgesia of AA rats were measured at various times after CFA administration. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-2 (IL-2) and interleukin-10 (IL-10) levels in serum were determined with ELISA. Histopathological changes in synoviocytes were examined under a microscope. Involvement of the cholinergic system in the effects of CTX was examined by pretreatment of animals with the alpha(7) nicotinic receptor (alpha(7)-nAChR) antagonist methyllycaconitine (MLA). RESULTS: CFA induced marked paw swelling and reduced thresholds of mechanical and cold-induced paw withdrawal. The levels of TNF-alpha, IL-1 and IL-2 in the serum of AA rats were increased, whereas the level of IL-10 was decreased. Histopathological examination of synoviocytes showed pronounced inflammation and accumulation of collagen. The administration of CTX (17.0 microg/kg, ip) significantly reduced paw swelling and mechanical and thermal hyperalgesia. CTX also reduced the production of TNF-alpha, IL-1, and IL-2 but increased the production of IL-10 and altered pathohistological changes. The analgesic and anti-inflammatory efficacy of CTX was significantly reduced by MLA (3 mg/kg, sc). CONCLUSION: These results indicate that CTX has a beneficial effect on CFA-induced arthritis by modulating the production of inflammatory cytokines. alpha(7)-nAChR appears to mediate the anti-nociceptive and anti-inflammatory actions of CTX.

Targeting alpha7-nicotinic receptor for the treatment of pleural mesothelioma.

Human malignant pleural mesothelioma (MPM) is a dreadful disease and there is still no standard therapy available for a consistent therapeutic approach. This research is aimed at the evaluation of the potential therapeutic effect of a specific nicotinic receptor (nAChR) antagonist, namely alpha-Cobratoxin (alpha-CbT). Its effectiveness was tested in mesothelioma cell lines and in primary mesothelioma cells in vitro, as well as in vivo, in orthotopically xenotransplanted NOD/SCID mice. Cells showed alpha7-nAChR expression and their growth was significantly inhibited by alpha-CbT. Severe induction of apoptosis was observed after exposure to alpha-CbT [IC(80-90)]. Apoptosis was characterised by: change in mitochondrial potential, caspase-3 cleavage, down-regulation of mRNA and protein for survivin, XIAP, IAP1, IAP2 and Bcl-XL, inhibition by caspase-3 inhibitor. In vivo, the alpha-CbT acute LD(50) was 0.15 mg/kg. The LD(100) [0.24 mg/kg] induced fatal respiratory failure and massive kidney necrosis. Phase II experiments with 0.12 ng/kg alpha-CbT (1/1000 of LD(10)) were done in 53 xenotransplanted mice, inhibiting tumour development as confirmed by chest X-ray examinations, autopsy and microscopical findings. The growth of human proliferating T lymphocytes and of mesothelial cells in primary culture was not affected by alpha-CbT. Non-immunogenic derivatives of the alpha-CbT molecule need to be developed for possible human use.

Alpha-cobratoxin as a possible therapy for multiple sclerosis: a review of the literature leading to its development for this application.

The use of snake venom in the treatment of multiple sclerosis has been, at best, controversial. The anecdotal reports for snake venom's beneficial effects in this condition may be supportable now by recent scientific evidence. Cobratoxin, a neurotoxin obtained from the venom of the Thailand cobra, has demonstrated several pharmacological activities that strongly support its use in this application. By employing a chemical detoxification step, the neurotoxin can be rendered safe for administration to humans with minimal side effects. This modified neurotoxin has demonstrated neuromodulatory, antiviral, and analgesic activity, elements associated with the multiple sclerosis condition. Modified cobratoxin has demonstrated potent immunosuppressive activity in acute and chronic animal models of the disease. The drug is under investigation for use in adrenomyeloneuropathy and clinical trials in Multiple sclerosis are planned.

Preparation, characterization and nasal delivery of alpha-cobrotoxin-loaded poly(lactide-co-glycolide)/polyanhydride microspheres.

In this study, alpha-cobrotoxin was incorporated into the microspheres composed of poly(lactide-co-glycolide) (PLGA) and poly[1,3-bis(p-carboxy-phenoxy) propane-co-p-(carboxyethylformamido) benzoic anhydride] (P(CPP:CEFB)) and intranasally delivered to model rats in order to improve its analgesic activity. The microspheres with high entrapment efficiency (>80%) and average diameter of about 25 microm could be prepared by a modified water-in-oil-in-oil (w/o/o) emulsion solvent evaporation method. Scanning electron micrograph (SEM) study indicated that P(CPP:CEFB) content played a considerable role on the morphology and degradation of the microspheres. The presence of P(CPP:CEFB) in the microspheres increased their residence time at the surface of the nasal rat mucosa. The toxicity of the composite microspheres to nasal mucosa was proved to be mild and reversible. A tail flick assay was used to evaluate the antinociceptive activity of the microspheres after nasal administration. Compared with the free alpha-cobrotoxin and PLGA microspheres, PLGA/P(CPP:CEFB) microspheres showed an apparent increase in the strength and duration of the antinociceptive effect at the same dose of alpha-cobrotoxin (80 microg/kg body weight).

A randomized controlled study of modified cobratoxin in adrenomyeloneuropathy.

Adrenomyeloneuropathy is a peroxisomal disorder that causes demyelination, with no proven therapy. Oral modified cobratoxin was assessed in a double-blind, randomized, crossover study of eight patients. Treatment was well tolerated. There were no significant improvements with therapy. The authors do not confirm previous anecdotal reports of dramatic improvement with modified cobratoxin.

Double-blind study of modified neurotoxin in motor neuron disease.

In a double-blind study, we evaluated the intramuscular administration of modified neurotoxin in 48 patients who were treated for 6 months. There was no evidence to support claims that patients receive any benefit from the neurotoxin. A significant number of patients had minor functional improvement, often of a transient nature, but these manifestations were more common in the placebo-treated patients than in those receiving neurotoxin. These variations in the course of motor neuron disease have not been stressed in the previous literature.