The Crotoxin:SBA-15 Complex Down-Regulates the Incidence and Intensity of Experimental Autoimmune Encephalomyelitis Through Peripheral and Central Actions.

Crotoxin (CTX), the main neurotoxin from Crotalus durissus terrificus snake venom, has anti-inflammatory, immunomodulatory and antinociceptive activities. However, the CTX-induced toxicity may compromise its use. Under this scenario, the use of nanoparticle such as nanostructured mesoporous silica (SBA-15) as a carrier might become a feasible approach to improve CTX safety. Here, we determined the benefits of SBA-15 on CTX-related neuroinflammatory and immunomodulatory properties during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis that replicates several histopathological and immunological features observed in humans. We showed that a single administration of CTX:SBA-15 (54 µg/kg) was more effective in reducing pain and ameliorated the clinical score (motor impairment) in EAE animals compared to the CTX-treated EAE group; therefore, improving the disease outcome. Of interest, CTX:SBA-15, but not unconjugated CTX, prevented EAE-induced atrophy and loss of muscle function. Further supporting an immune mechanism, CTX:SBA-15 treatment reduced both recruitment and proliferation of peripheral Th17 cells as well as diminished IL-17 expression and glial cells activation in the spinal cord in EAE animals when compared with CTX-treated EAE group. Finally, CTX:SBA-15, but not unconjugated CTX, prevented the EAE-induced cell infiltration in the CNS. These results provide evidence that SBA-15 maximizes the immunomodulatory and anti-inflammatory effects of CTX in an EAE model; therefore, suggesting that SBA-15 has the potential to improve CTX effectiveness in the treatment of MS.

Crotoxin Conjugated to SBA-15 Nanostructured Mesoporous Silica Induces Long-Last Analgesic Effect in the Neuropathic Pain Model in Mice.

Neuropathic pain is a disease caused by structural and functional plasticity in central and peripheral sensory pathways that produce alterations in nociceptive processing. Currently, pharmacological treatment for this condition remains a challenge. Crotoxin (CTX), the main neurotoxin of Crotalus durissus terrificus rattlesnake venom, has well described prolonged anti-inflammatory and antinociceptive activities. In spite of its potential benefits, the toxicity of CTX remains a limiting factor for its use. SBA-15 is an inert nanostructured mesoporous silica that, when used as a vehicle, may reduce toxicity and potentiate the activity of different compounds. Based on this, we propose to conjugate crotoxin with SBA-15 (CTX:SBA-15) in order to investigate if when adsorbed to silica, CTX would have its toxicity reduced and its analgesic effect enhanced in neuropathic pain induced by the partial sciatic nerve ligation (PSNL) model. SBA-15 enabled an increase of 35% of CTX dosage. Treatment with CTX:SBA-15 induced a long-lasting reduction of mechanical hypernociception, without modifying the previously known pathways involved in antinociception. Moreover, CTX:SBA-15 reduced IL-6 and increased IL-10 levels in the spinal cord. Surprisingly, the antinociceptive effect of CTX:SBA-15 was also observed after oral administration. These data indicate the potential use of the CTX:SBA-15 complex for neuropathic pain control and corroborates the protective potential of SBA-15.

Crotoxin in humans: analysis of the effects on extraocular and facial muscles / Crotoxina em humanos: estudo da ação em músculos extraoculares e faciais

PURPOSE: Crotoxin is the main neurotoxin of South American rattlesnake Crotalus durissus terrificus. The neurotoxic action is characterized by a presynaptic blockade. The purpose of this research is to assess the ability of crotoxin to induce temporary paralysis of extraocular and facial muscles in humans. METHODS: Doses of crotoxin used ranged from 2 to 5 units (U), each unit corresponding to one LD50. We first applied 2U of crotoxin in one of the extraocular muscles of 3 amaurotic individuals to be submitted to ocular evisceration. In the second stage, we applied crotoxin in 12 extraocular muscles of 9 patients with strabismic amblyopia. In the last stage, crotoxin was used in the treatment of blepharospasm in another 3 patients. RESULTS: No patient showed any systemic side effect or change in vision or any eye structure problem after the procedure. The only local side effects observed were slight conjunctival hyperemia, which recovered spontaneously. In 2 patients there was no change in ocular deviation after 2U crotoxin application. Limitation of the muscle action was observed in 8 of the 12 applications. The change in ocular deviation after application of 2U of crotoxin (9 injections) was in average 15.7 prism diopters (PD). When the dose was 4U (2 applications) the change was in average 37.5 PD and a single application of 5U produced a change of 16 PD in ocular deviation. This effect lasted from 1 to 3 months. Two of the 3 patients with blepharospasm had the hemifacial spasm improved with crotoxin, which returned after 2 months. CONCLUSIONS: This study provides data suggesting that crotoxin may be a useful new therapeutic option for the treatment of strabismus and blepharospasm. We expect that with further studies crotoxin could be an option for many other medical areas.

OBJETIVO: A crotoxina é a principal neurotoxina da cascavel sul-americana Crotalus durissus terrificus e sua ação neurotóxica caracteriza-se por um bloqueio pré-sináptico. O objetivo da pesquisa é avaliar a capacidade da crotoxina em induzir paralisia transitória de músculos extraoculares e faciais em seres humanos. MÉTODOS: As doses utilizadas de crotoxina foram de 2 a 5 unidades (U), sendo que cada unidade correspondia a uma DL-50. Na primeira etapa, aplicou-se 2U de crotoxina em músculos extraoculares de 3 indivíduos amauróticos, candidatos à evisceração. Na segunda etapa, realizaram-se 12 aplicações de crotoxina em músculos extraoculares de 9 indivíduos estrábicos e amblíopes. Na terceira e última etapa, utilizou-se a crotoxina para o tratamento do blefaroespasmo essencial em 3 indivíduos. RESULTADOS: Nenhum paciente demonstrou qualquer efeito sistêmico ou alteração da visão ou de qualquer estrutura ocular. O único efeito local adverso foi hiperemia conjuntival, que melhorou espontaneamente. Em 2 pacientes não houve alteração do desvio ocular após a aplicação de 2U de crotoxina. Observou-se em 8 das 12 aplicações, limitação do movimento ocular no campo de ação do músculo aplicado. A diminuição do desvio ocular com 2U crotoxina (9 aplicações) foi em média de 15,7 dioptrias prismáticas (DP); na dosagem de 4U (2 aplicações) foi em média de 37,5 DP e na única aplicação de 5U, obteve-se redução de 16 DP no desvio ocular. A alteração do alinhamento ocular manteve-se por 1 a 3 meses. Dois dos 3 pacientes portadores de blefaroespasmo apresentaram melhora dos espasmos hemifacias, os quais voltaram após 2 meses. CONCLUSÕES: Através dos resultados observados neste estudo, acreditamos que a crotoxina possa ser útil no tratamento do estrabismo e do blefaroespasmo. Novos estudos precisam ser realizados para confirmar a eficácia e a segurança da crotoxina como opção terapêutica para diversas áreas da medicina que atualmente utilizam a toxina botulínica.

Study of crotoxin on the induction of paralysis in extraocular muscle in animal model / Estudo da crotoxina na indução de paralisia da musculatura extraocular em modelo animal

PURPOSE: Crotoxin is the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus, capable of causing a blockade of the neurotransmitters at the neuromuscular junction. The objective of this study was to appraise the action and effectiveness of the crotoxin induced paralysis of the extraocular muscle and to compare its effects with the botulinum toxin type A (BT-A). METHODS: The crotoxin, with LD50 of 1.5 µg, was injected into the superior rectus muscle in ten New Zealand rabbits. The concentration variance was 0.015 up to 150 µg. Two rabbits received 2 units of botulinum toxin type A for comparative analysis. The evaluation of the paralysis was performed using serial electromyography. After the functional recovery of the muscles, which occurred after two months, six rabbits were sacrificed for anatomopathology study. RESULTS: The animals did not show any evidence of systemic toxicity. Transitory ptosis was observed in almost every animal and remained up to fourteen days. These toxins caused immediate blockade of the electrical potentials. The recovery was gradual in the average of one month with regeneration signs evident on the electromyography. The paralysis effect of the crotoxin on the muscle was proportional to its concentration. The changes with 1.5 µg crotoxin were similar to those produced by the botulinum toxin type A. The histopathology findings were localized to the site of the injection. No signs of muscle fiber's necrosis were seen in any sample. The alterations induced by crotoxin were also proportional to the concentration and similar to botulinum toxin type A in concentration of 1.5 µg. CONCLUSION: Crotoxin was able to induce transitory paralysis of the superior rectus muscle. This effect was characterized by reduction of action potentials and non-specific signs of fibrillation. Crotoxin, in concentration of 1.5 µg was able to induce similar effects as botulinum toxin type A.

OBJETIVO: A crotoxina é a principal toxina do veneno da cobra cascavel sul-americana Crotalus durissus terrificus e causa bloqueio da neurotransmissão na junção neuromuscular. O objetivo deste estudo foi avaliar a ação e aplicabilidade da crotoxina na indução de paralisia da musculatura extrínseca ocular, e comparar seus efeitos com os da toxina botulínica do tipo A (TB-A). MÉTODOS: A crotoxina, com DL50 de 1,5 µg, foi aplicada no músculo reto superior direito de dez coelhos da raça neozelandesa, em concentrações que variaram de 0,015 µg a 150 µg. Em dois coelhos, utilizou-se 2 unidades de toxina botulínica do tipo A para análise comparativa. A avaliação da paralisia foi realizada através de eletromiografia seriada. Após a recuperação, que ocorreu em dois meses, seis coelhos foram sacrificados para estudo anátomopatológico. RESULTADOS: Os animais não apresentaram sinais de intoxicação sistêmica. Ptose palpebral transitória foi observada em quase todos os animais e permaneceu por até 14 dias. As toxinas causaram um bloqueio imediato da captação dos potenciais elétricos. A recuperação foi gradativa no período aproximado de um mês, observando-se sinais evidentes de regeneração no registro eletromiográfico. Os efeitos da crotoxina na paralização do músculo injetado foram proporcionais à concentração. A crotoxina, na concentração de 1,5 µg, induziu alterações semelhantes às da toxina botulínica do tipo A. Os achados anátomo-patológicos foram localizados somente na região em que se aplicou as toxinas, não havendo necrose de fibras musculares em nenhuma amostra analisada. As alterações causadas pela crotoxina também foram proporcionais à concentração utilizada e similares a toxina botulínica do tipo A na concentração de 1,5 µg. CONCLUSÃO: A crotoxina foi capaz de induzir paralisia transitória do músculo reto superior. Este efeito foi caracterizado pela redução na amplitude dos potenciais de ação e sinais inespecíficos de fibrilação. Observou-se que a ação da crotoxina, em concentração de 1,5 µg, proporcionou efeito semelhante ao da toxina botulínica do tipo A.

Crotoxin enhances the antitumor activity of gefinitib (Iressa) in SK-MES-1 human lung squamous carcinoma cells.

Crotoxin (CrTX), a neurotoxin, is isolated from the venom of South American rattlesnakes and has potent antitumor activity. Here, we investigated the antitumor effect of CrTX on the SK-MES-1 human lung squamous cell carcinoma cell line that has acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. CrTX caused G1 arrest and p-JNK protein upregulation that resulted in apoptosis of SK-MES-1 cells. SP600125, a specific inhibitor of p-JNK, could rescue SK-MES-1 cells from CrTX-induced apoptosis. CrTX and gefinitib (Iressa) both inhibited the viability and proliferation of SK-MES-1 cells in a dose- and time-dependent manner. The combination of CrTX and Iressa significantly enhanced the antitumor activity of Iressa. In vivo studies revealed that CrTX caused increased damage to blood vessels and reduced tumor size when combined with Iressa. The present study showed that the JNK signal transduction pathway mediated the anti-apoptotic effect of CrTX, and furthermore, CrTX could enhance the antitumor effect of tyrosine kinase inhibitors in cells with acquired resistance.

Study of crotoxin on the induction of paralysis in extraocular muscle in animal model.

PURPOSE: Crotoxin is the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus, capable of causing a blockade of the neurotransmitters at the neuromuscular junction. The objective of this study was to appraise the action and effectiveness of the crotoxin induced paralysis of the extraocular muscle and to compare its effects with the botulinum toxin type A (BT-A). METHODS: The crotoxin, with LD50 of 1.5 µg, was injected into the superior rectus muscle in ten New Zealand rabbits. The concentration variance was 0.015 up to 150 µg. Two rabbits received 2 units of botulinum toxin type A for comparative analysis. The evaluation of the paralysis was performed using serial electromyography. After the functional recovery of the muscles, which occurred after two months, six rabbits were sacrificed for anatomopathology study. RESULTS: The animals did not show any evidence of systemic toxicity. Transitory ptosis was observed in almost every animal and remained up to fourteen days. These toxins caused immediate blockade of the electrical potentials. The recovery was gradual in the average of one month with regeneration signs evident on the electromyography. The paralysis effect of the crotoxin on the muscle was proportional to its concentration. The changes with 1.5 µg crotoxin were similar to those produced by the botulinum toxin type A. The histopathology findings were localized to the site of the injection. No signs of muscle fiber's necrosis were seen in any sample. The alterations induced by crotoxin were also proportional to the concentration and similar to botulinum toxin type A in concentration of 1.5 µg. CONCLUSION: Crotoxin was able to induce transitory paralysis of the superior rectus muscle. This effect was characterized by reduction of action potentials and non-specific signs of fibrillation. Crotoxin, in concentration of 1.5 µg was able to induce similar effects as botulinum toxin type A.

Crotoxin in humans: analysis of the effects on extraocular and facial muscles.

PURPOSE: Crotoxin is the main neurotoxin of South American rattlesnake Crotalus durissus terrificus. The neurotoxic action is characterized by a presynaptic blockade. The purpose of this research is to assess the ability of crotoxin to induce temporary paralysis of extraocular and facial muscles in humans. METHODS: Doses of crotoxin used ranged from 2 to 5 units (U), each unit corresponding to one LD50. We first applied 2U of crotoxin in one of the extraocular muscles of 3 amaurotic individuals to be submitted to ocular evisceration. In the second stage, we applied crotoxin in 12 extraocular muscles of 9 patients with strabismic amblyopia. In the last stage, crotoxin was used in the treatment of blepharospasm in another 3 patients. RESULTS: No patient showed any systemic side effect or change in vision or any eye structure problem after the procedure. The only local side effects observed were slight conjunctival hyperemia, which recovered spontaneously. In 2 patients there was no change in ocular deviation after 2U crotoxin application. Limitation of the muscle action was observed in 8 of the 12 applications. The change in ocular deviation after application of 2U of crotoxin (9 injections) was in average 15.7 prism diopters (PD). When the dose was 4U (2 applications) the change was in average 37.5 PD and a single application of 5U produced a change of 16 PD in ocular deviation. This effect lasted from 1 to 3 months. Two of the 3 patients with blepharospasm had the hemifacial spasm improved with crotoxin, which returned after 2 months. CONCLUSIONS: This study provides data suggesting that crotoxin may be a useful new therapeutic option for the treatment of strabismus and blepharospasm. We expect that with further studies crotoxin could be an option for many other medical areas.

Avaliação do efeito da crotoxina em animais portadores de encefalomielite autoimune experimental, um modelo de animais de esclerose múltipla / Evaluation of crotoxin effect in the experimental autoimune encephalomyelitis, an animal model of multiple sclerosis

A esclerose múltipla é uma doença inflamatória crônica, de origem autoimune, que acarreta diversas alterações motoras, cognitivas e sensitivas. Dentre as alterações sensitivas, a dor é um dos graves problemas que afetam pessoas portadoras desta doença, interferindo com diversos aspectos da vida do paciente. É importante ressaltar que a esclerose múltipla não tem cura, sendo que a terapêutica se concentra nas ações que retardam a progressão da doença e promovem o alívio dos sintomas, melhorando a qualidade da vida do paciente...

Multiple sclerosis is a Central Nervous System Inflamatory demyelinating disease that has as primary symptomps losses of sensory, cognitive and motor functions. Among the sensory alternations, pain is one of the major concern, afecting various aspects of the patients lives...

Vaccine site inflammation potentiates idiotype DNA vaccine-induced therapeutic T cell-, and not B cell-, dependent antilymphoma immunity.

Lymphoma idiotype protein vaccines have shown therapeutic potential in previous clinical studies, and results from a completed pivotal, phase 3 controlled trial are promising. However, streamlined production of these patient-specific vaccines is required for eventual clinical application. Here, we show that second-generation, chemokine-fused idiotype DNA vaccines, when combined with myotoxins that induced sterile inflammation with recruitment of antigen-presenting cells at vaccination sites, were exceptional in their ability to provoke memory antitumor immunity in mice, compared with several TLR agonists. The combined vaccination strategy elicited both antigen-specific T-cell responses and humoral immunity. Unexpectedly, vaccine-induced tumor protection was intact in B cell-deficient mice but was abrogated completely by T-cell depletion in vivo, suggesting T-cell dependence. Furthermore, the optimal effect of myotoxins was observed with fusion vaccines that specifically targeted antigen delivery to antigen-presenting cells and not with vaccines lacking a targeting moiety, suggesting that the rational vaccine design will require combination strategies with novel, proinflammatory agents and highly optimized molecular vaccine constructs. These studies also challenge the paradigm that antibody responses are the primary of idiotype-specific antitumor effects and support the optimization of idiotype vaccines designed to induce primarily T-cell immunity.

Opiate and acetylcholine-independent analgesic actions of crotoxin isolated from crotalus durissus terrificus venom.

The venom of Crotalus durissus terrificus is reported to have analgesic activity and the administration of Crotoxin (Cro) to cancer patients is reported to reduce the consumption of analgesics. This study investigated the analgesia induced by Cro and the effects of atropine and naloxone on the antinociceptive activity of Cro in mice and rats. The results showed that Cro at 66.5, 44.3 and 29.5microg/kg (ip) exhibited a dose-dependent analgesic action in mice using the hotplate and acetic acid writhing tests. Cro at 44.3microg/kg (ip) had significant analgesic action in the rat tail-flick test. In the mouse acetic acid-writhing test, intracerebral ventricular administration of Cro 0.3microg/kg produced marked analgesic effects. Microinjection of Cro (0.15microg/kg) into the periaqueductal gray area also elicited a robust analgesic action in rat hotplate test. Atropine at 0.5mg/kg (im) or 10mg/kg (ip) or naloxone at 3mg/kg (ip) failed to block the analgesic effects of Cro. These results suggest that Cro has analgesic effects mediated by an action on the central nervous system. The muscarinic and opioid receptors are not involved in the antinociceptive effects of Cro.

Effects of Crotalus durissus collilineatus venom in the isolated rat kidney.

Ophidian accidents caused by the subspecies Crotalus durissus are responsible for high morbity and mortality rates. Acute renal failure is a common complication observed in these accidents. The aim of the present study was to investigate the renal effects promoted by the venom of C. d. collilineatus and its fractions, crotoxin and phospholipase A2. C. d. collilineatus (Cdc; 30 microg mL(-1)), crotoxin (CTX; 10 microg mL(-1)) and phospholipase A2 (PLA2; 10 microg mL(-1)) were tested in isolated rat kidney. The first 30 min of each experiment were used as an internal control and Cdc or its fractions, CTX and PLA2 were added to the system after this period. All experiments lasted 120 min. The venom of Cdc decreased perfusion pressure (PP; control120 = 110.3 +/- 3.69 mmHg; Cdc120 = 96.7+/-8.1 mmHg), renal vascular resistance (RVR; control120 = 6.42+/-0.78 mmHg mL g(-1) min(-1); Cdc120 = 4.8+/-0.56 mmHg/mL g(-1) min(-1)), urinary flow (UF; control120 = 0.19+/-0.03 mL g(-1) min(-1); Cdc120 = 0.12 +/- 0.01 mL g(-1) min(-1)), and glomerular filtration rate (GFR; control120 = 0.79 +/- 0.07 mL g(-1) min(-1); Cdc120 = 0.53 +/- 0.09 mL g(-1) min(-1)), but had no effect on the percent of sodium tubular transport (%TNa+), percent of chloride tubular transport (%TK+) and percent of potassium tubular transport (%TCl-). CTX and PLA2 reduced the GFR, while UF, PP and RVR remained stable during the full 120 min of perfusion. Crotoxin administration also diminished the %TK+ (control120 = 69.94 +/- 6.49; CTX120 = 33.28 +/- 4.78) and %TCl- (control120 = 79.53 +/- 2.67; CTX120 = 64.62 +/- 6.93). PLA2 reduced the %TK+, but exerted no effect on the %TNa+ or on that of TCl-. In conclusion, the C. d. collilineatus venom altered the renal functional parameters evaluated. We suggest that crotoxin and phospholipase A2 were involved in this process, since the renal effects observed would be due to the synergistic action of the components of the venom.

Inflammatory oedema induced by phospholipases A2 isolated from Crotalus durissus sp. in the rat dorsal skin: a role for mast cells and sensory C-fibers.

The ability of the phospholipases A(2) (PLA(2)s) from Crotalus durissus cascavella, Crotalus durissus collilineatus and Crotalus durissus terrificus venoms and crotapotin to increase the vascular permeability in the rat skin as well as the contribution of both mast cells and sensory C-fibers have been investigated in this study. Vascular permeability was measured as the plasma extravascular accumulation at skin sites of intravenously injected 125I-human serum albumin. Intradermal injection of crotalic PLA(2)s (0.05-0.5 microg/site) in the rat skin resulted in dose-dependent increase in plasma extravascular whereas crotapotin (1 microg/site) failed to affect this response. Co-injection of crotapotin (1 microg/site) did not modify the increased vascular permeability induced by the PLA(2)s (0.05-0.5 microg/site). Previous treatment (30 min) of the animals with cyproheptadine (2 mg/kg, i.p.) markedly reduced PLA(2) (0.5 microg/site)-induced oedema. In rats treated neonatally with capsaicin to deplete neuropeptides, the plasma extravasation induced by all PLA(2)s (0.5 microg/site) was also significantly reduced. Similarly, the tachykinin NK(1) receptor antagonist SR140333 (1nmol/site) significantly reduced the PLA(2)-induced oedema. In addition, the combination of SR140333 with cyproheptadine further reduced the increased plasma extravasation by PLA(2) from C. d. cascavella venom, but not by PLA(2) from C. d. terrificus and C. d. collilineatus venoms. Our results suggest that increase in skin vascular permeability by crotalic PLA(2)s is mediated by activation of sensory C-fibers culminating in the release of substance P, as well as by activation of mast cells which in turn release amines such as histamine and serotonin.

Contribution of crotoxin for the inhibitory effect of Crotalus durissus terrificus snake venom on macrophage function.

Previous work of our group demonstrated that Crotalus durissus terrificus venom has a dual effect on macrophage function: it inhibits spreading and phagocytosis and stimulates hydrogen peroxide and nitric oxide production, antimicrobial activity and glucose and glutamine metabolism of these cells. Crotalid venom also induces analgesia and this effect is mediated by opioid receptors. The involvement of opioidergic mechanism and the determination of the active component responsible for the inhibitory effect of crotalid venom on macrophage function were investigated. The venom reduced the spreading and phagocytic activities of peritoneal macrophages. This effect was observed in vitro, 2 h after incubation of resident peritoneal macrophages with the venom, and in vivo, 2 h after subcutaneous injection of the venom. The inhibition of phagocytosis was not modified by naloxone, an antagonist of opioid receptors. Venom neutralization with crotalid antivenom abolished the inhibitory effect of the venom, indicating that venom toxins are involved in this effect. Crotoxin, the main toxin of crotalid venom, s.c. injected to rats or added to the medium of peritoneal cell incubation, inhibited macrophage function in a similar manner to that observed for crude venom. The present results suggest that crotoxin causes a direct inhibition of macrophage spreading and phagocytic activities and may contribute to the inhibitory effect of crotalid venom on macrophage function.

Acute lung injury in experimental pancreatitis in rats: pulmonary protective effects of crotapotin and N-acetylcysteine.

Respiratory complications are major factors contributing to death in acute pancreatitis. However, the mechanisms of these pulmonary complications are not completely elucidated. We studied the effects of pretreatment with purified crotapotin (a phospholipase A2 inhibitor), N-acetylcysteine (a reactive oxygen species inhibitor), and a combination of both on the pulmonary mechanical and morphometric changes secondary to severe acute necrohemorrhagic pancreatitis in Wistar rats. A total of 69 male Wistar rats were studied. Pancreatitis was induced by infusion of 0.5 mL of a 4% solution of sodium taurocholate into the biliopancreatic duct. Crotapotin, N-acetylcysteine, or a combination of both was given intraperitoneally 30 min before inducing pancreatitis. Data were compared with data from sham-operated animals with or without those pretreatments. The severity of pancreatic and pulmonary injuries was evaluated 4 h after inducing pancreatitis by morphometric and pulmonary mechanical studies. N-acetylcysteine prevented the development of alveolar edema, alveolar distention, and collapse. Crotapotin prevented alveolar distention and collapse, and pulmonary dynamic elastance increase. When used in combination, crotapotin and N-acetylcysteine prevented both pulmonary morphological and mechanical changes induced by acute pancreatitis, suggesting an increase in protective effect when these drugs are used together compared with individual effects. However, the severity of pancreatic necrosis and the increase in polymorphonuclear cells in alveolar septa induced by pancreatitis were not reduced by previous administration of crotapotin, N-acetylcysteine, or both. These results suggest that the protective effects of these drugs are probably due to an extra-pancreatic action in the circulation, or even directly in the lung.

30-day intravenous administration of VRCTC-310-ONCO in rabbits.

VRCTC-310-ONCO, an agent based on the snake phospholipase A2 (crotoxin), is currently under clinical development. After phase I study in patients by intramuscular administration, the interest of intravenous (IV) dosing arose. To evaluate IV administration of VRCTC-310-ONCO in rabbits, ten animals were subjected to surgical implant of fixed jugular catheter, by which they received daily IV doses of 0.03 mg/kg body weight of VRCTC-310-ONCO for 30 days (n = 8) or saline (n = 2). The procedure was well tolerated in all rabbits. One of the animals died after the sixth dose of VRCTC-310-ONCO with CNS involvement; two additional rabbits required dose-reduction. All other rabbits achieved 30 days of treatment and were sacrificed. All rabbits (even controls) developed lymphocytosis and mild anaemia, without changes in blood neutrophils. No changes were found in serum transaminases (GOT and GPT), cholesterol, triglycerides, and y-glutamyl transpeptidase. At necropsy, chronic granulation tissue was found surrounding the implant in all rabbits. VRCTC-3 10-ONCO-treated rabbits presented generalised and marked swelling of hepatocytes, with areas of cytoplasmic vacuolisation. No abnormalities were found in kidney, heart, lung, spleen, adrenal gland, uterus, testes and ovary. Additional studies with IV route for VRCTC-310-ONCO, including humans, are required to define its toxicity in the clinical setting.

Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions.

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Effect of membrane composition and of co-encapsulation of immunostimulants in a liposome-entrapped crotoxin.

Crotoxin isolated from the venom of Crotalus durissus terrificus (South American rattlesnake) was incorporated into liposomes by the dehydration-rehydration vesicle method using different membrane compositions and the co-encapsulation of immunostimulants. Crotoxin was also encapsulated into liposomes formed from a non-phospholipid amphiphile, a mixture of polyoxyethylene 2-cetyl ether, dicetyl phosphate and cholesterol. The preparations were characterized in relation to stability, toxicity and the protection of mice against whole venom after immunization. All liposome preparations were quite stable, retaining more than 75% of the originally encapsulated crotoxin after 1 week of incubation at physiological temperature. Co-encapsulation with lipopolysaccharide increased the leakage of crotoxin. In contrast, co-encapsulation of the lipid moiety of lipopolysaccharide did not influence the stability of liposomes. Toxicity of liposomes was dependent on membrane composition. Liposomes made with phospholipids that were resistant to phospholipase A(2) activity were less toxic. Mice immunized with three doses of the 1 x LD50 of crotoxin encapsulated into liposomes, and with associated immunostimulants, were protected against challenge with 8 x subcutaneous LD50 of C. durissus terrificus venom. Using the same immunization schedule, liposomes made from a non-phospholipid mixture and without immunostimulants achieved 100% protection.

One-month safety study of intraperitoneal VRCTC-310-Onco (crotoxin + cardiotoxin) in rats.

To evaluate the toxicity of VRCTC-310-Onco (Crotalus durissus terrificus crotoxin + cardiotoxin from Naja naja atra), 10 Sprague-Dawley rats were implanted with intraperitoneal slow-release devices and subjected to treatment with 0.5 microgram/g body weight/d for 14 days. Biochemical evidence at days 7 and 14 showed blood, muscular, renal and metabolic disturbance, mostly reversed by day 28. No significant changes were found in necropsy. The limited toxicity of i.p. VRCTC-310-Onco in rats deserves further study.

Tumor regression of advanced carcinomas following intra- and/or peri-tumoral inoculation with VRCTC-310 in humans: preliminary report of two cases.

The authors report their clinical experience with VRCTC-310 in two patients suffering with advanced cancer in which the skin was severely compromised. VRCTC-310 is a combination of the snake venoms crotoxin (CT) and cardiotoxin (CD). The local (peritumoral) treatment with the drug (0.O14 mg/kg/week during 6 weeks) provoked the complete disappearance of a relapsed skin squamous cell cancer in one patient. The other patient was an aged woman with local-advanced breast cancer (carcinoma en cuirasse) who was inoculated intra-and-peritumoral with VRCTC-310. After 6 weekly courses (0.014 mg/kg/week) with the drug a > 80% tumor reduction was seen. A 133 days follow-up demonstrated not only an objective complete response of the primary tumor mass, but the disappearance of supraclavicular tumor mass as well a significant reduction in lymphangitis. To our knowledge, this is the first communication about the in vivo antitumoral activity of VRCTC-310 when injected locally to humans. Further studies are now in progress.

Toxicity of the novel animal-derived anticancer agent, VRCTC-310: acute and subchronic studies in beagle dogs.

Acute and subchronic toxicities of VRCTC-310, a combination product of crotoxin (CT) and cardiotoxin (CD), which has shown antitumor activity in vivo, have been studied in Beagle dogs. Single i.m. doses of 0.25, 0.5 and 1.0 mg/kg resulted in dose-dependent local muscular toxicity consisting of myofiber atrophy, interstitial edema and macrophage infiltration. Also, AST, ALT and LDH levels increased on day 2, returning to normal values on days 6-8. Local lesions were absent after recovery on day 45. At 2.0 mg/kg, signs of neurotoxicity (ataxia) appeared, in addition to vomitus, salivation, hematuria and myotoxicity in tongue and diaphragm on day 8. Local lesions healed with fibrosis at the site of injection on day 45. Administration of fixed (0.025 and 0.05 mg/kg) or escalating (0.025-0.1 mg/kg) daily doses for 30 days also produced local muscular damage, which was absent at day 75. The increases in AST, ALT and LDH serum activities on days 2-4 were independent of dosing schedule and sharply decreased on day 8, despite continuation of treatment. An escalating dose schedule of 0.025-2.0 mg/kg showed local muscle damage at the site of injection on day 31, however, there were no lesions of myotoxicity in the tongue or diaphragm and no clinical signs of neurotoxicity were observed. Animals tolerated the subchronic treatment better than the acute. The resolution of serum enzymes to normal values during treatment may be attributed to a decrease of sensitivity to VRCTC-310-mediated myotoxic effects.