Literature Review on Crotalus durissus terrificus Toxins: From a Perspective of Structural Biology and Therapeutic Applications.

BACKGROUND: The venom of Crotalus durissus terrificus, as well as its fractions, has intrigued research groups worldwide who are working to isolate, characterize, and find possible biotechnological applications. A number of studies have elucidated that these fractions and their derivatives possess pharmacological properties, which can enable the development of new drug prototypes with anti-inflammatory, antinociceptive, antitumor, antiviral, and antiparasitic applications. OBJECTIVE: This review presents a systematic study on Crotalus durissus terrificus, the most notable crotalid subspecies in South America, focusing on the composition, toxicological mechanisms, structural aspects, and applications of the main venom toxins (convulxin, gyroxin, crotamine, crotoxin, and their subunits). CONCLUSION: The authors have found that research on this snake and its toxins is still an area of focus, despite that almost a century has passed since the isolation of crotoxin. Several applications of these proteins in the development of novel drugs and bioactive substances have also been demonstrated.

Crotoxin down-modulates pro-inflammatory cells and alleviates pain on the MOG35-55-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a Central Nervous System inflammatory demyelinating disease that has as primary symptoms losses of sensory and motor functions, including chronic pain. To date, however, few studies have investigated the mechanisms of chronic pain in animal models of MS since locomotor impairments render difficult its evaluation. It was previously demonstrated that in the MOG35-55-induced EAE, an animal model of MS, the hypernociception appears before the onset of motor disability, allowing for the study of these two phenomena separately. Here, we evaluated the effect of crotoxin (CTX), a neurotoxin isolated from the Crotalus durissus terrificus snake venom that displays, at non-toxic dose, antinociceptive, anti-inflammatory and immunomodulatory effects, in the pain and in symptoms progression of EAE. The pain threshold of female C57BL/6 mice decreased at the 4th day after immunization, while the first sign of disease appeared around the 11st-12nd days, coinciding with the onset of motor abnormalities. CTX (40 µg/kg, s.c.) administered in a single dose on the 5th day after immunization, induced a long-lasting analgesic effect (5 days), without interfering with the clinical signs of the disease. On the other hand, when crotoxin was administered for 5 consecutive days, from 5th-9th day after immunization, it induced analgesia and also reduced EAE progression. The antinociceptive effect of crotoxin was blocked by Boc-2 (0.5 mg/kg, i.p.), a selective antagonist of formyl peptide receptors, by NDGA (30 µg/kg, i.p.), a lipoxygenase inhibitor and by atropine sulfate (10 mg/kg, i.p.), an antagonist of muscarinic receptors, administered 30 min before CTX. CTX was also effective in decreasing EAE clinical signs even when administered after its onset. Regarding the interactions between neurons and immunocompetent cells, CTX, in vitro, was able to reduce T cell proliferation, decreasing Th1 and Th17 and increasing Treg cell differentiation. Furthermore, in EAE model, the treatment with 5 consecutive doses of CTX inhibited IFN-γ-producing T cells, GM-CSF-producing T cells, reduced the frequency of activated microglia/macrophages within the CNS and decreased the number of migrating cell to spinal cord and cerebellum at the peak of the disease. These results suggest that CTX is a potential treatment not only for pain alteration but also for clinical progression induced by the disease as well as an useful tool for the development of new therapeutic approaches for the multiple sclerosis control.

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.

Inhaled Biologicals for the Treatment of Cystic Fibrosis.

BACKGROUND: Cystic Fibrosis (CF), one of the most frequent genetic diseases, is characterized by the production of viscous mucus in several organs. In the lungs, mucus clogs the airways and traps bacteria, leading to recurrent/resistant infections and lung damage. For cystic fibrosis patients, respiratory failure is still lethal in early adulthood since available treatments display incomplete efficacy. OBJECTIVE: The objective of this review is to extend the current knowledge in the field of available treatments for cystic fibrosis. A special focus has been given to inhaled peptide-based drugs. METHODS: The current review is based on recent and/or relevant literature and patents already available in various scientific databases, which include PubMed, PubMed Central, Patentscope and Science Direct. The information obtained through these diverse databases is compiled, critically interpreted and presented in the current study. An in-depth but not systematic approach to the specific research question has been adopted. RESULTS: Recently, peptides have been proposed as possible pharmacologic agents for the treatment of respiratory diseases. Of note, peptides are suitable to be administered by inhalation to maximize efficacy and reduce systemic side effects. Moreover, innovative delivery carriers have been developed for drug administration through inhalation, allowing not only protection against proteolysis, but also a prolonged and controlled release. CONCLUSION: Here, we summarize newly patented peptides that have been developed in the last few years and advanced technologies for inhaled drug delivery to treat cystic fibrosis.

Walker 256 Tumor Growth Suppression by Crotoxin Involves Formyl Peptide Receptors and Lipoxin A4.

We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs.

Crotoxin from Crotalus durissus terrificus is able to down-modulate the acute intestinal inflammation in mice.

Inflammatory bowel diseases (IBD) is the result of dysregulation of mucosal innate and adaptive immune responses. Factors such as genetic, microbial and environmental are involved in the development of these disorders. Accordingly, animal models that mimic human diseases are tools for the understanding the immunological processes of the IBD as well as to evaluate new therapeutic strategies. Crotoxin (CTX) is the main component of Crotalus durissus terrificus snake venom and has an immunomodulatory effect. Thus, we aimed to evaluate the modulatory effect of CTX in a murine model of colitis induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS). The CTX was administered intraperitoneally 18 hours after the TNBS intrarectal instillation in BALB/c mice. The CTX administration resulted in decreased weight loss, disease activity index (DAI), macroscopic tissue damage, histopathological score and myeloperoxidase (MPO) activity analyzed after 4 days of acute TNBS colitis. Furthermore, the levels of TNF-α, IL-1ß and IL-6 were lower in colon tissue homogenates of TNBS-mice that received the CTX when compared with untreated TNBS mice. The analysis of distinct cell populations obtained from the intestinal lamina propria showed that CTX reduced the number of group 3 innate lymphoid cells (ILC3) and Th17 population; CTX decreased IL-17 secretion but did not alter the frequency of CD4+Tbet+ T cells induced by TNBS instillation in mice. In contrast, increased CD4+FoxP3+ cell population as well as secretion of TGF-ß, prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) was observed in TNBS-colitis mice treated with CTX compared with untreated TNBS-colitis mice. In conclusion, the CTX is able to modulate the intestinal acute inflammatory response induced by TNBS, resulting in the improvement of clinical status of the mice. This effect of CTX is complex and involves the suppression of the pro-inflammatory environment elicited by intrarectal instillation of TNBS due to the induction of a local anti-inflammatory profile in mice.

Growth inhibitory effects and molecular mechanisms of crotoxin treatment in esophageal Eca-109 cells and transplanted tumors in nude mice.

AIM: To investigate the antitumor actions of the Crotalus durissus neurotoxin (crotoxin) on human esophageal carcinoma (Eca-109) cells in vitro and transplanted esophageal Eca-109 tumors in nude mice. METHODS: The growth-inhibitory effect was analyzed in Eca-109 cells using MTT assay. Cell morphology changes in nuclei were observed using Hoechst 33342 staining, while apoptosis and cell cycle distribution were examined by flow cytometry. RT-PCR was used to measure the Bcl-2, p15, and caspase-3 p17 gene expression levels. A tumor transplantation model was established by inoculation of Eca-109 cells were into female Balb/c nude mice. Crotoxin (25, 50, and 100 mg/kg) was subcutaneously injected into the transplanted tumors every 2 d for a total of 10 injections. Tumor size and weight were measured. Bcl-2, p15, and caspase-3 p17 protein expression in transplanted tumors was analyzed using Western blotting. RESULTS: Crotoxin (25, 50, and 100 µg/mL) inhibited the growth of Eca-109 cells in a dose-dependent manner with inhibition rates of 22.9%, 35.8%, and 57.2%, respectively. Hoechst 33342 staining revealed apoptotic cells with pyknotic nuclear chromatin after crotoxin treatment. In Eca-109 cells, crotoxin induced apoptosis and G1 block, significantly upregulated the expression of p15 and caspase-3 p17 genes and downregulated the expression of Bcl-2 gene. Furthermore, crotoxin inhibited the growth of Eca-109 tumors in nude mice in a dose-dependent manner. Western blotting showed that crotoxin increased p15 and caspase-3 p17 protein levels and reduced Bcl-2 protein level in tumor specimens. CONCLUSION: Crotoxin inhibits the growth of Eca-109 cells in vitro via apoptosis induction and G1 block. Local administration of crotoxin inhibits the growth of subcutaneously transplanted Eca-109 cells in nude mice, possibly via increasing p15 and caspase-3 p17 protein expression and reducing Bcl-2 protein expression.

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...

Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549.

AIM: To assess the cytotoxic effect of crotoxin (CrTX), a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus, in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms. METHODS: A549 cells were treated with gradient concentrations of CrTX, and the cell cycle and apoptosis were analyzed using a flow cytometric assay. The changes of cellular effectors p53, caspase-3 and cleaved caspase-3, total P38MAPK and pP38MAPK were investigated using Western blot assays. A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo. RESULTS: Treatment of A549 cells with CrTX (25-200 µg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC(50)=78 µg/mL). Treatment with CrTX (25 µg/mL) for 24 h caused G1 arrest and induced cell apoptosis. CrTX (25 µg/mL) significantly increased the expression of wt p53, cleaved caspase-3 and phospho-P38MAPK. Pretreatment with the specific P38MAPK inhibitor SB203580 (5 µmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level, but G(1) arrest remained unchanged and highly expressed p53 sustained. Intraperitoneal injection of CrTX (10 µg/kg, twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth, and decreased MVD and VEGF levels. CONCLUSION: CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3, and by cell cycle arrest mediated by increased wt p53 expression. In addition, CrTX displayed anti-angiogenic effects in vivo.

The effect of treatment with crotapotin on the evolution of experimental autoimmune neuritis induced in Lewis rats.

Biomedical research in which venom components are being investigated for their potential as novel therapeutic agents has emerged as an interesting option. Crotapotin, which is purified from the venom of the rattlesnake Crotalus durissus terrificus, has been described as an anti-inflammatory agent that acts on the innate arm of the immune response. Here we have demonstrated that intraperitoneal administration of crotapotin significantly reduces the severity of experimental autoimmune neuritis (EAN), an experimental model for Guillain-Barré syndrome. The reduction of the severity of the disease is associated with a reduction in the mononuclear cells infiltrating the sciatic nerve and a significant decrease in the lymphocyte proliferative response to neuritogenic peptide.

Estrategias de la creencia en situaciones críticas: el cáncer y la crotoxina en Buenos Aires a mediados de los años ochenta / Strategies of the belief in critical situations: cancer and crotoxin in Buenos Aires in the 80's

Explora los avatares que acompañaron en Buenos Aires a mediados de la década de 1980 la movilización de enfermos de cáncer interesados en acceder a un tratamiento descalificado por el establishment médico y científico.(AU)

Estrategias de la creencia en situaciones críticas: el cáncer y la crotoxina en Buenos Aires a mediados de los años ochenta / Strategies of the belief in critical situations: cancer and crotoxin in Buenos Aires in the 80s

Explora los avatares que acompañaron en Buenos Aires a mediados de la década de 1980 la movilización de enfermos de cáncer interesados en acceder a un tratamiento descalificado por el establishment médico y científico.(AU)

Phase I and pharmacokinetics study of crotoxin (cytotoxic PLA(2), NSC-624244) in patients with advanced cancer.

A Phase I clinical trial was performed on patients with solid tumors refractory to conventional therapy. Crotoxin was administered i.m. for 30 consecutive days at doses ranging from 0.03 to 0.22 mg/m(2). Patients entered the study after providing a written informed consent. Although 26 patients were entered only 23 were evaluated. Reversible, nonlimiting neuromuscular toxicity evidenced as diplopia because of pareses of the external ocular muscles was present in 13 patients. It started at doses of 0.18 mg/m(2) and lasted from 2 to 6 h. These episodes did not require dose adjustment and disappeared in 1-3 weeks of treatment. Three patients experienced palpebral ptosis, nystagmus (grade 2), and anxiety (grade 2-3) at the dose-limiting toxicity of 0.22 mg/m(2). Also at dose-limiting toxicity, 1 patient showed nystagmus (grade 2) and anxiety (grade 3) without evidence of palpebral ptosis. Transient increases (grades 1-3) in the levels of creatinine kinase, aspartate aminotransferase, and alanine transaminase attributed to crotoxin myotoxicity were observed but returned to normal by the last week of treatment. At 0.21 mg/m(2) there was a case of grade-3 anaphylactic reaction on day 31, which required treatment. Hypersensitivity was regarded as an adverse drug-related reaction, and the patient was removed from the protocol. Two patients at different doses (0.12 mg/m(2) and 0.22 mg/m(2)) had sialorrhea. Four patients had asymptomatic transient increase in blood pressure (up to 20 mm Hg) 12 h after the first injection, which lasted 24 h. No treatment was required and toxicity did not reappear. Six patients experienced slight eosinophilia during the first 2 weeks. The maximum tolerated dose was set at 0.21 mg/m(2). Objective measurable partial responses (>50% reduction of tumor mass) were noted in 2 patients treated at 0.21 mg/m(2) and 1 at 0.12 mg/m(2). One patient (at 0.21 mg/m(2)) presented a complete response on day 110. Crotoxin pharmacokinetics showed rapid absorption from the injection site to blood (t(1/2 A) = 5.2 +/- 0.6 min). Plasma concentration reached a peak (C(max) = 0.79 +/- 0.1 ng/ml) at tau(max) = 19 +/- 3 min. The half-life of the distribution (alpha) phase is 22 +/- 2 min. Starting at 1.5 h after injection, the decrease in plasma concentration becomes slower, reaching 14 +/- 3 pg/ml 24 h after injection. The profile is dominated by the elimination (beta) phase with a half-life of 5.2 +/- 0.6 h. Consequently, 24 h after the injection ( approximately 5 half-life) 97% of the product was eliminated. The area under plasma concentration versus time curve was 0.19 +/- 0.05 microg/min/ml. Assuming availability (F) approximately 1, the clearance is C(L) = 26.3 +/- 7 ml/min, and the apparent volume of distribution is V(d) = 12 +/- 3 liter/kg. The recommended dose for a Phase II study is 0.18 mg/m(2).

Phase I study of VRCTC-310, a purified phospholipase A2 purified from snake venom, in patients with refractory cancer: safety and pharmacokinetic data.

A phase I study was performed to evaluate the maximum tolerated dose (MTD), safety profile and pharmacokinetic data with VRCTC-310, a natural product derived from purified snake venom fractions, with phospholipase A2 activity and inhibitory effects against human and murine tumor cell lines. Fifteen patients with refractory malignancies were entered after providing written informed consent. VRCTC-310 was administered as an intramuscular injection daily for 30 consecutive days. Doses were escalated from 0.0025 to 0.023 mg/kg. Toxicities included local pain at the injection site, eosinophilia, reversible diplopia and palpebral ptosis. Dose escalation was stopped at 0.023 mg/kg, when two patients had developed anaphylactoid reactions. Both cases had high VRCTC-310-specific IgG by EIA. MTD was 0.017 mg/kg and the recommended dose for phase II studies is 0.017 mg/kg. Stabilization was found in six patients.

Inhibition of carrageenin-induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2.

1. The effect of purified crotapotin, a non-toxic non-enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 micrograms/paw) and 5-hydroxytryptamine (5-HT) (3 micrograms/paw) in the rat hind-paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea-pig isolated lung were also investigated. 2. Subplantar co-injection of crotapotin (1 and 10 micrograms/paw) with carrageenin or injection of crotapotin (10 micrograms/paw) into the contralateral paw significantly inhibited the carrageenin-induced oedema. This inhibition was also observed when crotapotin (10-30 micrograms/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60 degrees C) failed to inhibit carrageenin-induced oedema. Subplantar injection of crotapotin (10 micrograms/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5-HT-induced oedema. 3. In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5-HT stores. 4. Crotapotin (30 micrograms/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 nM) and platelet activating factor (1 microM) in human platelet-rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml-1) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 microg/paw) did not affect the release of 6-oxo-prostaglandin Fla, and TXB2 induced by ovalbumin in sensitized guinea-pig isolated lungs.5. Our results indicate that the anti-inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo-oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity.

[The efficacy of the bothropic-crotalic antivenom in the neutralization of the main Bothrops jararacussu venom effects]. / A eficácia do antiveneno botrópico-crotálico na neutralização das principais atividades do veneno de Bothrops jararacussu.

Myonecrosis is one of the effects of Bothrops jararacussu venom, from which a myotoxin was isolated showing structural homology to phospholipase A2 (PLA2), but without enzymatic activity. Such myotoxic activity is also present in the Crotalus durissus terrifucus venom, and is attributed to crotoxin and to PLA2 (crotoxin B), the basic component of the crotoxin complex. The Bothrops jararacussu venom showed three proteins with immunologic identity to PLA2 from crotoxin. The bothropic (AB) and the bothropic/crotalic (AB/C) anti-venoms, two commercial polyvalent anti-venoms produced at Instituto Butantan, were compared in order to assess their capacity for neutralization of the lethal, hemorrhagic, coagulant and myotoxic activities of Bothrops jararacussu venom. Both anti-venoms showed the same level of hemorrhagic activity neutralization. However, AB/C was about three times more efficient than AB in neutralizing the myotoxic activity, and two times more potent for neutralization of lethality and coagulant activity of Bothrops jararacussu venom. These data suggest that the use of AB/C could be of value in the treatment of patients bitten by snakes of this species.