Photobiomodulation therapy on local effects induced by juvenile and adult venoms of Bothrops alternatus.

Bothrops snake envenomation is characterized by severe local manifestations such as pain, edema, inflammation, hemorrhage, and myonecrosis. Furthermore, it is described that venom from juvenile and adult snakes may have differences in their composition that can lead to differences in the evolution of the clinical manifestation of the victim. Photobiomodulation (PBM) has been shown to be an effective adjuvant therapy to serum therapy to reduce the local effects induced by bothropic snake venom. This study evaluated the effect of PBM on the local reaction, after Bothrops alternatus snake venom (BaV) injection, in its juvenile (BaJV) and adult (BaAV) stages. Balb/C mice were injected with the juvenile or adult venoms of BaV or saline solution (control group). PBM at a wavelength of 660 nm, 100 mW, 0.33 W/cm2, 40 s, and a 0.028 cm2 beam was applied transcutaneous to a single point with a radiant exposure of 4 J/cm2, 30 min after venom injection. Edema, inflammatory infiltrate, hyperalgesia, and myonecrosis were analyzed. Both venoms induced significant edema and myonecrosis in the gastrocnemius muscle. Hyperalgesia in the mice paw and a prominent leukocyte infiltrate into the peritoneum were also observed. PBM significantly reduced all evaluated parameters. In conclusion, PBM treatment was effective in reducing the local effects induced by B. alternatus venom at different stages of snake development and could be a useful tool as an adjuvant treatment for bothropic envenomation.

The potential of Brazilian native plant species used in the therapy for snakebites: A literature review.

Snakebite envenoming is a potentially fatal disease categorized as a neglected public health issue for not receiving the appropriate attention from national and international health authorities. The most affected people by this problem usually live in poor rural communities, where medical resources are often sparse and, in some instances, there is even a scarcity of serum therapy. The administration of the appropriate antivenom is the only specific treatment available, however it has limited efficacy against venom-induced local effects. In this scenario, various plant species are used as local first aid for the treatment of snakebite accidents in Brazil, and some of them can effectively inhibit lethality, neurotoxicity, hemorrhage, and venom enzymes activities. This review compiles a list of plants used in the treatment of snakebites in Brazil, focusing on the native Brazilian species registered in the databases Pubmed, Scielo, Scopus and Google Scholar. All these searches were limited to peer-reviewed journals written in English, with the exception of a few articles written in Portuguese. The most cited native plant species were Casearia sylvestris Sw., Eclipta prostrata (L.) L., Mikania glomerata Spreng., Schizolobium parahyba (Vell.) S.F.Blake and Dipteryx alata Vogel, all used to decrease the severity of toxic signs, inhibit proteolytic and hemorrhagic activities, thus increasing survival time and neutralizing myotoxicity effects. Different active compounds showing important activity against the snake venoms and their toxins include flavonoids, alkaloids and tannins. Although some limitations to the experimental studies with medicinal plants were observed, including lack of comparison with control drugs and unknown active extracts compounds, species with anti-venom characteristics are effective and considered as candidates for the development of adjuvants in the treatment of snake envenomation. Further studies on the chemistry and pharmacology of traditionally used plant species will help to understand the role that snakebite herbal remedies may display in local medical health systems. It might also contribute to the development of alternative or complementary treatments to reduce the number of severe disabilities and deaths.

Utilization of gallic acid to inhibit some toxic activities caused by Bothrops jararaca or B. jararacussu snake venoms.

Snake bite envenoming is a serious public health issue, affecting thousands of people worldwide every year, especially in rural communities of tropical and subtropical countries. Injection of venom into victims may cause hemorrhaging, blood coagulation imbalance, inflammation, pain, edema, muscle necrosis, and eventually, death. The official validated treatment recommended by governments is the administration of antivenom that efficiently prevents morbidity and mortality. However, this therapy does not effectively neutralize the local effects of Viperidae venoms which constitute one of the leading causes of disability or amputation of the affected limb. Thus, bioprospecting studies seeking for alternative therapies to complement antivenom should be encouraged, especially those investigating the blockage of local venomic toxicity. Plants produce a great diversity of metabolites with a wide range of pharmacological and biological properties. Therefore, the objective of this study was to assess the utilization of gallic acid, which is widely found in plants, against some toxic in vitro (coagulation, proteolytic, and hemolytic) or in vivo (edematogenic, hemorrhagic, and lethal) activities of Bothrops jararaca or B. jararacussu venom. Gallic acid was incubated with B. jararaca or B. jararacussu venom (incubation protocol), after which, in vitro or in vivo assays were performed. Additionally, a gel containing gallic acid was developed and topically applied over the skin of mice after injection of B. jararaca or B. jararacussu venom (treatment protocol), and then, a hemorrhagic assay was carried out. As a result, gallic acid inhibited the toxic activities, with variable efficacy, and the gallic acid gel neutralized B. jararaca or B. jararacussu venom-induced hemorrhagic activity. Gallic acid was devoid of in vitro toxicity as shown through a hemocompatibility test. Thus, these findings demonstrate the potential of gallic acid in the development of an alternative agent to treat victims of snake bites inflicted by Bothrops species.

Pharmacological re-assessment of traditional medicinal plants-derived inhibitors as antidotes against snakebite envenoming: A critical review.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional healers have used medicinal plants to treat snakebite envenomation worldwide; however, mostly without scientific validation. There have been many studies on the therapeutic potential of the natural products against snake envenomation. AIM OF THE STUDY: This review has highlighted snake venom inhibitory activity of bioactive compounds and peptides from plants that have found a traditional use in treating snakebite envenomation. We have systematically reviewed the scenario of different phases of natural snake venom inhibitors characterization covering a period from 1994 until the present and critically analysed the lacuna of the studies if any, and further scope for their translation from bench to bedside. MATERIALS AND METHODS: The medicinal plant-derived compounds used against snakebite therapy were reviewed from the available literature in public databases (Scopus, MEDLINE) from 1994 till 2020. The search words used were 'natural inhibitors against snakebite,' 'natural products as therapeutics against snakebite,' 'natural products as antidote against snake envenomation,' ' snake venom toxin natural inhibitors,' 'snake venom herbal inhibitors'. However, the scope of this review does not include computational (in silico) predictions without any wet laboratory validation and snake venom inhibitory activity of the crude plant extracts. In addition, we have also predicted the ADMET properties of the identified snake venom inhibitors to highlight their valuable pharmacokinetics for future clinical studies. RESULTS: The therapeutic application of plant-derived natural inhibitors to treat snakebite envenomation as an auxiliary to antivenom therapy has been gaining significant momentum. Pharmacological reassessment of the natural compounds derived from traditional medicinal plants has demonstrated inhibition of the principal toxic enzymes of snake venoms at various extents to curb the lethal and/or deleterious effects of venomous snakebite. Nevertheless, such molecules are yet to be commercialized for clinical application in the treatment of snakebite. There are many obstacles in the marketability of the plant-derived natural products as snake envenomation antidote and strategies must be explored for the translation of these compounds from drug candidates to their clinical application. CONCLUSION: In order to minimize the adverse implications of snake envenomation, strategies must be developed for the smooth transition of these plant-derived small molecule inhibitors from bench to bedside. In this article we have presented an inclusive review and have critically analysed natural products for their therapeutic potential against snake envenomation, and have proposed a road map for use of natural products as antidote against snakebite.

Benefits of Sebastiania hispida (Euphorbiaceae) extract and photobiomodulation therapy as potentially adjunctive strategies to be explored against snake envenoming.

The purpose of this study was to assess the topic use of Sebastiania hispida extract and low-level gallium-arsenide laser irradiation (GaAs, 904 nm) to reduce the local myonecrosis and edema of Bothrops moojeni snake venom-injected gastrocnemius. Wistar rats receiving intramuscular venom injection (VBm) were compared with saline control (S) and envenomed rats receiving local exposure to plant extract (VExt) or laser irradiation (VL). The phytochemistry and thin-layer chromatography of S. hispida extract indicated the presence of phenolic compounds like gallic acid and flavonoids including quercetin. Gastrocnemius of VExt and VL groups had a significant reduction of edema and creatine kinase (CK) activities and a greater Myogenin (MyoG) expression compared to VBm group, with the plant extract efficacy better than laser exposure. Reduction of edema and serum CK activities reflects a lessening of muscle damage, whereas the increase of MyoG indicates myoblast differentiation and acceleration of muscle repair. The S. hispida richness in phenolic compounds and flavonoids, such as the light modulatory ability to triggering a multitude of cell signalings likely underlie the positive outcomes. Our findings suggest both treatments as potential auxiliary tools to be explored in clinical trials in combination with anti-venom therapy after Bothropic snakebites.

The Search for Natural and Synthetic Inhibitors That Would Complement Antivenoms as Therapeutics for Snakebite Envenoming.

A global strategy, under the coordination of the World Health Organization, is being unfolded to reduce the impact of snakebite envenoming. One of the pillars of this strategy is to ensure safe and effective treatments. The mainstay in the therapy of snakebite envenoming is the administration of animal-derived antivenoms. In addition, new therapeutic options are being explored, including recombinant antibodies and natural and synthetic toxin inhibitors. In this review, snake venom toxins are classified in terms of their abundance and toxicity, and priority actions are being proposed in the search for snake venom metalloproteinase (SVMP), phospholipase A2 (PLA2), three-finger toxin (3FTx), and serine proteinase (SVSP) inhibitors. Natural inhibitors include compounds isolated from plants, animal sera, and mast cells, whereas synthetic inhibitors comprise a wide range of molecules of a variable chemical nature. Some of the most promising inhibitors, especially SVMP and PLA2 inhibitors, have been developed for other diseases and are being repurposed for snakebite envenoming. In addition, the search for drugs aimed at controlling endogenous processes generated in the course of envenoming is being pursued. The present review summarizes some of the most promising developments in this field and discusses issues that need to be considered for the effective translation of this knowledge to improve therapies for tackling snakebite envenoming.

Botanical treatments for snakebite in rural Ghana: A narrative review.

ETHNOPHARMACOLOGICAL RELEVANCE: In the countryside, there are some limitations with the use of venom antisera to manage snake bites. Due to poor access to healthcare and as a result of the difficulty in receiving treatment for cases of snake bites, most rural people in Ghana, a West African country, rely on plant medicine as a first aid to manage cases of venomous snakebite. This calls for more research into the species of plants used to medically manage snakebite envenomation. AIM OF THE STUDY: This review sought to present plants that are used in managing snakebite cases and also gather data supporting their use. METHODOLOGY: This is a systematic search and review of information obtained from textbooks and databases such as PubMed and ScienceDirect between January 1975 and August 2020. RESULTS: A search done identified 43 plant species and these were found to belong to 25 taxonomic families with the most frequent ones being, Fabaceae, Euphorbiaceae, Apocynaceae, and Solanaceae. Experimental data gathered indicate that among the many plants identified to be used to manage snakebites, only 5 were found with anti-venom in vitro and in vivo evidence-based data. CONCLUSION: Data collated hint that a few plant species identified namely Anacardium occidentale, Euphorbia hirta, Mimosa pudica, Musa paradisiaca and Mangifera indica, work by targeting diverse physiopathological and biochemical processes involved in the clinical manifestations of snakebites. This review has also unearthed knowledge gaps that can form the basis for broad investigations and development of these and other medicinal plants into useful anti-venom medications.

Comparative therapeutic index, lethal time and safety margin of various toxicants and snake antivenoms using newly derived and old formulas.

OBJECTIVE: The assessment of clinical efficacy and toxicity is very important in pharmacology and toxicology. The effects of psychostimulants (e.g. amphetamine), psychotomimetics (e.g. Cannabis sativus) and snake antivenoms are sometimes unpredictable even at lower doses, leading to serious intoxication and fatal consequences. Hence, there is need to re-assess some formulas for calculation of therapeutic index, lethal time and safety margin with a view to identifying therapeutic agents with remarkable toxicity potentials. RESULTS: The therapeutic index formula [Formula: see text] was derived from T1 = LD50/ED50 and ED50 = [Formula: see text]. Findings have shown that, therapeutic index is a function of death reversal (s), safety factor (10-4) and weight of animal (Wa). However, the new safety margin formula [Formula: see text] derived from LT50 = [Formula: see text] and MS = [Formula: see text] shows that safety margin is a function of cube root of ratio between LT50 and LD50 and ED100th. Concentration (k) of toxicant at the receptor [Formula: see text] derived from D1 × Tn = K and LD1 = [Formula: see text] shows that therapeutic index, lethal time and safety margin is a function of drug or toxicant concentration at the receptor, the drug-receptor interaction and dose of toxicant or drug administered at a particular time.

Detoxifying Action of Aqueous Extracts of Mucuna pruriens Seed and Mimosa pudica Root Against Venoms of Naja nigricollis and Bitis arietans.

BACKGROUND: The World Health Organization included snakebite envenomation among Neglected Tropical Diseases in 2017. The importance of natural products from plants is enormous, given that most prescribed drugs originate from plants. Among this is Mucuna pruriens and Mimosa pudica, with many registered patents asserting their health benefits. OBJECTIVE: This study investigated the in vitro neutralizing effects of Mucuna pruriens seed and Mimosa pudica root extracts on venoms of Naja nigricollis and Bitis arietans. METHODS: In mice, the LD50 and phytochemical analysis of M. pruriens and M. pudica plant extracts were carried out prior to the evaluation of their haemolytic and fibrinolytic effect. Their effects on the activities of phospholipase A2 (PLA2) were also assessed. RESULTS: At a concentration of 50 mg/ml, both plant extracts were found to neutralize the fibrinolytic activity of N. nigricollis, but 400 mg/ml was required to neutralize the fibrinolytic activity of B. arietans. In haemolytic studies, 50 mg/ml concentration of M. pruriens extract suppressed haemolysis caused by N. nigricollis venom by 70% but at the same concentration, M. pudica extract reduced haemolysis by 49.4%. M. pruriens, at 50 mg/ml concentration, only inhibited phospholipase A2 activity by 7.7% but higher concentrations up to 400mg/ml had no effect against the venom of N. nigricollis; at 200 mg/ml. M. pudica extract inhibited PLA2 activity by 23%. CONCLUSION: The results suggest that M. pruriens and M. pudica may be considered as promising antivenom agents for people living in a snake-bite prone environment.

Utilization of the Plant Clusia Fluminensis Planch & Triana Against Some Toxic Activities of the Venom of Bothrops jararaca and B. jararacussu Snake Venom Toxic Activities.

BACKGROUND: In Brazil, the Bothrops genus accounts for 87% of registered snakebites, which are characterized by hemorrhage, tissue necrosis, hemostatic disturbances, and death. The treatment recommended by governments is the administration of specific antivenoms. Although antivenom efficiently prevents venom-induced lethality, it has limited efficacy in terms of preventing local tissue damage. Thus, researchers are seeking alternative therapies able to inhibit the main toxic effects of venoms, without compromising safety. OBJECTIVE: The study aimed to test the ability of aqueous extracts of leaves, stems, and fruits of the plant Clusia fluminensis to neutralize some toxic effects induced by the venoms of Bothrops jararaca and Bothrops jararacussu. METHODS: The plant extracts were incubated with venoms for 30 min. at 25 °C, and then in vitro (coagulant and proteolytic) and in vivo (hemorrhagic, myotoxic, and edematogenic) activities were evaluated. In addition, the extracts were administered to animals (by oral, intravenous or subcutaneous routes) before or after the injection of venom samples, and then hemorrhage and edema assays were performed. In addition, a gel solution of the fruit extract was produced and tested in terms of reducing hemorrhage effects. A chemical prospection was performed to identify the main classes of compounds present in the extracts. RESULTS: All the extracts inhibited the activities of the two venoms, regardless of the experimental protocol or route of administration of the extracts. Moreover, the gel of the fruit extract inhibited the venom-induced-hemorrhage. The extracts comprised of tannins, flavonoids, saponins, steroids, and terpenoids. CONCLUSION: Antivenom properties of C. fluminensis extracts deserve further investigation in order to gain detailed knowledge regarding the neutralization profile of these extracts.

Anti-Ophidian Properties of Herbal Medicinal Plants: Could it be a Remedy for Snake Bite Envenomation?

Snake bite envenoming causes high rates of morbidity and mortality and is one of the serious health-related concerns all over the globe. Around 3200 species of snakes have been discovered till date. Amid these species, about 1300 species of snakes are venomous. On account of its severity, World Health Organization (WHO) recently included snakebite envenoming in the list of neglected tropical diseases. Immunotherapy has partially solved the issues related to snakebite envenomation. However, it is associated with numerous adverse effects, due to which alternative treatment strategies are required for the treatment of snakebite. Traditionally, a large repository of herbal medicinal plants is known to possess activity against snake venom. An exploration of the therapeutic benefits of these medicinal plants used for the treatment of snakebites reveals the presence of various potential phytochemicals. The aim of the present review is to provide an outline regarding poisonous snakes all over the world, various compositions of snake venom, adverse effects related to anti-snake venom and numerous medicinal plants used for the anti-ophidian activity.

Tabebuia aurea decreases hyperalgesia and neuronal injury induced by snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore is used as anti-inflammatory, analgesic and antiophidic in traditional medicine, though its pharmacological proprieties are still underexplored. In the bothropic envenoming, pain is a key symptom drove by an intense local inflammatory and neurotoxic event. The antivenom serum therapy is still the main treatment despite its poor local effects against pain and tissue injury. Furthermore, it is limited to ambulatorial niches, giving space for the search of new and more inclusive pharmacological approaches. AIM OF THE STUDY: evaluation of Tabebuia aurea hydroethanolic extract (HEETa) in hyperalgesia and neuronal injury induced by Bothrops mattogrossensis venom (VBm). MATERIALS AND METHODS: Stem barks from Tabebuia aurea were extracted with ethanol and water (7:3, v/v) to yield the extract HEETa. Then, HEETa was analyzed by LC-DAD-MS and its constituents were identified. Snake venoms were extracted from adult specimens of Bothrops mattogrossensis, lyophilized and kept at -20 °C until use. Male Swiss mice, weighting 20-25 g, were used to hyperalgesia (electronic von Frey), motor impairment (Rotarod test) and tissue injury evaluation (histopatology and ATF-3 immunohistochemistry). Therefore, three experimental groups were formed: VBm (1 pg, 1 ng, 0.3 µg, 1 µg, 3 and 6 µg/paw), HEETa orally (180, 540, 720, 810 or 1080 mg/kg; 10 mL/kg, 30 min prior VBm inoculation) and VBm neutralized (VBm: HEETa, 1:100 parts, respectively). In all set of experiments a control (saline group) was used. First, we made a dose-time-response course curve of VBm's induced hyperalgesia. Next, VBm maximum hyperalgesic dose was employed to perform HEETa orally dose-time-response course curve and analyses of VBm neutralized. Paw tissues for histopathology and DRGs were collected from animals inoculated with VBm maximum dose and treated with HEETa antihyperalgesic effective dose or neutralized VBm. Paws were extract two or 72 h after VBm inoculation and DRGs, in the maximum expected time expression of ATF-3 (72 h). RESULTS: From HEETa extract, glycosylated iridoids were identified, such as catalpol, minecoside, verminoside and specioside. VBm induced a time and dose dependent hyperalgesia with its highest effect seen with 3 µg/paw, 2 h after venom inoculation. HEETa effective dose (720 mg/kg) decreased significantly VBm induced hyperalgesia (3 µg/paw) with no motor impairment and signs of acute toxicity. HEETa antihyperalgesic action starts 1.5 h after VBm inoculation and lasted up until 2 h after VBm. Hyperalgesia wasn't reduced by VBm: HEETa neutralization. Histopathology revealed a large hemorragic field 2 h after VBm inoculation and an intense inflammatory infiltrate of polymorphonuclear cells at 72 h. Both HEETa orally and VBm: HEETa groups had a reduced inflammation at 72 h after VBm. Also, the venom significantly induced ATF-3 expression (35.37 ±â€¯3.25%) compared with saline group (4.18 ±â€¯0.68%) which was reduced in HEETa orally (25.87 ±â€¯2.57%) and VBm: HEETa (19.84 ±â€¯2.15%) groups. CONCLUSION: HEETa reduced the hyperalgesia and neuronal injury induced by VBm. These effects could be related to iridoid glycosides detected in HEETa and their intrinsic reported mechanism.

Rutin (quercetin-3-rutinoside) modulates the hemostatic disturbances and redox imbalance induced by Bothrops jararaca snake venom in mice.

Snakebites are a major Collective Health problem worldwide. In Brazil, Bothrops jararaca snake venom (BjV) evokes hemostatic disturbances, bleeding manifestations, and redox status imbalance. Specific antivenom therapy, although efficacious to revert most snakebite-induced manifestations, is incapable of treating secondary manifestations, such as oxidative/nitrosative stress. Searching for new complementary therapies that could attenuate physiological derangements triggered by envenomation, we elected to test quercetin-3-rutinoside (rutin) by its potential as both a potent antioxidant and a hemostasis modulatory compound. The activity of rutin was evaluated both on the biological activities of crude BjV in vitro, and in vivo by the ability of rutin (14.4 mg/kg b.w.) to modulate hematological, hemostatic and redox status markers altered by BjV injection (1.6 mg/kg b.w., s.c.) in mice. In vitro, rutin failed to inhibit BjV-induced platelet aggregation and biological activities of major BjV enzymes (metalloproteinases, phospholipases A2, serine proteases, and L-amino acid oxidases). On the other hand, rutin attenuated local hemorrhage, and the increase in reactive species, prevented the fall in RBC counts and fibrinogen levels, diminished tail bleeding and shortened prothrombin time (PT) evoked by envenomation. Furthermore, rutin reduced tissue factor (TF) activity and altered the protein expression of TF in liver, lungs, heart and skin. In conclusion, the disturbances in redox status and hemostatic system induced by B. jararaca envenomation were modulated by rutin, suggesting it has a great potential to be used as an ancillary therapeutic agent for snakebites.

Vibrational spectroscopy of muscular tissue intoxicated by snake venom and exposed to photobiomodulation therapy.

The pathogenesis of myonecrosis caused by myotoxins from bothropic venom is associated with local extracellular matrix (ECM) disintegration, hemorrhage, and inflammation. Search for alternative methods associated with serum therapy is mandatory to neutralize the fast development of local damage following snakebites. The experimental use of photobiomodulation therapy (PBMT) in murine models has shown promising results relative to structural and functional recovery from bothropic snakebite-induced myonecrosis. This study pioneered in using Raman and Fourier transform infrared (FTIR) spectroscopies to characterize biochemical alterations in the gastrocnemius that had been injected with Bothrops jararacussu venom and exposed to local PBMT. Results show that vibrational spectra from lyophilized and diluted venom (1307 cm -1) was also found in the envenomed gastrocnemius indicating venom presence in the unirradiated muscle 48 h post-injection; but any longer visible after PBMT at this time exposure or 72 h post-injection regardless irradiated or not. Raman and FTIR analyses indicated that the bands with higher area and intensity were 1657 and 1547 cm-1 and 1667 and 1452 cm-1, respectively; all are assignments for proteins, especially collagen, and are higher in the PBMT-exposed gastrocnemius. The infrared spectra suggest that laser treatment was able to change protein in tissue and that such change indicates collagen as the main target. We hypothesize that the findings reflect remodeling of ECM with key participation of collagen and faster tissue recovery for an anabolic condition.

Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa.

BACKGROUND: Antivenom is the treatment of choice for snakebite, which annually kills an estimated 32,000 people in sub-Saharan Africa and leaves approximately 100,000 survivors with permanent physical disabilities that exert a considerable socioeconomic burden. Over the past two decades, the high costs of the most polyspecifically-effective antivenoms have sequentially reduced demand, commercial manufacturing incentives and production volumes that have combined to create a continent-wide vacuum of effective snakebite therapy. This was quickly filled with new, less expensive antivenoms, many of which are of untested efficacy. Some of these successfully marketed antivenoms for Africa are inappropriately manufactured with venoms from non-African snakes and are dangerously ineffective. The uncertain efficacy of available antivenoms exacerbates the complexity of designing intervention measures to reduce the burden of snakebite in sub-Saharan Africa. The objective of this study was to preclinically determine the ability of antivenoms available in Kenya to neutralise the lethal effects of venoms from the most medically important snakes in East Africa. METHODS: We collected venom samples from the most medically important snakes in East Africa and determined their toxicity in a mouse model. Using a 'gold standard' comparison protocol, we preclinically tested the comparative venom-neutralising efficacy of four antivenoms available in Kenya with two antivenoms of clinically-proven efficacy. To explain the variant efficacies of these antivenoms we tested the IgG-venom binding characteristics of each antivenom using in vitro IgG titre, avidity and venom-protein specificity assays. We also measured the IgG concentration of each antivenom. FINDINGS: None of the six antivenoms are preclinically effective, at the doses tested, against all of the most medically important snakes of the region. The very limited snake polyspecific efficacy of two locally available antivenoms is of concern. In vitro assays of the abilities of 'test' antivenom IgGs to bind venom proteins were not substantially different from that of the 'gold standard' antivenoms. The least effective antivenoms had the lowest IgG content/vial. CONCLUSIONS: Manufacture-stated preclinical efficacy statements guide decision making by physicians and antivenom purchasers in sub-Saharan Africa. This is because of the lack of both clinical data on the efficacy of most of the many antivenoms used to treat patients and independent preclinical assessment. Our preclinical efficacy assessment of antivenoms available in Kenya identifies important limitations for two of the most commonly-used antivenoms, and that no antivenom is preclinically effective against all the regionally important snakes. The potential implication to snakebite treatment is of serious concern in Kenya and elsewhere in sub-Saharan Africa, and underscores the dilemma physicians face, the need for clinical data on antivenom efficacy and the medical and societal value of establishing independent preclinical antivenom-efficacy testing facilities throughout the continent.

Preclinical Evaluation of the Efficacy of Antivenoms for Snakebite Envenoming: State-of-the-Art and Challenges Ahead.

Animal-derived antivenoms constitute the mainstay in the therapy of snakebite envenoming. The efficacy of antivenoms to neutralize toxicity of medically-relevant snake venoms has to be demonstrated through meticulous preclinical testing before their introduction into the clinical setting. The gold standard in the preclinical assessment and quality control of antivenoms is the neutralization of venom-induced lethality. In addition, depending on the pathophysiological profile of snake venoms, the neutralization of other toxic activities has to be evaluated, such as hemorrhagic, myotoxic, edema-forming, dermonecrotic, in vitro coagulant, and defibrinogenating effects. There is a need to develop laboratory assays to evaluate neutralization of other relevant venom activities. The concept of the 3Rs (Replacement, Reduction, and Refinement) in Toxinology is of utmost importance, and some advances have been performed in their implementation. A significant leap forward in the study of the immunological reactivity of antivenoms against venoms has been the development of "antivenomics", which brings the analytical power of mass spectrometry to the evaluation of antivenoms. International partnerships are required to assess the preclinical efficacy of antivenoms against snake venoms in different regions of the world in order to have a detailed knowledge on the neutralizing profile of these immunotherapeutics.

Edema and Nociception Induced by Philodryas patagoniensis Venom in Mice: A Pharmacological Evaluation with Implications for the Accident Treatment.

We have investigated the mechanisms involved in the genesis of edema and nociception induced by Philodryas patagoniensis venom (PpV) injected into the footpad of mice. PpV induced dose-related edema and nociceptive effects. Pretreatment of mice with cyclooxygenase inhibitor (indomethacin), but not with cyclooxygenase 2 inhibitor (celecoxib) markedly inhibited both effects. Pretreatments with H1 receptor antagonist (promethazine) or with dual histamine-serotonin inhibitor (cyproheptadine) failed in inhibiting both effects. In groups pretreated with captopril (angiotensin-converting enzyme inhibitor) the edema was unaltered, but nociception was clearly increased, suggesting the participation of kinins in the pathophysiology of the nociception but not of the edema-forming effect of PpV. When PpV was treated with EDTA, the nociception was similar to the one induced by untreated venom, but edema was markedly reduced. We concluded that PpV-induced edema and nociception have cyclooxygenase eicosanoids as the main mediators and no participation of vasoactive amines. Kinins seem to participate in nociception but not in edema induced by PpV. The results also suggest that metalloproteinases are the main compounds responsible for the edema, but not for the nociception induced by this venom.

Protective Effect of the Plant Extracts of Erythroxylum sp. against Toxic Effects Induced by the Venom of Lachesis muta Snake.

Snake venoms are composed of a complex mixture of active proteins that induce toxic effects, such as edema, hemorrhage, and death. Lachesis muta has the highest lethality indices in Brazil. In most cases, antivenom fails to neutralize local effects, leading to disabilities in victims. Thus, alternative treatments are under investigation, and plant extracts are promising candidates. The objective of this work was to investigate the ability of crude extracts, fractions, or isolated products of Erythroxylum ovalifolium and Erythroxylum subsessile to neutralize some toxic effects of L. muta venom. All samples were mixed with L. muta venom, then in vivo (hemorrhage and edema) and in vitro (proteolysis, coagulation, and hemolysis) assays were performed. Overall, crude extracts or fractions of Erythroxylum spp. inhibited (20%-100%) toxic effects of the venom, but products achieved an inhibition of 4%-30%. However, when venom was injected into mice before the plant extracts, hemorrhage and edema were not inhibited by the samples. On the other hand, an inhibition of 5%-40% was obtained when extracts or products were given before venom injection. These results indicate that the extracts or products of Erythroxylum spp. could be a promising source of molecules able to treat local toxic effects of envenomation by L. muta venom, aiding in the development of new strategies for antivenom treatment.

Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom.

BACKGROUND: Snakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells. METHODOLOGY: C2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 µg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation. RESULTS: In non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom. CONCLUSION: LLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may be suggested as a possible mechanism mediating cytoprotection observed under laser irradiation.

Procoagulant snake venoms have differential effects in animal plasmas: Implications for antivenom testing in animal models.

BACKGROUND: Animal models are used to test toxic effects of snake venoms/toxins and the antivenom required to neutralise them. However, venoms that cause clinically relevant coagulopathy in humans may have differential effects in animals. We aimed to investigate the effect of different procoagulant snake venoms on various animal plasmas. METHODS: Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and D-dimer levels were measured in seven animal plasmas (human, rabbit, cat, guinea pig, pig, cow and rat). In vitro clotting times were then used to calculate the effective concentration (EC50) in each plasma for four snake venoms with different procoagulant toxins: Pseudonaja textilis, Daboia russelli, Echis carinatus and Calloselasma rhodostoma. RESULTS: Compared to human, PT and aPTT were similar for rat, rabbit and pig, but double for cat and cow, while guinea pig had similar aPTT but double PT. Fibrinogen and D-dimer levels were similar for all species. Human and rabbit plasmas had the lowest EC50 for P. textilis (0.1 and 0.4 µg/ml), D. russelli (0.4 and 0.1 µg/ml), E. carinatus (0.6 and 0.1 µg/ml) venoms respectively, while cat plasma had the lowest EC50 for C. rhodostoma (11 µg/ml) venom. Cow, rat, pig and guinea pig plasmas were highly resistant to all four venoms with EC50 10-fold that of human. CONCLUSIONS: Different animal plasmas have varying susceptibility to procoagulant venoms, and excepting rabbits, animal models are not appropriate to test procoagulant activity. In vitro assays on human plasma should instead be adopted for this purpose.