Efficacy Of Antivenom Research Articles

Comparative Analysis of the Enzymatic, Coagulant, and Neuromuscular Activities of Two Variants of Crotalus durissus ruruima Venom and Antivenom Efficacy

Background: We compared the enzymatic, coagulant, and neuromuscular activities of two variants (yellow—CDRy and white—CDRw) of Crotalus durissus ruruima venom with a sample of C. d. terrificus (CDT) venom and examined their neutralization by antivenom against CDT venom. Methods: The venoms were screened for enzymatic and coagulant activities using standard assays, and electrophoretic profiles were compared by SDS-PAGE. Neutralization was assessed by preincubating venoms with crotalic antivenom and assaying the residual activity. Results: SDS-PAGE showed that the venoms had similar electrophoretic profiles, with the main bands being phospholipase A2 (PLA2), serine proteinases, L-amino acid oxidase (LAAO), and phosphodiesterase. CDRy venom had the highest proteolytic and LAAO activities, CDRw venom had greater PLA2 and esterolytic activities at the highest quantity tested, and CDT had greater PLA2 activity than CDRy. CDRw and CDT venoms had similar proteolytic and LAAO activities, and CDRy and CDT venoms had comparable esterolytic activity. None of the venoms altered the prothrombin time (PT), but all of them decreased the activated partial thromboplastin time (aPPT); this activity was neutralized by antivenom. The minimum coagulant dose potency was CDRw >> CDRy > CDT. All venoms had thrombin-like activity that was attenuated by antivenom. CDRy and CDRw venoms showed α-fibrinogenolytic activity. All venoms partially cleaved the β-chain. CDRy and CDT venoms caused neuromuscular facilitation (enhanced muscle contractions) followed by complete blockade, whereas CDRw venom caused only blockade. Antivenom neutralized the neuromuscular activity to varying degrees. Conclusions: These findings indicate that while CDR and CDT venoms share similarities, they also differ in some enzymatic and biological activities and in neutralization by antivenom. Some of these differences could influence the clinical manifestations of envenomation by C. d. ruruima and their neutralization by the currently used therapeutic antivenom.

Breaking muscle: neurotoxic and myotoxic effects of Central American snake venoms and the relative efficacies of antivenom and varespladib

BackgroundThe snake genera Atropoides, Cerrophidion, and Metlapilcoatlus form a clade of neotropical pit vipers distributed across Mexico and Central America. This study evaluated the myotoxic and neurotoxic effects of nine species of Atropoides, Cerrophidion, and Metlapilcoatlus, and the neutralising efficacy of the ICP antivenom from Costa Rica against these effects, in the chick biventer cervicis nerve-muscle preparation. Given the prominence of PLA2s within the venom proteomes of these species, we also aimed to determine the neutralising potency of the PLA2 inhibitor, varespladib.ResultsAll venoms showed myotoxic and potential neurotoxic effects, with differential intra-genera and inter-genera potency. This variation was also seen in the antivenom ability to neutralise the muscle damaging pathophysiological effects observed. Variation was also seen in the relative response to the PLA2 inhibitor varespladib. While the myotoxic effects of M. mexicanus and M. nummifer venoms were effectively neutralised by varespladib, indicating myotoxicity is PLA2 mediated, those of C. godmani and M. olmec venoms were not, revealing that the myotoxicity is driven by non-PLA2 toxin types.ConclusionsThis study characterises the myotoxic and neurotoxic venom activity, as well as neutralisation of venom effects from the Atropoides, Cerrophidion, and Metlapilcoatlus clade of American crotalids. Our findings contribute significant clinical and evolutionary knowledge to a clade of poorly researched snakes. In addition, these results provide a platform for future research into the reciprocal interaction between ecological niche specialisation and venom evolution, as well as highlighting the need to test purified toxins to accurately evaluate the potential effects observed in these venoms.

Intraspecific venom variation in the medically important puff adder (Bitis arietans): Comparative venom gland transcriptomics, in vitro venom activity and immunological recognition by antivenom.

Variation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the ~310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy. We applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique. Through the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms. The large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region.

Isolation and Pharmacological Characterisation of Pre-Synaptic Neurotoxins from Thai and Javanese Russell's Viper (Daboia siamensis) Venoms.

The widespread geographical distribution of Russell's vipers (Daboia spp.) is associated with marked variations in the clinical outcomes of envenoming by species from different countries. This is likely to be due to differences in the quantity and potency of key toxins and, potentially, the presence or absence of some toxins in venoms across the geographical spectrum. In this study, we aimed to isolate and pharmacologically characterise the major neurotoxic components of D. siamensis venoms from Thailand and Java (Indonesia) and explore the efficacy of antivenom and a PLA2 inhibitor, Varespladib, against the neuromuscular activity. These data will provide insights into the link between venom components and likely clinical outcomes, as well as potential treatment strategies. Venoms were fractionated using RP-HPLC and the in vitro activity of isolated toxins assessed using the chick biventer cervicis nerve-muscle preparation. Two major PLA2 fractions (i.e., fractions 8 and 10) were isolated from each venom. Fraction 8 from both venoms produced pre-synaptic neurotoxicity and myotoxicity, whereas fraction 10 from both venoms was weakly neurotoxic. The removal of the two fractions from each venom abolished the in vitro neurotoxicity, and partially abolished myotoxicity, of the whole venom. A combination of the two fractions from each venom produced neurotoxic activity that was equivalent to the respective whole venom (10 µg/mL), but the myotoxic effects were not additive. The in vitro neurotoxicity of fraction 8 (100 nM) from each venom was prevented by the pre-administration of Thai Russell's viper monovalent antivenom (2× recommended concentration) or preincubation with Varespladib (100 nM). Additionally, the neurotoxicity produced by a combination of the two fractions was partially reversed by the addition of Varespladib (100-300 nM) 60 min after the fractions. The present study demonstrates that the in vitro skeletal muscle effects of Thai and Javanese D. siamensis venoms are primarily due to key PLA2 toxins in each venom.

Tiny but Mighty: Vipera ammodytes meridionalis (Eastern Long-Nosed Viper) Ontogenetic Venom Variations in Procoagulant Potency and the Impact on Antivenom Efficacies.

The Eastern Long-Nosed Viper (Vipera ammodytes meridionalis) is considered one of the most venomous snakes in Europe. However, it is unknown whether ontogenetic variation in venom effects occurs in this subspecies and how this may impact antivenom efficacy. In this study, we compared the procoagulant activities of V. a. meridionalis venom on human plasma between neonate and adult venom phenotypes. We also examined the efficacy of three antivenoms-Viperfav, ViperaTAb, and Inoserp Europe-across our neonate and adult venom samples. While both neonate and adult V. a. meridionalis venoms produced procoagulant effects, the effects produced by neonate venom were more potent. Consistent with this, neonate venom was a stronger activator of blood-clotting zymogens, converting them into their active forms, with a rank order of Factor X >> Factor VII > Factor XII. Conversely, the less potent adult venom had a rank order of FXII marginally more activated than Factor VII, and both much more so than Factor X. This adds to the growing body of evidence that activation of factors besides FII (prothrombin) and FX are significant variables in reptile venom-induced coagulopathy. Although all three examined antivenoms displayed effective neutralization of both neonate and adult V. a. meridionalis venoms, they generally showed higher efficacy on adult venom than on neonate venom. The ranking of antivenom efficacy against neonate venom, from the most effective to the least effective, were Viperfav, Inoserp Europe, ViperaTAb; for adult venom, the ranking was Inoserp Europe, Viperfav, ViperaTAb. Our data reveal ontogenetic variation in V. a meridionalis, but this difference may not be of clinical concern as antivenom was effective at neutralizing both adult and neonate venom phenotypes. Regardless, our results highlight a previously undocumented ontogenetic shift, likely driven by the documented difference in prey preference observed for this species across age classes.

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom

Taking the sting out of scorpions: Electrophysiological investigation of the relative efficacy of three antivenoms against medically significant Centruroides species

In this study, we report the innovative application of whole-cell patch-clamp electrophysiology in assessing broad-spectrum neutralisation by three different antivenoms, of venoms from the medically significant scorpion genus Centruroides. Envenomations by as many as 21 species from the Centruroides genus result in up to 300,000 envenomations per year in Mexico, which poses significant and potentially life-threatening pathophysiology. We first evaluated the in vitro manifestation of envenomation against two human voltage-gated sodium (hNaV) channel subtypes: hNaV1.4 and hNaV1.5, which are primarily expressed in skeletal muscles and cardiomyocytes, respectively. The neutralisation of venom activity was then characterised for three different antivenoms using a direct competition model against the more potent target, hNaV1.4. While broad-spectrum neutralisation was identified, variation in neutralisation arose for Centruroides elegans, C. limpidus, C. noxius and C. suffusus venoms, despite the presence of a number of these venoms within the immunising mixture. This raises questions regarding the truly “broad” neutralisation capacity of the antivenoms. This study not only extends previous validation of the in vitro investigation of antivenom efficacy utilising the whole-cell patch-clamp technique but also underscores the potential of this animal-free model in exploring cross-reactivity, experimental scalability, and most importantly, informing clinical management practices regarding the administration of antivenom in Mexico.

Immunoreactivity of eastern small eyed snake (Cryptophis nigrescens) venom towards species-specific antibodies of five medically important venomous Australian elapids.

The eastern small eyed snake (Cryptophis nigrescens; CN) is an uncommon cause of snakebite in Australia despite the widespread distribution of the snake along the east coast of Australia. Diagnosis of envenomation relies on identification of the snake which is often not possible with animal snakebite cases. This study examined the immunoreactivity profile of CN venom towards specific rabbit IgG made against the medically relevant snake venom immunotypes found in Australia (tiger, brown, black, death adder and taipan). A simultaneous sandwich ELISA format was used to quantify CN venom binding to venom specific Protein A purified rabbit IgG. The binding profiles demonstrated weak binding of CN venom to rabbit IgG made against both tiger (N. scutatus) and black snake (P. australis) venoms with approximately 0.19% and 0.069% cross reactivity, respectively. However, the concentration of venom likely to be present in the urine of CN envenomed patients and the low cross reactivity suggest that envenomed veterinary patients are unlikely to be detected in the commercial snake venom detection kit. It is possible that CN envenomation is more common but may be underdiagnosed where snake venom antigen detection is relied upon solely. Serum biochemical abnormalities also overlap with other snake species found in the same geographical area. In respect of antivenom therapy, administration of tiger snake antivenom is supported by the binding data, but due to the low cross reactivity multiple vials may be required. Limited clinical evidence also supports the efficacy of tiger snake antivenom for envenomation by CN.

Investigating Snake-Venom-Induced Dermonecrosis and Inflammation Using an Ex Vivo Human Skin Model.

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.

Comparative functional characterization and in vitro immunological cross-reactivity studies on Daboia russelii and Craspedocephalus malabaricus venom.

Snake venom is a complex mixture of organic and inorganic constituents, including proteins and peptides. Several studies showed that antivenom efficacy differs due to intra- and inter-species venom variation. In the current study, comparative functional characterization of major enzymatic proteins present in Craspedocephalus malabaricus and Daboia russelii venom was investigated through various in vitro and immunological cross-reactivity assays. The enzymatic assays revealed that hyaluronidase and phospholipase A2 activities were markedly higher in D. russelii. By contrast, fibrinogenolytic, fibrin clotting and L-amino acid oxidase activities were higher in C. malabaricus venom. ELISA results suggested that all the antivenoms had lower binding potential towards C. malabaricus venom. For D. russelii venom, the endpoint titration value was observed at 1:72 900 for all the antivenoms. In the case of C. malabaricus venom, the endpoint titration value was 1:2700, except for Biological E (1:8100). All these results, along with the avidity assays, indicate the strength of venom-antivenom interactions. Similarly, the western blot results suggest that all the antivenoms showed varied efficacies in binding and detecting the venom antigenic epitopes in both species. The results highlight the need for species-specific antivenom to better manage snakebite victims.

Ruthenium Antivenom Inhibits the Defibrinogenating Activity of Crotalus adamanteus Venom in Rabbits.

Eastern Diamondback Rattlesnake (Crotalus adamanteus) envenomation is a medical emergency encountered in the Southeastern United States. The venom contains a snake venom thrombin-like enzyme (SVTLE) that is defibrinogenating, causing coagulopathy without effects on platelets in humans. This investigation utilized thrombelastographic methods to document this coagulopathy kinetically on the molecular level in a rabbit model of envenomation via the analyses of whole blood samples without and with platelet inhibition. Subsequently, the administration of a novel ruthenium compound containing site-directed antivenom abrogated the coagulopathic effects of envenomation in whole blood without platelet inhibition and significantly diminished loss of coagulation in platelet-inhibited samples. This investigation provides coagulation kinetic insights into the molecular interactions and results of SVTLE on fibrinogen-dependent coagulation and confirmation of the efficacy of a ruthenium antivenom. These results serve as a rationale to investigate the coagulopathic effects of other venoms with this model and assess the efficacy of this site-directed antivenom.

Fasciotomy following North American pit viper envenomation in Texas 2004–2021

Introduction North American pit viper envenomation occurs over 4,000 times annually in the United States, with polyvalent Fab antivenom being the primary treatment. Fasciotomy is occasionally performed due to concerns about compartment syndrome. We utilized our direct access to Texas Poison Center Network data to create a new snakebite abstraction form and database on relevant available information between 2004 and 2021 and to identify, describe, and estimate the incidence of fasciotomy following pit viper envenomation in Texas. Methods We searched the Texas Poison Center Network database for cases during 2004–2021 using keywords such as fasciotomy, surgery, compartment pressure, and compartment syndrome. Descriptive statistics summarized the data. Results Of 16,911 reported envenomations, 0.69 percent involved fasciotomies (n = 117). Most common bite sites were digits/hands and lower extremities. Patients who underwent fasciotomy were typically male, aged 20–59, and 10 years younger than the total snakebite population. Only 6 percent of reported compartment syndrome cases had a compartment pressure measurement. Antivenom was administered in 101 (86.3 percent) cases, 92 (91.1 percent) of which received only Fab antivenom product. Patients with bites from rattlesnakes (47.9 percent) were associated with most fasciotomies Discussion Our findings suggest a potential increase in snakebite exposures, accompanied by a decrease in fasciotomies. Overall, copperheads constituted the majority of snakebites, but most fasciotomies were from rattlesnake envenomations (47.9 percent). In this cohort, compartment syndrome diagnosis and decisions regarding fasciotomy were primarily based on clinical evaluation/surgeon expertise without compartment pressure measurements. Despite the efficacy of antivenom, only 86.3 percent of patients in our study received antivenom. Conclusions Fasciotomy after North American pit viper envenomation in Texas is uncommon (0.69 percent) and has decreased over time, possibly due to increased antivenom use or surgeon comfort with nonsurgical management.

Standard Quality Characteristics and Efficacy of a New Third-Generation Antivenom Developed in Colombia Covering Micrurus spp. Venoms.

In Colombia, Micrurus snakebites are classified as severe according to the national clinical care guidelines and must be treated with specific antivenoms. Unfortunately, these types of antivenoms are scarce in certain areas of the country and are currently reported as an unavailable vital medicine. To address this issue, La Universidad de Antioquia, through its spin-off Tech Life Saving, is leading a project to develop third-generation polyvalent freeze-dried antivenom. The goal is to ensure access to this therapy, especially in rural and dispersed areas. This project aims to evaluate the physicochemical and preclinical parameters (standard quality characteristics) of a lab-scale anti-elapid antivenom batch. The antivenom is challenged against the venoms of several Micrurus species, including M. mipartitus, M. dumerilii, M. ancoralis, M. dissoleucus, M. lemniscatus, M. medemi, M. spixii, M. surinamensis, and M. isozonus, following the standard quality characteristics set by the World Health Organization (WHO). The antivenom demonstrates an appearance consistent with standards, 100% solubility within 4 min and 25 s, an extractable volume of 10.39 mL, a pH of 6.04, an albumin concentration of 0.377 mg/mL (equivalent to 1.22% of total protein), and a protein concentration of 30.97 mg/mL. Importantly, it maintains full integrity of its F(ab')2 fragments and exhibits purity over 98.5%. Furthermore, in mice toxicity evaluations, doses up to 15 mg/mouse show no toxic effects. The antivenom also demonstrates a significant recognition pattern against Micrurus venoms rich in phospholipase A2 (PLA2) content, as observed in M. dumerilii, M. dissoleucus, and M. isozonus. The effective dose 50 (ED50) indicates that a single vial (10 mL) can neutralize 2.33 mg of M. mipartitus venom and 3.99 mg of M. dumerilii venom. This new anti-elapid third-generation polyvalent and freeze-dried antivenom meets the physicochemical parameters set by the WHO and the regulators in Colombia. It demonstrates significant efficacy in neutralizing the venom of the most epidemiologically important Micrurus species in Colombia. Additionally, it recognizes seven other species of Micrurus venom with a higher affinity for venoms exhibiting PLA2 toxins. Fulfilling these parameters represents the first step toward proposing a new pharmacological alternative for treating snakebites in Colombia, particularly in dispersed rural areas, given that this antivenom is formulated as a freeze-dried product.

Biochemical and biological characterization of the venoms of Naja kaouthia and Naja mandalayensis from Myanmar and neutralization effects of BPI cobra antivenom

Snakebite is a neglected public health issue, with many scientific and medical issues to be solved. Cobras are among the most common venomous snakes in Myanmar and are responsible for a considerable number of severe snakebite envenoming. There are three species of cobra (Naja kaouthia, Naja mandalayensis and Ophiophagus hannah) in Myanmar. The study aims to characterize the N. kaouthia and N. mandalayensis venoms and to investigate the efficacy of anti-cobra antivenom (BPI) against the two venoms. Protein components and fibrinogenolytic activity were determined by SDS-PAGE. Enzymatic activities for PLA2, protease and acetylcholinesterase were determined by spectrophotometric method. Anticoagulant activity was determined by recalcification time of citrated human plasma. Myotoxicity, necrotizing activity, median lethal dose (LD50) and median effective dose (ED50) were determined by WHO recommended methods. The SDS-PAGE displayed the proteins and enzymes containing in two venoms were different. N. kaouthia venom exhibited more in PLA2, acetylcholinesterase, anticoagulant, fibrinogenolytic and necrotizing activities than N. mandalayensis venom. N. mandalayensis venom had more protease activity and myotoxicity than N. kaouthia venom. The median lethal dose (LD50) of N. kaouthia and N. mandalayensis venom was 4.33 μg/mouse and 5.04 μg/mouse respectively. Both venoms induced fibrinogen Aα chain degradation in 30 min (N. kaouthia) and in 6 h (N. mandalayensis). The same median effective dose (ED50) (19.56 μg/mouse) showed that anti-NK antivenom can neutralize against lethal effect of N. mandalayensis venom. It can also neutralize the protease activity, anticoagulant activity and fibrinogenolytic activity of both venoms. Immunodiffusion and immunoblotting studies showed that the antivenom recognized its homologous venom (N. kaouthia) and cross-reacted against the heterologous venom (N. mandalayensis). The anti-NK antivenom is suitable to use for N. mandalayensis bite if monospecific antivenom is not available.

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom

Mice are routinely used in snake venom research but are costly and subject to pain and suffering. The crustacean Artemia salina could be an alternative to mice, but data to support its adoption in snake venom research is limited. The aim of the present study was to evaluate the suitability of A. salina as a surrogate of mice in assessing the toxicity of venoms and the preclinical efficacy of antivenoms. The toxicity of venoms from 22 snakes of medical importance in sub–Saharan Africa was evaluated in mice (intraperitoneally; i.p. and intravenously; i.v.) and in A. salina. Subsequently, the capacity of a commercial antivenom to neutralize the toxicity of these venoms in mice and A. salina was investigated. There was a positive correlation between the i.v. median lethal doses (LD50s) and the i.p. LD50s in mice (r = 0.804; p < 0.0001), a moderate correlation between the i.v. LD50s in mice and the median lethal concentrations (LC50s) in A. salina (r = 0.606; p = 0.003), and a moderate correlation between the i.p. LD50s in mice and the LC50s in A. salina (r = 0.426; p = 0.048). Moreover, there was a strong correlation between the i.p. median effective doses (ED50s) and the i.v. ED50s in mice (r = 0.941, p < 0.0001), between the i.p. ED50s in mice and the ED50s in A. salina (r = 0.818, p < 0.0001), and between the i.v. ED50s in mice and the ED50s in A. salina (r = 0.972, p < 0.0001). These findings present A. salina as a promising candidate for reducing reliance on mice in snake venom research. Future investigations should build upon these findings, addressing potential limitations and expanding the scope of A. salina in venom research and antivenom development.

Ruthenium-based antivenom attenuates Crotalus atrox venom mediated coagulopathy in rabbits.

The Western diamondback rattlesnake ( Crotalus atrox ) is a medically important venomous snake in the Southwestern United States, injuring humans, and their companion animals. The goals of this investigation were to utilize a rabbit model of subcutaneous envenomation to assess Crotalus atrox venom coagulopathy and determine the efficacy of a ruthenium-containing antivenom (RA) in attenuating it. Sedated New Zealand White rabbits had viscoelastic measurements of whole blood coagulation kinetics obtained from ear artery samples. Crotalus atrox venom (4 mg/kg) was injected subcutaneously and changes in coagulation determined over three hours and compared to samples obtained prior to envenomation. Other rabbits had site-directed RA injected 5 min after venom injection. A significant decrease in the velocity of clot growth and thrombus strength was observed in animals injected with venom alone. Site-directed administration of RA resulted in no change in coagulation over the 3 h following venom injection. The interaction of antivenom administration and time was significantly different in the cases of clot growth velocity and strength. A novel rabbit model was used to define the toxicodynamic profile of coagulopathy of Crotalus atrox venom and demonstrate the efficacy of RA. Future investigation is planned involving other medically important venoms and RA administration.

Anti-inflammatory, healing and antiophidic potential of Jatropha mollissima (Pohl) Baill. (Euphorbiaceae): From popular use to pharmaceutical formulation in gel

Jatropha mollissima (Pohl) Baill. (Euphorbiaceae) is widely used in traditional medicine to treat inflammatory disorders. So, a topical gel containing the hydroethanolic extract of its leaves was developed and evaluated for its anti-inflammatory, wound healing, and antiophidic properties in mice. First, the chemical profile of different parts of the plant was characterized by liquid chromatography coupled to mass spectrometry (LC-MS) using molecular networking. In the leaf extract, 11 compounds were characterized, with a particular emphasis on the identification of flavonoids. The gel efficiently inhibited carrageenan-induced paw edema, as well as acute and chronic croton oil-induced ear edema models, thereby reducing inflammatory and oxidative parameters in inflamed tissues. Besides anti-inflammatory activity, the herbal gel showed significant wound healing activity. The edematogenic, hemorrhagic and dermonecrotic activities induced by Bothrops jararaca snake venom were effectively inhibited by the treatment with J. mollissima gel. The association with the herbal gel improved in up to 90% the efficacy of commercial snake antivenom in reduce venom-induced edema. Additionally, while antivenom was not able to inhibit venom-induced dermonecrosis, treatment with herbal gel reduced in 55% the dermonocrotic halo produced. These results demonstrate the pharmacological potential of the herbal gel containing J. mollissima extract, which could be a strong candidate for the development of herbal products that can be used to complement the current antivenom therapy against snake venom local toxicity.

A Comparison of the Efficacy of Antivenoms and Varespladib against the In Vitro Pre-Synaptic Neurotoxicity of Thai and Javanese Russell's Viper (Daboia spp.) Venoms.

The heterogeneity in venom composition and potency in disparate Eastern Russell's viper (Daboia siamensis) populations has repercussions for the efficacy of antivenoms. This is particularly pronounced in geographical areas in which the venom of the local species has not been well studied and locally produced antivenoms are unavailable. In such cases, alternative therapies following envenoming, which are not limited by species specificity, may be employed to complement antivenoms. We studied the neuromuscular activity of D. siamensis venom from Thailand and Java (Indonesia) and the ability of Thai antivenoms and/or Varespladib to prevent or reverse these effects. Both Thai and Javanese D. siamensis venoms displayed potent pre-synaptic neurotoxicity but weak myotoxicity in the chick biventer cervicis nerve-muscle preparation. Whilst the neurotoxicity induced by both venoms was abolished by the prior administration of Thai D. siamensis monovalent antivenom or pre-incubation with Varespladib, Thai neuro-polyvalent antivenom only produced partial protection when added prior to venom. Pre-synaptic neurotoxicity was not reversed by the post-venom addition of either antivenom 30 or 60 min after either venom. Varespladib, when added 60 min after venom, prevented further inhibition of indirect twitches. However, the subsequent addition of additional concentrations of Varespladib did not result in further recovery from neurotoxicity. The combination of Thai monovalent antivenom and Varespladib, added 60 min after venom, resulted in additional recovery of twitches caused by either Thai or Javanese venoms compared with antivenom alone. In conclusion, we have shown that Varespladib can prevent and partially reverse the pre-synaptic neurotoxicity induced by either Thai or Javanese D. siamensis venoms. The efficacy of Thai D. siamensis monovalent antivenom in reversing pre-synaptic neurotoxicity was significantly enhanced by its co-administration with Varespladib. Further work is required to establish the efficacy of Varespladib as a primary or adjunct therapy in human envenoming.

Evaluation of venom diversity and antivenom quality from the venom of long-term captive vs recently wild captured Pseudocerastes persicus snake: An In vitro and In vivo study

Snakebite envenomation is a life-threatening condition and antivenoms are used as the most effective treatment. Venom obtained from snakes in long-term captivity showed some variations in comparison to the venom of the wild snakes. The objective of this study is to compare the venom of the Pseudocerastes persicus under long-term captivity and wild conditions as well as the antivenom obtained from these venoms. We have analyzed venom samples and produced trivalent antivenoms using the venom of long-term captive (LTC) or recently wild-captured (RWC) Pseudocerastes persicus, and RWC Macrovipera lebetina, and Echis carinatus. The HPLC analysis revealed that the RWC snakes' venom had three peaks that were not present in the LTC snake's venom. Further analysis using MALDI-TOF and MS/MS showed that the fraction with a retention time (RT) of 14 min contained a toxin from the Kunitz-type serine protease inhibitor (KUT) class, while the fraction with RT 21 a peptide identified within the snake venom metalloproteinase (SVMP) class. The third peak was identified as a sphingolipid.Interestingly, the in vivo preclinical tests showed no significant differences in the effectiveness of the antivenoms. which could be due to the cross-immunogenicity or cross-reactivity between different toxins in the venom. According to our results, small variations in the venom composition of a species do not lead to a decrease in the efficacy of the polyvalent antivenom.

Immunological Cross-Reactivity and Preclinical Assessment of a Colombian Anticoral Antivenom against the Venoms of Three Micrurus Species.

Snakebite accident treatment requires the administration of antivenoms that provide efficacy and effectiveness against several snake venoms of the same genus or family. The low number of immunogenic components in venom mixtures that allow the production of antivenoms consequently gives them partial neutralization and a suboptimal pharmacological response. This study evaluates the immunorecognition and neutralizing efficacy of the polyvalent anticoral antivenom from the Instituto Nacional de Salud (INS) of Colombia against the heterologous endemic venoms of Micrurus medemi, and M. sangilensis, and M. helleri by assessing immunoreactivity through affinity chromatography, ELISA, Western blot, and neutralization capability. Immunorecognition towards the venoms of M. medemi and M. sangilensis showed values of 62% and 68% of the protein composition according to the immunoaffinity matrix, respectively. The analysis by Western blot depicted the highest recognition patterns for M. medemi, followed by M. sangilensis, and finally by M. helleri. These findings suggest that the venom compositions are closely related and exhibit similar recognition by the antivenom. According to enzyme immunoassays, M. helleri requires a higher amount of antivenom to achieve recognition than the others. Besides reinforcing the evaluation of INS antivenom capability, this work recommends the use of M. helleri in the production of Colombian antisera.