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There is an unmet need for economical snakebite therapies with long shelf lives that are effective even with delays in treatment. The orally bioavailable, heat-stable, secretory phospholipase A₂ (sPLA₂) inhibitor, LY333013, demonstrates antidotal characteristics for severe snakebite envenoming in both field and hospital use. A murine model of lethal envenoming by a Papuan taipan (Oxyuranus scutellatus) demonstrates that LY333013, even with delayed oral administration, improves the chances of survival. Furthermore, LY333013 improves the performance of antivenom even after it no longer reverses neurotoxic signs. Our study is the first demonstration that neurotoxicity from presynaptic venom sPLA2S can be treated successfully, even after the window of therapeutic antivenom has closed. These results suggest that sPLA₂ inhibitors have the potential to reduce death and disability and should be considered for the initial and adjunct treatment of snakebite envenoming. The scope and capacity of the sPLA2 inhibitors ability to achieve these endpoints requires further investigation and development efforts.

Antivenom is currently the standard-of-care treatment for snakebite envenoming, but its efficacy is limited by treatment delays, availability, and in many cases, species specificity. Many of the rapidly lethal effects of envenoming are caused by venom-derived toxins, such as phospholipase A2 (sPLA2); therefore, small molecule direct toxin inhibitors targeting these toxins may have utility as initial and adjunct therapies after envenoming. Varespladib (intravenous, IV) and varespladib-methyl (oral) have been shown to potently inhibit sPLA2s from snake venoms in murine and porcine models, thus supporting their further study as potential treatments for snakebite envenoming. In this pilot study, we tested the ability of these compounds to reverse neurotoxic effects of venom from the Australian and Papuan taipan (Oxyuranus scutellatus) subspecies in juvenile pigs (Sus domesticus). The mean survival time for control animals receiving Australian taipan venom (0.03 mg/kg, n = 3) was 331 min ± 15 min; for those receiving Papuan taipan venom (0.15 mg/kg, n = 3) it was 178 ± 31 min. Thirteen pigs received Australian taipan venom and treatment with either IV or oral varespladib (or with IV to oral transition) and all 13 survived the duration of the study (≥96 h). Eight pigs received Papuan taipan venom followed by treatment: Briefly: Two animals received antivenom immediately and survived to the end of the study. Two animals received antivenom treatment delayed 45 min from envenoming and died within 4 h. Two animals received similarly delayed antivenom treatment and were rescued by varespladib. Two animals were treated with varespladib alone after a 45-min delay. Treatment with varespladib only was effective but required repeat dosing over the course of the study. Findings highlight both the importance of early treatment and, as well, a half-life for the investigational inhibitors now in Phase II clinical trials for snakebite. Varespladib rapidly reversed weakness even when administered many hours post-envenoming and, overall, our results suggest that varespladib and varespladib-methyl could be efficacious tools in the treatment of sPLA2-induced weakness from Oxyuranus envenoming. Further clinical study as initial therapy and as potential method of rescue from some types of antivenom-resistant envenomings are supported by these data. [ABSTRACT FROM AUTHOR]

Introduction: Snake venom composition shows significant inter- and intra-species variation. In the case of the viperid species Bothrops atrox, responsible for the majority of snakebites in the Amazon region, geographical and ontogenetic variables affect venom composition, with ecological and medical implications. Previous studies had shown that venom from neonate and juvenile Bothrops specimens have a higher in vitro coagulant activity. The aim of this investigation was to assess the association of clinical outcomes, such as venom-induced coagulopathy and local complications, with B. atrox ontogenetic variables. Methods: This study explored the relationship between some clinical parameters in patients suffering envenomations by B. atrox in the Amazon and several morphometric parameters of the snake specimens causing the bites. Results: There were 248 specimens confirmed as agents of envenomation, mostly female snakes (70.5%) and classified as juveniles (62.7%). Patients bitten by neonates compared to adult snakes [OR = 2.70 (95%CI 1.15-6.37); p =.021] and by snakes with white tail tip [OR = 1.98 (95%CI 1.15–3.41); p =.013] were more likely to develop coagulopathy. Time from patient admission to the unclottable blood reversion was not affected by the snake gender (p =.214) or age (p =.254). Patients bitten by neonate (p =.024) or juvenile snakes (p <.0001) presented a lower frequency of moderate to severe edema, as compared to those bitten by adult snakes. In agreement with experimental observations, patients bitten by neonates and by snakes with a white tail tip were more likely to develop coagulopathy than those bitten by adult snakes. In contrast, envenomations by adult snakes were associated with a higher incidence of severe local edema. Conclusion: Despite these variations, no difference was observed in the time needed to recover blood clotting in these patients after Bothrops antivenom administration. [ABSTRACT FROM AUTHOR]

Snakebite envenoming (SBE) is a neglected tropical disease that kills and maims hundreds of thousands of people yearly, particularly in impoverished rural settings of the Global South. Understanding the complexity of SBE and tackling this disease demands a transdisciplinary, One Health approach. There is a long-standing research tradition on SBE in toxinology and human medicine. In contrast, other disciplines, such as veterinary medicine or social sciences, still need to be better developed in this field, especially in countries with a high incidence of SBE. Broadening the disciplinary landscape, connecting various research approaches, methods, and data across disciplines and sectors, and engaging with communities affected by SBE in implementing evidence-based solutions are needed. This review summarizes areas that require strengthening to better understand the complexity of SBE and to generate a robust body of knowledge to be translated into effective public health interventions. [Display omitted] • Snakebite envenoming has been traditionally studied within the field of Toxinology. • To grasp the complexity of snakebite envenoming it is necessary to broaden the disciplinary landscape. • Social sciences provide relevant tools to expand our understanding of the multiple factors associated with snakebites. • The search for transdisciplinarity will provide novel tools to understand and confront snakebite envenoming. [ABSTRACT FROM AUTHOR]

Micrurus clarki is an uncommon coral snake distributed from the Southeastern Pacific of Costa Rica to Western Colombia, for which no information on its venom could be found in the literature. Using a 'venomics' approach, proteins of at least nine families were identified, with a moderate predominance of three-finger toxins (3FTx; 48.2%) over phospholipase A2 (PLA2; 36.5%). Comparison of this venom profile with those of other Micrurus species suggests that it may represent a more balanced, 'intermediate' type within the dichotomy between 3FTx- and PLA2-predominant venoms. M. clarki venom was strongly cross-recognized and, accordingly, efficiently neutralized by an equine therapeutic antivenom against M. nigrocinctus, revealing their high antigenic similarity. Lethal activity for mice could be reproduced by a PLA2 venom fraction, but, unexpectedly, not by fractions corresponding to 3FTxs. The most abundant venom component, hereby named clarkitoxin-I, was identified as a short-chain (type I) 3FTx, devoid of lethal effect in mice, whose target remains to be defined. Its amino acid sequence of 66 residues shows high similarity with predicted sequences of venom gland transcripts described for M. fulvius, M. browni, and M. diastema. [ABSTRACT FROM AUTHOR]

Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact worldwide, mostly affecting poor people involved in agricultural activities in Africa, Asia, Latin America and Oceania. A key issue that complicates the treatment of snakebite envenomings is the poor availability of the only validated treatment for this disease, antivenoms. Antivenoms can be an efficacious treatment for snakebite envenoming, provided they are safe, effective, affordable, accessible and administered appropriately. The shortage of antivenoms in various regions, particularly in Sub-Saharan Africa and some parts of Asia, can be significantly alleviated by optimizing the use of current antivenoms and by the generation of novel polyspecific antivenoms having a wide spectrum of efficacy. Complementing preclinical testing of antivenom efficacy using in vivo and in vitro functional neutralization assays, developments in venomics and antivenomics are likely to revolutionize the design and preclinical assessment of antivenoms by being able to test new antivenom preparations and to predict their paraspecific neutralization to the level of species-specific toxins. [ABSTRACT FROM AUTHOR]

Abstract: Parenteral administration of animal-derived antivenoms constitutes the mainstay in the treatment of snakebite envenomings. Despite the fact that this therapy has been available for over a century, the detailed understanding of the neutralizing and immunoreactivity profiles of the majority of antivenoms is pending. Currently, a combination of preclinical neutralization tests and ‘antivenomics’, i.e. a proteomic-based assessment of antivenom immunoreactivity, provides a powerful analytical platform to investigate the preclinical efficacy of antivenoms. In this review, the studies performed on the polyvalent antivenom manufactured by Instituto Clodomiro Picado, Costa Rica, are summarized. This antivenom is prepared by immunizing horses with a mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys, and is used in Central America for the treatment of envenomings by viperid species. Overall, the antivenom shows a widespread pattern of immunological reactivity against homologous and heterologous venoms, which correlates with its ability to neutralize lethal, hemorrhagic, myotoxic, coagulant, defibrinogenating, phospholipase A2 and proteinase activities of viperid venoms. At the same time, antivenomics detected several venom components against which the antivenom shows only partial or negligible immunorecognition, such as low molecular mass vasoactive peptides, disintegrins, and some phospholipases A2, P-I metalloproteinases and serine proteinases. Such information can be used to design strategies for enhancing the antibody response of horses against poorly immunogenic, toxicologically-relevant venom components in order to further improve the efficacy of this antivenom. Biological significance: The timely parenteral administration of an appropriate antivenom remains, more than a century after the development of the first serum antivenimeux by Calmette and Phisalix and Bertrand, the only currently effective treatment for snakebite envenomings. A key technical issue in the generation of novel antivenoms is the design of optimized immunization venom mixtures that ensure that the resulting antidotes will be effective against the highest number of venoms from snakes of medical concern across the geographical range where they will be used. Antivenomics is a proteomics-based protocol developed to complement in vitro and in vivo standard preclinical tests in the qualitative and quantitative characterization of the immunological profile and the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. Antivenomics is translational venomics. The combination of antivenomics and neutralization assays represents a powerful analytical platform to investigate the efficacy of antivenoms at the molecular and preclinical levels. This article is part of a Special Issue entitled: Proteomics of non-model organisms. [Copyright &y& Elsevier]

Abstract: A polyspecific antivenom is used in Central America for the treatment of envenomings by viperid snakes. This antivenom is generated in horses hyperimmunized with a mixture of venoms from Bothrops asper, Crotalus simus and Lachesis stenophrys. The present study analyzed the ability of this antivenom to neutralize the venoms of three Central American viperid species of the ‘Porthidium group’, i.e. Porthidium nasutum, Porthidium ophryomegas and Cerrophidion sasai, formerly classified as Cerrophidion godmani. In addition, the immunorecognition of the components of these venoms was assessed by immunoaffinity antivenomics. The antivenom proved effective in neutralizing the lethal, hemorrhagic, myotoxic, phospholipase A2 (PLA2) and proteinase activities of the three venoms, albeit exhibiting quantitative differences in the values of the Median Effective Doses (ED50). Excepting for certain low molecular mass bands corresponding to disintegrins, and some PLA2s and PI-metalloproteinases, Western blotting and immunoaffinity chromatography revealed immunorecognition of most Porthidium and Cerrophidion venom proteins. In agreement with in vivo neutralization assays, immobilized antivenom IgGs showed higher immunocapturing activity of toxins from both Porthidium taxa than from C. sasai. Overall our results demonstrate a significant paraspecific protection of the Costa Rican polyspecific antivenom against the three venoms sampled. They also stress the need to search for novel ways to enhance the immune response of horses against several weakly immunogenic venom components. [Copyright &y& Elsevier]

Snakebite envenomings constitute a serious and neglected public health problem. Despite the fact that effective treatment exists, i.e. administration of animal-derived antivenoms, the availability and accessibility of these life-saving immunobiologicals is deficitary in various parts of the world, particularly in sub-Saharan Africa and some regions of Asia. This article discusses some of the problems that need to be circumvented in order to improve the availability and accessibility of antivenoms. The conglomerate of antivenom manufacturers is highly heterogeneous in terms of technological base, qualification of staff, implementation of Good Manufacturing Practices (GMPs), and volume of production. Therefore, improvements in antivenom quality and availability should be based on strategies tailored to the situation of each region or country; in this context, three different scenarios are discussed. Accessibility of antivenoms demands concerted efforts at multiple levels, including raising the awareness of public health authorities on the relevance of the problem, implementing innovative antivenom purchasing schemes, strengthening national distribution channels on the basis of robust epidemiological information, improving the cold chain and the provision of health services in remote rural settings, supporting the correct use of antivenoms, and promoting the involvement of local community organizations in various aspects of prevention and management. These tasks should be envisaged in terms of synergistic, interprogrammatic and intersectorial interventions, with the participation of many players. [ABSTRACT FROM AUTHOR]

Abstract: Venoms of several Naja species found in Sub-Saharan Africa, and commonly known as "spitting cobras", induce a predominantly cytotoxic pattern of envenomings that may evolve into tissue necrosis and gangrene. Cytotoxic components of their venoms have been identified as members of the three-finger toxin and phospholipase A2 protein families. In this study, an in vitro assay using the myogenic cell line C2C12, was utilized to compare the cytolytic activities of venoms from five species of spitting cobras: Naja nigricollis, Naja katiensis, Naja pallida, Naja nubiae, and Naja mossambica. These venoms were strongly cytotoxic, causing a 50% effect at ∼1.5 μg/well (15 μg/ml), except for N. katiensis venom, which required nearly twice this amount. Using the cell-based assay, the ability of an equine polyspecific antivenom (EchiTab-Plus-ICP) to neutralize cytotoxicity was assessed. The antivenom completely inhibited the cytotoxic activity of all five venoms, although high antivenom/venom ratios were needed. Neutralization curves displayed the following decreasing order of efficiency: N. nubiae > N. pallida > N. mossambica > N. nigricollis > N. katiensis. Results indicate that neutralizing antibodies toward toxins responsible for this particular effect are present in the antivenom, albeit in low titers. Fucoidan, a natural sulfated polysaccharide known to inhibit the toxic effects of some basic snake venom components, was unable to reduce cytotoxicity of Naja venoms. Results emphasize the need of enhancing the immunogenicity of low molecular mass toxins during antivenom production, as well as to search for useful toxin inhibitors which could complement antivenom therapy. [Copyright &y& Elsevier]

Abstract: The parenteral administration of antivenoms is the cornerstone of snakebite envenoming therapy. Efforts are made to ensure that antivenoms of adequate efficacy and safety are available world-wide. We address the main issues to be considered for the development and manufacture of improved antivenoms. Those include: (a) A knowledge-based composition design of venom mixtures used for immunization, based on biochemical, immunological, toxicological, taxonomic, clinical and epidemiological data; (b) a careful selection and adequate management of animals used for immunization; (c) well-designed immunization protocols; (d) sound innovations in plasma fractionation protocols to improve recovery, tolerability and stability of antivenoms; (e) the use of recombinant toxins as immunogens to generate antivenoms and the synthesis of engineered antibodies to substitute for animal-derived antivenoms; (f) scientific studies of the contribution of existing manufacturing steps to the inactivation or removal of viruses and other zoonotic pathogens; (g) the introduction of novel quality control tests; (h) the development of in vitro assays in substitution of in vivo tests to assess antivenom potency; and (i) scientifically-sound pre-clinical and clinical assessments of antivenoms. These tasks demand cooperative efforts at all main stages of antivenom development and production, and need concerted international partnerships between key stakeholders. [Copyright &y& Elsevier]

Abstract: Snakebite envenoming is a neglected public health challenge of compelling importance in many regions of the world, particularly sub-Saharan Africa, Asia, Latin America and Papua-New Guinea. Addressing the problem of snakebite effectively demands an integrated multifocal approach, targeting complex problems and involving many participants. It must comprise: [(a)] Acquisition of reliable information on the incidence and mortality attributable to snakebite envenoming, and the number of people left with permanent sequelae. [(b)] Improvements in production of effective and safe antivenoms, through strategies aimed at strengthening the technological capacity of antivenom manufacturing laboratories. [(c)] Increasing the capacity of low-income countries to produce specific immunogens (snake venoms) locally, and to perform their own quality control of antivenoms. [(d)] Commitments from regional producers to manufacture antivenoms for countries where antivenom production is not currently feasible. [(e)] Implementation of financial initiatives guaranteeing the acquisition of adequate volumes of antivenom at affordable prices in low-income countries. [(f)] Performance of collaborative studies on the safety and effectiveness of antivenoms assessed preclinically and by properly designed clinical trials. [(g)] Development of antivenom distribution programmes tailored to the real needs and epidemiological situations of rural areas in each country. [(h)] Permanent training programmes for health staff, particularly in rural areas where snakebites are frequent. [(i)] Implementation of programmes to support those people whose snakebites resulted in chronic disabilities. [(j)] Preventive and educational programmes at the community level, with the active involvement of local organizations and employing modern methods of health promotion. Such an integrated approach, currently being fostered by the Global Snake Bite Initiative of the International Society on Toxinology and by the World Health Organization, will help to alleviate the enormous burden of human suffering inflicted by snakebite envenoming. [Copyright &y& Elsevier]

Abstract: Venoms comprise mixtures of peptides and proteins tailored by Natural Selection to act on vital systems of the prey or victim. Here we review our proteomic protocols for uncoiling the composition, immunological profile, and evolution of snake venoms. Our long-term goal is to gain a deep insight of all viperid venom proteomes. Knowledge of the inter- and intraspecies ontogenetic, individual, and geographic venom variability has applied importance for the design of immunization protocols aimed at producing more effective polyspecific antivenoms. A practical consequence of assessing the cross-reactivity of heterologous antivenoms is the possibility of circumventing the restricted availability of species-specific antivenoms in some regions. Further, the high degree of target specificity makes toxins valuable scaffolds for drug development. [Copyright &y& Elsevier]

Abstract: The issues of antivenom stability and distribution, and the training of health staff in the correct use of antivenoms in Latin America were discussed in a workshop held at Instituto Clodomiro Picado, Costa Rica, in September 16–19, 2008, under the auspices of the program CYTED. Participants from public antivenom production laboratories of the region, together with representatives of the Ministries of Health, from Argentina, Paraguay, Brazil, Bolivia, Perú, Ecuador, Colombia, Venezuela, Panamá, Costa Rica and Nicaragua participated in the event. Technical advances in the study of antivenom stability and in the design of novel formulations aimed at generating products of higher stability were presented. In addition, antivenom acquisition and distribution systems in every country were presented and discussed, together with novel tools that could be useful for improving antivenom distribution, such as the software SIGEpi, developed by the Pan American Health Organization. The issue of the cold chain, as well as the most frequent causes of misuse of antivenoms in the region, were also analyzed. Finally, the experiences of training programs for health staff on the correct use of antivenoms in snakebite envenomation treatment in Latin America were presented. It was concluded that, in addition to the fostering of antivenom production and quality control, renewed efforts should be implemented at improving the stability, distribution and correct use of antivenoms in the region. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

The toxin composition of snake venoms and, thus, their functional activity, can vary between and within species. Intraspecific venom variation across a species' geographic range is a major concern for antivenom treatment of envenomations, particularly for countries like French Guiana that lack a locally produced antivenom. Bothrops asper and Bothrops atrox are the most medically significant species of snakes in Latin America, both producing a variety of clinical manifestations, including systemic bleeding. These pathophysiological actions are due to the activation by the venom of the blood clotting factors Factor X and prothrombin, thereby causing severe consumptive coagulopathy. Both species are extremely wide-ranging, and previous studies have shown their venoms to exhibit regional venom variation. In this study, we investigate the differential coagulotoxic effects on human plasma of six venoms (four B. asper and two B. atrox samples) from different geographic locations, spanning from Mexico to Peru. We assessed how the venom variation of these venom samples affects neutralisation by five regionally available antivenoms: Antivipmyn, Antivipmyn-Tri, PoliVal-ICP, Bothrofav, and Soro Antibotrópico (SAB). The results revealed both inter- and intraspecific variations in the clotting activity of the venoms. These variations in turn resulted in significant variation in antivenom efficacy against the coagulotoxic effects of these venoms. Due to variations in the venoms used in the antivenom production process, antivenoms differed in their species-specific or geographical neutralisation capacity. Some antivenoms (PoliVal-ICP, Bothrofav, and SAB) showed species-specific patterns of neutralisation, while another antivenom (Antivipmyn) showed geographic-specific patterns of neutralisation. This study adds to current knowledge of Bothrops venoms and also illustrates the importance of considering evolutionary biology when developing antivenoms. Therefore, these results have tangible, real-world implications by aiding evidence-based design of antivenoms for treatment of the envenomed patient. We stress that these in vitro studies must be backed by future in vivo studies and clinical trials before therapeutic guidelines are issued regarding specific antivenom use in a clinical setting. [ABSTRACT FROM AUTHOR]

A qualitative study was carried out in south-eastern Costa Rica on the circumstances and consequences of snakebite envenomings. This region has the highest incidence of snakebites and the lowest per capita and per family income in the country. There is a high degree of destitution and an unstable labor situation in the region. This study was based on semistructured interviews with 15 people who had suffered snakebite envenomings. This sample size was established on the basis of data saturation. Bites occurred mostly while doing agricultural work, either as salaried workers, as occasional workers, or working on their own. Although all people were attended in health centers of the public health system, and received antivenom free of charge, the majority of them did not receive compensation or rehabilitation upon discharge from the health facilities as a result of not being regular salaried workers. People described many difficulties as a consequence of these envenomings, such as permanent physical sequelae, including two amputations, psychological consequences, economic hardships, and difficulties for reinsertion into agricultural work. In spite of the significant advances that Costa Rica has made for reducing the impact of these envenomings, results reveal issues that require urgent attention by government and civil society organizations, to compensate for the physical, psychological, social, and economic consequences of these envenomings. [ABSTRACT FROM AUTHOR]

There is a clear, unmet need for effective, lightweight, shelf-stable and economical snakebite envenoming therapies that can be given rapidly after the time of a snake's bite and as adjuncts to antivenom therapies in the hospital setting. The sPLA2 inhibitor, LY315920, and its orally bioavailable prodrug, LY333013, demonstrate surprising efficacy and have the characteristics of an antidote with potential for both field and hospital use. The efficacy of the active pharmaceutical ingredient (LY315920) and its prodrug (LY333013) to treat experimental, lethal envenoming by Micrurus fulvius (Eastern coral snake) venom was tested using a porcine model. Inhibitors were administered by either intravenous or oral routes at different time intervals after venom injection. In some experiments, antivenom was also administered alone or in conjunction with LY333013. 14 of 14 animals (100%) receiving either LY315920 (intravenous) and/or LY333013 (oral) survived to the 120 h endpoint despite, in some protocols, the presence of severe neurotoxic signs. The study drugs demonstrated the ability to treat, rescue, and re-rescue animals with advanced manifestations of envenoming. Low molecular mass sPLA2 inhibitors were highly effective in preventing lethality following experimental envenoming by M. fulvius. These findings suggest the plausibility of a new therapeutic approach to snakebite envenoming, in this example, for the treatment of a coral snake species for which there are limitations in the availability of effective antivenom. [ABSTRACT FROM AUTHOR]

Abstract: A novel method of high performance liquid chromatography with UV detection for the quantification of caprylic acid in formulations of therapeutic immunoglobulins was developed and validated. Samples have interfering proteins that were removed by ultrafiltration in a centrifugal filter unit of 10kDa nominal molecular weight limit. Then, compounds present in ultrafiltrates were separated on an Eclipse XDB-C8 5μm column (150mm×4.6mm i.d.), using a mixture of acetonitrile–water (60:40, v/v) as the mobile phase at a flow rate of 1mL/min. The UV detection was performed at 210nm. The method was found to be precise and accurate, with a linearity range from 400μg/mL to 600μg/mL (r =0.9948). The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 13.46μg/mL and 44.85μg/mL, respectively. To illustrate the usefulness of the method in the in-process and final quality control for production of therapeutic immunoglobulin formulations, permeates obtained from the industrial diafiltration step in the manufacture of equine-derived snake antivenoms and ten batches of finished product were analyzed. [Copyright &y& Elsevier]

Abstract: Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid fractionation followed by β-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemical characteristics and anti-Bothrops asper venom antibody titers, except that ion-exchange purified antivenom had a lower protein concentration. Antivenoms fractionated by using β-propiolactone or filtration through quaternary ammonium membrane had a significantly reduced in vitro ACA. A preparation of caprylic acid-fractionated antivenom was heated in order to induce the formation of protein aggregates; however, its ACA was similar to non-heated antivenom. None of the antivenoms affected the hemolytic activity of serum complement in rabbits after a bolus intravenous administration. It is concluded that (a) β-propiolactone and quaternary ammonium membranes significantly reduce in vitro ACA of caprylic acid-fractionated equine antivenom, and (b) the validity of in vitro ACA as a predictor of EAR needs to be reexamined in clinical and experimental studies, since it may not adequately predict in vivo complement activation by antivenoms. [Copyright &y& Elsevier]

Introduction: It is estimated that 2 000 snakebites occur in Panama every year, 70 % of which are inflicted by Bothrops asper. Objective: To determine the biochemical and toxicologic effects and to assess the immunochemical characteristics of a reference pool of B. asper venom representative of Panama. Methods: The reference venom was prepared as a homogeneous mixture of the venoms obtained from 78 adult snakes collected in four geographic areas of Panama. Enzymatic and toxicological activities were assessed. The electrophoretic pattern was studied by SDS-PAGE. Immunoreactivity of various antivenoms was analyzed by Western blot. Results: B. asper reference venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating, proteinase and phospholipase A2 activities. SDS-PAGE showed the presence of protein bands with molecular weights ranging from 8 to 70 kDa, with the presence of predominant bands at ≈ 15 kDa and ≈ 30 to 66 kDa, which likely correspond to phospholipases A2 and metalloproteinases, respectively. Immunoblotting showed a high degree of recognition by various antivenoms, especially by antivenoms from Colombia and Costa Rica. Conclusions: Following recommendations by the World Health Organization, this reference venom of B. asper of Panama will become a useful tool for the preclinical evaluation of antivenoms distributed in this country. [ABSTRACT FROM AUTHOR]

Snakebite envenoming is a relevant public health problem in French Guiana, and Bothrops atrox is responsible for the vast majority of envenomings in this overseas French territory. The preclinical efficacy of freeze-dried antivenoms manufactured in Costa Rica (Polival-ICP®) and Mexico (Antivipmyn Tri®) was assessed against the lethal, hemorrhagic, in vitro coagulant, and myotoxic effects of Bothrops atrox venom from French Guiana. Antivenoms differ in protein concentration and in the type of active principle (IgG and F (ab') 2 , respectively). Polival-ICP® showed significantly higher neutralizing activity against lethal, hemorrhagic and in vitro coagulant activities of the venom. Antivenoms neutralized myotoxic effect to a similar extent. In the case of lethal activity, Antivipmyn Tri® did not neutralize the effect at the highest antivenom level tested (1 mg venom/mL antivenom). Image 10573 • Polival-ICP and Antivipmyn Tri antivenoms were assessed against Bothrops atrox venom from French Guiana. • Only Polival ICP neutralized the lethal activity of the venom. • Both antivenoms neutralized hemorrhagic, in vitro coagulant and myotoxic effects of venom. • Neutralizing potency against hemorrhagic and in vitro coagulant effects was higher for Polival-ICP antivenom. [ABSTRACT FROM AUTHOR]

Enhancement of antivenom immune responses in horses through adjuvant technology improves antivenom production efficiency, but substantial local reactogenicity associated with some traditional veterinary adjuvants limits their usability. To explore modern adjuvant systems suitable for generating antivenom responses in horses, we first assessed their physicochemical compatibility with Bothrops asper snake venom. Liposome and nanoparticle aluminum adjuvants exhibited changes in particle size and phospholipid content after mixing with venom, whereas squalene emulsion-based adjuvants remained stable. Next, we evaluated serum antibody response magnitude and neutralization capacity in horses immunized with adjuvant-containing Echis ocellatus , Bitis arietans , Naja nigricollis , and Dendroaspis polylepis venom preparations. Whereas all tested adjuvants elicited significant neutralization capacity against the viperid venoms, the greatest antibody responses were generated by a squalene-in-water emulsion, thus representing a promising novel alternative for antivenom production. [Display omitted] • Lack of antivenoms with neutralizing activity against different snake venoms is a challenge to snake envenomation treatment. • Modern vaccine adjuvant technology has the potential to improve antivenom production. • Physicochemical compatibility of various adjuvant formulations with snake venom was characterized. • Immune responses elicited by selected adjuvant-containing venom preparations were evaluated in immunized horses. [ABSTRACT FROM AUTHOR]

Snake antivenoms are formulations of animal immunoglobulins used in the treatment of snakebite envenomation. The general scheme for producing snake antivenoms has undergone few changes since its development more than a century ago; however, technological innovations have been introduced in the manufacturing process. These medicines must comply with identity, purity, safety and efficacy profiles, as requested by the current Good Manufacturing Practices (GMPs) applied to modern biopharmaceutical drugs. Industrial production of snake antivenoms comprises several stages, such as: 1) production of reference venom pools, 2) production of hyperimmune plasma, 3) purification of the antivenom immunoglobulins, 4) formulation of the antivenom, 5) stabilization of the formulation, and 6) quality control of in-process and final products. In this work, a general review of the existing technology used for the industrial manufacture of snake antivenoms is presented. [ABSTRACT FROM AUTHOR]

Bothrops lanceolatus is an endemic viperid species in the Lesser Caribbean island of Martinique. Envenomings by this species are characterized by local and systemic effects, among which the development of thrombosis in various organs is the most severe complication. An experimental toxicological characterization of this venom was performed using in vivo mouse tests and various in vitro assays. The venom induced lethal, local and systemic hemorrhagic, edema-forming, myotoxic, thrombocytopenic, proteinase and phospholipase A 2 activities. The preclinical efficacy of a batch of monospecific Bothrofav ® antivenom currently in use in Martinique was assessed. The antivenom was highly effective in the neutralization of all activities tested, in agreement with its described clinical efficacy. This batch of antivenom showed a higher preclinical efficacy as compared to a previous batch used in the past. [ABSTRACT FROM AUTHOR]

Haemorrhage is a common clinical manifestation in envenomings caused by bites from snakes of the family Viperidae. Therefore, knowing the haemorrhagic potential of venoms and the capacity of antivenoms to neutralise this effect are of paramount relevance in toxinology. The most widely used method for quantifying haemorrhage involves the intradermal injection of venom (or a mixture of venom/antivenom) in mice, and the assessment of the resulting haemorrhagic area in the inner side of the skin. Although this method allows a straightforward assessment of the haemorrhagic activity of a venom, it does not account for haemorrhagic lesions having a similar area but differing in the depth and intensity of haemorrhage. We have developed an approach that allows the assessment of both area and intensity of a venom-induced haemorrhagic lesion using computational tools and propose a unit to represent the combination of these two factors as a measure of haemorrhage intensity, namely haemorrhagic unit (HaU). A strong correlation was observed between haemoglobin extracted from a haemorrhagic lesion and the associated HaUs. The method was used to determine the haemorrhagic activity of the venoms of Bothrops asper , Echis ocellatus and Crotalus basiliscus and the haemorrhage neutralising capabilities of the three associated antivenoms. Overall, the ease of use, as well as the time involved in this new method, makes its implementation very feasible in the determination of haemorrhagic activity of venoms and its neutralisation by antivenoms in the murine model. [ABSTRACT FROM AUTHOR]

Saw-scaled vipers (genus Echis ) are one of the leading causes of snakebite morbidity and mortality in parts of Sub-Saharan Africa, the Middle East, and vast regions of Asia, constituting a public health burden exceeding that of almost any other snake genus globally. Venom-induced consumption coagulopathy, owing to the action of potent procoagulant toxins, is one of the most relevant clinical manifestations of envenomings by Echis spp. Clinical experience and prior studies examining a limited range of venoms and restricted antivenoms have demonstrated for some antivenoms an extreme lack of antivenom cross-reactivity between different species of this genus, sometimes resulting in catastrophic treatment failure. This study undertook the most comprehensive testing of Echis venom effects upon the coagulation of human plasma, and also the broadest examination of antivenom potency and cross-reactivity, to-date. 10 Echis species/populations and four antivenoms (two African, two Asian) were studied. The results indicate that the venoms are, in general, potently procoagulant but that the relative dependence on calcium or phospholipid cofactors is highly variable. Additionally, three out of the four antivenoms tested demonstrated only a very narrow taxonomic range of effectiveness in preventing coagulopathy, with only the SAIMR antivenom displaying significant levels of cross-reactivity. These results were in conflict with previous studies using prolonged preincubation of antivenom with venom to suggest effective cross-reactivity levels for the ICP Echi-Tab antivenom. These findings both inform upon potential clinical effects of envenomation in humans and highlight the extreme limitations of available treatment. It is hoped that this will spur efforts into the development of antivenoms with more comprehensive coverage for bites not only from wild snakes but also from specimens widely kept in zoological collections. [ABSTRACT FROM AUTHOR]

EchiTAb + ICP is a pan-African antivenom used for the treatment of snakebite envenomation in rural sub-Saharan African communities, where the cold chain can be difficult to maintain. To develop a formulation of EchiTAb + ICP that can be distributed and stored without refrigeration, we submitted three different formulations of EchiTAb + ICP: control (i.e. liquid antivenom formulated without stabilizer), liquid antivenom stabilized with sorbitol, and freeze-dried antivenom formulated with sucrose, to an accelerated stability study (i.e. 38 ± 2 °C and 75% relative humidity for 6 months). We analyzed changes in color, residual humidity, reconstitution time (for freeze-dried preparation), pH, osmolality, total protein concentration, antibody monomers content, turbidity, bacterial endotoxins, and pre-clinical neutralizing efficacy of the lethal effect of Echis ocellatus venom at 0, 3 and 6 months. In the control formulation, instability was evidenced by the development of a yellow coloration and an increment in aggregation and turbidity, without change in its neutralizing activity. The sorbitol-stabilized formulation did not develop marked aggregation or turbidity, but instability was evidenced by the development of yellow coloration and a drop in the neutralizing potency. The freeze-dried formulation maintained its neutralizing potency and did not show marked signs of instability, thus indicating that freeze-drying could confer EchiTAb + ICP with improved thermal stability required for distribution and storage at room temperature in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Bothrops asper is the medically most important venomous snake in Central America. In Panama, the country having the highest incidence of snakebites in Latin America, B. asper is widely distributed throughout the country and is responsible for the vast majority of snakebites. This study was performed to analyze whether there are variations in the toxicological profile and in some biochemical parameters between the venoms of B. asper from four different regions in Panama. The venoms showed a similar profile of lethal, hemorrhagic, in vitro coagulant, defibrinogenating, edema-forming, myotoxic and indirect hemolytic activities, with subtle quantitative variations between samples of some regions. The venoms also had similar SDS-PAGE patterns and reverse phase HPLC profiles. A polyvalent antivenom manufactured in Costa Rica, and regularly used in Panama, was effective in the neutralization of lethal activity of the venoms of the four populations, with Mean Effective Doses (ED 50 ) ranging from 5.98 to 9.72 mg venom/mL antivenom. In agreement, a widespread pattern of cross-reactivity between this antivenom and the four venoms was observed by immunoblotting. Overall, results highlight the lack of marked differences between the venoms of the various populations of B. asper in Panama, and that the antivenom from Costa Rica is effective in neutralizing lethality. [ABSTRACT FROM AUTHOR]

The assessment of the preclinical neutralizing ability of antivenoms in Latin America is necessary to determine their scope of efficacy. This study was aimed at analyzing the neutralizing efficacy of a polyspecific bothropic-crotalic antivenom manufactured by BIRMEX in Mexico against lethal, hemorrhagic, defibrinogenating and in vitro coagulant activities of the venoms of Bothrops jararaca (Brazil), B. atrox (Perú and Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia), and B. asper (Costa Rica). Standard laboratory tests to determine these activities were used. In agreement with previous studies with bothropic antivenoms in Latin America, a pattern of cross-neutralization of heterologous venoms was observed. However, the antivenom had low neutralizing potency against defibrinogenating effect of the venoms of B. atrox (Colombia) and B. asper (Costa Rica), and failed to neutralize the in vitro coagulant activity of the venom of B. asper (Costa Rica) at the highest antivenom/venom ratio tested. It is concluded that, with the exception of coagulant and defibrinogenating activities of B. asper (Costa Rica) venom, this antivenom neutralizes toxic effects of various Bothrops sp venoms. Future studies are necessary to assess the efficacy of this antivenom against other viperid venoms. Rev. Biol. Trop. 65 (1): 345-350. Epub 2017 March 01. [ABSTRACT FROM AUTHOR]

Freeze-drying is a process used to improve the stability of pharmaceutical proteins, including snake antivenoms. This additional step confers these with a higher stability in comparison to liquid formulations, especially in tropical regions where high temperatures could affect the activity of immunoglobulins. Currently, the knowledge about freeze-drying process conditions for snake antivenoms is very limited. Some of the scarce scientific works on this subject reported reconstitution times up to 90 min for these preparations, which could imply a delay in the beginning of the antivenom therapy at the clinical setting. Therefore there is a reasonable concern about whether freeze-dried antivenoms exhibit the desired attributes for solid pharmaceutical proteins. In this work, a physicochemical characterization of seven commercial freeze-dried snake antivenoms was performed based on tests recommended by the World Health Organization (WHO). No significant differences were observed between the products regarding macroscopic appearance of the solid cakes, reconstitution times, residual humidity and monomers content. On the other hand, total protein concentration, turbidity and electrophoretic profile were different among samples. Microscopic analysis by scanning electron microscopy showed no collapsed structure and, instead, most of the samples showed a characteristic protein morphology composed of smooth plates and channels. All the parameters tested in this study were according to literature recommendations and evidenced that, in spite of slight variations found for some products, formulation and freeze-drying conditions chosen by manufacturers are adequate to prevent aggregation and generate, in physicochemical terms, freeze-dried antivenoms of acceptable quality. [ABSTRACT FROM AUTHOR]

A chicken-derived antivenom (ChDAv) towards taipan snake ( Oxyuranus scutellatus ) venom was produced by purifying anti-taipan IgY from egg yolks of hens immunized with taipan venom. The productivity, antivenomic profile, neutralization ability, pharmacokinetic properties and immunogenicity of the ChDAv were compared with those of an antivenom produced in horses (EDAv). We found that 382 eggs are required to produce the mass of anti-taipan antibodies contained in one liter of equine hyperimmune plasma, and that 63 chickens would be needed to generate the amount of anti-taipan antibodies annually produced by one horse. It was estimated that, in Costa Rica, the production of anti-taipan antibodies could be 40% cheaper if chickens were used as immunoglobulin source, instead of horses. During antivenomic assessment, ChDAv showed lower ability to immunocapture the α subunit of taipoxin, the most important neurotoxin in the venom. ChDAv showed a lower ability to neutralize the coagulant and lethal activities of taipan venom. ChDAv was more immunogenic in rabbits than EDAv, probably due to the fact that chickens are phylogenetically more distant to rabbits than horses. This finding may explain why clearance from rabbit bloodstream was faster for chicken-IgY than for equine-IgG in a pharmacokinetic study. In conclusion, the production of anti-taipan antivenom was less effective when chicken egg yolks were used as source of immunoglobulins instead of horses. [ABSTRACT FROM AUTHOR]

The pathogenesis of dermonecrosis induced by the venom of the African spitting cobra Naja nigricollis was investigated in a mouse model. Intradermal injection of venom induced a macroscopic necrotic lesion. Histological examination revealed early edema of the dermis, followed by blistering, loss of skin appendages and reduction in cellularity. By 24 h, necrosis of the dermis was evident, sections of epidermis were lost, and a fibrinoid hyaline material filled the damaged areas. Abundant inflammatory infiltrate was present in the hypodermis and basal dermis, and there was an increment in the expression of matrix metalloproteinases (MMPs). Thrombi were observed in blood vessels. Abundant cells were present in the dermis by 7 days. By 14 and 28 days, re-epithelization had occurred, collagen was widespread in the dermis, and few skin appendages were present. The RP-HPLC fractions that reproduced the necrotic activity were composed of low molecular mass cytotoxins of the three-finger toxin family and, to a lesser extent, of phospholipases A 2 (PLA 2 ). Inhibition of PLA 2 of venom by p -bromophenacyl bromide did not reduce the area of necrosis, but modified the appearance of necrotic regions. Depletion of neutrophils and inhibition of venom metalloproteinases and tissue MMPs did not affect dermonecrosis. IgG and F(ab′) 2 antivenoms were effective in the neutralization of dermonecrosis when incubated with venom prior to injection. However, when antivenoms were administered immediately after venom injection, dermonecrosis was reduced only to a partial extent, underscoring the difficulties in neutralizing this effect with antivenoms. [ABSTRACT FROM AUTHOR]

Two antivenoms prepared by using Echis ocellatus , Bitis arietans and Naja nigricollis venoms from different locations in sub-Saharan Africa were compared for their neutralizing ability. Both antivenoms were similarly effective in the neutralization of the venoms of the three species from different locations. However in the case of E. ocellatus venom, antivenom prepared using venom from Nigerian specimens was more effective than antivenom prepared with venom from Cameroon specimens in the neutralization of coagulant activity. [ABSTRACT FROM AUTHOR]

Protein composition, toxicity, and neutralization of the venoms of Micrurus alleni and Micrurus mosquitensis , two sympatric monadal coral snakes found in humid environments of the Caribbean region of Costa Rica, were studied. Proteomic profiling revealed that these venoms display highly divergent compositions: the former dominated by three-finger toxins (3FTx) and the latter by phospholipases A 2 (PLA 2 ). Protein family abundances correlated with enzymatic and toxic characteristics of the venoms. Selective inhibition experiments showed that PLA 2 s play only a marginal role in the lethal effect of M. alleni venom, but have a major role in M. mosquitensis venom. Proteomic data gathered from other Micrurus species evidenced that the two divergent venom phenotypes are recurrent, and may constitute a general trend across New World elapids. Further, M. mosquitensis , but not M. alleni , venom contains PLA 2 -like/Kunitz-type inhibitor complex(es) that resemble the ASIC1a/2-activating MitTx heterodimeric toxin isolated from Micrurus tener venom. The evolutionary origin and adaptive relevance of the puzzling phenotypic variability of Micrurus venoms remain to be understood. An antivenom against the PLA 2 -predominant Micrurus nigrocinctus venom strongly cross-recognized and neutralized M. mosquitensis venom, but only weakly M. alleni venom. [ABSTRACT FROM AUTHOR]

Snakebite envenoming causes a heavy toll in sub-Saharan Africa in terms of mortality and sequelae. In the West African savannah, the viperid Echis ocellatus is responsible for the vast majority of bites. In the last decades, several new antivenoms have been introduced for the treatment of these envenomings, although the assessment of their preclinical efficacy against the venom of E. ocellatus has been studied only for some of them. This work analyzed comparatively the ability of four antivenoms (FAV Afrique, EchiTAb G, EchiTAB-Plus-ICP ® , and Inoserp™ Panafricain) to neutralize lethal, hemorrhagic, and in vitro coagulant activities of the venoms of E. ocellatus from Mali, Cameroon, and Nigeria. In addition, an immunoaffinity chromatography antivenomic protocol was used to assess the ability of the four antivenoms to bind to the proteins of these venoms. Results showed that all the antivenoms were effective in the neutralization of the three effects investigated, and were able to immunocapture, completely or partially, the most abundant components in the E. ocellatus venoms from the geographical origins sampled. Our observations also highlighted quantitative differences between antivenoms in their neutralizing and antivenomics profiles, especially regarding neutralization of in vitro coagulant activity, suggesting that different doses of these antivenoms are probably needed for an effective treatment of human envenomings by this species. [ABSTRACT FROM AUTHOR]

The potency of antivenoms is assessed by analyzing the neutralization of venom-induced lethality, and is expressed as the Median Effective Dose (ED 50 ). The present study was designed to investigate the pathophysiological mechanisms responsible for lethality induced by the venom of Bothrops asper , in the experimental conditions used for the evaluation of the neutralizing potency of antivenoms. Mice injected with 4 LD 50 s of venom by the intraperitoneal route died within ∼25 min with drastic alterations in the abdominal organs, characterized by hemorrhage, increment in plasma extravasation, and hemoconcentration, thus leading to hypovolemia and cardiovascular collapse. Snake venom metalloproteinases (SVMPs) play a predominat role in lethality, as judged by partial inhibition by the chelating agent CaNa 2 EDTA. When venom was mixed with antivenom, there was a venom/antivenom ratio at which hemorrhage was significantly reduced, but mice died at later time intervals with evident hemoconcentration, indicating that other components in addition to SVMPs also contribute to plasma extravasation and lethality. Pretreatment with the analgesic tramadol did not affect the outcome of the neutralization test, thus suggesting that prophylactic (precautionary) analgesia can be introduced in this assay. Neutralization of lethality in mice correlated with neutralization of in vitro coagulant activity in human plasma. [ABSTRACT FROM AUTHOR]

Freeze-drying is used to improve the long term stability of pharmaceutical proteins. Sugars and polyols have been successfully used in the stabilization of proteins. However, their use in the development of freeze-dried antivenoms has not been documented. In this work, whole IgG snake antivenom, purified from equine plasma, was formulated with different concentrations of sorbitol, sucrose or mannitol. The glass transition temperatures of frozen formulations, determined by Differential Scanning Calorimetry (DSC), ranged between −13.5 °C and −41 °C. In order to evaluate the effectiveness of the different stabilizers, the freeze-dried samples were subjected to an accelerated stability test at 40 ± 2 °C and 75 ± 5% relative humidity. After six months of storage at 40 °C, all the formulations presented the same residual humidity, but significant differences were observed in turbidity, reconstitution time and electrophoretic pattern. Moreover, all formulations, except antivenoms freeze-dried with mannitol, exhibited the same potency for the neutralization of lethal effect of Bothrops asper venom. The 5% (w:v) sucrose formulation exhibited the best stability among the samples tested, while mannitol and sorbitol formulations turned brown. These results suggest that sucrose is a better stabilizer than mannitol and sorbitol in the formulation of freeze-dried antivenoms under the studied conditions. [ABSTRACT FROM AUTHOR]

The toxicological profile of the venoms of the snakes Bothrops asper and Bothrops atrox from Ecuador was investigated, together with the venom of a population of B. asper formerly classified as ‘ Bothrops xanthogrammus’ . The three venoms exerted lethal, hemorrhagic, myotoxic, coagulant and defibrinogenating effects, in agreement with the characteristic toxicological profile of Bothrops sp venoms. A polyspecific antivenom (bothropic–crotalic–lachesic) manufactured in Costa Rica was assessed for its preclinical efficacy against the toxic activities of these Ecuadorian venoms. Antivenom was effective in the neutralization of the five activities tested in the three venoms. These observations are in agreement with previous reports on the extensive cross-reactivity and paraspecific neutralization of antivenoms manufactured in Latin America against the venoms of Bothrops sp snakes. [ABSTRACT FROM AUTHOR]

Abstract: Antivenom pharmacokinetics has been studied in heterologous models in which the animal species used as immunoglobulin source is different from that used as recipient. In these models, after intravenous administration of antivenom, the plasma concentration of immunoglobulins shows a rapid initial declining-phase followed by a slower terminal-phase, which has been associated with antivenom distribution and elimination, respectively. We have compared pharmacokinetic parameters for equine-derived antivenom in homologous (horse) and heterologous (cow) models. It was found that the maximum concentration is lower in cows than in horses. Additionally, the steady-state distribution volume is higher in cows as compared to horses. On the other hand, models were not different in the time required to reach the maximum concentration, the area under the concentration/time curve, the half-life of decay during the slowest phase, the systemic clearance and the mean residence time. Similar results were obtained in a rabbit model, in which the pharmacokinetics was also affected by passive immunization of rabbits with anti-equine IgG. We conclude that, in addition to other physiological differences (e.g. cardiac frequency, plasmatic volume, glomerular filtration rate, etc.) between animal models, the ability to remove foreign immunoglobulins might influence the way in which the plasma concentration of antivenom decreases over time, thereby distorting the pharmacokinetic predictions based on non-compartmental models. [Copyright &y& Elsevier]

Abstract: Purity is a characteristic that, together with effectiveness and safety, must be tested to determine the quality of biopharmaceutical products. In therapeutic immunoglobulins, such as human intravenous immunoglobulin (IVIG), purity is evaluated on the basis of physicochemical properties, and is usually assessed by chromatography and electrophoresis. However, in the case of antivenoms these methods fail to discriminate between antibodies towards venom antigens, which constitute the active substance, and antibodies towards non-venom antigens, which are the major impurities in most of the current formulations. The assessment of this aspect of purity requires the use of the immunochemical methods. In this study, it was demonstrated that antivenoms showing physicochemical purity higher than 90% might present immunochemical purity lower than 40%. It is proposed that a comprehensive analysis of antivenom purity should combine physicochemical and immunochemical parameters. In addition, these results are crucial to decide the more appropriate strategies to improve antivenom purity. Taking into account that the current methods of antivenom purification remove most non-antibodies proteins, we propose that efforts must be primarily directed to the improvement of immunization protocols to enhance the antibody response towards venom components in hyperimmunized animals, and secondarily, in the realm of immunoglobulin purification technology. [Copyright &y& Elsevier]

Abstract: The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A2 (PLA2), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA2s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey. [Copyright &y& Elsevier]

Abstract: Low pH treatment improves the tolerance to intravenous infusion, the stability, and the viral safety of various therapeutic immunoglobulins G preparations, but has never been evaluated for horse plasma-derived antivenoms. We have studied the impact of low pH formulation on the quality, safety, stability, potency and viral inactivation of a whole IgG antivenom used to treat viperid snake bite envenoming. Horse plasma-derived whole immunoglobulins purified by caprylic acid were incubated for 24 h at low pH in the presence of 4% sorbitol, then sterile-filtered and stored liquid at 2–8 °C. Appearance, aggregates, purity, safety tests in mice, venom antibody titre, and neutralization potency tests were controlled. Low pH treatment did not affect the physico-chemical characteristics, safety and potency of antivenom for at least 6 months of storage, but a major increase in aggregates was observed. In vitro antibody titre and in vivo neutralizing potency were maintained. There were ≥ 5.5 log inactivation of Herpes Simplex Virus-1, an enveloped virus, but no significant inactivation of the non-enveloped Poliovirus type 3. Low pH treatment appears feasible to improve the viral safety of antivenoms without affecting the neutralization potency. The possibility to formulate antivenoms at low pH requires further investigations to avoid formation of aggregates. [Copyright &y& Elsevier]