Review of North American pit viper antivenoms.

PURPOSE: For the first time in nearly 20 years, 2 antigen-binding fragment (Fab) antivenoms are available to treat patients who incur North American pit viper snakebites: Crotalidae polyvalent immune Fab (ovine), or simply FabAV; and Crotalidae immune F(ab')2 (equine), or simply F(ab')2. Pharmacists are in a key position for the selection, dosing, reconstitution, administration, and monitoring of antivenom therapy; however, they encounter inconsistent exposure and experience with these drugs. Thus, an updated review of the literature is necessary. METHODS: The search strategy and selection incorporated both controlled vocabulary terms and keywords to describe concepts relevant to the search. Retrieval was limited to literature published from 1997 to the present in English, Portuguese, or Spanish. RESULTS: Given the paucity of available prospective literature, the authors elected to include all prospective evidence to best describe the role of antivenom. For the primary literature review, manuscripts were excluded if they were observational studies, conference abstracts, narrative or opinion articles, letters to the editor, or in-progress studies. CONCLUSION: While there is limited evidence-based guidance on the superiority of F(ab')2 to FabAV, or vice versa, individual and regional considerations should contribute to formulary decisions. Pharmacists must play a role in the development of clinical pathways to ensure appropriate evaluation, supportive care, and antivenom procurement, administration, and monitoring.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper.

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.

Assessment of the anti-snakebite properties of extracts of Aniba fragrans Ducke (Lauraceae) used in folk medicine as complementary treatment in cases of envenomation by Bothrops atrox.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of leaves and bark of Aniba fragrans are used as tea (decoction) to treat snakebites in communities in the Brazilian Amazon. The aqueous extract of the leaves of A. fragrans has been proven to be effective against Bothrops venom, but only when pre-incubated with the venom. This study sought to assess the potential of different types of extract of this species to inhibit the biological activities of Bothrops atrox venom (BaV) when used the same way as in folk medicine. The main classes of secondary metabolites and the concentrations of phenolics in the extracts were also determined. MATERIALS AND METHODS: Four types of extract of A. fragrans were prepared: aqueous extract of the leaf (AEL), aqueous extract of the bark (AEB), hydroalcoholic leaf extract (HLE) and extract of the residue from hydrodistillation of the leaf (ERHL). The phytochemical profiles of the aqueous extracts were determined using thin layer chromatography (TLC), and the concentrations of phenolics were measured by colorimetric assays. To investigate the potential of the extracts to inhibit the biological activities of BaV, in vitro tests for antiphospholipase and antifibrinolytic activities were performed. In vivo tests for antihemorrhagic and antidefibrinating activities were also carried out, as well as antimicrobial tests for activity against the main bacteria found in the oral cavity of snakes. Interaction between the extracts and the proteins in BaV was assessed by electrophoresis (SDS-PAGE) and Western blot (WB). The cytotoxicity of the extracts was assessed in a strain of MRC-5 human fibroblasts. RESULTS: Terpenoids, flavonoids and condensed and hydrolysable tannins were detected in all the extracts. Metabolites such as coumarins, fatty acids and alkaloids were present in some extracts but not in others, indicating different phytochemical profiles. Phenolics content varied between extracts, and there were more tannins in AEB and HLE. In the in vitro tests, the extracts inhibited the phospholipase and fibrinolytic activities of BaV in the two ratios of venom to extract used. HLE exhibited effective antimicrobial action as it inhibited growth of 11 of the 15 bacteria investigated, including Morganella morganii, the main bacteria described in the oral cavity of snakes. The extracts failed to inhibit the defibrinating activity of BaV, and only the Bothrops antivenom had a significant effect (96.1%) on this activity. BaV-induced hemorrhage was completely inhibited by AEL and AEB when the pre-incubation (venom:extract) protocol was used. When administered orally, as in folk medicine, both AEB and AEL produced significant inhibition of hemorrhagic activity (maximum inhibition 46.5% and 39.2%, respectively). SDS-PAGE and WB of the extracts pre-incubated with BaV showed that the main proteins in the venom had been precipitated by the extracts. None of the four extracts showed cytotoxic effects in the tests carried out with a human fibroblast cell line. CONCLUSION: In addition to being effective in reducing hemorrhage when administered orally, the extracts displayed a high antimicrobial potential against microorganisms involved in secondary infections at the site of the snakebite. Once the extracts have been tested in accordance with the appropriate regulations, this species could potentially be used to produce a phytomedicine for complementary treatment of the secondary infections due to bacteria that aggravate the local signs and symptoms after snakebite envenomation.

Inhibition of local effects induced by Bothrops erythromelas snake venom: Assessment of the effectiveness of Brazilian polyvalent bothropic antivenom and aqueous leaf extract of Jatropha gossypiifolia.

Bothrops erythromelas is a snake of medical importance responsible for most of the venomous incidents in Northeastern Brazil. However, this species is not included in the pool of venoms that are used in the Brazilian polyvalent bothropic antivenom (BAv) production. Furthermore, it is well known that antivenom therapy has limited efficacy against venom-induced local effects, making the search for complementary alternatives to treat snakebites an important task. Jatropha gossypiifolia is a medicinal plant widely indicated in folk medicine as an antidote for snakebites, whose effectiveness against Bothrops jararaca venom (BjV) has been previously demonstrated in mice. In this context, this study assessed the effectiveness of the aqueous extract (AE) of this plant and of the BAv against local effects induced by B. erythromelas venom (BeV). Inhibition of BeV-induced edematogenic and hemorrhagic local effects was assayed in mice in pre-treatment (treatment prior to BeV injection) and post-treatment (treatment post-envenomation) protocols. Inhibition of proteolytic, phospholipase A2 (PLA2) and hyaluronidase enzymatic activities of BeV were evaluated in vitro. BAv cross-reactivity and estimation of antibody titers against BeV and BjV were assessed by Ouchterlony double diffusion test. The results show that in pre-treatment protocol AE and BAv presented very similar effects (about 70% of inhibition for edematogenic and 40% for hemorrhagic activities). However, BAv poorly inhibited edema and hemorrhage in post-envenomation protocol, whilst, in contrast, AE was significantly active even when used after BeV injection. AE was able to inhibit all the tested enzymatic activities of BeV, while BAv was active only against hyaluronidase activity, which could justify the low effectiveness of BAv against BeV-induced local effects in vivo. Ouchterlony's test showed positive cross-reactivity against BeV, but the antibody titers were slightly higher against BjV. Together, these data indicate that despite the presence of immunological cross-reactivity, Brazilian polyvalent bothropic antivenom presented low inhibitory potential against biological and enzymatic effects of BeV, illustrating the need for new strategies in the production of antivenom with broad neutralizing potential in the treatment of Bothrops spp. envenomation throughout the country. Together, the results highlight the antiophidic potential of J. gossypiifolia, suggesting that it can be considered a potential adjuvant in the treatment of bothropic envenomation local effects.

Plants used to treat snakebites in Santarém, western Pará, Brazil: an assessment of their effectiveness in inhibiting hemorrhagic activity induced by Bothrops jararaca venom.

ETHNOPHARMACOLOGICAL RELEVANCE: The poor distribution and limited availability of antivenoms in Brazil have led to greater use of plants to treat snakebites. Very often such plants are the only alternative available to riverside communities. MATERIALS AND METHODS: Direct questionnaire-based interviews were conducted with members of the Cucurunã, São Pedro and Alter do Chão communities in Santarém, Pará, Brazil. For each of the 12 most frequently mentioned species aqueous extracts were prepared and the phytochemical profiles determined by thin layer chromatography. The concentrations of phenolic compounds (tannins and flavonoids) in the aqueous extracts were determined by colorimetric assays. To assess inhibition of the hemorrhagic activity of Bothrops jararaca venom, solutions containing the venom mixed with aqueous extracts in the ratios 1:12 and 1:48 were tested (w/w). SDS-PAGE and Western blot were used to assess the action of the extracts on Bothrops jararaca venom. RESULTS: In all, 24 plants belonging to 19 families were mentioned in the survey as being used to treat snakebites. Leaves (84%), seeds (60.9%) and inner bark (53%) were cited as the most frequently used parts in folk remedies, which were usually prepared in the form of a decoction (62.5%), tincture (45%) or maceration (22.5%). Hemorrhage induced by Bothrops jararaca venom was completely inhibited by aqueous extracts of Bellucia dichotoma, Connarus favosus, Plathymenia reticulata and Philodendron megalophyllum, which had a high phenolic content and contained condensed and hydrolyzable tannins. The results of SDS-PAGE showed that some venom protein bands were not visible when the venom was preincubated with the extracts that had completely inhibited hemorrhagic activity of the venom. Western blot showed that the extracts did not have any enzymatic action on the proteins in the venom as it failed to detect low-molecular-weight bands, which are indicative of possible enzymatic cleavage. CONCLUSIONS: Traditional use of plants to treat snakebites is a common practice in the western region of Pará, Brazil. Our findings show that some plant extracts were able to inhibit snake venom-induced hemorrhage in vitro. In vivo studies are being carried out to validate the traditional use of these species to treat snakebites.

Evidence-based, multidisciplinary approach to the development of a crotalidae polyvalent antivenin (CroFab) protocol at a university hospital.

BACKGROUND: Several thousand people are bitten annually by venomous snakes in the US. While the development of ovine Crotalidae polyvalent immune Fab antivenin (FabAV) for Crotalinae snakebite envenomations has greatly changed the way this clinical presentation is treated, multiple issues complicate its use. From patient assessment and evaluation, to medication preparation and administration, to the management of adverse drug reactions, the use of this antidote carries with it multiple points of possible medication variances. The inappropriate use of this agent can result in adverse patient consequences and a significant financial burden for both the hospital and the patient. OBJECTIVE: To describe an evidence-based, multidisciplinary approach that was taken to ensure optimal, safe, and cost-effective treatment of patients with FabAV. METHODS: Following an analysis of the available literature, a multidisciplinary committee was formed to construct a protocol for use of FabAV. This group included clinical pharmacists, pharmacy administrators, emergency medicine physicians who specialized in wilderness medicine and pharmacy residents. RESULTS: A multidisciplinary FabAV usage protocol was constructed and implemented to ensure appropriate patient evaluation, FabAV use and preparation, monitoring, and follow-up. This protocol was based on the available literature and the expert opinion of the committee. Through the use of a 24-hour in-house pharmacy resident on-call system, clinical pharmacy services were provided to ensure a multidisciplinary approach to the care of these patients emergently. Although limited, initial data show that this approach is effective and may result in substantial cost savings. CONCLUSIONS: Initial results from implementation of a protocol for use of FabAV have limited inappropriate use, reduced medication wastage, and decreased costs.

Contribution of mast cells to the oedema induced by Bothrops moojeni snake venom and a pharmacological assessment of the inflammatory mediators involved.

The ability of Bothrops moojeni venom (BmV) to induce oedema in mice, the involvement of principal inflammatory mediators and mast cells (MCs) were investigated. The intraplantar injection of BmV (0.3-6 microg/paw) caused a dose- and time-dependent oedema with a peak between 30 and 60 min after venom injection (0.3-1 microg/paw), disappearing within 24h. Either MCs granule inhibition or depletion by cromoglycate or C48/80, respectively, markedly reduced BmV-induced oedema. MCs depletion by imatinib also reduced oedema. Intraperitoneal BmV injection (2.5-10 microg/site) induced MCs degranulation and release of PGD(2). Treatment with promethazine, cimetidine or thioperamide, histamine H1, H2 and H3/H4 receptor antagonists, respectively, markedly reduced the initial phase of oedema. Combined treatment with these antagonists further reduced, but not abrogated oedema. Indomethacin or eterocoxib (cyclooxygenase inhibitors) reduced oedema until 180 min, whereas zileuton (lipoxygenase inhibitor) affected this event until 60 min. Dexamethazone caused a long lasting reduction of oedema. However, L-NAME and aminoguanidine (NO synthase inhibitors) significantly increased BmV-induced oedema. In conclusion, BmV induces oedema, mediated by MCs degranulation, histamine by H1, H2, H3/H4 receptors, prostaglandins and leukotrienes, and down-regulated by NO. Partial neutralization of oedema was observed even when polyspecific bothropic antivenom was injected immediately after venom.

Immunological assessment of mice hyperimmunized with native and cobalt-60-irradiated Bothrops venoms

ELISA was used to evaluate, accompany, and compare the humoral immune response of Swiss mice during hyperimmunization with native and Cobalt-60-irradiated (60Co) venoms of Bothrops jararaca, Bothrops jararacussu and Bothrops moojeni. Potency and neutralization were evaluated by in vitro challenges. After hyperimmunization, immunity was observed by in vivo challenge, and the side effects were assessed. The animals immunization with one LD50 of each venom occurred on days 1, 15, 21, 30, and 45, when blood samples were collected; challenges happened on the 60th day. Results showed that ELISA was efficient in evaluating, accompanying and comparing mouse immune response during hyperimmunization. Serum titers produced with natural venom were similar to those produced with irradiated venom. Immunogenic capacity was maintained after 60Co-irradiation. The sera produced with native venom showed neutralizing potency and capacity similar to those of the sera produced with irradiated venom. All antibodies were able to neutralize five LD50 from these venoms. Clinical alterations were minimum during hyperimmunization with irradiated venom, however, necrosis and death occurred in animals inoculated with native venom

Immunization with native and cobalt 60-irradiated Crotalus durissus terrificus venom in swiss mice: assessment of the neutralizing potency of antisera

ELISA was used to evaluate, follow, and compare the humoral immune response of Swiss mice during hyperimmunization with natural and Cobalt 60-irradiated (60Co) Crotalus durissus terrificus venom. Potency and neutralization were evaluated by in vitro challenges. After hyperimmunization, immunity was observed by “in vivo” challenge and the side effects were assessed. The animals immunization with one LD50 of the venom was on days one, 15, 21, 30, and 45, when blood samples were collected; the challenges occurred on the 60th day. Results showed that ELISA was efficient in evaluating, following, and comparing mouse immune response during hyperimmunization. Serum titers produced with natural venom were similar to those produced with irradiated venom. Immunogenic capacity was maintained after 60Co irradiation. Serum produced from Crotalus durissus terrificus irradiated venom showed higher potency and neutralization capacity than that from natural venom. All antibodies were able to neutralize five LD50 from these venoms. Clinical alterations were minimum during hyperimmunization with irradiated venom

Role of TNF-alpha, IL-1beta and IL-6 in the local tissue damage induced by Bothrops asper snake venom: an experimental assessment in mice.

The role of the cytokines TNF-alpha, IL-1beta and IL-6 in the acute local pathological effects induced by Bothrops asper snake venom was assessed in mice. Intramuscular injection of this venom induced increments in IL-1beta and IL-6 in muscle, but no elevations of TNF-alpha were detected. Pentoxifylline (PTX), a methylxanthine derivative that inhibits the synthesis of TNF-alpha, and antibodies against these three cytokines were used to assess the role of these cytokines in venom-induced effects. As a control, PTX pretreatment was effective at abrogating lethality and serum TNF-alpha increments in mice subjected to endotoxemia induced by injection of Escherichia coli lipopolysaccharide, although it did not affect the increment in IL-1beta and IL-6 in such endotoxic model. PTX failed to reduce lethality, hemorrhage, myonecrosis, dermonecrosis and edema induced by B. asper venom. Moreover, pretreatment with anti-cytokine antibodies was also ineffective at reducing venom-induced myonecrosis and hemorrhage. It is concluded that TNF-alpha, IL-1beta and IL-6 do not have a significant role in the pathogenesis of the acute local pathological effects induced by B. asper venom in mice, although this does not exclude the possibility that these cytokines play a role in other aspects of venom-induced local pathology, as well as in the reparative and regenerative responses that take place after the onset of tissue damage.

Neutralization of four Peruvian Bothrops sp. snake venoms by polyvalent antivenoms produced in Perú and Costa Rica: preclinical assessment.

Envenomations after bites inflicted by snakes of the genus Bothrops constitute a public health hazard in Perú, and the intravenous administration of equine-derived antivenoms represents the only scientifically validated treatment. This study presents a preclinical assessment of the efficacy of two whole IgG antivenoms, prepared in Perú and Costa Rica, to neutralize the most relevant toxic effects induced by the venoms of Bothrops atrox, B. brazili, B. barnetti and B. pictus from Perú. Peruvian antivenom is produced by immunizing horses with Bothrops sp. venoms from this country, whereas the production of Costa Rican antivenom involves immunization with venoms from Central American snakes. The neutralization of lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating activities was evaluated in assays involving incubation of venom and antivenom prior to testing. Both antivenoms were effective in the neutralization of these effects, with quantitative variations in the values of effective dose 50% depending on the effects being studied. Peruvian antivenom was more effective in the neutralization of lethality induced by B. atrox and B. barnetti venoms. However, Peruvian antivenom failed to neutralize coagulant activity of B. barnetti venom and edema-forming activity of B. brazili venom, whereas neutralization was achieved by Costa Rican antivenom. It is concluded that an extensive immunological cross-reactivity exists between Bothrops sp. venoms from Perú and Costa Rica, and that both antivenoms are effective in the neutralization of these four venoms in a rodent model of envenoming.

Preclinical assessment of the ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa Rica to neutralize the venoms of North American snakes.

Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picado, Costa Rica, were assessed for their ability to neutralize various toxic activities of the venoms of North American snakes of the genera Crotalus, Agkistrodon and Micrurus, in assays involving preincubation of venom and antivenom. When the intraperitoneal route of injection was utilized, polyvalent (Crotalinae) antivenom was effective in the neutralization of the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus viridis viridis, Crotalus horridus atricaudatus, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus, whereas the venom of Crotalus scutulatus was not neutralized. When the intravenous route was used, results differed depending on the "challenge dose" of venom employed. Polyvalent antivenom neutralized all venoms when mice were challenged with 2 LD(50)s of venom. When 5 LD(50)s were used, antivenom neutralized the venoms of C. atrox, C. adamanteus, C. v. viridis and C. h. atricaudatus, being ineffective in the neutralization of C. scutulatus, A. c. contortrix and A. p. piscivorus. Polyvalent antivenom was effective in the neutralization of hemorrhagic and myotoxic activities of all venoms studied. It also neutralized coagulant activity of C. adamanteus venom, whereas most of the venoms were devoid of clotting activity on plasma in vitro. Moreover, it neutralized defibrinating activity of the only three venoms that induced this effect (i.e. C. adamanteus, A. c. contortrix and A. p. piscivorus). Anticoral (Elapidae) antivenom neutralized lethality induced by the venom of Micrurus fulvius, using either the intravenous or the intraperitoneal routes of injection. Moreover, it neutralized myotoxic effect of this venom as well. It is concluded that polyvalent antivenom neutralizes lethality and other activities of most of the crotaline venoms tested. However, since it is ineffective in neutralizing the lethal effect of C. scutulatus venom, it is suggested that a venom containing presynaptically-active neurotoxic phospholipases A(2) related to "mojave toxin" needs to be introduced in the immunizing mixture in order to increase the neutralizing scope of this product in North America. Anticoral antivenom is highly effective in the neutralization of the venom of M. fulvius.