Guillermo León

Pharmacokinetic-Pharmacodynamic Relationships of Immunoglobulin Therapy for Envenomation

Parenteral administration of horse- and sheep-derived antivenoms constitutes the cornerstone in the therapy of envenomations induced by animal bites and stings. Depending on the type of neutralising molecule, antivenoms are made of: (i) whole IgG molecules (150 kDa), (ii) F(ab′)2 immunoglobulin fragments (100 kDa) or (iii) Fab immunoglobulin fragments (50 kDa). Because of their variable molecular mass, these three types of antivenoms have different pharmacokinetic profiles. Fab fragments have the largest volume of distribution and readily reach extravascular compartments. They are catabolised mainly by the kidney, having a more rapid clearance than F(ab′)2 fragments and IgG. On the other hand, IgG molecules have a lower volume of distribution and a longer elimination half-life, showing the highest cycling through the interstitial spaces in the body. IgG elimination occurs mainly by extrarenai mechanisms. F(ab′)2 fragments display a pharmacokinetic profile intermediate between those of Fab fragments and IgG molecules.Such diverse pharmacokinetic properties have implications for the pharmacodynamics of these immunobiologicals, since a pronounced mismatch has been described between the pharmacokinetics of venoms and antivenoms. Some venoms, such as those of scorpions and elapid snakes, are rich in low-molecular-mass neurotoxins of high diffusibility and large volume of distribution that reach their tissue targets rapidly after injection. In contrast, venoms rich in high-molecular-mass toxins, such as those of viperid snakes, have a pharmacokinetic profile characterised by a rapid initial absorption followed by a slow absorption process from the site of venom injection. Such delayed absorption has been linked with recurrence of envenomation when antibody levels in blood decrease.This heterogeneity in pharmacokinetics and mechanism of action of venom components requires a detailed analysis of each venom-antivenom system in order to determine the most appropriate type of neutralising molecule for each particular venom. Besides having a high affinity for toxicologically relevant venom components, an ideal antivenom should possess a volume of distribution as similar as possible to that of the toxins being neutralised. Moreover, high levels of neutralising antibodies should remain in blood for a relatively prolonged time to assure neutralisation of toxins reaching the bloodstream later in the course of envenomation, and to promote redistribution of toxins from extravascular compartments to blood. Additional studies are required on different venoms and antivenoms in order to further understand the pharmacokinetic-pharmacodynamic relationships of antibodies and their fragments and to optimise the immunotherapy of envenomations.

A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms.

A randomized blinded clinical trial was performed in 53 patients bitten by Bothrops sp. and Porthidium sp. in Antioquia and Chocó, Colombia, in order to compare the efficacy and safety of two antivenoms made of whole IgG obtained by either ammonium sulphate (monovalent anti-B. atrox) or caprylic acid (polyvalent) fractionation. Additionally, antivenoms were compared by electrophoretic and chromatographic analyses and anticomplementary activity in vitro. With a protocol of 2, 4 and 6 antivenom vials for the treatment of mild, moderate and severe envenomings, respectively, both antivenoms were equally efficient to neutralize the most relevant signs of envenoming and to clear serum venom levels in patients from the first hour and later on. Three patients with severe envenoming and initially treated with less than six vials on admission had persistent or recurrent venom antigenemia within 12-48 h. Monovalent antivenom fractionated by ammonium sulphate precipitation had higher amounts of protein aggregates and nonimmunoglobulin proteins than polyvalent antivenom fractionated by caprylic acid precipitation. Both antivenoms presented anticomplementary activity in vitro, being higher in the monovalent product. In agreement, monovalent antivenom induced a significantly higher incidence of early antivenom reactions (52%) than polyvalent antivenom (25%).

Snake Venomics of African Spitting Cobras: Toxin Composition and Assessment of Congeneric Cross-Reactivity of the Pan-African EchiTAb-Plus-ICP Antivenom by Antivenomics and Neutralization Approaches

Venomic analysis of the venoms of Naja nigricollis, N. katiensis, N. nubiae, N. mossambica, and N. pallida revealed similar compositional trends. The high content of cytotoxins and PLA(2)s may account for the extensive tissue necrosis characteristic of the envenomings by these species. The high abundance of a type I α-neurotoxin in N. nubiae may be responsible for the high lethal toxicity of this venom (in rodents). The ability of EchiTAb-Plus-ICP antivenom to immunodeplete and neutralize the venoms of African spitting cobras was assessed by antivenomics and neutralization tests. It partially immunodepleted 3FTx and PLA(2)s and completely immunodepleted SVMPs and CRISPs in all venoms. The antivenom neutralized the dermonecrotic and PLA(2) activities of all African Naja venoms, whereas lethality was eliminated in the venoms of N. nigricollis, N. mossambica, and N. pallida but not in those of N. nubiae and N. katiensis. The lack of neutralization of lethality of N. nubiae venom may be of medical relevance only in relatively populous areas of the Saharan region. The impaired activity of EchiTAb-Plus-ICP against N. katiensis may not represent a major concern. This species is sympatric with N. nigricollis in many regions of Africa, although very few bites have been attributed to it.

Trends in Snakebite Envenomation Therapy: Scientific, Technological and Public Health Considerations

The therapy of snakebite envenomation has been based on the parenteral administration of animal-derived antivenoms. Despite the success of this treatment at reducing the impact of snakebite mortality and morbidity, mostly due to their capacity to neutralize systemically-acting toxins, antivenoms are of relatively low efficacy in the prevention of venom-induced local tissue damage, which often leads to permanent disability. The issue of safety also remains a concern, particularly for some antivenoms which induce a relatively high incidence of adverse reactions. Consequently, there is a need to improve the therapy of snakebite envenomations on the following lines: (a) the technologies to produce antivenoms require improvements aimed at obtaining more refined preparations of higher efficacy and safety, while being affordable for the public health systems of developing countries. (b) The growing knowledge on the biochemistry and toxicology of snake venoms should pave the way for the identification of natural and synthetic inhibitors of venom toxins, particularly of those involved in local tissue pathology. Such inhibitors might become a highly effective therapeutic tool for the abrogation of venom-induced local tissue damage. (c) A better knowledge of the inflammatory events secondary to venom actions may open the possibility of modulating such response, in order to prevent further tissue damage and to promote successful tissue repair and regeneration. A global partnership, involving many participants and combining scientific, technological and public health actions, is required to achieve a leap forward in the treatment of snakebite envenomations world-wide.

Antivenoms for the treatment of snakebite envenomings: The road ahead

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.

Comparative study on the ability of IgG and Fab sheep antivenoms to neutralize local hemorrhage, edema and myonecrosis induced by Bothrops asper (terciopelo) snake venom

The ability of sheep antivenoms, consisting of whole IgG molecules or Fab fragments, to neutralize local hemorrhage, edema and myonecrosis induced by Bothrops asper venom was comparatively studied in mice. The two antivenoms were produced from the same batch of hyperimmune plasma and were adjusted to the same neutralizing potency against these effects in assays where venom and antivenoms were incubated prior to injection. Thus, if differences are observed in experiments involving independent injection of venom and antivenoms, they would depend on the pharmacokinetic profiles of the products. Despite the observation that both antivenoms neutralized the three effects if preincubated with venom, neutralization was only partial when antivenoms were administered i.v. at various time intervals after envenomation. No significant differences were observed between IgG and Fab antivenoms concerning neutralization of hemorrhagic and edema-forming activities, whereas IgG antivenom was slightly more effective in neutralizing myotoxic activity in experiments involving independent injection of venom and antivenom. These results do not support the hypothesis that Fab fragments are more effective than whole IgG molecules in the neutralization of locally-acting toxins from B. asper venom.

Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries against medically-relevant Bothrops snake venoms.

Species of the genus Bothrops induce the vast majority of snakebite envenomings in Latin America. A preclinical study was performed in the context of a regional network of public laboratories involved in the production, quality control and development of antivenoms in Latin America. The ability of seven polyspecific antivenoms, produced in Argentina, Brazil, Peru, Bolivia, Colombia and Costa Rica, to neutralize lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities of the venoms of Bothrops neuwiedi (diporus) (Argentina), Bothrops jararaca (Brazil), B. neuwiedi (mattogrossensis) (Bolivia), Bothrops atrox (Peru and Colombia) and Bothrops asper (Costa Rica) was assessed using standard laboratory tests. Despite differences in the venom mixtures used in the immunization of animals for the production of these antivenoms, a pattern of extensive cross-neutralization was observed between these antivenoms and all the venoms tested, with quantitative differences in the values of effective doses. This study reveals the capacity of these antivenoms to neutralize, in preclinical tests, homologous and heterologous Bothrops venoms in Central and South America, and also highlight quantitative differences in the values of Median Effective Doses (ED50s) between the various antivenoms.

Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP) for the treatment of viper envenoming in sub-Saharan Africa

A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region.

Immune response towards snake venoms.

The immune response involves a complex repertoire of innate and adaptive responses to foreign agents in the organism. The present review focuses on the immune response to snake venoms, including those occurring in snakebite accidental envenomation, experimental vaccination and animal hyperimmunization for snake antivenom production. The following aspects are considered: (a) the structural characteristics of snake toxins and their relationship to immunogenicity, (b) the effects that factors such as administration route, venom dose, type of adjuvant, and individual and species characteristics of the immunized animal have on the immune response, (c) the initial venom-induced inflammatory response, (d) the process by which specific antibodies towards individual toxins are produced, and (e) the techniques currently used to evaluate the antibody response. Understanding the immune response to snake venoms is highly relevant for improving antivenom production and for gaining a more complete view of snakebite envenoming.

Impact of Regional Variation in Bothrops asper Snake Venom on the Design of Antivenoms: Integrating Antivenomics and Neutralization Approaches

Intraspecific snake venom variations have implications in the preparation of venom pools for the generation of antivenoms. The impact of such variation in the cross-reactivity of antivenoms against Bothrops asper venom was assessed by comparing two commercial and four experimental antivenoms. All antivenoms showed similar immunorecognition pattern toward the venoms from adult and neonate specimens. They completely immunodepleted most P-III snake venom metalloproteinases (SVMPs), l-amino acid oxidases, serine proteinases, DC fragments, cysteine-rich secretory proteins (CRISPs), and C-type lectin-like proteins, and partially immunodepleted medium-sized disintegrins, phospholipases A(2) (PLA(2)s), some serine proteinases, and P-I SVMPs. Although all antivenoms abrogated the lethal, hemorrhagic, coagulant, proteinase, and PLA(2) venoms activities, monospecific experimental antivenoms were more effective than the polyspecific experimental antivenom. In addition, the commercial antivenoms, produced in horses subjected to repeated immunization cycles, showed higher neutralization than experimental polyspecific antivenom, produced by a single round of immunization. Overall, a conspicuous pattern of cross-neutralization was evident for all effects by all antivenoms, and monospecific antivenoms raised against venom from the Caribbean population were effective against venom from the Pacific population, indicating that geographic variations in venom proteomes of B. asper from Costa Rica do not result in overt variations in immunological cross-reactivity between antivenoms.