Andrés Sánchez

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

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

Proteomic, toxicological and immunogenic characterization of Mexican west-coast rattlesnake (Crotalus basiliscus) venom and its immunological relatedness with the venom of Central American rattlesnake (Crotalus simus)

The venom of the Mexican west-coast rattlesnake (Crotalus basiliscus) was characterized for its protein composition, toxicological profile and immunogenic properties. This venom is composed of 68% Zn2+-dependent metalloproteinases (SVMPs), 14% phospholipases A2 (PLA2s), 11% serine proteinases, 4% SVMPs-inhibitor tripeptides (SVMP-ITs), 2% bradykinin-potentiating peptides (BPPs), 0.6% cysteine-rich secretory proteins (CRISPs), and 0.2% l-amino acid oxidases (LAAOs). SVMPs present in the venom are responsible for azocasein hydrolysis and hemorrhagic activity, but their contribution to the lethal activity of the venom in mice is masked by the neurotoxic activity of PLA2s, which in addition are also responsible for myotoxic activity. Despite its relatively high content of serine proteinases, the venom of C. basiliscus did not exert in vitro coagulant or in vivo defibrinogenating activities. The ability of antivenoms raised against the venoms of C. basiliscus and C. simus (from Costa Rica) to neutralize homologous and heterologous venoms revealed antigenic similarities between toxins of both venoms. Preclinical evaluation of an antivenom produced by using the venom of C. basiliscus as immunogen demonstrated that it is able to neutralize not only the most relevant toxic activities of C. basiliscus venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. BIOLOGICAL SIGNIFICANCE The Central American rattlesnake (Crotalus simus) is widely distributed from Mexico to west central Costa Rica, and induces an important number of envenomations in this region. On the other hand, the immunogenic mixture used by Laboratorios de Biológicos y Reactivos de Mexico S.A. (Birmex) to produce the snake antivenom more frequently used in Mexico does not include the venom of C. simus. This immunogenic mixture is composed by the venoms of the Fer-de-lance (Bothrops asper) and the Mexican west-coast rattlesnake (Crotalus basiliscus). We studied the protein composition, toxicological profile and immunogenic properties of the venom of C. basiliscus, and evaluated the ability of the Birmex antivenom to neutralize the venom of C. basiliscus and whether it cross-neutralizes the venom of C. simus from Costa Rica. Using proteomics analysis, in combination with in vitro and mouse tests, we determined that the venom of C. basiliscus is mainly composed by SVMPs, which confer proteolytic and hemorrhagic activities to the venom. Other major components of the venom of C. basiliscus are PLA2s, which are responsible for the myotoxic activity and are the main contributors to the lethal activity. Non-clotting SVSPs correspond to 11% of the venom. Minor components include SVMP-ITs, BPPs, CRISPs and LAAOs, which have not been associated with toxicity. The antibodies induced in horses by the venom of C. basiliscus are able to neutralize not only the most relevant toxic activities of the homologous venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. Our preclinical evaluation suggests that Birmex antivenom can be used to treat envenomations by Costa Rican adult C. simus snakebites, despite this venom not being included in the immunizing mixture.

Physicochemical characterization of commercial freeze-dried snake antivenoms

ABSTRACT 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. HIGHLIGHTSAntivenoms were different in protein concentration and turbidity, and varied slightly in SDS‐PAGE profile.SEM analysis showed no microscopic collapse of samples, suggesting adequate freeze‐drying process conditions.All the formulations analyzed met the quality control requirements established by WHO regulations.

Expanding the neutralization scope of the EchiTAb-plus-ICP antivenom to include venoms of elapids from Southern Africa

ABSTRACT EchiTAb‐plus‐ICP is an antivenom prepared from plasma of horses hyperimmunized with the venoms of the carpet viper (Echis ocellatus), the puff adder (Bitis arietans) and the black‐necked spitting cobra (Naja nigricollis). Therefore, the use of this antivenom has been limited to Western Africa. In order to expand the neutralization scope of EchiTAb‐plus‐ICP, we supplemented the immunogenic mixture with the venoms of B. arietans, the black mamba (Dendroaspis polylepis), the Mozambique spitting cobra (Naja mossambica), the snouted cobra (N. annulifera), and the rinkhals (Hemachatus haemachatus) from Swaziland. The ability of the expanded‐scope antivenom, hereby named EchiTAb + ICP, to neutralize the venoms of B. arietans, D. polylepis, N. mossambica and H. haemachatus was similar to those of FAV Afrique and the SVA African antivenoms. In comparison to the SAIMR antivenom, the expanded‐scope EchiTAb + ICP had lower ability to neutralize the venom of B. arietans, but similar ability to neutralize the venoms of D. polylepis, N. mossambica and H. haemachatus. Owing to its low protein concentration, the expanded‐scope EchiTAb + ICP had lower ability to neutralize the venom of N. annulifera than FAV Afrique and the SAIMR antivenoms. However, when formulated at a protein concentration as high as FAV Afrique and SAIMR antivenoms, the expanded‐scope EchiTAb + ICP showed similar capacity to neutralize this poorly immunogenic venom. Our results encourage the transition to the new EchiTAb + ICP antivenom, with an expanded neutralization scope that includes venoms of some of the most medically important elapids from Southern Africa. Clinical trials are required to determine the minimum effective‐safe dose of the new EchiTAb + ICP for each type of envenomation. HighlightsEchiTab‐plus‐ICP is an antivenom towards venoms of snakes from Western Africa.Venoms of elapids from Swaziland were included in the immunization mixture.The neutralization scope of the EchiTab‐plus‐ICP was expanded to include venoms of elapids from Southern Africa.The expanded‐scope antivenom has neutralization properties similar to FAV Afrique and the SVA African antivenoms.The clinical efficacy of the new EchiTab‐plus‐ICP in envenomations caused by elapids from Southern Africa is pending.

Effect of geographical variation of Echis ocellatus, Naja nigricollis and Bitis arietans venoms on their neutralization by homologous and heterologous antivenoms

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.

An improved technique for the assessment of venom-induced haemorrhage in a murine model

ABSTRACT 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. Graphical abstract Figure. No caption available. HighlightsA new method to quantify local haemorrhagic activity of venoms is described.The method assesses not only the area of haemorrhage but also the intensity.The method uses freely accessible computational tools.The method also allows the study of the neutralisation of haemorrhagic activity by antivenoms.

Proteomic and toxinological characterization of the venom of the South African Ringhals cobra Hemachatus haemachatus

The protein composition and toxinological profile of the venom of the African spitting elapid Hemachatus haemachatus (Ringhals) were characterized by bottom-up proteomics and functional in vitro and in vivo assays. Venom is composed of abundant three-finger toxins (3FTxs; 63.3%), followed by phospholipases A2 (PLA2s; 22.8%), snake venom metalloproteinases (SVMPs; 7.1%), cysteine-rich secretory proteins (CRISPs; 4.1%) and Kunitz type protease inhibitors (KTPIs; 1.5%). 3FTxs are the main responsible for lethality and myotoxicity in mice and in vitro anticoagulant activity. In contrast to closely related spitting species, whose venom 3FTxs induces dermonecrosis, the 3FTxs of H. haemachatus did not induce dermonecrotic activity. The venom showed in vitro PLA2 activity, and most likely PLA2s contribute to some extent in venom lethality, as judged by partial reduction in toxicity after inhibition of their catalytic activity. Despite its relatively high content of SVMPs, compared to most elapids, the venom of H. haemachatus did not exert hemorrhagic effect, proteolytic activity on azocasein or defibrinogenating activity. Toxicovenomic characterization of H. haemachatus venom revealed that RP-HPLC fractions with higher abundance of 3FTxs presented lethal activity, while fractions with high content of PLA2s did not, underscoring the role of 3FTxs in the pathophysiology caused by this venom. BIOLOGICAL SIGNIFICANCE The proteomic composition and toxinological profile of the venom of Ringhals snake, Hemachatus haemachatus, a cobra-like spitting snake endemic to southern Africa, were investigated. In vitro, Ringhals venom showed anticoagulant and phospholipase A2 activities, but was devoid of proteolytic activity on azocasein. In mice, venom induced lethality and myotoxicity, but no local hemorrhage or dermonecrosis. The lack of dermonecrotic activity is in sharp contrast to venoms of closely related spitting cobras which present a similar relative abundance of 3FTxs but are potently dermonecrotic. 3FTxs, the most abundant protein family in the venom, are predominantly responsible for toxic effects. PLA2 enzyme inactivation experiments suggest that H. haemachatus venom lethality is not dependent on PLA2s, but instead is more related to neurotoxic or cardiotoxic 3FTxs. The characterization of this venom, based on proteomic and toxicovenomic approaches, is useful for more in depth studies associated with biogeography, phylogeny, toxinology and antivenom efficacy towards the venom of this species, and its association with related elapids.

Effect of premedication with subcutaneous adrenaline on the pharmacokinetics and immunogenicity of equine whole IgG antivenom in a rabbit model

Subcutaneous administration of a low dose of adrenaline is used to prevent the early adverse reactions (EARs) induced by snake antivenoms. We used a rabbit model to study the effect of premedication with adrenaline on the potential of antivenoms to exert therapeutic effects and to induce late adverse reactions. We found that premedication with adrenaline did not change the heart rate or blood pressure of normal rabbits, but reduced the rise in temperature in rabbits previously sensitized with antivenom. Pharmacokinetic studies suggest that premedication with adrenaline does not affect the ability of the antivenom to exert the initial control of envenomation nor the susceptibility of rabbits to develop recurrence of antigenemia and envenomation. Our results also indicate that it is unlikely that premedication with adrenaline decreases the incidence of late reactions induced by the antivenom administration, although it reduces the extent of early reactions.

Current technology for the industrial manufacture of snake antivenoms

ABSTRACT 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. HighlightsSnakebite envenomation is an important public health problem around the world.Antivenoms are the only effective therapy to control snakebite envenomations.Antivenoms must comply with identity, purity, safety and efficacy profiles, according to GMPs.Antivenom manufacture involves several stages.A review of the technology used for the manufacture of snake antivenoms is presented.

Some educational applications of alpha spectrometry

The most difficult task in alpha spectrometry is perhaps making the source to be measured, but once sources are prepared, the gathering and analysis of results is an activity which can be developed in undergraduate laboratories. Energy calibration, determinations of counting yield and energy resolution, calculation of areas under the peaks in the spectrum (overlapping and well separated), determination of activities, etc., are frequently tasks that students find straightforward and informative. Neither is the cost of the experimental setup needed for such measurements excessive. Some educational applications of alpha spectrometry are described.