The application of laboratory-based analytical tools and techniques for the quality assessment and improvement of commercial antivenoms used in the treatment of snakebite envenomation.

Snakebite envenomation is a public health problem of high impact, particularly for the developing world. Antivenom, which contains whole or protease-digested immunoglobulin G, purified from the plasma of hyper-immunized animals (mainly horses), is the mainstay for the treatment of snakebite envenomation. The success of antivenom therapy depends upon its ability to abrogate or reduce the local and systemic toxicity of envenomation. In addition, antivenom administration must be safe for the patients. Therefore, antivenom manufacturers must ensure that these products are effective and safe in the treatment of envenomations. Antivenom efficacy and safety are determined by the physicochemical characteristics of formulations, purity of the immunoglobulin fragments and antibodies, presence of protein aggregates, endotoxin burden, preservative load, and batch to batch variation, as well as on the ability to neutralize the most important toxins of the venoms against which the antivenom is designed. In this context, recent studies have shown that laboratory-based simple analytical techniques, for example, size exclusion chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, mass spectrometry, immunological profiling including immuno-turbidimetry and enzyme-linked immunosorbent assays, Western blotting, immune-chromatographic technique coupled to mass spectrometry analysis, reverse-phase high performance liquid chromatography, spectrofluorometric analysis, in vitro neutralization of venom enzymatic activities, and other methodologies, can be applied for the assessment of antivenom quality, safety, stability, and efficacy. This article reviews the usefulness of different analytical techniques for the quality assessment of commercial antivenoms. It is suggested that these tests should be applied for screening the quality of commercial antivenoms before their preclinical and clinical assessment.

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium. The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium. The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms. Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.

Preclinical assessment of a polyspecific antivenom against the venoms of Cerrophidion sasai, Porthidium nasutum and Porthidium ophryomegas: Insights from combined antivenomics and neutralization assays.

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 A(2) (PLA(2)) and proteinase activities of the three venoms, albeit exhibiting quantitative differences in the values of the Median Effective Doses (ED(50)). Excepting for certain low molecular mass bands corresponding to disintegrins, and some PLA(2)s 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.

Assessment of the viral safety of antivenoms fractionated from equine plasma.

Antivenoms are preparations of intact or fragmented (F(ab')2 or Fab) immunoglobulin G (IgG) used in human medicine to treat the severe envenomings resulting from the bites and stings of various animals, such as snakes, spiders, scorpions, or marine animals, or from the contact with poisonous plants. They are obtained by fractionating plasma collected from immunized horses or, less frequently, sheep. Manufacturing processes usually include pepsin digestion at acid pH, papain digestion, ammonium sulphate precipitation, caprylic acid precipitation, heat coagulation and/or chromatography. Most production processes do not have deliberately introduced viral inactivation or removal treatments, but antivenoms have never been found to transmit viruses to humans. Nevertheless, the recent examples of zoonotic diseases highlight the need to perform a careful assessment of the viral safety of antivenoms. This paper reviews the characteristics of equine viruses of antivenoms and discusses the potential of some manufacturing steps to avoid risks of viral contamination. Analysis of production parameters indicate that acid pH treatments and caprylic acid precipitations, which have been validated for the manufacture of some human IgG products, appear to provide the best potential for viral inactivation of antivenoms. As many manufacturers of antivenoms located in developing countries lack the resources to conduct formal viral validation studies, it is hoped that this review will help in the scientific understanding of the viral safety factors of antivenoms, in the controlled implementation of the manufacturing steps with expected impact on viral safety, and in the overall reinforcement of good manufacturing practices of these essential therapeutic products.

Valoración de endotoxinas bacterianas en sueros antiofídicos de origen equino: optimización de métodos en el control de la calidad / Evaluation of bacterial endotoxins in equine antiophidic sera: quality control methods optimization

Se valoró la presencia de pirógenos en el antisuero contra venenos de serpientes de los genéros Bothrops, Crotalus y Lachesis elaborado por la planta de producción de sueros antiofídico del Instituto Nacional de Salud en Bogotá Colombia. Se evaluó materia prima, proceso y producto terminado utilizando las técnicas de pirógenos en conejos USP y limulus amebocito lisado (LAL). Los resultados por los dos métodos de análisis para los cinco lotes de materia prima fueron negativos a pirógenos y positivos para los ocho lotes analizados de producto terminado en los que se determinó por LAL niveles de endotoxina entre >13.87 UE/ml y > 24.7 UE/ml; superiores al permitido por la Farmacopea Americana USP XXIII, que fija un valor máximo de 1.67 UE/ml para este tipo de producto. Se estandarizó el uso de la técnica del LAL en el suero de origen equino utilizado como materia prima, el cual presenta inhibidores naturales para la técnica; mediante pretratamientos al plasma para la extracción de inhibidores aplicando diferente técnicas, con los siguientes resultados de mayor a menor capacidad de detección de endotoxina: técnica de sulfato de amonio hasta 0.125 UE/ml; técnica de dilución calor 0.21 UE/ml; técnica de cloroformo dilución 0.42 UE/ml y la técnica con ajuste de pH no detécto endotoxina en la muestra. Se valoró la potencialización de las muestras sometidas a extracción, con la adición de iones mono y divalentes. Se ensayaron los iones NaCl, MnCl, CaCl subíndice 2 y MgCl subíndice 2, en concentraciones de 0.02, 0.05, 0.08M. El mejor resultado se obtuvo con los iones MgCl subíndice 2 y CaCl subíndice 2 a 0.05M, asociado a la técnica de extracción de cloroformo y dilución, en la que aumentó la detección de endotoxina de 0.42 a 0.125 UE/ml con MgCl subíndice 2 y de 0.42 a 0.35 UE/ml con CaCl subíndice 2. Se concluyó que la técnica del LAL ajustada con la técnica sulfato de amonio y calor y la adición MgCL subíndice 2 a 0.05M, sirve como prueba de control para ser aplicada a la materia prima. En el estudio se demostró que durante el proceso de purificación de las inmunoglobulinas, el producto se contamina con endotoxinas bacterianas; fueron responsables de esta contaminación 11.44 por ciento de los materiales, 51.17 por ciento de los equipos y 2.35 por ciento de los reactivos utilizados. Se detecto endotoxinas en 64.96 por ciento de las mustras analizadas. La aplicación del método del LAL en la línea de proceso permitió...