Elsevier

Toxicon

Volume 215, August 2022, Pages 37-48
Toxicon

In vitro laboratory analyses of commercial anti-scorpion (Mesobuthus tamulus) antivenoms reveal their quality and safety but the prevalence of a low proportion of venom-specific antibodies

https://doi.org/10.1016/j.toxicon.2022.06.001Get rights and content

Highlights

  • Commercial anti-scorpion antivenoms (ASAs) manufactured in India contain F(ab’)2 with a trace quantity of immunoglobulin G.

  • Study showed that the ASAs were prepared according to the guidelines of the World Health Organization.

  • The ASAs were devoid of aggregate content and virus particles.

  • Biophysical analyses demonstrated the poor binding of venom with commercial ASAs.

  • Commercial ASAs contain less proportion of scorpion venom toxins-specific antibodies.

Abstract

Mesobuthus tamulus (Indian Red Scorpion) sting is a severe but neglected health issue in India. The accomplishment of in-patient scorpion sting management is highly dependent on the safety, efficacy, and homogeneity of scorpion antivenom preparation. Therefore, in this study, the above qualities of commercial anti-scorpion antivenoms manufactured in India were assessed by in vitro laboratory analyses. Biophysical characterization of venom by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size exclusion chromatography, and proteomics analysis demonstrated that anti-scorpion antivenoms (ASAs) mostly contain F(ab')2 molecules with a trace amount of undigested immunoglobulin (Ig) G. The physicochemical characterization, electron microscopy, and dynamic light scattering studies revealed that ASAs were prepared according to the guidelines of World Health Organization (WHO), and were devoid of aggregate content and virus particles. ASAs did not show IgE contamination and bacterial endotoxin but demonstrated moderate complement activation properties, which may have adverse effects in treated patients. Spectrofluorometric and atomic force microscopy analyses showed poor binding of venom with commercial ASAs. The percent of antibodies raised against the venom toxins in commercial ASAs was determined at the range of 5.3–6.3%, which is a reason for their poor efficacy. This study advocates the importance of in vitro laboratory analyses for assessing commercial antivenom's quality and safety parameters before their pre-clinical research and clinical use to treat Indian red scorpion sting.

Introduction

Scorpion sting is a severe but neglected public health issue in tropical and sub-tropical countries, causing 1.23 million stings globally, which results in 3250 deaths annually (Bawaskar, 1984; Chippaux and Goyffon, 2008). Notably, scorpions are the second most noxious animal after the snake, causing significant problems to children and adolescents. The Mesobuthus tamulus (Indian red scorpion) is reported as one of the lethal scorpions of the ecosphere, and epidemiological data show that this species is distributed in India, Sri Lanka, eastern Nepal, and eastern Pakistan (Badhe et al., 2007; Bhadani et al., 2006; Kovařík, 2007; Kularatne et al., 2015). In India, maximum mortalities due to scorpion sting is reported in Kerala, Tamil Nadu, Andhra Pradesh, western Maharashtra, Saurashtra, and Karnataka states (Agrawal et al., 2015; Bawaskar and Bawaskar, 1996, 1998; Yuvaraja et al., 2019).

M. tamulus venom is predominated (76.7%) by low molecular mass toxins bind to ion-channels, for example, Na+ and K+ channels affecting toxins (Das et al., 2020), which are mainly responsible for inducing pharmacological activity and clinical manifestations. Further, stimulation of α-adrenergic receptor resulted in myocardial dysfunction, pulmonary edema, tachycardia, hypertension, and cool extremities, and these pathophysiological conditions play a significant role in the pharmacology of Indian red scorpion sting (Bawaskar and Bawaskar, 1992; Dutta and Deshpande, 2011; Kumar et al., 2012; Rowan et al., 1992; SinghDeshpande, 2005; Strong et al., 2015). Treatment of M.tamulus sting is a medical emergency, and administration of specific anti-scorpion antivenom (ASA) is an inevitable choice for treatment in case of severe envenomation (Chippaux, 2012; Das et al., 2021). To date nineteen ASAs are manufactured worldwide including India for their intended use in humans and animals against scorpion sting (Laustsen et al., 2016). However, as shown in Supplementary Table S1, commercial ASA may show adverse effects due to inferior quality of the antivenom. Further, antivenom therapy may also be associated with limitations like its poor efficacy (Chippaux, 2012; David, 2005; Mukherjee and Mackessy, 2021; Patra et al., 2021a,b).

To eradicate the aforesaid problems, World Health Organization (WHO), in 2019 has amended the guidelines for safe and effective antivenom production and stated that the availability of antivenom in affected countries including efficient treatment with antivenom must be reinforced globally (WHO, 2019). Notably, the adverse manifestations are dependent on the purity, homogeneity, physico-chemical properties, level of microbial load (endotoxin), and the proportion of antibodies against venom-toxins in commercial antivenom (Patra et al., 2018, 2021a,b,c). It has been recommended that failure to meet a standard benchmark by commercial antivenom, as determined by in vitro laboratory assays, may lead to rejection of that particular batch of antivenom for pre-clinical testing (Patra et al., 2021b). Therefore, the determination of purity of the active constituent (IgG or mostly F(ab’)2) of antivenom including assessment of safety parameters, such as preservative content, endotoxin load, IgA and IgE content, Fc content, protein aggregation, and percentage of venom specific therapeutic antibodies have a great role on quality assessment of antivenom and subsequent successful in-patient treatment of scorpion sting. Sadly, the quality, efficacy, and safety of commercial ASAs against scorpion venom remain to be determined, which imposes a severe concern about the in-patient treatment of M. tamulus stings. The two leading antivenom manufacturing companies viz. Premium Serum and Vaccine Pvt. Ltd. (PSVPL), Pune, India, and Haffkine Biopharmaceutical Corp. Ltd. (HBC), Mumbai, India, produce equine ASA against M. tamulus venom, which the clinicians frequently use for the hospital management of scorpion stings (Bawaskar, 2005; Bawaskar and Bawaskar, 2011; Das et al., 2021; Pandi et al., 2014). In India, limited clinical studies have shown that these ASAs are not associated with adverse serum reactions; however, further in-depth region-wise clinical studies are necessary to verify this (Bawaskar and Bawaskar, 2011; Pandi et al., 2014). Consequently, we studied the in vitro laboratory assessment of safety, homogeneity, and quality of two commercial ASAs manufactured in India.

Section snippets

Chemicals and reagents

Lyophilized M. tamulus venom was a gift from PSVPL, Pune, India. Lyophilized commercial ASAs were purchased from HBC, Mumbai, India (batch No.: PRMSC-002, expiry date: Feb. 2022), and PSVPL, Pune, India (batch No.: SS170401, expiry date: Sept. 2021). As per the manufacturer's protocol, after reconstitution in 10 mL of sterile water, each mL of these ASAs should neutralize not less than of 1.0 mg of M. tamulus venom. Horse IgG and affinity-purified horse F(ab’)2, were purchased from BioRad, USA,

Physicochemical characterization of ASA

Some of the parameters to be used for determining the physicochemical properties of commercial antivenom are shown in Table 1 (reprinted with permission from Patra et al., 2021b). The lyophilized commercial ASAs (PSVPL and HBC) had a white and homogenous appearance (Supplementary Fig. S1a,b) and were entirely dissolved in de-ionized sterile water (provided with the ASA package) within 6–7 min, and they did not contain insoluble material because no precipitate material was observed post

Discussion

Laboratory-based quality assessment of commercial antivenom is an essential step for efficient treatment against envenomation/sting and minimizes adverse reactions (Zhou et al., 2013). For stabilizing the antivenom during transportation and storage (preferably without refrigeration in rural health centers) freeze-drying is an essential step. Following the WHO guidelines, the freeze-dried ASAs were dissolved in water within the recommended time limit (10 min) and no disperse material was

Conclusion

It may be recommended that the availability of safe antivenom must be guaranteed, particularly in developing countries, and manufacturing of these life saving drugs should be ensured globally. One of the most crucial criteria in preparing antivenom is the Good Manufacturing Practice (GMP) that can vouch for their quality and safety (WHO, 2019). The assessment of in vitro neutralization of lethality and toxic activities of venom can be regarded as a benchmark for assessing antivenom's efficacy;

Authors contributions

AKM conceived the idea. AKM and BD, AP designed the experiments. BD, AP, UP, and PD performed the experiments. BD, AP, and AKM wrote the manuscript. All the authors have read and approved the final version of the manuscript.

Ethical statement

No animal experiment was done in this study. The goat blood was collected from slaughter house. For collecting human blood, permission was obtained from Ethical Committee, Tezpur University.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors thank C-CAMP, NCBS, Bangalore, for LC-MS/MS analysis, Mayuri Bora, Department of Physics, Tezpur University for DLS analysis, Sophisticated Analytical Instrumentation Centre (SAIC), Tezpur University for TEM and FESEM analysis. The authors also thank SAIC of IASST for AFM analysis. This study received financial support from Research Innovation Grants from Tezpur University 2021 (DoRD/RIG/10–73/1592-A, S. No. 22).

References (77)

  • S.A. Kularatne et al.

    Clinico-epidemiology of stings and envenoming of Hottentotta tamulus (Scorpiones: buthidae), the Indian red scorpion from Jaffna Peninsula in northern Sri Lanka

    Toxicon

    (2015)
  • L. Kumar et al.

    Autopsy diagnosis of a death due to scorpion stinging–A case report

    J. Forensic Leg. Med.

    (2012)
  • P.S. Kumar et al.

    Characterization techniques for nanomaterials

  • G. Leon et al.

    Pathogenic mechanisms underlying adverse reactions induced by intravenous administration of snake antivenoms

    Toxicon

    (2013)
  • O. Lowry et al.

    Protein measurement with the Folin phenol reagent

    J. Biol. Chem.

    (1951)
  • B.K. Meyer et al.

    Antimicrobial preservative use in parenteral products: past and present

    J. Pharmaceut. Sci.

    (2007)
  • A.K. Mukherjee et al.

    A proteomic analysis of Pakistan Daboia russeli irusselii venom and assessment of potency of Indian polyvalent and monovalent antivenom

    J. Proteonomics

    (2016)
  • A.K. Mukherjee et al.

    Prevention and improvement of clinical management of snakebite in Southern Asian countries: a proposed road map

    Toxicon

    (2021)
  • D.E. Overcashier et al.

    Lyophilization of protein formulations in vials: investigation of the relationship between resistance to vapor flow during primary drying and small-scale product collapse

    J. Pharmaceut. Sci.

    (1999)
  • R. Panchagnula et al.

    Biopharmaceutics and pharmacokinetics in drug research

    Int. J. Pharm.

    (2000)
  • A. Patra et al.

    Assessment of quality, safety, and pre-clinical toxicity of an equine polyvalent anti-snake venom (Pan Africa): determination of immunological cross-reactivity of antivenom against venom samples of Elapidae and Viperidae snakes of Africa

    Toxicon

    (2018)
  • A. Patra et al.

    The in vitro laboratory tests and mass spectrometry-assisted quality assessment of commercial polyvalent antivenom raised against the ‘Big Four’ venomous snakes of India

    Toxicon

    (2021)
  • G. Pidde-Queiroz et al.

    Human complement activation and anaphylatoxins generation induced by snake venom toxins from Bothrops genus

    Mol. Immunol.

    (2010)
  • S.S. Rane et al.

    Validation of a simple RP-HPLC method developed for the quantification of meta-cresol in parathyroid hormones formulation

    Pharm. Methods.

    (2011)
  • E.G. Rowan et al.

    The effects of Indian red scorpion Buthus tamulus venom in vivo and in vitro

    Toxicon

    (1992)
  • K. Schersch et al.

    Systematic investigation of the effect of lyophilizate collapse on pharmaceutically relevant proteins I: stability after freeze-drying

    J. Pharmaceut. Sci.

    (2010)
  • S.M. Singh et al.

    Mechanisms of m-cresol-induced protein aggregation studied using a model protein cytochrome c

    J. Pharmaceut. Sci.

    (2011)
  • S.K. Singh et al.

    Intra-arterial injection of Mesobuthus tamulus venom elicits cardiorespiratory reflexes involving perivascular afferents

    Toxicon

    (2005)
  • D.C. Smith et al.

    An affinity purified ovine antivenom for the treatment of Vipera berus envenoming

    Toxicon

    (1992)
  • G. Solano et al.

    Assessing endotoxins in equine-derived snake antivenoms: comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL)

    Toxicon

    (2015)
  • J.J. Sung et al.

    Transmission electron microscopy as an orthogonal method to characterize protein aggregates

    J. Pharmaceut. Sci.

    (2015)
  • W. Wang

    Protein aggregation and its inhibition in biopharmaceutics

    Int. J. Pharm

    (2005)
  • G.D.J. Adams et al.

    Some implications of structural collapse during freeze-drying using Erwiniacaratovoral-asparaginase as a model

    J. Chem. Technol. Biotechnol.

    (1993)
  • A. Agrawal et al.

    Scorpion bite, a sting to the heart

    Indian J. Crit. Care Med.

    (2015)
  • J.Y. Ahn et al.

    Comparison of oven-drying methods for determination of moisture content in feed ingredients. Asian Austral

    J. Anim.

    (2014)
  • C.A. Ariaratnam et al.

    A new monospecific ovine Fab fragment antivenom for treatment of envenoming by the Sri Lankan Russell's viper (Daboia Russelii Russelii): a preliminary dose-finding

    Am. J. Trop. Med. Hyg.

    (1999)
  • R.V. Badhe et al.

    The action of red scorpion (Mesobuthus tamulus coconsis, Pocock) venom and its isolated protein fractions on blood sodium levels

    J. Venom. Anim. Toxins Incl. Trop. Dis.

    (2007)
  • H.S. Bawaskar

    Management of severe scorpion sting at rural settings: what is the role of scorpion antivenom

    J. Venom. Anim. Toxins Incl. Trop. Dis.

    (2005)
  • Cited by (3)

    • Ethnomedicines for the treatment of scorpion stings: A perspective study

      2023, Journal of Ethnopharmacology
      Citation Excerpt :

      Antivenom therapy is considered cardinal and controversial as a mode of immediate treatment for scorpion stings, as some clinical studies have shown no benefits from administering antivenom (Chippaux and Goyffon, 2008; Isbister and Bawaskar, 2014). In addition, the inability to neutralize the high proportion of low molecular mass toxins in scorpion venom (Das et al., 2020) is a significant disadvantage arising from a low titer of toxins-specific antibodies (Das et al., 2021, 2022). Further, the numerous side effects arising from antivenom therapy have also proven to be handicapped (Deshpande et al., 2008; Tuuri and Reynolds, 2011).

    View full text