*N. nigricollis*, and *N. haje*, respectively). Conversely, increasing inhibitor concentrations resulted in an increase in the percent inhibition for the same species (**Figure 2**).

The relatively high and comparable inhibition observed in *N. ashei*, *N. nigricollis*, and *N. haje* may confirm two things; first, the similarity in the venom proteomes of the cobras (both spitting and non-spitting) and second, the venom proteomes of Naja sp. contain more 3FTxs. ODs obtained for *B. arietans* and *D. polylepsis* suggest that there was no or minimal inhibition mainly because the venoms either contain little or none of the antigen of interest. Furthermore, the detection of envenoming by the inhibition ELISA model (LOD of 0.01 μg/mL) is consistent with previously reported ELISA methods, although different LODs were found.

#### *3.5.2 Small molecule therapeutics*

Venom components, particularly those of viperids, are known to contain distinctive active sites that usually depend on three amino acid residues. The ability therefore, of compounds to block such an active site makes it possible to functionally disable the venom component. Lately, various small molecule inhibitors have caused excitement, with the potential to expedite the utility of such compounds through molecular docking approaches given the already encouraging results available [15]. One of the most interesting small molecule therapeutics (SMTs) to have recently emerged is varespladib and its orally available prodrug format, methyl-varespladib. As a treatment drug initially meant for acute coronary syndrome, varespladib has been shown to successfully inhibit phospholipase A2 activity of various snake venoms obtained from different parts of the world. The effect of varespladib against venomderived PLA2s is relevant given their poor immunogenicity, which may elicit a poor immune response during immunization of animals as part of the development of conventional antivenoms. Consequently, such antivenom products obtained from poorly immunogenic components may not be very effective against PLA2s. In an experimental set up, pretreating mice with 4 mg/kg varespladib and then injecting the same with the lethal dose of *Micrulus fulvius* venom was shown to prolong survival for nearly 24 hours following, which the protective effect wore off and reduce signs of hemorrhage in the mice. Also, the co-injection of an LD50 of *Vipera belus* venom and

*Perspective Chapter: Diagnostic and Antivenom Immunotherapeutic Approaches… DOI: http://dx.doi.org/10.5772/intechopen.112147*

4 mg/kg varespladib subcutaneously successfully ensured the survival of 3/7 mice, while all controls died. When varespladib was given following a little delay in the injection of *V. belus* venom, the same result was produced. A 100% survival was also observed in treated mice after the intravenous administration of 8 mg/kg varespladib followed by the subcutaneous injection of an LD50 of *V. belus* venom. In yet another experiment, rats injected with *M. fulvius* venom subcutaneously were entirely rescued following the intravenous administration of varespladib within 5 minutes of the challenge. In addition, varespladib was shown to suppress the increase in PLA2 activity induced by the venom, as well as haemolysis of the venom [44].

In another study conducted quite recently, varespladib was reported to inhibit the *in vitro* PLA2-induced toxic effects of *Agkistrodon halys*, *Deinagkistrodon acutus, Naja atra,* and *Bungarus multicinctus*. At a dose of 4 mg/kg, varespladib resulted in a reduction in the density of hemorrhagic plagues provoked by both *D. acutus* and *A. halys* venoms and also decreased edema and hemorrhage instigated by all four venom samples *in vivo*, varespladib-treated mice recorded a 31–81% decrease in edema relative to control animals. Again, signs of muscle damage induced by the venoms including desmin degradation were reduced by varespladib. At median effective doses (ED50s) of 0.45 and 1.14 μg/g, the inhibition of venom-induced lethality by varespladib was found to be more effective in viperid venoms of *A. halys* and *D. acutus,* respectively, as opposed to those of elapids; 22.09 and 15.23 μg/g for *N. atra* and *B. multicinctus,* respectively [45]. Venoms from most snakes contain toxic components, especially PLA2s that act synergistically with other toxins. To this end, it could be conjectured that the administration of varespladib could potentially obstruct the synergistic effect of key toxins, resulting in the complete inhibition of venom toxicity. Nonetheless, the usefulness of varespladib may be limited because the extensive reliance on PLA2s does not apply to all snake venoms. For instance, venom from the genus *Dendroapsis* is nearly exclusively devoid of phospholipases A2; thus, it is not probable that varespladib would be beneficial against snakebites from this genus [46, 47]. However, whereas varespladib on its own may possess exciting applications, methyl-varespladib, its corresponding prodrug format, is available orally for administration, rendering it a potential first-choice candidate drug for protection. Therefore, when methyl-varespladib is used alone or combined with other drugs, it may be capable of providing some respite to victims, whiles efforts are made to access further antivenom treatment at appropriate health facilities [48].

Another example of a SMT that has shown great promise is the matrix metalloproteinase batimastat and its orally available prodrug format marimastat. The incubation of these SMTs with a challenge dose of *E. ocellatus* venom (4 LD50) and subsequent co-injection into the tail vein of mice resulted in prolonged survival, although it was not fully protective. However, administering batimastat abrogated the hemorrhagic, *in vitro* coagulant, defibrinogenating, and proteinase activities induced by *E. ocellatus* venom sourced from Cameroon. When *E. ocellatus* venom samples from Ghana were used, an increased abrogation in hemorrhagic activity was observed when batimastat was quickly administered. Conversely, the delayed administration of batimastat resulted in better abrogation of defibrinogenating activity, with the possibility of completely abrogating the effect following a 60-min delay in administration of the molecule. While batimastat proved to be more efficacious in abrogating hemorrhagic activity, defibrinogenating activity was more effectively abrogated by marimastat [49]. Other SMTs including acetylcholinesterase inhibitors (e.g., atropine and neostigmine) are being investigated with some encouraging results in the reduction of mortality among venoms of certain elapids. In addition, nanoparticles and C60 fullerene are also being investigated, with the latter showing antivenom features in an insect model [27].

#### *3.5.3 Protein, peptide, and oligomer-based technologies*

Apart from antivenom, protein, peptide, and oligomer-based therapies are being explored. Human monoclonal-based single-chain variable fragments and fully humanized monoclonal IgGs have been developed. There are indications that these technologies may possibly be crucial in inhibiting various snake venom components in the future given that they are associated with relatively less adverse reactions, as well as the potential to be competitive in terms of cost [27]. According to El-Aziz *et al.* [50], oligonucleotides are without most of the problems that usually characterize the immunization approach of antibody production including the use of animals, protracted production time, low immunogenicity mostly with small-sized toxins, and production cost, as well as treatment-related challenges such as requirement for refrigeration, specificity issues, reduced shelf-life, and immunogenicity of animalbased antibodies. In exploring the neutralization action of αC-conotoxin PrXA found in *Conus parius*, a species of cone snail by oligonucleotides, a sample of oligonucleotide tested demonstrated capacity to neutralize the *in vitro* activity of αC-conotoxin PrXA. Although at the doses tested, the oligonucleotide was not fully protective, it, however, provided extended survival. Nonetheless, at much higher concentrations, the oligonucleotide provided full protection. Furthermore, [44] argue that an added benefit that comes with the use of oligonucleotides in the laboratory is that the cost of smallscale synthesis is low for the purposes of research and development (R&D), allowing researchers to easily and rapidly appraise a wide variety of molecules at reduced cost. There is the need, however, for more studies to evaluate the cost of manufacturing oligonucleotides on a large scale before its evaluation for clinical-based application. Lakkappa et al. [27] also report that aptamers (short DNA or RNA sequences) have been demonstrated to neutralize cardiotoxins and α-bungarotoxin, as well as toxins present in corn snails. In addition, a large number of alternative binding scaffolds (AbScaffs) are being investigated for their therapeutic prospects in snakebite envenoming because they cost low to produce, highly stable, and easy to engineer.
