Fangs in the Ghats: Preclinical Insights into the Medical Importance of Pit Vipers from the Western Ghats.

The socioeconomic impact of snakebites in India is largely attributed to a subset of snake species commonly known as the 'big four'. However, envenoming by a range of other clinically important yet neglected snakes, a.k.a. the 'neglected many', also adds to this burden. The current approach of treating bites from these snakes with the 'big four' polyvalent antivenom is ineffective. While the medical significance of various species of cobras, saw-scaled vipers, and kraits is well-established, the clinical impact of pit vipers from regions such as the Western Ghats, northeastern India, and the Andaman and Nicobar Islands remains poorly understood. Amongst the many species of snakes found in the Western Ghats, the hump-nosed (Hypnale hypnale), Malabar (Craspedocephalus malabaricus), and bamboo (Craspedocephalus gramineus) pit vipers can potentially inflict severe envenoming. To evaluate the severity of toxicity inflicted by these snakes, we characterised their venom composition, biochemical and pharmacological activities, and toxicity- and morbidity-inducing potentials, including their ability to damage kidneys. Our findings highlight the therapeutic inadequacies of the Indian and Sri Lankan polyvalent antivenoms in neutralising the local and systemic toxicity resulting from pit viper envenomings.

The Middle Eastern Cousin: Comparative Venomics of Daboia palaestinae and Daboia russelii.

Among the medically most important snakes in the world, the species belonging to the genus Daboia have been attributed to the highest number of human envenomings, deaths and disabilities. Given their significant clinical relevance, the venoms of Russell's vipers (D. russelii and D. siamensis) have been the primary focus of research. In contrast, the composition, activity, ecology and evolution of venom of its congener, the Palestine viper (D. palaestinae), have remained largely understudied. Therefore, to unravel the factors responsible for the enhanced medical relevance of D. russelii in comparison to D. palaestinae, we comparatively evaluated their venom proteomes, biochemical activities, and mortality and morbidity inflicting potentials. Furthermore, the synthesis and regulation of venom in snakes have also remained underinvestigated, and the relative contribution of each venom gland remains unclear. We address this knowledge gap by sequencing the tissue transcriptomes of both venom glands of D. palaestinae, and comparatively evaluating their contribution to the secreted venom concoction. Our findings highlight the disparity in the venom composition, function and toxicities of the two Daboia species. We also show that toxin production is not partitioned between the two venom glands of D. palaestinae.

The Preclinical Evaluation of a Second-Generation Antivenom for Treating Snake Envenoming in India.

Snake envenoming afflicts the Indian subcontinent with the highest rates of mortality (47,000) and morbidity globally. The only effective treatment for snakebites is the administration of antivenom, which is produced by the hyperimmunisation of equines. Commercial Indian antivenoms, however, have been shown to exhibit a poor preclinical performance in neutralising venom, as a result of inter- and intrapopulation snake venom variation. Additionally, their poor dose effectiveness necessitates the administration of larger volumes of antivenom for treatment, leading to several harmful side effects in snakebite victims, including serum sickness and fatal anaphylaxis. In this study, we employed chromatographic purification to enhance the dose efficacy of commercial Indian antivenoms. The efficacy of this 'second-generation' antivenom was comparatively evaluated against six other marketed antivenoms using a number of in vitro and in vivo preclinical assays, which revealed its superior venom recognition capability. Enhanced purity also resulted in significant improvements in dose effectiveness, as the 'second-generation' antivenom exhibited a 3 to 4.5 times increased venom neutralisation potential. Furthermore, preclinical assays revealed the increased effectiveness of the 'second-generation' antivenom in countering morbid effects inflicted by the 'big four' Indian snakes. Thus, we demonstrate the role of simpler purification steps in significantly enhancing the effectiveness of snakebite therapy in regions that are most affected by snakebites.

Venomics of the Enigmatic Andaman Cobra (Naja sagittifera) and the Preclinical Failure of Indian Antivenoms in Andaman and Nicobar Islands.

The Andaman and Nicobar Islands are an abode to a diversity of flora and fauna, including the many endemic species of snakes, such as the elusive Andaman cobra (Naja sagittifera). However, the ecology and evolution of venomous snakes inhabiting these islands have remained entirely uninvestigated. This study aims to bridge this knowledge gap by investigating the evolutionary history of N. sagittifera and its venom proteomic, biochemical and toxicity profile. Phylogenetic reconstructions confirmed the close relationship between N. sagittifera and the Southeast Asian monocellate cobra (N. kaouthia). Overlooking this evolutionary history, a polyvalent antivenom manufactured using the venom of the spectacled cobra (N. naja) from mainland India is used for treating N. sagittifera envenomations. Comparative evaluation of venoms of these congeners revealed significant differences in their composition, functions and potencies. Given the close phylogenetic relatedness between N. sagittifera and N. kaouthia, we further assessed the cross-neutralising efficacy of Thai monovalent N. kaouthia antivenom against N. sagittifera venoms. Our findings revealed the inadequate preclinical performance of the Indian polyvalent and Thai monovalent antivenoms in neutralising N. sagittifera venoms. Moreover, the poor efficacy of the polyvalent antivenom against N. naja venom from southern India further revealed the critical need to manufacture region-specific Indian antivenoms.

Remarkable intrapopulation venom variability in the monocellate cobra (Naja kaouthia) unveils neglected aspects of India's snakebite problem.

Interpopulation venom variation has been widely documented in snakes across large geographical distances. This variability is known to markedly influence the effectiveness of snakebite therapy, as antivenoms manufactured against one population may not be effective against others. In contrast, the extent of intrapopulation venom variability, especially at finer geographical scales, remains largely uninvestigated. Moreover, given the historical focus on the 'big four' Indian snakes, our understanding of venom variation in medically important yet neglected snakes, such as the monocellate cobra (Naja kaouthia), remains unclear. To address this shortcoming, we investigated N. kaouthia venoms sampled across a small spatial scale (<50 km) in Eastern India. An interdisciplinary approach employed in this study unveiled considerable intrapopulation differences in the venom proteomic composition, pharmacological and biochemical activities, and toxicity profiles. Documentation of stark differences in venoms at such a finer geographical scale, despite the influence of similar ecological and environmental conditions, is intriguing. Furthermore, evaluation of in vitro and in vivo venom recognition and neutralisation potential of Indian polyvalent 'big four' antivenoms and Thai monovalent N. kaouthia antivenom revealed concerning deficiencies. These results highlight the negative impact of phylogenetic divergence and intrapopulation snake venom variation on the effectiveness of conventional antivenom therapy. SIGNIFICANCE: In contrast to our understanding of snake venom variation across large distances, which is theorised to be shaped by disparities in ecology and environment, intrapopulation variation at finer geographic scales remains scarcely investigated. Assessment of intrapopulation venom variability in Naja kaouthia at a small spatial scale (<50 km) in Eastern India unravelled considerable differences in venom compositions, activities and potencies. While the influence of subtle differences in prey preference and local adaptations cannot be ruled out, these findings, perhaps, also emphasise the role of accelerated molecular evolutionary regimes that rapidly introduce variations in evolutionarily younger lineages, such as advanced snakes. The inability of 'big four' Indian antivenoms and Thai N. kaouthia monovalent antivenom in countering these variations highlights the importance of phylogenetic considerations for the development of efficacious snakebite therapy. Thus, we provide valuable insights into the venoms of one of the most medically important yet neglected Indian snakes.

Biogeographic venom variation in Russell's viper (Daboia russelii) and the preclinical inefficacy of antivenom therapy in snakebite hotspots.

BACKGROUND: Snakebite in India results in over 58,000 fatalities and a vast number of morbidities annually. The majority of these clinically severe envenomings are attributed to Russell's viper (Daboia russelii), which has a near pan-India distribution. Unfortunately, despite its medical significance, the influence of biogeography on the composition and potency of venom from disparate D. russelii populations, and the repercussions of venom variation on the neutralisation efficacy of marketed Indian antivenoms, remain elusive. METHODS: Here, we employ an integrative approach comprising proteomic characterisation, biochemical analyses, pharmacological assessment, and venom toxicity profiling to elucidate the influence of varying ecology and environment on the pan-Indian populations of D. russelii. We then conducted in vitro venom recognition experiments and in vivo neutralisation assays to evaluate the efficacy of the commercial Indian antivenoms against the geographically disparate D. russelii populations. FINDINGS: We reveal significant intraspecific variation in the composition, biochemical and pharmacological activities and potencies of D. russelii venoms sourced from five distinct biogeographic zones across India. Contrary to our understanding of the consequences of venom variation on the effectiveness of snakebite therapy, commercial antivenom exhibited surprisingly similar neutralisation potencies against the majority of the investigated populations, with the exception of low preclinical efficacy against the semi-arid population from northern India. However, the ability of Indian antivenoms to counter the severe morbid effects of Daboia envenoming remains to be evaluated. CONCLUSION: The concerning lack of antivenom efficacy against the north Indian population of D. russelii, as well as against two other 'big four' snake species in nearby locations, underscores the pressing need to develop pan-India effective antivenoms with improved efficacy in high snakebite burden locales.

Biogeographical venom variation in the Indian spectacled cobra (Naja naja) underscores the pressing need for pan-India efficacious snakebite therapy.

BACKGROUND: Snake venom composition is dictated by various ecological and environmental factors, and can exhibit dramatic variation across geographically disparate populations of the same species. This molecular diversity can undermine the efficacy of snakebite treatments, as antivenoms produced against venom from one population may fail to neutralise others. India is the world's snakebite hotspot, with 58,000 fatalities and 140,000 morbidities occurring annually. Spectacled cobra (Naja naja) and Russell's viper (Daboia russelii) are known to cause the majority of these envenomations, in part due to their near country-wide distributions. However, the impact of differing ecologies and environment on their venom compositions has not been comprehensively studied. METHODS: Here, we used a multi-disciplinary approach consisting of venom proteomics, biochemical and pharmacological analyses, and in vivo research to comparatively analyse N. naja venoms across a broad region (>6000 km; seven populations) covering India's six distinct biogeographical zones. FINDINGS: By generating the most comprehensive pan-Indian proteomic and toxicity profiles to date, we unveil considerable differences in the composition, pharmacological effects and potencies of geographically-distinct venoms from this species and, through the use of immunological assays and preclinical experiments, demonstrate alarming repercussions on antivenom therapy. We find that commercially-available antivenom fails to effectively neutralise envenomations by the pan-Indian populations of N. naja, including a complete lack of neutralisation against the desert Naja population. CONCLUSION: Our findings highlight the significant influence of ecology and environment on snake venom composition and potency, and stress the pressing need to innovate pan-India effective antivenoms to safeguard the lives, limbs and livelihoods of the country's 200,000 annual snakebite victims.

A Wolf in Another Wolf's Clothing: Post-Genomic Regulation Dictates Venom Profiles of Medically-Important Cryptic Kraits in India.

The Common Krait (Bungarus caeruleus) shares a distribution range with many other 'phenotypically-similar' kraits across the Indian subcontinent. Despite several reports of fatal envenomings by other Bungarus species, commercial Indian antivenoms are only manufactured against B. caeruleus. It is, therefore, imperative to understand the distribution of genetically distinct lineages of kraits, the compositional differences in their venoms, and the consequent impact of venom variation on the (pre)clinical effectiveness of antivenom therapy. To address this knowledge gap, we conducted phylogenetic and comparative venomics investigations of kraits in Southern and Western India. Phylogenetic reconstructions using mitochondrial markers revealed a new species of krait, Romulus' krait (Bungarus romulusi sp. nov.), in Southern India. Additionally, we found that kraits with 17 mid-body dorsal scale rows in Western India do not represent a subspecies of the Sind Krait (B. sindanus walli) as previously believed, but are genetically very similar to B. sindanus in Pakistan. Furthermore, venom proteomics and comparative transcriptomics revealed completely contrasting venom profiles. While the venom gland transcriptomes of all three species were highly similar, venom proteomes and toxicity profiles differed significantly, suggesting the prominent role of post-genomic regulatory mechanisms in shaping the venoms of these cryptic kraits. In vitro venom recognition and in vivo neutralisation experiments revealed a strong negative impact of venom variability on the preclinical performance of commercial antivenoms. While the venom of B. caeruleus was neutralised as per the manufacturer's claim, performance against the venoms of B. sindanus and B. romulusi was poor, highlighting the need for regionally-effective antivenoms in India.

In Vivo Toxicity Profile of NN-32 and Nanogold Conjugated GNP-NN-32 from Indian Spectacled Cobra Venom.

BACKGROUND: NN-32 toxin, which was obtained from Naja naja venom and showed cytotoxicity on cancer cell lines. As the toxicity of NN-32 is the main hurdle in the process of drug development; hence, we have conjugated NN-32 toxin with gold nanoparticles (GNP-NN-32) in order to decrease the toxicity of NN-32 without reducing its efficacy, GNP-NN-32 alleviated the toxicity of NN-32 in in vitro studies during the course of earlier studies. In continuation, we are evaluating in vivo toxicity profile of NN-32 and GNP-NN-32 in the present study. OBJECTIVE: To study in vivo toxicity profile of NN-32 and nanogold conjugated GNP-NN-32 from Naja naja venom. MATERIALS AND METHODS: We have carried out in vivo acute toxicity study to determine LD50 dose of GNP-NN-32, in vivo sub-chronic toxicity for 30 days, haematology, serum biochemical parameters and histopathology study on various mice tissues and in vitro cellular and tissue toxicity studies. RESULTS: The LD50 dose of GNP-NN-32 was found to be 2.58 mg/kg (i.p.) in Swiss male albino mice. In vivo sub-chronic toxicity showed significantly reduced toxicity of GNP-NN-32 as compared to NN-32 alone. DISCUSSION: In vitro cellular toxicity studies on human lymphocyte and mouse peritoneal macrophage showed significant inhibition of cells by NN-32 alone. CONCLUSION: Conjugated GNP-NN-32 toxin showed less in vivo toxicity as compared to pure NN-32.

Anticancer potential of nanogold conjugated toxin GNP-NN-32 from Naja naja venom.

BACKGROUND: Cancer is the second most common fatal disease in the world, behind cardiovascular disorders in the first place. It accounts for around 0.3 million deaths per year in India due to the lack of proper diagnostic facilities, prevention and treatment. Current therapeutic methods do not provide adequate protection and affect normal cells along with cancerous ones. Thus, there is a need for some alternative therapeutic strategy, preferably from natural products, which have been traditionally used for treatment of various diseases in the country. METHODS: In this study, we have conjugated purified NN-32 toxin from Naja naja venom with gold nanoparticles and its anticancer potential was evaluated against human breast cancer cell lines. UV-Vis spectroscopy, dynamic light scattering, transmission electron microscopy, atomic force microscopy and zeta potential analysis were the techniques used for characterization of GNP-NN-32. RESULTS: GNP-NN-32 showed dose- and time-dependent cytotoxicity against breast cancer cell lines (MCF-7 and MDA-MB-231). NN-32 and GNP-NN-32 induced apoptosis in both breast cancer cell lines. The results of CFSE cell proliferation study revealed that NN-32 and GNP-NN-32 arrested cell division in both MCF-7 and MDA-MB-231 cell lines resulting in inhibition of proliferation of these cancer cells. CONCLUSION: GNP-NN-32 showed an anticancer potential against human breast cancer cell lines. Analysis of detailed chemical characterization along with its cytotoxic property might help to perceive a new dimension of the anti-cancer potential of GNP-NN-32 that will enhance its biomedical function in near future.

Anticancer potential of nanogold conjugated toxin GNP-NN-32 from Naja naja venom

Background: Cancer is the second most common fatal disease in the world, behind cardiovascular disorders in the first place. It accounts for around 0.3 million deaths per year in India due to the lack of proper diagnostic facilities, prevention and treatment. Current therapeutic methods do not provide adequate protection and affect normal cells along with cancerous ones. Thus, there is a need for some alternative therapeutic strategy, preferably from natural products, which have been traditionally used for treatment of various diseases in the country. Methods: In this study, we have conjugated purified NN-32 toxin from Naja naja venom with gold nanoparticles and its anticancer potential was evaluated against human breast cancer cell lines. UV-Vis spectroscopy, dynamic light scattering, transmission electron microscopy, atomic force microscopy and zeta potential analysis were the techniques used for characterization of GNP-NN-32. Results: GNP-NN-32 showed dose- and time-dependent cytotoxicity against breast cancer cell lines (MCF-7 and MDA-MB-231). NN-32 and GNP-NN-32 induced apoptosis in both breast cancer cell lines. The results of CFSE cell proliferation study revealed that NN-32 and GNP-NN-32 arrested cell division in both MCF-7 and MDA-MB-231 cell lines resulting in inhibition of proliferation of these cancer cells. Conclusion: GNP-NN-32 showed an anticancer potential against human breast cancer cell lines. Analysis of detailed chemical characterization along with its cytotoxic property might help to perceive a new dimension of the anti-cancer potential of GNP-NN-32 that will enhance its biomedical function in near future.(AU)

Cytotoxic activity of NN-32 toxin from Indian spectacled cobra venom on human breast cancer cell lines.

BACKGROUND: Breast cancer is the most common cancer which causes significant morbidity and mortality among women worldwide. Lack of medical facilities for early detection, therapeutic strategies for treatment and side effects due to pharmacological compounds have encompassed the need for new therapies mostly from natural sources. A lot of components have been identified from different snake venoms as therapeutic agents. A group of polypeptides (60-70 amino acid residues) called cytotoxins or cardiotoxins present in an elapid family of snakes have a wide variety of pharmaceutical actions and have the tendency to damage a wide variety of cells including cancerous cells. The aim of the present study was to evaluate the cytotoxic effect of NN-32 protein toxin purified from Indian Spectacled Cobra venom against human breast cancer cell lines (MCF-7 and MDA-MB-231). METHODS: The NN-32 toxin was purified by ion exchange chromatography and further by RP-HPLC. The potential anticancer effects of the NN-32 toxin on MCF-7 and MDA-MB-231 cells were evaluated using MTT, anti-proliferation, neutral red (NR) uptake and Lactate Dehydrogenase (LDH) release assay. RESULTS: The ion exchange chromatography showed various peaks among fraction no. 35 showing cytotoxic activity and this fraction showed a single peak with retention time 3.6 mins by HPLC using C18 column. The NN-32 toxin induced cytotoxicity in MCF-7 and MDA-MB-231 cells with the IC50 value of 2.5 and 6.7 µg/ml respectively. The NN-32 showed significant cytotoxicity to both the cell lines along with low cytotoxicity to MCF-10A (normal breast epithelial) cells. The cytotoxic effect was further confirmed by the anti-proliferative, NR uptake and LDH release assays. CONCLUSION: The purified toxin NN-32 from Naja naja venom showed cytotoxic activity against MCF-7 (ER+) and MDA-MB-231(ER-) cells in both dose dependent and time dependent manner.