Animal Venoms as Potential Source of Anticonvulsants

Abstract* Epilepsy affects millions of people worldwide, and there is an urgent need to develop safe and effective therapeutic agents. Animal venoms contain diverse bioactive compounds like proteins, peptides, and small molecules, which may possess medicinal properties against epilepsy. In recent years, research has shown that venoms from various organisms such as spiders, ants, bees, wasps, and conus snails have anticonvulsant and antiepileptic effects by targeting specific receptors and ion channels. This review underscores the significance of purified proteins and toxins from these sources as potential therapeutic agents for epilepsy. In conclusion, this review emphasizes the valuable role of animal venoms as a natural resource for further exploration in epilepsy treatment research.


Introduction
Epilepsy is a global epidemic that affects more than 50 million people worldwide, making it one of the most common neurological diseases. 1Almost 80% of epileptic patients come from low-and middle-income countries. 1The Task Force of the International League Against Epilepsy (ILAE) defined epileptic seizure as a transient occurrence of symptoms caused by abnormally excessive brain neuronal activity.Epilepsy is a brain disorder prone to epileptic seizures and consequences from cognitive, neurobiological, psychological, and social aspects. 2 There are several types of epileptic seizures, depending on the symptoms.One-third of the epileptic seizures occurred as generalized seizures, whereas twothirds began as focal seizures.Generalized seizures can be classified into six groups: tonic-clonic, myoclonic, atonic, tonic, clonic, and absence seizures. 3All these symptoms happen without warning and involve loss of consciousness on the patient.Tonic seizures are characterized by constant muscle contraction, while tremors in the limbs indicate clonic seizures.Tonic-clonic seizures have symptoms that combined both tonic and clonic seizures symptoms.The contraction of limb muscles is followed by limb extension and tremors with back arching for around ten to thirty seconds.
Epileptogenesis is the gradual development of epilepsy in a normal brain. 4The causes of epilepsy can be identified as structural or genetics.Structural causes of epilepsy include brain tumors, brain trauma, and other neurodegenerative diseases.Brain areas prone to trauma causing epileptogenesis include structures of the temporal lobe such as the amygdala, hippocampus, and piriform cortex. 5Brain electrical activity is usually non-synchronous in normal humans, while patients suffering from epileptic seizures will have excessive synchronized firing of neuronal firing, which causes a paroxysmal depolarizing shift.Neuronal biochemical receptors play an essential role in epileptogenesis, primarily glutamate receptors (ionotropic and metabotropic).When the receptors are activated, Ca2+ ions will increase within the area of the cells.Therefore, glutamate is seen as one of the most critical molecules in epileptogenesis as it causes excitotoxicity, i.e., neurons are severely depolarised. 4ilepsy treatment may include drugs, surgery, and even diet changes.The standard first-line therapy for patients newly diagnosed with epilepsy is usually by antiepileptic drugs (AEDs) such as phenobarbital, phenytoin, carbamazepine, or valproic acid.Second line medications such as oxcarbazepine, gabapentin, topiramate, felbamate, and lamotrigine are also applied in the treatment. 6No single AED is proved to be more effective than another, with all drugs producing some side effects depending on the individual treated.These drugs used have a 70% to 80% success rate in treating epilepsy, while the remaining 20% to 30% still suffer from seizures or more significant adverse effects. 7nce epilepsy is caused by the malfunction of ion channels, Gamma-aminobutyric acid (GABA)-ergic neuron inhibition, and excess glutamate release, most AED will target these specific pathways. 8Drugs that target ion channels include carbamazepine, oxcarbazepine, phenytoin, and lamotrigine, which block sodium channels. 9Gabapentin binds on voltage-dependent calcium channel proteins in rat models and is believed to have GABAergic effects.GABA-mediated inhibition is vital as the binding of GABA to their receptors leads to an influx of negatively charged chloride ions (Cl-) which in turn causes cell hyperpolarisation.Barbiturates and benzodiazepines treat epilepsy by targeting the GABAergic transmission and increasing chloride ion influx. 10Glutamate, which is a primary excitatory neurotransmitter were also believed to play a part in epilepto-genesis.Application of antagonists to the glutamate receptor such as N-methyl-Daspartate (NMDA) demonstrated anticonvulsant effects in animal models.Valproate, felbamate, and topiramate are NMDA receptor antagonists which are currently used.However, these drugs are known to present several undesirable side effects. 8

Animal venom as a potential source of anticonvulsants
The earth's flora and fauna are an important reservoir of naturally derived therapeutic agents.Since ancient times, humans have utilized these resources to treat diseases and improving wellbeing.One significant source is the venom and toxins from venomous and poisonous animals.The venom consists of proteins, enzymes, inorganic ions, and nucleotides, which can induce a plethora of effects, including necrosis, coagulation, haemorrhagic, and neurotoxicity depending on the type of venom. 11Targeted sites of venom are specific and diverse due to the different constitutions and may target receptors and channels such as neuro-junctions and ion channels. 124][15][16] In this literature review, the different types of venom used in treating epilepsy is discussed.

Spider venom (Table 1)
Neuroprotective potential of various venom fractions and components from venomous spider species has been identified (Table 1).Arylamine-enriched fraction isolated from the venom of funnel-web grass spider Agelenopsis aperta demonstrated significant anticonvulsant properties by antagonizing and blocking NMDA receptor and dihydropyridine (DHP) calcium channels. 17Overexcitation of NMDA receptors and DHP calcium channels were found to be responsible for the pathophysiology of epilepsy. 18,19In the study by Jackson and Parks, 17 arylamine-enriched venom fraction was administered both intravenously (i.v.) on male Sprague-Dawley rats induced with epilepsy using kainic acid (KA), picrotoxin (PIC), and bicuculline (BIC).Subjects administered with KA when paired with the venom fractions demonstrated less to nonconvulsive behavior until 70 minutes.Symptoms of seizures subsided after 4 hours with no deaths observed in the 10 subjects.Subjects administered with BIC and PIC paired with the venom fraction (i.v.) also shows closely similar observations and seem mildly sedated at the same time responsive to external stimuli.
Active fraction (PbTx1.2.3) from Brazillian orb-weaver spiders Parawixia bistrata demonstrated potential anticonvulsive activity in synaptosomes model from rat cerebral cortex. 20Glutamate and gamma-Amnobutyric acid (GABA) uptake balance is crucial in maintaining normal brain function.Functioning as the primary excitatory neurotransmitter in the brain, the increase of glutamate activity is critical in epileptogenesis. 21In contrast, GABA counterbalances neuronal excitation by acting as the primary inhibitory neurotransmitter.Hence, changes to GABA balance in the brain may trigger seizures. 22PbTx1.2.3 was demonstrated to increase glutamate uptake while inhibiting GABA uptake in cortical synaptosomes in a dose-dependent manner.Drugs that stimulate GABA concentration are potent anticonvulsants such as vigabatrin and tiagabine. 22Moreover, PbTx1.2.3 successfully protected neurons in the rat retinal glaucoma model from excitotoxic death. 20Therefore, PbTx1.2.3 may serve as a template for potential anticonvulsant and neuroprotection drug development.
FrPbAII is another compound found in P. bistrata venom, which was studied for anticonvulsive and anxiolytic properties.Bicuculline was injected into Area-tempestas of male Wistar rats to induce seizures and treated with FrPbAII.The toxin component significantly inhibited seizures elicited by bicuculline.In addition, FrPbAII was demonstrated to block GABAergic transporters that caused epilepsy. 23Animal behavior study using male Wistar rats also showed anticonvulsive properties of FrPbAII.Rats were induced with seizures using pentylenetetrazol and were treated with diazepam, nipecotic acid, and FrPbAII.All subjects treated with diazepam, nipecotic acid, and FrPbAII showed anticonvulsive properties with reduced immobility, increased exploratory walking, rearing, and head-dips than saline control pentylenetetrazole (PTZ)-induced seizure subjects. 24rawixin-10 is another polyamine found in P. bistrata venom which elicits anticonvulsive properties.Parawixin-10 was administered via i.c.v.injection using a cannula and kainic acid (KA), NMDA, and PTZ to induce seizures.Parawixin-10 demonstrated anticonvulsive properties with the lowest dose required for KA compared to NMDA and PTZ indicating that the mode of action for Parawixin-10 is through the inhibition of excitatory transmission of non-NMDA receptors.Neurochemistry data from the study also showed an increase in L-glutamate and glycine uptake, wherein elevated levels of both compounds are responsible for epilepsy.However, the important mode of action for Parawixin-10 has yet to be pinpointed and thus requires further investigation and studies. 25Additional study on P. bistrata venom fraction, RT10, which comprised of Parawixins 1 and 2, demonstrated neuroprotective effects on neuron-glia primary culture against high L-glutamate concentrations. 26

Scorpion venom
Several subtypes of α and β neurotoxins have been described from the venom of Asian scorpion Buthus martensi Karsch (BmK) (Table 2).These neurotoxins were found to modulate voltage-gated sodium channels (VGSCs). 28,29Antinociceptive effects from BmK venom were demonstrated by the activity of BmK I (α-like neurotoxin) and BmK IT2 (β-like neurotoxins). 30,31A study by Zhao, et al. 32 in PTZ and pilocarpine-induced epilepsy rats showed significant anticonvulsant activity from the administration of BmK IT2.The effects of BmK IT2 were attributed to its ability to modulate sodium channels in the hippocampus.A further study with a different toxin from BmK, Bmk-AS, demonstrated similar antiepileptic activities in PTZ and pilocarpine-induced epilepsy in the rat model. 33In another study, a novel peptide synthesised and derived from the Malaysian forest scorpion venom, Hetermetrus spinifer, was shown to reduce inflammation in PTZ-induced epilepsy in-vivo. 34The 25 amino acid peptide named HsTx2 alleviate epilepsy progression by targeting the circ_0001293/miR-8114/TGF-β2 axis, which is responsible for inflammation response in astrocytes. 34

Bee and Wasp venom
Bee and wasp venom is rich in compounds that act on various biological systems, especially in the nervous system.These compounds include peptides, amines, enzymes, and amino acids (Table 3). 35Both peptides and acyl polyamines have been studied to treat many neurology-related diseases.A single study on mellitin, a major component of the venom from Apis mellifera (honey bee), demonstrated anticonvulsant effects in bicuculine-induced seizures in rats. 36Mellitin could potentially induced its neuroprotective activities via activation of phospholipase A 2 and consequently modulating the GABAergic and glutamergic systems. 36Denatured crude venom from Polybia occidentalis (Yellow-banded Polybia wasp) identified dose-dependent effectiveness in treating KA-induced seizures rat model.This finding suggests that venom-induced anticonvulsive activities by antagonizing NMDA-glu receptors.Additional results include in-vivo anticonvulsive effects in rats with PIC-and BIC-induced seizures. 37Recent study on a venom peptide isolated from Polybia occidentalis, occidentalin-1202, pinpointed anti-seizure effects by blocking glutamate and kainic acid interaction with the kainite receptor. 38A different study on the venom of Polybia ignobilis (Brazilian social wasp) demonstrated a similar outcome by preventing seizures induced by KA, PIC, and BIC. 39w molecular weight fractions of the venom from Polybia paulista (Neotropical social wasp) exhibited anticonvulsant activities in 60% of animals (Wistar rats) induced with PTZ. 40Compared with diazepam treatment, both offer protection from seizures, but the venom demonstrated fewer sedation effects. 40Additionally, peptides isolated from Polybia paulista, Ppnp7 and neuropolybin, demonstrated tonic-clonic seizure protection in 80% of PTZ-induced rats. 41Agelaia vicina venom showed inhibition of GABA and glutamate uptake responsible during epileptogenesis.The venom targeted enzymes accountable for the regulation of ion flow, thus disrupting ion concentration gradient.Additionally, free amino acids were found to inhibit GABA and glutamate uptake. 42Philanthus triangulum F. wasp venom contains components such as β-PTX and δ-PTX, which show effects on animal glutamatergic neuro-junctions.These components were found to inhibit the uptake of glutamate in the neuromuscular synapse on locusts.A study showed a significant decrease in glutamate uptake on hippocampal slices of rats incubated with δ-PTX (74% decrease) and a less significant reduction with incubation in β-PTX (18%).Based on the findings, the venom from Philanthus triangulum F. wasps demonstrated potentials as a source of anticonvulsant agents due to its interference with glutamate uptake, a critical factor in epileptogenesis. 43Besides PTZ-induced seizures, a treatment using wasp venom peptide from Chartegellus communis (Social wasp), Chartergellus-CP1, showed neuroprotective effects in pilocarpine-induced epilepsy. 44In the study, electroencephalographic abnormalities that are associated with pilocarpine-induced seizures were significantly reduced upon administration of Charthergellus-CP1. 44

Ant venom
Ant venom contains an arsenal of molecules that ranges from proteins, peptides, free amino acids, alkaloids, formic acid, and several other organic molecules. 46Like other venomous organisms, the venom has been a subject of interest to uncover its properties and therapeutic potential, including as an anticonvulsive agent (Table 4).The venom of Dinoponera quadriceps (South American giant ant) has been studied for anticonvulsant effects.An initial study by Noga, et al. 47 demonstrated the pro-and anticonvulsant effects of the crude venom in-vivo.Injection of crude venom (DqTX) in the lateral ventricle region of mice brain showed procursive behavior and tonic-clonic seizures.
Interestingly, the effects were negated via pre-administration of the same but denatured venom (AbDq), hence, protected the animals against seizures and death. 47Similar results were noted in another study by Lopes, et al. 48Different administration routes of D. quadriceps venom in mice seizure models were responsible for neuroprotective or neurotoxic effects.Intraperitoneal administration of the venom demonstrated increased latency to seizure with an increased survival rate in the PTZ-induced seizure model.In contrast, endovascular treatment-induced neurotoxic effects by reducing the seizure latency time.In another study utilising PTZ-induced seizure model, three different peptides from Dinoponera quadriceps (M-PONTX-Dq3a, M-PONTX-Dq3b, and M-PONTX, Dq3c) also showed anticonvulsant properties by reducing neuroinflammation caused by reactive oxygen species. 38 a subsequent study by Noga, et al., 49 the anticonvulsant effects from the fractions of D. quadriceps crude venom were investigated.The crude venom was fractionated using high-performance liquid chromatography (HPLC) and yielded six fractions labelled DqTX1 -DqTX6.The fractions were administered intracerebroventricularly in BIC-induced seizure rats, and the anticonvulsant effects were determined.DqTX-6 showed significant neuroprotection with 62.5% protection from seizures and 100% protection from death.Since BIC induces seizures by interfering with GABA receptors, the mode of action for the DqTX-6 fraction is most likely to be GABAergic. 49 Decreased production of reactive oxygen and nitrogen species

Conus snail venom
Conopeptides from the venom of marine cone snails belonging to the genus Conus have been identified with potentials as antinociceptive, antiepileptic, neuroprotective, and cardioprotective agents. 50There are at least four peptide families with neuroprotective and cardioprotective activities from Conus snail venom, namely, conantokins, omega-conotoxin (ω-Conotoxin), mu-conotoxin (μ-Conotoxin), and kappa-conotoxin (κ-Conotoxin) (Table 5). 51Conantokins are of particular interest due to their potential in treating epilepsy.Seven types of conantokin peptides were identified, and all were described as NMDA receptor antagonists. 52nantokin-G (Con-G) is a well-studied conantokin from the venom of Geography cone snail Conus geographus.It was advanced into clinical trial phase 1 to treat intractable epilepsy and potential neuro-protectant in ischemic stroke. 535][56] Anticonvulsive mode of Con-G was via the dose-dependent inhibition of NMDA responses. 57Besides Con-G, Conantokin-R (Con-R) from Conus radiatus Conantokin-L (Con-L) from Conus lynceus showed significant anticonvulsant effects when administered intracerebroventricularly to subjects induced with Frings audiogenic seizures. 58,59Ziconotide, a synthetic derivative of ω-Conotoxin from Conus magus, demonstrated anticonvulsant and anxiolytic effects by potentially reducing extracellular concentrations of glutamate via N-type calcium channel inhibition. 60 Future directions The potential of venom as a source of anticonvulsants warrants further exploration and investigation, extending beyond its current focus to encompass other venomous animals which could potentially include snakes.Snake venom is a complex mixture of proteins, peptides, and small molecules that serve as a defence mechanism and aid digestion for snakes towards their prey.Despite its toxicity, snake venom can be an essential source of therapeutic compounds, as demonstrated by its potential in treating cardiovascular disease and as an anti-microbial, anti-viral, anti-fungal, and anticancer agent.61 To date, there is only a few studies the anticonvulsant potential of snake venom via Ophiophagus hannah (king cobra) and Micrurus mipartus (Costa Rican coral snake).Saha, et al. 62 isolated a non-protein toxin called KC-MMTx from O. hannah crude venom and demonstrated its neuroprotective activity in animal models of epilepsy.KC-MMTx presented significant protection against seizures induced by strychnine, PTZ, and yohimbine, and its potential in interfering with the GABAergic and glycine receptors was touted.Remarkably, toxins from Costa Rican coral snake (MmTX1 and MmTX2) were found to specifically target GABA receptors and significantly improve its sensitivity via allosteric binding.63 Moreover, the ability of snake venom toxins, particularly three-finger toxins (3FTXs), to bind with nicotinic acetylcholine receptors (nAChR) presents another promising avenue in this field.This is noteworthy because the overactivation of nAChR is frequently associated with tonic-clonic convulsions and epilepsy in mammals.[64][65][66][67] In addition to snake venoms, venoms from sea anemone could also be another important source of therapeutic proteins and peptides as anticonvulsants.68 Several sea anemone toxins extracted from Stichodactyla heliantus, Actinia equina, Anemonia eryhtrae, Anemonia sulcata, Bunodosoma caissarum, Bunodosoma granulifera and Heteractis magnifica were found to selectively target Kv1 channels.69 These findings hold particular importance as Kv1 ion channels play a clinically relevant role in the pathogenesis of neurological diseases, including epilepsy.70

Conclusions
Epilepsy remains one of the most critical health burdens in the world.However, the disease currently presents a significant challenge in discovering and developing therapeutic agents that are safe and effective.Venom-based therapeutics has been a subject of focus due to their potential to combat various disorders such as cardiovascular diseases and cancer.This present review demonstrates that animal venom could exert anticonvulsant effects by targeting and regulating specific receptors, modulating uptakes of molecules such as glutamate and glycine, and antagonizing ion channels.In addition, the advancement of proteomic and genomic technology has presented us with the ability to characterize and purify proteins with therapeutic potentials accurately.More proteins/compounds can be isolated and tested in-vivo for anticonvulsant activities.Animal venom represents a promising avenue for researchers to further explore and investigate its compounds as a source of potential antiepileptic/anticonvulsive agents.
This is a short review about Animal Venoms as Potential Source of Anticonvulsants written by Syafiq Asnawi Zainal Abidin, Anthony Kin Yip Liew, Iekhsan Othman, and Farooq Shaikh.Epilepsy is a global epidemic that affects more than 50 million people worldwide, making it one of the most common neurological diseases.Epilepsy remains one of the most critical health burdens in the world.The discovery of anti-epilepsy agents with high effect and low toxicity is still the most urgent need today and future.Animal venom is confirmed to be one significant and potential source of natural drugs including anticonvulsants.Overall, the theme of this review is very meaningful and interesting.Moreover, the structure and organization of this review is logically clear.From a scientific perspective, the article reviews and summarizes all anti-epileptic peptides and components derived from animal venom sources.Moreover, the direction and focus of future attention and research have also been proposed.All in all, I recommend that this review could be considered to be accepted after a revision.However, some minor questions still need to be modified.
The language of this review is good.However, there are still some errors in the following."synthesised" should be adjusted to "synthesized"; "in-vivo" should be adjusted to the italicized "in-vivo"; "mice brain" should be adjusted to "mouse brain"; "mice seizure models" should be adjusted to "mouse seizure models".

Is the topic of the review discussed comprehensively in the context of the current literature? Yes
Are all factual statements correct and adequately supported by citations?

Cletus Anes Ukwubile
University of Maiduguri, Maiduguri, Borno, Nigeria The author has rightly included in the review most essential parameters.This review draws the attention of the audience on the potentials of venom as anticonvulsant agent.
The language use was concise and interesting.However, there is the need to provide scientific names to some of the organisms that serve as venom sources.The use of only common names are scientifically incomplete.The author should also be specific in the conclusion.For instance more proteins/compounds can be..... Proteins are compounds.It should be More compounds be... epilepsy since there were no approved venom-based drugs available to treat epilepsy.Section 2.1 sub-heading: I suggest removing (Table 1) Page 4, Paragraph 1: If "Subjects" was used to refer to "rats", I suggest using the latter throughout Page 4, Paragraph 2: "A' of Aminobutyric should be written with lowercase font Table 2: Voltage-gated?Table 3: Could be moved to sub-section 2. Reviewer Expertise: Venomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Is the topic of the review discussed comprehensively in
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3 , Paragraph 1 : 3 , Paragraph 2 : 3 , Paragraph 3 :
the context of the current literature?Yes Are all factual statements correct and adequately supported by citations?Yes Is the review written in accessible language?Yes Are the conclusions drawn appropriate in the context of the current research literature?Yes Introduction Page Reference to back up the definition of epilepsy by the Task Force of the International League Against Epilepsy (ILAE).Page Ca2+, 2+ should be in superscript Page The first sentence could be supported with reference especially "diet changes as methods to treat epilepsy" Page 3, Paragraph 3: Cl-, -should be in superscript Page 3, Paragraph 5: I would suggest venom and venom toxins with potential effects against Page 13 of 15 F1000Research 2024, 13:225 Last updated: 08 JUL 2024

3 . 5 , Paragraph 1 : 5 ,
Page in vivo?Page Paragraph last: Apis mellifera should be in italicsThe authors' "Future directions" to explore the venoms of snakes and sea anemones is interesting, considering the role of neurotoxins (three-finger toxins) in snakebite envenoming victims.The interaction of this toxin at the neuromuscular junction inhibits presynaptic voltage-gated calcium channels by so doing causes paralysis of the snakebite victims.Is the topic of the review discussed comprehensively in the context of the current literature?YesAre all factual statements correct and adequately supported by citations?YesIs the review written in accessible language?YesAre the conclusions drawn appropriate in the context of the current research literature?YesCompeting Interests: No competing interests were disclosed.

Table 1 .
Anticonvulsive proteins/peptides from spider venom and the mechanism of action.

Table 2 .
Anticonvulsive proteins/peptides from scorpion venom and the mechanism of action.

Table 3 .
Anticonvulsive proteins/peptides from wasp and bee venom and the mechanism of action.

Table 4 .
Anticonvulsive protein/peptides from ant venom and the mechanism of action.

Table 5 .
Anticonvulsive proteins/peptides from cone snail venom and the mechanism of action.

Yes Is the review written in accessible language? Yes Are the conclusions drawn appropriate in the context of the current research literature? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 24 June 2024 https://doi.org/10.5256/f1000research.161164.r293539© 2024 Ukwubile C.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.