Exploring the Pain-Relieving Potential: Unveiling Antinociceptive Properties in Animal Venoms and Toxins

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors reviewed the analgesic potential of animal venoms and toxins, identifying 152 promising molecules targeting various pain-related receptors and ion channels. Comments,

1. How generalisable are rodent model findings to human physiology?
2. What are the limitations of focusing primarily on voltage-gated ion channels?
3. How do variations in venom composition affect your conclusions?
4. Could you elaborate on the pharmacokinetic challenges of these peptide-based drugs?
5. What are the main obstacles in developing non-invasive delivery systems?
6. How does seasonal variation in venom composition impact your findings?
7. Can you discuss the ethical considerations in venom sourcing?
8. How do environmental factors influence toxin potency and variability?
9. What are the limitations of current analytical techniques in characterizing complex venoms?
10. Could you expand on the economic feasibility of large-scale venom production?
11. How do genetic variations within species affect toxin efficacy?

Author Response

Response to Reviewer 1 Comments

 

Dear Reviewer,

Thank you for your careful review and insightful comments on my manuscript. I have found your feedback to be extremely helpful. All points raised have been addressed, and the appropriate revisions have been made to the text. My specific responses to each comment are outlined below.

 

Comments:

 

1. How generalisable are rodent model findings to human physiology?

 

R: Thank you for your pertinent question. Since most of the molecules mentioned were tested in mice or rats, it really makes sense to know if these results actually apply to human patients. Although rodent models (such as chronic constriction, formalin, and hot plate models) are the gold standard for initial screening, there are differences in the expression of ion channels and receptors between species. However, we can cite translational successes such as Prialt® (Ziconotide) and Halneuron® (Tetrodotoxin), which are real evidence that targets identified in animal models (calcium and sodium channels) are functionally relevant in humans. There is also the fact that nociceptive pain pathways are highly conserved among mammals, but chronic pain in humans has complex cognitive and psychological components that rodent reflex models do not fully capture. The findings in rodents are generalizable at the level of the "molecular mechanism of action," but clinical efficacy and the therapeutic window need validation in human tissues.

 

1. What are the limitations of focusing primarily on voltage-gated ion channels?

 

R: Your question is very relevant, given that most of your toxins interact with ion channels. We mentioned in the text that calcium channel blockers (CCBs) such as Pha1β toxin and Ziconotide can cause motor dysfunction or paralysis at high doses. This is the main limitation: the omnipresence of these channels in the nervous system. This review is not limited to ion channels alone, having identified 14 pharmacological targets, including opioid, serotonergic, and adrenergic receptors. We present alternatives when ion channel modulation fails or causes side effects.

 

1. How do variations in venom composition affect your conclusions?

 

R: This review focused on 152 specific molecules/toxins. Since the amino acid sequence of the toxin is already known (e.g., μ-conotoxin PIIIA), its pharmacological activity is constant, regardless of variations in the crude venom of the species. However, we know that significant geographic, ontogenetic, or dietary variations can affect the abundance of these molecules in the venom, impacting the initial discovery process. The use of Venomics (proteomics and transcriptomics) allows for robust identification of the molecules present, mitigating the risk of conclusions based on unrepresentative venom samples.

 

1. Could you elaborate on the pharmacokinetic challenges of these peptide-based drugs?

 

R: It is known that peptides have very different pharmacokinetic profiles from small molecules. The main barrier is degradation by systemic proteases and peptidases. Many identified toxins mentioned in the text possess disulfide bridges that confer some natural resistance, but still insufficient for prolonged circulation. Due to rapid renal clearance (due to their small size) and degradation, the plasma half-life is usually minutes to a few hours. We can cite Prialt® (Ziconotide) mentioned in the article as an example. It needs to be administered via intrathecal pump precisely because its systemic pharmacokinetics are unfavorable and it does not cross the blood-brain barrier (BBB).

 

1. What are the main obstacles in developing non-invasive delivery systems?

 

R: There are several obstacles to overcoming injectable/intrathecal administration. Among them are:

Gastrointestinal Barrier: For oral administration, the acidic pH of the stomach and digestive enzymes destroy peptides before absorption.

Epithelial Permeability: Peptides are generally large and hydrophilic, which hinders passive crossing of the cell membranes of the intestine or skin.

Blood-Brain Barrier (BBB): As many analgesic targets are in the Central Nervous System (CNS), the inability of most toxins to cross the BBB is the biggest obstacle for non-invasive systems (such as nasal sprays or patches).

The use of liposomes and micelles, as mentioned in the text, are examples of how nanotechnologies can protect the peptide from degradation and facilitate permeation.

 

This sentence was added to the text: The transition from parenteral administration to non-invasive routes faces significant challenges, including proteolytic and acid degradation in the gastrointestinal tract, low epithelial permeability due to the hydrophilic nature of peptides, and the restriction imposed by the blood-brain barrier. To mitigate these limitations, the use of nanocarriers, such as liposomes and micelles, emerges as a promising strategy to increase the metabolic stability and bioavailability of these molecules.

 

1. How does seasonal variation in venom composition impact your findings?

 

R: We believe we have already answered this question previously. Raw Material variability is extremely important and should always be taken into account for these studies. The composition of crude venom can change according to the season, diet, and age of the animal. This means that a study done in the summer may find a different concentration of toxin X than a winter study.

Just to cite the importance of these differences, our group developed a new drug (antivenom) for the treatment of envenomation by Africanized bees (http://dx.doi.org/10.3389/fimmu.2021.653151). For its development, it was necessary to study the seasonal differences present in the venom over several months (http://dx.doi.org/10.1016/j.toxicon.2010.03.023). Our review article focuses on isolated and sequenced toxins (152 specific molecules), thus minimizing the impact of seasonality. Once the toxin sequence is identified, it can be produced synthetically or recombinantly, ensuring standardization independent of environmental factors. Original research articles should always take this fact into account.

 

1. Can you discuss the ethical considerations in venom sourcing?

 

R: Excellent question. Reflection on animal welfare and legal access to biodiversity should always guide these studies. Venom extraction (milking) must follow animal welfare protocols (the 3Rs principle: Replacement, Reduction, Refinement). The use of crude venom is generally only used for an initial phase of the study. In the article, we highlight that the future lies in recombinant production and chemical synthesis. This eliminates the continued dependence on wild or captive animals.

 

This sentence was added to the text: "Furthermore, ethical considerations in the supply of venoms include compliance with animal welfare protocols and biodiversity access legislation. The transition to toxin production via recombinant technology or chemical synthesis, discussed in this review, is fundamental to reducing dependence on animal specimens and ensuring the sustainability of drug discovery."

 

1. How do environmental factors influence toxin potency and variability?

 

R: We believe we have already answered this question previously, highlighting its importance. Precise molecular characterization (the focus of our article) is superior to the study of crude venoms, as it ensures that the active molecule is identified and standardized, regardless of the environmental conditions where the animal was collected.

 

This sentence was added to the text: Environmental factors, such as diet and geographic variations, significantly influence the potency and composition of venoms, as observed in comparative studies. This variability highlights the importance of identifying isolated molecules to ensure the reproducibility of pharmacological effects.

 

1. What are the limitations of current analytical techniques in characterizing complex venoms?

 

R: Venoms contain hundreds of molecules in concentrations that vary by orders of magnitude. Techniques such as Mass Spectrometry (MS) may not detect potent toxins that are in very low abundance (the "noise" of major proteins masks the minor ones). Many analgesic toxins (such as conotoxins) have complex PTMs that make correct identification difficult using only transcriptomics. Therefore, there is a challenge in correlating transcriptomic data (what the gland expresses) with proteomics (what is actually in the venom), which can lead to false positives in the search for new drugs.

 

This sentence was added to the text: "Despite advances in 'venomics', technical limitations persist, especially in the detection of low-abundance toxins and in the characterization of complex post-translational modifications. The integration of transcriptomic and proteomic data is essential to overcome the gap between genetic identification and biological functionality of toxins."

 

1. Could you expand on the economic feasibility of large-scale venom production?

 

R: Direct extraction (milking) from animals is laborious, has high fixed costs for maintaining animal facilities, and generates low yields of specific toxins. In the case of scalability and standardization for pharmaceutical purposes, large-scale production depends on recombinant methods (in bacteria or yeast) or chemical synthesis. The cost of producing recombinant biopharmaceuticals can be competitive with traditional methods, offering the advantage of rigorous standardization (without batch-to-batch variation). We must also consider that the high specificity of toxins reduces "off-target" side effects, which can decrease losses in advanced clinical phases, making the investment attractive.

 

This sentence was added to the text: "Although the natural extraction of venoms faces scalability challenges and high operating costs, the economic viability of these drug candidates lies in the transition to recombinant production and chemical synthesis. These technologies allow the biotechnological standardization necessary for the pharmaceutical market, mitigating biological variability and reducing mass production costs in the long term."

 

1. How do genetic variations within species affect toxin efficacy?

 

R: This sentence was added to the text: "Intraspecific genetic variations, resulting from polymorphisms and differential gene regulation, can lead to the expression of isoforms with distinct pharmacological efficacies. Such genetic variability should be considered in the selection of lead molecules, as small structural alterations can significantly impact the interaction with molecular targets and the reproducibility of the observed analgesic effects."

 

All modifications to the text are marked in yellow to make them easier to locate. With these changes, we have aimed to comprehensively address the feedback. Please do not hesitate to let us know if anything further is needed.

 

We are truly grateful for your insightful comments, which have been invaluable to this manuscript's development.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, a comprehensive summary on animal venoms and toxins with antinociceptive activity at different physiological targets, ranging from voltage-gated channels to ligand-gated ion channels and receptors, including those with unresolved targets. However, the organization of the review needs substantial work. The review is littered with structural and factual errors. One stark error is the use of scientific species names that are not italicized in the manuscript despite the supplementary section having majority of them in italics. Also, the numbering of references in Tables S2 to S5 is incorrect that require major attention.    

 

Comments. 

 

Title.

As the review is about animal venoms and toxins derived from animal venoms, it is accurate to add “animal” to the title. Also, given the articles reviewed used animal models in their investigations, the term “analgesic” should not be used. The term ‘analgesic’ should be reserved for human studies whereas for animal models, “antinociceptive” would be appropriate. Replace “Exploring the Pain-Relieving Potential: Unveiling Analgesic Properties in Venoms and Toxins” with “Exploring the Pain-Relieving Potential: Unveiling Antinociceptive Properties in Animal Venoms and Toxins”

 

Abstract.

Line 19: Fix the edits.

 

Introduction.

Line 75: Replace “snails” with “snail”, “Conus magus” with “Conus magus”

Line 76: Reference 24 is about CNV1014802, also known by its generic name Raxatrigine and Reference 25 is about Halneuron, not Prialt.

Line 78. Insert Reference 25 after “…clinical studies”

Line 80: Replace “analgesic” with “antinociceptive”

Line 81-82: The statement “to identify those most likely to become new biopharmaceuticals on the market.” is misleading as one would expect a summary of toxins with potentials that have been identified as therapeutic agents.

 

Results and Discussion.

Line 88-90: The sentence “…were categorized according to Figure 1 into the following categories: venom characterization, clinical manifestations, envenoming, antimicrobial/parasite, plants, other therapeutic properties, thesis, books, congress, and evolution/genetics.”. How is this related to Figure 1?

Line 94-95: Replace “14 different types of pharmacologically related receptors” with “14 different types of ion channel/receptor targets”.

Figure 2: Abbreviations must be defined.

Line 116: Replace “1.1 to 3.3 Cav” with “1.1-1.4, 2.1-2.3 and 3.1-3.3 Cav)”

Line 122: Replace “Conus” with “Conus”

Line 124: The sentence “main component of the drug Prialt” is incorrect. Prialt referes to the commercial name of ziconotide/ω-conotoxin MVIIA.  References 24 and 25 have nothing to do with Prialt. Replace “calcium channels” with “Cav2.2”

Line 132: Replace “c-Conotoxin MVIIA” with “ω-conotoxin MVIIA”

Line 135: Replace “Conus sp.” with “Conus sp.”

Line 136: Replace “Bu8, C. bullatus” with “Bu8, derived from the venom of C. bullatus”

Line 137: Replace “Conotoxin” with “conotoxin”

Line 139: Replace “C. catus” with “C. catus”

Line 141: Replace “C. kinoshitai” with “C. kinoshitai”

Line 145: Replace “C. marmoreus” with “C. marmoreus”

Line 149-150: Replace “C. moncuri” with “C. moncuri”, “C. regularis” with “C. regularis” and “SO3- (C. striatus)” with “SO-3 (C. striatus)”. Replace “Cav channels” with “Cav2.2” RsXXIVA has relatively low potency (2.8uM) for Cav2.2. Thus, must mention it may target other channels as stated in the original paper.

Line 152: Replace “C. tesselatus” with “C. tessulatus” and “Nav” with “Nav1.8”

Line 154: Replace “In the group of spiders” with “In spiders” and “species” with “species produce toxins”

Line 155: Replace “Phoneutria nigriventer” with “Phoneutria nigriventer”

Line 162: Replace “analgesic” with “antinociceptive”

Line 165-166: Replace “CTX-MVIIA” with “ω-conotoxin MVIIA”

Line 168: Replace “calcium” with “Cav”

Line 170-171: Replace “only action” with “action only”

Line 175: Replace “NMDA” with “NMDA (N-methyl-D-aspartate)”

Line 179: Replace “genus Cyriopagopus” with “spider genus Cyriopagopus”

Line 182-183: Replace “IV” with “-IV” and “Ornithoctonus hainana” with “Ornithoctonus hainanaI”

Line 185: Delete “very” and specify the “high doses” used.

Line 186: Replace “Agelenopsis aperta” with “Agelenopsis aperta”

Line 187: Replace “calcium channel” with “Cav”

Line 189: Replace “Ceratogyrus darlini” with “Ceratogyrus darlingi”

Line 191: Replace “Chilobranchys jingzhao” with “Chilobrachys jingzhao”

Line 194: Replace “Phlogiellus sp.” with “Phlogiellus sp.”

Line 196: Replace “Thrixopelma pruriens” with “Thrixopelma pruriens”

Line 198: Replace “Davus fasciatus” with “Davus fasciatus”

Line 200: Grammostola porteri has been revised as a junior synonym of Grammostola rosea (World Spider Catalog). Consider replacing “Grammostola porteri” with “Grammostola rosea”

Line 207: Replace “Haplopelma lividum” with “Haplopelma lividum”

Line 210-211: Replace “Heteropoda venatoria” with “Heteropoda venatoria”

Line 215: Replace “Ornithoctonus huwena” with “O. huwena”

Line 216: Replace “ziconotide” with “ω-conotoxin MVIIA”

Line 218-219: Replace “Hysterocrates gigas that acts on CaV channels (specifically Cav2.3),” with “Hysterocrates gigas that acts on Cav2.3,” 

Line 222: Replace “Buthus sp.” with “Buthus sp.”

Line 223: Replace “Inducing” with “These toxins induce”

Line 225: Replace “Hottentotta franzwerneri” with “Hottentotta franzwerneri”

Line 226: Replace “receptors” with “channels”

Line 228: Replace “Heterometrus laoticus” with “Heterometrus laoticus”

Line 229-230: Replace “in the” with “into”

Line 231: Replace “It was observed an important” with “The crude venom induced”

Line 234: Replace “Gloydius ussuriensis” with “Gloydius ussuriensis”

Line 235: Delete “very”

Line 238: Replace “voltage-dependent potassium receptors” with “Kv channels”

Line 239: Replace “Tetradontidae” with “fish Tetradontidae”

Line 241: Replace “analgesic” with “antinociceptive”

Line 245-246: Replace “Trachycephalus typhonius and Bufo gargarizans” with “Trachycephalus typhonius and Bufo gargarizans”

Line 250: Replace “Hyla annectans” with “Hyla annectans”

Line 255: Replace “Naja atra” with “Naja atra”

Line 257: Replace “S. subspinipes multilans make” with “Scolopendra subspinipes mutilans produces”

Line 261: Replace “Opioids and Cannabinoids” with “Opioid and Cannabinoid Receptors”

Line 280: Replace “Phyllomedusa bicolor” with “Phyllomedusa bicolor”

Line 287: Replace “Phyllomedusa sp.” With “Phyllomedusa sp.”. Please confirm the suitability of Refernce 140.

Line 290: Replace “Hannalgesin” with “Hannalgesin from the venom of king cobra (Ophiophagus hannah)”

Line 296: Replace “Crotalus durissus terrificus, C.d.t” with “Crotalus durissus terrificus”

Line 306: Replace “analgesic” with “antinociceptive”

Line 309: Replace “crotamine” with “C. d. terrificus crotamine”

Line 310: Replace “voltage-gated sodium” with “Nav”

Line 312: Please elaborate more on “strong biological activity”.

Line 314: Replace “Naja naja atra” with “N. n. atra”. Why it’s named “Naja naja atra” here yet, in line 255, it’ called “Naja atra” (assuming they are the same species)?

Line 318: Replace “Micrurus lemniscatus” with “Micrurus lemniscatus”

Line 321: Replace “Cannabis sp,” with “Cannabis sp. and”

Line 331: Replace “et al” with “et al” and ”Conus” with “Conus”

Line 335: Replace “C. textile” with “C. textile”

Line 338-339: Replace “Phoneutria nigriventer” with “P. nigriventer”

Line 344: Replace “Bufo gargarizans” with “B. gargarizans”

Line 347-359: It would read better if this section was moved to line 314 (after the paragraph on crotamine) as they are related to toxins from the rattlesnake.

Line 362: Replace “TRPs (Transient Receptor Potential)” with “TRP (Transient Receptor Potential) Channels”

Line 364: Replace “Drosophila sp,” with “Drosophila sp.”

Line 375: Replace “genus Heteractis crispa” with “Heteractis crispa”

Line 377: Replace “consists” with “consist”

Line 382: Replace “Metridium senile” with “Metridium senile”

Line 383: Replace “in vitro and in vivo” with “in vitro and in vivo”

Line 391: Replace “Urticina eques” with “Urticina eques”

Line 394 and 399: Replace “Phoneutria nigriventer” with “P. nigriventer”

Line 401: Replace “ziconotide” with “ω-conotoxin MVIIA”

Line 404: Replace “c-conotoxin” with “ω-conotoxin”

Line 407: Replace “voltage-gated calcium ion” with “Cav”

Line 413: Replace “Grammostola rosea” with “G. rosea”

Line 417: Replace “in vitro” with “in vitro”

Line 420: Replace “Buthus martensii.” with “B. martensii.”

Line 424: Replace “crotalphine” with “crotalphine from C. d. terrificus”

Line 428: Replace “C. d. t.” with “C. d. terrificus”

Line 451: Replace “Heteractis crispa” with “H. crispa” and “Urticina grebenyi” with “U. grebelnyi”

Line 453: Replace “Dendroaspis polylepis” with “Dendroaspis polylepis”

Line 456: Replace “et al” with “et al”

Line 462: Replace “Nicotinic Receptors (nAChRs)” with “Nicotinic Acetylcholine Receptors (nAChRs)”

Line 463: nAChRs are not G protein-coupled receptors.

Line 467-468: The nAChR subtypes involved must be specified. Activation of the α7 subtype has been indeed proposed to mediate anti-inflammatory response, however the same response is putatively also achieved via the inhibition of α9-containing subtypes. 

Line 470-471: Replace “Epipedobates tricolor” with “Epipedobates tricolor”

Line 475: Replace “group” with “group,” and “analogue BuIA” with “α-conotoxin BuIA”. BuIA itself is capable of inducing antinociceptive effect. It seems inconsistent to mention “analogue BuIA” whereas the rest are “wild-type” toxins.

Line 477: Replace “Conus sp. (C. lividus, C. bullatus, C. regius, C. victoriae, C. marmoreus, C. literatus and C. generalis)” with “Conus sp. (C. lividus, C. bullatus, C. victoriae, C. marmoreus, C. litteratus and C. generalis, respectively)”

Line 478: Replace “nAChRs channels” with “nAChRs” and specify the subtype involved

Line 479: Insert References 219-227 after”pain.” Reference 225 may not be appropriate as it focuses on an analogue of RgIA.

Line 480: Replace “RgiIA from C. regius” with “RgIA”

Line 481: Replace “[219-227]” with “[224]”. See comment for line 479 on Reference 225.

Line 481-484: This section appears out of place as it talks about synthetic analogues of BuIA. See comment for line 475 on BuIA.

Line 485: Replace “snake’s” with “snake”

Line 486: This is the only time where “CT” is used. Consider delete. Replace “Naja naja atra” with “N. n. atra”.

Line 487: Replace “central nervous system (CNS)” with “CNS”

Line 488: Replace “Naja naja kaouthia” with “N. n. kaouthia”.

Line 494: Replace “Bufo bufo gargarizans” with “B. b. gargarizans”. Why it’s named “Bufo bufo gargarizans” here yet, in line 344, it’ called “Bufo gargarizans” (assuming they are the same species)?

Line 494-495: Replace “nicotinic receptors” with “nAChRs” and specify the subtype involved.

Line 495: Replace “Phoneutria nigriventer” with “P. nigriventer”

Line 499: Replace “GABA” with “γ-Aminobutyric acid (GABA)”

Line 514: Replace “in vitro and in vivo” with “in vitro and in vivo”

Line 515: Replace “voltage-gated calcium (Cav)” with “Cav” and “GABA” with “GABAB”

Line 517: Replace “toxins” with “conotoxins”

Line 519: Replace “injury” with “injury by modulating GABAB receptors”

Line 526: Delete “, which are called iGluRs”

Line 527-528: Delete “(N-methyl-D-aspartate)”

Line 536: Replace “cone snails of the genus Conus sp.” with “Conus cone snails”

Line 538: Replace “Conus achatinus” with “C. achatinus”

Line 540-541: Replace “Conus geographus” with “C. geographus”

Line 544: Replace “Trichonephila clavata” with “Trichonephila clavata”

Line 549: Replace “Phoneutria nigriventer” with “P. nigriventer”

Line 553: Replace “Tityus serrulatus” with “Tityus serrulatus”

Line 554: Replace “channels changing” with “receptors by changing” Specify the “changing”. Increased? Decreased?

Line 562: Alpha2-adrenoreceptor belongs to the adregneric receptor Section 2.9 is it not?

Line 572: The statement” related to pain DRG and peripheral glial and visceral organs” does not make sense.

Line 576: Replace “Geolycosa sp.” with “Geolycosa sp.”

Line 579: “[269,269]”? Shouldn’t it be “[268,269]”?

Line 580: Replace “Bufalin” with “B. gargarizans bufalin”

Line 590: Replace “Apis mellifera” with “The bee Apis mellifera”

Line 605-606: Replace “Freund's complete adjuvant” with “CFA” and “chronic constriction injury” with “CCI”

Line 607: Replace “Polybia occidentalis” with “Polybia occidentalis”

Line 621: Replace “Naja naja atra” with “N. n. atra”.

Line 634: Replace “dorsal root ganglion” with “DRG”

Line 635: Replace “central nervous system” with “CNS”

Line 637-638: Replace “CCK” with “CCKr” and “Cholecystokinin receptors” with “CCKr”

Line 640: Replace “Hyla caerulea” with “H. caerulea”

Line 644: Replace “CCK receptors” with “CCKr”

Line 658: Replace “bunodenosine” with “bunodosine” and “Bunodosoma cangicum” with “Bunodosoma cangicum”

Line 668: Replace “Acanthoscurria gomesiana” with “Acanthoscurria gomesiana”

Line 671: Replace “Brachypelma albopilosum” with “the curlyhair tarantula Brachypelma albopilosum”

Line 674: Replace “the Complete Freund's Adjuvant (CFA)” with “CFA”

Line 675: Replace “Apis mellifera” with “A. mellifera”

Line 683: Replace “Apis mellifera syriaca” with “A. m. syriaca”

Line 687: Replace “Parachartergus fraternus” with “Parachartergus fraternus”

Line 695: Replace “Pseudopolybia vespiceps” with “Pseudopolybia vespiceps”

Line 696: Replace “toxin” with “ant toxin” and “Pseudomyrmex triplarinus” with “Pseudomyrmex triplarinus”

Line 697: Replace “Dinoponera quadriceps” with “Dinoponera quadriceps”

Line 699: Replace “Paraponera clavata” with “the bullet ant Paraponera clavata”

Line 702: Replace “Buthus martensii.” with “B. martensii.”

Line 707: Replace “lepucetin” with “leptucin” and “Hemiscorpius lepturus” with “Hemiscorpius lepturus”

Line 710: Replace “Scolopendra subspinipes mutilans” with “S. s. mutilans”

Line 712-713: Replace “Stichodactyla mertensii, Stichodactyla gigantea, Pseudopterogorgia elisabethae, and Pelagia noctiluca” with “Stichodactyla mertensii, Stichodactyla gigantea, Pseudopterogorgia elisabethae, and Pelagia noctiluca”

Line 716-717: Replace “species of Conus sp., including Conus striatus, C. coronatus, C. virgo, and C. frigidus,” with “Conus sp. including C. striatus, C. coronatus, C. virgo, and C. frigidus,”

Line 717-718: Replace “C. imperialis” with “C. imperialis”

Line 719: Replace “Conus parvatus” with “C. parvatus”

Line 723: Replace “C. virgo and C. striatus” with “C. virgo and C. striatus”

Line 727: Replace “toad” with “the toad B. gargarizans”

Line 732: Replace “Hyla japonica” with “the Japanese tree frog H. japonica”

Line 734: Replace “Bufo gargarizans” with “B. gargarizans”.

Line 737: Replace “Neurotensin B” with “Neuropeptide B” and “Bufo gargarizans” with “B. gargarizans”.

Line 740: Replace “Bungarus fasciatus” with “Bungarus fasciatus”

Line 742: Replace “C. d. t.” with “C. d. terrificus”

Line 760: The sentence “Some of these toxins have been used to produce new antinociceptive drugs and are already in advanced stages of study” is misleading the examples given. Prialt is commercially available and it not new nor in advanced stages of study.

Line 762: Replace “Conus magus” with “C. magus”

Line 763-764: Replace “fishes of the Tetrodontinae” with “the fish Tetraodontidae”

Line 775: Replace “Venomics” with “venomics”

Line 780-795: Fix edits in Conclusions and Materials and Methods.

Line 1720: Fix the edit for Reference 352.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplementary Materials

Table S1

Page 1:

Column 1, lines 3-14 (Buthus martensii) can be merged, and Column 2 lines 3-14 can be merged

Replace “ANEPtoxin” with “ANEP”

 

Page 2:

Replace “tanetanus” with “tunetanus”

Replace “tesselatus” with “tessulatus”

Grammostola porteri has been revised as a junior synonym of Grammostola rosea (World Spider Catalog). Consider replacing “Grammostola porteri” with “Grammostola rosea”

Replace “Glovdius” with “Gloydius”

 

Page 3:

Column 1, lines 3-4 (Ornithoctonus huwena) can be merged, and Column 2 lines 3-4 can be merged

Column 1, lines 7-9 (Phoneutria nigriventer) can be merged, and Column 2 lines 7-9 can be merged

Replace “S. subspinipes mutilans” with “Scolopendra subspinipes mutilans”

Replace “Trachycephalus typhonius” with “Trachycephalus typhonius”

 

Table S2

Page 4:

Replace “[166,167]” with “[165,166]”

Column 1, lines 4-5 (Crotalus durissus terrificus) can be merged, and Column 2 lines 4-5 can be merged

Replace “Crotalus d. terrificus” with “Crotalus durissus terrificus”

Replace “[169-171]” with “[167-169]”

“Naja naja atra” or “Naja atra” as in Table S1?

Column 1, lines 9-10 can be merged, and Column 2 lines 9-10 can be merged

Replace “[165]” with “[164]”

Replace “[163,164]” with “[163]”

Please confirm if Reference 140 is suitable.

 

Table S3

Page 4:

Replace “[101,197]” with “[101,194]”

Replace “[195,196]” with “[192,193]”

 

Page 5:

Replace “Heteractis crispa” with “Heteractis crispa”

Replace “[183,184]” with “[181,182]”

Replace “Metridium senile” with “Metridium senile”

Replace “[185,186]” with “[183,184]”

Column 1, lines 4-5 (Phoneutria nigriventer) can be merged, and Column 2 lines 4-5 can be merged

Replace “[188,189]” with “[186,187]”

Replace “[190-194]” with “[55,188-191]”

Replace “[187]” with “[185]”

 

Table S4

Page 5:

Column 1, lines 1-2 (Apis mellifera) can be merged, and Column 2 lines 1-2 can be merged

Replace “[265,279-281]” with “[261,275-277]”

There is no mention in the text about Apis venom acting on NMDA receptors. Also the references are incorrect.

Replace “[274]” with “[270]”

“Bufo bufo gargaruzans” or “Bufo gargaruzans”?

Replace “[227]” with “[234]”

Replace “[308]” with “[304]”

Replace “achantinus” with “achatinus”

Replace “[258]” with “[254]”

Replace “Conus aulicus” with “Conus aulicus”

Replace “GABA” with “GABAB receptor”

Replace “[250]” with “[246]”

BuIA itself has analgesic activity. Why are analogues of BuIA mentioned?

Replace “[222]” with “[219]”

 

Page 6:

Replace “[223]” with “[220]”

Replace “NMDA” with “NMDA receptor”

Replace “[259,260]” with “[255,256]”

Replace “lt14a” with “Conotoxin lt14a”

Replace “[224]” with “[221]”

Replace “[225]” with “[222]”

Replace “[226]” with “[223]”

Replace “[227,228]” with “[224,225]”

Replace “[229,230]” with “[226,227]”

Replace “[209,210]” with “[206,207]”

nAChRs is out of aligment

Replace “[216,221]” with “[216,218]”

Recombinant is out of aligment

Replace “[272,273]” with “[268,269]”

Replace “[206,207]” with “[203,204]”

Replace “CCK” with “CCKr”

Replace “[302,303]” with “[298,299]”

Column 1, lines 13-14 (Naja naja atra) can be merged, and Column 2 lines 13-14 can be merged

Define “CNT”

Replace “[293]” with “[289]”

Replace “[232-236]” with “[229-233]”

 

Page 7:

Column 1, lines 2-3 (Phoneutria nigriventer) can be merged, and Column 2 lines 2-3 can be merged

Replace “[238]” with “[235]”

Replace “NMDA” with “NMDA receptor”

Replace “[263]” with “[259]”

Replace “AMPA” with “AMPA receptor”

Replace “[261,262]” with “[257,258]”

Replace “[264]” with “[260]”

Replace “[208]” with “[205]”

 

Table S5

Page 7:

Replace “found to interact no known receptors” with “with no known targets”

Replace “[309]” with “[305]”

Replace “[311-314]” with “[307-310]”

Replace “[315]” with “[311]”

Replace “[310]” with “[306]”

Column 1, lines 5-7 (Bufo gargaruzans) can be merged, and Column 2 lines 5-7 can be merged

Replace “[338-340]” with “[334-337]”

Replace “[343]” with “[339]”

Replace “Peptide B” with “Neuropeptide B”

Replace “[344]” with “[340]”

 

Page 8:

BF-4VS is not a toxin, but a fraction of snake venom. This must be clarified

Replace “[345]” with “[341]”

Column 1, lines 2-6 (Buthus martensii) can be merged, and Column 2 lines 2-6 can be merged

Replace “[322]” with “[318]”

Replace “[324]” with “[320]”

Replace “[325,326]” with “[321,322]”

Replace “[327]” with “[323]”

Replace “[323]” with “[319]”

Replace “[333]” with “[329]”

Replace “[334]” with “[330]”

Replace “[335]” with “[331]”

Replace “[336]” with “[332]”

Replace “[337]” with “[333]”

Replace “[346,347]” with “[342,343]”

Replace “[319,320]” with “[315,316]”

Replace “Lepucitin” with “Leptucin”

Replace “[328]” with “[324]”

Replace “[342]” with “[338]”

Replace “[317]” with “[313]”

Replace “[321]” with “[317]”

Replace “[332]” with “[328]”

Replace “[318]” with “[314]”

Replace “[316]” with “[312]”

Replace “[331]” with “[327]”

Replace “[341]” with “[337]”

 

Page 9:

Replace “[329]” with “[325]”

Replace “[330]” with “[326]”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Author Response

Response to Reviewer 2 Comments

 

Dear Reviewer,

Thank you for your careful review and insightful comments on my manuscript. I have found your feedback to be extremely helpful. All points raised have been addressed, and the appropriate revisions have been made to the text. My specific responses to each comment are outlined below.

Comments 1: As the review is about animal venoms and toxins derived from animal venoms, it is accurate to add “animal” to the title. Also, given the articles reviewed used animal models in their investigations, the term “analgesic” should not be used. The term ‘analgesic’ should be reserved for human studies whereas for animal models, “antinociceptive” would be appropriate. Replace “Exploring the Pain-Relieving Potential: Unveiling Analgesic Properties in Venoms and Toxins” with “Exploring the Pain-Relieving Potential: Unveiling Antinociceptive Properties in Animal Venoms and Toxins”

R: We have made the requested changes to the article title.

Comments 2: Line 19: Fix the edits. Line 780-795: Fix edits in Conclusions and Materials and Methods. Line 1720: Fix the edit for Reference 352.

R: The edits have been corrected.

Comments 3: All suggested replacements and merges.

 

R: All substitutions and merges suggested in both the manuscript and the supplementary material have been adopted.

 

Comments 4: Line 76: Reference 24 is about CNV1014802, also known by its generic name Raxatrigine and Reference 25 is about Halneuron, not Prialt.

R: The references in this paragraph have been corrected.

 

Comments 5: Line 78. Insert Reference 25 after “…clinical studies”

R: Reference 25 has been relocated to the suggested location.

Comments 6; Line 81-82: The statement “to identify those most likely to become new biopharmaceuticals on the market.” is misleading as one would expect a summary of toxins with potentials that have been identified as therapeutic agents.

R: The paragraph was rewritten as follows: Therefore, the main objective of this study was to present the antinociceptive mole-cules found in different animal groups, as well as their different mechanisms of action, and to identify those with the potential to be studied and become future biopharmaceuti-cals.

 

Comments 7: Line 88-90: The sentence “…were categorized according to Figure 1 into the following categories: venom characterization, clinical manifestations, envenoming, antimicrobial/parasite, plants, other therapeutic properties, thesis, books, congress, and evolution/genetics.”. How is this related to Figure 1?

R: We made a typo; the correct wording was "Figure 2".

Comments 8:  Figure 2: Abbreviations must be defined.

R: The abbreviations have been added to the caption.

 

Comments 9: Line 124: The sentence “main component of the drug Prialt” is incorrect. Prialt refers to the commercial name of ziconotide/ω-conotoxin MVIIA.  References 24 and 25 have nothing to do with Prialt. Replace “calcium channels” with “Cav2.2”

R: We rewrote this passage: Conus species are widely studied for producing toxins that interact with VGCs, among them, ω-Conotoxin MVIIA stands out. Popularly known as ziconotide, this peptide is the main component of the drug Prialt® [23].

 

Comments 10: Line 200: Grammostola porteri has been revised as a junior synonym of Grammostola rosea (World Spider Catalog). Consider replacing “Grammostola porteri” with “Grammostola rosea”

R: We have updated the correct nomenclature for the animal mentioned.

Comments 11: Line 235: Delete “very”

R: We deleted this word from the mentioned passage.

Comments 12: Line 287: Replace “Phyllomedusa sp.” With “Phyllomedusa sp.”. Please confirm the suitability of Refernce 140.

R: Citation 140 has a section that discusses the cited toxin, therefore its reference was changed.

Comments 13: Line 347-359: It would read better if this section was moved to line 314 (after the paragraph on crotamine) as they are related to toxins from the rattlesnake.

R: The paragraph was moved to the suggested location to make the text more coherent.

Comments 14: Line 463: nAChRs are not G protein-coupled receptors.

R: This information regarding the recipient has been corrected.

Comments 15: Line 467-468: The nAChR subtypes involved must be specified. Activation of the α7 subtype has been indeed proposed to mediate anti-inflammatory response, however the same response is putatively also achieved via the inhibition of α9-containing subtypes. 

R: The main pain-related subtypes in this receptor class have been added.

Comments 16: Line 479: Insert References 219-227 after”pain.” Reference 225 may not be appropriate as it focuses on an analogue of RgIA.

R: The citations within the paragraph have been relocated.

 

Comments 17: Line 481-484: This section appears out of place as it talks about synthetic analogues of BuIA. See comment for line 475 on BuIA.

R: We made this correction both in the body of the text and in the supplementary material.

 

Comments 18: Line 527-528: Delete “(N-methyl-D-aspartate)”

R: Thank you for letting us know; this information was repeated unnecessarily in the text.

 

Comments 19: Line 562: Alpha2-adrenoreceptor belongs to the adregneric receptor Section 2.9 is it not?

R: Indeed, the information belonged there, so we moved the paragraph to the appropriate topic.

Comments 20: Line 572: The statement” related to pain DRG and peripheral glial and visceral organs” does not make sense.

R: It was indeed a writing error; therefore, the articles used to write this passage were reread, and we rewrote the text as follows: Activation of these receptors is primarily related to controlled ATP release into the environment allowing the influx of hypertension and calcium into the cell, and has been associated with various types of pain, including neuropathic pain, cancer pain, allodynia, and acute inflammatory pain [262, 264, 265].

Comments 21: Line 760: The sentence “Some of these toxins have been used to produce new antinociceptive drugs and are already in advanced stages of study” is misleading the examples given. Prialt is commercially available and it not new nor in advanced stages of study.

R: We made this mistake in the paragraph and rewrote it in a way that makes more sense: Considering that most of the toxins found interact with ion channels, these may be strong candidates to become alternative biopharmaceutical targets. Some of these toxins are used in the production of analgesic drugs, such as Prialt®, derived from C. magus, and Halneuron®, produced from tetrodotoxin first found in fish of the Tetrodontinae family.

 

Comments 22: Line replacements 762, 763-764, 775, 780-785, 1720.

 

R: All substitutions were made in the text.

 

Comments 23: Supplementary Materials replacements

 

R: All substitutions were made in the text of Supplementary Materials tables.

 

 

 

All modifications to the text are marked in yellow to make them easier to locate. With these changes, we have aimed to comprehensively address the feedback. Please do not hesitate to let us know if anything further is needed.

 

We are truly grateful for your insightful comments, which have been invaluable to this manuscript's development.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The review article entitled "Exploring the Pain-Relieving Potential: Unveiling Analgesic Properties in Venoms and Toxins" is a thorough bibliographic investigation of the drug candidates derived from animal venoms and toxins that have potential pain relief effects. The research has been conducted appropriately and the manuscript is very well organized according to target receptor. The supporting information on the toxins are presented in a very detailed and useful manner, although a link to a database record (e.g. PubChem) could be as well useful. Overall, the review is very comprehensive and is presented in a way to attract the interest of the Toxins' readers. Considering that it meets the quality standards of MDPI, I can suggest its publication after the authors consider the following minor point to improve its quality and usefulness to the scientists in the field.

Figures

• Fig. 1: "Adr" and "Adn" should be changed to "ADR" and "ADN" according to the provided abbreviations, both in the figure, the legend, and throughout the manuscript.
• Fig. 2: Either the full receptor names or their abbreviation as provided in Fig. 1 should be used throughout the pie chart. Also, the labels of the bottom semicircle should be inverted so as to be legible. "NA" is not described in the legend and could be as well given as "N/A" == not available or "Unk." == unknown.
• Fig. 2 in Methods should be renumbered as Fig. 3

Section titles

• 2.2 - 2.13 titles should follow the format of 2.1, i.e. 2.x Receptor name (abbreviation). In this respect, the title of 2.2 should read the receptors and not the substrates.

• 2.14 should be corrected to "Not known target" or changed to "Unknown target".

Content-specific

Section 2.15 shall be a new section, i.e. Section 3, as it is not related to the previous subsections 2.x. Importantly, this part is rather weak and should be further elaborated in a concise, but more comprehensive way to demonstrate:

• The success of the antinociceptive drugs presented versus the challenges of the clinical candidates in separate paragraphs, including more information.
• The advantages and disadvantages of the toxins versus small-molecules as drug candidates. A minor point to consider here is that the size range between "5kDa and 500Da" should be better presented as "500 - 5,000 Da" so as to be more appropriate.  In this paragraph, the sentence "because small molecules ..." in lines 772-774 should be rewritten in a better way without vague generalizations. 
• A new paragraph in the end, should provide the efforts to improve the potential of toxins as drugs by providing clear examples of pharmacological optimization, routes of administration and other works toward this direction. This is perhaps the most interesting part to stimulate the research in the field.

Lastly, the abbreviations should be thoroughly checked so as to be consistent throughout the whole text.

Author Response

Response to Reviewer 3 Comments

 

Dear Reviewer,

Thank you for your careful review and insightful comments on my manuscript. I have found your feedback to be extremely helpful. All points raised have been addressed, and the appropriate revisions have been made to the text. My specific responses to each comment are outlined below.

 

Comments 1: Figures

• Fig. 1: "Adr" and "Adn" should be changed to "ADR" and "ADN" according to the provided abbreviations, both in the figure, the legend, and throughout the manuscript.
• Fig. 2: Either the full receptor names or their abbreviation as provided in Fig. 1 should be used throughout the pie chart. Also, the labels of the bottom semicircle should be inverted so as to be legible. "NA" is not described in the legend and could be as well given as "N/A" == not available or "Unk." == unknown.
• Fig. 2 in Methods should be renumbered as Fig. 3

 

R: There were indeed some errors and missing important information in the figures and their captions; we have made the corrections.

 

Comments 2: Section titles

• 2.2 - 2.13 titles should follow the format of 2.1, i.e. 2.x Receptor name (abbreviation). In this respect, the title of 2.2 should read the receptors and not the substrates.
• 2.14 should be corrected to "Not known target" or changed to "Unknown target".

R:  We standardized the titles in section 2.

 

 

Comments 3: Content-specific

3.1 Section 2.15 shall be a new section, i.e. Section 3, as it is not related to the previous subsections 2.x. Importantly, this part is rather weak and should be further elaborated in a concise, but more comprehensive way to demonstrate:

R: We removed item 2.15 from within section 2 and transformed it into section 3.

 

3.2 The success of the antinociceptive drugs presented versus the challenges of the clinical candidates in separate paragraphs, including more information.

 

R: Thank you for this insightful suggestion. We have added two separate paragraphs in the Trends and Challenges section to contrast the success of approved drugs like Ziconotide with the challenges faced by clinical candidates such as Tetrodotoxin and Nav-selective toxins. We specifically addressed the trade-offs between molecular precision and delivery limitations, as well as the 'translational gap' between preclinical rodent models and human clinical outcomes."

 

This sentence has been modified in the text for more clarity to the reader: “The clinical success of venom-derived analgesics is underscored by the high affinity and specificity of these toxins for ion channels, exemplified by Ziconotide (Prialt®)—a synthetic w-conotoxin MVIIA that targets N-type voltage-gated calcium channels (CaV2.2) to provide relief for refractory pain without opioid-related risks—and Tetrodotoxin (TTX/Halneuron®), which is utilized in palliative care. These molecules, shaped by millions of years of evolutionary refinement, achieve a level of molecular precision that traditional small-molecule drugs rarely attain, establishing venoms as essential 'biological blueprints' for developing biopharmaceuticals that effectively bypass conventional opioid pathways [23, 28].”

 

This sentence was added to the text: There are still several challenges facing clinical candidates. The success of Ziconotide, many promising candidates encounter significant hurdles during clinical trials. A major challenge is the narrow therapeutic index; for instance, many Nav1.7 or Nav1.8 blockers derived from spider and scorpion toxins show remarkable efficacy in rodent models but fail in humans due to off-target effects on the cardiovascular or neuromuscular systems. Furthermore, the delivery remains a bottleneck. While Ziconotide requires invasive intrathecal administration to reach its CNS targets, many newer candidates (such as modified w-conotoxins) have failed Phase II trials because they could not achieve stable therapeutic concentrations in human plasma due to rapid proteolytic degradation. The 'translational gap'—where molecules perform well in reflex-based animal pain models but fail to address the complex, multidimensional nature of human chronic pain—remains the primary obstacle for candidates currently in the pipeline.

 

 

3.3 The advantages and disadvantages of the toxins versus small-molecules as drug candidates. A minor point to consider here is that the size range between "5kDa and 500Da" should be better presented as "500 - 5,000 Da" so as to be more appropriate.  In this paragraph, the sentence "because small molecules ..." in lines 772-774 should be rewritten in a better way without vague generalizations.

 

R: We restructured this paragraph to make it a bit more specific:  Furthermore, most antinociceptive toxins, in addition to the drugs mentioned above, have a molecular mass of less than 5 kilodaltons (kDa) and greater than 500 daltons (Da). According to Muttenthaler et al., approximately 75% of drugs marketed in 2019 were based on low molecular weight molecules, 20% were biopharmaceuticals, and 5% were peptides [349]. The work of Wang et al. and Craik et al. identified that the future of research on molecules with pharmaceutical potential will focus on peptides located between sizes of 500 Da and 5000 Da, since there is a gap in the development of peptide-based drugs with intermediate sizes, which may yield different results from those found in small and large molecules, such as greater specificity and precision with their pharmacological tar-gets [350-351].

 

3.4 A new paragraph in the end, should provide the efforts to improve the potential of toxins as drugs by providing clear examples of pharmacological optimization, routes of administration and other works toward this direction. This is perhaps the most interesting part to stimulate the research in the field.

 

R: We strongly agree with the reviewer that highlighting efforts to optimize these molecules is essential to stimulate future research. We have added a new concluding paragraph to Conclusions that details pharmacological optimization strategies—such as the development of synthetic analogues and recombinant production—alongside innovative routes of administration (intranasal, topical) and the use of nanocarriers (micelles, liposomes) to enhance stability and safety.

 

This sentence was added to the text: "To fully realize the clinical potential of toxins as biopharmaceuticals, research is increasingly focused on pharmacological optimization and innovative delivery systems. Strategies such as the development of synthetic analogues with enhanced stability and selectivity, as demonstrated with KIIIA and AC1 conotoxins , and the use of recombinant technology for scalable production of molecules like Pha1$\beta$ and crotamine, are critical steps toward therapeutic viability. Furthermore, to overcome the limitations of invasive administration—typically associated with intrathecal delivery—new routes are being explored, including intranasal and topical applications that have shown promising antinociceptive effects in preclinical models. The integration of nanotechnology also plays a pivotal role; for instance, the association of toxins with micelles or liposomes has proven effective in protecting these peptides from degradation, facilitating tissue permeation, and significantly reducing motor or systemic side effects. These multifaceted efforts to refine 'biological blueprints' through bioengineering and nanotechnology are essential to bridging the translational gap and establishing toxins as the next generation of safe, non-opioid analgesics."

 

Comments 4: Lastly, the abbreviations should be thoroughly checked so as to be consistent throughout the whole text.

R: We double-checked all abbreviations throughout the text.

 

All modifications to the text are marked in yellow to make them easier to locate. With these changes, we have aimed to comprehensively address the feedback. Please do not hesitate to let us know if anything further is needed.

 

We are truly grateful for your insightful comments, which have been invaluable to this manuscript's development.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have addressed the comments to my satisfaction. No further corrections required.

Author Response

Dear Reviewer,

Thank you for your careful review and insightful comments on my manuscript.

 

Comments:

 

1. Authors have addressed the comments to my satisfaction. No further corrections required.

 

R: Thank you.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript is significantly improved. However, there are minor issues to be rectified.

 

Comments. 

 

Introduction

Line 74: Replace “venoms animals” with “in animal venom”

 

Results

Line 117: Replace “(.1-1.4,…)” with “(1.1-1.4,…)”

Line 124, 139-139, 144: Replace “ω-Conotoxin” with “ω-conotoxin”.

Line 161: Replace “In spider’s species produce toxins” with “In toxin-producing spiders”

Line 384: Replace “t exhibited” with “sea anemone Heteractis crispa exhibited”

Line 411: Replace “conotoxinMVIIA” with “conotoxin MVIIA”

Line 422: Replace “dorsal root ganglion” with “DRG”

Line 468: Replace “their” with “the”

Line 484: Replace “RgiIA” with “RgIA”

Line 491: Replace “central nervous system (CNS)” with “CNS”

Line 557: Replace “decreasingthe” with “decreasing the”

Line 764: Replace “(CaV2.2)” with “(Cav2.2)”

Line 770-771: The statement “The success of Ziconotide, many promising candidates encounter significant hurdles during clinical trials” does not make any sense.

Author Response

Dear Reviewer,

Thank you for your careful review and insightful comments on my manuscript. I have found your feedback to be extremely helpful. All points raised have been addressed, and the appropriate revisions have been made to the text (green mark). My specific responses to each comment are outlined below.

 

Comments:

The revised manuscript is significantly improved. However, there are minor issues to be rectified.

 

Introduction

Line 74: Replace “venoms animals” with “in animal venom”

R: The substitution was made in the text (green mark).

 

Results

Line 117: Replace “(.1-1.4,…)” with “(1.1-1.4,…)”

Line 124, 139-139, 144: Replace “ω-Conotoxin” with “ω-conotoxin”.

Line 161: Replace “In spider’s species produce toxins” with “In toxin-producing spiders”

Line 384: Replace “t exhibited” with “sea anemone Heteractis crispa exhibited”

Line 411: Replace “conotoxinMVIIA” with “conotoxin MVIIA”

Line 422: Replace “dorsal root ganglion” with “DRG”

Line 468: Replace “their” with “the”

Line 484: Replace “RgiIA” with “RgIA”

Line 491: Replace “central nervous system (CNS)” with “CNS”

Line 557: Replace “decreasingthe” with “decreasing the”

Line 764: Replace “(CaV2.2)” with “(Cav2.2)”

R: All substitutions were made in the text (green mark).

Line 770-771: The statement “The success of Ziconotide, many promising candidates encounter significant hurdles during clinical trials” does not make any sense.

R: The phrase was removed from the text.