Approximately 15 % of animal species worldwide have poison for purposes, such as prey capture and defense against predators [1,2]. Sometimes the terms poison and venom are considered synonyms. However, there is a difference between the two, specifically the delivery method. A poison is generated by specialized cells or tissues or is acquired from the diet, causing prey toxicity by ingestion or contact with the poisonous animal [3]. For its part, a venom is a secretion produced in a specialized gland in one animal and delivered to a target animal through the infliction of a wound, which contains molecules that disrupt normal physiological or biochemical processes [4].

Venoms are complex mixtures that vary depending on the producing species [5]. Various proteins and peptides usually constitute them [6]. Likewise, there are non-protein compounds such as carbohydrates, lipids, metal ions and other unidentified substances [7]. These compounds and their respective toxins have different pharmacological activities [3] and in their purified forms, offer nontoxic biological activities at picomolar to nanomolar concentrations [8]. Therefore, part of the scientific research has focused on its study as molecular research tools, innovative diagnostic and therapeutic methods and biopesticides, among others [1].

This situation led to the clinical exploitation of seven venom-derived drugs for diverse pathologies [9]. An example is captopril, an Angiotensin-Converting Enzyme (ACE) inhibitor for treating high blood pressure and heart failure. The molecule was isolated from the Bothrops jararaca snake. Moreover, exenatide is contemplated to treat type 2 diabetes mellitus, developed thanks to the isolation of exendin-4, a GLP-1 receptor agonist from the venom of the Gila monster (Heloderma suspectum) [10]. Other relevant drugs are tirofiban and eptifibatide, used as antiplatelets and derived from viper snakes (Echis carinatus and Sistrurus miliarius barbourin, respectively) [10-12] and bivalirudin, the synthetic version of hirudin (with anticoagulant purposes), a protein found on leeches, which potently inhibits thrombin [13]. The rest of the list involved ziconotide (analgesic for neuropathic pain) and batroxobin (anti-clotting agent) [9].

These drugs were approved by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) [3,11]. These approvals indicate that they managed to overcome all the stages for their respective commercialization. Therefore, making these compounds available to the population requires extensive preclinical and/or clinical trials, depending on the considerations. In this way, it will be possible to know their molecular target, mechanism of action, effective dose, potential adverse effects and other relevant parameters [3].

One of the most studied groups is insects. In ancient cultures, these organisms were utilized as medicine. Nowadays, the idea is to rediscover many natural products from them [14]. Thus, this research summarizes the recent studies on insect venoms and their possible therapeutic applications.

Generalities

Distinct venoms have been employed for centuries as traditional remedies for illnesses such as arthritis, cancers and gastrointestinal issues, administering small doses of whole mixtures to accomplish the therapeutic goals [15]. There are documents from Egypt and Greece, especially bees and scorpions' venom. Nonetheless, the first reliable documentation has been attributed to Aristotle, Hippocrates, Galen and Pliny the Elder, who recommended them as analgesic and for arthritis treatment [16]. Some venomous animals comprise cnidarians (jellyfishes, sea anemones and hydra), mollusks (cone snails), annelids (leeches), arthropods (spiders, scorpions, centipedes, bees and wasps, ants, ticks and horseflies, crustaceans), echinoderms (sea urchins and starfishes) and vertebrates (fishes, snakes, lizards and mammals) [17].

The problem that insect venoms usually present is related to many allergenic components. Such substances make genetically predisposed individuals sensitive, increasing the anaphylaxis risk in future exposures [18]. This type of immune process is due to type 1 hypersensitivity, which is mediated by IgE antibodies. These molecules induce mast cell degranulation. The manifestations produced can be local (swelling, redness, or itching) or systemic [19,20]. Such a situation can be remedied by identifying the epitopes [21] and their immunological characterization [22,23], to modify this type of allergic reaction.

On the other hand, there is a limitation for obtaining, producing and stabilizing the poison from these organisms [24]. The amounts that can be extracted are small. For example, about 30,000 insects are required to collect about 1 gram of wasp venom [25]. Therefore, new ways are sought to deal with such situations. One of them is applying biotechnology through recombinant DNA techniques [26] and transcriptomics and proteomics [27].

Several studies have been carried out to learn more about the therapeutic potential of insect venoms. The pharmacological groups involved are detailed below.

Antifibrinolytic

Protease inhibitors found in venom are divided according to the enzyme class inhibited (serine, cysteine, aspartic, or metalloprotease). A protease inhibitor was found in the bee Bombus Ignitus, named BiVMPI. The recombinant version of said protein was evaluated by fibrinolytic cleavage assay and a fibrin plate experiment. The recombinant BiVMPI inhibited plasmin activity in a dose- and time-dependent manner, demonstrating an antifibrinolytic function for the molecule [28].

In addition, evaluations of poisons produced by arthropods were done, specifically scorpions, spiders and ticks. These insects are distributed in urban centers because of the large availability of shelter and food [29]. With its venom proteins, preclinical evaluations have been developed to assess the inhibition of coagulation factors XII and XIIa. Some of them come from the ticks Ixodes ricinus and Haemaphysalis longicornis [30].

As a complement, there are studies with macromolecules of triatomine insects, a group of ectoparasites that use the blood of vertebrates [31]. From Triatoma infestans, a factor called infestin 4 (with activity against FXIIa) was obtained. Nevertheless, it also shows activity against plasmin, FXa, FIXa, FVIIa and thrombin. Therefore, a genetic modification of the sequence that is in contact with the active site of FXIIa has been suggested [30]. Therefore, there is a need for safer anticoagulants and FXII is being pursued as a target for developing such agents [32]. Although trials have been established with animal models, more exhaustive investigations must continue to understand the in vivo role of FXII and its participation in thrombosis [33].

AntiviraL

Scorpions' venoms have neurotoxins that affect their prey movement by acting on their ion channels. Besides, they contain many peptides without disulfide bonds that have antimicrobial activity. One molecule with this characteristic is LaPLA2-1, one of the phospholipase A2 proteins found in the scorpion Liocheles australasiae [34].

LAPLA2-1 demonstrated total inhibitory activity against the hepatitis C virus in a concentration-dependent manner from 0.1 to 100 ng/ml. Plus, its toxicity to the host cells was determined. The compound did not show cytotoxic effects at concentrations up to 100 ng/ml and only a marginal cytotoxic effect at 1000 ng/ml. This result indicates that the action observed is specific to the virus. Evaluations were made in other viruses (Trinidad 1751 strain of dengue virus and strain of Japanese encephalitis virus), with similar results regarding the degree of inhibition [34].

Another significant contributor in this area is the bee Apis mellifera. The results from an investigation related to the hepatitis C virus revealed that its venom inhibited the pathogen infection with a 50 % Inhibitory Concentration (IC50) of 0.05 ng/mL, while the 50 % cytotoxic concentration (CC50) was 20,000 ng/ml. So, there is the possibility of developing an efficient and safe drug against this virus [35].

Furthermore, in vitro studies evaluated its antiviral activity against the Human Immunodeficiency Virus (HIV). Its PLA2 and p3bv derivatives (with 21 to 35 amino acids of PLA2) could inhibit viral fusion and/or replication, presumably by different mechanisms. For the first component, it would be the inhibition of the HIV-1 entry step, while for the second, it would be the inhibition of the interaction between the CXCR4 chemokine co-receptor and the targeted cell [36,37]. Finally, some authors suggest the possibility of exploiting the PLA2 as biological markers of the COVID-19 severity [37].

Antibacterial

Antimicrobial peptides are usually short (20 to 60 amino acid residues, 2000 to 7000 Da), cationic amphipathic molecules with distinct amino acid compositions, structures and antimicrobial mechanisms. They are generally characterized by strong alkalinity, thermal stability and broad-spectrum antibacterial activity [38,39].

These compounds' biological activity is found in many organisms, where social species such as bees, wasps and ants stand out. Their poisons allow them to defend the colonies from predators' attacks and provide anticancer, antitumor, antioxidant, antifungal and antimicrobial properties [40]. Additionally, it can induce responses to their immune system, offering protection against bacterial infections [41].

As for A. mellifera, it has two compounds of great interest in this area: apitoxin (bee's venom) [42,43] and melittin (the main component of apitoxin) [16,44]. Both were investigated against Methicillin-Resistant Staphylococcus Aureus (MRSA) planktonic and biofilm states. The information obtained was that the Minimum Inhibitory Concentration (MIC) values ​​were 7.2 and 6.7 μg/ml, respectively and the Minimum Bactericidal Concentration (MBC) values were ​​28.7 and 26 μg/ml, respectively. However, none of these substances affected the production or release of this bacterium's enterotoxin [45].

Even with melittin, a preclinical model was developed in mice with third-degree burns, evaluating the eradication of Vancomycin-Resistant S. Aureus (VRSA). 50 clinical isolates collected from burn wound infections revealed values ​​of 0.125 to 2 μg/ml for MIC and 0.125 to 4 μg/ml for MBC. Likewise, it showed an optimal safety profile, as it did not cause toxicity at the dermal level or in vivo hemolysis [46].

Regarding this compound, it has been tested in hip replacement surgeries as an antibacterial coating to establish a synergistic effect with vancomycin. The cell viability of melittin in preosteoblast cell lines, along with the antimicrobial activity of the coating and the two substances administered individually against MRSA and VRSA bacterial strains isolated from burn wounds of two persons, were evaluated. Among the most representative findings were increased cell proliferation and MRSA and VRSA bacteria elimination when both substances were present [47]. For this reason, further development of these combinations is expected in the future.

On the other hand, one of the most frequently evaluated aspects is peptides' hydrophilic and hydrophobic properties [48]. Various peptide structures of insect venoms are under study, specifically eumenitin, lasiocepsin, lycosin 1, mastoparan B, panurgine1 and their modified C-terminal-NH2 analogs. There are differences between them in their degree of hydrophilicity and their hydrophobic moment. These characteristics generate diverse degrees of Escherichia coli ATP synthase inhibition, which is more significant for the analogs [40].

Wasps also play a prominent role in this development. A study on the Vespa velutina species determined the presence of four peptides with antimicrobial activity against Gram-positive bacteria (S. aureus, Bacillus subtilis and Enterococcus faecalis) and Gram-negative bacteria (E. coli and Klebsiella pneumoniae) at concentrations between 3.25 and 120 μM, depending on the microorganism. They were shown to be safe (hemolysis rates from 2.12 to 4.08 % at 120 μM) and significant antioxidant abilities against OH radicals at concentrations between 30 and 120 μM were observed [49].

As a complement, in ants such as Dinoponera quadriceps, a series of compounds called ponericins are found [50,51]. The synthesized peptides have shown antimicrobial activity against carbapenem-resistant Actinobacter baumannii, E. coli, K. pneumoniae and Pseudomonas aeruginosa with MICs between 5 and 10 μM and an optimal safety profile, as in other trials. The World Health Organization (WHO) advises that these microorganisms are part of a list of critical global priorities for new antibiotics discovery [50].

Plus, evaluation of four other substances in methicillin-sensitive and methicillin-resistant S. aureus strains was performed. Of these, one showed the best results, preventing biofilm formation at low concentrations (0.78 to 3.12 μM) and a short-time effect to observe the bacteria membrane disruption [52].

Antiparasitic

Ponericins derived from two other species of ants (Neoponera apicalis and Neoponera commutata) have been obtained and their anthelmintic potential against the nematode Haemonchus contortus, highly present in ruminants, has been appreciated. Five were characterized in their peptide sequences and three gave IC50 values ​​less than 6 μM. The minimum value of 2.8 μM is only four times lower than levamisole (commercial anthelmintic). Alone with this information, C57BL/6J mice were carried out for behavioral experiments. The injection of the synthetic ponericins induced spontaneous nocifensive behavior (licking, biting, scratching and shaking of the injected paw). Thus, they induced pain, showing its natural function of being a defensive substance against predators or for prey capture [51].

Another relevant disease is Chagas. It is a neglected illness transmitted by a triatomine insect and occasioned by Trypanosoma cruzi. About 10 million people are infected worldwide. Due to limited pharmacological development, antimicrobial peptides from the wasp Polybia paulista could be considered because of their trypanocidal activity. Of them, Polybia-CP revealed efficacy against the epimastigote (the proliferative form of T. cruzi), being higher after 24 hours of incubation (half maximal effective concentration or EC50 equals to 9.3 μmol/l) than the standard drug currently employed, benznidazole (EC50 equals to 218 μmol/l). Another essential detail is the non-observation of toxicity against the host cells (Rhesus monkey LLC-MK2 kidney cells) at the range of concentrations evaluated [53].

Oncolitic

Osteosarcoma is a bone tumor with a high degree of malignancy. This disorder can produce local damage and distant metastasis. In a preclinical study with BALB/Cnu/nu nude mice, a quantity of 143 B cells (human cells of this cancer) were inoculated subcutaneously and divided into groups of ten animals, to which a low, a medium, or a high melittin dose (160, 320 and 640 μg/kg/d, respectively) was applied. Moreover, there was a negative control (saline solution) and a positive control (doxorubicin). The treatment lasted for four courses (five days with one day between the two courses). The tumor volume was monitored every two days until the mice were sacrificed after four periods. The tumor tissues were removed and weighed and the lung tissues were examined. Melittin significantly inhibited the growth of tibia xenografts in the model and decreased the number of metastatic lung nodules. The treatment with melittin at medium and high concentrations attenuated the expression of Wnt/β-catenin signaling pathway-related factors in vivo. This pathway controls osteosarcoma incidence and progression [54].

In addition to its cytotoxic properties, this substance has immunomodulatory and cell cycle regulatory activities, affecting signaling cascades of toll-like receptors, cyclin D and Epidermal Growth Factor (EGF). Therefore, they have been studied in vitro and in vivo in ovarian, pancreatic, non-small cell lung, leukemic and hepatocellular carcinomas. Besides, strategies have been established to improve some characteristics, including linkers (increases its selectivity) and hydrogels (decreases its toxicity) [9].

Other in vitro tests have been developed with apamin, which is found in the venom of A. mellifera and has shown antitumor activity by inhibiting the migration of breast cancer cells. This effect is possible by specific binding to Ca2+-activated K+ activated channels [55].

For its part, chlorotoxin is a 36-residue peptide isolated from the venom of the scorpion Leiurus quinquestriatus. This molecule is a disulfide-rich one. It has a lot of relevant characteristics, such as potent and specific interactions with distinct physiological targets, enhanced stability and structural complexity compared to linear peptides. Because of these conditions, particularly selectivity toward glioblastoma, a product of chlorotoxin with 131I was obtained. The same is in a phase II clinical trial in people newly diagnosed with gliomas [56].

A product created with this peptide is associated with the Chimeric Antigen Receptor (CAR) T cell therapy. Animal models showed high selectivity and regression of glioblastoma tumors. The exact mechanism by which selectivity is obtained is still under study [56].

Furthermore, phase I clinical trials have been done in breast cancer, glioma and tumors of the Central Nervous System (SNC) and a phase II/III investigation with SNC cancer is under recruiting. The drug name is tozuleristide, a chlorotoxin derivative [57]. The research showed that the drug enabled real-time visualization between pathologically confirmed breast cancer and normal tissues. For its part, in glioma, it demonstrated its safety in doses of up to 30 mg [58].

Other research regarding the usefulness of tozuleristide as a fluorescent imaging agent was studied. 21 adults received the product intravenously before excision of skin tumors (both known and suspected). Fluorescence imaging procedures were determined before and 48 hours after administering the dose. The drug was well-tolerated without even identifying a maximum tolerable dose. Most tumors (four of five basal cell carcinomas and four melanomas) could be seen at doses of 3 to 12 mg [59].

Polybia-MPI, another peptide made up of 14 amino acids and isolated from the venom of the wasp P. paulista (which had shown antiparasitic activity), has been investigated for the treatment of leukemia. Its selective toxicity to human leukemia T lymphocytes is much higher than that of human primary lymphocytes [55].

At last, in vitro and in vivo investigations have revealed their utility in tumors (glioma, neuroblastoma, leukemia, lymphoma, breast, lung and prostate) regarding scorpion venoms. The mechanism of action embraces an apoptotic, antiproliferative action, the induction of cell cycle arrest and the inhibition of cancer progression [60].

Immunologic

One of the regular practices related to venoms is their immunotherapy. Such a procedure seeks to protect people against allergies caused by them [61]. It is indicated only to prevent anaphylactic reactions to stings in individuals with a history of previous systemic reactions and positive venom skin tests or serum venom-specific IgE serology [62]. Nonetheless, despite concerns about its security, it is common in some countries. In Korean and Chinese clinics, bee venom acupuncture is a standard procedure [20].

The results obtained have been satisfactory since it provides a practical therapeutic approach against various insects (77 to 84 % of bee venom allergy patients and 91 to 96 % of yellow jacket venom allergy patients) [63]. Therefore, empirical guidelines on the duration of appropriate treatment and variations of maintenance regimens have been done for multiple insects [64].

A buildup phase constitutes insect venom immunotherapy. During this first stage, growing amounts are administered until the maintenance dose of the allergen has been reached (1 day to 15 weeks). Then, the dose is administered every 4 to 6 weeks in the maintenance phase. After the second stage, the systemic reaction risk to a subsequent sting in most persons decreased from 70 to 3 % [65].

Yet, the immunomodulatory role of poisons on the immune system is complex. Regarding the venom of A. mellifera, the presence of PLA2 can promote the maturation of dendritic cells and its utilization, together with Tumor Necrosis Factor α (TNFα) and interleukin 1β (IL-1β), can induce the upregulation of costimulatory molecules (CD83 and CD86), of great importance for T lymphocyte stimulation [11].

PLA2 can also induce a Th2 response through IL-33 release. This cytokine is found in the skin and intestine. For this reason, the bee venom was contemplated a long time ago for allergic disorders (asthma) and autoimmune disease (rheumatoid arthritis) [11]. Regarding this last pathology, the venom of the bee Apis dorsata significantly reduced the levels of IL-6 and TNF-α, paw volume, arthritic index score and mortality, while increasing the body weight, dorsal flexion ability and stair climbing ability, compared to the control group in two models (Freund's adjuvant and collagen-induced arthritis) [66].

In a preclinical model, groups of seven animals had arthritis induced by applying Freund's Complete Adjuvant with olive oil. Three treatments of A. mellifera were at three doses (low: 2 mg/kg; moderate: 4 mg/kg; high: 20 mg/kg). Among the most important information obtained, low doses of venom decreased the levels of IL-1β, IL-6, TNF-α and Tumor Growth Factor β1 (TGF-β1), while interferon γ (IFN-γ) was increased. Likewise, the differences for the moderate and high doses were not statistically significant compared to the low ones, where anti-inflammatory and antioxidant effects were found [67].

Another disease in which studies have been performed is gout, related to high urate serum levels. As a complement, gouty arthritis is characterized by arthritis and the monosodium urate crystals accumulation in the intra-articular space, inducing inflammation. In a mouse model, groups of five animals were given A. mellifera poison (0.5 or 1 mg/kg) and apamin (0.5 or 1 mg/kg) after injection of monosodium urate crystals. After measuring the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, a significant decrease was found in the four groups with the therapies of interest compared to the control group injected with only the crystals. The most significant decrease was found for the groups where a dose of 1 mg/kg was applied [68].

Additionally, progress is being made at substances' delivery. In the case of melittin as a possible treatment for rheumatoid arthritis, its administration was evaluated through polymeric microneedles (micro-structured needles with a length of 200 to 1000 μm). The advantages are its ability to overcome the skin barrier non-invasive and pain-free and long enough to penetrate the skin without touching the nerves and blood vessels. This strategy was considered in a rat model, with outstanding results since both the paws and lymphatic organs showed a decrease in IL-17 and TNF-α levels and an increase in regulatory CD4 T cells [69]. In this way, this system can avoid the low compliance caused by the parenteral administration typically established in clinical practice [70].

Finally, apitoxin has been employed in preclinical models to treat ulcers induced by Nonsteroidal Anti-Inflammatory Drugs (NSAIDs). These drugs cause gastric mucosal eosinophilia. It is a gastrointestinal disorder characterized by marked diffuse eosinophilic infiltration in the lamina propria and mucosa of the bowel in cases of gastric ulcers. Groups of ten rats were administered bee venom after inducing ulcers with acetylsalicylic acid. This treatment was compared against ranitidine. A marked reduction in the ulcer index was found due to a series of venom properties (antioxidant, anti-inflammatory, anti-apoptotic and anti-secretory activities) [71].

Insect venoms have been of great importance since ancient times. There is currently research at preclinical and clinical levels to demonstrate its applicability in different therapeutic areas, thanks to its antifibrinolytic, antiviral, antibacterial, antiparasitic and immunological properties (for both cancer and autoimmune diseases). In this way, the commercialization of future therapies based on them or the products found in these mixtures is expected to grow considerably in the coming years.

In addition, it is clear from this research that nature is an essential source of resources that must be protected. Otherwise, possible therapeutic options to improve people's quality of life could be lost in the coming years.

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