Rodenticide

Abstract

The present disclosure describes novel synthetic peptide sequences based on cobra venom, which are suitable for use as cardiotoxic rodenticides.

Description

Technical Field

[0001] The present invention relates to synthetic peptides, rodenticides based on cobra snake venom, in particular cardiotoxins derived from snake venom, and the use of cardiotoxins as rodenticides. Background Art

[0002] Rats and mice are inevitably associated with human habitation, and this can have significant negative consequences, as rodents are known to consume agricultural products and stored resources. They cause damage to crops, cereals and grains, which account for almost 10% of the world's food crops, varying depending on the country (Buckle and Smith, (2015). Rodent Pests and their

[0003] control.2 nd Edition. Wallingford Oxfordshire. 330-345). They also transmit zoonotic diseases to humans and livestock and are known to chew and burrow, which can cause serious damage to buildings and power supplies (Van den Brink et al., (2018) In

[0004] Anticoagulant Rodenticides and Wildlife; Springer: Cham, Switzerland,

[0005] pp.1-9). Therefore, controlling rodents is a vital daily task worldwide, especially in agricultural environments and inside and outside buildings. Methods for managing and controlling rodent populations take several forms: improving sanitation and rodent-proofing areas, capturing rodents and moving them elsewhere, and lethal control tools such as mousetraps and rodenticides. The use of anticoagulants (ARs) has become the most attractive method for controlling rodents due to a combination of efficacy, ease of use, cost-effectiveness and the fact that they are relatively safe for operators (Jakob and Buckle, (2018). Emerging Topics in Ecotoxicology (Principles, Approaches and Perspectives), vol5. Springer, Cham.). The complex social organization of rodent populations and the neophobic behavior of rats make toxic behaviors with immediate effects ineffective (Modlinska and Stryjek, (2016) PLoS ONE 11 (6)). Anticoagulant rodenticides address this problem because they have a delayed onset of action and death usually occurs several days after consumption. Chronic or repeated exposure to AR causes death in rodents due to hemorrhage within 3 to 7 days.

[0006] Anticoagulant rodenticides (ARs) act by inhibiting vitamin K epoxide reductase (VKOR) and thereby depleting vitamin K-dependent coagulation factors (Ng WY et al. (2018) Journal of Medical Toxicology; 14 (3): 218-228). AR inhibits vitamin K(1)-2,3-epoxide reductase, thereby inhibiting the synthesis of vitamin K and subsequently inhibiting the synthesis of coagulation factors II, VII, IX and X (Hadler and Buckle, (1992) Proceedings of the Vert Pest Conference, 15 :149-155). The increased potency and duration of action of long-acting ARs is attributed to their greater affinity for vitamin K(1)-2,3-epoxide reductase and their ability to disrupt the vitamin K(1)-epoxide cycle at more than one point (Watt et al., (2005) Toxicology Reviews. 24 (4):259-69). Due to their high lipid solubility, they also have a long biological half-life and can be reabsorbed into the circulation via enterohepatic recirculation.

[0007] Warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl) chroman-2-one) was one of the first anticoagulant rodenticides (ARs) to be introduced to the market, but resistance has developed in rodents.

[0008] Although reported to have low toxicity, coumatetralin (4-hydroxy-3-(1,2,3,4-tetrahydronaphthalen-1-yl)chroman-2-one) is slightly more effective than warfarin due to its greater palatability. The compound was introduced when warfarin-resistant rat populations emerged and was used successfully for many years before resistance was reported in the United Kingdom and Denmark in the late 1960s.

[0009] Second-generation anticoagulant rodenticides (SGARs) were developed in the 1970s to overcome resistance in rats to first-generation anticoagulant rodenticides (FGARs). Second-generation anticoagulants are more toxic than first-generation ARs, and their increased potency is related to their greater affinity for vitamin K epoxide reductase (VKOR). Bromadiolone (3-[3-bromo[1,1'-biphenyl-4-yl)-3-hydroxy-1-phenylpropyl]-4-hydroxy-2H-1-benzopyran-2-one) and brodifacoum (3-[3-diphenyl-1,2,3,4-hydronaphthalen-1-yl]-4-hydroxycoumarin) were the first compounds introduced to the market, and the three most potent compounds are brodifacoum (3-[3-(4'-bromobiphenyl-4- coumarin), fluazifop (4-hydroxy-3-[l,2,3,4-tetrahydro-3-[4-(4-trifluoromethylbenzyloxy)phenyl]-l-naphthyl]coumarin) and thiazolin (3-[3-[4-(4-bromophenyl)phenyl]-1,2,3,4-tetrahydronaphthalen-1-yl]-2-hydroxythiochroman-4-one).

[0010] In the resistant rat population, VKOR is slightly altered, thus preventing proper binding of the rodenticide, which then fails to work (Thijssen, (1995) Pesticide Sciences 43 ; 73-78). The most common resistance mechanism is the result of a single nucleotide polymorphism in the VKORC1 gene (Rost et al. (2004) Nature. 427 (6974):537-41; Li et al. (2004) Nature 427 :541–544). The VKOR1 gene encodes the VKORC1 protein, which contains 163 amino acids, and specific amino acid substitutions in VKORC1 confer resistance to AR. It has been suggested that at least seven independent polymorphisms in the VKORC1 gene in Norway rats provide a genetic basis for resistance to anticoagulant rodenticides (Mooney et al. (2018) Nature. 8 :4543). Resistance has been observed to expand geographically and develop from warfarin and FGAR to more potent active substances SGAR (such as brodifacoum and fumarole). Resistance to different ARs is called cross-resistance and develops from FGAR to SGAR, so resistance to SGAR is always accompanied by resistance to FGAR (Mooney et al. (2018) supra).

[0011] The use of FGAR is now greatly restricted due to this phenomenon of genetic resistance; since 1958, numerous cases of resistance in brown rats have been reported in the United Kingdom, Europe, the United States, Canada, and Australia (Buckle et al. (1994) Proceedings of the Sixteenth Vertebrate Pest Conference. 7 ). Mutations in VKORC1 have been shown to cause two distinct genetic phenotypes; defective coagulation due to vitamin K-dependent coagulation factor deficiency type 2 (VKCFD2) and warfarin resistance (OMIM) (Rost et al. (2009) BMC genetics). Brodifacoum, fludioxonil and thiabendazim are currently the only active compounds that have shown efficacy in all resistant strains of rats and mice.

[0012] AR on the market is formulated into palatable baits for oral administration to target harmful rodents, and is usually based on grains made into blocks or pellets, with binders, flavorings and colorants added. Wax is added to some products to make them more durable and increase weather resistance elements (Horak et al. (2018) In Anticoagulant Rodenticides and Wildlife; Springer: Cham, Switzerland, pp. 87-108). Bait preparations rely on active intake of sufficient amounts to effectively combat target harmful rodents. Since AR poisoning deaths occur several days after ingestion, it is very common for rodents to continue to eat baits after ingesting a lethal dose, thereby increasing the concentration of anticoagulant rodenticides in their tissues. Unfortunately, this may also lead to undesirable primary exposure to other animals that can be exposed to the bait, and secondary oral exposure may also occur by ingesting animal tissues containing AR residues. This is dangerous for species that prey on rodents or feed on the carcasses of poisoned animals (Horak et al. (2018) Supra).

[0013] Unintentional exposure of non-target wildlife and domestic pets through primary or secondary poisoning, as well as reports of AR residues in predatory birds and non-target species are common worldwide. Primary exposure is a high risk for omnivores or herbivores because rodenticide bait formulations are cereal-based, while secondary poisoning is typically seen in scavengers or carnivores due to the consumption of tissues from animals carrying residual concentrations of AR (Fisher et al. (2019) Animals (Basel). 9(11):919). The higher toxicity and metabolic persistence of SGARs result in a higher risk of secondary mortality in non-target species (Erikson and Urban, (2004) Potential risks of Nine Rodenticides to Birds and Nontarget Mammals: A Comparative Approach; US Environmental Protection Agency: Washington, DC, USA, 2004).

[0014] The risk to non-target birds and other wildlife is influenced by species-specific susceptibility and the toxicokinetics of the compound used: for example, brodifacoum is highly toxic to birds and mammals (Eason et al. (2002) Ecotoxicology. 1 :35-48). Second generation anticoagulant rodenticides (SGARs) such as brodifacoum, bromadiolone and fenvalerate are extremely persistent in organs and tissues such as the liver, kidney and pancreas for at least 6 months and cause poisoning with a lethal dose delivered by a single feeding (Environmental Protection Agency 2004 (www.fluoridealert.org / pesticides / EPA-HQ-OPP-2006-0955-0005.pdf); Erickson and Urban, (2004) supra).

[0015] Poisoning caused by rodenticides is one of the most common types of poisoning in dogs treated by the Pet Poison Helpline (Pet Poison Helpline website, accessed 2021) and is reported to be one of the common causes of poisoning in dogs worldwide (Seljetun et al. (2020) Acta Veterinaria Scandinavica 62 :30). A study conducted in 2020 determined that brodifacoum and dapoxetine were present in dog feces for more than 700 and 650 days, respectively, after a single ingestion (Seljetun et al. (2020) supra). In one litter produced by exposed dogs, low fecal concentrations were detected 28 days after delivery. Dogs allowed to roam freely may be more likely to encounter rodent baits that may have been improperly placed or dragged into an area by rodents (Merola, (2002) Vet Med 97 :716-722).

[0016] Humans may be exposed to anticoagulants through rodenticide baits (Tran and King, (2016) Journal of Epidemiological Research. 2 (2); Watt et al. (2005) supra), and is usually taken orally; either accidentally or intentionally. Most human cases of exposure to AR involve young children, so the amounts ingested are small. Intentional ingestion of large amounts of AR can result in anticoagulant effects for weeks or months (Watt et al. (2005) supra). Ingestion of large amounts will cause epistaxis, extensive bruising, hematomas, and gastrointestinal bleeding, and severe blood loss may lead to hypovolemic shock and death.

[0017] Due to the emergence of rodent resistant strains and the environmental and non-target species risks of using second-generation anticoagulant rodenticides, the development of alternative rodenticides has become more urgent. Alternatives that reduce the risk to non-target species will be beneficial to the control of harmful rodents, and reducing persistence in the environment will be advantageous. It is obvious that rodent species are increasingly resistant to SGAR and FGAR, with brodifacoum being the only exception at present. Brodifacoum is extremely stable in the environment, has been shown to have consistent efficacy over a period of up to 30 days, and only one dose is required to induce death. The use of this more potent and more toxic rodenticide has become more common, which is usually harmful to the environment and other species. An alternative that can provide a primary target rodent, minimize the risk of primary and secondary exposure to non-target species (such as pets and predatory birds) and have a short half-life to reduce the chance of harmful effects on the environment will be the most attractive method for controlling rodents.

[0018] Therefore, it is in this context that the present invention is designed. Summary of the invention

[0019] In one aspect, the present invention is directed to a rodenticide comprising at least one compound that is cardiotoxic to rodents.

[0020] Preferably, at least one compound is selectively or specifically cardiotoxic. In other words, the at least one compound has no, substantially no, or no significant or detectable toxicity to other tissues, such as neurotoxicity or hemolytic activity.

[0021] In one embodiment, the compound has an excitatory or tachycardic, rather than an inhibitory, cardiotoxic effect. In other words, the compound causes a change in heart rate. Preferably, the compound increases heart rate and causes tachycardia. For the avoidance of doubt, inhibitory cardiotoxins slow down and / or stop heart rate, while excitatory cardiotoxins speed up heart rate, an effect known as tachycardia. Slowing down the heart rate causes a reduction in oxygen supply to essential organs (such as the brain), and therefore may cause death more quickly. Tachycardic cardiotoxins increase heart rate, exerting additional stress on the heart over time, and therefore are more likely to cause heart failure due to a heart attack. Ultimately, both will lead to the same result, i.e., the death of the animal, but through different mechanisms.

[0022] In a specific embodiment, the compound is an isolated amino acid sequence obtained or derived from a snake venom.

[0023] The advantage of using snake venom as a rodenticide is the impact on the environment. Snake venom is broken down by digestion and therefore does not persist in the food chain. In addition, because the poison is a biological composition, it will be quickly broken down in the environment and will not accumulate in, for example, soil. Therefore, the rodenticides contemplated by the present invention provide significant advantages over existing synthetic chemicals.

[0024] In one embodiment, the snake venom is obtained or derived from a species of the family Elapidae.

[0025] The elapid family (commonly known as cobras or elapids) is a family of venomous snakes characterized by permanent upright fangs at the front of the mouth. Most species have neurotoxins in their poisons for immobilization of prey and defense, which are directed through their hollow fangs, while some species may contain other toxic components in varying proportions. The main groups of toxins are phospholipase A2 (PLA2) and three-finger toxins (3FTx). Other toxic components in some species include cardiotoxins and cytotoxins, which cause cardiac dysfunction and cell damage, respectively.

[0026] Preferably, the venom is derived from or obtained from a species of cobra. Cobra is the common name for a variety of venomous snakes, most of which belong to the genus Naja. All members of this group have varying degrees of toxicity and are classified based on their rodent LD 50 Due to their high mortality and fatality rates, some cobras are considered the most dangerous snakes in the world.

[0027] In one embodiment, the amino acid sequence is cardiotoxic, preferably selectively cardiotoxic, as defined and described above. In contrast, the neurotoxic effects of snake venom are thought to be mediated through nicotinic acetylcholine receptors at the neuromuscular junction, which act to cause respiratory arrest and death.

[0028] It will be appreciated that isolated whole snake venom may be used. Such venom may be isolated from snakes by typical methods such as milking. Alternatively, the venom may be synthetic. Using intact or whole venom may minimize any additional processing and formulation required, since the protein is already in its native active form. However, a disadvantage is the additional presence of other proteins, including neurotoxins.

[0029] Another consideration is that the cytotoxicity of snake venom affects the mucous membranes of rodents, which would make the taste aversive to rodents.

[0030] Yet another consideration is the speed of lethal activity. Snake venom can kill mice within a minute, as seen during feeding. However, the sight of one or more dead rodents next to or near the baited food will deter other rodents from consuming the food, thus avoiding poisoning. Thus, a delayed lethal onset is desirable.

[0031] Therefore, as a result, another aspect of the present invention includes synthetic peptide sequences or artificial peptide sequences, which include sequences based on or derived from cobras or cobra three finger toxin (3FTx).

[0032] 3FTx is a protein superfamily of small toxin proteins found in snake venom. The second largest class of 3FTx proteins causes cardiomyocyte toxicity and can cause an increase in heart rate, ultimately leading to cardiac arrest. Short cardiotoxins (CTX) are single-chain polypeptides of 59-61 amino acids arranged in a three-finger fold composed of antiparallel β-strands, strengthened by 4 disulfide bonds and numerous hydrogen bonds. Long three-finger cytotoxins are approximately 81 amino acids. CTX are basic proteins with a hydrophobic three-finger ring flanked by cationic residues (mainly lysine and arginine) at their termini. The hydrophobic termini render CTX amphipathic, mediating its binding and insertion into anionic phospholipid membranes, leading to deleterious cellular events (such as pore formation and cell lysis), increasing intracellular Ca 2+ Ion influx and membrane depolarization and, importantly, internalization of the toxin leads to mitochondrial and lysosomal damage and a perturbation of the cellular cascade leading to cell death.

[0033] It should be understood that according to aspects of the present invention, whole cell toxins can be used. However, it is usually more economical to use only a portion. In addition, after production, shorter peptide sequences hardly need or need to fold or refold, or ensure correct folding. Therefore, it is advantageous to use peptides with only simple secondary structures. In addition, the secondary structure of the peptide can be designed to enhance certain properties, such as stability, thermal stability, bioavailability and species selectivity.

[0034] In a specific embodiment, the sequence is based on or derived from Loop I of a cobra or cobra three-finger toxin. It is believed that the cytotoxic effect of 3FTx resides in the Loop I sequence, as well as some amino acid residues at the base of Loop II (Menez et al. (1990) Biochimie 72: 575-588).

[0035] In one embodiment, the sequence is based on or derived from amino acid residues 1 to 30 of cobra three-finger toxin. Ideally, the sequence has between 10 and 15 amino acid residues, for example 13 amino acid residues. It should be understood that the ranges specified herein include all discrete numbers between the lower and upper numbers, for example 6, 11, 14, 21, 25 and 27 amino acids.

[0036] In one embodiment, the sequence has the following sequence:

[0037] LKC(H / N)KL(V / I)PX(V / A)(W / Y)KT [SEQ ID NO: 1].

[0038] For example, the sequence may have the following sequence and their variants:

[0039] LKCHKLVPPVWKT [SEQ ID NO: 2]

[0040] LKCNKLIPLAYKT [SEQ ID NO:3]

[0041] LKCHKLIPIAWKTK[SEQ ID NO:4].

[0042] It is helpful if the peptide includes one or more hydrophobic amino acids located at one or both ends of the sequence to aid membrane penetration. Additionally or alternatively, the sequence may include one or more amino acids that enhance solubility in water. It should be understood that the sequence may also be designed to include amino acids that enhance solubility in hydrophobic materials such as waxes.

[0043] In addition, the sequence may include one or more synthetic amino acids, may be pegylated and / or include a detectable label, such as a UV-fluorescent label. Labels detectable under UV light are particularly interesting because rodent urine is visible under UV light. Therefore, including a UV-fluorescent label can be used to assess and / or monitor the decline of rodent populations.

[0044] It will be appreciated that in this context, as described above, it is desirable if the sequence is cardiotoxic, preferably selectively cardiotoxic, thereby causing a change in the heart rate. Preferably, the change in heart rate is an increase, ie causing tachycardia.

[0045] The present invention also includes a rodenticide formulation comprising a synthetic peptide sequence as described above formulated with a carrier. The formulation may be a paste, solid, powder, gel or liquid, and may additionally comprise one or more of the following substances: wax (petroleum or plant-derived), cereals, grains, nuts, fruits, colorants, flavorings, herbs, spices, essential oils, oils, fats or other materials palatable to rodents. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will now be described in further detail with reference to the following non-limiting examples and accompanying drawings, in which:

[0047] Figure 1: Rodent cardiomyocyte and cardiac fibroblast toxicity data. Figure 1 shows the toxicity observed in neonatal rat cardiomyocytes (orange) and cardiac fibroblasts (blue) in response to treatment with a panel of purified cobra peptides at 12.5 μg / ml ( Figure 1A )、7.5μg / ml( Figure 1B ) and 6μg / ml( Figure 1C ) dosage treatment.

[0048] Figure 2 : Rodent erythrocyte toxicity data. Figure 2 The toxicity of purified Naja fractions to isolated rat erythrocytes (erythrocytes) is shown.

[0049] Figure 3 : Six exemplary lead fractions of human IPSC-derived cardiomyocyte toxicity. Figure 3 Shown are the % toxicity to human IPSC-derived cardiomyocytes following treatment with the six selected purified fractions at doses of 1 μg / ml, 2.5 μg / ml, 3.5 μg / ml, 6 μg / ml and 7.5 μg / ml.

[0050] Figure 4 : Thermal stability evaluation of six selected example components. Figure 4Shown are the thermal stability curves of six selected purified poison components. The components were heated at 37°C, 60°C, 80°C and 100°C and then applied to rat myocardial cells at a dose of 6 μg / ml for 24 hours. The amount of myocardial cell toxicity at each temperature was assessed and used to determine whether there was a loss of activity due to a lack of peptide thermal stability.

[0051] Figure 5 :SAR analysis. Figure 5 Schematic diagram showing the potential importance of amino acid residues for cardiomyocyte toxicity based on SAR.

[0052] Figure 6: The toxicity of synthetic peptides to neonatal rat cardiomyocytes and cardiac fibroblasts was evaluated using resazurin activity assay. Figure 6 shows the toxicity of three synthetically designed short peptides to neonatal rat cardiomyocytes ( Fig. 6A ) and cardiac fibroblasts ( Figure 6B ) toxicity.

[0053] Figure 7: Toxicity of synthetic peptides to human IPSC-derived cardiomyocytes and embryonic chick cardiomyocytes. Fig. 7A The toxicity of three synthetically designed short peptides to human IPSC-derived cardiomyocytes is shown; Figure 7B The toxicity of three synthetically designed short peptides to embryonic chicken cardiomyocytes is shown.

[0054] Figure 8: Fold changes in beating rate of neonatal rat and human IPSC-derived cardiomyocytes in response to treatment with synthetic peptides. Figure 8 shows the fold increase in beating rate in rat neonatal cardiomyocytes (orange) and human IPSC-derived cardiomyocytes (blue) in response to treatment with synthetic peptide 1 ( Fig. 8A )、Synthetic peptide 2( Figure 8B ) and synthetic peptide 3 ( Figure 8C ) processing.

[0055] Figure 9: Thermal stability of synthetic peptides evaluated by fold increase in beating rate of neonatal rat cardiomyocytes. Figure 9 shows that synthetic peptide 1 ( Fig.9A )、2( Fig. 9B ) and 3( Fig. 9C ), using the fold change in beating rate to detect loss of activity. All three synthetic components showed a slight loss of activity in response to heating to 100°C (blue) when compared to the same beating rate obtained after treatment with the unheated component (orange).

[0056] Fig.10 : Graphic illustration of how to perform a Caco-2 flux assay. DETAILED DESCRIPTION

[0057] sequence:

[0058] SEQ ID NO: 1 - Generic synthetic peptide sequence

[0059] SEQ ID NO:2—Synthetic 13-mer peptide 1

[0060] SEQ ID NO:3—Synthetic 13-mer peptide 2

[0061] SEQ ID NO:4—Synthetic 13-mer peptide 3

[0062] SEQ ID NO:5—sequence of component N.aan_I15_R4

[0063] SEQ ID NO:6—sequence of component N.nka_I18_R4

[0064] SEQ ID NO:7—Sequence of component N.naj_I25_R3

[0065] SEQ ID NO:8—sequence of component N.nub_I16_R4

[0066] SEQ ID NO:9 - Sequence of component N.nig_I19_R4

[0067] SEQ ID NO: 10 - Sequence of component N.atr_I28_R3

[0068] method

[0069] Cobra venom collection:

[0070] Toxins were extracted from twelve cobra species using a proprietary method, collected into new vials, quantified, lyophilized and stored in a -20 °C freezer until use.

[0071] Purification of cobra venom using two-dimensional HPLC:

[0072] Lyophilized cobra venom was reconstituted in HPLC grade H2O and used Protein concentration was determined using a DS11 spectrophotometer. The whole poison was diluted to 100 mg / ml in IE buffer A, and 100 μl was injected onto an Agilent 1100 HPLC for separation by ion exchange chromatography. The poison was separated using a gradient increase of IE buffer B over time. Peak detection parameters were set, and components were collected. The components were lyophilized, reconstituted in RP buffer A, and injected again onto an Agilent 1100 HPLC for separation using reverse phase chromatography. The components were separated using a gradient increase of RP buffer B over time. A set of two-dimensionally fractionated poison components was obtained by this method and lyophilized. The components were resuspended, quantified by spectrophotometry, and diluted to the same working protein concentration. Finally, the components were plated in a low protein binding 384-well plate in preparation for analytical screening.

[0073] Rodent Cardiomyocyte and Cardiac Fibroblast Isolation:

[0074] Hearts were collected from euthanized 1-2 day old newborn rat pups and transferred to filter sterilized ice-cold Hanks balanced salt solution (HBSS) containing 2% penicillin-streptomycin (Pen-Strep). The hearts were washed in HBSS to remove any non-cardiac tissue, then cut into 1-3 mm slices and placed in 40 ml of filter sterilized ice-cold 0.1% trypsin / HBSS. The heart slices in trypsin were placed in a large ice bucket and incubated overnight at 4°C on a shaker with gentle agitation to allow tissue digestion.

[0075] On the second day, all trypsin was aspirated from the tissue and the tissue was evenly transferred to a 2ml Eppendorf tube. The tissue was then further digested with a 37°C 1mg / ml collagenase / HBSS (filter sterilized) solution. For each collagenase digestion, 2ml of enzyme buffer was added to all Eppendorf tubes, and the tubes were incubated in a 37°C heating block with 400rpm stirring for 2 minutes. The supernatant of the first two digestions was discarded. After each subsequent digestion step, the tissue was gently ground using a pipette to release cells. Once grinding is complete, the supernatant is retained, added to a tube containing 10ml cell culture medium, and stored on ice.

[0076] The supernatant was spun through a Percoll gradient at 1800 g for 45 minutes, and all supernatants containing cells were filtered through a 250 μm cell strainer to remove any possible undigested tissue and myocytes separated from cardiac fibroblasts. The bands containing myocytes and the bands containing cardiac fibroblasts were collected separately, and both groups of cells were washed with HBSS and precipitated by centrifugation 3 times to remove the Percoll buffer, resuspended in fresh medium (DMEM:F12, 10% FCS, 2% PS), and the viable cell yield was determined using a hemocytometer. The cells were plated at 1×10 5 -2×10 5 cells / cm 2 The cells were plated at a seeding density of 100 μl in a 384-well plate. All the culture medium was topped up to 50 μl and the cells were returned to a humidified incubator at 37° C. and 5% CO 2 to settle and culture until spontaneous beating.

[0077] Isolation of Embryonic Chicken Cardiomyocytes:

[0078] Hearts were collected from euthanized 12-13 day old chicks and transferred to filter sterilized ice-cold Hanks balanced salt solution (HBSS) containing 2% Pen-Strep. Hearts were washed in HBSS to remove blood, any non-cardiac tissue was removed, and then cut into 1-3 mm slices and placed in 40 ml of filter sterilized ice-cold 0.05% trypsin / HBSS. Heart slices in trypsin were placed in a large ice bucket and incubated on a shaker for 1 hour with gentle agitation to allow tissue digestion.

[0079] All trypsin was aspirated from the tissue and discarded, and the tissue was evenly transferred to a 2ml Eppendorf tube. The tissue was then further digested using 1mg / ml collagenase II / 0.1% trypsin / HBSS (filter sterilization) solution. For each digestion, 2ml of enzyme buffer was added to all Eppendorf tubes, and the tubes were stirred and incubated for 3 minutes at 400rpm in a 37°C heating block. The supernatant of the first two digestions was discarded. After each subsequent digestion step, the tissue was gently ground using a Pasteur pipette to release cells. Once grinding is completed, the supernatant is retained, added to a tube containing 10ml cell culture medium (DMEM:F12, 10% FCS, 2% PS), and stored on ice.

[0080] All supernatants containing cells were filtered through a 250 μm cell strainer to remove any possible undigested tissue. The cell suspension was then pre-inoculated in a 75 cm 3The cells were then placed in a tissue culture flask for 2 hours to allow excess fibroblasts to settle and adhere. The supernatant was spun through a Percoll gradient at 1800 g for 1 hour, and the remaining supernatant containing the enriched myocytes was separated from the remaining cardiac fibroblasts. The bands containing immature cardiomyocytes were collected separately from the bands containing residual cardiac fibroblasts, and both groups of cells were washed with HBSS and precipitated by centrifugation 3 times to remove the Percoll buffer, resuspended in fresh culture medium (fibroblasts: DMEM:F12, 10% FCS, 2% PS; chicken myocytes: DMEM:F12, 10% horse serum, 1% FCS, 2% PS), and the viable cell yield was determined using a hemocytometer. The cells were plated at 1×10 5 -2×10 5 cells / cm 2 The cells were plated in 384-well plates at a seeding density of 100 μl. All the culture medium was topped up to 100 μl and the cells were returned to a humidified incubator at 37°C, 5% CO2 to settle and culture until spontaneous beating. Spontaneous beating of myocytes was observed 3 days after plating.

[0081] Purified toxicants screened for toxicity against rat cardiomyocytes and cardiac fibroblasts:

[0082] Neonatal rat cardiomyocytes and cardiac fibroblasts were treated with purified toxic components of suspected cardiotoxins at doses of 12.5 μg / ml, 7.5 μg / ml, and 6 μg / ml, and rat cardiomyocytes were treated with additional concentrations of 3.5 μg / ml, 2.5 μg / ml, 1 μg / ml, and 0.5 μg / ml, in a humidified incubator at 37°C, 5% CO2 for 2 hours. After 2 hours, resazurin was added to all dosing wells at a final working concentration of 160 μM. The fluorescence changes of resazurin dye were monitored over a period of 5 hours using a BMG Fluostar microplate reader. The percentage of inhibition of the two cell types in response to each component at each dose was calculated and plotted.

[0083] Screening of purified toxicant components for rat erythrocyte toxicity:

[0084] Toxic components were diluted to stock concentrations of 15 μg / mL and 1 μg / mL and plated in triplicate (100 μl / well) in V-bottom 96-well plates. In addition, serial dilutions of natural venom from black-necked spitting cobra (Naja nigricollis) and DMSO were used as toxicity controls.

[0085] Blood from postmortem rats was collected in PBS containing heparin. Red blood cells were diluted in PBS and counted using a hemocytometer. Red blood cells were diluted to a final concentration of 1 × 10 8Cells / ml were centrifuged at 300 g for 3 minutes and the supernatant containing white blood cells was removed. The red blood cell pellet was resuspended in an equal volume of PBS. 100 μL of the resuspended cells was added to all component wells and toxicity control wells to provide a final volume of 200 μL / well (1×10 7 red blood cells / well, 7.5 μg / ml and 0.5 μg / ml component working concentrations).

[0086] The erythrocytes were incubated in the components for 5 hours at 37°C, 5% CO2 to lyse or settle by gravity. After 5 hours, the supernatant was removed from all wells and plated in quadruplicate in a 384-well plate (30 μl / well). The absorbance of the plate was read at 620 nm. All replicates were averaged and the % lysis of the erythrocytes for the two doses was plotted.

[0087] Screening of purified toxic components for altering the beating rate of cardiomyocytes:

[0088] Neonatal rat cardiomyocytes were isolated by standard protocols and seeded at a density of 100,000 cells / 96 wells. Once the myocyte cultures were beating spontaneously in uniform sheets, they were videotaped well by well using a microscope camera to determine the beating rate / minute before dosing. Suspected cardiotoxin components were administered to the wells in duplicate at doses of 12 μg / ml, 6 μg / ml, 3 μg / ml, and 1 μg / ml. The cells were incubated in each component in a humidified incubator at 37°C, 5% CO2 for 1 hour. After 1 hour, the images of the cells were re-recorded to obtain the beating rate / minute after dosing. The fold change in beating frequency for each dosing well pair was calculated.

[0089] Human IPSC-derived cardiomyocytes were used to evaluate the effects of six example components on non-target species:

[0090] People's IPSC-derived cardiomyocytes were purchased from Axol Biosciences and cultured by their standard methods. In brief, cells were recovered from liquid nitrogen storage when needed, and cells were transferred dropwise to 10 ml preheated (37 ° C) plating medium (cardiomyocyte maintenance medium + 10% FCS + 10 μM ROCK inhibitor). Cells were centrifuged at room temperature for 5 minutes at 200 × g. Supernatant was aspirated from the cell pellet, and the cell pellet was gently resuspended in 1 ml of plate culture medium. Cells were counted using a hemocytometer and trypan blue staining. Cells were plated in 384-well plates pre-coated with fibronectin at a seeding density of 10-20,000 cells / well. Cells were incubated overnight in a humidified incubator at 37 ° C, 5% CO2, and then the plate culture medium was removed the next day and replaced with fresh maintenance medium (no FCS, no ROCK inhibitor). Partial medium changes were performed every 2 days until the cells formed a single continuous spontaneously beating cardiomyocyte sheet (7-10 days in culture).

[0091] Once spontaneously beating, cells were treated with six exemplary components diluted in maintenance medium to a final working concentration of 7.5 μg / ml, 6 μg / ml, 3.5 μg / ml, 2.5 μg / ml and 1 μg / ml. 30 μl of each component dilution was added to triplicate wells of the cells and incubated for 2 hours in a humidified incubator at 37°C, 5% CO2. After 2 hours, resazurin dye was added to all wells at a final working concentration of 160 μM and the plate was monitored for fluorescence changes over a 5 hour period using a BMG Labtech Fluostar microplate reader. The percent toxicity at each dose was calculated from the triplicate wells and plotted.

[0092] The thermal stability of six example purified poison components was evaluated:

[0093] The thermal stability of six example components was studied. Aliquots of each component were heated to 37°C (unheated), 60°C, 80°C, and 100°C in a heating block, and then treated with a dose of 6μg / ml in rat neonatal cardiomyocytes. The cardiomyocyte toxicity of each temperature was evaluated 24 hours after administration relative to similar aliquots of each component that were not heated before administration. After 24 hours, 160μM resazurin dye was added to all cells, and changes in cardiomyocyte activity / metabolism were monitored every hour after the dye was added until 5 hours. Additional measurements were recorded 24 hours after the addition of the dye.

[0094] Mass spectrometry analysis of six selected components:

[0095] 10 μg of six fractions showing different levels of cardiomyocyte toxicity and effects on beating rate were subjected to intact mass and peptide mapping mass spectrometry analysis (Peak Proteins Ltd, Alderley Park, Macclesfield). For intact mass spectrometry, 5 μg samples of each lyophilized fraction were reconstituted in 40 μl of 0.1% formic acid / 5% acetonitrile. 10 μl of sample was loaded onto a Sciex Exion liquid chromatograph and eluted with a 5-minute reverse phase gradient using 0.1% formic acid buffer A and 0.1% formic acid / 100% acetonitrile buffer B. RP HPLC was performed with 500 μl of flow throughout the gradient, starting at 5% buffer B, increasing to 45% B in 3 minutes, followed by a 95% B wash, and a 5% B balance. The column used was a Phenomenex Jupiter 5 μm, C4, 300A 50x2.1 mm. The liquid flow from the post is made to enter a Sciex X500B mass spectrometer, which is set to collect data in positive ion mode. In order to enable the eluent to be ionized, the source is set to 400 ° C, 5500 V under 50 psi gas. The TOF mass window of 500-3000 Da is collected and scanned with 0.5 s. The X500B mass spectrometer is calibrated with a positive calibration mixture, and the error of the experiment is estimated to be 0.5 Da. The obtained total ion chromatogram (TIC) is deconvoluted using BioToolKit software (Sciex).

[0096] For peptide mapping mass spectrometry, 5 μg of each lyophilized sample was reconstituted in 50 μl of 100 mM ammonium bicarbonate. 5 μl of 100 mM DTT in 100 mM ammonium bicarbonate solution was added to each sample, and the sample was heated at 65 ° C for 30 minutes. 5 μl of 500 mM iodoacetamide in 100 mM ammonium bicarbonate solution was added to all samples, and the samples were incubated for another 30 minutes at room temperature in the dark. 10 μl of 25 ng / μl trypsin in 50 mM ammonium bicarbonate solution was added to each sample, and they were incubated at 37 ° C overnight. Take 10 μl of complete digestion solution and mix with 10 μl of 0.1% trifluoroacetic acid (TFA). 10 μl of mixed sample was loaded onto Sciex Exion liquid chromatography and run for 10 minutes reverse phase gradient. Phenomenex Lunar1.6 μm, PS C18, 100A 150x2.1mm column was used. Buffer composition is consistent with intact mass MS (0.1% formic acid buffer A and 0.1% formic acid / 100% acetonitrile buffer B). Liquid flow is set to 300 μ l, and gradient elution is performed, starting from 5% buffer B, increasing to 45% B in 15 minutes, then washing with 95% buffer B, and the balance of 5% B, so that the liquid flow from the column enters the Sciex X500B mass spectrometer, which is set to collect data in positive ion mode. In order to enable the eluent to ionize, the source is set to 400 ° C, 5500V under 30psi gas. The TOF mass window of 300-1800Da is collected and scanned with 1.2s. Mass spectrometry (MSMS) data are collected using information-dependent acquisition methods, wherein each scan collects up to 10 MSMS. X500B is calibrated with a positive calibration mixture, and the error of the experiment is estimated to be 1ppm. Using the Swiss-Prot database, the resulting data from MSMS are analyzed using Mascot (MatrixScience).

[0097] SAR and synthetic peptide design:

[0098] Using the data obtained from the six fractions for mass spectrometry, structure activity relationship (SAR) analysis was performed, resulting in the design of three short synthetic peptides for further study. These three peptides were designed to exploit the different characteristics of the six exemplary fractions and to investigate their toxicity and beating effect properties.

[0099] • Peptide 1 - a peptide designed using the first 13 amino acids of component N.aan_I15_R4, which was shown to be non-toxic but caused an increase in the beating rate.

[0100] • Polypeptide 2 - a polypeptide designed using the first 13 amino acids of N.aan_I15_R4, but with substitutions made at important loci identified by SAR analysis to include amino acids important for both high beating rate and high toxicity.

[0101] • Polypeptide 3 - a polypeptide designed using the first 13 amino acids of N.aan_I15_R4, but with substitutions made at important loci identified by SAR analysis to include amino acids that are important for high beating rates and are non-toxic.

[0102] Screening of synthetic peptides for toxicity against rat cardiomyocytes, chicken cardiomyocytes, human IPSC-derived cardiomyocytes, and cardiac fibroblasts:

[0103] Rat neonatal cardiomyocytes, embryonic chicken cardiomyocytes, human IPSC-derived cardiomyocytes and rat cardiac fibroblasts were cultured. Synthetic peptides 1, 2 and 3 were diluted to a working concentration of 100 μg / ml, 10 μg / ml, 1 μg / ml and 0.1 μg / ml in a cell culture medium suitable for the optimal growth of each cell type. Toxicity controls were also included in the experiment, and were selected from the aforementioned identifications showing toxicity of the whole poison and poison cardiotoxin components. The cells from each of the three cell types were also measured in parallel with 100 μg / ml of the whole poison of black-necked spitting cobra (N.nigricollis) and 10 μg / ml and 1 μg / ml of the identified cardiotoxin components N.nka_I18_R4 and N.nig_I19_R4. All wells were incubated for 24 hours in each treatment. After 24 hours, resazurin dye was added to each well at a final concentration of 160 μM, and the changes in dye fluorescence were monitored and recorded over a 24-hour period.

[0104] Screening of synthetic peptides for altering cardiomyocyte beating rate:

[0105] The baseline beating rate of neonatal rat cardiomyocytes and human IPSC-derived cardiomyocytes to be treated with the three synthetic components was determined by performing three imaging microscopy on each well using a microscope camera. The average beating / minute (n=9) was calculated by triplicate readings of three separate wells. An aliquot of each peptide was taken to treat neonatal rat cardiomyocytes at a dose of 100 μg / ml, 10 μg / ml, 1 μg / ml and 0.1 μg / ml. The cells were returned to a humidified incubator at 37°C, 5% CO2 for 2 hours. After 2 hours, the beating rate of the treated cardiomyocytes was re-evaluated using the same method as used to obtain the beating data before administration. The process was repeated 24 hours after adding the synthetic peptide.

[0106] Embryonic chicken cardiomyocytes were cultured until spontaneous beating. Cells were recorded by image microscopy and a baseline beating rate of the cells before administration of the synthetic peptide was generated. The synthetic peptide was diluted in cardiomyocyte culture medium to a final working concentration of 100 μg / ml, 10 μg / ml, 1 μg / ml and 0.1 μg / ml. Chicken cardiomyocytes were dosed with the synthetic for 2 hours in a humidified cell culture incubator at 37°C, 5% CO2 before recording the beating rate again. The fold change in beating rate was calculated by dividing the beats / minute after synthetic administration by the beats / minute value before administration.

[0107] Screening of synthetic peptides for thermal stability:

[0108] The thermal stability of three designed synthetic peptides was studied. The baseline beating rate of neonatal rat cardiomyocytes to be treated with the synthetic components was determined by performing three image microscopy on each well using a microscope camera. The average beating / minute (n=9) was calculated from triplicate readings of three separate wells. An aliquot of each peptide was heated at 100°C in a heating block, and then the rodent cardiomyocytes were treated with doses of 100μg / ml, 10μg / ml, 1μg / ml and 0.1μg / ml. The cells were returned to an incubator at 37°C, 5% CO2 for 2 hours. After 2 hours, the beating rate of the treated cardiomyocytes was re-evaluated using the same method used to obtain the beating data before administration. The process was repeated 24 hours after the addition of the synthetic peptides. The fold change data of the beating rate of the cardiomyocytes after treatment with the heated synthetic components were compared with the fold change of the beating rate obtained with the previously unheated synthetic peptides to evaluate their thermal stability.

[0109] Evaluation of intestinal permeability of example short synthetic peptide 1 using Caco-2 permeability assay (bidirectional):

[0110] The cells were cultured at 1×10 5 cells / cm 2 Inoculate on Millipore Multiscreen Transwell plates. Cells were cultured in DMEM and the culture medium was changed every two or three days. On the 20th day, permeability studies were performed. Cell culture and analysis incubations were performed at 37°C, 5% CO2 atmosphere, 95% relative humidity. On the day of analysis, monolayers were prepared by rinsing the basolateral and apical surfaces twice with Hanks balanced salt solution (HBSS) warmed to 37°C at the desired pH. Cells were then incubated in the apical and basolateral chambers for 40 minutes with HBSS at the desired pH to stabilize physiological parameters.

[0111] The dosing solution was prepared by diluting the test compound with assay buffer to give a final test compound concentration of 10 μM or 100 μM (final water concentration ≤ 1% v / v). The dosing solution also contained the fluorescent integrity marker Lucifer Yellow. Assay standards were prepared from the aqueous dilutions of the test compound and transferred to the buffer to maintain an aqueous concentration of ≤ 1% v / v. The assay buffer consisted of supplemented HBSS pH 7.4.

[0112] To assess apical to basolateral (A2B) permeability, HBSS was removed from the apical chamber and replaced with test compound dosing solution. The apical chamber insert was then placed in a companion plate containing fresh buffer (containing ≤1% v / v water). To assess B2A permeability, HBSS was removed from the companion plate and replaced with test compound dosing solution. Fresh buffer (containing ≤1% v / v water) was added to the apical chamber insert, which was then placed in the companion plate.

[0113] At 120 minutes, the apical chamber insert and companion plate were separated and the apical and basolateral samples were diluted for analysis. The permeability of the test compounds was assessed in duplicate. Compounds with known permeability characteristics were run on each assay plate as controls.

[0114] The test and control compounds were quantified by LC-MS / MS cassette analysis using a 7-point calibration of appropriately diluted samples. Cyprotex universal analysis conditions were used. The initial concentration (C0) was determined by the dosing solution, and the experimental recovery was calculated by C0 and the top and bottom chamber concentrations.

[0115] Check the integrity of the polarized cell monolayer throughout the experiment by monitoring Lucifer Yellow penetration using fluorescence analysis. If the monolayer is not damaged, the penetration of this intercellular marker is low. If the Lucifer Yellow P measured in a particular well is app The value is higher than the acceptance limit (>1.0cm / s x10 6 ), but the P obtained by the test compound or positive control substrate in this well app If the results were qualitatively similar to those measured in the remaining replicate wells (within the Lucifer Yellow threshold), the cell monolayer was considered acceptable based on the scientific judgment of the responsible scientist. If this was not the case, the results for the affected monolayer were excluded.

[0116] Data Analysis:

[0117] The measured concentration (nM≡pmol / mL) of each test sample receiving well was multiplied by the volume of the receiving chamber (top = 0.09 mL, basolateral = 0.210 mL) to calculate the amount of probe substrate that permeated the monolayer (pmol). The measured amount in each receiving chamber was then divided by the incubation time (seconds) to give the transport rate of the test compound or positive control substrate, which was used to determine the apparent permeability (P) according to the equation given below: app ):

[0118]

[0119] Where dQ / dt is the permeation rate of the drug across the cell, C0 is the donor compartment concentration at time zero, and A is the area of ​​the cell monolayer. C0 is obtained from analysis of the dosing solution.

[0120] The discharge ratio (ER) is calculated from the average data of A to B and B to A. This is obtained by the following formula:

[0121]

[0122] The recovery (mass balance) of the test compound or positive control substrate was calculated by dividing the sum of the amounts (pmol) in the receiver and donor compartments after incubation by the amount in the initial donor solution at time zero (C0) and expressed as a percentage.

[0123] Four control compounds, atenolol (50% absorption in humans), antipyrine (97% absorption in humans), talinolol (P-gp substrate), and estrone 3-sulfate (BCRP substrate), were screened along with the test compounds.

[0124] result

[0125] Cardiomyocyte and Cardiac Fibroblast Toxicity Assays:

[0126] At a dose of 12.5 μg / ml, fourteen and eleven purified synthetic components showed greater than 50% toxicity to rat cardiomyocytes and cardiac fibroblasts, respectively ( Figure 1A ). Reducing the dose of the purified toxicant fractions resulted in loss of fibroblast toxicity for all fractions except one tested purified fraction (N.naj_I24_R2). Still, eight purified fractions were found to produce greater than 50% toxicity to rat cardiomyocytes at a dose of 6 μg / ml ( Figure 1C ). The data show that by choosing the correct dose, selectivity for cardiomyocytes over cardiac fibroblasts can be achieved. Interestingly, the purified fraction of the venom from Naja annulifera was not toxic to either cardiomyocytes or cardiac fibroblasts at any of the three doses tested.

[0127] Analytical studies of a panel of purified fractions suspected to be cobra cardiotoxins based on their HPLC peak elution times and profiles identified fractions that exhibited a wide range of varying levels of cytotoxic effects on neonatal rat cardiomyocytes and neonatal rat cardiac fibroblasts. The data also identified that the window between toxic and non-toxic doses was relatively narrow for many of the fractions, with many approaching 90-100% toxicity at doses of 12.5 μg / ml and complete loss of toxicity at approximately 1-2.5 μg / ml. In summary, neonatal rat cardiomyocytes showed greater susceptibility to many of the screened fractions than their dosed cardiac fibroblast counterparts. Fractions isolated from the venom of the beaked cobra, Naja annulifera (N. aan), exhibited little or no cytotoxic effects on cardiomyocytes or cardiac fibroblasts. Interestingly, this was the only graded cobra venom in which all of the screened fractions exhibited no toxic effects at any dose tested.

[0128] refer to Figure 1C The 6 μg / ml dose showed the greatest selectivity in toxic effects between the target rat cardiomyocytes and the non-target cardiac fibroblast control cell groups, with cardiac fibroblast toxicity almost abolished after treatment with all components except one (N.naj_I24_R2). Although fibroblast toxicity was completely abolished at 6 μg / ml, 40% or greater rodent cardiomyocyte toxicity was still observed for 12 of the tested components, with 8 showing greater than 70% toxicity.

[0129] Additional selectivity was still observed for the other fractions at higher tested doses, with fraction N.nub_I16_R3 ( Figure 1B ) and the highest tested dose of 12.5 μg / ml of component N.atr_I23_R3 ( Figure 1A ) achieved good selectivity between cardiomyocytes and cardiac fibroblasts.

[0130] This initial toxicity screening data supports the hypothesis that the components of cobra venom can be isolated and used for selective stress to rodent cardiomyocytes and therefore have the potential to be used as novel, unique rodenticides that exploit previously unstudied mechanisms of action. The selectivity data showed that the components were more selectively toxic to cardiomyocytes compared to cardiac fibroblasts (which are also present in heart tissue), which is encouraging and suggests that it may be possible to develop drugs that are highly specific and targeted to a single cell type.

[0131] The decision to perform additional screening of these purified fractions against another morphologically distinct cell type (rat erythrocytes) was important in evaluating whether the fractions might display undesirable compounding effects (e.g., hemolytic properties) that could be detrimental to the effectiveness of their synthetically designed forms.

[0132] Erythrocyte toxicity test:

[0133] refer to Figure 2 , erythrocyte lysis assays showed that none of the purified toxicant components caused rat erythrocyte death at doses of 0.5 μg / ml or 7.5 μg / ml. Interestingly, some purified components have previously been shown to be toxic to rat cardiomyocytes, rat cardiac fibroblasts, or both at a dose of 7.5 μg / ml, thus further adding to the evidence for a selective targeted mechanism of action. Black-necked spitting cobra natural venom was used as a control for erythrocyte toxicity and was shown to be able to lyse rat erythrocytes at doses of 50 μg / ml and 5 mg / ml.

[0134] No erythrocyte toxicity was observed for any of the tested components at the highest dose of 7.5 μg / ml, a dose at which a number of tested components were previously found to be cardiomyocyte toxic. This lack of toxicity supports the fact that all tested components did not display hemolytic properties, thus suggesting that the previously observed cardiomyocyte toxicity in response to treatment with these purified components was more likely mechanism of action rather than a generalized necrosis or apoptosis-induced effect. Treatment with natural venom from the black-necked spitting cobra as a control determined that venom from the cobra could cause erythrocyte lysis, supporting the finding that these purified components did not cause erythrocyte lysis.

[0135] Screening of purified toxic components for altering the beating rate of cardiomyocytes:

[0136] After 1 hour of administration, all the screened components (except the component from the cobra) at 12 μg / ml were toxic to rat cardiomyocytes, so no beating data after administration were obtained. Administration of each component at other concentrations produced different multiple increases in beating rate (see Table 1 below), confirming that the cobra venom components can indeed cause an increase in the beating rate of rodent cardiomyocytes. Among the screened components, eighteen components caused an increase in the beating rate of rat cardiomyocytes at at least one tested concentration. In the three components that did not cause an increase in beating rate (N.atr_I5_R5, N.kao_I18_R3 and N.nig_I4_R3), no cytotoxicity was observed in the previous resazurin analysis (see Figure 1) at any tested concentration, so combined, these components are unlikely to contain any compounds that actively affect cardiomyocyte function or activity. It was found that most components caused the greatest increase in rat cardiomyocyte beating rate at a dose of 6 μg / ml or 3.5 μg / ml, and the beating rate decreased at higher doses, which may be due to cytotoxicity and death of cells. Each component showed very different degrees of increase in rat cardiomyocyte beating rate, with a maximum fold increase of 2 times observed with N.nub_I17_R2 and a maximum fold increase of 13.75 times observed with N.naj_I25_R3, indicating that the active substances in each component have very different activity levels on cells.

[0137] Table 1

[0138]

[0139]

[0140] - The components did not cause an increase in the beating rate at any of the concentrations tested.

[0141] Selection of six example components and confirmation of their toxicity and cardiomyocyte beating rate effects:

[0142] Using the cardiomyocyte and cardiac fibroblast toxicity data, as well as the beating rate change data obtained before and after dosing, six fractions were selected for further characterization. Fractions with different myocyte and non-myocyte toxicity profiles were selected to allow identification of amino acid residues that are particularly important for cardiomyocyte toxic effects by mass spectrometry and SAR. The yield of each purified fraction was also considered as an attribution factor for selection.

[0143] The six components were selected based on the following reasons:

[0144] N.aan_I15_R4: showed no visible or detectable toxicity to rat cardiomyocytes or cardiac fibroblasts at doses up to 12.5 μg / ml. Caused a greater than 3.5-fold increase in beating rate at a dose of 6 μg / ml. No cytotoxicity, but increased the contraction rate of rat cardiomyocytes.

[0145] N.atr_I28_R3: showed rat cardiomyocyte and cardiac fibroblast toxicity at doses of 3 μg / ml and above and 7.5 μg / ml and above, respectively. Caused a greater than 5-fold increase in beating rate at a dose of 6 μg / ml. Has high cytotoxicity to specific and non-specific cell types and caused a significant increase in the contraction rate of rat cardiomyocytes.

[0146] N.nub_I16_R3: showed rat cardiomyocyte and cardiac fibroblast toxicity at doses of 7.5 μg / ml and above and 12.5 μg / ml, respectively. Caused a greater than 6-fold increase in beating rate at a dose of 6 μg / ml. Was less cytotoxic to specific and non-specific cell types than N.atr_I28_R3 (required higher doses), and caused a significant increase in the contraction rate of rat cardiomyocytes.

[0147] N.nka_I18_R4: showed rat cardiomyocyte and cardiac fibroblast toxicity at doses of 3 μg / ml and above and 7.5 μg / ml and above, respectively. Caused a greater than 7-fold increase in beating rate at a dose of 6 μg / ml. Has high cytotoxicity to specific and non-specific cell types and caused a great increase in the contraction rate of rat cardiomyocytes.

[0148] N.nig_I19_R4: showed rat cardiomyocyte and cardiac fibroblast toxicity at doses of 7.5 μg / ml and above and 12.5 μg / ml, respectively. Caused a greater than 5.5-fold increase in beating rate at a dose of 6 μg / ml. Observed similar cytotoxicity profiles to specific and nonspecific cell types as with N.nub_I16_R3, and caused a similar increase in contraction rate of rat cardiomyocytes.

[0149] N.naj_I25_R3: showed rat cardiomyocyte and cardiac fibroblast toxicity at doses of 6 μg / ml and above 12.5 μg / ml, respectively. Caused a greater than 8.5-fold increase in beating rate at a dose of 6 μg / ml. Was more toxic to rat cardiomyocytes than N.nub_I16_R3 and N.nig_I19_R4, but had similar cardiac fibroblast toxicity. Caused a significant increase in the contraction rate of rat cardiomyocytes.

[0150] Human IPSC-derived cardiomyocytes were used to evaluate the effects of six example components on non-specific species:

[0151] Human induced pluripotent stem cell (IPSC) derived cardiomyocytes were purchased from Axol Bioscience and cultured directly. After 48 hours, the cells began to show the formation of a spontaneously beating cell monolayer. After 7 days in culture, the cells were ready for the evaluation of potential human cardiomyocyte toxicity in response to treatment with our six selected cardiotoxin components.

[0152] Human IPSC-derived cardiomyocytes were dosed with serial dilutions of each of the six toxicant components (7.5 μg / ml, 6 μg / ml, 3.5 μg / ml, 2.5 μg / ml, and 1 μg / ml) in triplicate for 2 hours at 37°C, 5% CO2, followed by the addition of resazurin dye and monitoring of color and fluorescence changes over the next 5 hours. The percentage of toxicity for each component at all doses was calculated and plotted ( Figure 3 ).

[0153] Five of the six selected fractions showed cytotoxic activity against human IPSC-derived cardiomyocytes, with only fraction N.ann_I15_R4 consistently showing low levels of cytotoxicity at all five doses tested. A clear dose-dependent effect was observed in response to treatment with the five other purified toxicant fractions, with higher doses causing higher levels of cardiomyocyte toxicity.

[0154] The resazurin data showed that human IPSC cardiomyocytes responded to treatment with the five selected components with a good dose response curve (see Figure 3 As previously seen in neonatal rodent cardiomyocytes, there appeared to be no observable cardiomyocyte toxicity in response to treatment with fraction N.aan_I15_R4.

[0155] The toxicity data for human IPSC cells appear to indicate a higher level of toxicity in response to certain components in these cells than previously observed in rat neonatal cardiomyocytes, with the cells showing some toxicity at doses below those observed with rodent cardiomyocytes. However, components N.atr_I28_R3 and N.nka_I18_R4 showed a reduction in toxicity at 3.5 μg / ml in human IPSC-derived cells compared to the levels of toxicity previously observed with rodent cardiomyocytes.

[0156] Notably, these cells are not mature cardiomyocytes, but rather human stem cells that have been programmed to differentiate into cardiomyocyte-like cells. Human IPSCs have a different cell origin than rodent cardiomyocytes that are directly isolated from animals. Because they require a delicate balance of growth factors to trigger their differentiation, they may show greater susceptibility to environmental stressors and changes.

[0157] The thermal stability of six selected purified poison fractions was evaluated:

[0158] See also Figure 4 Overall, all purified fractions showed good thermal stability, retaining strong cytotoxic activity after treatment of the fractions at temperatures up to 100°C. After treatment of fractions N.nub_I16_R3 and N.atr_I28_R3 at 100°C, slight reductions in toxicity (approximately 5-10%) were observed when compared to their activity at 37°C (without heat treatment), however these reductions were considered negligible. Fraction N.ann_I15_R4 continued to show below-average cytotoxicity at all four tested temperatures, which is consistent with previous toxicity screening results.

[0159] Analysis of the thermal stability of the six selected components revealed that they appear to be thermally stable up to 100°C with little or no decrease in their cardiomyocyte toxicity (see Figure 4 ). The components with slightly lower direct toxicity at the beginning (N.ann_15_R4 and N.nub_I16_R3) showed a slightly more significant decrease in activity, however, at the highest heating temperature of 100°C, this still only amounted to a loss of about 10% in cytotoxicity. Component N.ann_I15_R4 continued to show low levels of cardiomyocyte toxicity at all tested temperatures. However, this is not surprising, as it has always shown that the direct cytotoxicity to rat cardiomyocytes is much lower than any of the other five selected components. The components N.nka_I18_R4, N.nig_I19_R4 and N.naj_I25_R3 did not change significantly in activity in response to any of the tested temperature treatments, and the same cytotoxic activity was observed at 100°C as at 37°C. The data show that the active ingredients in each component can withstand temperatures exceeding 100°C with little or no change in their stability and activity.

[0160] The active compound in the final product needs to be able to exhibit good thermal stability to withstand incorporation into hot liquid wax (~70°C) and retain its active properties after the cooling and solidification process.

[0161] Mass spectrometric identification of six selected components:

[0162] Six components that showed cardiotoxic effects in rat neonatal cardiomyocytes were selected and sent for mass spectrometry analysis. Intact mass analysis combined with peptide mapping confirmed that all six selected components were cardiotoxins, matching the obtained sequences with cardiotoxins reported in the Uniport and NCBI protein databases. Three of the selected components had high percentage match coverage of cardiotoxins already present in the database (indicating that they were most likely these specific cytotoxins).

[0163] Certain regions of the sequence are highly conserved among all cardiotoxins, most likely for structural integrity.

[0164] Amino acids that matched the mass spectra are in bold. Conserved amino acids in the cardiotoxin sequence in Uniprot (but not matched in the acquired mass spectrometry data) are in Underline Amino acid substitutions are highlighted in grey.

[0165] N.aan_I15_R4: (match to Cobra cytotoxin 1) ≈ 91.6% match:

[0166]

[0167] N.nka_I18_R4: (matches to Naja naja cytotoxin 8) ≈ 96.6% match:

[0168]

[0169] N.naj_I25_R3: (matches Indian cobra cytotoxin 10) ≈ 75% match:

[0170]

[0171] Although the other three sequences produced a lower percentage of matches due to the lack of information characterizing cardiotoxins from those species in the database, the most likely sequence could still be predicted. By using sequence data obtained from mass spectrometry, sequences of cardiotoxins from closely related species (in uniport / NCBI), and sequences of conserved regions in the sequence structure of all cardiotoxins, the possible sequences of some of the amino acids in these cardiotoxins could be interpreted. Only amino acids that were shown in the database to have high variability at specific loci and for which there was no mass spectrometry sequence data had not yet been confirmed (black amino acids).

[0172] Matching amino acids are in bold. Conserved amino acids that do not match the peptide are in Underline Amino acid substitutions are highlighted in grey.

[0173] N.nub_I16_R4: (matches Naja pallida cytotoxin 1) ≈ 65% match:

[0174]

[0175] N.nig_I19_R4: (with Mozambique spitting cobra (Naja mossambica) cytotoxin 4 matches) ≈ 43.3% match:

[0176]

[0177] N.atr_I28_R3: (with 5 matches of Naja atra cytotoxin)≈46.6% match:

[0178]

[0179] SAR and synthetic peptide design:

[0180] Using the above sequences, structure activity relationship (SAR) analysis was performed to determine which amino acid residues may be the most important factors causing the changes observed in cardiomyocyte toxicity, nonspecific cardiac fibroblast toxicity, and changes in fold increase in beating frequency in cardiomyocytes after drug administration.

[0181] Figure 5 The highlighted amino acids identified by SAR for their importance to cardiomyocyte toxicity are shown in . SAR revealed that most of the important amino acids are within the first 30 amino acids of the sequence, supporting the conclusion that the beginnings of loops 1 and 2 of the cardiotoxin structure are most important for cardiomyocyte toxicity. Using this information, these were proposed as regions of the cardiotoxin sequence from which short active synthetic peptides could be designed.

[0182] Three short synthetic peptides were designed using SAR analysis. Peptide 1 was designed using the first 13 amino acids of cardiotoxin N.aan_I15_R4 (matched to cobra cardiotoxin 1 by mass spectrometry analysis) as this is the only cardiotoxin studied that showed a large increase in cardiomyocyte beating rate without causing cytotoxicity and was therefore a good choice of starting scaffold to ensure that the synthetic peptide had low levels of direct toxicity:

[0183] Compound 1: LKCHKLVPPVWKT [SEQ ID NO: 2].

[0184] Synthetic peptide 2 was designed using the first 13 amino acids of the cardiotoxin N.aan_I15_R4 as the overall peptide framework, but those amino acids shown by SAR analysis to cause high beating rates and high toxicity were substituted into the sequence.

[0185] Synthesis 2:

[0186] The amino acids in bold are the amino acids that the SAR shows are important for high toxicity. The underlined amino acids are the amino acids that the SAR shows are important for high beating rates.

[0187] Finally, Synthesis 3 was designed using the first 13 amino acids of the cardiotoxin N.aan_I15_R4 as the overall peptide framework, but those amino acids that were shown by SAR analysis to be important (non-toxic) only for high beating rates in other studied cardiotoxins were substituted into the sequence. These amino acid substitutions into the sequence were found to cause poor solubility of the peptide in water, so an additional lysine (K) was added to the end of the peptide sequence to improve solubility.

[0188] Synthesis 3:

[0189] The amino acids in bold are the amino acids that the SAR showed to be important for low toxicity. The underlined amino acids are the amino acids that the SAR showed to be important for low beating rate.

[0190] Screening of synthetic peptides for toxicity against rat cardiomyocytes and rat cardiac fibroblasts:

[0191] All three short synthetically designed peptides showed no cytotoxic effects on cardiomyocytes or cardiac fibroblasts at any of the four screening doses (0.1 μg / ml, 1 μg / ml, 10 μg / ml and 100 μg / ml). Treatment with natural venom of black-necked spitting cobra at a dose of 100 μg / ml and purified fractions N.nka_I18_R4 and N.nig_I19_R4 at a dose of 10 μg / ml consistently showed high levels of toxicity (≈90%) in both rat cardiomyocytes and cardiac fibroblasts.

[0192] After 24 hours of treatment with the three synthetic peptides, the neonatal rat cardiomyocytes (see Fig. 6A ) or cardiac fibroblasts (see Figure 6B ) had no observable toxicity. The toxicity data showed that the three synthetic peptides showed no cytotoxic activity against cardiomyocytes and non-myocyte cell types.

[0193] This synthetic design approach helped to eliminate the toxicity previously observed with other cytotoxic components, which produced peptides equivalent to only the first finger of the native cardiotoxin, combined with the use of the amino acid sequence from component N.ann_I15_R4 (a non-cytotoxic cardiotoxin) as the backbone for the construction of all three synthetics. 100 μg / ml of the black-necked spitting cobra whole venom and 10 μg / ml of the cardiotoxin containing components N.nka_I18_R4 and N.nig_I19_R4 consistently showed high levels of cytotoxicity against both cell types tested.

[0194] This data has shown that by designing a shortened synthetic version of the peptide that includes only the amino acids from loop 1 of the cardiotoxin structure, the undesirable cytotoxic effects observed with the full-length cardiotoxin were engineered away.

[0195] Selectivity Screening: Screening Synthetic Peptides for Human IPSC Cardiomyocyte Toxicity:

[0196] Referring to Figure 7, all three short synthetically designed peptides showed no cytotoxic effects against any type of cardiomyocytes at any of the 4 screening doses (0.1 μg / ml, 1 μg / ml, 10 μg / ml and 100 μg / ml). Treatment with 100 μg / ml dose of black-necked spitting cobra natural venom and 10 μg / ml dose of purified components N.nig_I19_R4 and N.nka_I18_R4 consistently resulted in high levels of toxicity (≈90%) against myocytes of both species.

[0197] The three synthetic components were screened against commercially available human IPSC-derived cardiomyocytes and isolated embryonic chick cardiomyocytes to determine if they exhibited any human or avian specific cytotoxicity. After a 24-hour dosing period, no cytotoxicity was observed in human IPSCs ( Fig. 7A ) or embryonic chick cardiomyocytes ( Figure 7B ), no direct toxicity was observed in response to any of the three synthetically designed peptides.

[0198] Toxicity was still observed in these cells in response to treatment with two natural cardiotoxin components, a previously purified poison and a natural poison from the black-necked spitting cobra. The data suggest that cytotoxicity to human cardiomyocytes is not a feature of the synthetic peptides designed at this stage.

[0199] Screening of synthetic peptides for altering the beating rate of rodent and human IPSC cardiomyocytes:

[0200] With reference to Figure 8, the increase in the exponential beating rate caused by all three peptides in rat cardiomyocytes is greater than that of human IPSC-derived cardiomyocytes, in which almost no increase in response to treatment with any dose was seen. Synthetic peptide 1 caused the maximum multiple increase in beating rate in rat cardiomyocytes, with a dose of 100 μg / ml causing an 8-fold increase in the measured beating rate. Synthetic peptides 2 and 3 caused a 3.5-fold and 3-fold peak increase in the beating rate of rat neonatal cardiomyocytes, respectively.

[0201] Neonatal rodent cardiomyocytes and human IPSC-derived cardiomyocytes were dosed with the three synthetically designed peptides and their changes in beating rate in response to 2 hours of dosing were evaluated. After 2 hours of dosing at 100 μg / ml, 10 1 and 0.1 μg / ml, rodent cardiomyocytes were seen to increase their beating rate in response to all three synthetic components at all tested doses. Synthesis 2 ( Figure 8B ) showed an increase in beating rate approximately three-fold above pre-dose in response to all four concentrations tested, whereas Synthesis 3 ( Figure 8C ) saw an approximately two-fold increase in beating rate for the three lower concentrations tested, increasing to three-fold after treatment with the highest tested dose of 100 μg / ml. Fig. 8A ) shows the maximum fold increase in beating rate for the three tested synthetic compounds, with 3.5-fold, 4-fold, 5-fold and 8-fold increases in beating rate at 0.1 μg / ml, 1 μg / ml, 10 μg / ml and 100 μg / ml, respectively. A three-fold or greater increase in heart rate in response to a synthetic component could have catastrophic effects on the efficient function of the rodent heart.

[0202] The effects of three synthetic peptides on human IPSC-derived cardiomyocytes were also assessed to determine whether they show any activity to non-target species. After administration in rat cells with the same 2-hour time period and the same four doses (100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml), compared with the observed effects on rat cardiomyocytes, synthetic peptides seem to show almost no or no effect on human IPSC-derived cardiomyocytes. All three synthetic peptides do not cause the increase in the beating rate of human IPSC cardiomyocytes at a dosage of 0.1 μg / ml, 1 μg / ml and 10 μg / ml. Synthetic peptides 1 and 2 seem to cause a slight increase in the beating rate of human cardiomyocytes, which is 1.5 times the beating rate before administration. However, when using synthetic peptide 1 at the same dose, a greater increase was observed in rodent cardiomyocytes, and by utilizing the large difference window observed between the two species, there is the possibility of selecting a dose that only shows selectivity to rodent cardiomyocytes.

[0203] None of the compounds were found to increase the beating rate of chicken cardiomyocytes at any concentration tested ( Fig. 8A -C), which was also seen previously for human IPSC cardiomyocytes. The data generated from the effects of three synthetically designed peptides show that these peptides exhibit preferential activity against rodent cardiomyocytes when compared to their human or chicken derived counterparts. These differences are promising to reduce the potential problem of side effects occurring in non-target species of large mammals or birds in the event of accidental exposure to these novel peptides, and suggest that there is an even larger dosing window between target and non-target species when considering differences in circulating blood volume and heart size.

[0204] Screening of synthetic peptides for thermal stability:

[0205] Referring to Figure 9, all three synthetic peptides showed a slight loss of activity in response to heating to 100°C compared to the same beating rate obtained after treatment with the unheated components, however, despite heating to 100°C, the components still retained most of their activity. Fig.9A ) still performed best in terms of increasing the beating rate. After being treated with 100μg / ml, 10μg / ml, 1μg / ml and 0.1μg / ml of peptide, the heat-treated component still caused the beating rate to increase by 7 times, 4 times, 3 times and 3 times respectively.

[0206] Synthesis 2( Fig. 9B ) continued to increase the beating rate by about 3.5-fold at 100 μg / ml and appeared to show greater thermal stability at higher concentrations. Decreased activity was observed in peptide 2 at doses of 10 μg / ml, 1 μg / ml, and 0.1 μg / ml, however all doses still caused a 2.5-fold or greater increase in the beating rate of rat neonatal cardiomyocytes. Synthesis 3 ( Fig. 9C ) shows that the minimum multiple increase in the beating rate after heating and the maximum multiple increase reaches 2.5 times.

[0207] Evaluating the beat enhancement and thermal stability data for all three peptides, Synthetic Peptide 1 appeared to perform best, causing the greatest increase in the beating rate of rat cardiomyocytes, while not causing a similar increase in beating rate in human IPSC-derived cardiomyocytes. Peptide 1 also continued to show good activity on rat neonatal cardiomyocytes when heated to a temperature of 100°C, indicating that it exhibited excellent thermal stability characteristics. Considering all the data for the three synthetic peptides, Synthetic Peptide 1 was selected for further evaluation of its intestinal permeability and was sent for Caco-2 assay analysis.

[0208] Evaluation of intestinal osmotic permeability of example short synthetic peptide 1 using the Caco-2 permeability assay:

[0209] like Fig.10As shown, Caco-2 cells grow as monolayer in Transwell inserts on permeable membranes.Inserts are inserted into the holes of plates to form apical chambers (above cells) and basal chambers (below cells), with permeable membranes between the two layers.Then compounds are added to apical chambers or basal chambers, and the passage into another chamber is monitored.The transfer indication cell uptake of the test compound from the apical chamber like the basal chamber, simulated compounds transfer from the intestinal cavity to the bloodstream by uptake receptors (direction A).The transfer indication compound of the compound from the basal chamber to the apical chamber is discharged, and it is shown that, although the compound can pass through the intestinal wall, it is transported back into the intestinal cavity (direction B) by the discharge receptors found on the surface of the intestinal cell.Then the value obtained by A2B and B2A permeability calculates the discharge ratio.Good compounds for intestinal absorption should show permeability in the A2B direction, and show low permeability in the B2A direction.

[0210] The control compound performed as expected in the assay and all P app and discharge ratios were within acceptable limits (Table 2).

[0211] Table 2: Caco-2 permeability positive control data

[0212]

[0213] Rep = replicate; n = number of replicates; ND = not detected; NA = not applicable

[0214] a The concentration of the test compound sample from the receiver compartment of replicate 1 was lower than that of LQS A2B.

[0215] b Replicate 2 A2B did not detect the test compound in the receiver compartment.

[0216] The data for DMVTL061120 (synthetic peptide 1) at 10 μM showed that the compound could not be detected at lower concentrations in the standard curve, so the sample concentration from the Caco-2 experiment was increased to 100 μM. The data for DMVTL061120 at 100 μM are shown in Table 3.

[0217] Table 3: Caco-2 permeability DMVTL061120 data (synthetic peptide 1)

[0218]

[0219] Rep = parallel determination; n = number of parallel determinations

[0220] Average P values ​​of DMVTL061120 (synthetic peptide 1) in the A2B and B2A directions app The values ​​are 4.60×10 -6 cms -1and 0.633×10 -6 cms -1 The excretion ratio was 0.137, and the average recoveries A2B and B2A were 45.5% and 96.6%, respectively. The excretion ratio of 0.137 indicated that the compound was not a substrate for the excretion transporter, but could be a possible substrate for the uptake transporter because the excretion ratio was <1.

[0221] The findings from the Caco-2 flux assays indicate that synthetic peptide 1 can be readily taken up and absorbed by intestinal cells. The data for the apical to basolateral (A2B) direction indicate that the peptide may be a possible substrate for uptake transport and should therefore be able to efficiently cross the intestinal lining for uptake into the bloodstream. There was some loss of peptide recovery in the A2B direction, suggesting that some of the peptide may interact with or be retained or internalized by the intestinal cells themselves.

[0222] The data from the basolateral to apical (B2A) direction showed that almost no peptide was detected passing in this direction, thus supporting that the peptide is not easily expelled from the basolateral cellular environment by the efflux transporter. This is important information because it suggests that once internalized and passed through the lining of the intestinal wall, the peptide is not easily pumped back into the intestinal lumen, and therefore has a greater chance of being absorbed into the bloodstream once absorbed through the intestinal lining.

[0223] Using fluorescence analysis during the experiment, the introduction of Lucifer Yellow permeabilization enabled monitoring of the integrity of the polarized cell monolayer. Lucifer Yellow monitoring enabled determination that no toxicity of synthetic peptide 1 to Caco-2 cells was observed at a dose of 100 μM. The fact that the cell monolayer remained intact after treatment with the synthetic peptide is critical as it suggests that the synthetic peptide is unlikely to cause gastrointestinal cell damage and thus gastric upset upon ingestion. Therefore, it can be inferred that ingestion of peptide 1 is unlikely to cause bait rejection in its rodent target.

[0224] In summary, SAR identified amino acids important for cardiomyocyte toxicity and revealed that most of the important amino acids are located within the first 30 amino acids of the sequence, supporting that the beginnings of loops 1 and 2 of the cardiotoxin structure are most important for cardiomyocyte toxicity. Using this information, we proposed these as regions of the cardiotoxin sequence from which we could prepare short active synthetic peptides.

[0225] Using mass spectrometry data and structure activity relationship (SAR) analysis of the six components studied for use in cardiomyocytes, three short synthetic peptides were designed:

[0226] Peptide 1 - designed using the first 13 amino acids of component N.aan_I15_R4, which showed no toxicity but caused an increase in beating rate.

[0227] Peptide 2 - designed to include amino acids important for both high beating rate and high toxicity, but with modifications that increase toxicity and increase beating rate

[0228] Peptide 3 - designed with amino acids important for high beating rate and non-toxic, plus an additional lysine for solubility.

[0229] The designed peptide sequences were compared to public database sequences using the Basic Local Alignment Search Tool (BLAST) for proteins (BLASTP) from NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). These are unrestricted searches, so results from all matching organisms will be displayed.

[0230] Peptide 1

[0231] BLASTP analysis showed that the sequence had 100% identity to cytotoxin 1 from Naja annulifera and Najanivea, and 82% identity to the closest non-cobra sequence: a hypothetical protein from the wild potato Solanum commersonii.

[0232] Peptide 2

[0233] Although peptide 2 is modified, it has 100% identity to 10 Indian cobra (Naja naja) cytotoxins and 3 Bengal cobra (Naja kaouthia) sequences. It has 84% ​​identity to the closest non-cobra sequence: Russell's viper (Daboia russelii) cytotoxin. The closest non-snake sequence is only 76% similar to the sequence, with 90% identity to this portion (Clostridium tarantellae).

[0234] Peptide 3

[0235] This peptide has no unique matches in the NCBI database, with the closest entry covering 92% of the sequence and having 84% identity to the Indian cobra (Naja naja) cytotoxin. The closest non-cobra sequence is from Clostridia bacterium, which only fully matches 57% of the sequence.

[0236] It should be understood that these peptides can be modified to improve bioavailability and alter key features to ensure that the compound is selective for rodents and does not persist in the environment. These modifications may include C-terminal amides, D amino acids, and unnatural amino acids.

[0237] Thus, short synthetic peptides have been generated that show functional activity against rodent cardiomyocytes. The generation of short active synthetic peptides is advantageous over large-scale purification of full-length cardiotoxins from toxicants, as the use of short active synthetic peptides will greatly reduce the time scale and cost when (commercial) rodenticide production is scaled up.

[0238] All three peptides showed no toxicity towards cardiomyocytes and non-myocyte cell types, good selectivity between rodent and human cardiomyocytes and good thermal stability, as well as enhancement of the beating rate of rodent cardiomyocytes.

Claims

1. A synthetic peptide sequence comprising a sequence based on or derived from loop I of a cobra or cobra three-finger toxin.

2. A synthetic peptide sequence according to claim 1, wherein the sequence is based on or derived from amino acid residues 1 to 30 of the three-finger toxin.

3. A synthetic peptide sequence according to claim 1 or claim 2, wherein the sequence comprises 10 to 15 amino acid residues, preferably 13 amino acid residues.

4. A synthetic peptide sequence according to any one of claims 1 to 3, wherein the sequence has the following sequence: LKC(H / N)KL(V / I)PX(V / A)(W / Y)KTC [SEQ ID NO: 1].

5. The synthetic peptide sequence according to claim 4, wherein the sequence has the sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and variants thereof.

6. The synthetic peptide sequence according to any one of claims 1 to 5, wherein the sequence comprises one or more hydrophobic amino acids located at one or both ends of the sequence.

7. A synthetic peptide sequence according to any one of claims 1 to 6, wherein the sequence comprises one or more amino acids that enhance solubility in water.

8. A synthetic peptide sequence according to any one of claims 1 to 7, wherein the sequence comprises one or more synthetic amino acids.

9. The synthetic peptide sequence according to any one of claims 1 to 8, wherein the sequence is pegylated.

10. The synthetic peptide sequence according to any one of claims 1 to 9, wherein the sequence comprises a detectable tag, preferably a UV-fluorescent tag.

11. The synthetic peptide sequence according to any one of claims 1 to 10, wherein the sequence is cardiotoxic, preferably selectively cardiotoxic.

12. The synthetic peptide sequence according to any one of claims 1 to 11, wherein the sequence causes a change in heart rate.

13. The synthetic peptide sequence according to claim 12, wherein the compound induces tachycardia and increases heart rate.

14. A rodenticide comprising at least one compound that is cardiotoxic to rodents.

15. The rodenticide of claim 14, wherein the compound has selective cardiotoxicity.

16. A rodenticide according to claim 14 or claim 15, wherein the compound has an excitotoxic cardiotoxic effect.

17. The rodenticide according to any one of claims 14 to 16, wherein the compound causes a change in heart rate.

18. The rodenticide of claim 17, wherein the compound causes tachycardia and increases heart rate.

19. A rodenticide according to any one of claims 14 to 18, wherein the compound is an isolated amino acid sequence obtained or derived from snake venom, preferably from a species of the family Elapidae.

20. A rodenticide according to claim 19, wherein the snake venom is derived or obtained from a species of cobra, preferably from the genus Naja.

21. The rodenticide according to any one of claims 14 to 20, wherein the compound is the compound according to any one of claims 1 to 13.

22. A rodenticide comprising an isolated amino acid sequence derived from or obtained from the venom of a species of the family Elapidae.

23. A rodenticide according to claim 22, wherein the snake venom is derived or obtained from a species of cobra, preferably a species of the genus Naja.

24. A rodenticide according to claim 22 or claim 23, wherein the amino acid sequence is cardiotoxic.

25. The rodenticide of any one of claims 22 to 24, wherein the sequence is selectively cardiotoxic.

26. A rodenticide according to claim 24 or claim 25, wherein the compound has an excitotoxic effect.

27. The rodenticide according to any one of claims 24 to 26, wherein the compound causes a change in heart rate.

28. The rodenticide of claim 27, wherein the compound causes tachycardia and increases heart rate.

29. The rodenticide according to any one of claims 22 to 28, wherein the isolated amino acid sequence is a synthetic peptide sequence according to any one of claims 1 to 13.

30. A rodenticide formulation comprising the synthetic peptide sequence of any one of claims 1 to 13 and a carrier.

31. The rodenticide formulation of claim 30, wherein the formulation is formulated as a paste, solid, powder, gel or liquid.

32. A rodenticide formulation according to claim 30 or claim 31, wherein the formulation further comprises one or more of the following: petroleum or plant derived waxes, cereals, grains, nuts, fruits, colorants, flavorings, herbs, spices, essential oils, oils, fats or other materials palatable to rodents.

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