Rodenticide

Abstract

Novel synthetic peptide sequences based on Elapidae snake venom suitable for use as cardiotoxic rodenticides are described.

Description

[Technical field]

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

[0002] It is well known that rats destroy agricultural products and stored resources. They destroy crops, grains and cereals, accounting for nearly 10% of the world's grain harvest, depending on the country (Buckle and Smith, (2015). Rodent Pests and their control.2 ndEdition.Wallingford Oxfordshire.330-345). Rats are also known to transmit zoonotic diseases to humans and livestock, and cause significant damage to buildings and power supplies by gnawing and burrowing (Van den Brink,et al,(2018)In Anticoagulant Rodenticides and Wildlife;Springer:Cham,Switzerland,pp.1-9). Therefore, rodent control is important and routine worldwide, especially in agricultural environments and in and around buildings. Methods for controlling and eliminating rodent populations take several forms, including improved sanitation and establishment of rodent-proof areas, rodent capture and relocation, and lethal control measures such as traps and rodenticides. The use of anticoagulants (ARs) has become the most attractive approach for rodent control due to their effectiveness, ease of use, cost-effectiveness, and relative safety for workers. (Jakob and Buckle,(2018).Emerging Topics in Ecotoxicology(Principles,Approaches and Perspectives),vol 5.Springer,Cham.). The sophisticated social organization and novel behavior of rodents makes fast-acting toxicity ineffective. (Modlinska and Stryjek,(2016)PLoS ONE 11(6)). Anticoagulant rodenticides address this issue because they are slow-acting and usually result in death within a few days after ingestion. With prolonged or repeated exposure to AR, mice die from bleeding within 3-7 days.

[0003] Anticoagulant rodenticides (ARs) reduce vitamin K-dependent coagulation factors by inhibiting vitamin K epoxide reductase (VKOR). (Ng WY,et al(2018)Journal of Medical Toxicology;14(3):218-228). ARs inhibit vitamin K(1)-2,3-epoxide reductase, thus inhibiting the synthesis of vitamin K and, consequently, the synthesis of blood coagulation factors II, VII, IX, and X. (Hadler and Buckle,(1992)Proceedings of the Vert Pest Conference,15:149-155). The increased efficacy and duration of action of long-acting ARs is due to their higher 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). In addition, due to their high lipid solubility, they have long biological half-lives and are reabsorbed into the circulation via the enterohepatic circulation.

[0004] Warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl)chromen-2-one) was one of the first commercially available anticoagulant rodenticides (AR), but resistance has developed in rodents.

[0005] Coumatetralyl (4-hydroxy-3-(1,2,3,4-tetrahydronaphthalen-1-yl)chromen-2-one) is slightly more effective than warfarin due to its greater palatability, despite its reported lower toxicity. This compound was introduced following the emergence of warfarin-resistant rats and was used successfully for many years until resistance was reported in the UK and Denmark in the late 1960s.

[0006] Second-generation anticoagulant rodenticides (SGARs) were developed in the 1970s to overcome the development of resistance to first-generation anticoagulant rodenticides (FGARs) in rats. These rodenticides are more acutely toxic than first-generation ARs, and their enhanced potency is related to their higher 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 difenacoum (3-[3-p-diphenyl-1,2,3,4-hydronaphth-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-yl)-2H-pyran-2-one) and difenacoum (3-[3-p-diphenyl-1,2,3,4-hydronaphth-1-yl]-4-hydroxycoumarin). These are: flocoumafen (4-hydroxy-3-[l,2,3,4-tetrahydro-3-[4-(4-trifluoromethylbenzyloxy)phenyl-l-napthylcoumarin), and difethialone (3-[3-(4-bromophenyl)phenyl]-1,2,3,4-tetrahydronaphthalen-1-yl]-2-hydroxythiochromen-4-one).

[0007] In resistant rat populations, VKOR is slightly altered, preventing it from binding correctly to rodenticides and rendering them ineffective (Thijssen,(1995)Pesticide Sciences 43;73-78). The most common resistance mechanism is the result of single nucleotide polymorphisms 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 VKOR1C protein, which contains 163 amino acids, and specific amino acid substitutions in VKORC1 have been shown to confer resistance to AR. At least seven independent polymorphisms exist in the VKORC1 gene in Norway rats, suggesting that they may provide a genetic basis for anticoagulant rodenticide resistance (Mooney,et al(2018)Nature.8:4543). Resistance has been observed to spread geographically and progress from warfarin and FGARs to more potent active agents, SGARs, such as bromadiolone and difenacoom. Resistance to different ARs is called cross-resistance and evolves from FGARs to SGARs, and as a result, resistance to SGARs is always accompanied by resistance to FGARs (Mooney, et al (2018) supra).

[0008] The use of FGAR is now largely limited by this phenomenon of genetic resistance, and since 1958 large cases of resistance in brown rats have been reported in the UK, Europe, USA, Canada and Australia (Buckle, et al (1994) Proceedings of the Sixteenth Vertebrate Pest Conference. 7). Mutations in VKORC1 have been shown to cause two different heritable phenotypes: blood coagulation failure due to vitamin K-dependent coagulation factor deficiency type 2 (VKCFD2) and warfarin resistance (OMIM) (Rost, et al (2009) BMC genetics). Brodifacoum, flocumafen and difethialone are currently the only active compounds that have shown efficacy in all resistant strains of rats and mice.

[0009] Commercially available ARs are formulated into palatable baits for oral administration to target rodent pests, usually based on grains processed into blocks or pellets, with binders, flavorings, and colorants. Wax is added to some products to make them last longer and add an element of weather resistance (Horak, et al (2018) In Anticoagulant Rodenticides and Wildlife; Springer: Cham, Switzerland, pp. 87-108). Bait formulations require voluntary ingestion of sufficient quantities to be effective against the target pests. Since death from AR poisoning occurs several days after ingestion, it is quite typical for rodents to continue eating the bait after ingesting a lethal dose, increasing the concentration of anticoagulant rodenticide in their tissues. Unfortunately, in this case, other animals that have access to the bait may receive unwanted primary exposure, and secondary oral exposure may occur through ingestion of animal tissues containing AR residues. This poses a danger to species that prey on rodents or scavenge on the carcasses of poisoned animals (Horak, et al. (2018) supra).

[0010] There have been widespread reports worldwide of unintentional exposure of non-target wildlife and domestic pets to ARs, resulting in residues of ARs in predatory birds and non-target species, through primary or secondary poisoning. Primary exposure is a high risk for omnivorous or herbivorous animals due to the cereal-based nature of rodenticide baits, while secondary exposure is characterized by scavenging or predatory animals as a result of ingestion of animal tissues carrying AR residue concentrations. (Fisher,et al(2019)Animals(Basel).9(11):919). The high toxicity and metabolic persistence of SGARs pose a high risk of secondary mortality to 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).

[0011] Risks to non-target birds and other wildlife are influenced by species-specific susceptibility and the toxicokinetics of the compounds 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 difenacoum, are highly persistent in organs and tissues, including the liver, kidney, and pancreas, for at least six months and are toxic at lethal doses in a single feeding (Environmental Protection Agency 2004 (www.fluoridealert.org / pesticides / EPA-HQ-OPP-2006-0955-0005.pdf); Erickson and Urban, (2004) supra).

[0012] Rodenticide poisoning is one of the most common dog poisonings managed by the Pet Poison Helpline (Pet poison helpline website, accessed in 2021) and is a commonly reported cause of poisoning in dogs worldwide (Seljetun, et al (2020) Acta Veterinaria Scandinavica 62:30). A study conducted in 2020 determined that brodifacoum could be present in dog feces for more than 700 days and difenacoum for more than 650 days after a single vaccination (Seljetun, et al (2020) supra). Low levels of brodifacoum were detected in puppies born to exposed dogs for 28 days after birth. Dogs that are allowed to roam free are likely to encounter improperly placed rat baits or baits that have been dragged by rats (Merola, (2002) Vet Med 97:716-722).

[0013] Humans can be exposed to anticoagulants through rodenticide bait (Tran and King,(2016)Journal of Epidemiological Research.2(2);Watt,et al(2005)supra), usually by ingestion, either accidentally or intentionally. Most human exposure to AR occurs in young children, resulting in small doses. Intentional ingestion of large amounts of AR can result in an anticoagulant state lasting for weeks to months (Watt,et al(2005)supra). Large doses can cause nosebleeds, extensive bruising, hematomas, and gastrointestinal bleeding, leading to hypovolemic shock and death from severe bleeding. Summary of the Invention

[0014] Due to the emergence of resistant rodent strains and the risk to the environment and non-target species from the use of second-generation anticoagulant rodenticides, there is an urgent need to develop alternative rodenticides. Alternatives that reduce the risk to non-target species are beneficial for the control of pest rodents, and reducing their persistence in the environment is advantageous. It is clear that rodent species are becoming resistant to SGARs as well as FGARs, with brodifacoum being the only exception to date. Brodifacoum has been shown to be extremely stable in the environment and to have stable efficacy for up to 30 days. It has become common to use these more potent and toxic rodenticides, which are typically harmful to the environment and other species. As an alternative to such rodenticides, rodenticides that primarily target the desired rodents, minimize the risk of primary and secondary exposure to non-target species such as pets and predatory birds, and have a short half-life and reduced harmful effects on the environment, would be the most attractive method of rodent control.

[0015] It is against this background that the present invention was devised.

[0016] In one embodiment, the present invention relates to a rodenticide comprising at least one compound that is cardiotoxic to rodents.

[0017] Preferably, at least one compound exhibits selective or specific cardiotoxicity. In other words, at least one compound has no, essentially no, or no significant or detectable toxicity to other tissues, such as neurotoxicity or hemolytic activity.

[0018] In one embodiment, the compound has an excitatory or tachycardial cardiotoxic effect rather than an inhibitory one. In other words, the compound causes changes in heart rate. Preferably, the compound increases the heart rate and causes tachycardia. For the avoidance of doubt, an inhibitory cardiotoxin slows and / or stops the heart rate, whereas an excitatory cardiotoxin speeds up the heart rate, an effect called tachycardia. A slower heart rate reduces the oxygen supply to vital organs (such as the brain), making death more likely to occur sooner. A tachycardial cardiotoxin increases the heart rate, putting more strain on the heart over time and making it more likely to develop heart failure due to a heart attack. Ultimately, both have the same outcome, the death of the animal, but by different mechanisms.

[0019] In certain embodiments, the compound is an isolated amino acid sequence obtained or derived from a snake venom.

[0020] The advantage of using snake venom as a rodenticide is its impact on the environment. Snake venom is broken down by digestion and therefore does not remain in the food chain. Furthermore, since the venom is a biological composition, it is quickly broken down in the environment and does not accumulate in soil, etc. Thus, the rodenticide contemplated by the present invention offers a significant advantage over existing synthetic chemicals.

[0021] In one embodiment, the snake venom is obtained or derived from a species of the Elapidae family.

[0022] Elapidae (commonly referred to as cobras) are a family of venomous snakes characterized by permanent fangs at the front of the mouth. Most species contain neurotoxins in their venom, which are secreted through hollow fangs to immobilize prey and for defense. The main toxins are phospholipase A2 (PLA2) and three-finger toxin (3FTx). Other toxic components include cardiotoxins and cytotoxins, which cause cardiac dysfunction and cell damage, respectively.

[0023] Preferably, the snake venom is derived from or obtained from a species of cobra. Cobra is the common name for various snakes of the Elapidae family, most of which belong to the genus Cobra. All members of this group are more or less venomous and are known to cause LD in rats. 50 Some are considered to be among the most dangerous snakes in the world due to their bite size and patient mortality rate.

[0024] In one embodiment, the amino acid sequence is cardiotoxic, preferably selectively cardiotoxic, as defined and described herein. In contrast, the neurotoxic effect of snake venom is recognized to act via nicotinic acetylcholine receptors at the neuromuscular junction, the effect being respiratory arrest and death.

[0025] It should be understood 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 complete or whole venom minimizes the need for additional processing and formulation, since the proteins are already in their native active form. However, the drawback is the presence of other proteins, including neurotoxins.

[0026] Another possibility is that the cytotoxicity of snake venom affects the mucous membranes of the mice, causing them to dislike the taste.

[0027] An additional consideration is the speed of the killing action. Snake venom can kill a rat within a minute, as occurs during feeding. However, the presence of one or more dead rats next to or near a baited bait will deter other rats from eating the bait, and therefore the venom. It is therefore desirable to delay the lethal action.

[0028] Therefore, a further aspect of the present invention encompasses synthetic or artificial peptide sequences that include sequences based on or derived from Elapidae or cobra three-finger venom (3FTx).

[0029] 3FTx is a superfamily of low molecular weight toxin proteins found in snake venoms. The second largest class of 3FTx proteins causes toxicity in cardiomyocytes, leading to increased heart rate and ultimately cardiac arrest. Short cardiotoxins (CTXs) are single-chain polypeptides of 59–61 amino acids arranged in a three-finger fold consisting of antiparallel β-strands, strengthened by four disulfide bonds and numerous hydrogen bonds. Long three-finger cytotoxins are approximately 81 amino acids long. CTXs are basic proteins with a hydrophobic three-finger loop, the ends of which are flanked by cationic residues (mainly lysine and arginine). The hydrophobic tail confers amphipathic properties of CTX, mediating its binding and insertion into anionic phospholipid membranes, leading to deleterious cellular events such as pore formation and lysis, as well as the upregulation of intracellular Ca. 2+ This leads to increased ion influx, membrane depolarization, and, importantly, toxin internalization leading to mitochondrial and lysosomal damage, resulting in impaired cellular cascades leading to cell death.

[0030] It is understood that the entire cytotoxin can be used in accordance with the present invention. However, it is generally more economical to use only a portion. Furthermore, shorter peptide sequences require less folding or refolding or provision for correct folding after production. Therefore, it is advantageous to use peptides with only simple secondary structures. Furthermore, the secondary structure of the peptide can be designed to enhance certain properties, such as stability, thermal stability, bioavailability, species selectivity, etc.

[0031] In certain embodiments, the sequence is based on or derived from loop I of an elapid or cobra three-finger toxin. The cytotoxic effect of 3FTx is believed to reside in the sequence of loop I, together with several amino acid residues at the base of loop II (Menez et al (1990) Biochimie 72:575-588).

[0032] In one embodiment, the sequence is based on or derived from amino acid residues 1-30 of the cobra three-toed toxin. Ideally, the sequence has 10-15 amino acid residues, for example 13 amino acid residues. It is understood that the ranges defined herein encompass all discrete numbers between the lower and upper limits, such as 6, 11, 14, 21, 25 and 27 amino acids.

[0033] In one embodiment, the sequence has the following sequence: LKC(H / N)KL(V / I)PX(V / A)(W / Y)KT[SEQ ID NO:1]

[0034] For example, the sequence can have the following sequences and variants thereof: LKCHKLVPPVWKT [SEQ ID NO: 2] LKCNKLIPLAYKT [SEQ ID NO: 3] LKCHKLIPIAWKTK [SEQ ID NO: 4]

[0035] It is useful for the peptide to contain one or more hydrophobic amino acids at one or both ends of the sequence to aid in membrane penetration. Additionally or alternatively, the sequence may contain one or more amino acids that enhance solubility in water. It is understood that the sequence may also be designed to contain amino acids that enhance solubility in hydrophobic materials such as waxes.

[0036] Additionally, the sequence may include one or more synthetic amino acids, may be PEGylated, and / or may include a detectable tag, such as a UV fluorescent tag. Tags detectable under UV light are of particular interest, since rodent urine is visible under UV light. By including a UV fluorescent tag, the reduction of rodent populations may be assessed and / or monitored.

[0037] It is understood here that ideally the sequence is cardiotoxic, preferably selectively cardiotoxic, and induces a change in heart rate as described herein above, preferably causing an increase, i.e. tachycardia.

[0038] The present invention also encompasses rodenticides comprising the synthetic peptide sequences described herein above formulated with a carrier. The formulation may be a paste, solid, powder, gel or liquid and may further include one or more of the following: waxes (petroleum or vegetable derived), grains, nuts, fruits, colors, flavors, herbs, spices, essential oils, oils, lipids or other rodent-palatable materials.

[0039] The present invention will now be described in further detail with reference to the following non-limiting examples and figures. [Brief description of the drawings]

[0040] [Figure 1A] Figure 1 shows toxicity data for rodent cardiomyocytes and cardiac fibroblasts. Figure 1A shows the toxicity observed in neonatal rat cardiomyocytes (orange) and cardiac fibroblasts (blue) when treated with the purified cobra peptide panel at a dose of 12.5 μg / ml. [Figure 1B] Figure 1B shows toxicity data for rodent cardiomyocytes and cardiac fibroblasts. Figure 1B shows the toxicity observed in neonatal rat cardiomyocytes (orange) and cardiac fibroblasts (blue) when treated with a 7.5 μg / ml dose of the purified cobra peptide panel. [Figure 1C] Toxicity data for rodent cardiomyocytes and cardiac fibroblasts. Figure 1C shows the toxicity observed in neonatal rat cardiomyocytes (orange) and cardiac fibroblasts (blue) when treated with the purified cobra peptide panel at a dose of 6 μg / ml (Figure 1C). [Diagram 2] Figure 2 shows rodent erythrotoxicity data. Figure 2 shows the toxicity of purified Cobra fractions to isolated rat erythrocytes (red blood cells). [Diagram 3] Figure 3 shows the toxicity of six major fractions to human iPSC-derived cardiomyocytes. Figure 3 shows the % toxicity to human iPSC-derived cardiomyocytes after treatment with six purified fractions at doses of 1, 2.5, 3.5, 6, and 7.5 μg / ml. [Figure 4]Figure 4 shows the thermal stability profile of six selected major fractions. The fractions were heated at 37°C, 60°C, 80°C, and 100°C and then administered to rat cardiomyocytes at 6 μg / ml for 24 hours. The amount of cardiomyocyte toxicity at each temperature was assessed and used to determine if there was a reduction in activity due to lack of thermal stability of the peptide. [Diagram 5] Figure 5 shows SAR analysis.Figure 5 is a schematic showing amino acid residues highlighted by SAR as potentially important for cardiomyocyte toxicity. [Figure 6A] 6 shows the toxicity of synthetic peptides to neonatal rat cardiomyocytes and cardiac fibroblasts assessed using a resazurin survival assay: Figure 6A shows the toxicity of three synthetic peptides to neonatal rat cardiomyocytes. [Figure 6B] Figure 6B shows the toxicity of synthetic peptides to neonatal rat cardiomyocytes and cardiac fibroblasts using the resazurin survival assay. Figure 6B shows the toxicity of three synthetic peptides to cardiac fibroblasts. [Figure 7A] Figure 7 shows the toxicity of synthetic peptides to human IPSC-derived and chick embryo cardiomyocytes Figure 7A shows the toxicity of three synthetic peptides to human IPSC-derived cardiomyocytes. [Figure 7B] Figure 7B shows the toxicity of synthetic peptides to human IPSC-derived and chick embryo cardiomyocytes.Figure 7B shows the toxicity of three synthetic peptides to chick embryo cardiomyocytes. [Figure 8A] Figure 8 shows the fold change in beating rate in neonatal rat and human IPSC-derived cardiomyocytes in response to synthetic peptide treatment. Figure 8A shows the fold increase in beating rate in neonatal rat cardiomyocytes (orange) and human IPSC-derived cardiomyocytes (blue) in response to synthetic peptide 1 treatment. [Figure 8B] Figure 8B shows the fold change in beating rate in neonatal rat and human IPSC-derived cardiomyocytes in response to synthetic peptide treatment. Figure 8B shows the fold increase in beating rate in neonatal rat cardiomyocytes (orange) and human IPSC-derived cardiomyocytes (blue) in response to synthetic peptide 2 treatment. [Figure 8C] Figure 8C shows the fold change in beating rate in neonatal rat and human IPSC-derived cardiomyocytes in response to synthetic peptide treatment. Figure 8C shows the fold increase in beating rate in neonatal rat cardiomyocytes (orange) and human IPSC-derived cardiomyocytes (blue) in response to synthetic peptide 3 treatment. [Figure 9A] Figure 9 shows the thermal stability of synthetic peptides as assessed by the fold increase in neonatal rat cardiomyocyte beating rate. Figure 9A shows the thermal stability of synthetic peptide 1, using the fold change in beating rate to detect loss of activity. [Figure 9B] Figure 9B shows the thermal stability of synthetic peptide 2 as assessed by the fold increase in neonatal rat cardiomyocyte beating rate.Figure 9B shows the thermal stability of synthetic peptide 2, using the fold change in beating rate to detect loss of activity. [Figure 9C] Figure 9C shows the thermal stability of synthetic peptides as assessed by the fold increase in neonatal rat cardiomyocyte beating rate. Figure 9C shows the thermal stability of synthetic peptide 3, using the fold change in beating rate to detect loss of activity. All three synthetic fractions 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 non-heated fraction (orange). [Figure 10] Schematic diagram of how to perform a Caco-2 flux assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] array: SEQ ID NO:1 - Generic synthetic peptide sequence SEQ ID NO:2 - Synthetic 13-mer peptide 1 SEQ ID NO:3 - Synthetic 13-mer peptide 2 SEQ ID NO:4 - Synthetic 13-mer peptide 3 SEQ ID NO:5 - Sequence of fraction N.aan_I15_R4 SEQ ID NO:6 - Sequence of fraction N.nka_I18_R4 SEQ ID NO:7 - Sequence of fraction N.naj_I25_R3 SEQ ID NO:8 - Sequence of fraction N.nub_I16_R4 SEQ ID NO:9 - Sequence of fraction N.nig_I19_R4 SEQ ID NO:10 - Sequence of fraction N.atr_I28_R3 EXAMPLES

[0042] method Cobra Venom Recovery: Venom was extracted from 12 cobra species using a proprietary method, collected into fresh vials, quantified, freeze-dried, and stored in a -20°C freezer until use.

[0043] Purification of cobra venom using two-dimensional HPLC: Lyophilized cobra venom was reconstituted in HPLC grade H2O and protein concentration was measured using a DeNovix® DS11 spectrophotometer. Total venom was diluted to 100mg / ml in IE Buffer A and 100μl was injected onto an Agilent 1100 HPLC for separation by ion exchange chromatography. Venom was separated using an increasing gradient of IE Buffer B over time. Peak detection parameters were set and fractions were collected. Fractions were lyophilized, reconstituted in RP Buffer A and reinjected onto the Agilent 1100 HPLC for separation using reversed phase chromatography. Fractions were separated using an increasing gradient of RP Buffer B over time. From this process a panel of venom components fractionated in 2D was obtained and lyophilized. Fractions were resuspended, quantified on a spectrophotometer and diluted to the same working protein concentration. Finally, fractions were plated out onto low protein binding 384-well plates and available for assay screening.

[0044] Isolation of rodent cardiomyocytes and cardiac fibroblasts: Hearts were harvested from euthanized 1-2 day old neonatal rats and transferred to ice-cold Hanks Balanced Salt Solution (HBSS) containing filter-sterilized 2% penicillin-strep. Hearts were washed with HBSS to remove non-cardiac tissue, then cut into 1-3 mm pieces and placed in 40 ml of ice-cold, filter-sterilized 0.1% trypsin / HBSS. Heart pieces in trypsin were placed in a large ice bucket and incubated overnight at 4°C on a rocker with gentle agitation to digest the tissue.

[0045] The next day, all trypsin was aspirated from the tissue and the tissue was transferred evenly into 2 ml Eppendorf tubes. The tissue was then further digested with 1 mg / ml collagenase / HBSS (filter-sterilized) solution at 37 °C. For each collagenase digestion, 2 ml of enzyme buffer was added to all Eppendorf tubes and the tubes were incubated in a heating block at 37 °C with 400 rpm agitation for 2 min. The supernatants of the first two digestions were discarded. After each of the following digestion steps, the tissue was gently triturated with a pipette to release the cells. Upon completion of trituration, the supernatant was retained and added to a tube containing 10 ml cell culture medium and stored on ice.

[0046] All cell-containing supernatants were filtered through a 250 μm cell strainer to remove any potentially undigested tissue, and cardiomyocytes and cardiac fibroblasts were separated by spinning the supernatant through a Percoll gradient at 1800 g for 45 min. The bands containing cardiomyocytes and the bands containing cardiac fibroblasts were collected separately, and both cells were washed with HBSS and centrifuged three times to remove the Percoll buffer, after which they were resuspended in fresh medium (DMEM:F12, 10% FCS, 2% PS) and viable cell yield was measured using a hemocytometer. Cells were plated in 384-well plates at 1x10 5 ~2x10 5 cells / cm 2 All media was replenished to 50 μl and cells were returned to a humidified 37°C, 5% CO2 incubator and grown statically until spontaneously beating.

[0047] Isolation of chick embryonic cardiomyocytes: Hearts were collected from euthanized 12-13 day old embryonic chicks and transferred to filter-sterilized ice-cold Hank's Balanced Salt Solution (HBSS) containing 2% Pen-strep. Hearts were washed with HBSS to remove blood and non-cardiac tissue, then cut into 1-3 mm pieces and placed in 40 ml of filter-sterilized ice-cold 0.05% trypsin / HBSS. Heart pieces in trypsin were placed in a large ice bucket and incubated on a rocker with gentle agitation for 1 h to digest the tissue.

[0048] All trypsin was aspirated from the tissue and discarded, and the tissue was transferred evenly into 2 ml Eppendorf tubes. The tissue was then further digested using a 1 mg / ml collagenase II / 0.1% trypsin / HBSS (filter-sterilized) solution. For each digestion, 2 ml of enzyme buffer was added to all Eppendorf tubes, and the tubes were incubated in a 37°C heating block with 400 rpm agitation for 3 minutes. The supernatants from the first two digestions were discarded. After each of the following digestion steps, the tissue was gently triturated with a Pasteur pipette to release the cells. Once trituration was complete, the supernatant was retained and added to a tube containing 10 ml of cell culture medium (DMEMF12, 10% FCS, 2% PS) and stored on ice.

[0049] All cell-containing supernatants were filtered through a 250 μm cell strainer to remove any potentially undigested tissue. The cell suspension was then filtered through a 75 cm 3The cells were pre-seeded in 1x10 tissue culture flasks and incubated at 37°C, 5% CO2 for 2 hours to allow excess fibroblasts to settle and adhere. The remaining supernatant containing enriched cardiomyocytes was separated from the remaining cardiac fibroblasts by spinning at 1800g for 1 hour through a Percoll gradient. The band containing immature cardiomyocytes was collected separately from the band containing remaining cardiac fibroblasts, and both sets of cells were washed with HBSS, centrifuged three times to remove the Percoll buffer, suspended in fresh culture medium (fibroblasts: DMEM:F12, 10% FCS, 2% PS; chicken myocytes: DMEM:F12, 10% horse serum, 1% FCS, 2% PS) and viable cell yield was measured using a hemocytometer. Cells were plated at 1x10 in 384-well plates. 5 ~2x10 5 cells / cm 2 All media was replenished to 100 μl and the cells were returned to a humidified incubator at 37°C, 5% CO2, and cultured until they began to beat spontaneously. Myocytes were observed to be beating spontaneously 3 days after plating.

[0050] Rat cardiomyocyte and cardiac fibroblast cell toxicity screening of purified venom fractions: Neonatal rat cardiomyocytes and cardiac fibroblasts were treated with the purified venom fractions of suspected cardiotoxins at doses of 12.5, 7.5, and 6 μg / ml for 2 hours in a humidified incubator at 37°C and 5% CO2, and rat cardiomyocytes were treated with additional concentrations of 3.5, 2.5, 1, and 0.5 μg / ml. After 2 hours, resazurin was added to all wells at a final working concentration of 160 μM. Changes in resazurin dye fluorescence were monitored over a 5-hour period using a BMG Fluostar plate reader. Percent inhibition of both cell types in response to each fraction at each dose was calculated and graphed.

[0051] Rat erythrocyte toxicity screening of purified venom fractions: Venom fractions 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. Serial dilutions of crude venom from the black-necked cobra (Naja nigricollis) (N. nig) and DMSO were also plated as toxicity controls.

[0052] Blood from rats was collected postmortem in PBS containing heparin. Red blood cells were diluted with PBS and counted using a hemocytometer. Red blood cells were counted at 1x10 8 The cells were diluted to a final concentration of cells / ml and spun at 300g for 3 min and the supernatant containing the white blood cells was removed. The red blood cell pellet was suspended in an equal volume of PBS. 100 μl of resuspended cells were added to all fractions and to toxicity control wells, giving a final volume of 200 μl / well (1x10 7 erythrocytes / well, 7.5 μg / ml and 0.5 μg / ml fractional working concentrations).

[0053] Erythrocytes were incubated at 37°C, 5% CO2 for 5 hours and then allowed to lyse or settle by gravity. After 5 hours, supernatants were removed from all wells and plated in quadruplicate in 384-well plates (30 μl / well). All replicates were averaged and the percentage of erythrocyte lysis at both doses was graphed.

[0054] Screening of purified venom fractions for changes in cardiomyocyte beating rate: Neonatal rat cardiomyocytes were isolated according to standard protocols and seeded at a density of 100,000 cells / 96 wells. Once the cardiomyocyte cultures were beating spontaneously in uniform sheets, videos were taken of each well using a microscope camera to measure the number of beats / min before treatment. Wells were treated in duplicate with suspected cardiotoxin fractions at doses of 12, 6, 3, and 1 μg / ml. Cells were incubated in each fraction for 1 h in a humidified incubator at 37°C and 5% CO2. After 1 h, the video of the cells was re-recorded and the number of beats / min after treatment was determined. A fold change in beating frequency was calculated for each pair of treatment wells.

[0055] Evaluating the effects of six major fractions on non-target species using human iPSC-derived cardiomyocytes: Human IPSC-derived cardiomyocytes were purchased from Axol Biosciences and cultured according to the company's standard methods. Briefly, when needed, cells were retrieved from liquid nitrogen storage and dropped into 10 ml of plating medium (cardiomyocyte maintenance medium + 10% FCS + 10 μM ROCK inhibitor) pre-warmed to 37 °C. The supernatant was aspirated from the cell pellet, and the cell pellet was gently suspended in 1 ml of plating medium. Cells were counted using a hemocytometer and trypan blue staining. Cells were plated at a seeding density of 10–20,000 cells / well in fibronectin-precoated 384-well plates. Cells were cultured overnight in a humidified incubator at 37 °C and 5% CO2, after which the medium was removed the next day and replaced with fresh maintenance medium (without FCS and without ROCK inhibitor). The medium was partially replaced every 2 days until the cells formed a continuous sheet of spontaneously beating cardiomyocytes (7–10 days of culture).

[0056] After cells were beating spontaneously, the six major fractions were diluted in maintenance medium to give final working concentrations of 7.5, 6, 3.5, 2.5, and 1 μg / ml. 30 μl of each fraction dilution was added to triplicate wells of cells and incubated for 2 hours at 37°C, 5% CO2 in a humidified incubator. After 2 hours, resazurin dye was added to all wells at a final working concentration of 160 μM, and the change in plate fluorescence was monitored over a 5-hour period using a BMG Labtech Fluostar plate reader. Percent toxicity was calculated from triplicate wells at each dose and graphed.

[0057] Assessment of the heat stability of the six major purified venom fractions: The thermal stability of the six main fractions was investigated. Aliquots of each fraction were heated in a heat block to 37°C (unheated), 60°C, 80°C, and 100°C before being administered to rat neonatal cardiomyocyte cells at a dose of 6 μg / ml. Cardiomyocyte cytotoxicity was assessed 24 hours after administration at each temperature against similar aliquots of each fraction that had been unheated prior to administration. After 24 hours, 160 μM resazurin dye was added to all cells and changes in cardiomyocyte cell viability / metabolism were monitored hourly up to 5 hours after dye addition. Further measurements were recorded 24 hours after dye addition.

[0058] Mass spectrometry of six selected fractions: Ten micrograms of six fractions that showed different levels of myocardial cytotoxicity and impact on heart rate were subjected to Intact Mass and Peptide Mapping mass spectrometry (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. Ten micrograms of sample were loaded onto a Sciex Exion liquid chromatograph and run over a 5-minute reversed-phase gradient with 0.1% formic acid buffer A and 0.1% formic acid / 100% acetonitrile buffer B. The RP HPLC was run with a 500-μl flow rate and a gradient increasing from 5% buffer B to 45% buffer B over 3 minutes, followed by a wash with 95% B and equilibration with 5% B. The flow from the column was run on a Sciex X500B mass spectrometer and set to collect data in positive ion mode. The source was set at 400 °C, 5500 V, and gas at 50 psi to allow ionization of the eluate. A TOF mass window of 500–3000 Da was collected and scanned in 0.5 s. The X500B mass spectrometer was calibrated with a positive calibration mix, and the experimental error was estimated to be 0.5 Da. The resulting total ion chromatograms (TICs) were deconvoluted using BioToolKit software (Sciex).

[0059] 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 was added to each sample and the samples were heated at 65 °C for 30 min. 5 μl of 500 mM iodoacetamide in 100 mM ammonium bicarbonate was added to all samples and reincubated at room temperature in the dark for 30 min. 10 μl of 25 ng / μl trypsin in 50 mM ammonium bicarbonate was added to each sample and incubated overnight at 37 °C. 10 μl of the complete digest was taken and mixed with 10 μl of 0.1% trifluoroacetic acid (TFA). 10 μl of the mixed sample was loaded onto a Sciex Exion liquid chromatograph and a 10 min reversed phase gradient was performed. A Phenomenex Lunar 1.6 μm, PS C18, 100A 150x2.1 mm column was used. The buffer composition was as per intact mass MS (0.1% formic acid buffer A, 0.1% formic acid / 100% acetonitrile buffer B). The flow was set to 300 μl with a gradient starting from 5% buffer B and increasing to 45% B over 15 min. To allow ionization of the eluent, the source was set at 400 °C, 5500 V, and 30 psi gas. A TOF mass window of 300–1800 Da was collected and scanned in 1.2 s. Mass spec mass spec (MSMS) data were collected using an information-dependent acquisition method collecting up to 10 MSMS per scan. The X500B was calibrated with a positive calibration mix and the experimental error was estimated to be 1 ppm. Data obtained from MSMS were analyzed using Mascot (MatrixScience) using the Swiss-Prot database.

[0060] SAR and synthetic peptide design: Data obtained from the six fractions sent to mass spectrometry were used to perform structure-activity relationship (SAR) analyses from which three short synthetic peptides were designed for further study. The three peptides were designed to take advantage of different properties of the six main fractions examined for toxicity and pulsatile effect properties.

[0061] Peptide 1 - designed using the first 13 amino acids of the N.aan_I15_R4 fraction, did not show toxicity but caused an increase in beating rate.

[0062] Peptide 2 - designed using the first 13 amino acids of N.aan_I15_R4 but substituted at key sites identified by SAR analysis to include amino acids important for both high beating rate and high toxicity.

[0063] Peptide 3 - designed using the first 13 amino acids of N.aan_I15_R4, but substituted at key sites identified by SAR analysis to include amino acids important for high beating rate and non-toxicity.

[0064] Screening of synthetic peptides for toxicity to rat cardiomyocytes, chicken cardiomyocytes, human iPSC-derived cardiomyocytes and cardiac fibroblasts: Neonatal rat cardiomyocytes, embryonic chicken cardiomyocytes, human iPSC-derived cardiomyocytes and rat cardiac fibroblasts were cultured. Synthetic peptides 1, 2 and 3 were diluted to working concentrations of 100, 10, 1 and 0.1 μg / ml in cell culture medium appropriate for optimal growth of each cell type. Toxicity controls were also included in the experiment and were selected from total venom and venom cardiotoxin fractions previously identified to be toxic. Duplicate wells of cells from each of the three cell types were also treated with 100 μg / ml of N. niger total venom and 10 μg / ml and 1 μg / ml of the identified cardiotoxin fractions N. nka_I18_R4 and N. ng_I19_R4. All wells were incubated with each treatment for 24 hours. After 24 hours, resazurin dye was added to each well at a final concentration of 160 μM and changes in dye fluorescence were monitored and recorded over a 24 hour period.

[0065] Screening for synthetic peptides that alter cardiomyocyte beating rate: The baseline beating rate of neonatal rat and human IPSC-derived cardiomyocytes treated with the three synthetic fractions was determined by triplicate video microscopy of each well using a microscope camera. The average beats / min was calculated from triplicate measurements of three separate wells (n=9). Aliquots of each peptide were taken to treat neonatal rat cardiomyocyte cells at doses of 100, 10, 1, and 0.1 μg / ml. The cells were returned to a humidified incubator at 37°C and 5% CO2 for 2 hours. After 2 hours, the beating rate of treated cardiomyocytes was reassessed using the same method used to obtain pre-treatment beating data. This process was repeated 24 hours after synthetic peptide addition.

[0066] Embryonic chicken cardiomyocytes were cultured until they beat spontaneously. The cells were recorded by video microscopy to generate a baseline beating rate for cells pretreated with synthetic peptides. Synthetic peptides were diluted in cardiomyocyte culture medium to final working concentrations of 100, 10, 1 and 0.1 μg / ml. Chicken cardiomyocytes were treated with synthetic peptides for 2 hours in a humidified cell culture incubator at 37°C and 5% CO2, after which the beating rate was recorded again. The change in beating rate was calculated as the number of beats / min after synthetic treatment divided by the number of beats / min before treatment.

[0067] Screening of synthetic peptides for thermal stability: The thermal stability of the three designed synthetic peptides was examined. The baseline beating rate of neonatal rat cardiomyocytes treated with the synthetic fractions was determined by triplicate video microscopy of each well using a microscope camera. An aliquot of each peptide was heated in a heat block at 100°C and then treated with rodent cardiomyocyte cells at doses of 100, 10, 1, and 0.1 μg / ml. The cells were returned to a 37°C, 5% CO2 incubator for 2 hours. After 2 hours, the beating rate of the treated cardiomyocyte cells was reassessed using the same method used to obtain the pre-treatment beating rate data. This process was repeated 24 hours after the addition of the synthetic peptides. The data of fold change in cardiomyocyte beating rate after treatment with the heated synthetic fractions was compared to the previously obtained fold change in beating rate of the non-heated synthetic peptides to assess their thermal stability.

[0068] Assessment of intestinal permeability of major short synthetic peptide 1 using Caco-2 permeability assay (bidirectional): 1x10 cells 5 cells / cm 2 Cells were seeded in Millipore Multiscreen Transwell plates at 100°C for 24 h. Cells were cultured in DMEM and the medium was changed every 2–3 days. Permeability tests were performed on day 20. Cell culture and assay incubations were performed at 37°C in an atmosphere of 95% relative humidity and 5% CO2. On the day of the assay, monolayers were prepared by washing both the basolateral and apical surfaces twice with Hanks' Balanced Salt Solution (HBSS) of the desired pH pre-warmed to 37°C. Afterwards, cells were incubated in HBSS of the desired pH for 40 min in both the apical and basolateral compartments to stabilize physiological parameters.

[0069] Dosing solutions were prepared by diluting test compounds in assay buffer to give final test compound concentrations of 10 μM or 100 μM (final water concentration ≦1% v / v). Lucifer Yellow, a fluorescent integrity marker, was also included in the dosing solutions. Analytical standards were prepared from water dilutions of the test compounds and transferred to buffer, keeping the water concentration ≦1% v / v. Buffers were supplemented with HBSS pH 7.4.

[0070] To assess apical to basolateral (A2B) permeability, HBSS was removed from the apical compartment and replaced with test compound dosing solution. The apical compartment insert was then placed into 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 compartment insert before placing it into the companion plate.

[0071] After 120 min, the apical compartment inserts and companion plate were separated and apical and basolateral samples were diluted and analyzed. Permeability of test compounds was assessed in duplicate. Compounds with known permeability properties were run as controls on each assay plate.

[0072] Test and control compounds were quantified by appropriate sample dilution and LC-MS / MS cassette analysis using a seven-point calibration. Cyprotex universal analysis conditions were used. Starting concentrations (C0) were determined from the dosing solution, and experimental recoveries were calculated from C0 and both the apical and basolateral compartment concentrations.

[0073] Throughout the experiment, the integrity of the polarized cell monolayer was checked by fluorescent monitoring of the permeation of Lucifer Yellow. The permeation of this paracellular marker is low if the monolayer is intact. The Papp value of Lucifer Yellow determined in a given well was within the acceptable limit (>1.0 cm / sx10 6 ) but the resulting Papp value for the test substance or positive control substrate in that well was qualitatively similar (within the Lucifer Yellow threshold) to the Papp values ​​measured in the remaining replicate wells, the cell monolayer was considered to be within the acceptable range based on the scientific judgment of the responsible scientist. Otherwise, results from the affected monolayer were excluded.

[0074] Data Analysis: The amount of probe substrate (pmol) that permeates the monolayer is calculated by multiplying the determined concentration (nM ≡ pmol / mL) in each test sample receiver well by the volume of the receiver compartment (apical = 0.09 mL, basolateral = 0.210 mL). The amount measured in each receiver compartment is then divided by the incubation time (seconds) to determine the transport rate of the test compound or positive control substrate, which is then used to calculate the apparent permeability (P) according to the following equation: app ) to determine:

number

[0075] where dQ / dt is the permeation rate of drug across the cell, C0 is the donor compartment concentration at time zero, and A is the area of ​​the cell monolayer. C0 was obtained from analysis of the dosing solution. The Emission Ratio (ER) was calculated from the average A vs. B and average B vs. A data, which is derived from:

number

[0076] 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.

[0077] Four control compounds, atenolol (human absorption rate: 50%), antipyrine (human absorption rate: 97%), talinolol (P-gp substrate), and estrone-3-sulfate (BCRP substrate), were screened simultaneously with the test compounds.

[0078] result Cardiomyocyte and cardiac fibroblast toxicity assays: At a dose of 12.5 μg / ml, 14 and 11 of the purified synthetic fractions were toxic to rat cardiomyocytes and cardiac fibroblasts, respectively (Figure 1A). Reducing the dose of the purified venom fractions abolished fibroblast toxicity for all but one of the purified fractions tested (N.naj_I24_R2). Eight of the purified fractions were found to cause 50% or more toxicity to rat cardiomyocytes even at a dose of 6 μg / ml (Figure 1C). This data indicates that selectivity for cardiomyocytes over cardiac fibroblasts can be achieved by choosing the appropriate dose. Interestingly, the fractions purified from the venom of Naja annulifera showed no toxicity to either cardiomyocytes or cardiac fibroblasts at any of the three doses tested.

[0079] Profiling of a panel of purified fractions suspected to be cobra cardiotoxins based on their HPLC peak elution times and profiles confirmed that the fractions displayed a wide range of different levels of cytotoxic effects on both neonatal rat cardiomyocytes and neonatal rat cardiac fibroblasts. Furthermore, the window between toxic and non-toxic doses for many fractions was relatively narrow, with toxicity approaching 90-100% for many fractions at a dose of 12.5 μg / ml and complete abolition of toxicity at doses around 1-2.5 μg / ml. Overall, neonatal rat cardiomyocytes were more sensitive to many of the fractions screened than were administered cardiac fibroblasts. Fractions isolated from the venom of N. aan displayed little cytotoxic effects on either cardiomyocytes or cardiac fibroblasts. Interestingly, this cobra venom was the only fractionated cobra venom in which all screened fractions displayed no toxic effects at any dose tested.

[0080] Referring to Figure 1C, the 6 μg / ml dose showed the greatest selectivity in toxic effects between targeted rat cardiomyocytes and non-targeted cardiac fibroblast control cell groups, with cardiac fibroblast toxicity nearly abolished upon treatment with all but one fraction (N.naj_I24_R2). While fibroblast toxicity was completely abolished at 6 μg / ml, murine cardiomyocyte cytotoxicity was observed at 40% or greater for 12 of the fractions tested, with 8 of these exhibiting 70% or greater toxicity.

[0081] Further selectivity was observed at higher doses for the other fractions, with the N.nub_I16_R3 fraction giving good selectivity between cardiomyocytes and cardiac fibroblasts at a dose of 7.5 μg / ml (Fig. 1B) and the N.atr_I23_R3 fraction giving this selectivity at the highest dose tested of 12.5 μg / ml (Fig. 1A).

[0082] This initial toxicity screening data supports the hypothesis that components of cobra venom can be isolated and used to selectively stress rodent cardiomyocyte cells, thus potentially applying these components as novel and unique rodenticides utilizing previously unexplored mechanisms of action. The selectivity data are encouraging, as they are more selectively toxic to cardiomyocytes than to cardiac fibroblasts, which are also present in cardiac tissue, and suggest the possibility of developing highly specific and targeted single cell types.

[0083] Additional screening of these purified fractions against another morphologically distinct cell type, rat erythrocytes, was deemed important to assess whether the fractions exhibited undesirable combination effects, such as hemolytic properties, that could compromise the efficacy of synthetically engineered versions of these fractions.

[0084] Erythrotoxicity Assay: Referring to Figure 2, in an erythrocyte lysis assay, none of the purified venom fractions killed rat erythrocytes at either the 0.5 μg / ml or 7.5 μg / ml doses. Interestingly, the 7.5 μg / ml dose of some purified fractions had previously been shown to be toxic to rat cardiac myocytes, rat cardiac fibroblasts, or both. Crude venom of the black-necked cobra was used as a control for erythrocyte toxicity and was shown to lyse rat erythrocytes at doses of 50 μg / ml and 5 mg / ml.

[0085] No erythrotoxicity was observed with any of the fractions at the highest dose of 7.5 μg / ml, a dose previously found to be cytotoxic with many of the fractions tested. This lack of toxicity supports the absence of hemolytic properties in all fractions tested. Thus, it suggests that the cytotoxicity previously observed in response to treatment with these purified fractions is likely a mechanistic effect, rather than a general necrotic or apoptotic induction effect. Treatment with crude black-necked cobra venom as a control confirmed that cobra venom can induce erythrolysis, but not these purified fractions.

[0086] Screening of purified venom fractions for changes in cardiomyocyte beating rate: One hour after administration, all fractions screened (except for the fraction from N. aan) were toxic to rat cardiomyocytes at 12 μg / ml, so no post-administration beating data were available. Administration of other concentrations of each fraction resulted in various fold increases in beating rate (see Table 1 below), confirming that the cobra venom fractions do indeed increase the beating rate of murine cardiomyocyte cells. Of the fractions screened, 18 increased the beating rate of rat cardiomyocytes at at least one tested concentration. Of the three fractions that did not cause an increase in beating rate (N. atr_I5_R5, N. kao_I18_R3, and N. nig_I4_R3), no observable cytotoxicity was observed at any tested concentration in the previous resazurin assay (see Figure 1), so it is unlikely that these fractions contain compounds that actually positively affect cardiomyocyte function or viability. Most fractions were found to increase rat cardiomyocyte beating rate the most at doses of 6 μg / ml or 3.5 μg / ml, with higher doses resulting in a decrease in beating rate, likely due to cell cytotoxicity and death. The fractions showed widely different increases in rat cardiomyocyte beating rate, with the lowest maximum fold increase observed in N.nub_I17_R2 being 2-fold, and the highest maximum fold increase observed in N.naj_I25_R3 being 13.75-fold, indicating that the active substances in each fraction have widely different levels of activity on the cells. [Table 1] The - fraction did not cause an increase in beating rate at any of the concentrations tested.

[0087] Selection of six major fractions and identification of their toxicity and effects on cardiomyocyte beating rate: Using toxicity data for cardiomyocytes and cardiac fibroblasts, as well as data on changes in beating rate before and after administration, six fractions were selected for further characterization. Fractions with different toxicity profiles for cardiomyocytes and non-cardiomyocytes were pursued to allow identification of amino acid residues important for cardiomyocyte toxic effects by mass spectrometry and SAR. The yield of each purified fraction was also considered as a factor in the selection.

[0088] The six fractions were selected for the following reasons.

[0089] N.aan_I15_R4: showed no obvious or detectable toxicity to rat cardiomyocytes and cardiac fibroblasts at doses up to 12.5μg / ml. At a dose of 6μg / ml, the heart rate increased by more than 3.5 times. It has no cytotoxicity but can increase the contraction rate of rat cardiomyocytes.

[0090] N.atr_I28_R3: Toxic to rat cardiomyocytes at doses of 3 μg / ml and above, and toxic to cardiac fibroblasts at doses of 7.5 μg / ml and above. At a dose of 6 μg / ml, heart rate increased more than five-fold. Highly cytotoxic to specific and non-specific cell types, and strongly increases the contraction rate of rat cardiomyocytes.

[0091] N.nub_I16_R3: Toxic to rat cardiomyocytes at doses of 7.5 μg / ml and above, and toxic to cardiac fibroblasts at a dose of 12.5 μg / ml. At a dose of 6 μg / ml, the heart rate increased more than six-fold. It was less cytotoxic to specific and non-specific cell types than N.atr_I28_R3 (higher doses were required), and strongly increased the contraction rate of rat cardiomyocytes.

[0092] N.nka_I18_R4: Toxic to rat cardiomyocytes at doses of 3 μg / ml and above, and toxic to cardiac fibroblasts at doses of 7.5 μg / ml and above. At a dose of 6 μg / ml, heart rate increased more than 7-fold. Highly cytotoxic to specific and non-specific cell types, and highly increased contraction rate of rat cardiomyocytes.

[0093] N.nig_I19_R4: was toxic to rat cardiomyocytes at doses of 7.5 μg / ml and above, and to cardiac fibroblasts at a dose of 12.5 μg / ml. At a dose of 6 μg / ml, it increased heart rate by more than 5.5-fold. It showed a similar cytotoxicity profile to N.nub_I16_R3 against specific and non-specific cell types, and similarly increased the contraction rate of rat cardiomyocytes.

[0094] N.naj_I25_R3: showed toxicity to rat cardiomyocytes at doses of 6μg / ml and above, and to cardiac fibroblasts at a dose of 12.5μg / ml. At a dose of 6μg / ml, the heart rate increased by more than 8.5 times. Its toxicity to rat cardiomyocytes was stronger than that of N.nub_I16_R3 and N.nig_I19_R4, but its toxicity to cardiac fibroblasts was comparable. It highly increased the contraction rate of rat cardiomyocytes.

[0095] Evaluation of the non-specific species-specific effects of the six major fractions using human iPSC-derived cardiomyocytes: Human induced pluripotent stem cell (IPSC) derived cardiomyocytes were purchased from Axol Bioscience and cultured as instructed. After 48 hours, the cells began to show the formation of a spontaneously beating cell monolayer. After 7 days of culture, the cells were ready to be used to evaluate potential human cardiomyocyte toxicity in response to treatment with six selected cardiotoxin fractions.

[0096] Human iPSC-derived cardiomyocytes were treated with serial dilutions of the six toxic fractions (7.5, 6, 3.5, 2.5, 1 μg / ml) in triplicate for 2 h at 37 °C and 5% CO2, after which resazurin dye was added and the color and fluorescence changes were monitored for 5 h. The toxicity rates of each fraction at all doses were calculated and graphed (Figure 3).

[0097] Five of the six selected fractions exhibited cytotoxic activity against human IPSC-derived cardiomyocytes, but only the N.ann_I15_R4 fraction continued to show low levels of cytotoxicity across all five doses tested. A clear dose-dependent effect was observed for treatment with the other five purified venom fractions, with higher doses inducing higher levels of cardiomyocyte toxicity.

[0098] Resazurin data demonstrated an excellent dose-response profile in human iPSC cardiomyocytes in response to treatment with the five selected fractions (see Figure 3). As previously seen in neonatal rodent cardiomyocytes, no cardiomyocyte cytotoxicity appears to be observed in response to treatment with fraction N.aan_I15_R4.

[0099] The toxicity data for human IPSC cells appears to suggest a higher level of toxicity in these cells in response to some fractions than previously observed in rat neonatal cardiomyocytes. However, fractions N.atr_I28_R3 and N.nka_I18_R4 showed reduced toxicity at 3.5 μg / ml in human IPSC-derived cells compared to the toxicity levels previously seen in rodent cardiomyocytes.

[0100] It is worth noting that these cells are not mature cardiomyocytes but human stem cells programmed to differentiate into cardiomyocyte-like cells. Human iPSC cells have a different cellular origin than rodent cardiomyocytes isolated directly from animals. They require a fine balance of growth factors to trigger differentiation and may therefore be more sensitive to environmental stresses and changes.

[0101] Assessment of the heat stability of the six purified venom fractions: Referring to Figure 4, overall, all purified fractions exhibited good thermal stability characteristics, with strong cytotoxic activity maintained upon treatment of fractions at temperatures as high as 100 °C. A slight decrease in toxicity (approximately 5-10%) was observed when N. nub_I16_R3 and N. atr_I28_R3 fractions were treated at 100 °C, compared to activity at 37 °C (no heat treatment), but these decreases were considered negligible. Fraction N. ann_I15_R4 continued to exhibit lower than average levels of cytotoxicity at all four temperatures tested, consistent with previous toxicity screening results.

[0102] Analysis of the thermal stability of the six selected fractions revealed that they were thermally stable up to temperatures as high as 100°C with little or no decrease in cardiomyocyte cytotoxicity (see Figure 4). Fractions with slightly lower tonal toxicity (N.ann_15_R4 and N.nub_I16_R3) showed a slightly more pronounced decrease in activity, but still only a 10% decrease in cytotoxicity at the highest heating temperature of 100°C. Fraction N.ann_I15_R4 continued to show low levels of cardiomyocyte cytotoxicity at all temperatures tested. However, this is not surprising as this fraction has been consistently shown to be much less cytotoxic to rat cardiomyocytes than any of the other five fractions. No significant changes in the activity of fractions N.nka_I18_R4, N.nig_I19_R4 and N.naj_I25_R3 were observed upon treatment at any of the tested temperatures, and cytotoxic activity was observed at 100°C similar to that at 37°C. This data suggests that the active components contained in each fraction show no or little change in stability and activity even at temperatures above 100°C.

[0103] The active compounds contained in the final product need to be able to exhibit good thermal stability in order to survive incorporation into the hot liquid wax (~70°C) and retain their active properties after the cooling and solidification process.

[0104] Mass spectrometry identification of six selected fractions: Six fractions that were shown to exhibit cardiotoxic effects in neonatal rat cardiomyocytes were selected and subjected to mass spectrometry. Intact mass spectrometry coupled with peptide mapping confirmed that all six selected fractions were cardiotoxins, and the sequences obtained matched cardiotoxins reported in the Uniport and NCBI protein databases. Three of the selected fractions had a high percentage match with cardiotoxins already present in the databases.

[0105] Certain regions of the sequence are highly conserved across all cardiotoxins, most likely due to structural integrity.

[0106] Amino acids with mass spectrometry matches are shown in bold. Underlines indicate amino acids that are conserved among cardiotoxin sequences in Uniprot (but did not match in the mass spectrometry data obtained). Grey highlights indicate amino acid substitutions. N.aan_I15_R4: (matches Naja annulifera cytotoxin 1) Nearly 91.6% match: [ka] N.nka_I18_R4: (matches Indian cobra (Naja naja) cytotoxin 8) Nearly 96.6% match: [ka] N.naj_I25_R3: (matches Indian cobra (Naja naja) cytotoxin 10) Nearly 75% match: [ka]

[0107] The other three sequences obtained a lower percentage of matches due to the lack of characterized cardiotoxins from those species in the database, but it was still possible to predict the most likely sequences. By using sequence data obtained from mass spectrometry, sequences of cardiotoxins from related species (uniport / NCBI), and sequences of conserved regions across all cardiotoxin sequence structures, it was possible to describe the likely sequences of amino acids for some of these cardiotoxins. Only amino acids that showed high variability at certain sites in the database and lacked sequence data by mass spectrometry remain unidentified (amino acids in black).

[0108] Bold indicates matching amino acids, underline indicates conserved but mismatched amino acids in the peptide, and grey highlight indicates amino acid substitutions. N.nub_I16_R4: (matches Naja pallida cytotoxin 1) Nearly 65% ​​match: [ka] N.nig_I19_R4: (identical to Naja mossambica cytotoxin 4 with amino acid substitution) Approximately 43.3% identical: [ka] N.atr_I28_R3: (Identifies with cytotoxin 5 of the Taiwan cobra (Naja atra) with amino acid substitution) Approximately 46.6% identity: [ka]

[0109] SAR and synthetic peptide design: Using the above sequences, structure-activity relationship (SAR) analysis was performed to determine the amino acid residues that were likely to be most important in causing the observed changes in cardiomyocyte cytotoxicity, nonspecific cardiac fibroblast cytotoxicity, and fold change in increase in cardiomyocyte beating frequency following administration.

[0110] SAR identified the highlighted amino acids in Figure 5 as important for cardiomyocyte toxicity. SAR results revealed that most of the important amino acids were located within the first 30 amino acids of the sequence, supporting that loop 1 and the beginning of loop 2 of the cardiotoxin structure are most important for cardiomyocyte toxicity. Using this information, we proposed regions of the cardiotoxin sequence for designing short active synthetic peptides. 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 (identified by mass spectrometry to N. longissimus cardiotoxin 1). This was the only investigated cardiotoxin that significantly increased cardiomyocyte beating rate without causing cytotoxicity, and therefore was a good choice as a starting scaffold to secure synthetic peptides with low toxicity: [ka]

[0111] Synthetic peptide 2 was designed using the first 13 amino acids of the cardiotoxin N. aan_I15_R4 as the backbone of the entire peptide, but substituting amino acids in the sequence that were shown by SAR analysis to cause high heart rate and high toxicity. [ka]

[0112] Amino acids in bold are those whose SAR we showed to be important for high toxicity. Amino acids in underline are those whose SAR we showed to be important for high beating rate.

[0113] Finally, synthetic compound 3 was designed using the first 13 amino acids of the cardiotoxin N.aan_I15_R4 as the backbone for the entire peptide, but substituting amino acids into the sequence that were shown by SAR analysis to be important only for high beat rate (without toxicity) in other investigated cardiotoxins. Substituting these amino acids into the sequence was found to worsen the aqueous solubility of the peptide, so lysines (K) were added to the ends of the peptide sequence to improve solubility. [ka]

[0114] Amino acids in bold demonstrate SAR importance for low toxicity. Amino acids in underline demonstrate SAR importance for low beating rate.

[0115] Screening of synthetic peptides for toxicity in rat cardiomyocytes and rat cardiac fibroblasts: The three synthetically designed short peptides showed no cytotoxicity to cardiomyocytes and cardiac fibroblasts at any of the four doses (0.1, 1, 10, 100 μg / ml). Treatment with crude venom of N. niger at a dose of 100 μg / ml and purified fractions N. nka_I18_R4 and N. ng_I19_R4 at a dose of 10 μg / ml continued to show high levels of toxicity (approximately 90%) in both rat cardiomyocytes and cardiac fibroblasts.

[0116] After 24 h of treatment with the three synthetic peptides, no toxicity was observed in either neonatal rat cardiomyocytes (see Figure 6A) or cardiac fibroblasts (see Figure 6B). The toxicity data indicate that the three synthetic peptides do not exhibit cytotoxic activity against both cardiomyocyte and non-cardiomyocyte types.

[0117] The synthetic design process of generating peptides representing only the first finger of the native cardiotoxin, along with using the amino acid sequence of N. ann_I15_R4 fraction (a non-cytotoxic cardiotoxin) as a scaffold to create three synthetic compounds, helped to eliminate the toxicity previously seen with other cytotoxic fractions. Total S. cerevisiae venom at 100 μg / ml and cardiotoxin-containing fractions N. nka_I18_R4 and N. ng_I19_R4 at 10 μg / ml continued to show high levels of cytotoxicity against both cell types tested.

[0118] The data indicate that by designing a truncated synthetic version of the peptide that contains only amino acids from loop 1 of the cardiotoxin structure, the undesirable cytotoxic effects observed with the full-length cardiotoxin can be engineered away.

[0119] Screening for selectivity: Screening synthetic peptides for human iPSC cardiomyocyte toxicity: Referring to Figure 7, the three short synthetic peptides did not show any cytotoxic effect on either type of cardiomyocytes at any of the four doses screened (0.1, 1, 10, 100 μg / ml). Treatment with crude N. nigricans venom at a dose of 100 μg / ml and purified fractions N. nig_I19_R4 and N. nka_I18_R4 at a dose of 10 μg / ml continued to show high levels of toxicity (approximately 90%) against cardiomyocytes of both species.

[0120] The three synthetic fractions were screened against commercially available human IPSC-derived cardiomyocytes and isolated chick embryo cardiomyocytes to determine whether they exhibited human or avian specific cytotoxicity. After a 24 hour dosing time frame, no obvious cytotoxicity was observed for any dose of the three synthetic peptides tested in either human IPSCs (Figure 7A) or chick embryo cardiomyocytes (Figure 7B).

[0121] Toxicity was observed in these cells in response to treatment with two natural cardiotoxin fractions from the black-necked cobra, one previously purified and one crude venom. This data indicates that human cardiac cell toxicity is not a feature of the synthetic peptides designed at this stage.

[0122] Screening of synthetic peptides for alterations in rodent and human iPSC cardiomyocyte beating rate: Referring to Figure 8, all three peptides caused a greater fold increase in beating rate in rat cardiomyocytes than in human IPSC-derived cardiomyocytes, with little increase seen with treatment at any dose in human IPSC-derived cardiomyocytes. Synthetic peptide 1 caused the greatest increase in beating rate in rat cardiomyocytes, resulting in an 8-fold increase in beating rate measured at the 100 μg / ml dose. Synthetic peptides 2 and 3 caused a peak increase of 3.5-fold and 3-fold, respectively, in beating rate in neonatal rat cardiomyocytes.

[0123] Neonatal rodent cardiomyocytes and human IPSC-derived cardiomyocytes were treated with the three synthetic peptides and assessed for changes in beating rate in response to a 2-hour treatment. After a 2-hour treatment at 100, 101, and 0.1 μg / ml, murine cardiomyocytes showed an increase in beating rate in response to all three synthetic fractions at all doses tested. Synthetic 2 (Figure 8B) showed an approximately 3-fold increase in beating rate over pre-treatment for all four concentrations tested. Meanwhile, synthetic 3 (Figure 8C) showed an approximately 2-fold increase in beating rate over the three lower concentrations tested, with a 3-fold increase after treatment with the highest dose tested, 100 μg / ml. Synthetic 1 (Figure 8A) showed the greatest increase in beating rate among the three synthetic compounds tested, with 3.5-, 4-, 5-, and 8-fold increases at 0.1, 1, 10, and 100 μg / ml, respectively. A greater than three-fold increase in heart rate in response to synthetic fractions would likely have a devastating effect on the efficient functioning of the rodent heart.

[0124] The effects of the three synthetic peptides were also evaluated in human IPSC-derived cardiomyocytes to determine whether they had activity against off-target species. After administration at the same four doses (100, 10, 1, and 0.1 μg / ml) as examined in rat cells over the same 2-hour time frame, the synthetic peptides appeared to have little to no effect on human IPSC-derived cardiomyocytes compared to the effects observed on rat cardiomyocytes. All three synthetic peptides did not cause an increase in the beating rate of human IPSC cardiomyocytes at doses of 0.1, 1, and 10 μg / ml. Synthetic peptides 1 and 2 appeared to slightly increase the beating rate of human cardiomyocytes to 1.5-fold above pre-treatment. However, at the same dose with synthetic peptide 1, a much larger increase was observed in rodent cardiomyocytes, suggesting that the large differences observed between the two species could be exploited to select doses that are selective only for rodent cardiomyocytes.

[0125] All synthetic peptides were found to not induce an increase in beating rate in chicken cardiomyocytes at any of the concentrations tested, as was also previously seen in human iPSC cardiomyocytes (Figure 8A-C). Data examining the effects of three synthetic peptides indicate that the peptides exhibit preferential activity on rodent cardiomyocytes compared to those of human or chicken origin. These differences are promising in terms of mitigating potential issues of side effects occurring in off-target species of larger mammals and birds if accidentally exposed to these novel peptides, and suggest an even larger dosing window between target and non-target species given differences in circulating blood volume and heart size.

[0126] Screening of synthetic peptides for thermal stability: Referring to Figure 9, all three synthetic peptides showed a slight decrease in activity in response to heating to 100°C compared to the same beating rates obtained after treatment with the non-heated fraction, however the fractions still retained most of their activity despite being heated to 100°C. Synthetic peptide 1 (Figure 9A) still performed the best in terms of increasing beating rate, inducing a 7-, 4-, 3-, and 3-fold increase in beating rate in the heat-treated fraction after treatment with 100, 10, 1, and 0.1 μg / ml of peptide, respectively.

[0127] Synthetic material 2 (Figure 9B) continued to cause an approximately 3.5-fold increase in beating rate at 100 μg / ml and appeared to exhibit greater thermal stability at higher concentrations. Peptide 2 caused a greater than 2.5-fold increase in beating rate in neonatal rat cardiomyocytes at all doses, although decreased activity was observed at doses of 10, 1, and 0.1 μg / ml. Synthetic material 3 (Figure 9C) caused the lowest fold increase in heart rate after heating, with a maximum increase of 2.5-fold.

[0128] The beating rate increase and thermostability data of all three peptides were evaluated and synthetic peptide 1 performed the best, eliciting the greatest increase in beating rate in rat cardiomyocytes, but did not elicit a similar increase in beating rate in human IPSC-derived cardiomyocytes. Peptide 1 also continued to show good activity on rat neonatal cardiomyocytes even when heated to 100°C, indicating its excellent thermostability properties. Taking into account all the data of the three synthetic peptides, synthetic peptide 1 was selected and subjected to Caco-2 assay analysis to further evaluate its intestinal permeability.

[0129] Assessment of intestinal permeability of major short synthetic peptide 1 using the Caco-2 permeability assay: As shown in Figure 10, Caco-2 cells were cultured in a monolayer in a Transwell insert on a permeable membrane. The insert was inserted into a well of a plate to form an apical chamber (above the cells) and a basolateral chamber (below the cells), with a permeable membrane sandwiched between the two layers. Compounds were then added to either the apical or basolateral chamber, and passage to the other chamber was monitored. Migration of the test substance from the apical to the basolateral chamber indicates uptake into the cells, simulating transfer from the intestinal lumen to the bloodstream by uptake receptors (direction A). Movement of the compound from the basolateral to the apical chamber indicates efflux of the compound, suggesting that the compound may pass through the intestinal wall, while being returned to the intestinal lumen by efflux receptors present on the intestinal cell surface (direction B). Efflux ratios were then calculated from the values ​​obtained for A2B and B2A transmission. Compounds suitable for intestinal absorption should be permeable in the A2B direction, but less permeable in the B2A direction.

[0130] The control compounds behaved as expected in the assay, and all Papp and efflux ratio values ​​were within acceptable limits, where available (Table 2). Table 2: Caco-2 permeability positive control data [Table 2] Rep = replicates; n = number of replicates; ND = not detected; NA = not applicable a Test compound sample concentrations from the receiver compartment of replicate 1 below LQS A2B. b No test compound was detected in the receiver compartment of replicate 2 A2B.

[0131] Data for DMVTL061120 (synthetic peptide 1) at 10 μM showed that the compound was not detectable at the lower concentrations in the standard curve, therefore the sample concentration for the Caco-2 experiments was increased to 100 μM. Data for DMVTL061120 at 100 μM are shown in Table 3. Table 3: Caco-2 permeability DMVTL061120 data (synthetic peptide 1) [Table 3] Rep = replicas; n = number of replicas

[0132] For DMVTL061120 (synthetic peptide 1), the values ​​were 4.60 and 0.633x10 for the A2B and B2A orientations, respectively. -6 cms -1 Mean Papp values ​​of 0.137 were obtained with mean recoveries of 45.5% for A2B and 96.6% for B2A. The efflux ratio of 0.137 suggests that this compound is not a substrate for efflux transporters, but since the efflux ratio was <1, it may be a substrate for uptake transporters.

[0133] The results of the Caco-2 flux assay suggested that synthetic peptide 1 could be readily taken up and absorbed by intestinal cells. The data from the apical to basolateral (A2B) direction suggested that the peptide could be a substrate for uptake transport and, as such, should be able to efficiently cross the intestinal membrane for uptake into the bloodstream. There was some loss of peptide recovery in the A2B direction, suggesting that a portion of the peptide may interact with or be retained or internalized by the intestinal cells themselves.

[0134] The basolateral to apical (B2A) data supported that the peptide is not easily exported from the basolateral cellular environment by efflux transporters, as very little of the peptide was detected passing in this direction, which is important information as it suggests that once the peptide has crossed the inner wall of the intestinal wall, it is less likely to be pumped back into the lumen and, once absorbed through the inner wall, it is more likely to be absorbed into the bloodstream.

[0135] The integrity of the polarized cell monolayer could be monitored by adding Lucifer Yellow permeation during the experiment using fluorometric analysis. Monitoring Lucifer Yellow allowed us to determine that no toxicity to Caco-2 cells by synthetic peptide 1 was observed at a dose of 100 μM. The fact that the cell monolayer remained intact upon treatment with the synthetic peptide is important because it suggests that the synthetic peptide is unlikely to damage gastrointestinal cells and thus unlikely to cause gastric upset when ingested. Therefore, it can be inferred that ingestion of peptide 1 would not cause rodent targets to shy away from food.

[0136] In summary, SAR identified amino acids important for cardiomyocyte toxicity, revealed that most of the critical amino acids are located within the first 30 amino acids of the sequence, and supported that loop 1 and the beginning of loop 2 of the cardiotoxin structure are most important for cardiomyocyte toxicity. Using this information, we proposed regions of the cardiotoxin sequence for generating short active synthetic peptides. Using mass spectrometry data obtained for the six investigated cardiomyocyte fractions and structure-activity relationship (SAR) analysis, three short synthetic peptides were designed:

[0137] Peptide 1-designed using the first 13 amino acids of fraction N.aan_I15_R4, showed no toxicity but induced an increase in the beating rate.

[0138] Peptide 2 - designed to contain amino acids important for both high beating rate and high toxicity, but modified to increase toxicity and increase beating rate.

[0139] Peptide 3 - Designed to have high beating rate and key amino acids without toxicity, with added lysine for solubility.

[0140] The designed peptide sequences were compared to public database sequences using NCBI's Basic Local Alignment Search Tool (BLAST) for proteins (BLASTP) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). These are unconstrained searches, so hits from all matching organisms are displayed. Peptide 1

[0141] BLASTP analysis showed that the sequence was 100% identical to cytotoxin 1 from the long-legged cobra (Naja annulifera) and the Cape cobra (Naja nivea), and the closest non-cobra sequence showed 82% identity to a hypothetical protein from the wild potato (Solanum commersonii). Peptide 2

[0142] Peptide 2 is modified but 100% identical to 10 Naja naja cytotoxins and 3 Naja kaouthia sequences. The closest non-cobra sequence shows 84% ​​identity to the Russell's viper (Daboia russelii) cytotoxin. The closest non-snake sequence is only 76% similar to a sequence (Clostridium tarantellae) with 90% identity to the segment. Peptide 3

[0143] This peptide has no unique matches in the NCBI databank, with the closest entry covering 92% of the sequence with 84% identity to the cytotoxin of the Indian cobra. The closest non-cobra sequence is from a Clostridia bacterium, with only 57% exact sequence matches.

[0144] It will be appreciated that these peptides can be modified to improve bioavailability, altering key characteristics to make the compounds selective for rodent activity and less likely to persist in the environment. These modifications include C-terminal amides, D-amino acids and unnatural amino acids.

[0145] In this way, short synthetic peptides were generated that showed functional activity on rodent cardiomyocytes. The generation of short, active synthetic peptides is preferred over large-scale purification of full-length venom-derived cardiotoxins, as their use significantly reduces both time and costs when scaling up to (commercially) manufacture rodenticides.

[0146] All three peptides showed no toxicity to cardiomyocytes and non-cardiomyocytes, good selectivity between rodent and human cardiomyocytes, good thermal stability, and increased the beating rate of rodent cardiomyocytes.

Claims

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

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

3. The synthetic peptide sequence according to claim 1, wherein said sequence has 10 to 15 amino acid residues, preferably 13 amino acid residues.

4. The sequence is LKC(H / N)KL(V / I)PX(V / A)(W / Y)KTC [SEQ ID NO: 1]; or SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and variants thereof 2. The synthetic peptide sequence of claim 1 having the sequence:

5. The sequence is a) one or more hydrophobic amino acids at one or both termini of said sequence; b) one or more amino acids to increase solubility in water; c) one or more synthetic amino acids, and / or d) a detectable tag, preferably a UV fluorescent tag 2. The synthetic peptide sequence of claim 1, comprising:

6. The synthetic peptide sequence of claim 1, wherein the sequence is PEGylated.

7. 2. The synthetic peptide sequence according to claim 1, wherein said sequence is cardiotoxic, preferably selectively cardiotoxic.

8. the sequence causes a change in heart rate; or 2. The synthetic peptide sequence of claim 1, wherein the compound is tachycardic and increases heart rate.

9. A rodenticide comprising at least one compound that is cardiotoxic, optionally selectively cardiotoxic, to rodents.

10. 10. The rodenticide of claim 9, wherein said compound has an excitocardiotoxic effect.

11. 10. The rodenticide of claim 9, wherein said compound causes a change in heart rate, and optionally said compound is tachycardic and increases heart rate.

12. 10. The rodenticide of claim 9, wherein the compound is an isolated amino acid sequence obtained or derived from snake venom, optionally from a species of the Elapidae family.

13. 13. The rodenticide of claim 12, wherein the snake venom is from a species of cobra, optionally from the genus Cobra.

14. 10. The rodenticide of claim 9, wherein the compound is a synthetic peptide sequence based on or including a sequence derived from loop I of an elapid or cobra three-finger toxin.

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

16. 16. The rodenticide of claim 15, wherein the snake venom is derived from or obtained from a species of cobra, optionally a species of the genus Cobra.

17. 16. The rodenticide of claim 15, wherein said amino acid sequence is cardiotoxic, optionally, selectively cardiotoxic.

18. The rodenticide of claim 15, wherein the amino acid sequence has an excitatory cardiotoxic effect.

19. The rodenticide of claim 15, wherein the amino acid sequence causes a change in heart rate or the amino acid sequence is tachycardic and increases heart rate.

20. 16. The rodenticide of claim 15, wherein the isolated amino acid sequence is a synthetic peptide sequence that includes a sequence based on or derived from loop I of an elapidae or cobra three-finger toxin.

21. A rodenticide formulation comprising a synthetic peptide sequence including a sequence based on or derived from loop I of a three-finger toxin of an elapidae or cobra, and a carrier.

22. 22. The rodenticide formulation of claim 21 formulated as a paste, solid, powder, gel or liquid.

23. 22. The rodenticide formulation of claim 21, further comprising one or more of waxes (petroleum-derived or plant-derived), grains, nuts, fruits, colors, flavors, herbs, spices, essential oils, oils, lipids or other rodent-palatable materials.

24. The rodenticide of claim 15, wherein the sequence is based on or derived from amino acid residues 1 to 30 of a three-finger toxin.

25. A rodenticide according to claim 15, wherein the sequence has 10 to 15 amino acid residues, preferably 13 amino acid residues.

26. The sequence of claim 1, LKC(H / N)KL(V / I)PX(V / A)(W / Y)KTC [SEQ ID NO: 1]; or SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and variants thereof 16. The rodenticide of claim 15, having the sequence:

27. The sequence of claim 1, a) one or more hydrophobic amino acids at one or both termini of said sequence; b) one or more amino acids to increase solubility in water; c) one or more synthetic amino acids, and / or d) a detectable tag, preferably a UV fluorescent tag 16. The rodenticide of claim 15, comprising:

28. The rodenticide of claim 15, wherein the sequence is PEGylated.

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