Use of avermectin, and rodenticide formulation

Abstract

The present invention relates to the technical field of rodent pest control. Disclosed are the use of avermectin and a rodenticide formulation. The avermectin has a structure represented by formula (I). The present invention uses a strategy of "new use for old drugs", uses commercially available pesticides as candidate compounds, and uses mouse thrombin as a target to single out pesticide avermectin strongly bound to thrombin, which can activate the activity of mouse thrombin and promote blood coagulation. The present invention has the advantages of low cost, good rodenticidal killing effect, acute and chronic efficacy, good palatability, easy degradation, and the like.

Description

Application of abamectin and rodenticide formulations

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202411955321.7, filed on December 27, 2024, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of rodent control technology, specifically to the application of avermectin and rodenticide formulations. Background Technology

[0004] Rodents are a diverse group of rodent mammals with remarkable adaptability and reproductive capacity. However, their ever-increasing populations now pose a significant threat to global agriculture, the environment, and human health. Therefore, controlling and preventing rodent infestations is essential.

[0005] Currently, the dominant rodenticides on the market are mainly anticoagulants, such as warfarin and bromadiolone, which are first- and second-generation anticoagulant rodenticides. However, with the long-term and large-scale use of anticoagulant rodenticides, rodent resistance is gradually increasing. Resistant rodents have been found worldwide. For example, sequencing of brown rats and roof rats has revealed the evolution and spread of resistance to anticoagulant rodenticides. In some countries, strong resistance to second-generation anticoagulant rodenticides has also been reported. Meanwhile, the rodent marmot (squirrel family) frequently burrows and grazes on vegetation in grasslands and mountains, potentially leading to soil erosion and grassland degradation; it is also one of the main natural hosts of Yersinia pestis, which can transmit deadly diseases. Therefore, there is an urgent need to develop new rodenticides to control harmful rodents.

[0006] Anticoagulant rodenticides primarily kill rats by disrupting their blood clotting mechanism. Vitamin K cyclooxygenoreductase is a key target of these rodenticides; inhibiting its activity causes massive hemorrhage and death in rats. Thrombin is a crucial factor in the rat blood clotting mechanism within the coagulation pathway and holds potential as a target for developing novel rodenticides. However, there are currently no reports on rodenticides targeting thrombin. Summary of the Invention

[0007] The present invention aims to provide a rodenticide technical material that is low in cost, palatable, and has a significant rodenticidal effect.

[0008] To achieve the above objectives, a first aspect of the present invention provides the use of avermectin or its agriculturally chemically acceptable salts, prodrugs, hydrates, solvates, and metabolites in rodenticides; the avermectin having the structure shown in formula (I):

[0009] In formula (I), R is methyl and / or ethyl.

[0010] A second aspect of the present invention provides a rodenticide formulation containing excipients and a rodenticide-effective amount of an active ingredient; the active ingredient includes avermectin as described in the first aspect, or an agriculturally chemically acceptable salt, prodrug, hydrate, solvate, or metabolite thereof.

[0011] This invention provides a drug for preventing and / or killing rodent pests, especially rats.

[0012] This invention utilizes the strategy of "repurposing existing drugs" by using commercially available pesticides as candidate compounds and screening avermectin, a pesticide that strongly binds to thrombin, as a target of mouse thrombin. Aavermectin can activate thrombin activity in mice and promote blood coagulation. Furthermore, avermectin has both acute and chronic toxicity to mice and is characterized by low cost, good palatability, and significant rodenticide effect. Attached Figure Description

[0013] Figure 1 shows the interaction results between avermectin and thrombin obtained by surface plasmon resonance method in Example 1.

[0014] Figure 2 shows the interaction results of avermectin and thrombin measured using the micro-thermal surge method in Example 2.

[0015] Figure 3 shows the interaction results between avermectin and thrombin obtained by biological layer interferometry in Example 3.

[0016] Figure 4 shows the effect of avermectin on thrombin activity in Example 4.

[0017] Figure 5 shows the effects of avermectin and argatroban on thrombin activity in Example 5.

[0018] Figure 6 shows the effect of avermectin on thrombin time in mouse blood in Example 6.

[0019] Figure 7 shows the effect of avermectin on prothrombin time in mouse blood in Example 7.

[0020] Figure 8 shows the effect of avermectin on the activation time of partial thromboplastin in mouse blood in Example 8.

[0021] Figure 9 shows the LD50 of avermectin in mice within 24 hours in Example 9. 50 The toxicity effect results are shown in the figure.

[0022] Figure 10 shows the LD50 of avermectin in mice in Example 9. 50 The trend of mortality rate from toxicity over time.

[0023] Figure 11 shows the effect of avermectin on the food intake coefficient of mice in Example 10.

[0024] Figure 12 shows the trend of mortality rate of mice after ingestion of avermectin over time in Example 11.

[0025] Figure 13 shows the trend of mortality rate of rats after ingestion of avermectin over time in Example 12. Detailed Implementation

[0026] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0027] As previously stated, a first aspect of the present invention provides the use of avermectin or its agriculturally chemically acceptable salts, prodrugs, hydrates, solvates, and metabolites in rodenticides; the avermectin having the structure shown in formula (I):

[0028] In formula (I), R is methyl and / or ethyl.

[0029] In this invention, when R is methyl, the structure shown in formula (I) is avermectin B. 1b When R is ethyl, the structure shown in formula (I) is avermectin B. 1a .

[0030] Preferably, in the avermectin, the content of the compound in which R is an ethyl group is 80wt%-100wt%.

[0031] Preferably, the pests include at least one of the following: brown rat, yellow-breasted rat, yellow-haired rat, ground squirrel, pinnate rat, house mouse, striped field mouse, Brandt's field mouse, oriental field mouse, Daurian ground squirrel, brown field mouse, and Chinese mole rat.

[0032] According to another preferred embodiment, the pest includes marmots. The marmots include, but are not limited to, non-protected animals such as the Himalayan marmot and the grey marmot.

[0033] The abamectin or its agriculturally chemically acceptable salts, prodrugs, hydrates, solvates, and metabolites provided by this invention have good control and / or killing effects on rodent pests. These rodent pests include, but are not limited to, at least one of the following: Squirrels (such as Himalayan marmots, grey marmots, and other non-protected animals), Beavers, Hamsters, Bamboo Rats, Dormice, Flying Sciuridae, Porcupines, and Forest Flying Sciuridae.

[0034] This invention does not impose any particular requirements on the preparation method of avermectin or its agriculturally chemically acceptable salts, prodrugs, hydrates, solvates, or metabolites. They can be prepared using methods known in the art or can be obtained commercially. This invention will not elaborate further here, and those skilled in the art should not understand this as a limitation of the invention.

[0035] Furthermore, it should be noted that the avermectin described in this invention can be a fermentation product of *Streptomyces avermitilis* isolated from soil, which generally mainly contains compounds with the structure shown in formula (I) where R is ethyl, and the content of such compounds is above 80 wt%; it also generally contains a small amount of compounds with the structure shown in formula (I) where R is methyl. Therefore, all avermectins known in the art are within the scope of protection of this invention, and those skilled in the art should not understand them as limitations on this invention.

[0036] As previously described, a second aspect of the present invention provides a rodenticide formulation containing excipients and a rodenticide-effective amount of an active ingredient; the active ingredient includes avermectin as described in the first aspect, or an agriculturally chemically acceptable salt, prodrug, hydrate, solvate, or metabolite thereof.

[0037] Preferably, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.0001%-100 wt%. Preferably, the content of the active ingredient is 0.0001%-50 wt%. Preferably, the content of the active ingredient is 0.0001%-25 wt%. Preferably, the content of the active ingredient is 0.0001%-25 wt%. Preferably, the content of the active ingredient is 0.0001%-10 wt%. Preferably, the content of the active ingredient is 0.0001%-9 wt%. Preferably, the content of the active ingredient is 0.0001%-10 wt%, preferably, the content of the active ingredient is 0.0001%-5 wt%. Preferably, the content of the active ingredient is 0.001%-5 wt%. Preferably, the content of the active ingredient is 0.0001%-5 wt%. Preferably, the content of the active ingredient is 0.001%-1 wt%. Preferably, the content of the active ingredient is 0.001%-0.05 wt%. More preferably, the content of the active ingredient is 0.001%-0.03 wt%.

[0038] Preferably, the content of the active ingredient is 0.01-1 wt% based on the total weight of the rodenticide formulation.

[0039] The auxiliary materials mentioned in this invention include, but are not limited to, at least one of starch, yeast products, oligosaccharides, proteins, dietary fiber, fillings, spices, seasonings, plant and animal extracts, beverage concentrates, meat, and amino acids.

[0040] Preferably, the auxiliary materials include at least one of oils, emulsifiers, food additives, grains, or starches.

[0041] Preferably, based on the total weight of the rodenticide formulation, the excipients include 0.1wt%-30wt% of oils, 0.5wt%-5wt% of emulsifiers, 1wt%-5wt% of food additives, and the remainder is grains and / or starch.

[0042] Preferably, the oil is edible oil and / or fat.

[0043] Preferably, the emulsifier includes at least one of glyceryl monostearate, sodium stearoyl lactylate, and soybean lecithin.

[0044] Preferably, the food additive includes at least one of sucrose, glucose, sodium saccharin, monosodium glutamate, sodium chloride, and sodium benzoate.

[0045] Preferably, the grain includes at least one of wheat, corn, soybean, rice seeds and their processed products.

[0046] According to a particularly preferred embodiment, the rodenticide formulation contains excipients and the active ingredient; the excipients include oils, emulsifiers, food additives, and grains and / or starch; based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-1 wt%, the content of the oil is 0.1-5 wt%, the content of the emulsifier is 0.5-2 wt%, the content of the food additive is 1-5 wt%, and the balance is grains and / or starch.

[0047] Preferably, the dosage form of the rodenticide formulation is selected from at least one of hydration agents, wettable powders, powders, granules, sustained-release agents, microcapsules, suspensions, and emulsions.

[0048] According to a preferred embodiment, the rodenticide formulation is in powder form, and the excipients are starch, corn, soybean, and oil. More preferably, in this preferred embodiment, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of starch is 0.01-99.98 wt%, and the total content of corn, soybean, and oil is 0.01-99.98 wt%.

[0049] According to a preferred embodiment, the rodenticide formulation is a hydrated formulation, and the excipients are proteins, yeast products, and oligosaccharides. More preferably, in this preferred embodiment, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of the protein is 0.01-99.98 wt%, and the total content of the yeast product and oligosaccharides is 0.01-99.98 wt%.

[0050] According to a preferred embodiment, the rodenticide formulation is a wettable powder, and the excipients are dietary fiber, cornmeal, rapeseed, and peanuts. More preferably, in this preferred embodiment, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of the dietary fiber is 0.01-99.98 wt%, and the total content of cornmeal, rapeseed, and peanuts is 0.01-99.98 wt%.

[0051] According to a preferred embodiment, the rodenticide formulation is a suspension concentrate, and the excipients are cottonseed, protein, sunflower, and soybean. More preferably, in this preferred embodiment, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of cottonseed is 0.01-99.98 wt%, and the total content of protein, sunflower, and soybean is 0.01-99.98 wt%.

[0052] According to a preferred embodiment, the rodenticide formulation is an emulsion, and the excipients are corn, ryegrass, clover, and pumpkin. More preferably, in this preferred embodiment, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of corn is 0.01-99.98 wt%, and the total content of ryegrass, clover, and pumpkin is 0.01-99.98 wt%.

[0053] According to a preferred embodiment, the rodenticide formulation is a microcapsule formulation, and the excipients are legumes, fish meal, meat meal, and eggs. More preferably, in this preferred embodiment, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of legumes is 0.01-99.98 wt%, and the total content of fish meal, meat meal, and eggs is 0.01-99.98 wt%.

[0054] According to another preferred embodiment, the rodenticide formulation is in granule form, and the excipients are meat and bone meal, grains, potatoes, barley, and wheat. More preferably, in this preferred embodiment, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of the meat and bone meal is 0.01-99.98 wt%, and the total content of grains, potatoes, barley, and wheat is 0.01-99.98 wt%.

[0055] According to another preferred embodiment, the rodenticide formulation is a sustained-release formulation, and the excipients are sorghum, soybean, plant protein, and seafood. More preferably, based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-99.98 wt%, the content of sorghum is 0.01-99.98 wt%, and the total content of soybean, plant protein, and seafood is 0.01-99.98 wt%.

[0056] The present invention will be described in detail below through examples. In the following examples, unless otherwise stated, the raw materials used are all commercially available products.

[0057] The following avermectin is a product purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., wherein the concentration of the compound with R representing an ethyl group as shown in formula (I) is 98 wt%.

[0058] Example 1

[0059] This test example illustrates the determination of molecular interactions between avermectin and thrombin using surface plasmon resonance (SPR, Biacore 3000 instrument).

[0060] Experiments were conducted using an SPR molecular interaction instrument: First, thrombin protein was coupled to the surface of the Classic series CM5 sensor chip according to the instructions. Avermectin sterile PBS buffer solutions (pH 7.1-7.4, DMSO content 1 v / v) with final concentrations of 100 μM, 80 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, and 10 μM were prepared; the control group consisted of PBS buffer solution without avermectin (DMSO content 1 v / v). The solutions were then transferred to the instrument's sample chamber for SPR testing.

[0061] Combined analysis was performed using Biacore Insight software. Figure 1 shows the interaction results of avermectin and thrombin measured by surface plasmon resonance in Example 1. The test results show that avermectin and thrombin have a good interaction, as shown in Figure 1, with a dissociation constant (K). d The M value is 36 μM, indicating good interaction.

[0062] Example 2

[0063] This test example illustrates the determination of molecular interactions between avermectin and thrombin using the micro-thermal surge apparatus method (MST, instrument model MONOLITHNT.115).

[0064] Thrombin protein was labeled using RED-NHS second-generation dye. A sterile PBS buffer solution (pH 7.1-7.4, DMSO content 1% by volume) with a final concentration of 2 mM avermectin was prepared; incubated in a 60°C water bath for 10 minutes, and vortexed to mix thoroughly. The solution was then centrifuged at room temperature (25°C, the same below) (12000 rpm) for 10 minutes, and the supernatant was collected. Following the MST procedure, the solution was diluted proportionally and transferred to sterile, enzyme-free, low-adsorption 200 μL PCR tubes; the thermophoretic activity of thrombin protein interacting with different concentrations of avermectin was detected using an MST instrument.

[0065] The MO.Affinity analysis software was used for combined analysis to obtain K. d Figure 2 shows the interaction results of avermectin and thrombin measured using the micro-thermal surge method in Example 2, along with other parameters. As can be seen from Figure 2, the MST test revealed that K... d The value was 39 μM, indicating that avermectin and thrombin have a good interaction, which is similar to the results of the surface plasmon resonance method.

[0066] Example 3

[0067] This test case illustrates the determination of molecular interactions between avermectin and thrombin using the biolayer interference method (BLI, instrument model OCTETR8).

[0068] Following the BLI experimental instructions, biotinylation and labeling experiments of thrombin protein were performed to obtain biotin-labeled thrombin protein. Two SSA sensors were pre-wetted in sterile PBS buffer (pH 7.1-7.4) for 10 min, and then immersed in sterile PBS buffer (pH 7.1-7.4) and biotinylated thrombin protein samples, respectively. Avermectin sterile PBS buffer solutions (pH 7.1-7.4, DMSO content 1 v / v) with final concentrations of 100 μM, 80 μM, 60 μM, 40 μM, 20 μM, and 10 μM were prepared as experimental groups; the control group used PBS buffer solution without avermectin (DMSO content 1 v / v). The solutions were transferred to a sample plate, and the interference pattern on the surface of the biosensor tip was measured.

[0069] Binding constants and kinetic parameters were obtained by analyzing the binding spectrum using Octet Bli discovery software. Figure 3 shows the interaction results of avermectin and thrombin measured by biolayer interferometry in Example 3. As shown in Figure 3, the Kd value of avermectin binding to thrombin is 43 μM.

[0070] Therefore, the three interactions in Example 1, Example 2, and Example 3 above all demonstrate that avermectin and thrombin have a good interaction.

[0071] Example 4

[0072] This test case illustrates the effect of avermectin on thrombin activity.

[0073] Prepare 500 mL of Tris-HCl buffer (50 mM, pH 7.4) and NaCl solution (150 mM); prepare avermectin solutions with gradient concentrations of 200 μM, 150 μM, 100 μM, and 50 μM (DMSO:buffer = 1:99, v / v). Prepare solutions of thrombin protein and thrombin substrate S-2238 to final concentrations of 1 U / mL and 1.5 mM (DMSO:buffer = 1:99, v / v). Mix 50 μL of avermectin solution and blank buffer with 50 μL of 150 mM sodium chloride solution and incubate at 37°C for 2 min. Then, incubate 50 μL of thrombin solution (1 U / mL) at 37°C for 1 min. Prepare a 96-well microplate. Aspirate all the incubated solution into each well of the microplate and immediately add 50 μL of S-2238 solution. Place the sample in a microplate reader at 37°C and monitor the absorbance at 405 nm. Perform three replicate tests for each sample concentration and take the average value.

[0074] Table 1 shows the thrombin activation efficiency of avermectin at concentrations of 200 μM, 150 μM, 100 μM and 50 μM.

[0075] The formula for calculating activation efficiency is:

[0076] Where, k n k represents the slope of the experimental groups at different concentrations. 空白 The slope representing the blank group;

[0077] Table 1

[0078] As shown in Table 1, the activation efficiencies of avermectin at concentrations of 200 μM, 150 μM, 100 μM, and 50 μM were 75.63%, 65.01%, 53.34%, and 47.8%, respectively. Figure 4 shows the effect of avermectin on thrombin activity in Example 4. As can be seen in Figure 4, the slope of the curve gradually increases with increasing avermectin concentration compared to the blank control, indicating that avermectin may act as a thrombin agonist.

[0079] Example 5

[0080] This test case illustrates the effects of avermectin and argatroban (a human thrombin inhibitor, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., hereinafter the same) on thrombin activity.

[0081] Prepare 500 mL of Tris-HCl buffer (50 mM, pH 7.4) and NaCl solution (150 mM); prepare 200 μM and 100 μM avermectin solutions and 100 μM and 10 μM argatroban solutions (DMSO:buffer = 1:99, v / v). Prepare solutions of thrombin protein and thrombin substrate S-2238 to final concentrations of 1 U / mL and 1.5 mM respectively (DMSO:buffer = 1:99, v / v). Mix 50 μL of avermectin solution, argatroban solution, and blank buffer with 50 μL of 150 mM sodium chloride solution and incubate at 37°C for 2 min. Then, take 50 μL of thrombin solution (1 U / mL) and incubate at 37°C for 1 min. Prepare a 96-well microplate. Aspirate all the incubated solution into each well of the microplate and immediately add 50 μL of S-2238 solution. Place the sample in a microplate reader at 37°C and monitor the absorbance at 405 nm. Perform three replicate tests for each sample concentration and take the average value.

[0082] Table 2 shows the effects of abamectin at concentrations of 200 μM and 100 μM and argatroban at concentrations of 100 μM and 10 μM on thrombin activation (the calculation method is the same as in Test Example 4).

[0083] Table 2

[0084] Argatroban is a known commercial thrombin inhibitor. From the results in Table 2, it can be seen that its inhibition rates of thrombin at concentrations of 100 μM and 10 μM are 97.38% and 77.02% respectively. In contrast, at concentrations of 200 μM and 100 μM, the activation rates of abamectin are 88.01% and 62.43% respectively. Figure 5 shows the effects of abamectin and argatroban on thrombin activity in Test Example 5. As can be seen in Figure 5, the slope of the abamectin curve is opposite to that of argatroban. These results indicate that abamectin has a significant activating effect on thrombin rather than an inhibitory effect.

[0085] Test Example 6

[0086] This test example is used to illustrate the effect of abamectin on the thrombin time in mice (Kunming closed colony mice (SPF grade), weighing (20 ± 10) g, 6 - 8 weeks old, half male and half female, purchased from the Experimental Animal Management Center of Southern Medical University [Animal Production License Number: SCXK(Yue)2021 - 0041; all mice were routinely raised in an environment with an average temperature of (24 ± 0.5) °C, humidity of (55 ± 5)%, and light / dark (12 / 12) h).

[0087] Take 1.8 mL of fresh venous blood from the eyes of mice, mix it gently with 0.2 mL of sodium citrate anticoagulant (109 mM) at a volume ratio of 9:1. Prepare abamectin solutions with gradient concentrations of 400 μM, 100 μM, 50 μM, and 10 μM (DMSO: physiological saline = 1:99, volume ratio) for standby; prepare a 10 μM argatroban solution (DMSO: physiological saline = 1:99, volume ratio); take 100 μL of the plasma to be tested, and then add the same volume of the abamectin solution or argatroban solution (positive control group), and incubate at 37 °C for 5 min. Quickly add 100 μL of the thrombin solution incubated at 37 °C, start timing immediately, and then tilt the centrifuge tube every 5 s until it is observed that the plasma no longer flows, which is the plasma coagulation time. Each sample is repeated 2 - 3 times and the average value is taken.

[0088] Table 3 shows the effects of abamectin at concentrations of 400 μM, 100 μM, 50 μM, 10 μM, 0 μM and argatroban at a concentration of 10 μM on the thrombin time of mouse blood.

[0089] Table 3

[0090] As can be seen from Table 3, at concentrations of 400 μM, 100 μM, 50 μM, and 10 μM, the thrombin times after treatment with avermectin were 36.01 s, 49.12 s, 57.56 s, and 58.99 s, respectively, while the thrombin time of the blank control group (0 μM concentration) was 62.66 s, and the thrombin time of the positive control drug argatroban was greater than 5 min. Figure 6 shows the effect of avermectin on the thrombin time in the blood of mice in Test Example 6, where a, b, and c represent significance level markings (different letters corresponding to different concentrations indicate significant differences). As shown in Figure 6, through error analysis, the inventors found that there were significant differences in the thrombin time of avermectin compared with the blank control and the positive control drug. This indicates that avermectin can significantly accelerate the formation of fibrin and shorten the coagulation time of mouse blood.

[0091] Test Example 7

[0092] This test example is used to illustrate the effect of avermectin on the prothrombin time in white mice (Kunming closed colony mice (SPF grade), weighing (20 ± 10) g, 6 - 8 weeks old, half male and half female, purchased from the Experimental Animal Management Center of Southern Medical University [Animal Production License Number: SCXK(Yue)2021 - 0041]; all mice were conventionally raised in an environment with an average temperature of (24 ± 0.5) °C, humidity of (55 ± 5)%, and light / dark (12 / 12) h).

[0093] Take 1.8 mL of fresh venous blood from the eyes of white mice, mix it gently with 0.2 mL of sodium citrate anticoagulant (109 mM) at a volume ratio of 9:1. Prepare avermectin solutions with gradient concentrations of 400 μM, 100 μM, 50 μM, and 10 μM (DMSO: physiological saline = 1:99, volume ratio) for standby; prepare a 10 μM argatroban solution (DMSO: physiological saline = 1:99, volume ratio); take 100 μL of the plasma to be tested, and then add the same volume of the avermectin solution or the argatroban solution (positive control group); for the blank group, respectively pipette 0.1 mL of the prepared avermectin solution, argatroban solution, and the blank group, and mix well with the plasma to be tested containing prothrombin reagent, and let it stand at 37 °C for 5 min. Then add 0.1 mL of CaCl2 (25 mM) solution at 37 °C, start timing immediately, continuously invert and mix well, observe and record the appearance time of filamentous fibrin in the centrifuge tube, and repeat 3 times to take the average value.

[0094] Table 4 shows the effect of avermectin at concentrations of 400 μM, 100 μM, 50 μM, 10 μM, 0 μM and 10 μM argatroban on the prothrombin time in the blood of mice.

[0095] Table 4

[0096] Table 4 shows that at avermectin concentrations of 10 μM, 50 μM, 100 μM, and 400 μM, the average prothrombin times measured in three replicates were 10.75 s, 10.5 s, 8.93 s, and 8.63 s, respectively, while the prothrombin time of the blank control group (0 μM concentration) was 14.55 s, and the prothrombin time of the positive control drug argatroban was greater than 5 min. Figure 7 shows the effect of avermectin on the prothrombin time in the blood of mice in Test Example 7, where a, b, bc, cd, d represent significant difference markings (different letters corresponding to different concentrations represent significant differences). As shown in Figure 7, through error analysis, the inventor found that there were significant differences in the prothrombin time of avermectin compared with the blank control group and the positive control drug. This indicates that avermectin can significantly accelerate the process of prothrombin conversion to thrombin, thereby shortening the blood coagulation time.

[0097] Test Example 8

[0098] This test example is used to illustrate the effect of avermectin on the activated partial thromboplastin time in white mice (Kunming closed colony mice (SPF grade), weighing (20 ± 10) g, 6 - 8 weeks old, half male and half female, purchased from the Experimental Animal Management Center of Southern Medical University [Animal Production License Number: SCXK(Yue)2021 - 0041; all mice were conventionally raised in an environment with an average temperature of (24 ± 0.5) °C, humidity of (55 ± 5)%, and light / dark (12 / 12) h).

[0099] 1.8 mL of fresh venous blood was collected from the eyeballs of mice and mixed with 0.2 mL of sodium citrate anticoagulant (109 mM) at a volume ratio of 9:1. A bacitracin solution with gradient concentrations of 400 μM, 100 μM, 50 μM, and 10 μM (DMSO:water = 1:99, volume ratio) was prepared for later use. A 10 μM argatroban solution (DMSO:water = 1:99, volume ratio) was also prepared. 100 μL of the plasma to be tested was taken, and then the same volume of either bacitracin solution or argatroban solution (positive control) was added. For the blank control, 0.1 mL of each of the prepared bacitracin solution, argatroban solution (positive control), and blank control were respectively mixed with the plasma containing prothrombin reagent, thoroughly aspirated and incubated at 37°C for 5 min. Then add 0.1 mL of 37℃ CaCl2 (25 mM) solution, start timing immediately, continuously invert and mix, observe and record the time of appearance of filamentous fibrin in the centrifuge tube, repeat 3 times and take the average value. Invert and mix the activated partial thromboplastin ellagic acid solution, and allow the temperature to rise to room temperature; take 0.1 mL each of the test plasma (including normal control plasma) and activated partial thromboplastin ellagic acid solution, and mix thoroughly with a pipette. Take 0.1 mL of avermectin solution, blank group and positive control respectively, and mix thoroughly with the test plasma containing activated partial thromboplastin reagent, and incubate at 37℃ for 5 min. Then immediately take 0.1 mL of 37℃ CaCl2 (25 mM) solution, start timing immediately, continuously invert and mix every 5 seconds, observe and record the time of appearance of fibrin filaments, repeat 3 times and take the average value.

[0100] Table 5 shows the effects of avermectin at concentrations of 400 μM, 100 μM, 50 μM, 10 μM, and 0 μM, and argatroban at a concentration of 10 μM, on the activated partial thromboplastin time in the blood of mice.

[0101] Table 5

[0102] As can be seen from Table 5, at the dose concentrations of 10 μM, 50 μM, 100 μM, and 400 μM of avermectin, the average activated partial thromboplastin time measured in three replicates was 31.88 s, 29.39 s, 29.13 s, and 24.75 s, respectively, while the time of the blank control group (0 μM concentration) was 36.26 s, and the positive control drug argatroban extended the blood coagulation time to more than 5 min. Figure 8 shows the effect of avermectin on the activated partial thromboplastin time in the blood of mice in Test Example 8, where a, b, bc, c, d represent significant difference markings (different corresponding letters at different concentrations represent significant differences). As shown in Figure 8, through error analysis of the measured data, the inventor found that there were significant differences in the activated partial thromboplastin time of avermectin compared with the blank control group and the positive control drug. This indicates that avermectin can significantly shorten the activated partial thromboplastin time, thereby accelerating the blood coagulation process.

[0103] Test Example 9

[0104] This test example is used to illustrate the LD 50 of avermectin on white mice (Kunming closed colony mice (SPF level), body weight (20 ± 10) g, 4 - 6 weeks old, half male and half female (female not pregnant), purchased from the Experimental Animal Management Center of Southern Medical University [Animal Production License Number: SCXK(Yue)2021 - 0041]; all mice were conventionally raised in an environment with an average temperature of (24 ± 0.5) °C, humidity of (55 ± 5)%, and light / dark (12 / 12) h).

[0105] Prepare sesame oil solutions of avermectin at 50 mg / kg, 40 mg / kg, 20 mg / kg, 10 mg / kg, and 5 mg / kg; a 50 mg / kg warfarin (warfarin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., the same below) sesame oil solution was used as the positive control group; sesame oil without the drug was used as the blank control.

[0106] Select similar white mice, half male and half female, put them into mouse cages, a total of 7 groups, with 6 white mice in each group.灌胃 is translated as "administer by gavage", so this sentence should be "Administer 0.5 ml of the prepared drug solution by gavage into the stomach of the white mice; raise and observe for one week, observe the symptoms of the white mice after drug administration with death as the main target, and count the mortality rate every 24 h. Figure 9 shows the LD 50 toxicity effect of avermectin on mice within 24 h in Test Example 9. As shown in Figure 9, after drug administration, deaths began to occur in the mice within the first day (24 h) after the drug was administered to the mice. Among them, at the high concentration of 50 mg / kg dose, all the mice died within the first day. This shows that avermectin has relatively high toxicity to mice within 24 h.

[0107] Table 6 shows the number of dead mice in the treatment groups of abamectin and warfarin (50 mg / kg) at concentrations of 50 mg / kg, 40 mg / kg, 20 mg / kg, 10 mg / kg, 5 mg / kg, and 0 mg / kg.

[0108] Table 6

[0109] From the data in Table 6, it can be seen that at high concentration doses of abamectin, the number of dead mice is higher and the death time is shorter. At a low concentration of 5 mg / kg, mice began to show death within 72 h. For the positive control group of warfarin at a dose concentration of 50 mg / kg, all mice had died within 72 h, while the blank control group (0 mg / kg) had no effect. To further evaluate the toxicity of abamectin, the inventors continued to count the mortality rate of the mice 4 days later. Figure 10 shows the trend of the mortality rate of abamectin on mice LD 50 toxicity over time; as shown in the results of Figure 10, at a low dose of 10 mg / kg, mice also showed death within 7 days. This indicates that abamectin at low concentrations also has a toxic effect on mice, and abamectin has the characteristics of chronic rodent control.

[0110] Test Example 10

[0111] This test example is used to illustrate the effect of abamectin on the food intake coefficient of white mice (Kunming closed colony mice (SPF grade), weighing (20±10) g, 4 - 6 weeks old, half male and half female (female not pregnant), purchased from the Experimental Animal Management Center of Southern Medical University [Animal Production License Number: SCXK(Yue)2021 - 0041]; all mice were conventionally raised in an environment with an average temperature of (24±0.5) °C, humidity of (55±5)%, and light / dark (12 / 12) h).

[0112] Seven groups of six mice were selected, with equal numbers of males and females placed in each cage. A certain amount of avermectin was dissolved in sesame oil. The feed (a type of experimental mouse growth and reproduction feed, purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.) contained the following components: moisture content 0.001%-10 wt%; crude protein content 0.001%-20 wt%; crude fiber content 0.001%-5 wt%; crude fat content 0.001%-4 wt%; crude ash content 0.001%-8 wt%; total calcium content 1%-1.8 wt%; total phosphorus content 0.6%-1.2 wt%; and total lysine content 1%. Poison baits containing 0.05wt%, 0.03wt%, 0.015wt%, and 0.01wt% abamectin were prepared by mixing 32%-100wt% methionine + cystine (total content of 0.78%-100wt%), wheat flour (purchased from Yihai Kerry Food Marketing Co., Ltd., with wheat flour content exceeding 90wt%; yeast accounting for 0.001%-6wt% of the total; and food additives sodium bicarbonate and calcium dihydrogen nitrate accounting for 0.001%-1wt%). The feed content was ≤60wt%, and the wheat flour content was <40wt%. 0.05wt% rodenticide (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was used as a positive control, and poison baits were prepared using the same method. Poison baits without the rodenticide served as a blank control. During the experiment, mice were simultaneously provided with poisoned bait containing avermectin and poisoned bait without the drug, along with clean water. The locations of the two types of bait were changed periodically to allow the mice ample opportunity to choose their food. After 2 days, they were fed normally. The feeding coefficient was calculated by recording and observing the symptoms of the mice after ingesting the poisoned bait and the number of deaths, as well as recording the consumption of poisoned bait containing and without the drug over 2 days.

[0113] Table 7 shows the feeding coefficients of avermectin at mass fractions of 0.01 wt%, 0.015 wt%, 0.03 wt%, and 0.05 wt% and 0.05 wt% and rodenticide at 0.05 wt% over two days.

[0114] The formula for calculating the gluten intake coefficient is:

[0115] The consumption amount in the formula represents mass.

[0116] Table 7

[0117] As can be seen from Table 7, by calculating the feeding coefficient through the formula, it is found that the feeding coefficients of the abamectin baits with mass fractions of 0.05%, 0.03%, 0.015% and 0.01% are 0.990, 0.957, 0.987 and 0.913 respectively. The feeding coefficient of the bait with 0.05% warfarin, the first-generation anticoagulant rodenticide, is 0.960, and that of the bait without the agent as a blank control is 0.988. The overall average feeding coefficient is 0.965. Figure 11 shows the effect of abamectin on the feeding coefficient of mice in Test Example 10; Figure 11 shows that the overall feeding coefficient is close to 1. By analyzing the data, it is found that abamectin has good palatability for mice and does not cause mice to refuse to eat; that is, the abamectin technical has good palatability for mice.

[0118] Test Example 11

[0119] This test example is used to illustrate the poisoning effect of abamectin on white mice (Kunming closed colony mice (SPF level), weighing (20±10) g, 4-6 weeks old, half male and half female (females must not be pregnant), purchased from the Laboratory Animal Management Center of Southern Medical University [Animal Production License Number: SCXK(Yue)2021-0041]; all mice are routinely raised in an environment with an average temperature of (24±0.5) °C, humidity of (55±5)%, and light / dark of (12 / 12) h).

[0120] Seven groups of six mice were selected, with equal numbers of males and females placed in each cage. A certain amount of ivermectin was dissolved in sesame oil. The feed (a laboratory mouse growth and reproduction feed purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., containing 0.001%-10wt% moisture, 0.001%-20wt% crude protein, 0.001%-5wt% crude fiber, 0.001%-4wt% crude fat, and 0.001%-8wt% crude ash; with a total calcium content of 1%-1.8wt% and a total phosphorus content of...) was also prepared. Poisoned baits containing 0.05wt%, 0.03wt%, 0.015wt%, and 0.01wt% avermectin were prepared by mixing 0.6%-1.2wt% lysine, 1.32%-100wt% methionine + cystine, and corn starch (purchased from Hubei Yucheng E-commerce Co., Ltd., with a carbohydrate content of 28wt%). The feed content was ≤60wt%, and the corn starch content was <40wt%. A 0.05wt% rodenticide (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was used as a positive control, and poisoned baits were prepared using the same method. Poisoned baits without the rodenticide served as a blank control. During the experiment, mice were provided only with the feed (including the two poisoned baits and the rodenticide-free bait) and clean water. The mortality rate was calculated by recording and observing the symptoms and the number of mice that died after ingesting the poisoned baits over 7 days.

[0121] Table 8 shows the toxicity test results of avermectin at mass fractions of 0.01%, 0.015%, 0.03%, and 0.05% and 0.05% acetamiprid on mice.

[0122] Table 8

[0123] Table 8 shows that mice fed with 0.05 wt% avermectin technical grade bait were the first to show mortality. Mice fed with 0.03 wt% bait showed mortality starting on day 5. At 0.015 wt% bait, mortality occurred on days 6-7, with 3 mice dying. The positive control, rodenticide, showed mortality starting on day 5, while the blank control mice were unaffected. Figure 12 shows the mortality rate of mice after avermectin ingestion over time in Example 11. The high-concentration avermectin bait group reached its peak mortality first, with final mortality rates of 100%, 100%, 50%, and 30% at 0.05 wt%, 0.03 wt%, 0.015 wt%, and 0.01 wt% avermectin technical grade baits, respectively. The mortality rate of the control drug, 0.05 wt% rodenticide, was 100%, and no mice in the blank control group showed mortality.

[0124] Test Example 12

[0125] This test example is used to illustrate the poisoning effect of avermectin on rats (Kunming closed colony rats (SPF grade), weighing (140±10) g, 6-8 weeks old, half male and half female (females should not be pregnant), purchased from the Experimental Animal Management Center of Southern Medical University [Animal Production License Number: SCXK(Yue)2021-0041]; all mice are routinely raised in an environment with an average temperature of (24±0.5) °C, humidity of (55±5)%, and light / dark (12 / 12) h).

[0126] Select similar rats, half male and half female, and place them in cages. There are a total of 5 groups, with 6 rats in each group. Weigh a certain amount of avermectin and dissolve it in sesame oil. Mix the feed (the feed type belongs to the experimental rat growth and reproduction feed, purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd. Among them, the moisture content is 0.001%-10wt%; the crude protein content is 0.001%-20wt%; the crude fiber content is 0.001%-5wt%; the crude fat content is 0.001%-4wt%; the crude ash content is 0.001%-8wt%. Among them, the total calcium content accounts for 1%-1.8wt%; the total phosphorus content is 0.6%-1.2wt%; the total lysine content is 1.32%-100wt%; the total methionine + cystine content is 0.78%-100wt%) and corn starch (purchased from Hubei Yucheng E-commerce Co., Ltd., with a carbohydrate content of 28wt%) to make baits containing 0.06wt%, 0.03wt%, and 0.006wt% of avermectin respectively (where the feed content is ≤60wt% and the corn starch content is <40wt%). Use 0.06wt% warfarin as a positive control drug (warfarin is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and prepare baits in the same way. The bait without the drug is used as a blank control. During the experiment, only provide the rats with bait (including the two types of poisoned baits and the bait without the drug) and clean water. By recording and observing the symptoms and the number of deaths of the rats within 7 days after ingesting the poisoned bait, calculate the mortality rate.

[0127] Table 9 shows the poisoning test results of avermectin with mass fractions of 0.06%, 0.03%, and 0.006% and 0.06% warfarin on rats.

[0128] Table 9

[0129] As shown in Table 9, the rats containing 0.06 wt% avermectin technical grade were the first to show mortality. At a dose of 0.03 wt%, mortality began on the second day. At a dose of 0.006 wt%, mortality occurred between days 3 and 5, with a total of 3 rats dying. In contrast, the positive control drug, rodenticide, showed mortality on the fifth day, while the blank control rats were unaffected.

[0130] Figure 13 shows the trend of mortality rate of rats after ingestion of avermectin in Example 12 over time. As can be seen from Figure 13, the high-concentration avermectin bait group reached its peak mortality rate first, with final mortality rates of 100%, 100%, and 50% at avermectin concentrations of 0.06 wt%, 0.03 wt%, and 0.006 wt%, respectively. In contrast, the mortality rate of the control drug, 0.05 wt% rodenticide, was 100%, while no deaths occurred in the blank control group.

[0131] Example 13

[0132] This test case illustrates the effects of avermectin on the acute toxicity of marmots (Himalayan marmot, a non-protected animal, weighing approximately 3-4 kg, male); all marmots were raised in a temperature (15-25) °C, light / dark (16 / 8h) environment.

[0133] A poisoned bait containing 0.5 w% abamectin (98.5 w% corn flour and 1.0 w% soybean oil) was prepared, with untreated feed serving as a blank control.

[0134] Two groups of four marmot mice with similar appearances were selected and placed in cages. The symptoms of mortality in the marmots were observed, and the mortality rate was recorded. In this experiment, the treated group of marmots died within 6 hours after feeding, and all died within 12 hours. This indicates that avermectin has high toxicity to marmots within 12 hours.

[0135] Table 10 shows the number of marmot deaths in the 0.5 w% avermectin treatment group.

[0136] Table 10

[0137] As shown in Table 10, after marmots consumed bait containing 0.5 w% avermectin, the mortality rate reached 50% within 6 hours and all of them died within 12 hours; no deaths occurred in the blank control group.

[0138] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. The use of avermectin or its agriculturally chemically acceptable salts, prodrugs, hydrates, solvates, and metabolites in the control and / or killing of rodent pests; the avermectin having the structure shown in formula (I): in, In formula (I), R is methyl and / or ethyl; Preferably, in the avermectin, the content of the compound in which R is an ethyl group is 80wt%-100wt%.

2. The application according to claim 1, characterized in that, The pests include at least one of the following: brown rat, yellow-breasted rat, yellow-haired rat, house rat, pinnate rat, house mouse, striped field mouse, Brandt's field mouse, oriental field mouse, Daurian ground squirrel, brown field mouse, and Chinese mole rat.

3. The application according to claim 1, characterized in that, The pests mentioned include marmots.

4. A rodenticide formulation, characterized in that, The rodenticide formulation contains excipients and a rodenticide-effective amount of active ingredient; the active ingredient includes avermectin as described in claim 1 or its agriculturally chemically acceptable salts, prodrugs, hydrates, solvates, and metabolites.

5. The rodenticide formulation according to claim 4, characterized in that, Based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.001-100 wt%. Preferably, the content of the active ingredient is 0.01-1 wt% based on the total weight of the rodenticide formulation.

6. The rodenticide formulation according to claim 5, characterized in that, The auxiliary materials include at least one of oils, emulsifiers, food additives, grains, or starches; Preferably, based on the total weight of the rodenticide formulation, the excipients include 0.1wt%-30wt% of oils, 0.5wt%-5wt% of emulsifiers, 1wt%-5wt% of food additives, and the remainder is grains and / or starch.

7. The rodenticide formulation according to claim 6, characterized in that, The oils and fats are edible oils and / or fats.

8. The rodenticide formulation according to claim 6, characterized in that, The emulsifier includes at least one of glyceryl monostearate, sodium stearoyl lactylate, and soybean lecithin; And / or, the food additive includes at least one of sucrose, glucose, sodium saccharin, monosodium glutamate, sodium chloride, and sodium benzoate.

9. The rodenticide formulation according to claim 6, characterized in that, The grains include at least one of wheat, corn, soybean, rice seeds and their processed products.

10. The rodenticide formulation according to any one of claims 4-9, characterized in that, The rodenticide formulation contains excipients and the active ingredient; the excipients include oils, emulsifiers, food additives, and grains and / or starch; based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-10 wt%, the content of the oil is 0.1-5 wt%, the content of the emulsifier is 0.5-2 wt%, the content of the food additive is 1-5 wt%, and the remainder is grains and / or starch; Preferably, the rodenticide formulation contains excipients and the active ingredient; the excipients include oils, emulsifiers, food additives, and grains and / or starch; based on the total weight of the rodenticide formulation, the content of the active ingredient is 0.01-1 wt%, the content of the oil is 0.1-5 wt%, the content of the emulsifier is 0.5-2 wt%, the content of the food additive is 1-5 wt%, and the remainder is grains and / or starch.

11. The rodenticide formulation according to any one of claims 4-10, characterized in that, The dosage form of the rodenticide formulation is selected from at least one of the following: hydration agents, wettable powders, powders, granules, sustained-release agents, microcapsules, suspensions, and emulsions.

Images