Pest control composition

Abstract

Provide improved means for transdermally delivering a pesticidal or other toxic agent from a composition containing the toxic agent to a target animal, particularly into its bloodstream. **Solution**: A composition for killing animals, such as rodents, comprising (i) one or more toxic agents toxic to the animal, such as cholecalciferol or 25-hydroxy cholecalciferol (vitamin D3), as a toxic component, and (ii) a carrier system for the toxic component, wherein the carrier system (ii) comprises (a) at least one solvent or dispersion medium for the toxic component, such as an alcohol like ethanol, and (b) at least one skin disrupting component, such as dimethyl sulfoxide (DMSO). The composition enhances the skin penetration of the toxicant into or through the skin of the animal where the composition is sprayed or delivered, for example, in a trap or other device. ​

Description

[Technical Field]

[0001] This invention relates to a composition for controlling vermin. More specifically, exclusively However, this invention relates to a composition for killing or injuring pests, such as rodents or other animals. Furthermore, relating to a method for controlling pests using these compositions, and further relating to a method for controlling pests, Apparatus comprising these compositions for use in a method for killing or injuring pests, preferably Regarding. [Background technology]

[0002] In many agricultural, social, and public health sectors, pest control is widely needed. Among the pests are various types of animals, such as rodents and harmful organisms (pests). In addition to various other mammals or vertebrates classified as such, such as rodents, Marsupials, leporids, and mustelids ) may be included.

[0003] Traps for capturing, incapacitating, and / or killing pests are well known and are used in many places around the world. It is widely used in many places. Similarly, various chemical or biological agents are used to harm Various pest-killing compositions and rodenticides for use in pest control by poisoning animals. The product is well known and widely used. Mistargeting of pesticides and humans Frequent reports concerning the risks of unwanted or inadvertent exposure to other animals, such as livestock. Due to concerns, the use of unprotected poisons for pest control is gradually disappearing, and selective traps are becoming more common. It is becoming increasingly common to use them in combination with selective poisons.

[0004] While controlling pests for public health and agricultural reasons is permitted, animal welfare is a concern. Considering this has become an increasingly important requirement, and with this in mind, in recent years To be used for pest control, more humane traps and pesticides, especially more efficient and less painful ones. Traps and pesticides have shown various advancements.

[0005] Pest control is desired in certain social areas and locations that are difficult to access due to their wide geographical extent. And in agricultural situations, for example, not only for the removal of killed animals but also for the replenishment of poison, Further consideration should be given to making the management and monitoring of traps or other poison baiting devices easier and more frequent. Often, this is necessary. The degree to which poisons are used is an important factor for efficient pest control management. Therefore, even with a low intake amount, and thus a low total intake rate, it is efficient, and such toxins are not needed. To reduce the cost of manufacturing and deploying at any given time and place, and to create more effective poisons. The development of the product is desired.

[0006] One known method for capturing, incapacitating, and / or killing pests, such as rodents. The trap is the present inventors' published international patent application, International Publication No. 2010 / 106352 Disclosed in issue number. The disclosed device is a mechanical trap for inflicting poison on target animals. It is a form that combines a pheromone component to attract target animals into the trap. By applying a certain dose of a poison to an animal, typically externally, i.e., to the animal's skin, The intended killing objective is achieved. The effective toxicity of the poison is that it kills animals systemically. It relies on its ability to penetrate the skin of animals where it can exert its function, and thereby enter the animal's bloodstream. The mechanism involves the chemical and / or biological properties of the drug and the animal under study. may be dependent on

[0007] Another known disclosure is in New Zealand Patent No. 548082 (Agnew ), in which a topical pesticidal composition for killing various non - human pest animals is proposed. This composition comprises a specific toxin agent, namely cholecalciferol or 25 - hydroxy cholecalciferol (also known as vitamin D3), and at least one carrier that acts to deliver the toxin agent transdermally to the target animal, such as combinations with various alcohols (especially absolute ethanol), glycols, or certain other solvent species. including.

[0008] In the research of the inventors after the above - mentioned disclosure, the inventors have found that the degree and efficiency of penetration of the pesticidal toxin agent into the skin of animals or into the bloodstream of animals through the skin are important factors for determining the efficiency of exerting the systemic killing function of the intended target animal by a predetermined toxin agent or a predetermined amount. However, hitherto, there has been little or no publication or study on how the degree to which a predetermined poison or toxin agent penetrates the skin of the target animal, and moreover, how such penetration levels can be improved or optimized for any predetermined agent or its dosage that can be applied externally to the animal. The recent research of the inventors has focused on this area and led to the present invention.

[0009] In particular, through the research of the inventors, the inventors have surprisingly found that, as proposed in New Zealand Patent No. 548082, cholecalciferol, which has been used in conventional formulations Many rodenticides, including cyferol (or 25-hydroxycholecalciferol) Or, a venom-killing agent used in an alcohol-based composition in a known rat trap or other animal killing device. When doing so, the effectiveness of percutaneous penetration of toxic components into the animal's bloodstream is particularly important for achieving the desired effect. From this perspective, we found it to be far from ideal or optimal. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Brochure for International Patent Application Publication No. 2010 / 106352 [Patent Document 2] New Zealand Patent No. 548082 [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] Therefore, the object of the present invention is to provide a combination of animal-killing agents or other toxins containing the toxin. An improved method for delivering the product percutaneously to target animals, particularly into their bloodstream. The present inventors have, surprisingly, found a novel method for toxins. By using a carrier system, the transdermal delivery level of the toxin agent is improved or optimized, or it may improve upon newly recognized shortcomings in known prior art toxin delivery compositions. They discovered that. [Means for solving the problem]

[0012] Therefore, in a first embodiment, the present invention is a composition for killing animals, (i) A toxic component comprising one or more agents that are toxic to animals, one dose of the composition When applied to the skin of the animal, the composition contains in an amount sufficient to kill the animal. The toxic components present, (ii) Carrier systems for toxic components, Includes, Here, the carrier system (ii) is (a) at least one solvent or dispersion medium for the toxic component, (b) One selected from the group consisting of sulfoxides, amides, hydrocarbons, ketones, and ethers A skin disrupting component containing more than one substance (nt component) and, Includes, A skin-disrupting component (b) and a solvent or dispersion medium (a) These are different chemical species, At least one skin disturbance component (b) is approximately 20% to 90% by weight of the carrier system itself. The amount present in the composition, and the amount of skin disturbing component (b) into the animal's skin and / or This refers to the passage of toxic components through animal skin in the absence of at least one skin-disrupting component. Sufficient to cause disturbance to the animal's skin to promote or enhance it compared to the combination. The present invention provides a composition.

[0013] At least one skin-disrupting component of the carrier system enters and / or passes through the skin of an animal. It is a substance that promotes the skin penetration of one or more toxins, and is present in such quantities. Preferred substances or amounts of the composition in this embodiment of the present invention, preferred embodiments and actual A more detailed explanation, related to the implementation examples, is provided below.

[0014] In a second embodiment, the present invention relates to a composition according to the first embodiment of the present invention or any other substance The composition according to the application form or example is to be delivered to the skin of an animal, preferably topically. The present invention provides a method for killing animals, including its application to [a specific condition].

[0015] In a third aspect, the present invention provides a method for delivering one or more toxins percutaneously to an animal, preferably or a method of delivery to the bloodstream of an animal, the composition according to the first aspect of the present invention or A method comprising applying a composition according to any embodiment or example to the skin of an animal. provide.

[0016] In a fourth embodiment, the present invention relates to a composition according to the first embodiment of the present invention or any other embodiment The present invention provides the application form or examples for use as an animal-killing agent or animal-killing composition.

[0017] In a fifth embodiment, the present invention relates to a composition or embodiment according to a first aspect of the present invention. Alternatively, one or more of the toxins from the examples are delivered to the animal percutaneously, preferably into the bloodstream. This provides a use for the purpose of administering one or more toxic agents to an animal. More specifically, this embodiment of use involves administering one or more toxic agents to an animal. Use of a carrier system for one or more toxins, preferably for delivery into the bloodstream of an animal. A composition comprising one or more toxins and a carrier system for application to the skin of an animal. Provided in or in a composition, this carrier system (a) at least one solvent or dispersion medium for one or more toxin agents, (b) Selected from the group consisting of sulfoxides, amides, hydrocarbons, ketones, and ethers. A skin-disrupting component comprising one or more substances, Includes, A skin-disrupting component (b) and a solvent or dispersion medium (a) These are different chemical species, At least one skin disturbance component (b) is approximately 20% to 90% by weight of the carrier system itself. The amount present in the composition, and the amount of skin disturbing component (b) into the animal's skin and / or This refers to the passage of toxic components through animal skin in the absence of at least one skin-disrupting component. Sufficient to cause disturbance to the animal's skin to promote or enhance it compared to the combination. This includes use.

[0018] In a sixth aspect of the present invention, an animal is subjected to the delivery of one or more toxins to the skin of an animal. A device for killing is provided, and this device, (i) an enclosure in which animals can enter, (ii) A composition according to the first aspect of the present invention or a composition according to an embodiment or example A container means containing the supply, (ii) a delivery means for delivering a certain amount of the composition to the skin of an animal, Includes.

[0019] Further features, embodiments, and examples of the present invention in various aspects are shown below. This will become clear from the following detailed description of the present invention, including the examples provided. [Brief explanation of the drawing]

[0020] [Figure 1] This figure shows a comparison of various chemiosmotic enhancements for the delivery of cholecalciferol through synthetic membranes. [Figure 2] This figure shows the measured osmotic rates of cholecalciferol when dissolved in DMSO and ethanol in different proportions. [Figure 3] This figure shows the degradation of cholecalciferol when sealed in an amber-colored bottle and placed in an accelerated stability cabinet at 25°C ± 2°C / 60% RH ± 5%. [Figure 4] This figure shows the results obtained by investigating the freezing point of DMSO and ethanol as a cosolvent. [Figure 5]This figure shows the viscosity-enhancing effect of preferred thickeners, as well as cholecalciferol itself, on penetration enhancers. [Figure 6] This figure shows a comparison of chemiosmotic enhancements for cholecalciferol delivery through synthetic membranes in a cellulose tube in vitro model. [Figure 7] This figure shows the enhanced penetration of cholecalciferol using different DMSO and ethanol ratios in the same model as Figure 6. [Figure 8] This figure shows a comparison of chemiosmotic enhancements for cholecalciferol delivery via synthetic membranes in a diffusing cell in vitro model. [Figure 9] Figure 8 shows the dose-response correlation between cholecalciferol concentration and diffusion ratio using a diffusion cell model. [Figure 10(a)] This figure shows the survival rate for transdermal cholecalciferol preparations with increased chemical penetration. Figure 10(a) shows the survival graph for 20% (w / v) cholecalciferol in 90:10 DMSO / ethanol. [Figure 10(b)] Figure 10(b) shows the survival graph for 20% (w / v) cholecalciferol in a 90:10 DMSO / oleic acid mixture. [Figure 10(c)] Figure 10(c) shows a survival graph for 40% (w / v) cholecalciferol in 70:30 DMSO / ethanol. [Figure 10(d)] Figure 10(d) shows a survival graph for 20% (w / v) cholecalciferol in 100% ethanol. [Figure 10(e)] Figure 10(e) shows a survival graph for 40% (w / v) cholecalciferol in 100% ethanol. [Figure 10(f)] Figure 10(f) is a summary of mortality and time to endpoint for all formulations shown in Figures 10(a) to 10(e). [Figure 11(a)]Figure 10 shows a pain rating table for each set of five animals exposed to each formulation used. Figure 11(a) shows the pain ratings for experimental set 1. [Figure 11(b)] Figure 11(b) shows the pain ratings for experimental set 2. [Figure 11(c)] Figure 11(c) shows the pain ratings for Experiment Set 3. [Figure 11(d)] Figure 11(d) shows the pain ratings for experimental set 4. [Figure 11(e)] Figure 11(e) shows the pain ratings for experimental set 5. [Figure 12] Figure 11 shows the average pain at the endpoint for all animals used in the previous figure. [Figure 13(a)] This figure shows the survival analysis using a dose-fixed method. Figure 13(a) is the survival graph for 9% and 20% (w / v) cholecalciferol formulations. Other formulations were not included because they showed a 0% mortality rate. [Figure 13(b)] Figure 13(b) shows the mortality rate and average time to the endpoint for all formulations. [Figure 14(a)] This figure shows the pain rating and rat body weight for each of the five formulations tested using a fixed-dose treatment protocol. [Figure 14(b)] This figure shows the pain rating and rat body weight for each of the five formulations tested using a fixed-dose treatment protocol. [Figure 14(c)] This figure shows the pain rating and rat body weight for each of the five formulations tested using a fixed-dose treatment protocol. [Figure 14(d)] This figure shows the pain rating and rat body weight for each of the five formulations tested using a fixed-dose treatment protocol. [Figure 14(e)] This figure shows the pain rating and rat body weight for each of the five formulations tested using a fixed-dose treatment protocol. [Figure 15]This figure shows the results of an in-vitro experiment corresponding to Example 2, demonstrating the degree of delivery of various alternative toxic substances through synthetic membranes facilitated by the exemplary composition of the present invention. [Figure 16] This figure shows the results of an in-vitro experiment corresponding to Example 2, demonstrating the degree of delivery of various alternative toxic substances through synthetic membranes facilitated by the exemplary composition of the present invention. [Figure 17] This is a perspective view of a first embodiment of a delivery device or pest trap useful for delivering the composition according to the present invention to a target animal. [Figure 18] Figure 17 is a partial cross-sectional view of the device or trap. [Figure 19] This is a perspective end view of a second embodiment of a delivery device or pest trap useful for delivering the composition according to the present invention to a target animal. [Figure 20] Figure 19 is a perspective side view of the device or trap. [Figure 21] This is a perspective end view of a third embodiment of a delivery device or pest trap useful for delivering the composition according to the present invention to a target animal. [Figure 22] Figure 21 is a perspective side view of the device or trap. [Modes for carrying out the invention]

[0021] The compositions of the present invention, as well as methods and uses of the compositions, are applicable to various types of animals. It can be used to kill or injure any animal, especially those classified as pests or harmful organisms. Typically, animals are vertebrates, especially mammals, more preferably relatively small mammals. In this specification, the terms "kill" or "kill" refer to the complete (relatively short or comparative) killing of an animal. Not only does it kill through its long-lasting systemic toxicity, but it is also likely to be killed by human intervention. This may include the animals becoming incapacitated, apathetic, paralyzed, or unconscious after being released.

[0022] Animals that can be killed particularly effectively by using the present invention include pests, such as rats. Rodents such as mice and other rodents, as well as marsupials, rabbits and weasels. Examples of other pest species include members of the group to be classified. The present invention relates to these group It can also be effectively used to kill or incapacitate species other than the pygmy barb.

[0023] [Toxic ingredients] According to the present invention, the composition comprises one or more substances that are toxic to the animal to be killed, i.e., the target animal. It contains toxic components, including activators (toxic agents). Any suitable toxic agent or poison may be a toxin. It can be used as a drug.

[0024] However, in certain embodiments, one or more toxins specific to the target animal, that is, Furthermore, it is substantially toxic only to the target animals, and is not substantially toxic to animals other than the target animals. It may be preferable to select non-toxic substances. In this way, harm or damage to other species may occur. Killing of the target species is achieved without causing harm. In particular, by trapping and killing a designated target species. If two or more animals can access the device used for this purpose, it is likely that toxic components will be accidentally exposed. The killing of the target species is achieved without causing harm or injury to other species that may come into contact with it.

[0025] Various known poisons or toxins, alone or in combination, affect the toxicity of the compositions according to the present invention. It can be used as an ingredient. Suitable toxins for use in the present invention include various known This includes poisons, toxins, or pathogenic drugs, whether natural or synthetic. It is also good to use it alone or in combination of two or more such drugs. stomach.

[0026] Toxin agents particularly preferred for use in the present invention are lipid-soluble (i.e., substantially insoluble in water) ) could be a toxin. The reason is that being lipid-soluble means that such a toxin can affect the target. Dissolve in water and in the environment at a predetermined geographical location where the object is applied or at a location away from there. This is because it helps prevent it from easily leaking out.

[0027] Suitable toxins that can be used in the present invention include cholecalciferol and 25-hydroxy Cholecalciferol, calciferol, ergocalciferol, anticoagulants, metals Arsenic compounds, α-naphthylthiourea, arsenic compounds, barium compounds, thallium compounds, bromegate Tallinn, Chloralose (e.g., α-Chloralose), Crimidine, 1,3-difluoro- 2-Propanol, Endrin, Fluoroacetamide, Fosacetim, White Phosphorus, Pyryl Phosphate N, sililoside, sodium fluoroacetate, strychnine, tetramethylenedisulfate One or more of the following are listed: tramine, hydrogen cyanide, sodium cyanide, and potassium cyanide. Anticoagulants include warfarin, coumatetralyl, brodifacom, and difenaco. Um, furocumafen, chlorophacinone, pindone, diphacinone, difethiaone Examples include , and coumarin. Metallic phosphides include aluminum phosphide, calcium phosphide. It may contain um, magnesium phosphide, and zinc phosphide. Other suitable toxins include 1 Examples include more than one biological agent, such as bacterial or viral toxins.

[0028] For use in this invention, it may be particularly useful for killing rodents such as rats. A particularly preferred toxin agent is cholecalciferol or its hydroxylated derivatives. 5-hydroxycholecalciferol (both of these compounds are vitamin D3) It is a precursor, often referred to as a vitamin D3 precursor.

[0029] Toxic components may be present in the composition of the present invention in any appropriate amount. Preferably, in the composition The amount of the toxic component is preferably such that a single dose of the composition is administered to the animal in order to kill the target animal. It can be selected to be sufficient and effective to kill or injure with a single application.

[0030] In a preferred embodiment, the amount of the toxic component in the composition is about 0.001% by weight of the composition. ~Approximately 90% by weight, more preferably approximately 0.01% by weight to approximately 75% by weight of the composition, and even more Preferably about 0.1% to about 70% by weight of the composition, and more preferably about 1% by weight of the composition It is approximately 60% by weight.

[0031] In many practical embodiments, the delivered composition is used in a predetermined apparatus according to the present invention. In use, the concentration of toxic components in the composition is determined by the predetermined single dose or application of the composition. The required lethal dose of the toxic component is delivered to the designated target animal, preferably with a large lethal dose. It may be preferable that the formulation be designed so that any excess amount is not delivered. Thus, only one dose of a toxic component sufficient to kill or injure a designated target animal. The product is delivered, and the concentration of the toxic component contained therein is suitable for both lethal efficacy and economic considerations. It can be formulated to be optimized for each individual.

[0032] In embodiments of the present invention, the toxic component is present in the composition in any various suitable physical form. It can be incorporated into. For example, (i) The toxic component is the solvent / dispersion medium component of the carrier system, or any other component of the carrier system itself. They may also be dissolved in a solution, for example, as dissociated ionic species within it; or (ii) Toxic components may be dispersed as particles, for example, toxic components or toxic components as appropriate. The resulting suspension, emulsion, or colloidal mixture with one or more other components in the entire composition. Alternatively, in the form of a solution, in the carrier system (or simply in its solvent / dispersion component, or in the carrier system) It may be dispersed (in any other component of itself); or (iii) Toxic components are present in the composition or its carrier system, possibly simply in the solvent / In the dispersion medium component, or in any other component of the carrier system itself, any other suitable physical form In this state, it may be incorporated together with one or more other components of the whole composition. Appropriate Other possible physical forms include, for example, liposomes (e.g., one or more) (formed by a bilayer containing phospholipids), micelles (for example, one or more surfactants or (This includes phospholipids), complexes (for example, hydrogen bonds or disulfide bonds with another molecule) (Through physical interactions such as, etc.), salts (e.g., toxic components and / or dissolves of toxic components) (including changes in associated ions with skin-disrupting components that alter their decomposition or solubility characteristics), nanoparticles (e.g.) For example, lactic acid of any molecular weight with one or both of the toxic or skin-disrupting components. -Precipitated using glycolic acid copolymer-PLGA and polyvinyl alcohol-PVA (including nano-sized layers), or ferroids (e.g., vitamin F ethyl ester (2.8) %w / v), Cremophor® EL (1%w / v) and D-α-Tocoff Examples include particles or entities that have the properties of ester (0.2% w / v).

[0033] [Carrier system] According to the present invention, the carrier system of the composition is (a) at least one solvent or dispersion medium for the toxic component, (b) One selected from the group consisting of sulfoxides, amides, hydrocarbons, ketones, and ethers A skin-disrupting component comprising more than one substance, Includes, A skin-disrupting component (b) and a solvent or dispersion medium (a) These are different chemical species, At least one skin disturbance component (b) is approximately 20% to 90% by weight of the carrier system itself. The amount present in the composition, and the amount of skin disturbing component (b) into the animal's skin and / or This refers to the passage of toxic components through animal skin in the absence of at least one skin-disrupting component. Sufficient to cause disturbance to the animal's skin to promote or enhance it compared to the combination. ru.

[0034] Therefore, in the composition of the present invention, at least one solvent or dispersion medium and at least Both are skin-disrupting components, but they are different chemical species.

[0035] [Solvent or dispersion medium] A solvent or dispersion medium for toxic components acts as a solvent or dispersion medium for toxic components. You can choose from any one or more suitable materials.

[0036] In a preferred embodiment, the solvent or dispersion medium is one or more liquid solvents for the toxic component. A medium and / or dispersion medium, wherein one or more toxins are dissolved in them and / or It is dispersed. Examples of suitable solvents include one or more combinations of the following: One of the following combinations is possible: • Alcohols, such as ethanol, isopropanol, methanol, benzyl alcohol Ru, • Glycols, for example, diethylene glycol, propylene glycol, butyl diglyceride call, • Glycol ethers, for example, diethylene glycol monomethyl ether, diethylene Glycol monoethyl ether, diethylene glycol mono-n-butyl ether, Glycerol formal, • Polyethylene glycol, • Liquid polyoxyethylene glycol, • Pyrrolidone, for example, N-methylpyrrolidone, 2-pyrrolidone, ·acetone, Acetonitrile, • Amides, for example, dimethylacetamide, dimethylformamide, monomethylacetamide Mido, • Phthalates, such as diethyl phthalate.

[0037] A particularly preferred solvent or dispersion medium is a solvent for one or more toxins, particularly preferably E It's Tanor.

[0038] The solvent or dispersion medium may be present in the support system of the composition according to the present invention in any appropriate amount, The amount is preferably sufficient to dissolve or disperse the toxic component in the overall composition. It is possible. In some embodiments, a suitable amount of solvent or dispersion medium is about the amount of the carrier system itself. 1% by weight to approximately 99% by weight, more preferably about 5% by weight to approximately 80 or 90% by weight of the carrier system itself. Amount %, more preferably about 10 or 25% by weight to about 60 or 70% by weight of the carrier system itself It is a percentage.

[0039] [Skin disrupting ingredients] The skin-disrupting components used in the compositions of the present invention are sulfoxides, amides, hydrocarbons, and keto It contains one or more substances selected from the group consisting of ethers. Such substances are toxic. The passage of such skin-disrupting components into and / or through the skin of animals To promote or enhance compared to when it is not present, the skin of an animal, preferably a target animal. It is used in amounts that act to block or alter the physical and / or chemical properties of the skin. Such skin disruptors are preferably permanent or irreversible to the skin. Damaging animal skin in a way that causes cell death It is not. Instead, such skin-disrupting substances are used as components of the composition of the present invention. When defined as a condition that simply alters the behavior of the skin, particularly pain, inflammation, and swelling of the skin, Alternatively, it does not substantially cause skin cell death, and therefore simply increases reversible interlaminar cell exchange. (This is distinct from the cellular and inter-layers.) This results in several irreversible cell exchanges occurring between individual lipid layers of the skin.

[0040] Our research has shown that damage caused by skin-disrupting components used in accordance with the present invention is Rather than causing it, it is simply a disruptive, accompanying change in the properties of the skin that occurs through the skin cells. It was shown to cause an increase or prolongation of the level of movement or transfer of toxic substances. This improves the rate and efficiency of uptake of toxins into the animal bloodstream, thereby improving the absorption of a predetermined amount of toxins. This can lead to enhanced toxic effects.

[0041] The inventors have identified preferred species of substances that may be suitable for use as skin disturbance components. It was found to be an aprotic solvent. Therefore, the composition of the present invention is suitable for skin scratching. Preferred examples of such aprotic solvents used in accordance with the present invention as disruptive components include This could be one of the following, or a combination of two or more of the following: • Sulfoxides, for example, dimethyl sulfoxide (DMSO), decylmethyl sulfoxide Sid, Amides, for example, dimethylacetamide, dimethylformamide, Hydrocarbons, for example, hexane, toluene, • Ketones, for example, acetone, Ethers, for example, diethyl ether.

[0042] A particularly preferred skin-disrupting ingredient is dimethyl sulfoxide (DMSO).

[0043] The skin-disrupting component produces the above-mentioned skin-disrupting effect within the carrier system of the composition according to the present invention. It may be present in an appropriate amount. According to the present invention, this amount is about 20% by weight of the carrier system itself. It is approximately 90% by weight. In some embodiments, the amount of skin-disrupting component is more preferably This can be 30 or 40% to about 70 or 80% by weight of the carrier system itself.

[0044] Our research has shown that in the composition according to the present invention, such skin-disrupting components can be used in such a manner. It is predicted that using it at relatively high doses will hinder the uptake and passage of toxic molecules. To facilitate the passage of toxic components through the skin of target animals without causing substantial skin damage or We found it to be particularly effective and useful for enhancement.

[0045] The skin disturbance component (b) of the carrier system (ii) is preferably another component, in particular, the carrier system (i) i) The solvent or dispersion medium component (a) (and possibly the toxic component (i)) in the same phase as the present It may be present in the composition. For example, the various components of the composition may be substantially single-phase, homogeneous or in or as a homogeneous solution, dispersion, emulsion or colloidal composition It can exist.

[0046] Alternatively, the skin disturbance component (b) of the carrier system (ii) is the other component of the composition of the present invention. Components, in particular one of the solvent or dispersion medium components of the carrier system (ii) (a) (and optionally toxic Component (i)) can exist as a separate, distinct, or different phase, or within a phase. For example , the solvent or dispersion medium component (a) or skin disturbance component (b) of the carrier system (ii) (if applicable) (Also, toxic component (i) is present in the composition as a component of the solution layer, while the carrier system ( ii) The solvent or dispersion component (a) or the skin-disrupting component (b) may be toxic. (i) together with the components of the solution phase is a dispersed phase, emulsion phase, or co It can be provided as a roid phase component.

[0047] Furthermore, alternatively or additionally, and as mentioned above, various carrier systems for toxic components Ingredients such as, for example, skin-disrupting ingredients may be present in the composition of the present invention as liposomes, micelles, It exists to include at least one phase having the properties of a complex, salt, nanoparticle, or ferroid. I will.

[0048] [Optional additional components depending on the composition] In some embodiments of the present invention, the animal-killing composition comprises one or more attractant compounds, for example. It can be provided in combination with one or more pheromones (e.g., sex pheromones). Such pheromones or other attractants may be present as additional components of the composition itself. , or alternatively, separately, that is, independently of the same apparatus as the animal-killing composition of the present invention itself. To deliver to and / or in close proximity to, inside or around, The composition may be provided separately.

[0049] One or more pheromones or other attractant compounds are provided as additional components of the composition itself. Therefore, any appropriate amount, for example, about 0.0001% by weight to about 1.2% relative to the weight of the composition. 3, 4 or 5% by weight, more preferably about 0.001% by weight relative to the weight of the composition. It may be included in amounts of 0.01, 0.05, or even 0.1% by weight.

[0050] When used, one or more attractant compounds, such as pheromones, are the attractants of the animal-killing composition. It can be selected to be specific to the target animal species. Therefore, appropriate One or more of the following can be used as attractants: (i) Pheromones, for example, relatively high molecular weight (e.g., about 200,000 to about 300, A lipid-based pheromone of type 000), preferably one or more related target mammals or pheromones specific to vertebrates, especially rodents, such as rats or mice; Taha (ii) One or more animals selected from the group consisting of marsupials, rabbits, and mustelids. Pheromones that are specific to other animals; or (iii) Attractant compounds, for example, relatively low molecular weight (e.g., hundreds of orders of magnitude, e.g., about 50 The region is 0, and preferably one or more related target animals, such as mammals or vertebrae. An attractant compound specific to vertebrates, especially rodents.

[0051] Specific examples of suitable attractants or pheromones include squalene, 2-heptano n, 4-ethylphenol, E,E-β-farnesene, E-α-farnesene, R,R Dehydro-exo-brevicomin and S-2-sec-butyl-dihydrothiazole One of the following could be cited.

[0052] In the composition according to the present invention, one or more additional optional auxiliary components may be added as desired. Alternatively, if necessary, preferably in small amounts, for example, about 1, 2, or 5 of the total composition. It may be included in the composition in an amount of 10% by weight or less.

[0053] Such optional additional components may include one or more of the following: (i) Skin penetration enhancers: - For example, facilitating the passage of one or more active ingredients into and / or through the skin. Or one or more substances known to be used to facilitate skin penetration. Appropriate examples of agents include one or more of the following, or a combination of two or more: : • Fatty acids, for example, oleic acid, linoleic acid, linolenic acid, lauric acid, 2-pyrrolidone, • Propylene glycol, Alcohols, for example, ethanol, isopropyl alcohol, methanol, benzyl alcohol. Alcohols, fatty alcohols (e.g., saturated or unsaturated C8-C8) 14 alcohol), • Glycols, for example, diethylene glycol, propylene glycol, butyl diglyceride call, • Glycol ethers, for example, diethylene glycol monomethyl ether, diethylene Diethylene glycol monoethyl ether and diethylene glycol mono-n-butyl ether , Glycerol formal, • Polyethylene glycol, • Liquid polyoxyethylene glycol, Pyrrolidones, for example, N-methylpyrrolidone (NMP), 2-pyrrolidone (2P), ·acetone, Acetonitrile, • Amides, for example, dimethylacetamide, dimethylformamide, monomethylacetamide Amides, N,N-diethyl-meth-toluamide (DEET), • Phthalate esters, for example, diethyl phthalate, • Phospholipids, for example, phosphatidylcholine, hydrogenated soybean phospholipids, • Natural oils, for example, emu oil, • Azone (1-dodecyl azacycloheptan-2-one or laurocaprum), • Terpenes and terpenoids, • Essential oils, such as eucalyptus oil, kenopoi oil, and ylang-ylang, ·urea, ·water.

[0054] A preferred skin penetration enhancer from the above preferred list is already present in the composition. The substance is the same as the essential component for the carrier system of the solvent or dispersion medium component (or) toxic component. In terms of application, the substance may possess both functions.

[0055] (ii) Humectants: For example, one or more surfactants, such as anions, cationics, amphoteric, or amphoteric ions. (This refers to a nonionic surfactant.) (iii) Viscosity modifiers: For example, one or more thickening agents or diluting components, especially viscosity enhancers, particularly one or more The substance is preferably substantially insoluble in water compared to the main organic material (i.e., a material that is substantially insoluble in water). It is soluble and can increase viscosity (i.e., the viscosity of a substance that does not contain the substance). (compared to PEG-40), for example, polyethylene glycol, for example, PEG200. (iv) cosolvent; For example, one or more protic or aprotic solvents, such as water. (v) Other additional substances: For example, one more emulsifier, stabilizer, preservative, emollient, fragrance, colorant, or Dyes, pH adjusters, gel-forming agents, foam-forming agents, pharmaceuticals. (vi) Delivery-enhancing ingredients: For example, one or more aerosol propellants. Examples of such propellants are well known in the art.

[0056] [Delivery device for delivering compositions] According to one aspect of the present invention, by delivering one or more toxins to the skin of an animal, the animal A device for killing is (i) an enclosure in which animals can enter, (ii) A composition according to the first aspect of the present invention or a composition according to an embodiment or example of the present invention A storage means containing the supply, (ii) a delivery means for delivering a certain amount of the composition to the skin of an animal, Includes.

[0057] Such a device can be of any known type, structure, and method of operation. A suitable device like the one described is described in International Publication No. 2010 of the International Patent Application filed earlier by the inventors. Disclosed and described in patent application / 106352, the contents of which are referred to herein by reference. It is incorporated into the target animal. Regarding delivery devices for delivering toxic substances such as ticides, various other configurations and The design is known in the art and is applicable for use with the compositions of the present invention. This is easily understood and utilized by those skilled in the art.

[0058] Therefore, a preferred apparatus used to deliver the composition according to the present invention In this case, the enclosure may preferably be a trap enclosure, and in some cases, the enclosure may contain a trap. Attractions are facilitated by delivered bait, attractants, or pheromones, and the enclosure A target animal could get inside.

[0059] Preferred delivery means include sprays, aerosols, or other dispensing means. These factors preferably allow an appropriate amount of the liquid composition, for example, a single dose, to be administered to a target animal. A sufficient quantity to kill, from inside the enclosure, onto the target animal once it enters the enclosure, especially It is delivered locally to the skin of the target animal. Such as a spray, aerosol, or other fraction Appropriate configurations and operating mechanisms for distribution means are, for example, in International Publication No. 2010 / 106. From the description in Patent No. 352 (the disclosure thereof is incorporated herein by reference), or target animals From other known pest control devices for delivering toxic substances, such as biological agents, the technology It is well known in the field.

[0060] The appropriate volume or weight of the composition may be applied to the aforementioned amount or dosage, and their volumes or The weight is, for example, the concentration of the toxic component in the composition and / or the size of the delivery means and / Alternatively, it may be selected based on its properties. As a non-limiting example, for example, about 0.1 ml (or From g) about 5 or 10 ml (or g), more preferably about 0.5 ml (or g) A dose of approximately 2, 3, 4, or 5 ml (or g) is used in many embodiments of the present invention. In finished products, they can be appropriately or generally arranged.

[0061] The composition itself is available in liquid form, for example, in the form of a spray or aerosol, or alternatively. It can be delivered in the form of a gel or foam. For the latter purpose, a suitable gel-forming agent or A foaming agent can be used, and this is well known and easily utilized within the scope of the knowledge of those skilled in the art. It is possible.

[0062] The storage means preferably supplies the composition to the delivery means as needed or when required. It is connected to a delivery means so as to deliver toxic substances to target animals, known pest control devices. It could be any appropriate kind in that context.

[0063] [Preparation of the composition according to the present invention] The compositions according to the present invention can be prepared by conventional methods using conventional apparatus and experimental techniques. .

[0064] In particular, in many embodiments, various components of the composition are widely used in the art. It can be simply mixed using well-understood and commonly used known preparation chemical methods.

[0065] In other embodiments, for example, the composition may be a solution, suspension, emulsion, colloidal mixture or This refers to one or more phases in the form of a solution, liposomes, micelles, complexes, nanoparticles, or ferroids. When including, various components of various phases are generally available and well understood and used by those skilled in the art. The final composition is similarly prepared and combined using known chemical techniques. It is possible.

[0066] Detailed examples of suitable preparation techniques used to produce the compositions according to the present invention are described below. This is illustrated in the examples.

[0067] The following examples further illustrate aspects of the present invention and its preferred features and embodiments. This invention is presented and interpreted in order to limit the scope of the invention as defined in the attached claims. It cannot be explained away. [Examples]

[0068] <Example 1 - Composition> The rodenticide composition according to the present invention, administered in a single dose volume of 1 ml, is described below using the following formulations: It was prepared by simply mixing the specified components. Ingredients Quantity Vitamin D3 (Cholecalciferol) 0.09g ethanol * 0.40ml DMSO * 0.40ml PEG200 0.15ml water ** 0.04 ml * The composition is ethanol / DMSO in a 50:50 ratio, diluted to 1 ml. ** The amount of water present may not be a desirable component of the composition, but it is quite expensive. Since it cannot be removed without applying a certain agent, it is acceptable.

[0069] The composition had a viscosity of 4.02 centipoise and a pH of 7.

[0070] <Example 2 - In vitro optimization of transdermal delivery of cholecalciferol> (A) Introduction Cholecalciferol (vitamin D3) is toxic to nocturnal small mammals at low doses. However, since the upper limit of the permissible limit is relatively high for humans and birds, humans and many It is a safe and effective rodenticide for non-target vertebrates and mammals. To test and optimize calciferol delivery, we are using synthetic membranes for in-vitr The test involved using various chemopreservatives that are expected to be useful as skin-disrupting components. The procedure was performed. In order to propose an effective and functional transdermal formulation suitable for use in the present invention, the freezing point, Physical parameters of the formulation, such as viscosity and stability, were also investigated.

[0071] (B) Materials and Methods B.1 Materials European Pharmacopoeia grade cholecalciferol and dimethyl sulfoxide (DMSO) F Purchased from agron UK Ltd (UK). Contains ethanol and o Leic acid is produced by Fisher Scientific UK Ltd (Loughboro). Purchased from ugh, UK, while 2-pyrrolidone (2P) is from Sigma-Aldri Purchased from ch Co (St. Louis, MO, USA). These are all experimental reagents. It was graded. The receiver phase for the in-vitro test was ethanol (Fisher). Scientific UK Ltd (Loughborough, UK)), Pollier Teylene glycol (mwt200, Sigma-Aldrich Co(St. Louis It is composed of s,MO,USA) and water. Cellulose membrane (Visking tubin) g) Fisher Scientific UK Ltd (Loughboroug) Purchased from h,UK). The thermocouple used for the freezing point test was Ktype (RS Comp Onents Ltd, Northants, UK) is the company, while temperature measurement is done by Datalo Using Gar (RS Components Ltd, Northerns, UK) I recorded it.

[0072] B.2 Method B.2.1 In-vitro optimization testing Regenerated cellulose dialysis tube (Visking tubing, Fisher Scient) ific UK Ltd (Loughborough, UK) has been used in synthetic films. (Corrigan, OI, Farvar, MA) ,& Higuchi, WI (1980), “Drug member e transport enhancement using high energy y drug polyvinylpyrrolidone (PVP) co-preci pitates”, International Journal of Pharma ceutics, 5, 229-238; Haigh, JM, & Smith (S MIT), EW (1994), “The selection and use of natural and synthetic membranes for i n vitro diffusion experiments”,European Journal of Pharmaceutical Sciences,2(5-6 ),311-330; Wang, T., Kasichaya nula), S., & Gu, X. (2006), “In vitro perm eation of repellent DEET and sunscreen o xybenzone across three artificial membrane nes”, International Journal of Pharmaceut ics,310(1-2),110-7,doi:10.1016 / j.ijpharm .2005.11.039); Wissing, Sa, & Müller Muller, RH (2002), “Solid lipid nanopa rticles as carrier for sunscreens:in vit ro release and in vivo skin penetration” ,Journal of Controlled Release:Official Journal of the Controlled Release Society y,81(3),225-33). The tube was cut into small pieces and one end was closed. 1 ml of each preparation was used. The solution was then dispensed into a dialysis tube, and the tube was placed in a 50 ml centrifuge tube containing 45 ml of receiver phase. Cholecalciferol is a hydrophobic compound and is substantially insoluble in water, at 6% (v / v) Using an ethanol aqueous solution (1:9 (v / v)) containing PEG200 as the receiver phase Sampling of the receiver phase was performed every hour for the first four hours, and every two hours thereafter. At each sample collection point, 5 ml of the receiver phase was removed and replaced with the stock receiver phase. Of the 5 ml extracted, 1 ml was diluted eight times in succession (256-fold dilution), and the result was obtained by HPLC. The analysis was performed. The temperature of the receiver phase was maintained at 26°C by immersing the centrifuge tube in a hot water bath. Three copies were used for each formulation.

[0073] B.2.1.1 Penetration enhancers and formulation preparations In total, five types of penetration enhancers—DMSO, oleic acid, ethanol, 2P, and water—are used to create a membrane. Selected to improve the transport of cholecalciferol through the pharmacoosmotic agent, for example, DMSO (Stoughton, RB, & Fritz) ch), W. (1964), “Influence of Dimethylsulfo xide(DMSO)on Human Percutaneous Absorpti on, Arch Dermatol, 90(5), 512-517) is an antiviral agent. It has been shown to enhance the penetration of compounds such as steroids and antibiotics ( Williams, AC, & Barry, BW (20 12),“Penetration enhancers”,Advanced Dru g Delivery Reviews,64,128-137;doi:10.101 (6 / j.addr.2012.09.032). DMSO disrupts lipid channels in the stratum corneum. It disrupts the process and promotes the intercellular passage of penetrating agents. Organic solvents such as ethanol penetrate the stratum corneum and lipids. While extracting, the same method is used to increase penetration. Fatty acids such as oleic acid also penetrate the stratum corneum. It has been shown that creating a storage facility where the permeable agent can be moved improves skin permeability. (Larrucea, E., Arellano, a, Sa Santoyo, S., & Ygartua, P. (2001) ,“Combined effect of oleic acid and prop ylene glycol on the percutaneous penetra tion of tenoxicam and its retention in t he skin”,European Journal of Pharmaceuti cs and Bio pharmaceutics:Official Journa l of Arbeitsgemeinschaft fur Pharmazeuti sche Verfahrenstechnik eV,52(2),113-9; Meshulam, Y., Kadar, T., Wenigger (W engier), A., Dachir, S., & Levy, A (1993), “Transdermal penetration of phys. ostigmine:Effects of oleic acid enhancer ”,Drug Development Research,28(4),510-51 5; Moreira, TS; de Sousa, VP .,& Pierre, MBR (2010), “A novel tr ansdermal delivery system for the anti-i nflammatory lumiracoxib:influence of ole ic acid on in vitro percutaneous absorpt ion and in vivo potential cutaneous irri tation”,AAPS PharmSciTech,11(2),621-9;do i:10.1208 / s12249-010-9420-1).

[0074] B.2.1.2 Membrane preparation Before use, coat the cellulose membrane with 2% sodium bicarbonate (Sigma-Aldrich Co(St. Louis, MO, USA) and 1 mM ethylenediaminetetraacetic acid in distilled water The cellulose membrane was washed with a 1L washing solution consisting of (EDTA). The cellulose membrane was placed in the solution and the temperature was adjusted. The temperature was raised to 80°C, and the solution was then maintained at that temperature for 30 minutes (m). After washing, the film was rinsed with distilled water. It was maintained in a bath of distilled water for up to 5 days and then used. This was in accordance with the manufacturer's guidelines (Medicell International Ltd, 200 4).

[0075] B.2.1.3 Data Analysis For each formulation used in the in-vitro test, the drug flux (J s ) of the formulation was calculated. This value was obtained after plotting the cumulative drug concentration in the receiver chamber against time and using the following equation (Barry, B.W. (1983), “Derma tological Formulations”, pp. 49-94, New Yor k, NY, Marcel Dekker Inc.; Gwak, H.S., & Chun, I.K. (2002), “Effect of vehicl es and penetration enhancers on the in v itro percutaneous absorption of tenoxica m through hairless mouse skin”, Internati onal Journal of Pharmaceutics, 236(1-2), 5 7-64):

Number

[0076] B.2.1.4 HPLC analysis Quantitative analysis of all cholecalciferol formulations is performed using a diode array and high-performance liquid Prominence Modular HPLC (S) was performed using whole-body chromatography. Himadzu Corporation, Japan. The detection wavelength is set to 265 nm. Determined. Luna 3μ NH2100A column (Phenomenex, Chesh (ire, UK), Dimensions: 150 x 4.6 mm, NH23 mm ID security glass It was used with a cord and holder. The total flow rate was 2 ml / min, and the runtime was 6 minutes. The mobile phase consisted of 99:1 (v / v) hexane / isopropanol. For the first 3 minutes, a gradient ratio of 1:99 to 50:50 (v / v) is used, and thereafter, the mobile phase is used. The original ratio was restored. The concentration of cholecalciferol was changed to 26 for cholecalciferol. Standard curve created at 5nm (r 2 Using the equation based on the gradient of =0.999, each assembly I calculated the answer to (i).

[0077] B.2.2 Physical optimization In addition to determining the appropriate penetration enhancer, the physical properties of the formulation are important for achieving both efficacy and practicality. Optimizing quality is also necessary. For this purpose, the formulation is evaluated and perfected. Therefore, a series of physical parameters, namely stability, freezing point, viscosity, and solubility, are also considered. Identified. By optimizing these parameters, it is considered that the selected chemical permeation enhancer will act most effectively.

[0078] B.2.2.2 Stability Test The stability of colecalciferol in the selected permeation enhancer is an important consideration when analyzing the physical stability of the formulation. An unstable formulation causes the decomposition of colecalciferol and delivers less than the lethal dose. Colecalciferol is reported to be sensitive to light, heat, and air and unstable in solutions without antioxidants (British Pharmacopoeia. (2012), “Colecalciferol” , British Pharmacopoeia). Crystalline colecalciferol decomposes under conditions that promote oxidation (Huber, W., & Barlow, O.W. (1943), “ Chemical and biological stability of cry stalline vitamins D2 and D3 and their de rivatives”, Journal of Biological Chemist ry, 149, 125 - 137); Examination of the decomposition of the crystalline form showed that 35% and 85% decreases in potency were suggested when maintained at 40°C / 45% RH and 40°C / 85% RH for 7 days, respectively (Grady, L.T., & Thacker, K.D. (1980), “Stability of solid drugs : Degradation of ergocalciferol and chole calciferol at high humidity and elevated temperature”, Journal of Pharmaceutical "temperatures", Journal of Pharmaceutical Sciences, 69(9), 1099 - 1102). Conversely, cholecalciferol in surfactants and oils is said to be stable for a long time at 40°C. To evaluate the stability of cholecalciferol when combined with selected penetration enhancers, an accelerated stability test was conducted. A total of six formulations using various types and concentrations of permeation enhancers were stored in an accelerated stability cabinet at 25°C ± 2°C / 60% RH ± 5%. Each formulation contained 10% (w / v) of cholecalciferol. Each formulation was prepared one day before the scheduled start date of the stability test and left in a water bath / shaker at 25°C overnight to fully dissolve cholecalciferol . Amber bottles were used to prevent photodegradation, and parafilm was used to close around the screw caps to prevent oxidative decomposition by air . At each sample collection point, the lid was removed, 200 μl was collected, and the sample was serially diluted 8 times (256 - fold dilution) for HPLC analysis .

[0079] B.2.2.2.1 Freezing Point Test Regarding the actual use of rodenticide formulations containing cholecalciferol, it is necessary to determine the freezing point of the formulation. Rodenticides are exposed to a variety of temperature ranges, and if an undesired phase change occurs , the rodenticide may lose its effectiveness. To determine the maximum freezing point of formulations containing penetration enhancers, the formulations were frozen in an - 80°C freezer. Each of the eight selected formulations was added to a separate well of a 96 - well plate at a volume of 1 ml. The freezing curve and the corresponding freezing point were observed using a K - type thermocouple. This process was repeated three times, and the average value was calculated. Deionized water and DMSO were used to determine the accuracy of the thermocouple .

[0080] ​​​​​ B.2.2.3 Viscosity Test Topical application can take many forms, from liquids to powders. However, active ingredients in the skin... When the purpose of application is to increase the bioavailability of a drug, theoretically, the drug is more effective. The gel-like formulation to be released is specified (Aulton, ME (2007)). ,“Aulton's Pharmaceutics:the design and manufacture of medicines”,Edinburgh;New York: Churchill Livingstone). In contrast, Cholecalci In the case of ferol, simple mixing with ethanol allows for sufficient skin penetration. Ethanol has a relatively low density of 0.805-0.812 g / cm³. 3 (British Pharmacopiea,(2013),“Ethanol (96 percent )”, January 29, 2013 at http: / / www.pharmacopoeia.c o.uk / bp2013 / ixbin / bp.cgi?a=display&r=5r9 (Read from wILbW9es&n=457&id=7614&tab=search) This results in a "watery" formulation. Increasing the viscosity seems necessary to optimize the formulation. To increase viscosity, a thickening agent is necessary. Therefore, the optimal penetration enhancer must be identified. To that end, a series of thickening agents were tested for suitability and thickening effect. Viscosity measurements were performed using a 1.6mm diameter tubing. Using a capillary column, automated Micro Viscometer (Anto The measurement was performed at Paar, St. Albans, UK. Before measurement, the density of each formulation was measured. (Sartorius Mechatronics UK Ltd, Surrey, UK The viscosity of the thickener was calculated by recording the mass while adding a known volume of solution to the substance. In addition to quantification, the thickening effect of cholecalciferol was also quantified. Both density and viscosity measurements were performed at specific time points. The temperature was recorded using a thermometer and kept within the range of 20-21°C.

[0081] B.2.2.4 Solubility Test The solubility of cholecalciferol in the proposed formulation was tested with reference to the British Pharmacopoeia. Test the solution, and if it is less than 1 ml / g, it is extremely soluble. If it is soluble, it was classified as freely soluble. To determine whether cholecalciferol dissolves well in the relevant formulation, 0.99 ml The preparation was added to 1 g of cholecalciferol. The solution was placed in a water bath and incubated at 20°C for 24 hours. The conditions were maintained. After visually observing each solution, three samples were prepared for each solution and diluted 12 times in succession. The solution was then diluted 4096 times and analyzed by HPLC. The concentration was measured and recorded. Analysis or visual inspection confirmed that cholecalciferol was sufficiently dissolved. If it is suggested that the solution has not been dissolved, add another 1 ml of the formulation and perform HPLC. The test process was repeated until all three measurements matched.

[0082] (C) Result C.1 In-vitro optimization Figure 1 shows the appropriate carrier system for the penetration enhancer, i.e., cholecalciferol. The results obtained from in vitro studies are shown. All formulations contain 10% (w / v) cholecal. Cyferol was used; a total of 1 ml was used for each formulation as shown in Table 1 below. The diffusion area is approximately 40 cm². 2 That was the case.

[0083] From the results, it is suggested that the fastest diffusion of colecalciferol through the membrane is obtained with a 90:10 (v / v) mixture of DMSO / ethanol. The remaining penetration enhancers suggest similar penetration rates. Interestingly, the second-best formulation is a combination of 90:10 (v / v) DMSO / oleic acid, which also had a high proportion of DMSO. Therefore, it seems that the co-solvent of DMSO / ethanol should be further investigated. / v) DMSO / oleic acid, which also had a high proportion of DMSO. Therefore, it seems that the co-solvent of DMSO / ethanol should be further investigated.

[0084]

Table 1

[0085] Figure 2 shows the measured penetration rates when colecalciferol was dissolved in different ratios of DMSO and ethanol. From the previous experiment, it was thought that DMSO and ethanol could increase the penetration rate of colecalciferol. However, the optimal ratio was unknown, so a second experiment was carried out using various ratios of DMSO / ethanol. Figure 2 shows the enhanced penetration of colecalciferol using different ratios of DMSO and ethanol. All solutions contained 10% (w / v) colecalciferol; a total of 1 ml was used in each formulation shown in Table 1A below. From the results of Figure 2, when DMSO and ethanol are dissolved in various ratios within the range of 50:50 (v / v) to 90:10 (v / v), there is a significant enhancement in the penetration of colecalciferol.

[0086] Figure 2 shows the enhanced penetration of colecalciferol using different ratios of DMSO and ethanol. All solutions contained 10% (w / v) colecalciferol; a total of 1 ml was used in each formulation shown in Table 1A below. Figure 2 shows the enhanced penetration of colecalciferol using different ratios of DMSO and ethanol. All solutions contained 10% (w / v) colecalciferol; a total of 1 ml was used in each formulation shown in Table 1A below. Figure 2 shows the enhanced penetration of colecalciferol using different ratios of DMSO and ethanol. All solutions contained 10% (w / v) colecalciferol; a total of 1 ml was used in each formulation shown in Table 1A below.

[0087]

Table 2

[0088] From the results of Figure 2, when DMSO and ethanol are dissolved in various ratios within the range of 50:50 (v / v) to 90:10 (v / v), there is a significant enhancement in the penetration of colecalciferol. This suggests that there are no significant differences.

[0089] C.2 Stability Test Figure 3 shows the results of cholecalcifer when dissolved in various ratios of DMSO and ethanol. The results of the accelerated stability test of the drug are shown. All formulations contain 10% (w / v) cholecalcifone. It contains ferol. Figure 3 shows it sealed in an amber bottle in an accelerated stabilization cabinet at 25°C. This shows the degradation of cholecalciferol when placed at ±2℃ / 60%RH±5%. DM By using various ratios of SO and ethanol, and by including DMSO, cholecalcium We checked whether an increase in ferrol degradation occurred.

[0090] The results showed that after a period of more than 50 days, 27.11% (+ / -1.29%) of cholecalci were present. Ferrol degradation occurred in all formulations, with 16.83% occurring in the first 10 days. This suggests that decomposition of + / -1.48% occurs. The previously published ethanol preparation ( Agnew, WR (2011), “Topical Pestici Compared to "de Formulation" (US patent), the DMSO content accelerates decomposition. Connecting doesn't lead to anything.

[0091] C.3 Freezing Point Test These in vitro tests showed that the mixture of DMSO and ethanol passed through the synthetic membrane. Therefore, the most effective penetration enhancer for transporting cholecalciferol is a carrier system. It was suggested that the acceptable freezing points for ethanol and DMSO are -114°C, respectively. The temperatures are 6°C and 18.3°C. High concentrations of DMSO are very effective as a penetration enhancer. However, using it in high doses is not practical. Commercial use of this formulation is prohibited for outdoor use. Therefore, this indicates that the formulation must be in the liquid phase over a range of temperatures. On the other hand, Ethanol has a low freezing point. Therefore, in order to determine the ratio at which the formulation becomes practical, 2 We investigated the freezing points of different solvent combinations.

[0092] Figure 4 shows the results obtained by investigating the freezing point. These results are related to the solute (in this case, the active compound). This shows the maximum freezing point when the freezing point decreases due to the addition of (minutes). In addition to these measurements, the experiment In this system, the freezing point of deionized water at 0.32 ± 0.11°C was measured.

[0093] Includes 50 / 50 (v / v) and 60 / 40 (v / v) DMSO / ethanol mixtures. The freezing points for the formulations are -35.45+ / -1.26℃ and -20.06+ / -1.8℃, respectively. The temperature was 3°C. All remaining mixtures tested were frozen at temperatures above -7°C.

[0094] C.4 Viscosity Test Selected natural rubber and semi-synthetic materials were used as additives to the formulation. Generally, These compounds can exert a significant effect on viscosity at relatively low concentrations, thus providing a rational explanation. It is an ideal thickening agent. However, since cholecalciferol is a lipophilic compound... The solvent used is an organic solvent, and many water-soluble thickeners are unsuitable, or at least optimal. or may not be suitable. Table 2 below lists ingredients that are unsuitable or relatively unsuitable for the formulation. The selection of natural rubber and semi-synthetic materials is shown below. Table 2 shows the materials that are used as thickeners. This shows inappropriate test materials.

[0095] [Table 3]

[0096] A more preferred alternative to these materials is polyethylene glycol, within a certain range It can yield a molecular weight and is soluble in organic solvents.

[0097] Figure 5 below shows the viscosity-enhancing effect of PEG200 on a penetration enhancer and cholecalciferol. It exhibits its own thickening effect, and the interaction between cholecalciferol and the thickening agent with the chemical penetration enhancer. As shown, all viscosity measurements are expressed as the solute percentage in 50:50 DMSO / ethanol. It has an SD of + / - 0.003 mPa.s.

[0098] Table 3 below shows the effects of combining both cholecalciferol and PEG200 on the viscosity of the formulation. This suggests a thickening effect. The maximum amount of PEG200 is 15% (v / v), and this This makes it possible to use a 50:50 DMSO / ethanol penetration enhancer, and cholecalcifer This promotes the complete dissolution of the compound.

[0099] [Table 4]

[0100] High molecular weight PEGs can also be used, but increasing the molecular weight leads to an increase in the freezing point. Therefore PEG200 was identified as a preferred thickening agent.

[0101] C.5 Solubility Test Table 4 below shows the penetration enhancer (50:50 (v / v) DMSO / ethanol) and the proposed Approximate values ​​for both the thickening agent (15% (v / v) PEG200) and the penetration enhancer. The solubility and corresponding classifications are shown. Ethanol is also included in the table as a reference. From the results, It is suggested that solubility decreases when PEG200 is added to the formulation.

[0102] [Table 5]

[0103] For approximate solubility, first add 0.99 ml of solvent to 1 g of cholecalciferol. Next, the calculation was performed by HPLC analysis on three separate samples obtained from the solution. The process is repeated until the same concentration is suggested in three samples analyzed by HPLC, and then the substance is added at that point. The total volume is calculated by determining the amount of cholecalciferol in the vial by HPLC. Diluted. The classification of ethanol is obtained from the British Pharmacopoeia (British Ph armacopiea (2013), “Colecalciferol”, 2013 On January 30th, http: / / www.pharmacopoeia.co.uk / bp2 013 / ixbin / bp.cgi?a=display&r=jFUlqg0blYj (Read from &n=3&id=7803&tab=search).

[0104] (D) Discussion Agnew (Agnew, WR (2010), “T "Opical pesticide formulation", International Publication No. 2010 / 071450 A1 (World Intellectual Property Organization) states that ethanol contains 20% (w / v) corn. It is proposed that transdermal formulations containing recalciferol demonstrate efficacy in vivo. This study investigated improved formulations that produce an increased drug flux of cholecalciferol. We are using an in-vitro method to demonstrate this.

[0105] The results showed that formulations containing a high percentage (50-90%) of DMSO were effective in drug transmission through the artificial membrane. This suggests an increase in Lux measurements. In the initial test, ethanol (0.13 + / -0.0035 mg / cm 2 Compared to h), 90:10(v / v)DMSO / ethanol Nol (0.25 + / - 0.019 mg / cm³) 2 h) or 90:10(v / v)DMS O / oleic acid (0.18 + / - 0.022 mg / cm³) 2 In case h), the drug flux It was suggested that it would increase. Further studies showed that the increase in drug flux was lower in What happens at DMSO concentration in Tanol (50:50 (v / v) DMSO / ethanol) This suggests a ratio of 0.19+ / -0.011 between DMSO and ethanol. The combination shows improved flux compared to previously published ethanol formulations. Therefore, it was selected as a preferred chemical penetration enhancer, i.e., a carrier system.

[0106] Addition of water to ethanol preparations (0.16 + / - 0.023 mg / cm³) 2 h) and 2P Preparations containing (0.083+ / -0.017 mg / cm³) 2 h) contains only ethanol. 0.13+ / -0.0035 mg / cm³ 2 h) No substantial difference was observed compared to the formulation. Therefore, these chemical penetration enhancers were excluded from the remaining tests. The agent, especially oleic acid, has worked well in other tests (Larruce a) E., Arellano, a, Santoyo, S., & Ygartua, P. (2001), “Combined effect t of oleic acid and propylene glycol on the percutaneous penetration of tenoxica m and its retention in the skin”,Europe n Journal of Pharmaceutics and Biopharmaceuticals ceutics:Official Journal of Arbeitsgemei nschaft fur Pharmazeutische Verfahrenste Chnik eV, 52(2), 113-9; Meshulam, Y. Kadar, T., Wengier, A., Dahia chir), S., & Levy, A. (1993), “Transder mal penetration of physostigmine:Effects of oleic acid enhancer”,Drug Developmentmen t Research, 28(4), 510-515; Moreira, T S. de Sousa, VP, & Pierre, M .BR(2010), “A novel transdermal deliver y system for the anti-inflammatory luminaire acoxib:influence of oleic acid on in vit ro percutaneous absorption and in vivo p otential cutaneous irritation”,AAPS Phar mSciTech,11(2),621-9,doi:10.1208 / s12249- (010-9420-1), the results showed that DMSO was better than cholecalciferol. This suggests that it is a suitable alternative material. These results are partly due to the artificial membrane used. It's possible. Oleic acid and 2P act to create storage sites in the stratum corneum, allowing for invasive movement. It is suggested that it provides a wider pathway for the substance. On the other hand, water has a hydrating effect on the skin. This suggests that hydrophilic compounds increase the drug flux. These infiltration mechanisms do not occur in artificial membranes such as lurose membranes: however, Cholecalciferol is hydrophobic (essentially insoluble in water), so it penetrates the lipid film of the stratum corneum. And it can move freely. Therefore, the most advantageous thing about cholecalciferol The mechanism is that the membrane can be disturbed. This situation is likely to occur again in cellulose membranes. This revealed that DMSO increases the pore size of the membrane, leading to rapid absorption of cholecalciferol into the receiver phase. This is accelerating the spread.

[0107] While DMSO is widely recognized as a penetration enhancer, it is generally not used for transdermal delivery of pharmaceuticals. It is not used for this purpose. DMSO can mildly irritate the skin and produce an unpleasant taste in the mouth. However, the purpose of this preparation is for use as a rodenticide. Therefore, these elements are important. It is important, but it should be considered in comparison to its effects. An alternative chemical penetration enhancer is this This is intended to prolong the effects of calciferol, and these initial problems can be ignored.

[0108] In addition to chemical problems related to DMSO, physical problems may also exist. The freezing point of DMSO is... Its temperature is 18.3°C, and it is a liquid at room temperature, but a solid below that temperature. For practical purposes, rodenticides must be usable in a wide variety of environments, one of which is in low-temperature conditions. Yes. This can be particularly problematic if the intended target is a nocturnal species, and the temperature of the night can be a factor. A decrease in this factor can lead to a high percentage of DMSO preparations losing their effectiveness. To lower the freezing point of the preparation, A series of DMSO / ethanol ratios were investigated to enhance osmosis and improve the low freezing point of ethanol (-11°C). A ratio was determined that allows the use of both 4.6℃ and 50:50 (v / v Focusing on the DMSO / ethanol ratio, further tests were conducted to establish a basis for a preferred formulation. Ta.

[0109] According to drug penetration theory, formulations with gel-like homogeneity can conform to the contours of the skin. This is indicated to increase the bioavailability of the active ingredient. Therefore, formulation Increasing the viscosity was considered desirable to improve the drug flux. Regarding the thickening agents used, tests were conducted to increase viscosity. However, determining the appropriate agent proved difficult. This proved difficult with formulations composed almost entirely of organic solvents. Ultimately, PEG200 was preferred. It stood out as an excellent thickening agent. However, to obtain a significant effect on viscosity, high A ratio of 15% (v / v) was required. This amount corresponds to 0.1-1% (w / v) in natural gum. The viscosity increases significantly in the concentration range of v), which is particularly high.

[0110] The last two parameters considered are useful for objective implementation that has room for improvement. The solubility of luciferol in ethanol is defined as easily soluble, at 1-10 ml / g means that it can be used to completely dissolve the solute. Agnew )(Agnew, WR (2010), “Topical pesti "Cide Formulation", International Publication No. 2010 / 071450 A1, World The formulation proposed by the World Intellectual Property Organization is a high-dose cholecalciferol at 20% (w / v). It is important that a solution is required and that no loss of solubility is induced when changes occur in the formulation. In this regard, cholecalciferol was slightly soluble in all the formulations tested. This suggests that the delivery of an effective dose is not hindered by the formulation process.

[0111] Finally, for commercially available products, product stability must be confirmed over a defined period of time. No. Regarding this parameter, the results show that after 50 days at 25℃±2℃ / 60%RH±5% This suggests a 27.11% (+ / -1.29%) decrease in ability. Although Ferrol is sensitive to light, heat, and air, the use of an amber-colored bottle reduces its effectiveness. Photodegradation, which causes the following, is eliminated. Oxidation, heat, or a combination of these two factors leads to decreased performance. It is suggested that these are contributing factors. Various options are available to eliminate these problems. Yes, for example, filled under nitrogen or some other inert gas to counteract oxidative decomposition. Possible methods include carrying out manufacturing operations such as those mentioned above, or adding antioxidants chemically. It is possible.

[0112] (E) Conclusion Results from in vitro studies for the optimization of cholecalciferol suggest that DMSO and A carrier system containing ethanol delivers the drug flux of cholecalciferol through an artificial membrane. Compared to the previously released ethanol formulation, this suggests an increase (in ethanol 0.13+ / -0.0035 mg / cm³ 2 Compared to h, 50:50(v / v)DMSO / Ethanol: 0.19+ / -0.011 mg / cm³ 2 h).

[0113] Parameters such as freezing point and viscosity were also adjusted to make the formulation effective and functional. The ratio of DMSO to ethanol achieved a freezing point of -35.45+ / -1.26°C without any solute. It was manipulated to do so (50 / 50 (v / v) DMSO / ethanol). On the other hand, 15% The addition of PEG200 increases the viscosity of the formulation to 9% (w / v) cholecalciferol. The concentration increased to 2.58 mPa.s for the formulation containing the substance. It is easily soluble (1-10 ml / g) and This solubility classification was maintained in this formulation as well, and it was possible to dissolve an effective amount in the solution.

[0114] <Example 3 - In vitro, in rat skin uptake of cholecalciferol> -vivo model> (A) Introduction In this example, two in vitro models and cholecalciferro through the dermis were used. We present corresponding in-vivo data designed to optimize the delivery of the message.

[0115] (B) Materials and Methods B.1 Materials European Pharmacopoeia grade cholecalciferol, supplied by Fagron UK Ltd (Newcas Purchased from Tyne, UK. Penetration enhancers: ethanol, oleic acid, And dimethyl sulfoxide (DMSO) is a Fisher Scientific U Purchased from K Ltd (Loughborough, UK), while 2-pyrrolidone, Purchased from Sigma-Aldrich Co (St. Louis, MO, USA), All of these were experimental reagent grade. Methylcellulose (Fisher Scientif ic UK Ltd (Loughborough, UK) and polyethylene glyco (mwt200, PEG200)(Sigma-Aldrich Co(St.Lou) (is, MO, USA)) was used as a viscosity modifier. The sheba phase is ethanol (Fisher Scientific UK Ltd (Lou ghborough, UK), polyethylene glycol (mwt200, Sigma- It is composed of Aldrich Co (St. Louis, MO, USA) and deionized water. Regenerated cellulose dialysis membrane (Visking tubing, Fisher Science) Tific UK Ltd (Loughborough, UK) is used in synthetic films. Kita (Corrigan, OI, Farvar, MA) .,& Higuchi, WI (1980), “Drug membra ne transport enhancement using high energy gy drug polyvinylpyrrolidone (PVP) co-prec ipitates”, International Journal of Pharm aceutics, 5, 229-238; Haigh, JM, & Smith Smith), EW (1994), “The selection and use of natural and synthetic membranes for in vitro diffusion experiments”,European Journal of Pharmaceutical Sciences,2(5- 6), 311-330; Wang, T., Kasichay anula), S., & Gu, X. (2006), “In vitro per meation of repellent DEET and sunscreen oxybenzone across three artificial members anes”, International Journal of Pharmaceu tics,310(1-2),110-7,doi:10.1016 / j.ijphar m.2005.11.039; Wissing, Sa, & Müller Muller, RH (2002), “Solid lipid nanopa rticles as carrier for sunscreens:in vit ro release and in vivo skin penetration” ,Journal of Controlled Release:Official Journal of the Controlled Release Society y,81(3),225-33).

[0116] B.2 Method B.2.1 Transmission Test B.2.1.1 Cellulose tube in-vitro model The tube was cut into small pieces and one end was closed. 1 ml of each preparation was dispensed into the dialysis tube, and the tube was 45 m long. It was placed in a 50 ml centrifuge tube containing l of the receiver phase. Cholecalciferol is hydrophobic. It is a compound, substantially insoluble in water, and contains 6% (v / v) PEG200 ethanol. A 10:90 (v / v) aqueous solution of Nol was used as the receiver phase. Sampling of the receiver phase was performed as follows: The sampling was performed every hour for the first four hours, and then every two hours thereafter. At each sample collection point, 5m The receiver phase of l was removed and replaced with the stock receiver phase. 200 μg out of 5 ml. l was extracted, diluted, and analyzed by HPLC. The receiver phase temperature was controlled by placing the centrifuge tube in a hot water bath. The temperature was maintained at 26°C ± 2°C by immersion. Three replicas were used for each formulation. there was.

[0117] B.2.1.2 In-vitro model of diffusing cells Cut the membrane into 5x5cm squares and place them between the donor and receiver chambers of the fixed diffusion cells. (Ingham Group, Aston University, UK). Each formulation Regarding this, 15 ml of receiver solution is placed in the receiver chamber, while 5 ml of assay... The substance was distributed to the donor. 6% (v / v) PEG200 ethanol aqueous solution (1 A 0:90 (v / v) receiver phase was used. Sampling of the receiver phase was performed for the first 5 hours. The procedure was performed every hour, and then every two hours thereafter. At each sample collection point, 5 ml of receiver phase was collected. It was removed and replaced with the stock receiver phase. 200 μl was extracted from the 5 ml and diluted. Then, HPLC analysis was performed. The receiver phase temperature was set to 37°C+ using a heated stirring plate. The temperature was maintained at -2°C.

[0118] B.2.1.3 Formulations Two batches of the five formulations were tested in a cellulose tube model. One set consisting of the five formulations was... We tested a series of chemical penetration enhancers at various concentrations to determine the optimal one. Two batches of the agent were tested in a series of DMSO / ethanol cosolvents.

[0119] Two batches of the formulation were used in a diffusing cell model; the first batch showed optimal chemiosmotic enhancement. To determine the agent, a series of penetration enhancers at various concentrations were investigated. In the second batch, this The calciferol concentration was changed.

[0120] B.2.1.4 Enhancement of Chemo-Osmotics Chemical penetration enhancers, for example, DMSO (Stoughton, RB, & Fritsch, W. (1964), “Influence of dimethylsulfoxide(DMSO)on human percutan eous absorption”,Arch Dermatol,90(5),512 -517) enhances the penetration of compounds such as antivirals, steroids, and antibiotics. It has been shown that (Williams, AC, & Barry) Barry), BW (2012), “Penetration Enhancers ”,Advanced Drug Delivery Reviews,64,128- 137). DMSO and the sulfoxide family disrupt the stratum corneum and penetrate the intercellular spaces of the penetrating agents. It facilitates passage. Organic solvents such as ethanol extract lipids from the stratum corneum. It increases penetration. Fatty acids such as oleic acid also allow the penetrating agent to move into the stratum corneum. It has been shown that creating storage facilities improves skin penetration (Larr ucea), E., Arellano, A., Santoyo ,S.,& Ygartua,P.(2001),“Combined e ffect of oleic acid and propylene glycol on the percutaneous penetration of teno xicam and its retention in the skin”,Eur open Journal of Pharmaceutics and Bioph armaceutics,52(2),113-9;Meshulam, Y., Kadar, T., Wengier, A., Dahia Dachir), S., & Levy, A. (1993), “Translated ermal penetration of physostigmine:Effec ts of oleic acid enhancer”,Drug Developm ent Research, 28(4), 510-515; Moreira TS, de Sousa, VP, & Pierre ,MBR(2010),“A novel transdermal deliv. ery system for the anti-inflammatory lum iracoxib:influence of oleic acid on in v itro percutaneous absorption and in vivo potential cutaneous irritation”,AAPS Ph armSciTech, 11(2), 621-9). DMSO, ethanol, water, 2-p Loridone and oleic acid were selected as promising penetration enhancers.

[0121] B.2.1.5 Membrane preparation Before use, coat the cellulose membrane with 2% (w / v) sodium bicarbonate (Sigma-Ald Rich Co (St. Louis, MO, USA) and 1 mM ethylenedioxide in distilled water The cellulose membrane was washed with a 1 L washing solution consisting of tetraacetic acid (EDTA). The temperature was then raised to 80°C, and the solution was maintained at that temperature for 30m. After cleaning, the membrane was distilled. Rinse with water and keep in a distilled water bath for up to 5 days before use. This is according to the manufacturer's guidelines. This is in accordance with the (Medicell International Ltd, 2 004).

[0122] B.2.1.6 Data Analysis For each formulation used in the in vitro study, the drug flux of the formulation (J s ) This was calculated. This value was obtained by plotting the cumulative drug concentration in the receiver chamber against time. The following equation was obtained using the following equation (Barry, BW (1983), “Derma tological formulations”,pp.49-94,New York k, NY, Marcel Dekker Inc.; Gwak, HS, & Chun, IK (2002), “Effect of Vehicle es and penetration enhancers on the in v itro percutaneous absorption of tenoxica m through hairless mouse skin”,Internati onal Journal of Pharmaceutics,236(1-2),5 7-64):

number

[0123] B.2.1.7 HPLC analysis Quantitative analysis of all cholecalciferol formulations is performed using a UV diode array. The analysis was performed using Prominence Modular HPLC (rapid liquid chromatography). (Shimadzu Corporation, Japan)). Detection wavelength: 265nm Set to: Luna 3μ NH2100A column (Phenomenex, Che Shire, UK, Dimensions: 150 x 4.6 mm, NH23 mm ID Security - Used with guard and holder. Total flow rate was 2 ml / min, runtime was 6 minutes. Yes, it was. The mobile phase consisted of 99:1 (v / v) hexane / isopropanol (H (PLC grade). The first 3 minutes of the run should be 99:1~50:50 (v / v) hexane / I Using the gradient ratio of sopropanol, the mobile phase is then returned to its original ratio of 99:1 (v / v). I switched back to xan / isopropanol. I changed the cholecalciferol concentration back to cholecalciferol Standard curve created at 265nm (r 2 The equation derived from the gradient (=0.999) is used. Then, calculations were performed for each assay.

[0124] B.2.2 In-vivo testing B.2.2.1 Animal Husbandry All in vivo studies are conducted by Cellvax Pharma (Paris, Fran). The experiment was conducted at ce). The experimental protocol was followed by Ministere de L'enseigne. ment Superieur de la Recherche(ComEth An Approved by ses / ENVA / UPEC 16). 7-10 week old male Spra The gue Dawley rat (Harlan, France) was used in the protocol. Each rat was weighed between 250 and 350 g and tested for SOPF (Specific and Opportunistic Diseases). (Without the original substance) The animals were in a controlled environment with controlled ambient conditions and light, and polyethylene It was placed in a cage. Lighting hours were from 7:00 to 19:00, and temperature and humidity were controlled accordingly. The conditions were maintained at 21+ / -1°C and 70% RH. The animals were periodically supplied with food and water. In the reprogramming osmotic enhancement test (Protocol 1), each animal was housed individually. In the answer test (protocol 2), animals are housed in groups of two and three, and their fur is shaved. Each animal was identified. All animals were acclimatized to the laboratory for at least one week before the start of the experiment. Ta.

[0125] B.2.2.2 Protocol 1 (Screening Test) The screening test protocol is for evaluating the effectiveness of rodenticides (PP 1 / 113(2)). The European Plant Protection Organization (EPPO, 1998) was incorporated into the European Plant Protection Organization (EPPO, 1998). Planned by the Plant Protection Organization (EPPO) Based on established guidelines. Regarding the testing of the new rodenticide, for brown rats (Rattus Five mice (Mus musculus) (Mus norvegicus) A screening test using male experimental species has been proposed. A total of five formulations will be used. Each test was conducted on five animals.

[0126] The method for applying percutaneous rodenticides is the Organisation for Economic Co-operation and Development (Organisation for Economic Co-operation and Development Based on the OECD guideline 434 (OECD, 2004), the formulation was used in animals. 10cm from the nape of the neck 2 It was applied to the area. In this protocol, the hair in the application area was shaved. Although it is advised to protect that area, this measure is preferred to be applied "while using" the product. This was not included in this study.

[0127] B.2.2.3 Protocol 2 (Fixed Dose Procedure) ure)) To identify the optimal chemical penetration enhancing composition, it is necessary to determine the optimal dose of the active ingredient. The number of animals and test conditions are also included in the evaluation of the effectiveness of rodenticides (PP 1 / 113(2))(EP Based on the screening tests suggested in PO, 1998. The OECD has indicated acute skin Detailed guidelines on skin toxicity calculations (OECD Guideline 402) (OECD, This shows the 1987 data, where the LD50 is calculated from the dose-response curve of the active ingredient. However, the Directive does not recommend this protocol, therefore Dosage-fixing method (OECD guidelines 420, 434) (OECD, 2001, 200) 4) was used. The dosages used in this method were 5, 50, 300 and 2000 mg / kg. It corresponds to.

[0128] B.2.2.4 Data Analysis The directive states that for a rodenticide to be considered for the control of rodents, that rodenticide must be "sufficient" The EPPO guidelines state that it must be proven to be "effective." Regarding the evaluation of efficacy, for screening trials, the formulation that shows a 100% mortality rate... This indicates that it can be effective as a rodenticide.

[0129] In addition to mortality, the animals' suffering was reduced 2-3 times a day for 5 days. They were observed, and then observed twice a day for a further 9 days. Formulations with a mortality rate of less than 100% were... , it was deemed ineffective. The suffering of animals during the protocol was observed by Wolfenson et al. (Wolf Wolfensohn, S., & Lloyd, M. (2003) ),“Handbook of Laboratory Animal Management ent and Welfare” (3rd ed.), Blackwell Publ. Using the pain rating scale proposed by ishing Ltd., future field experiments We also added items related to evoked responses, which we considered to be important aspects of the experiment, to this chart and observed them.

[0130] In dose-fixed methods, body weight data is used to measure the effect of sublethal doses on animals. They collected the data. After the dose-fixing method was completed, the rodenticide was used in the OECD (OECD, 2001, 2 A classification was given based on the LD50 cutoff value indicated by 004).

[0131] (C) Result C.1 Transmission results We investigated the effect of chemosmotic enhancers on the delivery of cholecalciferol through rat dermis. To achieve this, we built two in-vitro models. The following results show that the process is efficient over time. This suggests the cumulative amount of cholecalciferol in the receiver phase. Each figure corresponds to The table shows the calculated drug flux values ​​determined from the steady-state gradient.

[0132] C.1.1 Cellulose tube in-vitro model C.1.1.1 Enhancement of Chemo-Osmotics Figure 6 shows the cholecalciferol recovered from the receiver phase plotted against time. This shows the cumulative amount. In total, there are 5 penetration-promoting chemicals: DMSO, ethanol, oleic acid, 2- Pyrrolidone and water were selected from the literature. The results showed that DMSO / ethanol in a 90:1 ratio was suitable. The maximum diffusion rate of cholecalciferol through the membrane can be obtained with a 0(v / v) mixture. This suggests that the remaining penetration enhancers have comparable penetration rates. Both agents exhibit the highest calculated drug flux.

[0133] Figure 6 shows a comparison of chemiosmotic enhancements for the delivery of cholecalciferol through synthetic membranes. As shown, all formulations (shown in Table 5 below) contain 10% (w / v) cholecalciferol. Used; a total of 1 ml was used for each formulation. The diffusion area was approximately 40 cm². 2 That was the case.

[0134] [Table 6]

[0135] C.1.1.2 Enhanced osmotic flow of DMSO / ethanol cosolvent Referring to Figure 7, this figure shows DMSO and ethanol in various ratios (as shown in Table 6 below). The measured penetration rate of cholecalciferol when using Nol as a penetration enhancer. As shown, all solutions contain 10% (w / v) cholecalciferol; the total is 1 ml. It was used in each formulation. From the aforementioned experiments, DMSO and ethanol cosolvents were found to be effective against drug flocculation. It was found that it increases the number of. However, the optimal ratio is unknown, and therefore, In experiment 2, various ratios of DMSO / ethanol were used.

[0136] [Table 7]

[0137] The results show that the ratio of DMSO to ethanol is 50:50 (v / v) to 90:10 (v / v). When used as a penetration enhancer in various ratios within the range, cholecalciferol's penetration enhancement This suggests there is no significant difference between the strong and weaker levels.

[0138] C.1.2 In-vitro model of diffusing cells C.1.2.1 Enhancement of Chemo-Osmotics Referring to Figure 8, this figure shows cholecalcif through a synthetic membrane using a diffusion cell arrangement. Figure 8 shows the diffusion of cholecalciferol. Figure 8 shows the chemical reaction for delivery of cholecalciferol through a synthetic membrane. This shows a comparison of osmotic enhancement agents. A total of 5 ml of the formulation was distributed to the donor phase. The dotted line represents the calculated results. The best fit line from the drug flux is shown in Table 7 below. A combination of cellulose concentration and chemical osmosis enhancer was used. (Cellulose tube in-vitro model) Based on the results, formulations containing a high percentage of DMSO were compared to ethanol formulations. I considered it.

[0139] The results showed that both cholecalciferol concentrations (20% and 40% (w / v)) Therefore, the ethanol medium increased the drug flux compared to the DMSO / ethanol cosolvent. This suggests that they did so.

[0140] [Table 8]

[0141] C.1.2.3 Dose-response Referring to Figure 9, this figure shows the cholecalciferol concentration and using a diffusion cell model. The dose-response correlation relating to the diffusion ratio is shown. A total of 5 ml was added to each donor chamber. All formulations were made to contain the indicated amount of cholecalciferol, and 15% (v / v)P A medium containing EG200 and a 50 / 50 cosolvent of DMSO / ethanol was used. did.

[0142] Figure 9 suggests dose-response correlations measured by a diffusion cell in vitro model. A series of cholecalciferols including 20%, 9%, 1.5%, and 0.15% (w / v) The concentration of the substance was investigated (as shown in Table 8 below). The concentration was obtained using a dose-fixed method, and the oral toxicity was determined. Sex was determined (OECD Guideline 420) (OECD, 2001). From the results, 20 It is suggested that a %(w / v) cholecalciferol concentration produces the maximum drug flux. All formulations will contain 15% (v / v) PEG200 in a 50:50 (v / v) ratio. DMSO / ethanol was used as the cosolvent.

[0143] [Table 9]

[0144] C.2 In-vivo results The following is Directive 9 for enabling in vitro models and measuring the efficacy of formulations. This is the result of an in-vivo study referencing 8 / 8 / EC (European Commission, 1998). In the study, a screening protocol was developed to determine whether the formulation was "sufficiently effective". A dose-fixing method designed to determine the amount of cholecalciferol required for an effective dose. ,carried out.

[0145] C.2.1 Screening Test A screening protocol was performed on a total of five formulations. These formulations were in-v In ITR studies, it showed a higher drug flux compared to other chemiosmotic enhancers. These are two fabrications using DMOS / ethanol cosolvent as shown in the cellulose tube model. The agent, two formulations using ethanol as shown in the diffusion cell model, and the rest are both in -A formulation consisting of DMSO and oleic acid, highlighted in the in vitro model. The procedure was performed with different cholecalciferol concentrations, with 40% (w / v) mixed with ethanol in a 70:3 ratio. 0 (v / v) DMSO / ethanol cosolvent, 20% (w / v) ethanol and 90 :10(v / v)DMSO / ethanol and 90:10(v / v)DMSO / oleic acid It was used in the following: A thickening agent (methylcellulose) was added to all formulations containing DMSO. 1% (w / v) is used in 90:10 (v / v) DMSO / ethanol and 70:30 (v / v) Added to DMSO / ethanol preparations. On the other hand, 0.75% (w / v) was added in a 90:10 ratio. Added to v / v)DMSO / oleate preparations. Mortality and time to endpoint. Records were kept for each formulation. Each formulation was administered to 5 animals, with a volume of 1 ml given to each animal. It was administered.

[0146] Referring to Figure 10, this figure shows the effects of transdermal cholecalciferol with increased chemical penetration. The survival rate for the formulation is shown. (a) 20% in 90:10 DMSO / ethanol (b) Survival graph against cholecalciferol (w / v); 90:10 DMSO / Oray Survival graph in 20% (w / v) cholecalciferol in nic acid; (c)70: Survival of 40% (w / v) cholecalciferol in 30 DMSO / ethanol Graph; (d) In contrast to 20% (w / v) cholecalciferol in 100% ethanol Survival graph; (e) In contrast to 40% cholecalciferol in 100% ethanol Survival graph; n=5 was used in all experiments, (f) mortality rate for all formulations Summary of the time to the endpoint.

[0147] Figure 10 shows a design to improve the transport of cholecalciferol through rat skin. The survival rates for each of the five formulations are shown. From the results, only the formulation containing DMSO survived. This suggests a 100% mortality rate. In accordance with EPPO guidelines, these Further research may be considered on the formulations of 20% and 40% (w / v) cholecalcife. Ethanol preparations containing rolls resulted in mortality rates of 20% and 60%, respectively; Therefore, these formulations are not considered effective as rodenticides, and further research is not justified. The results showed that the presence of DMSO compared to ethanol preparations resulted in a lower cholecalciferol content. The improved penetration suggests that it can lead to a 100% mortality rate within 5 days of application. To evaluate the effects of the formulation on animals, and to quantify all pain and suffering. To that end, pain levels were assessed 2-3 times a day using a pain rating scale. We considered five main areas that could be presented. These areas include: general findings, findings at the application site, These include natural behavior, stimulated behavior, and food and water intake. A score was assigned, allowing for a maximum of 15 points of pain. A score of 0 indicated that the formulation was ineffective. This indicates a slight change in behavior. Scores of 1-5 indicate a minor change, while scores of 5-10 indicate a moderate change. This suggests a change in behavior, while scores above 10 indicate significant changes in behavior such as hair stabilization and unconsciousness. This suggests that...

[0148] Referring to Figure 11, this figure shows the pain rating table for each set of five animals exposed to each formulation. Figure 11(a) shows the pain ratings for experimental set 1. Figure 11(b) shows the actual The pain ratings for Experiment Set 2 are shown. Figure 11(c) shows the pain ratings for Experiment Set 3. The points are shown. Figure 11(d) shows the pain ratings for experimental set 4. and Figure 11(e ) shows the pain rating for experimental set 5.

[0149] Figure 11 shows the quantitative results of pain obtained from the pain rating scale. The animals were administered the drug continuously. The graph shows the level of pain experienced by each animal and formulation for each administration cycle shown. The results showed that the average pain at the endpoint was 5-10, and this result indicates that The average pain level of 2 in the case of 20% (w / v) cholecalciferol in Tanol is quite different. However, this formulation resulted in only a 20% mortality rate. These results The effect is moderate changes in behavior in rodents, such as decreased mobility, as well as changes in food intake. This suggests that the water level has decreased, among other things.

[0150] Figure 12 shows the average pain at the endpoint for all animals. Here, 0 means there is no pain, and 15 means the maximum pain, and in this case the animal's hair is standing up. This indicates a significant decrease in mobility and a decrease in food and water intake. The endpoint of the experiment also includes 20% (w / v) cholecalciferol in ethanol. In this case, mild suffering was suggested to occur in only 20% of the animals that died.

[0151] C.2.2 Dose fixation technique A total of five formulations were used in the dose-fixing method. Here, the cholecalciferol concentration was set to O The GHS classification and effects were modified according to the ECD Guideline 420 (OECD, 2001). The minimum dose required to maintain the condition was determined. The concentrations were 20%, 9%, 1.5%, and It consists of 0.15% (w / v) and 15% (v / v) PEG200, making it a 50:50 (v / v) Administered in 1 ml volume of DMSO / ethanol cosolvent. As a negative control, 15% ( A 50:50 DMSO / ethanol cosolvent containing v / v PEG200 was used. Mortality rate, The time to death and the degree of suffering were recorded for each animal. DMS was used as a cosolvent osmotic enhancer. We used 0 / ethanol because, based on the results of the screening protocol, this This is because it was suggested that there is evidence for further research on the formulation. PEG200 was added to the formulation. In addition, the formulation was made thicker to promote adhesion to animals.

[0152] Referring to Figure 13, this figure shows the survival analysis using a dose-fixed method. Figure 13(a) shows 9 Survival graphs for % and 20% (w / v) cholecalciferol preparations are shown. The formulation was not included to show a 0% mortality rate. Figure 13(b) shows the results for all formulations. This shows the mortality rate and mean time to the endpoint; formulations for 0%, 0.15%, and 1%. In the 5% of cases, none of the patients showed symptoms of death.

[0153] Figure 13 shows the survival analysis, mortality rate, and time to death obtained using the dose-fixed method. Of the five formulations examined, only 9% and 20% (w / v) cholecalciferol were found to be effective. These formulations show a 100% mortality rate, and therefore should be considered as rodenticides in accordance with the directive. These formulations are suggested to be effective within minutes. These formulations were administered in a screening protocol. It also shows a reduction in the time to the endpoint compared to the DMSO / ethanol cosolvent (90 : 10 DMSO / 20% (w / v) cholecalciferol in ethanol within 5 days 0% mortality rate, 9% (w / v) cholecalciferol in 50:50 DMSO / ethanol (100% mortality rate within 3 days). The negative control group showed no lethality, suggesting that Choleca It can be concluded that luciferol is responsible for the lethal effect.

[0154] Referring to Figure 14, this figure shows the results for each of the five formulations tested with a fixed dose processing protocol. The pain rating and rat body weight for the treatment are shown. All rats received the indicated amount of cholecalcifer. A 1 ml formulation containing roll was administered. Figure 14(a) shows the pain ratings for the control group. Figure 14(b) shows the average rat body weight. Figure 14(b) shows the pain experienced by the group exposed to 0.15% (w / v). The scores and average rat body weight are shown. Mild distress was observed, but weight gain was observed in the rats. Figure 14(c) shows the distress scores and averages of the group exposed to 1.5% (w / v) cholecalciferol. The average rat body weight is shown. The rats show increased pain, and their body weight decreases before slightly increasing. , signs of recovery were suggested. Figure 14(d) shows exposure to 9% (w / v) cholecalciferol. The distress scores and average rat body weight of the exposed group are shown. The result was a sharp increase in distress after 24 hours. This suggests a significant decrease in body weight. Figure 14(e) shows the suffering of the group exposed to 20%(w / v). Pain scores and average rat body weight are shown. The results show a rapid increase in pain and average rat body weight after 24 hours. This suggests a decrease in weight.

[0155] Pain quantification is performed for each screening protocol, and further weight data is collected. Measurements were taken to obtain further information about the effects of luciferol. Negative control and 0.15% (w / v) cholecalciferol doses showed no or minimal change in behavior. He was incited. This is consistent with weight gain suggested by healthy food and water intake. 9%( Significant changes in behavior were observed at w / v and 20% (w / v). However, during the first day... Only slight changes were observed in between, followed by a rapid deterioration. 9% (w / v) choleca Luciferol doses showed a 100% mortality rate within 3 days. (1.5% w / v) At cholecalciferol doses, moderate changes in behavior were observed, but in the analysis of mean body weight... A weight gain was observed five days later, suggesting that the animal was beginning to recover. Due to the apparent toxicity demonstrated within this dose range, the formulation is classified as GHS Category 2 (OECD). Guideline 434 - Proposal for a new draft guideline 434 new draft guideline 434)-Acute skin toxicity-Dose fixation method) It is classified as such.

[0156] (D) Discussion Previous reports on the in vivo delivery of cholecalciferol indicated that ethanol was used. It has been suggested that it is an effective penetration enhancer and carrier substance (Agnew) ), WR (2010), “Topical pesticide formula "ion", International Publication No. 2010 / 071450 A1, World Intellectual Property Organization; Agnu -(Agnew),WR(2011),“Topical pesticide f "ormulation", U.S. Patent Application Publication No. 2011 / 0257135 A1, United States (Patent). The use of ethanol is because cholecalciferol is "easily soluble" in organic solvents (Br itish Pharmacopoeia.(2012), “Colecalcifer "ol" (British Pharmacopoeia) is logically sound, and large amounts of cholecalciferol are delivered in the formulation. It is possible. High concentrations of cholecalciferol create a high diffusion gradient across the dermis. This promotes transdermal delivery. However, these reports are not applicable to rats of unknown body weight. This is based on findings from applying it alone. Therefore, the published data is based on EP. To justify the effectiveness of the PO Guidelines (EPPO, 1998), that is, the Directive. It is not sufficient.

[0157] Furthermore, in order to investigate the transdermal delivery of cholecalciferol as a rodenticide, the formulation was screened. We developed two in-vitro models for leaning. In both models, first The apparatus described in the published research was used. As a cellulose tube model, (Alton) (Aulton),ME(2007),“Aulton's Pharmaceut ics: The design and manufacture of medicine ines”,Edinburgh;New York,Churchill Livin gstone; Barry, BW (1983), “Dermatolog ical formulations”, pp.49-94, New York, NY, Use first what is listed in Marcel Dekker Inc., then, Further testing was conducted using a diffusion cell apparatus, as has been used in several studies. When using the Rose tube model, cholecalciferol in DMSO / ethanol cosolvent (10% w / v cholecalciferol) vs. 0.25+ / -0.019 mg / c m 2 The drug flux of h was achieved. The eta proposed by Agnew Nol preparations (Agnew, WR (2010), “Topical p “Esticide formulation”, International Publication No. 2010 / 071450 A No. 1, World Intellectual Property Organization; Agnew, WR (2011), “To “Pical pesticide formulation”, U.S. Patent Application Publication No. 20 11 / 0257135 A1 (US Patent) was also tested using this model. 0.13 + / -0.0035 mg / cm 2 Drugs with low h (10% w / v cholecalciferol) It became a flux. However, when these formulations were tested in a diffusion cell model, Contrary results were observed (at a 20% (w / v) cholecalciferol concentration, 90:10v / vDMSO / ethanol: 3.46+ / -0.033 mg / cm³ 2 h drug flush For oxalic acid and ethanol: 2.57 + / - 0.16 mg / cm³ 2 h drug flac (S). In the diffusion cell model, two cholecalciferol concentrations (20% and 40% w / Regarding v), the drug flux of ethanol preparations compared to DMSO / ethanol cosolvents This suggests that it is higher. The differences between the models are probably due to the diffusion area. The cellulose tube model is 40cm 2 It has a diffusion area, while the diffusion cell model is small 2.54cm 2 It has an area of ​​. DMSO diffuses due to membrane disturbance and increased pore size. To promote the diffusion. The increased diffusion area within the cellulose bag is sufficient to distinguish the DMSO formulation. However, the diffusion cell model is insufficient. Alternative driving mechanisms are needed within the diffusion cell model. This may be present and could be causing inconsistent results between models.

[0158] In addition to in-vitro data, enable any model and the necessary information according to the instructions. In vivo studies were also conducted to obtain experimental efficacy data. (EPPO Guidelines) (P1 / 113(2))(EPPO(1998), “Efficacy evaluation ion of rodenticides Laboratory tests for evaluation of the toxicity and acceptability ility of rodenticides and rodenticide pr "eparations" (PP1 / 113(2)) is considered to be when the rodenticide is considered "sufficiently effective". For this to be possible, a 100% mortality rate must be observed in the screening protocol. They state that they do not. The screening protocol involves DMSO as a cosolvent and an ethanol preparation. To compare them and to determine whether there is evidence for further research on these formulations. It was helpful for both. Ethanol containing 20% ​​and 40% (w / v) cholecalciferol. The formulations showed mortality rates of 20% and 60%, respectively, during the screening protocol. On the other hand, all formulations containing DMSO have a 100% mortality rate (90:10(v / v )DMSO / ethanol, 70:30(v / v)DMSO / ethanol and 90:10 (v / v)DMSO / oleic acid) was shown. All formulations were lethal within 5 days of application. This caused a moderate change in behavior. According to EEPO guidelines, ethanol-based Because the formulation does not produce sufficient lethality, it is not intended for use as a rodenticide. These studies and Agnew's research (Agnew (Agnew),WR(2010),“Topical pesticide fo "rmulation", International Publication No. 2010 / 071450 A1, World Intellectual Property Association Seki; Agnew, WR (2011), “Topical pesti "Cide formulation", U.S. Patent Application Publication No. 2011 / 0257135 The difference from A1 (US Patent) may lie in the differences between rodent species. In this test, body weight They use Sprague Dawley rodents weighing 250-350g, while A Agnew's patent describes a Norway rat (Ra) of unknown weight. Although *Ttus norvegious* is used, Norway rats are generally Body weight is lower than in the experimental group. The result is a 20% (w / v) cholecalciferol dose. This means it would be more powerful against rodents with less body weight. The in vivo results from the cellulose tube in vitro model also showed that This suggests a closer correlation with n-vivo results.

[0159] The second protocol is based on OECD Guideline 420 (OECD (2001), OECD Based on the dose-fixing method shown in Guideline 420 - Acute Oral Toxicity - Dose-Fixing Method The study was conducted using four different doses of cholecalciferol. The drug is designed for both determining the GHS category to which it belongs and determining its efficacy at low doses. Low doses of 0.15% (w / v) cholecalciferol showed no evidence of toxicity and negative effects. The same applies to light, so in reality, a substantial amount of cholecalciferol is responsible for the lethal effect. This suggests that the obvious toxicity is observed in 15% (v / v) PEG200 at 50:50 (v / v) 1.5% (w / v) cholecalciferol in 1 ml of DMSO / ethanol This was demonstrated in rodents administered with the drug. This indicates that it falls under acute cutaneous toxicity GHS category 2 (OECD). Guideline 434) (OECD (2004), OECD Guideline 434 - New Guide This suggests a proposal for line proposal 434 (acute skin toxicity - dose-fixed method). 9% The fact that a 100% mortality rate was observed with (w / v) cholecalciferol doses indicates that this specific situation is particularly relevant. This suggests that the formulation is sufficiently effective for use as a commercial rodenticide. A 100% mortality rate was shown within 3 days at this specific dose, but this was a screening trial. The results were faster than those shown. However, the pain score deteriorated rapidly in rodents 24 hours after administration. This suggests that...

[0160] In vivo studies have shown that a cellulose tube in vitro model can be used for comparing formulations. This suggests that it shows more accurate results. The reason is that this model and in-viv The study suggests that DMSO has a higher drug flux and is the most effective. Because it demonstrates this.

[0161] DMSO and oleic acid have not been previously tested together with cholecalciferol. However, these are related to compounds such as anti-inflammatory drugs (Gwak, H. S., & Chun, IK (2002), “Effect of veh icles and penetration enhancers on the i n vitro percutaneous absorption of tenox icam through hairless mouse skin”,Intern ational Journal of Pharmaceutics,236(1-2 ), 57-64; Larrucea, E., Arellano A., Santoyo, S., & Ygartua, P. 2001), “Combined effect of oleic acid and propylene glycol on the percutaneous pe netration of tenoxicam and its retention in the skin”,European Journal of Pharma Ceutics and Biopharmaceutics,52(2),113-9 Meshullam, Y., Kadar, T., Wenniger (Wengier), A., Dachir, S., & Levy , A. (1993), “Transdermal penetration of ph ysostigmine:Effects of oleic acid enhanc er”,Drug Development Research,28(4),510- 515; Moreira, TS, de Sousa, V .P.,& Pierre, MBR (2010), “A novel transdermal delivery system for the anti -inflammatory lumiracoxib:influence of o leic acid on in vitro percutaneous absor ption and in vivo potential cutaneous ir ”,AAPS PharmSciTech,11(2),621-9) It has been shown to improve penetration (Williams, AC, & Barry, BW (2012), “Penetration Enh ancers”,Advanced Drug Delivery Reviews,6 4,128-137). DMSO has been proven to be a penetration enhancer, but it does not penetrate the skin. Due to the incidental effect of producing an unpleasant taste in the mouth during use, which reduces its use, It is not used in this application. This may be a point to consider in many transdermal delivery applications. However, when it comes to its use as a rat poison, the time it takes to kill and the humaneness of the formulation are the priorities. In this regard, both the transmission test and the in vivo results indicate that cholecalcifer This suggests that it is the most effective penetration enhancer investigated for rolls, and further investigations are needed. This justifies carrying out the attack.

[0162] Finally, these results suggest that transdermal delivery of cholecalciferol is an anticoagulant-based feeding method. This suggests that alternative methods may be available. Feeding methods have been used successfully. However, this method relies on the rodent ingesting a lethal amount of food. This feeding is not always successful. It cannot be said that it is effective, and it may cause resistance to anticoagulants. Transdermal application for a single dose is This could be a more efficient way to deliver toxins to the skin: the optimized minimum dose can reach the environment. This could reduce wasteful and unnecessary exposure. The difficulty in this effort lies in the application of rodenticides. However, devices that can be used for skin delivery have been proposed (Goode, S). .L.(2010), “Vertebrate Trap”, International release No. 2010 / 10 (6352 A1, World Intellectual Property Organization). Therefore, considering this point, Korecal Transdermal delivery of cyferol is a feasible alternative to anticoagulant feeding.

[0163] (E) Conclusion The objective of this research is the transdermal delivery of cholecalciferol for use as a rodenticide. The objective was to identify appropriate chemiosmothers to promote this process.

[0164] Through both in-vitro and in-vivo studies, 9% (w / v) of this compound was found. 1 ml dose containing calciferol: 50:50 (v / v) DMSO / ethanol 15 %(v / v), PEG200 medium (257 mg / kg per 0.350 kg rat) (equivalent to a certain quantity) based on the efficacy experiment guidelines suggested in Directive 98 / 8 / EC It has been proven to be sufficiently effective. This dose, when applied to 5 animals, lasts for 3 days. It resulted in a 100% mortality rate within the population. Administration involves applying 1 ml of the formulation to the nape of the animal's neck. This research proposes an alternative method to the common method of using rodenticide diets containing anticoagulants. To provide.

[0165] In parallel with experimental efficacy evaluations, dose-fixed methods are also used to determine the GHS classification of the formulation. This was done to achieve obvious toxicity, and the dose of 1.5% (w / v) cholecalciferol was 1 ml. The classification was determined based on the five animals to which the method was applied, and they were classified as Category 2.

[0166] Prior to in-vivo research, two in-vitro models were developed using protein formulations. Developed for screening purposes. The cellulose tube model is DMSO / ethanol soluble. The medium suggests that cholecalciferol promotes high flux, while the diffusion cells The model suggested that the ethanol formulation increased flux. Cellulose tube model The data showed a higher correlation with in-vivo data, suggesting a more accurate pattern. It can be done.

[0167] This study classified transdermal delivery of cholecalciferol as sufficiently effective based on the instructions. This provides an alternative to anticoagulant dietary methods that differ in both active ingredients and delivery methods. This suggests that...

[0168] <Example 4 - Alternative Toxic Substances> To demonstrate the degree of the ability to promote delivery through synthetic films by using the compositions of the present invention, various For alternative toxic substances, in-vitro experiments corresponding to Example 2 were conducted. The toxic substances identified were the anticoagulant warfarin and diphenacome.

[0169] The results are shown in Figures 15 and 16 and Table 9 below.

[0170] [Table 10]

[0171] The above results suggest that warfarin exhibits the highest drug flux. However, Rufarin is most effective in small daily doses.

[0172] <Example 5 - Example of a delivery device> Regarding a delivery device for delivering the composition of the present invention to a target animal for killing the target animal, Actual examples of these devices are shown in Figures 17-22 of the attached drawings. These devices are the inventions of the inventors. Vertebrates disclosed in the previous international patent application, International Publication No. 2010 / 106352 These correspond to some exemplary embodiments of the trap, but are provided only as non-exclusive examples in this application. To quote and refer to: a certain dose or amount of a pest-killing or animal-killing composition (or other poison) Any known delivery device that acts by delivering a composition containing a sexually transmitted drug to an animal. Alternatively, a rodent (or other animal) trap may be used to deliver the composition of the present invention in a similar manner. It should be understood that it can be used in the corresponding format.

[0173] Figure 17 shows a vertebrate trap, that is, in this embodiment, an enclosure 2 and a pressurized propellant or The diagram shows a rodent trap 1 including a carrier gas containment 3. The containment 3 is further provided according to the present invention. It contains a supply of animal-killing composition and is applied to the surface of animals entering enclosure 2. , and are prepared to be delivered into enclosure 2. Enclosure 2 has a first open end 4 and a second open end It includes a hollow tubular member having 5. The enclosure 2 is supported by supports 7 and 8, and the base 6 It is mounted on top.

[0174] The gas containment unit 3 is mounted on the enclosure 2 by clips 10 fixed to the enclosure 2. The containment unit 3 includes the nozzle 11. The nozzle 11 is connected by a connecting element 13 to a conduit. Alternatively, it is connected to tube 12. The connecting element 13 is, for example, a screw connection or The nozzle 11 is detachably connected by appropriate connection means.

[0175] The containment vessel 3 is equipped with a pressure sensor 17 that can sense the gas pressure inside the containment vessel 3, and a base and control A radio signal indicating the gas pressure of the containment unit 3 can be transmitted to a station or monitoring station (not shown). It further includes a wireless transmitter 18, provided that any suitable form of sensor and wireless device Please understand that it may be used.

[0176] Enclosure 2 further includes a GPS receiver 19 in combination with a wireless transmitter 20, The transmitter 20 then transmits a radio signal indicating the location of the trap to the base, control station, or monitoring station. Again, any alternative and appropriate localization means and associated transmitters may be used. I hope you understand this.

[0177] Regarding Figure 18, this figure shows a partial cross-sectional view of the trap enclosure 2 in Figure 17, and the conduit 12 It is connected to the discharge chamber 14 located below enclosure 2. The discharge chamber 14 is It communicates with the inside of the enclosure 2 via vent 15. The vent 15 is pressurized. Multiple (a plurality) that can pass the contents through the chamber 14 to the enclosure 2. Includes a laminated member having holes (ty or). Chamber 14 is the chamber for the contents of the containment 3. It also houses a discharge section 16 that controls the flow to 14. The discharge section 16 is an activation device (not shown) It is connected to an actuator (not shown) that moves in response to instructions received from (Zu). Enclosure Furthermore, the system includes a detector 21, which is a motion sensor that detects the presence of vertebrates such as rodents. It also includes the following. However, please understand that any suitable detector may be used.

[0178] The trap described above or the composition delivery devices shown in Figures 17 and 18 can be modified in various ways. For example, The tubular member of enclosure 2 has a curved central section located between its first open end and second open end. It may have parts. An example of such a configuration is shown in Figure 22, which will be discussed below. The details of the construction form are shown. In another modified example, one of the tubular members of enclosure 2 or (preferably) (i) Both open end portions extend from one side of the end portion to the opposite side. This may include the upper barrier bars 31, 32. Such barrier bars may be, for example, at the end of a tubular member. It is installed in a pore that extends through the wall of the part. The barrier bar is preferably a tooth movement The enclosure is positioned in such a way that it is practically impossible for an animal larger than the object to enter enclosure 2. Examples of such configurations using barriers like the one shown below are shown and discussed in Figure 19. The details of the embodiment shown in Figure 22 are also shown.

[0179] Figures 19 and 20 show a second embodiment of the rodent trap 1. The same reference figures are used for both. The embodiments are used for the same features. In the second embodiment, pressurized The gas containment unit 3 is received in a port 50 formed within the base 6. It includes a screw hole for securely receiving the screw-type nozzle 11 of the containment 3. Conduit or Tube 12 extends from port 50 to discharge chamber 14 (hidden in these diagrams) (is present). Furthermore, in this embodiment, the vent 15 is located within the wall of enclosure 2, and the olive Replacement by discharge orifice 51 connected to discharge chamber 14 by discharge conduit 52 Barrier bars 31 and 32 extend vertically and are located adjacent to each other at each end of enclosure 2. It is.

[0180] In the third embodiment shown in Figures 21 and 22, the base 6 is replaced with the operating housing 70. The housing is equipped with a port 71 for receiving the nozzle 11 of the containment unit 3. In this configuration, port 71 includes a screw hole that engages complementarily with the screw-type nozzle 11 of the storage container 3. The housing 70 encloses the contents of the container (including the animal-killing composition according to the present invention) and 2 It includes a channel (not shown) for transferring to the discharge chamber below.

[0181] When used, the storage container 3 containing the composition of the present invention is connected to the connecting element 13 or port 50 Alternatively, it is connected to one of the 71. The trap control device periodically activates the release unit. Then, a certain amount of the composition is released from the containment 3 into the enclosure 2, into the release chamber 14 and vent. It is mounted to be released via 15 or the orifice 51. For example, When the composition contains an attractant or pheromone component, the component is preferably air-transmitting. Therefore, it can be emitted from enclosure 2 into the surrounding air to attract rodents into the trap. Alternatively or additionally, some other suitable forms of bait can be used to lure rodents into the trap. It can be placed inside an enclosure.

[0182] When a rodent enters enclosure 2, the first sensor is activated and a signal is sent to the control device. If the first sensor continues to operate, the second sensor is activated, and the control unit moves the discharge unit. The control device then causes the animal-killing composition to flow from the pressurized containment container 3 into the enclosure 2. The release portion is released for a predetermined period of time equivalent to a dose sufficient to lethally poison a rodent. It is installed to operate. Once the predetermined time has elapsed, the discharge part returns to its original position and guides The pipe 12 is closed, preventing further composition 2 from entering the enclosure 2. The control device is located After a set amount of time has elapsed, the system will again act on the signals received by the first and second sensors. It is programmed to do so. This gives the rodent time to leave the trap and kill it. This prevents the delivery of more toxic components of the composition to the composition than is necessary for the wound to occur.

[0183] As described above, various alternative types, configurations, and arrangements of the above-mentioned delivery devices are in line with the present invention. Furthermore, the delivery or method or use of the composition in various embodiments in various aspects thereof. It can be used for the implementation of its intended purpose.

[0184] The above describes preferred embodiments, features, and aspects of the present invention, as well as illustrative examples thereof. These are merely non-limiting examples, and various modifications are described and discussed in detail in those embodiments. , that can be done from the features and manner, and on the other hand, that variations thereof may also be made, as per the attached claims. It should be understood that this falls within the scope of the present invention as defined by [the relevant definition].

Claims

1. Targets selected from the group consisting of rodents, marsupials, rabbits, and mustelids. A liquid composition for killing or injuring harmful animals, (i) an amount of 0.001% to 90% by weight relative to the weight of the composition, the target pest A toxic component comprising one or more toxins toxic to animals, wherein a certain dose of the composition The substance is sufficient to kill or injure the target pest animal when applied to its skin. The amount of the toxic component present in the aforementioned composition, (ii) Carrier system for the toxic component and Includes, Here, the carrier system (ii) is (a) Alcohol, glycol, glycol ether, glycerol formal, poly Ethylene glycol, liquid polyoxyethylene glycol, pyrrolidone, acetone, acetone Selected from one or more compounds among tonitriles, amides, and phthalates, A liquid solvent for the toxic component in an amount of 1% to 99% by weight relative to the weight of the carrier system. or a dispersion medium, (b) The passage of the toxic component into and / or through the skin of the target pest animal An amount of 20% to 90% by weight relative to the weight of the carrier system in order to promote or enhance One or more substances selected from the group consisting of sulfoxides, amides, hydrocarbons, ketones, and ethers. Skin-disrupting ingredients, A composition characterized by containing the following:

2. The composition according to claim 1, wherein the toxic component (i) is substantially water-insoluble. A composition characterized by the following.

3. A composition according to claim 1 or 2, wherein the one or more toxin agents are cholecalcife Roll, 25-hydroxycholecalciferol, vitamin D ３ , calciferol, e Lugocalciferol, anticoagulants, metal phosphides, α-naphthylthiourea, arsenic compounds, Barium compounds, thallium compounds, bromethalin, chloralose, crimidine, 1,3- Difluoro-2-propanol, endrin, fluoroacetamide, fosacetim, white limonyl phosphate N, pyrinulone, siriloside, sodium fluoroacetate, strychnine, tetramethyl Disulfotetramine, hydrogen cyanide, sodium cyanide, potassium cyanide, and A composition characterized by being selected from one or more toxins of bacteria or viruses.

4. The composition according to claim 3, wherein the one or more toxin agents is cholecalciferol, 25-Hydroxycholecalciferol, Vitamin D ３ warfarin, diphenacome A composition characterized by being selected from one or more of the following.

5. A composition according to any one of claims 1 to 4, wherein the toxic component (i) is the set A composition characterized by being present in the composition in an amount of 0.01% to 75% by weight of the product. thing.

6. The composition according to claim 5, wherein the toxic component (i) is 0.1% by weight of the composition. A composition characterized by being present in the aforementioned composition in an amount of ~70% by weight.

7. The composition according to claim 6, wherein the toxic component (i) is 1% to 6% by weight of the composition. A composition characterized by being present in the aforementioned composition in an amount of 0% by weight.

8. A composition according to any one of claims 1 to 7, The concentration of the toxic component (i) in the composition is such that a single dose of the composition is sufficient to reach the target pest animal. An amount of the poison sufficient to be lethal to an object, but not exceeding the minimum lethal dose by a large margin. A composition characterized by being selected to contain physical components.

9. A composition according to any one of claims 1 to 8, wherein the skin disturbance component (b) is 1 A composition characterized by containing one or more sulfoxides.

10. The composition according to claim 9, wherein the skin disturbance component (b) is dimethyl sulfoxide A composition characterized by containing (DMSO).

11. A composition according to any one of claims 1 to 10, wherein the skin disturbance component (b) is The composition contains an amount of 30% to 80% by weight relative to the weight of the carrier system (ii). A composition characterized by the following.

12. The composition according to claim 11, wherein the skin disturbance component (b) is the carrier system (ii) A composition characterized by being present in an amount of 40% to 70% by weight relative to the weight of [the substance].

13. A composition according to any one of claims 1 to 12, wherein the solvent of the carrier system (ii) The medium or dispersion medium (a) is 10 or 25% by weight to 6% of the weight of the carrier system (ii). A composition characterized by being present in an amount of 0 or 70% by weight.

14. A composition according to any one of claims 1 to 13, comprising one or more pheromones or A composition characterized by further comprising other attracting compounds.

15. A composition according to any one of claims 1 to 14, Up to 10% by weight relative to the total weight of the composition (i) Skin penetration enhancers, (ii) Wetting agent, (iii) Viscosity modifier, (iv) cosolvent, (v) One or more emulsifiers, stabilizers, preservatives, emollients, fragrances, colorants, or pigments pH adjusters, gel-forming agents, foam-forming agents, and other additional substances selected from pharmaceuticals, (vi) A delivery-enhancing component selected from one or more aerosol propellants, A composition characterized by further comprising one or more additional components selected from any of the following. 。

16. Targets selected from the group consisting of rodents, marsupials, rabbits, and mustelids. A method for killing harmful animals, wherein the liquid composition described in any one of claims 1 to 15 is used. A method characterized by including delivery to the skin of the target pest animal.

17. One or more toxins are used in a group consisting of rodents, marsupials, rabbits, and weasels. A device for killing target pest animals by delivering it to the skin of selected target pest animals. It is placed, (i) an enclosure into which the target pest animal can enter, (ii) A storage container containing a supply of the liquid composition according to any one of claims 1 to 15 Instruments and means, (iii) A delivery means for delivering a lethal dose of the liquid composition to the skin of the target pest animal. and, An apparatus characterized by including

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