A water-soluble afoxolaner complex, a method for producing thereof and veterinary antiparasitic drugs comprising the same

Abstract

The claimed group of inventions relates to a field of veterinary medicine and concerns a water-soluble supramolecular complex with afoxolaner, a method for production thereof, and veterinary drugs comprising said complex as a main active component to prevent or to treat parasite infections in animals. The inventive complex is a fine powder having a particle size of less than 1 micron that is an inclusion of afoxolaner molecules into the structure of 2-hydroxypropyl-β-cyclodextrin, stabilized by polysorbate 80 and may be used in veterinary drugs having antiparasite effect for effective oral and parenteral administration.

Description

[0001] A WATER-SOLUBLE AFOXOLANER COMPLEX, A METHOD FOR PRODUCING THEREOF AND VETERINARY ANTIP ARASITIC

[0002] DRUGS COMPRISING THE SAME

[0003] FIELD OF THE INVENTION

[0004] The claimed group of inventions relates to a field of veterinary medicine and concerns a water-soluble supramolecular complex with afoxolaner, a method for production thereof, and veterinary drugs comprising said complex as a main active component to prevent or to treat parasite infections in animals caused by ectoparasites and / or endoparasites.

[0005] Afoxolaner belongs to a class of isoxazolines, it is used to treat and to prevent flea infestation, as well as to treat and to control various ixodic. infestations.

[0006] Isoxazoline compounds are known in this field of art, and their production and use as antiparasitic agents are described, e.g., in international applications W02005085216 dated 17.01.2008, W02009002809 dated 31.12.2008,

[0007] W02009024541 dated 26.02.2009, W02009003075 dated 31.12.2008,

[0008] W02010070068 dated 24.06.2010 and W02010079077 dated 15.07.2010.

[0009] It is known that this class of compounds has distinctive properties of activity against parasite insects and acarides, such as fleas and mites.

[0010] A particularly active compound is isoxazoline compound, 4-[5-[3-chloro-5- (trifluoromethyl)phenyl]-4,5-dihydro-5-(trifluoromethyl)-3-isoxazolyl]-N- [2-oxo- 2-[(2,2,2-trifluoroethyl)amino]ethyl]-l-naphthalene carboxamide, which is known under a non-patented name afoxolaner and described in the application W02007079162A1 dated 12.07.2007. However, the efficiency of use of isoxazoline compounds, in particular, afoxolaner, in a pure form is limited by its bioavailability, since it is not water-soluble and its pure form cannot be delivered to body tissues by means of a water-based blood, but it rather forms regular solutions only in organic solvents, thereby making it necessary to include additional excipients to pharmaceutical compositions based thereon, and the excipients are usually belong to highly toxic compounds.

[0011] An alternative approach may be to use dispersions of afoxolaner with various solubilizers and surfactants to transport the active substance to the body tissues. However, presently known compositions facilitate formation of dispersions having a particle size of more than 10 microns, thereby affecting the bioavailability of the active component.

[0012] The application WO2013119442A1 dated 15.08.2013 teaches parasitocidal oral veterinary compositions comprising systemically-acting active agents being at least one isoxazoline active agent, in particular, afoxolaner, in combination with a pharmaceutically acceptable carrier. According to this solution, in order to provide effective delivery of the active agent by oral administration, it is introduced into a solid or a liquid matrix, where, in order to achieve matrix characteristics, ingredients are used, such as, e.g., hydroxypropyl starch or hydroxypropyl cellulose, while surfactants, in particular, polysorbates, are used as a matrix complex stabilizer. This invention proposes enhanced methods for eliminating, controlling and preventing parasite infections and infestations in animals, the methods comprise oral administration of the inventive composition to the animal in a need thereof. However, the compositions being used facilitate formation of dispersions having a particle size of afoxolaner of more than 10 microns, thereby affecting the bioavailability of the active component. Besides, use of these compositions for parenteral administration is problematic.

[0013] The application WO2019091936A1 dated 16.05.2019 teaches pharmaceutical compositions of isoxazoline for injections and their use against parasite infestation, where various suspension agents and lubricants are used to obtain dispersions having active substance inclusions. Carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum etc. are selected as excipients for production of aqueous suspensions. Natural phosphatides, e.g., lecithin, or products of condensation of alkylene oxide with fatty acids, e.g., polyoxyethylene stearate, or products of condensation of ethylene oxide with long-chain aliphatic alcohols, e.g., heptadecaethyleneoxycetanol etc., are selected as dispersing agents or lubricants. Their use in the complex with the compound facilitates their solubilization, while in order to stabilize the obtained solubilized comples, a surfactant is used, and the surfactant is selected from sorbitol fatty acids ethers (Spans), polyoxyethylene sorbitol fatty acids esters (polysorbates / Tweens), castor oil polyoxyethylene derivatives (Cremaphors), polyoxyethylene stearates or lecithines.

[0014] The obtained composition is a stable complex of isoxazoline compound, in particular, afoxolaner, having a particle size from about 25 microns to about 250 microns, that is used to create injection compositions having an antiparasitic effect. At this particle size of the active agent in the suspension or in the injection solution, the effective dose of afoxolaner in the drug is from 5 to 10 mg / kg.

[0015] Therefore, at present, there is a need to provide a water-soluble afoxolaner composition having a reduced particle size in order to increase its bioavailability, thus, also to reduce the effective dose and amount of the active agent.

[0016] SUMMARY OF THE INVENTION

[0017] The claimed invention attempts to provide a novel supramolecular afoxolaner complex having an increased water solubility and enhanced stability, as well as to develop veterinary drugs comprising this complex which have a high chemical and physical stability and a reduced toxic effect, and which are suitable both for oral and parenteral administration. With regard to components of the proposed complex, the following is known.

[0018] Upon development of the claimed complex composition, the problem of afoxolaner solubility has been resolved by making an attempt to include cyclodextrin into its formulation. Cyclodextrines are cyclic oligosaccharides comprising 6 (a-cyclodextrin), 7 ( -cyclodextrin) or 8 (y-cyclodextrin) glucopyranose units. Structural organization of a molecule of cyclodextrines is such that it comprises an individual external hydrophilic portion that increases water solubility and an individual internal portion that forms a cavity. Hydrophobic areas of most of molecules are able to penetrate into the hydrophobic cavity, thereby forming so-called “inclusion complexes”. Therefore, use of cyclodextrines and derivatives thereof in most cases provide a significant increase of solubility of drugs. Besides, it is believed that the most important result that is achieved by the presence of cyclodextrines in pharmacological formulations is increase of the bioavailability of active components which allows to use them in concentrations lower than usual ones, which is essential both in terms of additional reduction of the drug toxicity and in terms of a possible reduction of its price.

[0019] In order to create the novel complex, 2-hydroxypropyl- -cyclodextrin is selected, since it is well studied, non-toxic for mammals and commercially available, and its pharmacological use is disclosed, particularly, in publications (Loftsson, T. and Brewster M E, “Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization.” Sd J Pharm, 1996, 85 (10). P. 1017-25. ; Abdel-Rahman, S M, et al., “Single-dose pharmacokinetics of intravenous itraconazole and hydroxypropyl-beta-cyclodextrin in infants, children, and adolescents. Antimicrob A”).

[0020] Polysorbate surfactants are used to stabilize the complex and to minimize its segregation and sedimentation of the active agent. It is known that the polysorbate surfactants demonstrate advantageous characteristics for isoxazoline compositions and upon contact with water, they are able to create micelles around the solubilized compound, thus, to maintain the solubilized compound in the aqueous medium.

[0021] In order to develop the claimed complex, polysorbate 80 is used that is known as Tween 80 and that is commercially available or may be obtained by methods which are known in this field of the art.

[0022] Therefore, the claimed invention is based on a task to develop an inclusion complex with afoxolaner that is based on use of 2-hydroxypropyl-P-cyclodextrin as a molecular packer of hydrophobic afoxolaner molecules, while polysorbate 80 is used as the stabilizer of the complex.

[0023] According to the invention, a supramolecular complex in a form of a finely dispersed powder is obtained, the powder having a particle size of less than 1 micron and characterized by the following composition of components (wt.%): afoxolaner - 1 - 10%,

[0024] 2-hydroxypropyl-P-cyclodextrin - 50-98%, polysorbate 80 - 1-40%, which makes said complex suitable both for preparing solutions for instillation use and / or injection administration and for forming solid forms.

[0025] The claimed complex may be obtained only under strict adherence to the Applicant’s method for production thereof which constitutes a subject matter of the claimed invention as well.

[0026] A method for producing the inventive complex comprises the following steps of: mixing afoxolaner solutions and 2-hydroxypropyl-P- cyclodextrin , while fulfilling a mass ratio in the solutions 2-hydroxypropyl- P-cyclodextrin / afoxolaner of 1 40 storing the mixture of solutions at a room temperature during at least 24 hours adding a polysorbate 80 solution to the stored mixture of the solutions at a mass ratio polysorbate 80 / mixture of solutions of afoxolaner and 2-hydroxypropyl-p-cyclodextrin of 1- 0; drying the obtained mixture until a solvent is completely evaporated and a dry residue is obtained.

[0027] In a further aspect of the claimed invention, a veterinary antiparasitic drug is claimed, the drug comprises said supramolecular complex of afoxolaner and suitable fillers and / or binder and / or preservatives in ratios which are suitable for forming solid forms, in particular, as dragees, tablets, powder, capsules etc.

[0028] According to an exemplary embodiment, the veterinary drug may further comprise excipients, such as antioxidants and / or food additives having meat flavor and taste. At the same time, the complex content in a single solid form is selected such that it comprises 2-4 mg of afoxolaner per 1 kg of the animal’s body weight.

[0029] A below-mentioned table provides an exemplary solid dosage form comprising the supramolecular complex of afoxolaner for preventing or treating parasite infections and infestations in animals caused by ectoparasites.

[0030] Table 1 Formulation of the oral tablet with the supramolecular complex of afoxolaner, based on 100 mg of the formulation

[0031] As it can be seen from above, the weight of the complex is almost one half of the tablet weight, and the afoxolaner content in the complex is about 2.5 mg which is optimal and sufficient dose per 1 kg of animal weight for achievement of the antiparasitic effect.

[0032] According to a further aspect of the claimed invention, a veterinary antiparasitic drug is claimed, the drug comprises said supramolecular complex of afoxolaner and is a colloidal solution in an isotonic medium comprising a weight fraction of afoxolaner in a range of 0.05 0.5 %. The isotonic medium may be 9% sodium chloride solution or 5% glucose solution. Said solvent may be used both for injections and for parenteral use, in particular, for drip skin application.

[0033] In order to enhance the therapeutic effect of afoxolaner and to enlarge the spectrum of management of parasites with one dosage form, another aspect of the invention is a veterinary drug being a solid dosage form that further comprises one or more active anthelminthic agents. According to the invention, said veterinary antiparasitic drug comprises a composition of the supramolecular complex of afoxolaner with an anthelminthic drug at a mass ratio of anthelminthic drug / afoxolaner complex of 1- 0, while providing the afoxolaner content therein of 2-4 mg based on 1 kg of animal body weight. Preservatives and / or antioxidants and / or food additives may be used as the excipients in ratios which are suitable to form solid forms, in particular, as dragees, tablets, powder, capsules. Milbemycin oxime or pyrantel or praziquantel or febantel or combinations thereof is / are used as the anthelminthic drug.

[0034] A below-mentioned Table 3 provides an exemplary solid dosage form comprising afoxolaner and anthelminthic drugs for preventing or treating parasite infections and infestations in animals caused both by ectoparasites and endoparasites.

[0035] Table 2

[0036] Formulation of the oral tablet with the supramolecular complex of afoxolaner and anthelminthic active substances, based on 100 mg of the formulation A qualitative formulation of the tablet is provided below taking a 0.4 mg tablet as example:

[0037] Water-soluble complex of afoxolaner - 0.2 g

[0038] (content of afoxolaner in the complex ~ 0.01 g)

[0039] Milbemycin oxime - 0.003 g

[0040] Praziquantel - 0.017 g

[0041] Food additives, antioxidant, preservative and filler - the remainder.

[0042] IMPLEMENTATION OF THE INVENTION

[0043] Implementation of the invention and achievement of the technical effect are confirmed by the following examples.

[0044] Example 1.

[0045] The supramolecular complex is produced in the following way

[0046] Preparing initial solutions in 96% ethyl alcohol:

[0047] 1. solution of afoxolaner having a concentration of 2 g / 1;

[0048] 2. solution of 2-hydroxypropyl- cyclodextrin having a concentration of 400 g / 1;

[0049] 3. solution of polysorbate 80 having a concentration of 20 g / 1.

[0050] Pouring the solution 1 and the solution 2 into a glass vessel in such amounts so as to maintain the ratio of 2-hydroxypropyl-0-cyclodextrin / afoxolaner being 1-M0 in the solutions, and storing the vessel in a dark place at a room temperature for at least 24 hours.

[0051] After 24 hours of holding in the dark, an estimated amount of the solution 3 is poured into the vessel, while maintaining the ratio, and blending a mixture of alcohol solutions for at least 15 minutes.

[0052] Placing the obtained mixture to an evaporating flask of a rotary evaporator and evaporating the same in vacuo to dryness at a water bath temperature of 50°C until complete removal of the solvent. Taking the obtained powder of the formed water-soluble supramolecular complex out from the flask and placing it into a sealed container and storing at a temperature of 4 35 °C in a dark place.

[0053] This results in a dry white powder that is easily soluble in water, while forming sub-microdispersions. Example 2.

[0054] Solubility test of the supramolecular complex of afoxolaner.

[0055] To study the solubility of the obtained substance, a dynamic light scattering method in a water solution was used, and results thereof are shown in the Table 3.

[0056] Table 3

[0057] According to the results of dynamic light scattering of 5% of the water solution of the afoxolaner complex as produced by the claimed method, it has been found that hydrodynamic diameters of particles are in a range of from 0.082 to 0.683 microns, and the average value is 0.469 microns. This indicates that the obtained complex has a higher bioavailability as compared to known complexes having afoxolaner inclusions, where particles have a diameter of 10 and more microns. Example 3.

[0058] Study of the efficiency of a pharmaceutical drug based on the supramolecular complex of afoxolaner according to the claimed invention.

[0059] Mammals, in particular, dogs, may have a large number of external and internal parasites. Ixodidae are the most widespread ones, and they occur in Europe, particularly, in Ukraine as well: Ixodes ricinis, Rhipicephalus sanguineus, Dermacentor reticulatis. Also, Sarcoptidae (Otodectes cynotis, Notoedres cati, Sarcoptes canis) and Trombidiformes (Demodex spp., Cheyletiella spp..) pose a threat.

[0060] In order to establish insectoacaricidal and ovicidal efficiency of the claimed complex, studies in vitro and in vivo of the pharmaceutical drug action have been conducted with its content on said types of parasites.

[0061] The studies have been conducted in the veterinary medicine laboratory “NOVA PLUS” LTD in Kharkiv city, animal charity (city of Balakliia) and veterinary medicine clinics of Kharkiv.

[0062] 108 dogs aged from 5 months to 6 years and having a body weight of 5-40 kg were enrolled to the study. The animals were held in typical crates at air temperature of 24±1,5°C, relative humidity of 40-70 %, air movement of 0.2-0.5 m / sec. The animals were fed according to a regimen approved in the crate.

[0063] Study design:

[0064] 1) clinical examination of the animals in the crate of Balakliia city (Kharkiv region) and private veterinary medicine clinics (Kharkiv city), establishment of a preliminary diagnosis, sampling ectoparasites and endoparasites in dogs for laboratory test, continuous clinical observation of physiological condition of the study animals;

[0065] 2) microscopic studies in order to find pathogens of invasive diseases in the biological material, their identification, establishment of extensity and intensity of invasions (II) in dogs; 3) formation of study and control groups of animals;

[0066] 4) use of the drug individually and directly by the animal, holding the animals in the crate and veterinary medicine clinics, sampling the biological material for the laboratory test in vivo when affected by ectoparasite insects, Ixodidae and Sarcoptidae after 1, 2, 3, 7, 14, 21 and 28 days and conduction of pelage and skin inspection as well as when affected by helminths (nematodes, cestodes) after 7 and 14 days and study of animals’ feces. Determination of extensefficiency (EE) and intensefficiency (IE) of the drug.

[0067] 5) daily clinical examination of health of the study animals during the entire experiment.

[0068] Materials, instrumentation and reagents used: laboratory vessels, analytical weights, vials, magnifying glass, binocular magnifying glass, light and digital microscope, freezer, Petri dishes, mounting and cover glass slides, reagents for microscopic tests (10% NaOH solution, 70% alcohol, alcohol-ether, chloroform, rosin, castor oil), aspirators for collection of arthropods, flasks for collection of arthropods, sealed polyethylene small bags, tweezers and lancet for collection of mites, skin samples from the study animals, cover glass slides, overlapping glass slides, McMaster chamber, reagents for scatoscopy tests (ammonium nitrate solution having a density of 1.32 g / ml; saturated sodium chloride solution having a density of 1.2 g / ml).

[0069] Test 1. Determination of acaricidal activity of the drug in in vivo studies on dogs.

[0070] Dogs of various breed served as study objects, aged from 5 months to 6 years, body weight of from 5 to 45 kg; the animals are held in a housed manner, in crates.

[0071] Diagnosis of infestation with dermestids mites of Otodectes cynotis species was established based on clinical features and acarological study methods by means of microscopy of scrapes taken from dog ear areas affected by parasites. Therewith, a parasitological damage index was determined - intensity of invasion before and after the treatment, and the drug efficiency was determined according to regulations of the European Medicines Agency and the World Association for the Advancement of Veterinary Parasitology.

[0072] During clinical observation of dogs and acarological study, skin damages of internal part of ear auricles and external auditory passages by the dermestids mites of Otodectes cynotis species were determined.

[0073] The dogs were divided into two groups. In the study group of dogs, before use of the drug in a form of tablets, the average intensity of invasion with the mites of Otodectes cynotis species was 34.2±6.2 units per animal, while in the control group of dogs, the average intensity of invasion with the mites of Otodectes cynotis species was 30.6±6.8 units per animal.

[0074] The study drug in tablets was used in the study group of dogs at the otoacariasis invasion according to the Table 1 with consideration of the animal body weight and, thus, the tablet weight, while the drug was not used in the second control group.

[0075] Study data are provided in the Table 4.

[0076] Table 4

[0077] Evaluation of acaricidal activity of the drug during invasion of the mites of Otodectes cynotis species in dogs (M±m) It is seen from the data provided in the Table 4 that in the first day, the efficiency of the drug in the form of tablets for dogs in the study group of dogs was 59.75% at the average intensity of invasion with the mites of Otodectes cynotis species of 12.8±3.4 units per animal, while in the control group of dogs, the average intensity of invasion with the mites of Otodectes cynotis species was 31.8±6.7 units per animal.

[0078] At the second day of the test, the efficiency of the drug for dogs in the animal study group was 95.03% at the average intensity of invasion with the mites of Otodectes cynotis species of 1.6±0.2 units per animal.

[0079] At the third day of the test, during observation of the internal surface of the ear auricle of dogs and acarological laboratory test being a microscopy of scrapes taken from the ear auricle, the dermestids mites of Otodectes cynotis species were not found in the animal study group.

[0080] During acarological tests of the dogs, at 7th, 14th, 2 Ind and 28th days after use of the drug in the form of the tablet, no reinfestation of the dermestids mites of Otodectes cynotis species was found in the animal study group.

[0081] Results of the conducted tests indicate that the drug in the form of the tablet having the claimed afoxolaner content in the supramolecular complex has outstanding acaricidal activity relative to the dermestids mites of Otodectes cynotis species. Therefore, the drug for the dogs is effective against Sarcoptidae invasions in dogs.

[0082] Test 2. Evaluation of acaricidal activity of the drug at Ixodidae invasion.

[0083] Diagnosis of infestation with parasitiformes being ectoparasites of the Ixodidae family (Ixodes spp., Dermacentor spp., Rhipicephalus spp.) was established based on clinical features, acarological test methods being observation of skin and body hair coat for presence of Ixodidae. Therewith, a parasitological damage index was determined before the treatment and after the treatment (after 1, 2, 3, 7, 14, 21 and 28 days) and the efficiency of the drugs was determined according to regulations of the European Medicines Agency and the World Association for the Advancement of Veterinary Parasitology.

[0084] Upon observation of the animal body with a naked eye and by means of the magnifying glass, Ixodidae were found in 14 dogs: Ixodes spp., Dermacentor spp., Rhipicephalus spp. (images and nymphs) which were adhered. In the study group of dogs, before use of the drug in the form of the tablet, the average intensity of the Ixodidae invasion was 22.5±3.7 units per animal (Table 5).

[0085] In the study group of dogs with the Ixodidae invasion, the study drug in the form of the tablet was used according to the manufacturer’s recommendations (considering the animal body weight and, thus, the tablet weight).

[0086] It is seen from the data provided in the Table 5 that in the first day, the efficiency of the drug in the form of the tablet for dogs in the animal study group was 58.02% at the average intensity of the Ixodidae invasion of 10.2±2.6 units per animal.

[0087] Table 5 Evaluation of acaricidal efficiency of the drug in the form of the tablets at the Ixodidae invasion of Ixodes spp., Dermacentor spp., Rhipicephalus spp. in dogs

[0088] (M±m)

[0089] At the second day of the test, the efficiency of the drug in the form of the tablet for dogs in the animal study group was 93.76 % at the average intensity of the Ixodidae invasion of 1.5±0.3 units per animal. At the third day of the test, upon pelage and skin inspection of the dogs and acarological test, no mites of the species: Ixodes spp., Dermacentor spp., Rhipicephalus spp. (images and nymphs) were found in the animals of the study group.

[0090] During further animal observation and acarological pelage and skin inspection, at 7th, 14th, 2 Ind and 28th days after use of the drug in the form of the tablet, no Ixodidae were found in the study group of dogs.

[0091] At the same time, the obtained results indicate that the drug in the form of the tablets for dogs possesses outstanding acaricidal activity against parasitiformes. Duration of the protective action of the drug against Ixodidae of the species: Ixodes spp., Dermacentor spp., Rhipicephalus spp. (imagos and nymphs) in dogs was not less than 28 days.

[0092] Test 3. Evaluation of therapeutic efficiency of the drug in the form of a solution for external use in adult dogs and puppies.

[0093] Within the veterinary clinic in Kharkiv city, a clinical examination of dogs and puppies was conducted. Scratches were taken from various areas of the affected skin of 10 dogs aged from 3 to 5 years and from puppies aged from 8 to 10 months for isolating pathogens in laboratory conditions.

[0094] In adult dogs (n=5), scratches of deep layers of dry skin and samples of fell out hair were taken in the affected locuses for demodecosis test.

[0095] Mites Demodex canis were detected by microscopic methods. Invasion intensity was from 37 to 40 items per 1 cm2of the skin.

[0096] In puppies (n=5), mites Otodectes cynotis were detected in amount of from 7 to 11 items per single scratch.

[0097] When otoacariasis is suspected, scabs from internal surface of the ear auricles were taken for the test. In order to establish the otoacariasis diagnosis, a vital method by Pryselkova D.R. and a method by Alfymova A.V. were used. The obtained material was tested microscopically for the presence of mites: a mite body was flat, oval shaped, gray-yellow colored; ovicells were oval shaped under 2 mm, coated with a thin shell.

[0098] During conduction of the tests, the animals were subjected to manipulations according to existing documents which regulate organization of works using animals in experiments and compliance with principles of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986).

[0099] Acarological and entomological tests were conducted two times before prescription of the drug, on day 7, 15 after treatment with the drug.

[0100] In case of demodecosis in the animals, the scratches from the affected skin were collected after 28, 45, 56 and 78 days from the start of the treatment.

[0101] Besides, blood samples were taken from 5 test animals from the group before, on day 7 and on day 15 after start of the insectoacaricide treatment. The blood samples for hematological and biochemical tests were taken under fasted conditions from a subcutaneous forearm vein. In case of demodecosis, the blood samples were collected before, after 28, 45, 56 and 78 days from the start of the treatment.

[0102] Evaluation of a functional state of the organism of the test animals (before treatment) throughout the experiment was conducted with determination of clinical and biochemical parameters in the blood according to common methods. Values of the blood parameters of the animals with clinical features of the disease before start of the treatment were considered according to the above-mentioned schemes.

[0103] Results of the tests were processed statistically using a software package Microsoft Excel 2003 (for Windows XP), and a probability of the obtained results were evaluated according to the Student’s test.

[0104] Study on adult dogs aged from 3 to 5 years

[0105] Clinical studies of the drug in the form of a solution for external use by spotting were conducted on adult dogs in the veterinary clinic in Kharkiv. Before conduction of the experiments, anamnestic data of the dogs was collected, clinical material for the laboratory studies was taken and infection diseases were excluded.

[0106] In adult animals (n=5), demodecosis was detected in a squamosal form: the affected animals had circular, hairless areas of skin having a diameter of from 1 to 20 mm which were located on superciliary arches, forehead, nose, lips, limbs.

[0107] Demodecosis diagnosis was established in a complex way with consideration of the anamnestic data, clinical features and results of microscopic test of deep skin scrapings.

[0108] In order to study the insectoacaricide effect of the drug in the form of a solution for external use by spotting according to the principle of analogues, a study group of adult dogs aged from 3 to 5 years (n=10) and having a weight of from 10 to 20 kg was formed: study group - 5 animals suffering from demodecosis were treated with the drug in the form of the solution for external use by spotting externally, once, by dripping onto a dry unaffected animal skin by means of a dropper pipette (having a volume of 1.5 ml).

[0109] The drug was applied to the affected areas of the animals suffering from demodecosis and it was spread by finger tips in a glove, while covering up to 1 cm2of healthy skin. The treatment was performed 4-fold with 10 days interval until achievement of clinical recovery of the animal which was confirmed by two negative results of acarological tests.

[0110] It should be noted that a complex therapy was applied to the dogs suffering from demodecosis as follows: antimicrobial drug Enrofloxacin in a dose of 50 mg per 10 kg of body weight during 10 days; Activyl-3 (probiotics for dogs) in a oral dose of 3 g per animal with food during 14 days, hepatoprotective agent Divopride in a dose of 2 tables thrice a day during 14 days and vitamin Decavit in a dose of 1 ml with drinking water 1 time per 5 days during 14 days.

[0111] Use of the acaricidal treatment in the dogs suffering from demodecosis has shown that on day 28 of the treatment, the amount of mites was gradually decreased in the study group by 1.8 times, on day 45 - by 2.8 times, on day 56 - by 9.3 times respectively (p<0.05). On day 78 of the experiment, the dogs of the study group were completely free of mites (Table 6).

[0112] During the demodecosis treatment process, it was noted that the dogs of the study group had increased intensefficiency during the entire study period: 64.9 % on day 45, 89.2 % on day 56 respectively relative to initial values. Table 6

[0113] Efficiency of the drug based on the supramolecular complex with afoxolaner in the form of the solution for external use by spotting for treatment of demodecosis in dogs aged from 3 to 5 years (M±m; n=5) On days 28 and 45, extensefficiency of the drug in the form of the solution for external use in the study group was 0, since all dogs remained affected by demodexes. Extensefficiency increase was observed after 56 days in the study group up to 60.0%. At the end of the study period (day 78), for the demodecosis of the dogs, therapeutic efficiency of the drug in the form of the solution for external use by spotting was 100%.

[0114] Results of clinical and biochemical studies of blood of the study dogs before and after conduction of the acaricidal treatment are provided in Table 7. In particular, before treatment, the total concentration of hemoglobin, red blood cell count in blood and glucose concentration in blood serum of the dogs were lower than reference, while white blood cell count in blood and concentration of total proteins, ALT and AST activity in blood serum were higher than reference, which indicated that there is an inflammation, a hepatotoxic activity, hemorrhage and derangement of metabolism in the organism of suffering dogs.

[0115] Table 7

[0116] Level of hematologic and biochemical blood parameters during treatment of demodecosis in dogs aged from 3 to 5 years (M±m; n=5) Starting from day 45 and till the end of the experiment, clinical and biochemical parameters of blood of dogs in the study group gained physiological level or were at its lower threshold:

[0117] - upon application of the solution for external use by spotting on the skin, the total hemoglobin concentration was 34.0% increased (p<0.05) relative to the parameter before treatment in average, red blood cell count was 22.8% increased, glucose concentration was 38.0% increased, while white blood cell count was 26.4% decreased (p<0.05), the total protein count was 7.0% decreased, ALT activity was 60.1% decreased and AST activity was 56.6 % decreased.

[0118] Study on puppies aged from 8 to 10 months

[0119] Clinical studies of the drug in the form of a solution for external use by spotting were conducted on puppies in the veterinary clinic in Kharkiv. During examination of the puppies, a localization and damaged area, a nature of change of skin cover, a presence of itching of affected areas of the skin, as well as data about time of occurrence and disease progression type were mandatory taken into account. During clinical examination, otoacariasis was noted in 5 puppies.

[0120] Otoacariasis in 5 puppies was present as psychic tension, frequent head shaking, chafing upon a carpet, corners of furniture, as well as frequent scratching of ears with their paws. Scabs of ochre-brown color were gradually appeared in the ear auricles and earwax of unpleasant odor was largely accumulated.

[0121] Diagnostics for otoacariasis was conducted with consideration of clinical picture of the disease and microscopy of samples taken from the affected ear auricles of the animals.

[0122] The insectoacaricidal activity of the studied drug in the form of the solution for external use by spotting was determined on the study group (n=5) of puppies aged from 8 to 10 months and having a weight of from 4 to 10 kg. In 5 animals suffering from otoacariasis, the external auditory passage was cleaned from crusts and scabs, and then 3 drops of the drug were instilled into each ear. The ear auricle was folded in half in longitudinal direction and its base was massaged. The treatment against otoacariasis was repeated 2 times with interval of 5 days.

[0123] In otoacariasis, after 7 days, dead and 3 and 2 units of alive mites were detected in the scratch which indicated that the invasion intensity was decreased by 3.7 times respectively in the study group (p<0.05). After repeated treatment, on day 15 of the experiment, both alive and dead mites were absent in the scratches from the animals of the study group (Table 8).

[0124] Table 8

[0125] Efficiency of the drug in the form of the solution for external use by spotting for treatment of ear dermestids in puppies aged from 8 to 10 months (M±m; n=5)

[0126] At day 7 after the therapeutic measures began, the intensefficiency of the drug in the form of the solution for external use by spotting relative to the mites in the study group was increased up to 72.7% respectively, while at day 15 of the study in the study group, this parameter was 100%.

[0127] At day 7 after the therapeutic measures began, the extensefficiency of the drug in the form of the solution for external use by spotting was increased up to 80.0% of the mites respectively, and it gained 100% after 15 days after deacarization.

[0128] Results of clinical and biochemical studies of blood of the study puppies before and after conduction of the insectoacaricidal treatment are provided in Table 9. Table 9

[0129] Level of hematologic and biochemical blood parameters during treatment of otoacariasis in puppies aged from 8 to 10 months (M±m; n=10) In particular, before treatment, the total hemoglobin concentration and the red blood cell count in the blood of the dogs were slightly higher than the reference, while the white blood cell count in the blood and the total protein concentration, urea, ALT and AST activity in the blood serum were slightly higher or were within the reference range, which indicated that there is inflammation, insignificant hepatotoxic activity and hemorrhage in the organisms of the affected dogs.

[0130] Starting from day 7 and till the end of the experiment, clinical and biochemical parameters of blood of dogs in the study group gained physiological level:

[0131] - upon application of the solution for external use by spotting on the skin of the puppies, when the animals were suffering from otoacariasis, the total hemoglobin concentration was 12.1% increased (p<0.05) relative to the parameter before treatment in average, red blood cell count was 51.8% increased, while white blood cell count was 23.3% decreased (p<0.05), the total protein concentration was 5.4% decreased, the urea concentration was 27.8% decreased, ALT activity was 51.3% decreased and AST activity was 70.3% decreased.

[0132] The mentioned tests confirm that the claimed afoxolaner complex both in the form of tablets and liquid ensures at least 70% efficiency against the target parasite.

[0133] This indicates that as compared to the known afoxolaner inclusion complexes, where the particles have a diameter of 10 and more microns, and the effective dose of the active drug must be from 5 to 10 mg / kg, the obtained complex is effective at reduced dosage, which indicates its high bioavailability and confirms that it may be used for effective oral and parenteral administration.

Claims

CLAIMS1. A water-soluble antiparasitic supramolecular complex comprising an effective amount of a solubilized afoxolaner as an active component and a surface-active agent, wherein 2-hydroxypropyl-0-cyclodextrin is used as a solubilizing agent, while polysorbate 80 is used as the surface-active agent, and the complex is a fine powder having a particle size of less than 1 micron at a ratio of components as follows (wt. %): afoxolaner - 1 - 10%, 2-hydroxypropyl-0-cyclodextrin - 50-98%, polysorbate 80 - 1-40%.

2. The complex according to claim 1, wherein it is suitable for preparing a solid oral dosage form.

3. The complex according to claim 1, wherein it is suitable for preparing a solution for instillation use.

4. A method for producing a water-soluble antiparasitic complex according to claims 1-3, the method comprises steps of mixing solutions of afoxolaner and 2- hydroxypropyl-0-cyclodextrin in ethyl aclohol at a mass ratio in the solutions of 2- hydroxypropyl- -cyclodextrin / afoxolaner of 1- 0, storing a mixture of the solutions at a room temperature during at least 24 hours, and stabilizing the mixture of the solutions with an alcohol solution of polysorbate 80 at a mass ratio of polysorbate 80 / the mixture of the solutions of afoxolaner and 2- hydroxypropyl-0-cyclodextrin of 1- 0, followed by evaporating an obtained mixture until a solvent is completely evaporated and a dry residue is obtained.

5. A veterinary drug comprising an antiparasitic complex according to claim 1 and suitable fillers and / or binders and / or preservatives in ratios which are suitable to form solid forms, in particular, as dragees, tablets, powder, capsules.

6. The veterinary drug according to claim 5, wherein it comprises excipients such as antioxidants and / or food additives having a meat flavor and taste.

7. A veterinary drug comprising an antiparasitic complex according to claim 1 and the drug is a colloidal solution in an isotonic medium and comprises a mass fraction of afoxolaner in a range of 0.05 0,5 %.

8. A veterinary drug comprising an antiparasitic complex according to claim 1 and an anthelminthic drug at a mass ratio of anthelminthic drug / afoxolaner complex of O.

9. The veterinary drug according to claim 8, wherein milbemycin oxime or pyrantel or praziquantel or febantel or a combination thereof is as the anthelminthic drug.

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