RU2096044C1 - Veterinary implantable preparation for regulation of biological rhythm in animals - Google Patents

Abstract

FIELD: veterinary medicine, veterinary science. SUBSTANCE: preparation has an active component melatonin or melatonin in combination with psychotropic substances, biodestructable polymeric base and plasticizing agent. Invention claims two medicinal composition of veterinary preparation of the following composition, %: melatonin 10-40,; cyacrine (polymer), 89.95-55.5; and plasticizing agent, 0.05-4.5; melatonin, 7-25; psychotropic agent, 7-25; cyacrine (polymer), 85.95-45.; and plasticizing agent, 0.05-4.5. Preparation is used for the accelerated maturation of hair in fur animals. EFFECT: enhanced effectiveness of preparation. 3 cl, 5 tbl

Description

 The invention relates to veterinary medicine to veterinary drugs used in fur farming to accelerate the maturation of the hair in fur animals, namely, a new veterinary product and its dosage form.

 It is known that fur animals, due to the short period of their domestication, are most sensitive to changes in the external environment and have a more pronounced biological rhythm: when the length of daylight is reduced, the animals begin to prepare for winter living conditions, change their hairline for winter, and when elongated, for reproduction. In recent years, biologically active drugs are increasingly being used to regulate the biological rhythm of fur animals.

 Numerous studies have found that with a decrease in daylight hours in animals, the secretion of melatonin of the hormone of the pineal gland (pineal gland), which regulates the biological rhythm of animal vital processes caused by changes in the length of daylight, is enhanced. In the late 70s, early 80s, Australian scientists described a method for artificially changing the photoperiod when animals (sheep, goats, etc.) were introduced melatonin (Pat. N 78305/81 Australia); the best effect is achieved with the introduction of melatonin subcutaneously (Pat. N 38111/85 Australia). However, these methods of implantation in animals had a number of significant drawbacks, one of which was the high content of melatonin in the blood plasma of animals in the initial phase of its isolation from the implant, 100 to 1000 times higher than the normal night level, which led to negative consequences.

 More attractive is the technical solution (US Pat. No. 4882137, USA) (analogue), which allows the manufacture of implants containing melatonin or related chemicals of the Indole class, a polymer carrier and the coating for the implant from a physiologically compatible polymer.

 The veterinary implant (analogue) has a cylindrical shape: length 3 10 mm, diameter 2 3 mm.

 The implant releases the active ingredient in an amount sufficient to maintain the desired level of melatonin in the blood and for a period sufficient to regulate seasonal sexual activity. The implant allows you to maintain the desired level of release of melatonin for at least 3 weeks. The active ingredient, for example melatonin, can be 25-98% by weight, the polymeric carrier 11 75% by weight (1 70 polymer base, 1 10% granulating agent, 0.5 to 5% compressive "lubricant"). In General, the veterinary drug in the shell according to patent N 4882137, USA consists of 2.5 to 100 mg of melatonin, 2.5 to 5 mg of ethyl cellulose and 0.2 to 0.3 mg of a “lubricant” of a pharmaceutical agent for controlling the rate of release of the active ingredient (usually , dibasic calcium phosphate dihydrate); has a double coating of 0.01 to 1.0 mg of ethyl cellulose with the addition of 0.2 mg of polyvinylpyrrolidone. As the film-forming polymer, an acrylic polymer or a copolymer of acrylic and methacrylic acids and their derivatives can be used.

The above veterinary preparation based on melatonin in the shell as applied to fur animals also has a number of significant drawbacks:
insufficient prolongation of melatonin (not more than 3 weeks);
low efficiency ("response" of the drug is not more than 40 50%);
multicomponent composition;
multi-stage and technically complex method of manufacturing the drug.

 Since 1984, in Russia, the Research Institute of Fur Breeding has begun the search and study of biologically active preparations to reduce the period for growing fur animals and to obtain products that are not inferior in quality to the skins at standard terms for slaughtering animals in November December. In particular, an implantable dosage form “melacryl” was developed and tested, containing melatonin and a polymer carrier, a copolymer of ethyl cyanoacrylate and 2-ethoxyethyl-α-cyanoacrylate, which provides a metered dose of melatonin into the body of fur animals (Rapoport O.L. Rabbit breeding, N 4, 1990 , p. 9) (prototype). The treatment of animals with the drug was carried out from June 25 to July 10, molting in fur animals began 30 to 40 days earlier than usual. The slaughter dates from November December shifted to late September and early October.

 It should be emphasized that the use of the well-known implant "Melacryl" is possible only with mandatory diets containing (per 100 kcal / g) (table. 1).

 However, the domestic veterinary implantable drug considered above containing melatonin (Melacryl) has a number of significant drawbacks that sharply limit its consumption.

1. Insufficient prolonging effectiveness of the drug, namely either insufficiently complete “release” of melatonin from the implant (40–60%), or too fast “release” of melatonin in the initial period of implant administration, which determined the level of “response” of the drug as 30–60%
2. Inconsistency of the molecular weight of the polymer base with the required values, which leads to the fact that only 30% of melatonin forms a solid solution with a polymer base, 70% of melatonin is in a crystalline state, which ultimately explains the partial biodegradation of the implant and, as a result, a low level "response" of the drug.

Thus, the veterinary drugs considered above, the regulators of the biological rhythm of animals have common significant disadvantages, namely
contain a partially biodegradable polymer base;
low efficiency (response rate 30-60%)
In order to eliminate the shortcomings of the known veterinary preparations based on melatonin of the regulators of animal biological rhythms used in animal husbandry and fur farming, the task was to create a new veterinary implantable agent for regulating the biological rhythm of animals with a high effect on a living organism ("response" to 100% )

The problem is solved in that the veterinary preparation includes an active substance, a biodegradable polymer base and a plasticizer. As the active substance, it is proposed to use:
a) melatonin is a synthetic analogue of the hormone of the pineal gland or its derivatives;
b) melatonin mixed with compounds of the drug group of psychotropic drugs: antipsychotics and tranquilizers.

From the group of antipsychotics can be used:
derivatives of phenothiazine (chlorpromazine, propazine, frenolone, etc.),
thioxanthene derivatives (chlorprotixen and others),
derivatives of butyrophenone and diphenylbutylpiperidine (haloperidol, metorin, etc.),
derivatives of indole (carbidine),
antipsychotics of various chemical groups (azaleptin, sulpiride, etc.).

From the group of tranquilizers can be used:
derivatives of benzodiazepine (phenazepam, nozepam, mezapam, etc.),
carbamino esters of substituted propanediol (meprotan and others),
diphenylmethane derivatives (amisyl),
tranquilizers of various chemical groups (mebicar, trioxazine, etc.).

 As you know, in conditions of supertechnological congestion of animals in large farms, when various etiological factors of a stressful nature constantly affect animal organisms, prerequisites are created for the occurrence of disorders of the autonomic innervation of organs and the central nervous system. As a result, dystrophic processes develop in the internal organs, mitotic activity of cells weakens, the growth and development of the body slows down, and the reproductive functions of fur animals are disrupted.

 In order to prevent the development of disorders of the central and autonomic nervous system in order to reduce the level of emotional tension and fear, it is proposed to introduce psychotropic drugs in addition to melatonin in the composition of the veterinary drug to regulate the biological rhythm of animals: from the group of antipsychotic drugs to study the effectiveness of the claimed veterinary drug, chlorpromazine is selected, from the group of tranquilizers - phenazepam.

 Aminazine is one of the main representatives of antipsychotics. One of the main features of the action of chlorpromazine on the central nervous system is a relatively strong sedative effect. An important property of chlorpromazine is its blocking effect on central adrenergic and dopaminergic receptors. It reduces or completely eliminates the increase in blood pressure and other effects caused by adrenoline and adrenomimetic substances. With the help of chlorpromazine, it is possible to stop various types of psychomotor agitation, to reduce fear and anxiety.

 As an example, the compositions of preparations containing melatonin and phenazepam, as well as melatonin and chlorpromazine are considered. Phenazepam is a highly effective tranquilizer. It surpasses other tranquilizers in the strength of tranquilizing and anxiolytic actions; also has a pronounced anticonvulsant, muscle relaxant and hypnotic effect. In addition, remaining a tranquilizer, phenazepam can compete with certain antipsychotics in terms of sedative and mainly anti-anxiety effects (M.D. Mashkovsky, Medicines, part 1, p. 64, Vilnius, 1993), therefore, all morphological tests were carried out using an example a drug containing melatonin and phenazepam.

 As a polymer carrier, a biodegradable polymer of a specific molecular weight based on ethyl cyanoacrylate monomer cyacrine-EO with the addition of a plasticizer is proposed.

 A veterinary preparation in the form of two compositions is claimed.

The drug "Melapol":
a) melatonin 10 40%
cyacrine-EO (polymer) 89.95 55.5%
plasticizer 0.05 4.5%
b) melatonin 7 25%
psychotropic drug 7 25%
cyacrine-EO (polymer) 85.95 45.5%
plasticizer 0.05 4.5%
The use of the proposed dosage forms of veterinary implantable drugs for the regulation of biological rhythms of animals in the literature is not described.

 As plasticizers, esters (ethyl, propyl, butyl, allyl, phenyl) of phthalic acid, alkyl cyanoacetates, triacetin can be used. The exclusion of 2-ethoxyethyl-a-cyanoacrylate from the copolymer of ethyl cyanoacrylate and 2-ethoxyethyl-a-cyanoacrylate polymer carrier of the veterinary preparation of the prototype and the introduction of a plasticizer lead to the original polymer structure, which, when tested for biodegradability, showed a discrete and even melatine property over 60 days into the blood of animals.

 The introduction of a psychotropic drug along with melatonin into the biodegradable polymer led to an unexpected (non-obvious) result - an increase in the body weight of fur-bearing animals and, as a result, an increase in the size of the skins.

 The introduction of the active principle below the lower limit does not provide the expected effect, above the upper limit violates the stability and effectiveness of the dosage form. The content of the polymer based on cyacrine-EO below the lower limit does not provide the necessary prolonging properties of the drug.

 The introduction of plasticizers can improve the physico-mechanical characteristics of the drug and eliminate the appearance of phase heterogeneity of the structure of the drug. The use of a plasticizer above the upper limit leads to a violation of the strength characteristics of the drug.

 The name "Melapol" was assigned to the drug by the Department of Veterinary Medicine of the Russian Federation as a temporary instruction for use on 11/01/93.

 The dosage form of both compositions are cylindrical tablets with a mass of 0.035 ± 0.005 g, a diameter of 2.0 to 2.4 mm, from yellow-green to dark brown.

 Compared with the known veterinary drugs based on melatonin, the proposed veterinary preparations have significant advantages.

1. Highly effective drugs are biodegradable, ie. completely soluble in biological environments of living organisms, which contributes to 100% "response" of drugs;
2. The use of drugs does not require changes in diets;
3. The use of drugs in fur farming allows animals to be slaughtered 2 3 months earlier than the standard slaughter terms and get fur products with an area of 10 15% more than the standard.

 The proposed veterinary preparations were experimentally tested in a number of animal farms of the Republic of Tatarstan.

 The high effectiveness of drugs as regulators of the biological rhythm of animals is confirmed by the following examples.

 Example 1. Evaluation of the effectiveness of veterinary implantable preparations containing melatonin and phenazepam; melatonin and chlorpromazine.

To assess the effectiveness of the claimed veterinary drugs, the kinetics of the "release" of the active substance from the implant in the medium of a blood substitute solution (LIPC fluid) of the composition was studied:
sodium chloride 15.0 g
potassium chloride 0.2 g
sodium bicarbonate 0.1 g
calcium chloride 0.2 g
distilled water up to 1 l
To prevent microbial spoilage with the solution, 0.07% preservative was added to the solution during the study.

The test sample is a known veterinary preparation according to US Pat. N 4882137, USA (analogue) in an amount of 10 mg was placed in a 50 ml volumetric flask, 50 ml of LIPC liquid with a preservative was added, the flask was closed with a stopper and placed in a thermostat at a temperature of 37 ^o C. 5 ml samples were taken at 2, 10, 20, 30, 40, 50, 60 days. After sampling each sample, 5 ml of LIPC liquid with a preservative was added to a volumetric flask. The content of the active substance in the samples was determined by gas-liquid chromatography on a Waters chromatograph (LC / GPC-244) or any other model equipped with a UV spectrophotometric detector.

Experiment Conditions:
wavelength, nm 254
stationary phase, sorbent silica gel With ₁₈ grain size 5 18 microns
eluent acetonitrile / water (50% by weight)
temperature 25 ^o C
Example 2

 Similar to example 1, the tests were subjected to the well-known veterinary preparation "Melacryl" (prototype).

 Example 3

Similar to example 1, the tests were subjected to the claimed veterinary preparation composition:
melatonin 10%
cyacrine-EO (polymer) 89.95%
plasticizer 0.05%
Example 4

Similar to example 1, the tests were subjected to the claimed veterinary preparation composition:
melatonin 40%
cyacrine-EO (polymer) 55.5%
plasticizer 4.5%
Example 5

Similar to example 1, the tests were subjected to the claimed veterinary preparation composition:
melatonin 7%
phenazepam 7%
cyacrine-EO (polymer) 85.95%
plasticizer 0.05%
Example 6

Similar to example 1, the tests were subjected to the claimed veterinary preparation composition:
melatonin 25%
phenazepam 25%
cyacrine-EO (polymer) 45.5%
plasticizer 4.5%
Example 7

Similar to example 1, the tests were subjected to the claimed veterinary preparation composition:
melatonin 7%
chlorpromazine 7%
cyacrine-EO (polymer) 89.95%
plasticizer 0.05%
Example 8

Similar to example 1, the tests were subjected to the claimed veterinary preparation composition:
melatonin 25%
chlorpromazine 25%
cyacrine-EO (polymer) 45.5%
plasticizer 4.5%
Thus, samples of veterinary preparations made according to US Pat. N 4882137, USA (analogue), a sample of the well-known drug "Melacryl" (prototype) and samples of the claimed veterinary drugs (examples 1 to 8). All samples were placed in a blood substitute solution at the same time. The test time was 60 days. The results are presented in table. 2.

 As can be seen from the data presented in table. 2, too rapid "release" of melatonin from the implant of known veterinary drugs in the initial period determines their low efficiency (response rate from 30 to 55%, which is confirmed by experimental tests (see table. 3)). A biodegradable polymer base containing a cyacrine-EO polymer and a plasticizer provides a fairly uniform "release" of melatonin and melatonin in a mixture with phenazepam and chlorpromazine in the inventive veterinary preparations, which determines 100% "response" of the drug (see table. 3).

 Example 9. Tests of preparations of experimental batches of "Melapol" containing melatonin; melatonin with phenazepam; melatonin with chlorpromazine.

 In the animal farms "Koshchakovsky" and "Biryulinsky" of the Republic of Tatarstan, tests were conducted of pilot batches of Melapol preparations containing melatonin; melatonin with phenazepam and melatonin with chlorpromazine, on minks and arctic foxes (the preparations are described in examples 3-8).

 The effectiveness of the claimed drugs was evaluated by comparing with the effectiveness of known drugs containing melatonin (the compositions are indicated in examples 1, 2). As a control group, animals that were not treated with a preparation containing melatonin were used.

Drugs were administered subcutaneously in the area between the shoulder blades. Mink was injected with 1 granule, foxes with 2 granules. The animals of the main herd were processed from June 3 to 5, young animals from June 20 to July 1. The animals of the main herd were slaughtered in the period from September 5 to 10, young animals from September 20 to October 1. Side effects and complications when using the drug were not observed. At the site of implantation of the drug, inflammatory processes in the outer and inner layers of the skin and subcutaneous tissue were absent. Weighting of body weight carried out before slaughter of animals treated with the claimed preparations containing melatonin with phenazepam and melatonin with chlorpromazine, of the compositions indicated in examples 5, 6, 7, 8, showed that the weight of the animals in the experimental groups was greater than in the control. The difference was from 8 to 15%
Furs after dressing did not differ in quality from furs that were not processed by “melapolus” animals and slaughtered during the period from November 5 to December 5. The test results are given in table. 3.

 Example 10. Conducting morphological studies of the nature of the effects of prolonged-action veterinary preparations containing melatonin (the drug "Melapol") and melatonin with phenazepam on the organs and tissues of fur animals.

The organs and tissues of 20 minks belonging to the Biryulinsky fur farm of the Republic of Tatarstan were subjected to morphological tests. Animals were killed on days 7, 12, 26, 44. after subcutaneous administration of Melapol preparations containing melatonin and a mixture of melatonin with phenazepam, of the following composition:
a) melatonin 40%
cyacrine-EO (polymer) 55.5%
plasticizer 4.5%
b) melatonin 25%
phenazepam 25%
cyacrine-EO (polymer) 45.5%
plasticizer 4.5%
In each study period, 5 animals were slaughtered. Clinical observations in experimental minks in the first 7 to 12 days. after the introduction of the drug, a decrease in anxiety and irritability was noted, there was an elongation of sleep while maintaining appetite. Studies using histological, histochemical and electron microscopic methods have established the following changes. For 7 days. Experience in the cytoplasm of cells of supraoptic, paraventricular nuclei of the anterior and ventromedial and dorsomedial nuclei of the middle hypothalamus noted the accumulation of a homo-positive substance. At 12, 26 days. After pharmacological loading, small and large vacuoles were found in the cytoplasm of the neurosecretory cells of the hypothalamus, as well as deposits of a large number of formazan particles, which indicated increased secretion of tropic hormones, an increase in the activity of the enzyme monoamine oxidase and an increase in the processes of oxidative deamination. The numerous basophilic and eosinophilic endocrinocytes of the adenohypophysis during these periods revealed the accumulation of a large number of large and small electron-dense granules, the expansion of the endoplasmic reticulum cyst profiles, which characterized the increased level of biosynthesis and the emission of somatotropic and thyrotropic hormones into the circulation. In the study of the epithelium of the gastrointestinal tract, pancreas, liver, kidneys, as well as lymphoid organs on days 26, 44. Experience has shown an increase in signs of anabolism in the form of an increase in the number of mitoses, polyploidization of nuclei, increased biosynthesis of ribonucleoproteins, glycosaminoglycans, cationic proteins, and the appearance of numerous interepithelial lymphocytes.

 The use of the drug "Melapol" with phenazepam did not cause the appearance of alterative changes in the structures of the central nervous system, parenchyma of the liver, pancreas, kidneys, lymphoid organs and mucous membrane of the gastrointestinal tract.

 In order to assess the nature of the influence of the claimed veterinary preparation containing melatonin and melatonin with phenazepam on the functional activity of the most important organ of the peripheral immune system of the spleen organism, the dynamics of changes in the morphological parameters of the organ was studied.

 It was found that the use of the drug "Melapol" (with melatonin) enhances the lymphoproliferative function of the spleen, starting from 26 days. experience (table. 4). The effect was manifested by an increase in the mass of lymphoid tissue, which was expressed as an increase in the germinal centers and the lymphatic follicles themselves. A tendency towards an increase in the number of mitosis figures in germinative centers in the absence of pathological forms of dividing cells was found. Accordingly, by the end of the experiment (44 days), an increase in the number of lymphofollicles with pronounced germinative centers and an increase in their relative area were observed. In minks exposed to the implant with melatonin and phenazepam, for 44 days. the estimated parameters (Table 5, paragraph 1-8) were significantly higher (with respect to Melapol) and especially those (paragraphs 1, 3, 4 of Table 5) that characterized the organ proliferative potential. (Statistical processing of the research results was carried out according to the method: Dyakonov V.P. Handbook of calculations on microcalculators. Ed. 3rd, additional and revised. M. Nauka, 1989, 464 p.). The effect of melatonin with phenazepam did not cause significant fluctuations in the content of circulating immune complexes in the blood of minks. The results obtained indicate a high level of morphological and functional potential of the lymphoid organs to a full-fledged immune response after exposure to the drug compared with control animals.

Thus, the use of the claimed veterinary preparations containing melatonin and melatonin in combination with phenazepam, under conditions predisposing in fur animals to vegetative neuroses, diencephalic paroxysms, helped stabilize the neurohormonal regulation of metabolic processes in the body, markedly increased proliferative and biosynthetic epithelial reactions studied organs, which, in combination with increased formation of somatropic and thyroid-stimulating hormones of the adenohypophysis, caused fold increase in body weight of animals, leading to increased size of the skins for 10 to 15%
Morphological studies were carried out in accordance with the methods (Rapoport Ya. L. Morphological basis of immunogenesis (immunomorphology) Arch. Pat. 1957, N 2, p. 3 19; Fontalin LN Immunological reactivity of lymphoid organs and stock. L. Medicine, 1967, 210 pp. Fridenstein A.Ya. Chertkov I. L. Cellular basis of immunity M. Medicine, 1969, 256 pp. Bart I. Selezenka Anatomy, Physiology, Pathology and Clinic. Budapest: Publishing House of Academic Sciences of Hungary, 1976 , 264 pp. Petrov RV Immunology. M. Medicine, 1983, 367 pp. Avtandilov G.G. Yabluchansky N.I. Gubenko V.G. System stereometry in the study of the pathological process. M. Medicine, 1981, 192 pp. Z. Loyda, R. Gossrau, T. Schiebler. Histochemistry of enzymes. Laboratory methods. M. Mir, 1982, 272 pp.).

 Thus, the above results demonstrate the high efficiency of a prolonged-action veterinary drug for regulating the biological rhythms of animals containing melatonin and psychotropic drugs.

 Based on the results of the tests, the Department of Veterinary Medicine of the Russian Federation (temporary instruction from 01.11.93) and the General Directorate of Veterinary Medicine of the Cabinet of Ministers of the RT (conclusion N 1146 from 04/28/95) recommended to allow the use of the claimed preparation in fur farming.

 The active substance of the inventive veterinary medicine (melatonin is a substance) is synthesized by known methods (Yakhontov L.N. Glushkov R.G. Synthetic drugs. M. Medicine, 1983). Used psychotropic substances are substances produced by the medical industry for the manufacture of dosage forms. The remaining components are harmless and are also approved for use in medicine and veterinary medicine (V.V. Korshak, A.M. Polyakova, M.S. Shapiro. Cyanoacrylate glue and its use. Journal of VCO named after D.I. Mendeleev, vol. XIV , N 1, 1969, p. 51; VR Belkin. Materials of the 4th Scientific session of the Research Institute of Clinical and Experimental Surgery. M. 1967, p. 238).

 Industrial applicability. The inventive veterinary product is used in veterinary medicine. The release of the first pilot industrial series is planned in 1995 by the Vetta Small Scientific-Production Enterprise at the All-Russian Scientific Research Veterinary Institute (VNIVI) of Kazan (license No. 9 dated May 5, 1995).

Claims (2)

 1. A veterinary implantable preparation for regulating the biological rhythm of animals, containing melatonin and a biodegradable polymer base, characterized in that it additionally contains a plasticizer, and as a biodegradable polymer base, the polymer is cacacrine EO in the following ratio of components, wt. Melatonin 10 40
Tsiakrin-EO (polymer) 89.95 55.5
Plasticizer 0.05 4.5
2. The drug according to claim 1, characterized in that phthalic acid esters, alkyl cyanoacetates, triacetin are used as a plasticizer.  3. The drug according to claims 1 and 2, characterized in that it additionally contains psychotropic drugs in the following ratio of components, wt. Melatonin 7 25
Psychotropic drug 7 25
Tsiakrin-EO (polymer) 85.95 45.5
Softener 0.05 4.5o

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