RU2092185C1 - Vaccine for suppression of sexual function in mammals - Google Patents

Abstract

FIELD: agriculture, biotechnology. SUBSTANCE: invention relates to vaccine that has a peptide conjugate of the formula: Pyro-Glu-X-Ser-Try-Y-Leu-Arg-Pro-Z where: X - Try or His-Try; Y - D-Ala or D-Lys and Z - NHC₂H₅ or -OH with tetra- and/or octamer of lysine, or bovine serum albumin in the parent or capsulated form. Stearate or aluminium hydrogel is used as an acceptable vehicle. Vaccine decreases sexual reflexes and excitement of animals but retains or increases the growth rate. EFFECT: enhanced effectiveness of vaccine. 4 cl, 3 tbl

Description

 The invention relates to veterinary medicine, namely to vaccines that regulate the sexual behavior of farm and domestic animals. Vaccines can be used in industrial complexes for the cultivation and feeding of animals, in rental groups and farms.

 Male animals raised for meat give higher growth rates with lower bone and fat content in the carcass. But they are more susceptible to stress, they are difficult (and even associated with risk) to maintain and carry out various preventive treatments with tethered maintenance, even more difficult with free-group and even dangerous with grazing. Significant difficulties with group maintenance are associated with frequent clarifications of hierarchical relationships, manifestations of sexual reflexes. All this leads to injuries, to premature culling, forced slaughter, shortage of products. In addition, males, such as boars, have a specific unpleasant smell of meat, which dramatically reduces its taste and marketability. No less concern is given to domestic animals during periods (seasons) of sexual activity.

 Females due to the presence of their sexual cycles periodically during the extra phase (hunting) exhibit strong excitement, anxiety, aggressiveness, accompanied by loss of appetite and even refusal to eat (this is especially pronounced in pigs), which ultimately leads to a decrease in productivity .

 It is known that surgical castration of animals significantly reduces the content of sex steroids in the blood and, as a result, their aggressiveness, excitability, irritability, resulting in reduced injuries and cases of premature rejection (Fomichev Yu.P. Levantin D.L. Dziuba N.F. . and others. Production of livestock products on an industrial basis. Collection of scientific works. VIZH Dubrovitsy. 1975. Issue 2. p.103-111).

 However, the use of surgical castration reduces the growth rate of gobies by 7.8% and reduces the carcass yield of carcasses, contributes to an increase in fat content in the carcass by 15.5% and a decrease in protein by 6-7%. Surgical castration of female individuals is a cavity operation and is difficult to perform.

 In the last decade, much attention has been paid to the development of specific vaccines to control the sexual activity of animals. Since sexual activity is a physiological process that depends on the concentration of sex hormones in the body, it can be almost completely blocked or suppressed with the help of specific antibodies generated during the immune response to the vaccine administered and binding to endogenous gonadoliberin (immunocastration, immunoneutralization, immunocontraception, biological castration). Immunocastrated animals behave similarly to surgically neutered animals. The advantage of such vaccines is the ease of administration and the ability to quickly carry out mass processing of animals. In addition, unlike surgical castration, the risk of bleeding, infections and injuries is eliminated.

 It has been shown that the active immunization of various animals against gonadoliberin in the case of males leads to atrophy of the testes, decreased secretion of testosterone and the disappearance of sphematozoids, and in the case of females, to cycle disorders and ovarian regression. In a comparative study of surgically castrated calves and immunologically castrated calves by the method of active immunization against gonadoliberin, it was found that the immunocastrates are close to uncastrated animals in terms of their skeleton, since their anabolic effect of the testes is not completely eliminated (Robertson et.al. Veterinary Record, 1982, III, 529-531).

 Australian patent AI-B-11017/88 (PCT application 88/05308, class A 61 K 39/295) describes an anti-reproductive hormone, which is an immunogenic conjugate of piptid protein, in which the peptide is a sequence selected in the group consisting of any continuous fragments of 5, 6 or 7 amino acids of the decapeptide.

PiroGlu-Gis-Three-Ser-Tir-Gli-Lei-Arg-Pro-Gli-NH ₂ ,
and the protein is selected in the group consisting of serum albumin, thyroglobulin, egg albumin, gelatin, serum globulin. The vaccine based on this conjugate contains a suitable immunoadjuvant, such as diethylaminoethyldextran or its emulsion in mineral oil. All amino acids, with the exception of glycine, which is not optically active, are in the L configuration.

In US patent N 3963691 (1976, 260-112,5LH) describes synthetic antigens related to gonadoliberin, which when introduced into the body of mammals, such as rabbits, causes the formation of antibodies to it. The presence of such antibodies controls the secretion of gonadoliberin and other hormones under its control, causing changes in the ovarian cycle and other important pituitary and gonadal effects. These antigens are useful in veterinary medicine as an alternative to ovarian removal in domestic animals such as cats and dogs. They are administered by injection, resulting in the formation of antibodies to gonadoliberin and the production of gonadotropins by the pituitary gland is suppressed. The antigen is obtained by chemically combining the peptide bond with a suitable carrier. Examples of suitable carriers are polymers derived from L-amino acids or a mixture of D- and L-amino acids, the preferred carrier is poly-L-lysine. The patent describes antigens of the general formula: Glu-His-Three-Ser-Tyr-Gli-Lei-Arg-Pro-Gli-NH ₂ bound to the carrier by a peptide bond between the carboxyl group of the ligand and the amino groups of the carrier, in particular the ε amino groups of poly-L lysine. The antigen is administered to domestic animals every 5-7 months at a dose of about 1 mg / kg body weight, subcutaneously or intramuscularly.

 European Patent Application No. 0293530 (CL CO 7 K 7/20, 1987) describes gonadoliberin analogs having the amino acid sequence PyroGlu-His-Three-Ser-Tyr-X-Lei-Arg-Pro-Gly-YZ, where X is Gly or a D-amino acid, for example D-Ala, Y is one or more amino acid residues that may be the same or different, and Z is Cis or Tyr, so that the conformation of said analogue in solution is almost similar to that of native gonadoliberin. Analogs can be used for vaccines that reduce the fertility of mammals, either as such, or in the form of conjugates with polypeptide carriers. The preparation of such conjugates (gonadobiberin analog carrier) can be carried out by modifying one or more lysine side chains of the polypeptide carrier with maleimide. Examples of preferred carriers include purified protein derivatives of tetanus toxin and tuberculin toxoid, the latter being preferred.

 A disadvantage of the known vaccines is their high cost, lack of trials or insufficiently high efficiency of their use by farm animals.

 The aim of the invention is to expand the arsenal of vaccines devoid of these disadvantages and their scope.

The problem is solved using a vaccine to suppress sexual function in mammals, consisting of a conjugate of a peptide analogue of gonadoliberin with a pharmaceutically acceptable immunogenic carrier, characterized in that it contains the amino acid sequence of the formula:
PirGlu-X-Ser-Tyr-Y-Lei-Arg-Pro-Z,
where X Three or His-Three, Y D-Ala or D-Lys and Z NHC ₂ H ₅ or OH, which is conjugated to the tetra-and / or octamer of lysine or bovine serum albumin in its original or encapsulated form, and an injectable excipient is pharmaceutically acceptable vaccine forms of stearate or aluminum hydrogel.

 The proposed vaccines were tested on young gobies and mature pigs. So, calves were injected with 3 mg of conjugate in one milliliter of aluminum hydrogel twice at the age of 5.5 and 8.5 months. Mature pigs were treated three times, during three sexual cycles, 1.5 mg conjugate also in aluminum hydrogel. At the same time, a decrease in the concentration of sex steroids was observed in the blood of animals, leading to a decrease in their aggressiveness, stress reactivity, manifestation of sexual reflexes, and at the same time to an increase in growth rate or its preservation at the level of intimate animals, as well as the elimination of signs associated with the estrous cycle period females.

 The following are examples illustrating the preparation of conjugates and vaccines according to the invention, as well as their use in raising domestic animals. The examples are for illustration only and are not restrictive.

Example 1. The conjugate with the structure
N ^a , N ^e - [(PyroGlu-His-Three-Ser-Tir-D-Ala-Lei-Arg-Pro) ₂ Lys] ₄ (Lys) ₂ -LysNH ₂ (conjugate 1).

Cooled to 0 ^o C solution of 496 mg (0.4 mmol) PiroGlu-Gis-Tri-Ser-Tyr-D-Ala-Leu-Arg-Pro-OH in 10.7 ml of dimethylformamide (DMF) was added a cooled solution of 50 mg (0.05 mmol) of lysine tetramer (LIZ) ₄ (Lys) ₂ Lys-OH and 137.5 mg (0.48 mmol) of phosphoric acid diphenylether azide (C ₆ H ₅ O) ₂ P (O) N ₃ in 0 4 ml of DMF.

A flask with a reaction mass for 30 minutes was placed in a freezer (-10 ^° C), and then stirred at 20 ^° C for 48 hours. During the reaction, a pH of about 8-8.5 was maintained with triethylamine.

 After completion of the reaction, 250 ml of ethyl acetate was added to the mixture. A white precipitate conjugate. Filter and dry in air.

 The dry product is dissolved in 15 ml of DMF and precipitated with 300 ml of ethyl acetate. 550 mg of conjugate are obtained.

 The degree of conversion during the reaction is controlled by thin layer chromatography (TLC). The dry conjugate contains 83.7 weight by UV absorption at 280 nm. gonadoliberin analog.

где БСА бычий сывороточный альбумин, являющийся природным лизинсодержащим продуктом, и конъюгирование которого с октапептидом происходит также через ε аминогруппы лизинов (конъюгат II).Example 2. The conjugate with the structure

where BSA is bovine serum albumin, which is a natural lysine-containing product, and whose conjugation with octapeptide also occurs through the ε amino groups of lysines (conjugate II).

 There were several variants of the bovine serum albumin conjugate octamer, differing in the method of crosslinking the octapeptide with albumin, which are described by the same chemical formula. One example is given as an example.

To a cooled to 0 ^o C solution of 490 mg (0.382 mmol) PiroGlu-Tri-Ser-Tyr-D-Lys-Leu-Arg-Pro-NHC ₂ H ₅ in 18 ml of DMF was added with stirring, cooled to 0 ^o C solution of 200 mg (0.418 mmol) of adipic acid dipentafluorophenyl ester (C ₆ F ₅ OOC- (CH ₂ ) ₄ -COOC ₆ F ₅ ) in 3 ml of DMF. Stirring is continued until the reaction is complete ≈ 1 h (the reaction mass becomes transparent).

To a solution of bovine serum albumin (BSA (400 mg, ≈ 6 mmol) in 30 ml of DMF) is added a solution of the obtained activated peptide. The reaction mixture was stirred for 48 hours at 4 ^° C. N-methylmorpholine was added during the reaction mixture to maintain a pH of 7.5-8.0.

After the reaction, the solution is filtered and the resulting conjugate is purified by dialysis, which is carried out for 48 hours at 4 ^o C.

 The conjugate solution thus obtained was purified by filtration through a membrane and lyophilized. 310 mg of conjugate are obtained.

Example 3. The conjugate with the structure
[N ^a , N ^e - (PiroGlu-Gis-Tri-Ser-Tir-D-Ala-Lei-Arg-Pro) ₂ Liz] ₈ (Liz) ₄ (Liz) ₂ -Liz-Ala-NHC ₂ H ₅ ( conjugate III).

To a solution cooled to 0 ^° C, 496 mg (0.4 mmol) of PiroGlu-His-Tri-Ser-Tir-D-Ala-Lei-Arg-Pro-OH in 6 ml of DMF, a cooled solution of 102 mg (50 mmol) of octamer is added lysine (Lys) ₈ (Lys) ₄ (Lys) ₂ Lys-Ala-NHC ₂ H ₅ and 137.5 mg (480 mmol) of phosphoric acid diphenyl ether azide (C ₆ H ₅ O) ₂ P (O) N ₃ in 4 ml of DMF.

The flask with the reaction mass was placed in a freezer for 30 minutes, and then stirred at 4 ^° C for 36 hours. During the reaction, pH ≈ 8.0-8.5 was maintained with triethylamine.

 After completion of the reaction, 200 ml of ethyl acetate was added to the mixture. A white precipitate conjugate. 505 mg of conjugate is obtained.

 The dry conjugate contains UV absorbance at 280 nm 78 weight. gonadoliberin analog.

 Example 4. The test of the effect of vaccines based on synthetic analogues of gonadoliberin conjugated with an immunogenic carrier - a heterologous protein (bovine serum albumin is a natural lysine-containing product) or a synthetic carrier polylysine on estrus (hunting) retention in mature pigs.

 Two vaccines, based on conjugates of synthetic analogues of gonadobiberin, were tested to show signs of hunting in pigs.

 The experiment was conducted on three groups of sexually mature pigs with 10 animals each.

 Group I animals were kept according to the technology used in the farm, with a placebo injection (saline with 25% aluminum hydrogel), 1 1.2 ml (control).

 Group II animals received a vaccine based on conjugate 1 (Example 1) over three reproductive cycles, one injection per cycle. An injection form of the vaccine was prepared as follows. Conjugate 1 was dissolved in saline with the addition of 25% aluminum hydrogel (total volume 1-1.2 ml containing 1.5 mg of conjugate per injection, option 1).

 Group III animals received a vaccine based on conjugate II (example 2). A vaccine for injection was prepared, as in option 1, by dissolving the conjugate in saline with the addition of 25% aluminum hydrogel (total volume 1-1.2 ml containing 1.5 mg of conjugate per injection) and was also administered over three sexual cycles, one injection into the cycle (option II).

 Due to the fact that the amino acid sequence used is a biological analogue of gonadoliberin, its conjugates cause the formation of antibodies that bind endogenous gonadoliberin, which leads to a decrease in the concentration of gonadoliberin in the blood, which in turn leads to a decrease in the synthesis and secretion of sexual steroids and ultimately to estrus retention, which was observed in the experiment.2 In sexually mature pigs treated with vaccines based on conjugate I, as well as conjugate II, estrus delay was observed during three reproductive cycles. The pigs were calm, showed no outward signs of hunting. In the control group, in pigs, regularity of reproductive cycles was observed.

 Thus, the tested vaccines, which include peptide-peptide conjugates of synthetic analogues of gonadoliberin, are able to delay estrus in mature individuals, in particular pigs.

 Example 5. The effect of vaccines based on peptide conjugates containing synthetic analogues of gonadoliberin with a homologous lysine-containing protein, bovine serum albumin, or synthetic polylysine octamer lysine (conjugates II and III, examples 2 and 3) on the state of gonads and the concentration of sex steroids was studied. animal behavior and their growth rate when introduced into the pre-mature period of life and at the beginning of puberty.

 Two vaccines were tested to lower testosterone and estradiol levels in the blood, followed by changes in animal behavior and maintaining growth rates.

3 groups of gobies were formed, 5 animals each:
Group I (control) the introduction of a placebo (aluminum hydrogel in saline);
Group II (option I) the introduction of a vaccine based on conjugate II (example 2);
Group III (option II) the introduction of a vaccine based on conjugate III (example 3).

 The vaccine was injected into the neck at 3 mg per head with 1 ml of aluminum hydrogel in saline solution at the age of 5.5 months (puberty) and at 8.5 months (onset of puberty).

 The animals were on a ration designed to receive 1200 g of the average daily gain in live weight, the content was tethered.

 To determine the effect of vaccines on the development of testes and the secretion of sex steroids, monthly blood concentrations of testosterone, estradiol, and intravital determination of testicle weight were performed. At the end of the experiment, at 10.5 months, when the animals were slaughtered, pieces of testicular tissue were taken from 5 participants in the testes for morphological studies. From time to time, the animals were driven into the pen for visual observation of their behavior. In order to study the effect of vaccines on the growth rate of animals, their live weight was determined monthly. At the end of the experiment, animals were slaughtered and the effect of vaccines on the morphological composition of the carcass and the chemical composition of meat was revealed.

The intravital determination of the testis mass did not reveal significant differences, with the exception of a decrease in their weight at 8.5 months of age in groups II and III by 9.8 and 5.5%, respectively
Morphological examination of testicular tissue taken from animals at 10.5 months of age showed significant changes in it under the influence of the administered conjugate. Spermatogenesis disorders, possibly irreversible, occurred in the seminiferous tubules. In addition, their atrophy was observed, as well as a partial destruction of specific organ structures, degeneration of interstitial tissue into connective tissue. The aforementioned indicates a violation of the function of sperm and hormone-producing structures of the testicle apparatus, which are under the control of gonadotropins.

 Intravital changes in testicular tissue are confirmed by monthly changes in the concentration of sex steroids (testosterone and estradiol, table 1).

 The prevailing ratio of sex hormones and, apparently, a certain ratio of different forms of androgens, as well as intermediate products of steroid synthesis and their metabolism, having different androgenic and anabolic activity, contributed not only to the formation of a certain state of the reproductive structures of the body, but also caused the buildup and strengthening of muscle tissue and bones and, in addition, also influenced the concentration of other hormones, such as growth hormone. The concentration of growth hormone increased with greater intensity in animals that received the claimed vaccine up to 7.5 months of age (respectively at 6.5 and 7.5 months in group I 67.3 and 126.3% in group II 70.7 and 222.8 % in III 124.6 and 185.9%).

 Therefore, the obtained status of these hormones when growing gobies using the proposed biosterilization vaccines introduced a corrective change in the growth rate of gobies, which is confirmed by their dynamics of increase in live weight (Table 2).

 Over the period of the experiment, the average daily gain in live weight in gobies of the control group was 1229 g, in variant I 1315 (107.0%) and in the second 1385 (112.7% compared with gobies in the control group).

 Gobies treated with the claimed vaccines, according to visual observations, were less aggressive, reacted more calmly to external stimuli, had less manifestation of sexual reflexes, they were easier to maintain, and it was easier to carry out the procedures planned for the study.

 Example 6. To confirm the positive effect of vaccines for biosterellization based on synthetic analogs of gonadoliberin on the growth rate and behavioral reactions of animals, we studied the introduction of the vaccine to sexually mature animals that are in group loose housing and having a growth rate due to the feeding and keeping conditions typical for most ordinary farms conducting cattle rearing and fattening.

 A vaccine was tested, the peptide part of which, as in examples 4 and 5, is represented by the nonapeptide as a synthetic analogue of gonadoliberin. However, unlike the vaccines used in Examples 4 and 5, the preparation tested in this example consisted of two conjugates I and III of equal weight (examples 1 and 3). The claimed vaccine differed from those proposed in examples 4 and 5 and the administered form. A mixture of conjugates, a component of the vaccine was used in prolonged form. The conjugates were introduced into capsules prepared on the basis of bovine serum albumin by a known method. The filler for the injectable form of the aluminum stearate vaccine was also different.

 Gobies with a live weight of 263 kg were put to experience. Under the experience were 50 goals. In the winter period with severe frosts (end of November - end of January), the animals were in an insulated room with drafts. Content group, loose, 25 goals in a group. Group feeding with constant access to feed with a feeding front sufficient to feed all groups of animals at the same time. Silo at the rate of 10-15 kg per head per day was set in the feeders from the tractor trolley in two doses: morning and evening. Combined feed of 1-2 kg animals received intermittently. The cottage depended on its availability on the farm. Gobies had constant access to water.

 2 groups were formed.

Group I (control) intact animals;
Group II (using the claimed vaccine) a single injection of the vaccine (encapsulated immunogenic conjugates of the synthetic analogue of gonadoliberin), an equal weight mixture of conjugates I and II (examples 1 and 3), the carrier of which is aluminum stearate, 1.5 ml per head containing 6 mg of a mixture of conjugates in a dose. In order to control the growth rate, animals were weighed monthly.

 The vaccinated animals were characterized by a calmer disposition, it was easier to catch and fix them, with less risk they took blood, weighed, serviced. To a lesser extent, sexual reflexes were manifested.

 To identify the effect of the vaccine on the endocrine system responsible for sexual motivation, a morphological study of testis tissue synthesizing hormones and spermogenesis was performed. To do this, 1.5 months after the introduction of the vaccine, the calves were castrated in the control and immunized groups and tissue samples of testicles were taken for analysis.

 The results of control weighings showed that the animals had a low, but different growth rate in the groups.

 Table 3 presents data on changes in live weight and daily average growth in intact animals and vaccinated.

 As can be seen from the data in Table 3, over the period of the experiment the control animals gave a zero increase in live weight. For the 1st month of the experiment, the bulls had 200 g of growth per head per day, but for the 2nd month of the experiment, a plumb was obtained and as a result the animals had a live weight equal to the set weight. Vaccinated animals for the 1st month of the experiment gave an average daily increase in live weight equal to 467 g per head, while maintaining the trend of a positive increase in live weight for the 2nd month of experience. As a result, over the period of the experiment, the average daily gain in live weight was 246 g per head.

 Morphological examination of testicular tissue showed that despite the fact that the vaccine was administered to sexually mature gobies, when hormone and sperm-producing structures were already formed, the vaccine introduced did affect the structure of the tissue. The initial stages of replacing secretory tissue with connective tissue are noted. Already this, apparently, was enough to disrupt endocrine secretion, which led to impaired spermogenesis, synthesis and secretion of sex hormones, which in turn led to changes in the behavioral reactions of animals.

 These changes confirm the chain of changes in the body that was identified and confirmed using biochemical and morphological studies in example 5.

 As a result, the general totality of the altered traits in the body led to a higher growth rate of calves reared using the proposed vaccine. Against the background of zero increase in live weight in gobies of the control group for two months of experience, the introduction of the vaccine led to an additional increase in live weight for this period by 246 g per head per day.

Claims (4)

1. A vaccine for suppressing sexual function in mammals, consisting of a conjugate of a peptide conjugate of a gonadoliberin analog with a polypeptide and a physiologically acceptable carrier, characterized in that it contains an amino acid sequence of the formula as a peptide
PiroGlu-X-Ser-Tir-U-Lei-Arg-Pro-Z,
where X Gis is Three or Three;
U-D-Ala or -D-Liz;
Z NHC ₂ H ₅ or —OH,
and as the polypeptide, the tetra- or octamer of lysine or bovine serum albumin.  2. The vaccine according to claim 1, characterized in that it contains a mixture of conjugates.  3. The vaccine according to claim 1, characterized in that the conjugate is used in its original form, and aluminum hydrogel from the carriers is used.  4. The vaccine according to claim 1, characterized in that the conjugate is used in encapsulated form, and aluminum stearate from the carriers.

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