SU683603A3 - Process for the preparation of polypeptides exhibiting contraceptional activity - Google Patents

Description

This invention relates to medical practice and relates to the preparation of contraceptives.

The proposed polypeptides — novel compounds and the method for their preparation — have not been described in the patent and scientific literature.

The aim of the invention is to obtain polypeptides with contraceptive action.

This goal is achieved by the fact that the polypeptide component isolated from follicle-stimulating hormone, lute-stimulating hormone or their p subunit, human placental lactogen, human prolactin, human chorionic gonadotropin, 20-30 or 30-39 amino peptide consisting of C-terminal

residues of L-sub-unit of human chorionic gonadotropin, the general formula

X-Ser-Ser-Ser-Lys-Ala-Prq-Pro-ProSer-Leu-Pro-Ser-y,

where X denotes;

Ser-Lys-Glu-Pro-Leu-Arg-Pro-Arg -Cys-Arg-Pro-lle-Asn-Ala-Thr-Leu-Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val- Cys 2.9 lle-Thr-Val-Asn-Thr-Thr-lle-Cys - A. ln4-O

-Gly-Try-Cys-Pro-Thr-Met-Thr-Arg-Val50 - Leu-Gln-Gly-Val-Leu-Pro-Ala-Leu-Pro60

-Gln-Val-Val-Cys-Asn-Try-Arg-Asp-v iilo-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-G.i.y50

-Cys-Pro-Arg-Gly-Val-Asn-Pro-Val-Vfal-Ser-Tyr-Ala-Val-Ala-L .eu-Ser-Cyg, - Gln90 - Cys-Ala-Leu-Cy, 4 -Arg-Arg-Ser-Thr Thr-Asp-: ys-Gly-Gly-Pro-Lys-Asp-nis1-IQ

-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Fhe-Gln-Asp-Ser,

OR Ser-Lys-Gln-Pro-Leu-Arg-ProHO

Arg-Cys-Arg-Pro- Pe-Asn-Al a-Thr Leu20

-Ala-Val-Glu-Lys-Glu-GJy-Cys-Pro-Val29

-Cys-He-Thr-Val-Asn-Thr-Thr-lle-Cys3 6 AQ -Ala-Gly-Tyr-Cys-Pro-Thr-Met-Thr-Arg-Val-Leu-Gln Gly-Val-Leu- Pro-Ala-Leu-Pro-Glx-Leu-Val-Cys-Asn Tyr-Arg Asp. ", R, ° -Val-Arg-Phe-Glu-Ser-Ile-Arg-Leu-Pro-GIy-Cys-Pro-Arg Gly-Val-Asn Pro-Val-a ° I - Ser-Tyr-Ala -Val-AIa-Leu-Ser-: ys-GlnKfys-Ala-Leu-Cys-Arg- (Arg) -Ser-Thr-Thr-Asp-Cys-Gly-Gly-Pro-Lys-Asp-llis-Pro- Leu-Thr-cTs-AsF-Asp-Pro-Arg-Phe-Gln-Asp, or Asp-AsfT-Pro-Arg-Phe-Gln-Asp-Ser-Ser-Ser-slr-Lys-Ala-Pro-Pro -Pro-Ser-Leu-Pro-Ser-Pro-Ser, PP-LCH-BRG V And JJ-J., or Thr, Cys-Asp-Asp-Pro-Arg-Phe - -. -G1 -Ai Phe-Gln-AsD,, 1 ... or Phe-Gln-Asp-Ser ,, ,,, „,„., or Asp-Asp-Pro-Arg-Phe-Gln-Asp, or Asp-Asp-Pro-Arg-Phe-Gln-Asp „or Asp- Ilis-Pro-Leu-Thr-Cys-Asp-Ac ry-Prn Ara-Php-rln-Ast Ser Asp-Pro Arg Phe-Cln Asp-Ser, and U Drests U Denotes. -Pro-slr-Arg-Leu-Pro-Gly-Pro-sfr ,, „,,,„ - Р, о-, г „,„ Whether P ro-sir-Ar, -Leu-Po-Iy-Prot, ml-t1 g, -, G.1 -Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln p ber-Leu go, "," o, or Arg-Leu-Pro-Gly-Pro-Ser- Asp FU-p -P-t -p-P inr Fro- le i.eu Fr p, t g, gh g, or Pro-Ser-Arg-Leu-Pro-Gly-Pro, „. - „„, -Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln, or Pro-Ser-Arg-Leu-Pro-Gly-Pro36034, -Pro-Asx-Thr-Pro-Ile-Leu-Pro -Gln-Ser-Leu-Pro, or Pro-Ser-ftrg-Leu-Pro-Gly-Pro-Pro-Asx-Ttir-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro, Pro-Ser- Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln. "Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asp-Thr-Pro-Ile- Leu-Pro-Gln20 - e-P ° Pro-Ser-Arg-Leu-ProGly-Pro-Pro-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro / is not reacted with containing diazo groups, such. diazosulfonyl acid, dextR dinitrophenol, trinitrophenol, S-acetonemercaptohydrate anhydride, succinic acid or direct or branched 30%, natural proteins, sucrose, copolymerized with epichlorohydrin, polyglucose a, homologous to serum albumin, hemocyanin, ayy, ay, a, and hemocyanin, akin homology, a homologous to serum albumin, hemocyanin, ay, a, and homologous serum albumin, a homologous serum albumin, hemogan, and you apply, you can also apply, and homologous serum alkanine, polymorphism 40 polypeptide structures, carried out as follows, 5 dfs, as Hemocyanin from Keyhole limpet, containing 40 free amino groups, are prepared in a buf® solution, as phosphate buffer, in a solution of chloride Atri R R K solution modifying group is added toluoll: diisocyanate reagent diluted 45dioksanom from about 1-10 to 1-40 Number 1 of toluene to be added to the solution, the stake may ° "/ a YGoG °; Leray -;. G, aTG ° TeT, 1: G..Gg, .T-, (+ 10) ° C, preferably 0-4 ° C, in those; 2-2 hours. Excess toluene diisocyanate is removed by centrifugation, 5 the precipitate is washed with phosphate the buffer, the remaining substances are combined, the activated solution of the modifying group is then combined with the hormonal hormone 60 polypeptides to be conjugated and, the 6U polypeptide is dissolved in the same phosphate buffer (5-30 mg / ml), the entire amount of modifier and polypeptide is combined with molar dS ratio desired in the conjugate product. The combined solutions are reacted at 30-50 ° C, preferably 35-40 ° C, for 3-6 hours. The modified polypeptide and the free unattached polypeptide can be separated by conventional methods, for example, by gel filtration. As an indicator, picogram quantities of L-labeled polypeptide are added to the reaction mixture during the coupling reaction, and the amount obtained by conjugation of the polypeptide with modifying groups (molar ratio) is determined by the amount of radioactivity. Methods for modifying hormones, non-hormonal proteins and their fragments (unmodified polypeptides) include a combination reaction with npHr-ieHeHKef-i water-soluble carbodiimide. The amino groups of the unmodified polypeptide are first preferably protected by acetylation, then this unmodified (acetylated) polypeptide is conjugated with a modifier, such as a natural protein modifier, for example, a homocyanin from Keyhole limpet, homologous albumin with an angle, and the like. or dextrans, FicoII agents or polytyrosine, preferably in the presence of guanidine, such as guanidine HCI, used as an activating reagent 10-ethyl-3 (3-dimethylaminopropyl) -carbidiimide. In the case of the use of the agent PicoII 70, it is first treated with ethylene diamine in order to achieve a more effective final combination. Treatment with ethylenediamine is carried out in solution or dioxane, at room temperature and pH, preferably 10-11, for VJ 1 / 4-2 hours. The combination of the unmodified polypeptide with the modifier is carried out in a solution, for example, glycine methyl ether, maintaining the pH 4- 5, preferably 4.5-4.8, at room temperature for 2-8, preferably 5 hours. The resulting modified polypeptide can be purified by conventional methods by column chromatography. Immunity-inducing substances are obtained by polymerizing an unmodified polypeptide using bifunctional imidoesters. Imido esters (dimethyl adipimidate, dimethyl suberimidate idethyl lonimidate) can be used to make a polymer. Polymerization is usually carried out at room temperature in an aqueous solution at pH 9-12, preferably about 10-11 for 1 / 4-2 hours. Immunity-inducing substances can also be obtained by dimerization through a sulfide bond formed by the oxidation of a thiol group to cis -the residue, using iodobenzene acid and reaction at room temperature for 10-40 minutes Modified polypeptides can also be obtained using glutaraldehyde as a conjugating agent. Commercial glutaral dialdehyde does not actually contain free glutaral dialdehyde, but consists of a very complex mixture of polymers containing a large amount of at, p - unsaturated degidy. By interacting with natural protein modifying agents, such as homologous serum alumina, these polymers form a stable bond with one another, pst. CPO of the amino group, Ostapich and al-hydroxy groups of the free. This prom:;: (the weft product is then filled with non-qualified polypeptides in the presence of an alkali metal borohydride, such as sodium pyohydride. This intermediate product is formed at a pH of 7-10, preferably 8-9, at a temperature close to room temperature. Modified polypeptide The reaction is also obtained at room temperature after about 1 / 4-2 hours. The 1 o-product obtained is isolated in its pure form by conventional methods (gel physical therapy, dialysis, and lyophilization: eL). Saccharin-polymerized Frogoll 70 or dextre nT 70 is prepared for use in conjugation by treating cyanuric acid with halogen-anhydride as cyanuric acid chloride to form a dihalo triazipyl adduct.The reaction is carried out in a solvent such as dimethylformamide, at 0-20 C., preferably 10-15. ° C, approximately 1/24 h. The resulting intermediate} 5 is dialyzed until complete removal of halitic ions, is lyophilized and treated with unmodified polypeptides at pH 8-11, preferably 9-10, F for 5-12 hours at 15-35 ° C, usually with cc v natn temperature The resulting modified polypeptide can be isolated by known methods. The above-mentioned polymerized sugar modifiers can also be treated with alkali metal periodates at pH 3-6, 3A-C for 1/24 h, after which the resulting intermediate product is subjected to a coupling reaction with an unmodified polypeptide at pH 7-11, preferably 8-10, for about 1 / 4-2 hours at a temperature of 15-80 ° C, preferably 20 ° C. The resulting immunity substance is recovered by the above techniques.

Modifying groups can only be attached to certain amino acid moieties, in particular, they form a chemical bond only with histidine / arginine and triozinovmi and lysine moieties. Thus, depending on the size and chemical type required of this modified polypeptide, it would not be difficult for a specialist to calculate the maximum number of modifying groups that can be combined with the polypeptide. Several modifying groups can join each other and then join one amino acid part.

Polypeptides that have a contraceptive effect are introduced into the body of an animal or patient, preferably with a physiologically compatible filler, given injections. They can be administered with conventional excipients or agents, as appropriate, containing other standard excipients, in the form I of injectable solutions or suspensions, preferably for intramuscular administration.

The amount of modified polypeptide to be injected depends on cs. however, satisfactory results are achieved when standard doses of 0.1–50 mg are administered to a large mammal from 1 to 5 times at intervals of 1 to 5 weeks.

Example 1. During at least one menstrual cycle, adult female baboons were studied for the purpose of obtaining samples and samples of urinary estrogens, plasma, progestin and, in some cases, urinary hormone anterior lobe of the hypophysis GPDR. Only animals that gave normal samples of these hormones were subjected to unification.

The biological activity of the anterior pituitary gland hormone and the ovarian follicle stimulating activity in a woman of the chorionic hormone is determined using an exhaustion test. The ovary is ascorbic acid, and the follicle-stimulating hormone is tested with an ovary enlargement test.

Hormones are modified into antigens by combining with g apten at different ratios of hapten and hormone. In this process, the protein hormone serves as a carrier, and the hapten is connected to it through diazo bonds. Although a number of hapten groups were attached to different hormones, the same basic process is used for any combination. For the production of IgF degrading antigens, the best number of hapten groups per hormone molecule is 15-35.

The main reaction consists in the diazotization of the hapten (sulfamic acid) by adding it to a 0.11 n solution. hydrochloric acid followed by the slow addition of this solution dropwise to a 1% solution of NaNOa with constant stirring at 4 ° C. Diazotization is considered complete when free HNO is detected in the reaction mixture. The reaction is carried out at + 4 ° C, the optimum temperatures are about 0-6 ° C.

The hapteno-protein compound is made by dissolving protein hormone in an alkaline buffer, pH. The diazotized hapten is slowly added to the hormone solution with constant stirring at 4 ° C. After adding the entire hapten, the pH is finally adjusted to 8.0, 1-2 hours, stirred and incubated overnight at 4 ° C. The mixture is carefully dialyzed 6-8 days into distilled water in order to remove unreacted hapten.

Although the number of diazo groups per hormonal molecule can be controlled by the number of interacting moles of hapten and hormone, a parallel control test with sulfanilic acid with a labeled S atom is carried out simultaneously with each diazotization in order to clarify the composition of hapten-protein samples. In the control test, the same hormone preparation was used as for immunization. At the end of the reaction, an aliquot is taken from the reaction mixture, and the residue is dialyzed carefully. Equal volumes of dialyzed and non-dialyzed solutions are measured by liquid scintillation. After comparing the dialyzed and non-dialyzed samples, the hapten molecules associated with each hormone molecule are counted; unreacted hapten is removed by dialysis. In accordance with this calculation, the molecular weight of 30.00 is determined for all gonadotropin preparations.

After dialysis, hapten hormones are lyophilized and stored at 4 ° C. Diazo-CG (35 groups per molecule) and hormone of the anterior lobe of the human pituitary (26 groups per molecule) are tested biologically using the ovarian depletion method with ascorbic acid; it was found that 62% and 85%, respectively, of the activity of the unchanged hormones from which they were derived, remained. None of the other hormones have been tested for biological activity.

Ways of immunization. Female baboons are subjected to the first immunization for 3-5 days of the menstrual cycle, the second and third injections in a week. The fourth injection is carried out 2-3 weeks after the third. Several animals get the fifth injection 70-80 days after the first one. All antigens are injected subcutaneously in a suspension of mannio da manoleate or arachid oil. For each injection, the doses of antigen range from 3-5 mg. Injection sequences are monitored daily for 5 days after each and F1unization for local reactions. Control action of immunization For at least one menstrual cycle before immunization and for the entire period of immunization before the effect of treatment, urine samples are collected around the clock every day and uninterruptedly - serum samples. The urinary hormone anterior pituitary HPDG, urinary estrogens and plasma progestins are measured. Antibodies were detected in serum samples obtained after immunization by the interaction of 0.2 ml of diluted serum (1: 1000) in phosphate buffered saline (pH 7.4) and 0.5% of normal baboon serum with 250 p of the hormone with labeled atoms. The syringes are reacted with an unmodified immunizing hormone and the unmodified hormone of the anterior pituitary baboon for the purpose of detecting antibodies. As an antigen indicator, a purified hormone preparation of the anterior lobe of the pituitary baboon is used. Complexes of antigen-antibodies are precipitated with the help of sheep antibabuin gamma-globulin 24 hours after incubation at 4 ° C. Antibody levels are expressed as linking a hormone to labeled atoms. Significant antibody levels are capable of attaching 5.0 mg or more of an antigen to labeled atoms. The immune sera are fractionated by gel filtration on Sephadex G-200 to determine the ratio of the ZdM antibodies to 3gG in baboon serum. Since the 3gG fraction contains a certain amount of OdA and 3gD antibodies, only TlgM and total titer are determined. The ZDM fraction from the column is reacted with 3-hormones and the ability to form a bond is determined. The volumes of fractionated serum were adjusted so that antibody levels could be compared with serum levels. Obtaining antibodies. At the injection sites, there were no noticeable reactions after any immunization. In 4 cases, a slight hardening of the tissue (2-3 cm in diameter) was observed, when mannide manoleate was used as a carrier, but the red note and the tumor disappeared within 4-5 days. Within 3–5 weeks, antibodies against the immunizing antigen were detected in all animals. The size, duration, and crossover response of these antibodies is fixed. In total, higher levels were observed with alien gonadotropin immunization than with homologous immunization. The crossover of injected antibodies with a GPDG baboon was studied in each animal. The crossover of immune sera at peak levels was recorded. Although a relatively high activity of antibodies against human GPDG was observed, the response to baboon's GPDG was relatively small. An intermediate cross-reaction with anti-liver GPDG was observed, as well as a high degree of cross-reactivity with anti-liver GPDG. The diazo-human follicle-stimulating hormone FSH has been found to be more, antigenic in the baboon. The duration of antibody formation was longer with human and sheep gonadotropin immunization than with monkey. Antibody level peaks were typically observed when antibodies moved in principle to the 3gG type. Early antibodies possessed a large amount of DgM type and, as a rule, greater cross-reactivity relative to sheep GPDG. The change in the amount of all dM antibodies was recorded from the very beginning of the detection of antibodies. Significant cross-reactivity to baboon GPDG was observed in anti-human gonadotropins, when EDM was abundant, but quickly subsided, when their ivy serums moved to almost. This differential cross-reactivity did not occur during immunization with monkeys and baboons and hormones Immunization, sheep GPDG caused a temporary change in reactivity when moving from to DgG. The effect on the menstrual cycle. The effects of immunization on the menstrual cycle are determined by observing changes in the swelling of the skin of the genital organs and changes in the level of the hormones of the pituitary and / or ovaries. The delay in ovulation is determined relative to the expected period obtained from the control cycle. One animal immunized with human CG showed no ovulation delay; in another, immunized with human FSH, the lag was only one cycle. In two animals immunized with human GPDG, and in two animals immunized with human CG, the ovulation rate was equal to two menstrual cycles. In the third animal, immunized with human GPDG, ovulation of e-paedala for 86 days. Sheep GPDG caused a delay of 88 days. Immunization with diazo or baboon GPDG causes prolonged interruption of the menstrual cycle. The lag in ovulitis in two animals immunized with monkey GPDG is 146 and 122 days, and in animals immunized with modified baboon GPDG, ovulation is observed 224 and 210 days later. The effects on the specific properties of the hormone after immunization with a single animal to human GPDG were registered. The interval between changes was considered a cycle. Samples and samples of urinary astrogens and progestin plasma showed that during the cycle of Ig-1, the duration of 85 days, no ovulation occurred. The level of urinary estrogens rose during the treatment period, but the estrogens did not produce a typical reaction. Plasma progestins did not increase until approximately the 19th day of the first cycle after treatment. The reactions of estrogen and progestins did not go beyond the normal during the second cycle after treatment. Antibody levels increased approximately from the 35th day of the treatment cycle to the 289th day, starting with the first detection of antibodies. During the study of this animal, the GPG test was not available, and no data were obtained regarding the level of this hormone in the plasma or urine. Samples of the hormone were recorded after immunization with diazobabuinovogo GPDG. In this animal, antibody levels were lower and existed, as a rule, for a shorter period of time than when immunized with ps and human gonadotropins. During the treatment cycle, the levels of estrogen in the urine and plasma progestins corresponded to normal samples, but were quantitatively lower than normal, and urine GPDG patterns markedly changed due to diazo-GPDG injections. During this period, there were no signs of ovulation. The first cycle after treatment was 246 days. During this cycle, levels of GPDG and estrogens in the urine increased for 35-41 days, but were not observed due to the next increase in the level of progestin plasma, which would mean ovulation. For the next 42 days No significant increase in the level of any of the three hormones was observed until the 231st day, when a strong increase in the levels of estrogen and HPDG in the urine was observed. Three days later, the level of plasma progestins increased, indicating the presence of a functioning yellow body of the ovary. The second menstrual cycle after immunization was lasting nopv, endocrine samples were normal. Antibodies against unmodified baboon GPDG reached a maximum level of about 70 days after treatment; this level remained relatively constant until the 190th day, when a steady deviation was observed. On the 215th day of this cycle, antibodies could almost not be detected. 16 days after this period, a peak of GPDG was found, corresponding to a normal increase in the level in the middle of the cycle. Starting from this moment, the function of the pituitary axis ichnikovoy animal was normalnoy.Gormonalnye animal specimens with other geterologovymi gonadotropin immunization used were similar to those in animals immunized with human GPDG and other animals immunized monkey or baboon Nyima GPDG reacted similarly with animals immunized with baboon GPDG. The results obtained on baboons show that by modifying the hormone of reproduction through the processes outlined above, antigenicity is attached to it and that the antibodies thus obtained neutralize natural endogenous hormones if the natural hormone is obtained from an animal species immunized with a modified hormone. Example 2. Human HGH is present only in the body of a pregnant woman, the GPDG hormone is immunologically and biologically identical to the human CG hormone, although there are chemical differences between them. Since they are biologically identical, the effectiveness of this immunological process can be evaluated by nyTSvi injecting modified human CG into the body of a non-pregnant woman, controlling the levels of HPDG in the blood. The resulting antibodies neutralize both GPDG and modified human CG. There is a sample of levels of GPD in women, it is mostly constant until the middle of the period between the menstrual period of Mtj, immediately before ovulation; In slow ovulation, the level of GPDG increases greatly, helping to induce ovulation. l diyu By monitoring the level of GPD1 and the level of antibodies, it can be seen whether the process caused or did not cause the formation of anti-. bodies capable of neutralizing the endogenous hormone of reproduction, namely GPDG. . The experience produced on the 27-year-old wives shine. Hormone biol obtained, purified, modified. Modified human hormone hormone was then introduced into the body of this woman. It is well known that antibodies against human chronic hormone react identically to GPDG. The effect of immunization was increased, mainly by controlling the levels of the hormone GPDG in the blood. Then the results were processed. Getting a hormone. Clinical human CG derived from the urine of a pregnant woman has an immunological dilution rate of 2,600 immune units / mg. In this preparation, contaminating substances were detected. Purification is performed by chromatography and elution. The fraction is dialyzed and lyophilized. The strongest fraction contains l 7600 immune units / mg, however, it gives a heterogeneous reaction when gel electrophoresis on oleyl acrylamide. The fraction is purified further by gel filtration. Elution showed two major protein peaks. The strongest human CG was detected at the first peak and is characterized by an immunological dilution rate of 13,670 immunological units / mg. The fraction is subjected to liacrylamide gel electrophoresis. Further purification from gelfiltration shows the lack of heterogeneity of human chronic hepatitis at this stage. The research materials are processed according to the above method. The contaminating substances of this purified human CG are tested using, for use in identification, one sample is subjected to interaction with the bacteria against some proteins that give a strong contamination. These proteins include FSH, human growth hormone, full of human serum, human protein, transferrin, alpha-1-globulin, alpha-2-globulin and orosomucoid. No association of purified human CG was detected with any immune serum at a dilution of 1:50. These negative results indicate that contamination by any one of them was below 0.005%. Modification of the hormone. The hormone is modified by the binding pathway to the hapten (sulfanilazo). In accordance with this method, hapten molecules bind with the protein of the chain to the amino group of the aliphatic or aromatic portion of the hapten. The number of hapten molecules linked to each human CG molecule (ha-hCG) can be regulated; in the present case, 40 haptenic groups per hCG molecule are used to produce an immunizing antigen. After the hapten binding process, ha-hCGH is sterilized and tested. The patient has already given birth to several children; reproduction properties were terminated by voluntary bilateral removal of the fallopian tube. The patient was healthy, menstruation was regular. She was subjected to a full medical examination with subsequent laboratory evaluation of the data obtained, including hemogram, urinalysis, latex fixation and a Pap smear (R. smear). Allergies never suffered. In order to demonstrate the normal function of the pituitary-ovary axis before immunization, starting from the first day of menstruation, blood was drawn every second day for 10 days, then every day for days and finally every second day before the next menstruation. Qualitative analyzes of serum FSH, GPDG, estrone, extradiol and progesterone were performed. These studies showed ovulation. 10 mg of ha-hCG antigen are dissolved in 1.0 ml of solution and emulsified with the same amount of oil. Before injection, skin tests are made with notches on the antigen and the carrier. Immunization is carried out in the phase of formation of the corpus luteum of the ovary in order to prevent over-ovulation due to injected hCG. The patient is given four injections over each two weeks. The first two are introduced into the oil subcutaneously (1.0 ml in the upper part of each arm); the last two injections are administered in the solution only through the intestines. After each injection, the blood pressure is checked, as well as the possible allergic reaction of the patient. Two weeks after the first injection, blood is taken every week to determine the presence of humoral and cellular antibodies. After the end of the immunization process, blood is collected in a manner similar to the method in the control cycle in order to confirm the effect of immunization on the hormonal distribution of the menstrual cycle. Since anti-hCG antibodies react identically to GPDG, they are monitored as indicators of the effectiveness of the process. The third cycle is examined similarly 6 months after the first immunization. At the end of the study, laboratory evaluations were repeated. Control serum and posttreatment serum samples were tested.

for the presence of FSH, GPDG, estrina, estradiol and progesterone.

Results. The ratios between the pituitary serum and gonadal hormones in the patient's control cycles were fixed. After two injections in the patient's body, antibody titers against ChCH were detected. During the immunization, menstruation took place at regular intervals.

After the first injection of mannide in manrleate, itching and swelling appeared at the injection site. Subsequent injections through the intestine in the solution did not result in any reactions, from which it was concluded that the local reactions were due to mannide mannide. Tests of lymphocyte transformation on plasma samples gave negative results.

During the cycle after treatment, the follicular zero line and the levels of GPDG. the phases of formation of the yellow body of the ovary did not change much. Compared with the usual large increase in the middle of the cycle, there were only very slight increases in the level of GPDG. The distribution of FSH in the cycle after treatment was normal HiiM. This indicates that the antibodies neutralized the action of endogenous GPD1.

The patient was examined for one more cycle approximately 6 months after the first immunization. Significant antibody titers were detected, indicating a slight increase in GPDG in the middle of the cycle, whereas FSH was mostly normal. Thus, the specificity of anti-ChCH-antibodies to GPDG, but not to FSH was proved.

Example 3. Additionally investigated a 29-year-old woman. Analogously to example 2, the hormone was obtained, purified, modified, and administered to a patient who was under control and examined as described in example 2.

The results did not differ from those obtained in Example 2, except for the fact that estrone and estradiol levels were generally normal. The patient developed significant antibody titers in the last segment of the post-immunization cycle; in the cycle studied every six months, the patient did not have significant increases in mid-cycle GPD1 levels.

Example 4 o A 29-year-old woman was examined. The hormone is obtained, purified and modified, and administered (as in Example 2) to the patient, which is observed and examined as in Example 2.

The results do not differ from those obtained in Example 2, except that the follicular zero and the levels of the GPDG phase of the formation of the corpus luteum of the egg in the post-immunization cycle were significantly reduced; no increase in the levels of the GPDG was observed in the middle of the cycle; estrone levels are elevated during the follicular phase of the post-immunization cycle; During the six months of the study, no significant increase in mid-cycle levels of HPDG was observed.

Example 5. A 35-year-old woman is examined. The hormone is obtained, purified is modified, and, analogously to example 2, the patient is administered, which is controlled and examined as in example 2.

The results do not differ from those obtained in example 2, except that in the post-immunization period the follicular line is zero and the levels of HPDG in the formation phase of the yellow body of the egg are markedly reduced; there is a very slight increase in GPDG levels in the middle of the cycle, the levels of FSH in the post-immunization period are reduced, the levels of estrone and estradiol are reduced during the follicular phase of the post-immunization period.

Example 6. Additionally, a woman of 28 years old is examined. The hormone is obtained, purified, modified, and, analogously to Example 2, administered to a patient, which is monitored and examined as Example 2.

The results do not differ from those obtained in Example 2, except that in the post-immunization cycle the follicular line is zero and the levels of HPDG in the formation phase of the yellow body of the egg are markedly reduced; no GPGG peaks observed in the middle of the cycle; in the follicular phase of the post-immunization cycle, estrone levels are elevated; in the six-month post-immunization cycle, no significant increase in the level of GPDG was found in the middle of the cycle. - Example 7. Additionally, investigate another 28-year-old woman. The hormone is obtained, purified, modified, and analogous to example 2, is introduced into the patient's body, which is controlled and examined as in example 2.

The results do not differ from those obtained in Example 2, except that up to the first three injections no anti-hCG antibody titers were detected, in the post-immunization cycle the follicular zero line and the levels of HPDG formation of the yellow body of the egg; there are no peaks or increases in the middle of the GPDG cycle; estrone levels are increased during the follicular phase; no significant antibody titers were detected in the six-month cycle.

All the above examples show the feasibility of introducing modified hormones to neutralize the endogenous multiplication hormone.

Example 8. A modified natural reproduction hormone, when introduced into an animal of the species from which the hormone was obtained, causes the formation of antibodies that neutralize the effect of the unmodified endogenous natural hormone in the animal's organism. In Examples 1-7, FSH, HCG, and GPDG are used.

Purified placental lactogen preparation is obtained from placenta baboons. The placents are extracted, purified by chromatography on a column, and the purity is controlled by polyacrylamide gel, electrophoresis, and immunity testing by radioactive isotopes. The material obtained is characterized by a high degree of purity, and the method of testing with radioactive isotopes has shown the absence of contamination with other placental hormones.

The placental lactogen of the baboon (PLB) is modified by attaching a sulfanilic acid salt to the diazonium salt in analogy to Example 1. In this case, the number of diazo molecules on the PLB is equal to ieTCH 15. The immunization processes do not differ from those in Example 1.

Within 4-6 weeks in the tube after the first injection of the diazo-PLB, levels of antibodies against natural unmodified PLB were detected in 6 female baboons. Levels rose to a certain height for 8–10 weeks and remained in that position for several months. Hormonal measurements showed that immunization did not cause changes in the normal menstrual cycle. Since PLB usually only occurs in a state of pregnancy, this fact was not unexpected.

i

All 6 females mate with samtsotl,

studied for fertility, 3 times. (once during three different cycles during the period fertile; ti). After each pairing, a conception is made by hormonal measurement. Out of 13 cases of mating in 13 cases, conception occurred, proved by an appropriate test. In fertilized animals, menstruation appears 7–12 days later compared to normal. Subsequent hormonal measurements showed that in all 6 females, 13 fertilizations ended in miscarriages approximately a week after the expected menstrual flow.

These results show that antibodies produced in an animal after immunization do not have

effects on the menstrual cycle in unfertilized females, but upon fertilization, they neutralize the baboon placenta lactogen in the baboon placenta followed by miscarriage very soon after conception.

When modifying FSH, samotomedian, growth hormone or angiotension II using diaeosulfanyl acid or troynitrofenol, the results obtained when the purified modified polypeptide is administered to the male organism indicate stimulation of antibodies that neutralize all modified polypeptides or part

them, like the corresponding endogenous

polypeptide.

Example 9. The experiments and studies listed in the above described examples of measurements were carried out on baboons, Zhi iBOTHHe were adults and capable

to breed. In the study of experimental organisms, highly purified hCG p -sub units are used as a preparation of biological activity less than 1.0 immune units / mg. Animals are immunized with 14-26 moles / mole

polypeptide associated with diazasulfanilic acid subunits manoleate mannide.

Antibody levels are determined by

links to diluted sera with a D antigen with a labeled atom. Cross-reactivity and immune sera are measured by direct attachment of antigens.

with a labeled atom and immunity test using a radioactive isotope displacement method. The contraceptive effect in actively immunized animals is determined by

mating females with males capable of breeding. The effect on the body of pregnant baboons passively immunized with either sheep or baboon anti- (b-hCG is determined by controlling serum levels of go45 for dotropin and steroid sex hormones before and after immunization.

8 female baboons are immunized with a modified p-sub-unit of 50% HPC. Significant levels of antibodies were found in all animals.

The result of immunization of baboons with a modified hCh subunit

55 were high levels of antibodies reacting to ChGG, human GPG and baboon CG, but not to baboon GPDG. All animals remain capable of ovulation, but after a number of

60 matings do not fertilize. Passive immunization of pregnant women not immunized to pregnant women with anti-sheep-serum sheep serum causes a violation of pregnancy (abortion)

65 for 36-44 hours

EXAMPLE 10 A hemocyanin solution was made from Keyhole limpet (KHL) (7 mg / ml) in 0.05 mol of sodium phosphate buffer in 0.2 mol sodium chloride, pH 7.5. Insoluble particles are removed by centrifugation. To 1 ml of this solution, 20 µl of a toluene diisocyanate reagent diluted with dioxane (1/30) is added, and the amount is basically equivalent to the amount of moles of lysyl residues in KLH peptide molecules.

After 40 minutes with toluene diisocyanate-activated, the KHL solution is combined with 0.5 mg of synthetic (L-HCC-peptide, which is previously dissolved in 25 µl of 0.05 M sodium phosphate buffer in 0.02 M sodium chloride, pH 7.5. The mixture is incubated for 4 hours. The resulting product is purified by gel filtration.

Example 11. 1 g 70 is dissolved in 1 ml of a common solution and 1 ml of 2 M ethylenediamine (the pH is adjusted to 10 with hydrochloric acid). The solution above is baked at room temperature in a water bath, stirred with a magnetic stirrer, add

4g cyanogen bromide dissolved in 8 ml of dioxane. The acidity of the mixture is maintained at a pH of 10-10, .5 for 8 minutes by adding drops of 2, sodium hydroxide solution. For addition: add another 2 ml of a 2 M solution of ethylene diamine, pH 10, while continuing to stir at room temperature for

30 min., The product is purified by passing through a biogel column.

Example 12. 2 mg of a compound containing a picogram of 3-labeled adduct and 1.6 mg of KLH are dissolved in 1 ml of 1 M glycine methyl ester in 5 M guanidine hydrochloride. Then 19.1 mg of ethyl dimethylaminopropylcarbodiimide is added to the solution. Acidity is reduced to. pH 4.75 and maintained at this value for 5 h at room temperature with the help of 1 and. sodium chloride. The conjugated KLH-peptide is purified by passing a biogel through the column (2.2x28 cm), equilibrated with 0.2 M sodium chloride &

Example 13. At room temperature, in a continuously stirred solution of 1 mol of the polypeptide (1-20 mg / ml) in 0.1 M sodium phosphate in portions of 2 g are added every

5 min 3 mol of solid bifunctional imido ester dihydrochloride, 0.1N is added to maintain pH 10.5. hydroxide, sodium. After 1 hour, upon completion of the addition of the diimidoester, a polymerization product according to the invention is obtained.

Example 14. To a solution (20 mg / ml) of homologous serum albumin in 0.1 M borate buffer, pH 8.5, a 1000% molar excess of a 25% aqueous solution of glutaral dialdehyde was added at room temperature. Excess dialdehyde is removed by gel filtration in water using biogel p2. The material collected in the empty space is lyophilized, dry. PRODUCT REPEATED

dissolved in 0.1 M borate buffer, pH 8.5 (20 mg / ml), mixed with an appropriate amount of polypeptide (20 mg / ml) in the same buffer at room temperature. After 20 min, sodium borohydride was added in a 250% molar excess of polypeptide-1X. The reaction is complete after 1 hour. The conjugated product is purified by gel filtration on a 0 p-60 biogel column, dialyzed to remove salt and lyophilized.

Example 15. When TeMnepatype below 16 ° C for 2 h stir

1g Fico 11 70, 500 mg of NaHCO, 3 g of 5 Tsiakur chloride, 20 ml of water and

80 ml of dimethylformamide. The product is distilled against distilled water until chlorine is removed, then lyophilized. The polypeptide (2 mg) containing

0 minimum amount of 3-labeled analogue, incubated with 1 mg of this product in 0.25 ml of 0.2 M sodium borate buffer, pH 9.5, for 1 hour at 20 ° C, after which the product is isolated on

5 column of biogel (2.2 x 28 cm).

When carrying out the above reaction using dextran T 70 instead of Ficoll 70, a corresponding modified polypeptide is obtained,

"Suitable according to the present invention.

Example 16.1 g of ficoll 70, 1.2 g of sodium periodate, 0.42 g of potassium chloride are dissolved in 1.5 ml of 1 M sodium acetate solution, pH 4.5, and

 incubated at 37 ° C for 1 h.

2 mg (588 moles) of a polypeptide mixed with a minimum amount of a 3-labeled analogue are incubated with

2mg of superbatch product

0 0.3 ml of 0.2 M borate buffer, pH 9.5, at 55 ° C for 1 hour. The reaction mixture is then rapidly cooled in an ice bath, after which 1 mg of sodium borohydride is added. The recovery reaction is prevented by passing the product through a p-60 biogel column (2.2 X 28 cm), equilibrated and eluted with 0.2 M sodium chloride. Example 17. Several rabbits were immunized with various synthetic peptides conjugated to form different modifying groups. After two or three doses of immunization at intervals from 3 to

Five to five weeks of sera from experimental animals are evaluated by determining their ability to in vial radioactively labeled HCG. The specificity of this bond is investigated by the reaction of these same sera with respect to the other protein hormones labeled in the usual way, in particular, the LH appendage of the brain. Serum is further evaluated by determining its ability to inhibit the biological effect of HCG exogenously administered to animals in a biological test. Thus, an increase in the weight of the uterus of pregnant rats, as a reaction to the prescribed dose of HCG, is subject to registration. The HCG dose is administered subcutaneously in a physiological solution of 5-shot sodium salt for 3 days, then the animal is killed in order to remove the uterus on the fourth day. The weight of the uterus increases in accordance with the increase in the amount of hormone injection. When assessing the effect of antisera, regardless of the subcutaneous administration of the hormone, different amounts of the test serum are also administered intraperitoneally. This contributes to the rapid uptake of anti-shortcuts to the 5th flow of the rat and allows it to interact with the hormone when the latter enters the blood. If antiserum is capable of interacting with the hormone, to prevent stimulation of the uterus, Sterotka is considered an effective biological inhibitor of the action of the hormone.

Example 18. Iodobenzoic acid dissolved in a slight excess of 1 n. Sodium hydroxide is added to the peptide in phosphate buffer with an ordinary solution at pH 7.0. After 30 minutes at room temperature, the polypeptide dimer is purified by gel filtration.

Example 19. Chilled in an ice bath and vigorously stirred sheep OG globulin (0.23 ml) (g / ml) in 0.05 M phosphate buffer at pH 7.5, 50 µl of toluene diisocyanate in dioxane (1: ten). After 40 minutes, the excess toluene diisocyanate is removed by centrifugation (0 ° C, 10 minutes, 10 g), the precipitate is washed twice. The combined remaining products were added to 7.7 mg of peptide dissolved in 0.8 ml of PBS, pH 7.5. The mixture is stirred at room temperature for 10 minutes and then incubated at 37 ° C for 4 hours. The conjugated product is purified by dialysis.

The proposed method allows to obtain polypeptides that can be used as contraceptives.

Claims (1)

Invention Formula A method for producing polypeptides having a contraceptive effect characterized in that polypeptide component isolated from follicle-stimulating hormone, luteostimulating hormone or their 5-subunits, human placental lactogen, human prolactin, human chorionic gonadotropin, 20-30 or 30-39 amino peptide consisting of C-terminal residues of p -sub-unit human chorionic gonadotropin, the general formula X-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Y, where X is: Ser-Lys-Glu-Pro-Leu-Arg-Pro-ArgioIt -Cys-Arg-Pro-Ile-Asn-Ala-Thr-Leu-Ala20 -Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-Cys29 -Ile-Thr-Val Asn-Thr-Thr-Ile-; ys-Ala4C -Gly-Try-Cys-Pro-Thr-Met-Thr-Arg-Val5-0 -Leu-Gln-Cly-Val-Leu-Pro-Ala-Leu-Pro60 -Gln-Val-Val-Cys-Asn-Try-Arg-Asp-Val70 -Arg-Phe-Glu-Ser-lle-Arg-Leu-Pro-Glyec -Cys-Pro-Arg-Gly-Val-As-Pro-Val-Val-Ser-Tyr-Ala-Val-Ala-Leu-Ser-Cys-Gln9O -Cys-Ala-Leu-Cys-Arg-Arg-Ser-Thr-Thr- (00 Asp-Cys-Gly-Gly-Pro-Lys-Asp-II i.s-Pro110 -Leu-Thr-: ys-Asp-Asp-Pro-Arg-Phe-Gln-Asp-Ser, or Ser-Lys-Gin-Pro-Leu-Arg-Pro- oif -Arg-Cys-Arg-Pro-lle-Asn-Ala-Thr Leu20 -Ala-Val-Glu-Lys-Glu-Gly-Cys-Pro-Val-24 - ". -Cys-lle-Thr-Val-Asn-Thr-Thr-Ile-Cys40 -Ala-Gly-Tyr-Cys-Pro-Thr-Met-Thr-Arg50 -Val-Leu-Cln-Gly-Val-Leu-Pro-Ala-Leubo -Pro-Glx-Leu-Val-Cys-Asn-Tyr-Arg-Asp10 -Val-Arg-Phe-Glu-Ser-lle-Arg-Leu-Pro-Gly-Cys-Pro-Arg-Gly-Val-Asn-Pro-ValBo -Val-Ser-Tyr-Ala-Val-Ala-Leu-Ser-Oys-Gln-Cys-Ala-Leu-Cys-Arg- (Arg) -Ser100, -Thr-Thr-Asp-Cys-Gly-Gly-Pro-Lys-Asp-Ills-Pro-Leu-Thr-Cys-Asp-Asp-Pro-Arg-Phe-Gln-Asp, or Asp-Asp-Pro- Arg-Phe-Gin-Asp-H Ser-Ser-Ser-Ser-Lys-Ala-Pro-Pro-Pro-Ser-Leu-Pro-Ser-Pro-Ser, or Gin-Asp-Ser, or Thr, Cys -Asp-Asp-Pro-Arg-Phe-Gln-Asp, or Phe-Gln-Asp, or Phe-Gln-Asp-Ser, or Asp-Asp-Pro-Arg-Phe-Gln-Asp, or Aap-Asp -Pro-Arg-Phe-Gln-Aspili Asp-Ilis-Pro-Leu-Thr-Cys-Asp -Asp-Pro-Arg-Phe-Gln-Asp-Ser, a Y means: Pro-Ser-Arg-Leu- Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln, or Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro -Gln-Se 147 -Leu-Pro, or Arg-Leu-Pro-Gly-Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln, or Pro-Ser-Arg-Leu-Pro-Gly- Pro-Ser-Asp-Thr-Prp-lle-Leu-Pro-Gln, j or Pro-Ser-Arg Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-Ile-Leu-Pro-Gln-Ser -Leu-Pro, or Pro-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asx-Thr-Pro-lle-Leu-Pro-Gln-Ser-Leu-Pro, or Pro-Ser-Arg-Leu -Pro-Gly-Pro-Ser-Asp-Thr-Pro-lle-Leu-Pro-Gln, or Gly, or Pr o-Ser-Arg-Leu-Pro-Gly-Pro-Pro-Asp-Thr-Pro-Ile-Leu-Pro-Gln-Ser-Leu-Pro, Pro-Ser-Arg-Leu-Pro-Gly-Pro- Pro-Asp-Thr-Pro-lle-Leu-Pro-Gln-Ser-Leu-Pro is reacted with a compound containing diazo groups, such as diazosulphonyl acid, dextran, dinitrophenol, trinitrophenol, S-acetamercaptocytic acid anhydride, polytyrosine my or branched, natural proteins, sucrose copolymerized with epichlorohydrin, polyglucose, homologous serum albumin, hemocyanin from Keyhole limpet or thyroglobulin, which are introduced in position 2-40 of the polypeptide structure.