前苏联专利SU1331433A3 Method of production of conjugate compounds of haptens and muramylpeptides

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专利摘要:
The invention relates to peptide derivatives, in particular, the conjugated compound hapten and muramyl peptide of the formula: M-acetylmuramyl-yl-aryl-D-isoglutaminyl-L-lysyl LH-RH (SGM), which has biological activity and can be used in medicine. The goal is to create active substances of the specified class. Synthesis of SGM is carried out from the hormone LH-RH in acidic form and N-acetyl-muramyl-1-alanyl-0-isoglutamyl-1-lysine at a mass ratio of 1: 1, in the aqueous formamide medium by the carbodiimide method in the presence of 1-hydroxyben - Zotriazole. Isolation of SGM is carried out using molecular sieves. The tests of SGM show that it possesses high immunogenic activity at the level of the corresponding muramsht tripeptide, but at the same time it exhibits lower pyrogenicity. 2 tab. CO 00 00 4 ;: 00 oo
公开号:SU1331433A3
申请号:SU833568453
申请日:1983-03-15
公开日:1987-08-15
发明作者:Одибер Франсуаз；Карелли Клод；Шеди Луи；Лефрансьер Пьер；Левель Мишель；Шоай Жан
申请人:Ажанс Насьональ Де Валоризасьон Де Ля Решерш (Анвар) (Фирма)；
IPC主号:

专利说明:

113
The invention relates to a method for producing new conjugated compounds of haptens and muramyl peptides, biologically active compounds that can be used in medicine.
The purpose of the invention is a method of obtaining new conjugated compounds of haptens and muramyl peptides, which have immunogenic activity with low pyrogenicity.
Synthesis of (LH-RH) -MTP. 3 mg of LH-RH, taken in acid form, is added to 242 mg of hydrochloride M-eyl-M - (3-methylaminpropyl) -carbodiimide (ECD 1) and to 43 mg of 1-hydroxybenzotriazole (HOBT), using 0.4 ml of dimethylformamide (DMF) as medium. The incubation is carried out with magnetic stirring for 7 hours at ambient temperature. Then 3.24 mg of MTP (M-acetylmuramsh1-1-alanyl -0-isoglutaminyl-1-lysine), mixed with 0.3 ml of distilled water, and left the mixture to continue the reaction with magnetic stirring for 48 hours at ambient temperature ayuschey environment. The reaction products are dissolved in a 0.15 M NaCl solution and the resulting conjugated compounds are recovered by fractionation through a Sephadex G 10 molecular sieve. Conjugated compounds are washed out in dead volume. The peptide content of muraminic acid is determined. It was found that 93% of the MTPs entered into a coupling reaction: the number of MTP molecules refers to the number of LH-RH molecules as about 1: 1.
The synthesis of the MTP conjugate compound is a tetanic (tetanus) toxin (fulfilled as a comparative example).
Mix 16 mg of MTP, 0.15 ml of DMF, more than 0.0 ml of PBS with a pH of 7.4 (phosphate bucket), then 300 g of ECD 1, 1 mg of HOBT, and 2 mg of tetanic toxin solution. The mixture was allowed to react for 15 minutes with magnetic stirring and at ambient temperature in the dark. Then, 300 g of ECD1, 1 mg of HOBT and 2 mg of tetanic toxin are added to this mixture; the reaction proceeds for another 15 minutes. This operation is repeated 9 times. Then
32
It is estimated that 20 mg of tetanic toxin was used. At the end of the reaction, the conjugate is recovered by chromatography on a Sephadex G75 column in PBS. The conjugate is contained at the top of the wash. Relevant Tetanic Toxin Definitions
and MTP was carried out according to the method Fo-1 i p and Re i SS i g.
Immunization with LH-RH coupled to muramyl peptide and introduced into a saline solution or additive (FIA).
On days 1, 2, 4 and 29, subcutaneously, 0.1 ml of the following solution was administered subcutaneously to a group of young Swiss males that were 6 weeks old:
1) 50 g LH-RH emulsified in addition to complet de Freund;
2) 50 fj.r LH-RH emulsified in FIA;
3) 8 ft ft LH-RH conjugated to 4.3 ft g MDP and emulsified in
FIA;
4) 50 LH-RH in PBS;
5) 8.6 g LH-RH conjugated to 4.3 f g MDP in PBS.
Three additional groups of mice, taken for comparison, are administered in the same way as either PCA, or FiA, or PB $, which contain neither antigen nor MDP.
Before using antigen
(LH-RH pure or combined) was adsorbed on 50% Ruhr (polyvinylpyrrolidone) in 0.9% NaCl, and incubated for 2 hours; then the mixture was subjected
emulsification with FIA or FCA, were added to PBS.
0.1 ml of the compound containing 12.5% Ruhr was administered subcutaneously to mice.
On the 23rd and 50th day, all children were healthy, they were killed, and their serum was collected and combined into groups. The testes of mice and seminal vesicles were analyzed, weighed, after
which they were placed in a Boin solution and prepared for histological examination.
Antibody Antibody Detection (LH-RH).
Antibodies anti-, LH-RH) are detected as follows. 50 liters / l of cheese, undissolved before testing, 50 liters of normal slurry and hare dissolved at 1 / 80th in
PBS, and 10 L (LH-RH). Iodine-125 labeled approximately 10,000 decays per minute (dpm). Incubation proceeded for 72 hours at 4 ° C. Then add 150 p. and suspension, cooled on ice, of activated carbon coated with dextran (obtained by adding 250 mg of active carbon in I00 ml of dextran solution in 0.01 M solution of PBS). The mixture is left at 4 ° C for 5 minutes. After centrifugation, carried out at 2500 rpm for 20 minutes, the substance that appeared on the surface of the mixture is decanted and its radioactivity is determined by the method of Haber. A preliminary study indicates that dextran-coated coal can adsorb up to 95% (LH-RH) pure under the specified conditions.
Detection of anti-MTP antibodies.
The presence of anti-MTP antibodies is monitored by a hemolytic test carried out on micro-dosing plates having recesses in the form of (Cooke Engenering), as follows: shorting is destroyed by inactivation for 30 minutes at 56 ° C. 50) u l of a double-diluted short are mixed with 50 | c. l pbs. Then, 20 | U g of the conjugate compound, fresh blood beads-MTP, having a concentration of 2.5%, are introduced into the mixture, and the mixture is left to incubate on plates at 37 ° C for 15 minutes: finally, 20 l of normal shortening are added guinea pig diluted with PBS to a ratio of 1:20 and again
This mixture is incubated for
37 ° C for 30 minutes before noticing the results.
Pyrogenicity rating.
The experiments were carried out on New Zealand rabbits weighing from 2.5 to 2.8 kg. The temperature values were determined using a probe with a thermistor of rabbits injected into a straight bristle to a depth of about 8 cm and connected to the thermometer model No. TEZ, Copenhagen, Dani. Rabbits were kept and observed in a climate chamber at 20 ° C. Injections were administered only to those animals whose temperature remained stable for the preceding 30 minutes. Temperature values were determined within 3 hours after the injection of the composition, the pyrogenicity of which was studied. The results were expressed in terms of the average temperature change (C) obtained from the rabbit within 3 hours after the intravenous administration of the composition under study. The minimum pyrogen dose corresponds to the amount causing the temperature to rise by,
Immunization experiments.
The results shown in Table 1 express the ratio of the number of strokes per minute (srm) of a surface substance to the number of srm total and multiplied by 100.
Table 1
Treatment
I
Fixation%
LH-RH 50g + FCA30.5
LH-RH 50g + FIA2.5
LH-RH 50 kg + PBS10
(LH-RH) -MTP g + PBS63
0 5
0

In the absence of antigen, no fixation in the short of comparable animals that received an injection of FCA, FIA or PBS (measured fixation) at a maximum of 2.3% (not indicative) is noticed. However, antibodies were formed when 50 g (LH-RH) emulsified in FCA was used (30.5% fixation). No indicative result is observed in the group of animals that received (LH-RH) in the FIA (fixation 2.5%, a weak result obtained using PBS (10%) does not make sense. A significant result was obtained in the study of serum obtained from animals immunized with the conjugation of MTP- (LH-RH), despite the fact that this compound contains only 8.6 | U g (LH-RH) and 4.4 fi g MTP. Antibodies are even increased when an equal number the MTP- (LH-RH) conjugate compound is introduced in an aqueous medium. These results indicate that the MTP- conjugated compounds ( LH-RH) are both more effective than compound (LH-RH) with FCA, as with the FIA and with PBS.
Histological analysis.
The average weight of the testes increased slightly (from 252 to 279 mg) as a result of the introduction of animals from 5 to 50, IG (LH-RH) in PBS. Although this increase is significant (p 0.05), it is not associated with any histological change. Conversely, the same number of antigens introduced into the FCA results in a significant increase in the weight of the seeds and a decrease in sperm production and atrophy of the seed canals in some cases. Conversely, in animals that received the conjugate compound MTP- (LH-RH) taken in solution in PBS, the weight of the testes is lower (p 0.01) than in animals from other groups. Moreover, a histological analysis of the testes showed that only in this group of animals atrophy was observed, noting immediately the interstitial cells and the seed canals, which were practically deprived of germinative cells, as noted in 9 of 10 outlets.
Determination of anti-MTP antibodies.
The same sera were combined to use in determining the activity of fixation.125 1 LH-RH were also tested for anti-MTP antibody content using a hemolytic test with conjugated red blood compounds and MTP. Aiti-MTP rabbit serum obtained after hyperimmunization performed with the conjugated alpha-methyl ester-MTP compound and the shortening of albumin bull was used as a comparative antigen. Then as
According to the rabbit hemolysis reaction in rabbit anti-MTP until a final dissolution of 1: 30.720, no anti-MTP antibodies are noted in any group of animals treated with the conjugate compound MTP- (LH-RH).
Detection of the possible pyrogenicity of the studied conjugated compounds with the compared molecules.
The results shown in Table 2 indicate the lack of pyrogenicity of the proposed compounds compared to pyrogenicity of either the muramyl peptide itself when it is in an isolated state or the conjugate compound that the same muramyl peptide can form with a natural antigen, for example with a theatrical toxin. Conjugated compound t (LH-RH) -MTP does not significantly increase the temperature of the animal, even when the relative dose of MTP in the conjugate compound is approximately 9.3 ju g MTP. These results are comparable to those obtained under the same conditions using comparative conjugated compounds (TT-MTP), which cause a significant increase in temperature, even when the relative content of MTP in the conjugated compound administered to an animal is 100 times lower than amount of the proposed compound.
In addition, higher doses of the conjugate compound (LH-RH) -MTP, which includes a relative amount of 77 jU g MTP, do not show a marked temperature in the animal.
table 2
1,, 6
3.4-1.8
17, 3
86 -46
86
The sign / refers to the combination in the studied conjugate compounds between the relative doses in these conjugate compounds, on the one hand, with the hormone, and on the other hand, with MTP.
The results obtained indicate a low pyrogenicity of the conjugated compound hapten with muramyl peptide obtained by the proposed method: LH-RH-M-acetylmuramyl-1-alanyl-0-isoglutamyl-1-lysyl.

权利要求:
Claims (1)
[1]
The invention The method of producing conjugated compounds of haptens and muramyl peptides of the general formula: M-acetylmuramyl-1 Compiler V. Volkova Editor A. Dolinich Tehred M. Khodanich Proofreader S. Cherni
Order 3596/58 Circulation 347Subscription
VNIIPI USSR State Committee
for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5
Production and printing company, Uzhgorod, st. Project, 4
2
0.1, 0.6
0.3, 0.7
1,3
0.1 0.2 0.4 0.3 1.2
alanyl-0-isoglutamine-1-lysyl LLH-RH, which consists in the fact that the hormone LH-RH in an acid form is introduced into interaction with M-acetylmuramyl-1-alanyl-0-isoglutaminyl-1-lysyl at a weight ratio of 1: 1 in a water-formamide medium using the carbomide method in the presence of 1-hydroxybenzotriazole, followed by allocation of the target product on a molecular sieve,

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法律状态:

优先权:

申请号 | 申请日 | 专利标题

FR8204353A|FR2522967B1|1982-03-15|1982-03-15|CONJUGATES OF HAPTENES AND MURAMYL-PEPTIDES, WITH IMMUNOGENIC ACTIVITY AND COMPOSITIONS CONTAINING THEM|

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