Combination of peptides

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to combinations of peptides in each case with the same sequence length (SEQL) which can be prepared in a stable reproducible quality and quantity of a mixture (A) containing a number of x amino acid with protected acid groups or a number of z peptides with the acid groups protected by the protective groups and the activated amino groups, with the amino acids in the mixture (A) found in a specific molar ratio, and a mixture (B), containing a number of y amino acids with the amino groups protected by the protective groups, with a molar ratio of the amino acids of the mixture (B) being the same as the molar ratio of the amino acids of the mixture (A), and the number x=y, and x is a figure from 11 to 18.

EFFECT: new combinations of the peptides are presented.

13 cl, 2 dwg, 1 ex

 

This invention relates to a combination of peptides, the method of synthesis of a combination of peptides, as well as the library of peptides.

From European patent descriptions 0983080 B1 is already known synthetic, statistical combination of thymus peptides and methods of their application as drugs immune and/or endocrine actions. Preparations containing combinations of the peptides of the thymus gland, receive consecutive accession chemically suitable derivatives of amino acids to peptides with short circuit, and receive characteristic of the tissue of the thymus gland proportion and combination of amino acids, so that the drug reaches increase depending on the dosage proliferation of human lymphocytes. The starting point for synthetic statistical combinations of peptides in the thymus gland is the analysis of individual peptides from partial protein hydrolysate of the thymus gland. Reaction of amino acids characteristic of the tissue of the thymus gland proportions and combinations of synthetic amino acids synthesize known from the analysis of the partial hydrolysate peptides, which in their totality form a typical synthetic statistical combination of peptides of the thymus gland, and thus it is possible to eliminate the risk to which Owego rabies.

From German published patent specifications 10327518 A1 is known a drug that contains a combination of synthetic peptides, and the way they are received. In this invention we are talking about a partial hydrolysate of human tissue, and the result of receiving a partial hydrolyzate depends on the specific enzyme combinations, which means that the result is controlled by means of a targeted selection of enzymes in specific combinations.

Based on the above-mentioned state of the art, the authors of the present invention has set itself the task to provide a combination of peptides in stable reproducible quality and quantity, which is the basis of the drug for treatment method.

In addition, the objective of the invention is providing a method for the synthesis of a combination of peptides, in which the combination of peptides can be produced in a reproducible manner with respect to its quantity and quality.

Finally, the objective of the invention is to provide a library of peptides, the content of which corresponds to the combination of the peptides with the same sequence length and specific and established quantitative ratio of amino acids.

A combination of peptides according to the invention solves the second problem concerning the combination of peptides in stable reproducible quality and quantity by means of the distinguishing features of claim 1 claims. Further, the combination of peptides characterized in that the peptide with the same in each case, the length of the sequence (SEQL) can be obtained from a mixture (A)containing

- the number of x amino acids with protected acid groups or the number z of peptides protected with protective groups, acid groups and the activated amino groups, and amino acids in the mixture (A) are in particular the prescribed molar ratio,

and the mixture (B)containing

the number y of the amino acids protected with protective groups, amino groups, and the molar ratio of amino acids in the mixture (B) is the same as the molar ratio of amino acids in the mixture (A), and thus x=y.

According to the method according to the invention have found that by connecting the two mixtures of amino acids A and B, which contain a matching number of amino acids, the molar ratio can be adjusted, which means that two mixtures of amino acids, identical in amino acid composition, except for their protected groups, react with each other or, respectively, then, the synthesized peptides from a mixture of A and B are protected with protective groups, acid groups, and activated amine groups react with a mixture of B; it is possible to provide qualitatively and quantitatively reproducible combination of peptides with peptides of the same length is posledovatelnosti as the basis for, for example, therapeutically effective drug. For the evaluation of stable quality and the number of combinations of peptides is presented in this paper figure 2, which shows a comparison of 3 independently of each of the obtained combinations of peptides (GKL-02) in their chromatographic separation (chromatogram). For example, the results of the applicants a drug called GKL-02 molar ratio tetrapeptides that means molar ratio in the library tetrapeptides, which consists of a mixture of 11 amino acids, for example, is for the amino acids aspartic acid (Asp) of 8.33 mol%, to the amino acid glutamic acid (Glu) at 9.53 mol%, to the amino acid Proline (Pro) 15,18 mol% and glycine (Gly) 37,10 mol%, etc. of a combination of peptides according to the invention can be applied both natural and synthetic amino acids, or a combination of natural and synthetic amino acids.

It is preferable to provide that the combination of a peptide consisting of 11 amino acids, could be combined with 11 identical to the amino acid groups of amino acids or, further, a peptide derived from the 11 amino acids and 11 are identical in their amino acid groups of amino acids, could be combined with 11 identical amino acids, etc. This means that peptides from dipeptides (SEQL=2) to the peptides with the infinite (∞) size (SEL≥2) can be combined. The smallest combination of peptides according to the invention can be called, respectively, the dipeptide z=(x*y), and therefore, a peptide derived from a mixture of A with x number of amino acids and mixture B with the number y of amino acids consisting of two amino acids, and the synthesis of the acid groups of the amino acids mixture A and the amino group of the amino acid mixture B is protected with protective groups, which after synthesis of the dipeptide are separated, and the number x is the same as the number y.

Particularly preferably, you can get tetrapeptide, which consist of four amino acids. Many tetrapeptide are pharmacologically active and often show affinity and specificity to a variety of receptors. Both linear and cyclic tetrapeptide may be preferable, and cyclic tetrapeptide can be formed using four peptide bond or by using covalent bonds. Examples of tetrapeptides are tuftsin (L-threonyl-L-lysyl-L-prolyl-L-arginine), tissue hormone phagocytosis, colleagues (glycyl-L-glutaminyl-L-prolyl-L-arginine) with a similar effect as tuftsin, " (Lys-Pro-Pro-Arg), which is N-terminal tetrapeptide cystatin C and antagonists tuftsin, endomorphin (H-Tyr-Pro-Trp-Phe-NH2) and (H-Tyr-Pro-Phe-Phe-NH2), peptidase with high affinity and specificity to opioid receptors, which are located in the centre of the social and the peripheral nervous system and, for example, tyrosine MIF-1 (Tyr-Pro-Leu-Gly-NH2), which is more endogenous opioid modulator. In General, a combination of peptides used in the field of Oncology, immunology, endocrinology and dermatology. And combinations of peptides used in the field of neurology as described for endomorphin, and in other clinical areas.

To a mixture of amino acids A and B can react with each other in a given direction with the formation of the peptide, the side functional groups, as described above, must be protected by protective groups and protective groups are then separated. To do this, depending on the synthetic strategy used is suitable protected amino acids - structural elements. For example, for the synthesis used the following protective groups for functional groups of amino acids: fluorenylmethoxycarbonyl (Fmoc) and tertbutyloxycarbonyl (Boc) amino groups, complex benzyl ester (OBzl), and complex tributyl ester (OtBu) for acid groups, 2,2,4,6,7-pentamethylcyclopentadiene-5-sulfonyl (Pbf), (2,2,5,7,8-pentamethylchroman-6-sulfonyl) (Pmc), trityl (Trt), nitro (NO2as protection for the functional groups in the side chain. Preferably used Boc-amino acids with complex benzyl esters of amino acids, hydrochloride or hydrotroilite. The standard reaction of compounds such as, for example the EP, N-ethyl-N'-diisopropylcarbodiimide (EDC, HCl), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylpropylenediamine) TBTU can also be used for the synthesis of peptides. Peptides are formed gradually, and at the end of the synthesis of all protective groups are separated.

Preferably, the peptide synthesis is carried out in solution, but also assumes the synthesis in solid phase. In the first stage of the synthesis, as has been described, a mixture of 11 complex benzyl esters of amino acids (Mixture A) is reacted with an appropriate mixture of 11 Boc-amino acids (Mixture B). After separation of the Boc protective group in the second stage, again the reaction occurs with the same mixture of 11 Boc-amino acids (Mixture B). After separation of the Boc protective group formed through the above stages, the combination of tripeptides (library tripeptides) again reacts with a mixture of 11 amino acids, the functional amino group which is protected groups benzyloxycarbonyl (Z). The protective group Z at the end is separated by hydrogenation with hydrogen. After the formation of the library tetrapeptides and separation of all the protective groups of the intermediate product lyophilizer. To the obtained devoid of protection library tetrapeptides it is preferable to stabilize and to improve functionality or specificity, add a mixture of natural the actual amino acids in the following proportions: 100 g ± 25 g of peptides: 80 g ± 20 g of a mixture of amino acids. In addition, the library, which means, the combination of tetrapeptides, dissolved in water and optionally mixed with aqueous solutions of amino acids H-Asp-OH 2,41 mol%; H-Thr-OH 9,26 mol%; H-Ser-OH 8,61 mol%; H-Glu-OH 6,44 mol%; H-Pro-OH 2,30 mol%; H-Gly-OH 4,94 mol%; H-Ala-OH of 11.69 mol%; H-Cys-OH.HCl 0.47 mol%; H-Val-OH 6,76 mol%; H-Met-OH 3,24 mol%; H-Ile-OH of 4.25 mol%; H-Leu-OH 14,15 mol%; H-Tyr-OH 0.36 mol%; H-Phe-OH 4,27 mol%; H-His-OH.HCl.H2O 1,22 mol%; H-Lys-OH.HCl 10,36 mol% and H-Arg-Ol-1.HCP 9.28 are mol% and then lyophilizer. Of course, depending on the application of combinations of peptides in drug, it is assumed the other mixture as described above was added a mixture of amino acids, also in freely chosen molar ratio.

Mentioned at the beginning of this work the problem of providing a method of synthesis for the preparation of a combination of peptides, in which a combination of peptides can be obtained reproducibly regarding quality and quantity, are solved by the distinguishing signs of clause 8 of the claims.

According to the method according to the invention the method of synthesis of a combination of peptides is characterized by the fact that peptides with every time the same sequence length (SEQL) are synthesized from the mixture (A)containing

- the number of x amino acids with protected acid groups or the number z of peptides protected with protective groups, acid groups and the activated amino groups, and amino acids from a mixture of (A) are the specific prescribed molar ratio,

and the mixture (B)containing

the number y of the amino acids protected with protective groups, amino groups, and the molar ratio of amino acids in the mixture (B) is the same as the molar ratio of the mixture (A), and with x=y.

Preferably, the method according to the invention serves for the synthesis of combinations of peptides on PP. from 1 to 6 or, respectively, to obtain a library of peptides on p. 14, so that to avoid repetition, we describe the advantages of a combination of peptides according to the invention in the previous section and the following description of a library of peptides.

In a preferred embodiment of the method according to the invention, however, the authors reiterate that preferably the addition of a mixture of natural amino acids in aqueous solution, while in the previous phase after phase synthesis of peptides, the functional groups of the side chains are activated by the Department of protective groups and the following intermediate stage before mixing the synthesized peptides with a mixture of natural amino acids, dissolved in water, the synthesized peptides lyophilizer and after mixing with the mixture of natural amino acids again lyophilizer, in order to obtain a powder.

Mentioned at the beginning of the task to provide a library of peptides, which contains a combination of peptides with dinasovoy in each case the length of the sequence and specific and established quantitative ratio of amino acids, solve using a library of peptides with distinctive features at 14, which presents a library of peptides containing a combination of peptides with the same in each case, the sequence length of SEQL≥2, consisting of a combination 2*11, 2*12, 2*13, 2*14, 2*15, 2*16, 2*17 or 2*18 amino acids for SEQL=2 or, respectively, consisting of a combination (SEQL=2)*11 to ∞, *12 to ∞, *13 to ∞, *14 to ∞, *15 to ∞, *16 to ∞, *17 to ∞ or *18 to ∞ amino acids for SEQL>2, which are specific prescribed molar ratio, and ∞ represents, for example, ((SEQL=2)*11)*11, (((SEQL=2)*11)*11)*11 etc.

In order to avoid repetitions in relation to the library of peptides is recommended to describe the advantages of a combination of peptides according to the invention or, respectively, the synthesis method according to the invention.

The invention presented and described in detail using the following figures and examples. It should be note that the figures and examples are only descriptive and are not intended to limit the invention in any form.

Examples

1. Synthesis of tetrapeptide/library tetrapeptides

1.1. Reagents, solvents and chemical reagents

Hydroxybenzotriazole monohydrate (HOBt·H2O); O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylethylenediamine (TBTU), N,N-diisopropylethylamine (DIPEA); ethyl acetate (EtOAc); N,N-di is malformed (DMF); toluene; triperoxonane acid (TFA); glacial acetic acid (AcOH); palladium (Pd), 10% carbon, mixed with 50% water; distilled water, and sodium bicarbonate (NaHCO3).

2. Stage of synthesis 1: Boc-AA-OH+H-AA-OBzl·TOS or ·HCl→Boc-AA-AA-OBzl

2.1. Raw materials

Mixture B

- Boc-Ala-OH, Boc-Arg(NO2)-OH, Boc-Asp(OBzl)-OH, Boc-Glu(OBzl)-OH, Boc-Gly-OH, Boc-Ile-OH·1/2H2O, Boc-Leu-OH·H2O, Boc-Lys(Z)-OH, Boc-Phe-OH, Boc-Pro-OH, Boc-Val-OH.

A mixture of A

- H-Ala-OBzl·TOS, H-Arg(NO2)-OBzl·TOS, H-Asp(OBzl)-OBzl·TOS, H-Glu(OBzl)-OBzl TOS H-Gly-OBzl·TOS, H-Ile-OBzl·TOS, H-Leu-OBzl·TOS, H-Lys(Z)-OBzl·HCl, Phe-OBzl·HCl, H-Pro-OBzl·HCl, H-Val-OBzl·TOS.

2.2. The method of synthesis

A reaction chamber, for example, 250-l tank, lined with glass, is placed under a vacuum of from 0.6 to 0.9 mbar (61,2 to 91,8 PA), purged with nitrogen and load DMF (18.4 kg). Then add the following source materials: Boc-Asp(OBzl) - OH (553,771 g), Boc-Glu(OBzl)-OH (661,065 g), Boc-Pro-OH (671,664 g), Boc-Gly-OH (1335,986 g), Boc-Ala-OH (455,612 g), Boc-Val-OH (258,295 g), Boc-He-OH·1/2H2O (152,469 g), Boc-Lys(Z)-OH (376,679 g), Boc-Arg(NO2)-OH (235,826 g), Boc-Leu-OH·H2O (7,789 g), Boc-Phe-OH (38,337 g).

The second reaction chamber, for example 250-l tank, lined with glass, is placed under a vacuum of from 0.6 to 0.9 mbar (61,2 to 91,8 PA), purged with nitrogen and load DMF (19,4 kg). Then add the following tozilaty complex benzyl ester amino acids or, respectively, hydrochloride:

H-Asp(OBzl)-OBzl·TOS (831,556 g), H-Glu(OBzl)-OBzl·TOS (978,910 g), H-Pro-OBzl·HCl (754,260 g), H-Gly-OBzl·TOS (2573,058 g), H-Ala-OBzl·TOS (846,208 g), (H-Val-OBzl TOS (451,43 g), H-Ile-OBzl TOS (249,683 g), (H-Lys(Z)OBzl·HCl (402,887 g), H-Arg(NO2)-OBzl 2 TOS (483,12 g), H-Leu-OBzl·TOS (12,294 g), (H-Phe-OBzl·HCl (42,162 g).

The contents of the first reactor is transferred to the second reactor. At low blowing nitrogen added HOBt monohydrate (3,78 kg) at 20-25°C and the mixture at 4-6°C is stirred, until then, until it is fully dissolved. To this solution was added TBTU (7,92 kg), resulting in the suspension. To the solution was added DIPEA (9,996 kg), while the temperature is kept below 15°C. the temperature was Then raised to 25°C and the mixture is approximately 15-20 hours mix, then hold azeotropic distillation with toluene (2×14 kg), in order to remove all of DMF, followed by removal of toluene with EtOAc (2×9.3 kg). After re-supplements EtOAc (28 kg), the reaction mixture was extracted with 8% solution of NaHCO3(51 l) and then with water (30 kg) and concentrated under vacuum to obtain an oily residue, which is used in the next stage of the synthesis of 2.

3. Stage synthesis 2: Boc-AA-AA-OBzl - H-AA-AA-OBzl·TFA

3.1. Raw materials

Oily residue after the stage of synthesis 1 and TFA

3.2. The method of synthesis

The reaction vessel, for example a 250 l capacity, glass-fronted containing oily residue after the previous stage, is placed under a vacuum of from 0.6 to 0.9 mbar (61,2 to 91,8 PA) and after purification by nitrogen load triperoxonane acid (TFA, 30,4 kg) for 10-30 min, while the temperature is kept below 30°C. Concentration at 45°C leads to the formation of an oily residue, to which is added toluene (32,8 kg), in order to remove the remaining TFA using azeotropic distillation. To ensure complete removal, gradually add EtOAc (9,8 kg) and distilled water (10,3 kg). Then the reaction mixture was concentrated to obtain oil and kept under vacuum at 35°C. This oil is used in the next stage of the synthesis.

4. Stage synthesis 3: Boc-AA-OH + H-AA-AA-OBzl·TFA - Boc-AA-AA-AA-OBzl

4.1. Raw materials

Oily residue after synthesis step 2 and Boc-Ala-OH Boc-Arg(NO2)-OH Boc-Asp(OBzl)-OH Boc-Glu(OBzl)-OH, Boc-Gly-OH, Boc-Ile-OH·1/2 H2O, Boc-Leu-OH·H2O, Boc-Lys(Z)-OH, Boc-Phe-OH, Boc-Pro-OH, Boc-Val-OH.

4.2. The method of synthesis

The reaction vessel, for example a 250 l capacity, glass-fronted, placed under a vacuum of from 0.6 to 0.9 mbar (61,2 to 91,8 PA), purged with nitrogen and load DMF (17,4 kg). At room temperature, add the following products:

BocAsp(OBzl)-OH (553,771 g), Boc-Glu(OBzl)-OH (661,065 g), Boc-Pro-OH (671,664 g), BocGly-OH (1335,986 g), Boc-Ala-OH (455,612 g), Boc-Val-OH (258,295 g), Boc-Ile-OH 1/2H2O (152,469 g), Boc-Lys(Z)-OH (376,679 g), Boc-Arg(NO2)-OH (235,826 g), Boc-Leu-OH·H2O (7,789 g), Boc-Phe-OH (38,337 g).

The second vessel, lined with glass containing oily residue from the previous stage, down the vacuum and purified nitrogen. Then add DMF (17,4 kg) and the mixture is stirred to obtain a homogeneous solution. Sod is rimae the first reactor is transferred to the second reactor and added HOBt monohydrate (3,78 kg) and the total mixture is stirred until complete dissolution of all components. At the moment (complete dissolution) the solution is cooled to 5°C and added HBTU (7,92 kg), followed by addition of DIPEA (9,996 kg), during which the temperature is kept below 10°C. This solution was concentrated at about 55°C under vacuum to obtain an oily residue, which was subjected to azeotropic distillation by addition of toluene, EtOAc and water and then use a vacuum then concentrated. To the resulting oily residue add EtOAc and the organic solution extracted several times from 8% solution of sodium bicarbonate (NaHCO3), and then from the water, and then evaporated to obtain an oily residue, which is used in the next stage.

5. Stage synthesis 4: Boc-AA-AA-AA-OBzl - H-AA-AA-AA-OBzl·TFA

5.1. Raw materials

Oily residue after synthesis step 3 and TFA.

5.2. The method of synthesis

To the reaction vessel, for example 250-l tanks, glass-fronted containing oily residue after the previous stage, striking a vacuum of from 0.6 to 0.9 mbar (61,2 to 91,8 PA) and after treatment with nitrogen this capacity load triperoxonane acid (TFA, 30,4 kg) for 10-30 min at a temperature of about 25-30°C. by evaporation at 45°C to obtain an oily residue, to which is added toluene (32,8 kg), and the mixture concentrated. To ensure complete removal of TFA, gradually we use the t EtOAc (9,8 kg) and distilled water (10,3 kg). The reaction mixture was concentrated to obtain oil and under vacuum at 35°C hold. This mixture is used in the next step 5.

6. Stage synthesis 5: Z-AA-OH+H-AA-AA-AA-OBzl·TFA-Z-AA-AA-AA-AA-OBzl

6.1. Raw materials

Z-Ala-OH, Z-Arg(NO2)-OH, Z-Asp(OBzl)-OH, Z-Glu(OBzl)-OH, Z-Gly-OH, Z-Ile-OH, Z-Leu-OH, Z-Lys(Z)-OH, Z-Phe-OH, 1-Pro-OH, Z-Val-OH and oily residue after synthesis step 4: H-AA-AA-AA-OBzl·TFA.

6.2. The method of synthesis

The reaction vessel, for example a 250 l capacity, glass-fronted, in an atmosphere of nitrogen load DMF (17,4 kg) and at room temperature the contents stirred. Next, add the following protected Nα-carboxybenzene amino acids (1-AA-OH):

Z-Asp(OBzl)-OH (612,017 g), Z-Glu(OBzl)-OH (727,726 g), Z-Pro-OH (777,819 g), Z-Gly-OH (1595,434 g), Z-Ala-OH (537,531 g), Z-Val-OH (298,741 g), Z-Ile-OH (168,344 g), Z-Lys(Z) - OH (410,362 g), Z-Arg(NO2)-OH (260,943 g), Z-Leu-OH (8,289 g) and Z-Phe-OH (43,253 g). Second, for example 250 l capacity, glass-fronted, bring the vacuum from 0.6 to 0.9 mbar (61,2 to 91,8 PA) and purified nitrogen. This capacity load oily residue after stage 4, also H-AA-AA-AAOBzl·TFA and DMF (19,4 kg) and the mixture is stirred to obtain a homogeneous solution. The contents of the first vessel (reactor) is completely transferred to the second reactor.

At room temperature add HOBt monohydrate (3,78 kg) and the mixture is stirred until the whole HOBt monohydrate is dissolved. At low blowing nitrogen slowly (60-90 minutes) to relax the Ute TBTU (7,92 kg) and DIPEA (9,996 kg) at a temperature of about 4-6°C and continuously measured pH values from 6.5 to 7.0, to provide a pH value within the given range.

The concentration of this solution at about 55°C under vacuum yields an oily residue, which azeotrope is distilled by adding and concentration after each stage with the following solvents: toluene, EtOAc/water. To the oily residue, obtained after evaporation of the last solution, add EtOAc and this solution is extracted several times with an 8% solution of sodium bicarbonate (NaHCO3) and water. This combined organic phase is concentrated to obtain oil, which is used in the next step 6.

7. Stage synthesis 6: Z-AA-AA-AA-AA-OBzl H-AA-AA-AA-AA-OH

7.1. Raw materials

The oil remaining after stage 5: Z-AA-AA-AA-AA-OBzl.

7.2. The method of synthesis

To the oily residue from the previous stage 5 add AcOH (86,3 kg) and this mixture at about 25°C and stirred until complete dissolution. Then this solution hydronaut.

For this purpose the reactor for hydrogenation (for example, 630 l) load the catalyst Pd (10% on C, 50% H2O) and using fusion inactivate. Library tetrapeptide Z-AA-AA-AA-AA-OBzl in AcOH fully placed in the reactor containing the catalyst Pd. Miss hydrogen gas and hydrogenation takes place at a maximum temperature of 40°C and from 2500 to 3000 mbar (from 255 to 306 kPa) (absolute pressure).

After the reaction, the reaction mixture purified by filtration and clean filtrate is evaporated to obtain the oil, which then use the gradual adding large amounts of EtOAc processed and evaporated. The oil obtained is dissolved in water and this solution (approximately 8-12%) in the next stage 7 lyophilizer.

8. Stage synthesis 7: Lyophilization of tetrapeptides H-AA-AA-AA-AA-OH

8.1. Raw materials

The aqueous solution after stage 6: H-AA-AA-AA-AA-OH in H2O.

8.2. The method of synthesis

Lyophilization is as follows:

- Sublimemovies: the Solution is cooled to -40°C for 3 hours and Then produce a temperature increase from 40°C to 50°C under vacuum at 200 mcbar within 48 h;

The desorption Temperature is maintained at 50°C for 20 mcbar within 24 hours

9. Stage synthesis 8: H-AA-AA-AA-AA-OH + H-AA-OH -> H-AA-AA-AA-AA-OH, H-AA-OH

9.1. Raw materials

H-Asp-OH, H-Thr-OH, H-Ser-OH, H-Glu-OH, H-Pro-OH, H-Gly-OH, H-Ala-OH, H-Cys·HCl, H-Val-OH, H-Met-OH, H-Ile-OH, H-Leu-OH, H-Tyr-OH, H-Phe-OH, H-His-OH·H2O·HCl, HLys-OH·HCl, H-Arg-OH·HCl.

9.2. The method of synthesis

The purified nitrogen, coated glass container of water (for example, 250 l) add dried tetrapeptide (A g) from the previous stage and add the following L-amino acids: H-Asp-OH (1,559 g x (f)), H-Thr-OH (5,364 g x (f)), H-Ser-OH (4,402 g x (f)), H-Glu-OH (4,610 g x (f)), H-Pro-OH (1,285 g x (f)), H-Gly-OH (1,803 g x (f)), H-Ala-OH (5,066 g x (f)), HCys·HCl (0361 g x (f)), H-Val-OH (3,848 g x (f)), H-Met-OH (2,348 g x f)), H-Ile-OH (2,711 g x (f)), H-Leu-OH (9,028 g x (f)), H-Tyr-OH (MX 0.317 g x (f)), H-Phe-OH (3,429 g x (f)), H-His-OH, H2O·HCl (1,242 g x (f)), H-Lys-OH·HCl (9,210 g x (f)), H-Arg-OH·HCl (9,512 g x (f)), and (f)=A g lyophilized library tetrapeptidesfound/g library peptidesnorm batch.

At a temperature of about 40°C the resulting solution was stirred until complete dissolution. Then the temperature is reduced to 20-25°C and the solution during the night mix. Then filter the solution of peptides, in order to clean the filtrate at the next stage 9 to liofilizirovanny.

10. Stage synthesis 9: Lyophilization GKLO2; medicine

10.1. Raw materials: the Filtrate after stage 8

10.2. The method of synthesis

Lyophilization is as follows:

- Sublimemovies: the Solution is cooled to -40°C for 3 hours and Then produce a temperature increase from 40°C to 50°C under vacuum at 200 mcbar within 48 h;

The desorption Temperature is maintained at 50°C for 20 mcbar within 24 hours

The substance obtained after stage 9, which is applicable to both medical valid drug may subsequently from powder form to compress into tablets, filled into capsules or packaged in other forms and stored at temperatures below -15°C.

The figures shown:

Figure 1: Block diagram of the synthesis process described in example 1

Figure 2: Chromatogram after the existing one after the other parties GKL-02 internal standard.

Figure 1 shows a block diagram of the synthesis process library tetrapeptide according to the invention or, respectively, tetrapeptide according to the invention. As a comment to the block diagram to prevent recurrence it is recommended that the above description of example 1.

Figure 2 shows a chromatogram successive batches GKL-02: party 1=GKL02 SC02808L1; part 2=GKL02 15935-AA/2 and party 3=GKL02 15507-AA/9. Using internal standards (see arrows) for all three parties consecutive identical samples detected identical peaks, from which we may conclude that exceptional consistency, which means that the reproducibility of libraries of peptides that are obtained by the method according to the invention. In particular, it shows the chromatogram are proof of consistent, reproducible quality and quantity appropriate to the invention combinations of peptides.

1. The combination of peptides, wherein the peptides with the same in each case, the length of the sequence (SEQL) can be obtained from a mixture (A)containing
the number of x amino acids with protected acid groups or the number z of peptides protected with protective groups, acid groups and the activated amino groups, and amino acids in the mixture (A) are in a prescribed molar ratio,
and mixtures (the), contains
the number y of the amino acids protected with protective groups, amino groups, and the molar ratio of amino acids mixture (B) is the same as the molar ratio of amino acids mixture (A), and
the number x=y, and x is a number from 11 to 18 inclusive, and where the peptides are cancer, immunological, dermatologically and/or endocrinologically effective.

2. A combination of peptides according to claim 1, characterized in that the functional groups of the side chains of the amino acids protected with protective groups.

3. A combination of peptides according to claim 1, characterized in that SEQL≥2.

4. A combination of peptides according to claim 3, characterized in that SEQL=2=z=(x*y), and acid groups, amino groups and functional groups of the side chains of the amino acids are activated.

5. A combination of peptides according to claim 4, characterized in that SEQL>2=(x*y)*y to ∞, and acid groups, amino groups and functional groups of the side chains of the amino acids are activated, and ∞ is a ((x*y)*y)*y, (((x*y)*y)*y)*y, and so on

6. A combination of peptides according to claim 1, characterized in that the peptides add a mixture of natural amino acids in a ratio of 100 g ± 25 g of peptide: 80 g ± 20 g of a mixture of amino acids.

7. The method of synthesis of a combination of peptides, characterized in that synthesize peptides with the same in each case, the length of the sequence (SEQL), which can the be obtained from a mixture of (A), contains
the number of x amino acids with protected acid groups or the number z of peptides protected with protective groups, acid groups and the activated amino groups, and amino acids in the mixture (A) are in a prescribed molar ratio,
and the mixture (B)containing
the number y of the amino acids protected with protective groups, amino groups, and the molar ratio of amino acids mixture (B) is the same as the molar ratio of amino acids mixture (A), and x=y, and
peptides with SEQL=2 synthesized from a mixture of (a) and (b), each of which contains 11, 12, 13, 14, 15, 16, 17 or 18 amino acids.

8. The method according to claim 7, characterized in that the peptides with SEQL>2 are synthesized from peptides with SEQL≥2 and a mixture (B)and the acid group of the protected peptides and their amino group is activated.

9. The method according to any of claims 7 or 8, characterized in that 11, 12, 13, 14, 15, 16, 17 or 18 amino acids mixture (A) are in a molar ratio, which is the same as for the amino acids mixture (B).

10. The method according to claim 7 or 8, characterized in that the amino acids or peptides with SEQL≥2 mixture (A) used in further synthesis with a mixture (B), and the functional groups of the side chains of the amino acids mixture (B) is protected with protective groups.

11. The method according to claim 10, characterized in that after the synthesis of peptides with SEQL≥2 protected acid groups, is aminogruppy and functional groups of the side chains by the Department of protective groups activate, and characterized in that in the intermediate stage, the peptides lyophilizer and in aqueous solution is mixed with a mixture of natural amino acids, and the ratio of peptides to the amino acid mixture of 100 g ± 25 g of peptide: 80 g ± 20 g of a mixture of amino acids, and/or in the next stage, the mixture of peptides with amino acids lyophilizer.

12. Library of peptides obtained by the method according to any of claims 7-11, contains a combination of peptides with the same in each case, the sequence length of SEQL≥2, consisting of a combination 2*11, 2*12, 2*13, 2*14, 2*15, 2*16, 2*17 or 2*18 amino acids for SEQL=2 or, respectively, consisting of a combination (SEQL=2)*11 to ∞, *12 to ∞, *13 to ∞, *14 to ∞, *15 to ∞, *16 to ∞, *17 to ∞ or *18 to ∞ amino acids for SEQL>2, which are in a prescribed molar ratio, and ∞ represents, for example ((SEQL=2)*11)*11, (((SEQL=2)*11)*11)*11 etc. where the peptides are cancer, immunological, dermatologically and/or endocrinologically effective.

13. Applying a combination of peptides according to any one of claims 1 to 6 for medicinal products for applications in Oncology, immunology, endocrinology, dermatology or neurology.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. A biomass of the gram-negative bacteria Salmonella typhi fam. Enterobacteriacea is prepared. A cell wall peptidoglycane (CWP) of the bacteria is recovered by biomass extraction in 45% aqueous phenol at temperature 70-90°C or in aqueous solution of ionic or non-ionic detergents at temperature 37-100°C. That is followed by preparative enzymatic hydrolysis for insoluble CWP cleavage with the use of lysozyme at pH 4.5 - 8.9 and temperature 10 - 37°C. Simultaneously, a pharmacologically acceptable mixture of substances is removed by dialysis from the reaction mixture with the use of semipermeable membranes for ultrafiltration with cut-off size up to 5 kDa. The mixture of substances is also recovered by means of column gel-chromatography, particularly preparative gel chromatography on Sephadex or TSK gel columns. The end product yield is 320 mg.

EFFECT: method enables producing the pharmacologically acceptable mixture of substances containing the following ingredients: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid) (GMtri); β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine) (GMtetra); and GMtetra dimer (diGMtetra) wherein a bond of monomer residues of GMtetra is enabled by a carboxyl group of terminal D-alanine of one GMtetra residue and ω-aminogroup of meso-diaminopimelic acid of the other GMtetra residue with the coupled tetrapeptide resides positioned in various polysaccharide chains.

4 cl, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method for producing total water-soluble proteolytic enzymes from dried milky sap of green papaya fruits provides water extraction of a raw material at room temperature and with mixing, separation of solid and liquid phases, and recovery of a purified product. An aqueous extract produced after separation of the solid and liquid phases is post-filtered through a filter of pore size (0.45-2.0) mcm. The product is dried by a spraying technique to prepare a purified product in the form of a dry extract of proteolytic activity not less than 3.5 PU/mg of the preparation and the protein content 0.43-0.50 mg/mg of the preparation. The prepared dry extract is water-dissolved that is followed by sterilisation filtration and lyophilisation of the solution.

EFFECT: method allows preparing a product of high proteolytic activity and protein content.

2 cl, 1 ex

FIELD: medicine.

SUBSTANCE: claimed is biopolymer with expressed immunostimulating effect, consisting of highly molecular fragment of peptidoglican of cell wall of Gram-negative bacteria, whose repeating link (RL) represents tetrasaccharide 4-O-{4-O-[4-O-(N-acetyl-β-D-glucosaminyl)-N-acetyl-β-D-muramyl]-N-acetyl-β-D-glucosaminyl}-N-acetyl-D-muramic acid, to which on carboxyl groups of both residues of muramic acid bound is tetrapeptide residue: N-(L-alanyl-D-isoglutaminyl-meso-diaminopimelonyl-D-alanine). Binding between RL is perfomed due to octapeptide bridges (dwg.1), number of RL constituting from 5 to 20 or their combination. Also claimed are method of biopolymer obtaining, based on it pharmaceutical immunostimulating composition and its application in methods of stimulating immune system of mammals and non-specific protection against bacterial infections.

EFFECT: possibility of application in immunopharmacology, in particular, for creation of medications of supporting therapy, intended for enhancing immune resistance of organism.

11 cl, 2 dwg, 2 tbl, 5 ex

FIELD: medicine, veterinary science.

SUBSTANCE: invention concerns veterinary medicine. Currently, for differentiating nonspecific tuberculin reactions in cattle, dried purified tuberculin is used for mammals and "КАМ" with considering the sensitisation pattern by the reaction intensity. A common complex allergen is produced by protein settling from M scrofulaceum No. 12-C and M intracellulare No. 13-H strain cultures with added allergen produced from Corynebacterium xerosis N1911, in amount 1350 units of activity. The presence of coryneformic bacteria allergen in the "КАМ" composition improves the efficacy of a simultaneous dried purified tuberculin test for mammals in differentiating the nonspecific coryneformic bacteria reactions.

EFFECT: use of the declared allergen allows to prevent unreasonable slaughter, as well as further diagnostic finding expenses.

2 tbl

FIELD: biology, medicine, organic chemistry.

SUBSTANCE: invention proposes compound of the general formula (I): wherein A means effector group; L means a linker link; B represents Skulachev-ion Sk or charged hydrophobic peptide. Compound can be used in preparing a pharmaceutical composition for target (directed) delivery of active substances in mitochondria carried out by electrochemical potential of hydrogen ions into mitochondria. Also, invention can be useful in treatment of diseases and states associated with disturbance of normal function of mitochondria, in particular, diseases associated with increased formation of free radicals and active forms of oxygen. The claimed invention owing to directed accumulation of biologically active substance in mitochondria provides enhancing the effectiveness of substance, to decrease total dose, probability and strength of adverse effects.

EFFECT: improved and valuable properties of method and pharmaceutical composition.

26 cl, 14 dwg, 16 ex

FIELD: bioengineering; genetic engineering; medicine; methods of production casamino acids.

SUBSTANCE: the invention is pertaining to the field of bioengineering, genetic engineering, medicine, in particular, to the methods of production of components for nutrient mediums from hydrolysates of animal protein. The invention offers the method of production of casamino acids by the method of the gel permeation chromatography of the hydrolyzed crude acid casein with the contents of the general nitrogen - 0.7-0.95 g in 100 ml of the solution and concentration - 6-10 % on Sefadex G-15, eluating by a distilled water of fractions of an eluate, selection of the active fractions of an eluate by a spectophotometery of portions of the eluate (D254), evaporation of the active fractions under vacuum at the temperature of no more than 55°C. The method allows to simplify the process of production of casamino acids, to reduce its cost and also to obtain casamino acids possessing the high growth- stimulating activity.

EFFECT: the invention ensures simplification of the process of production of casamino acids, reduction of its cost and also production of casamino acids possessing the high growth- stimulating activity.

2 cl, 2 dwg, 1 tbl, 1 ex

The invention relates to biotechnology, can be used in medical practice for a polypeptide, which is excreted through the kidneys and does not contain in its original form Fc-region of IgG

The invention relates to medicine, in particular to new drugs, containing immunoactive proteins, and is intended for the prevention and/or treatment of septic shock

The invention relates to preparative and process biochemistry for the production of biologically active chemical compounds, protein product used in cell biology and in medicine and veterinary medicine

FIELD: chemistry.

SUBSTANCE: invention relates to biosensors and can be used in studying proteins via a luminescence technique. A protein luminescence-activating hybrid complex is obtained through ultrasonic treatment of a protein which contains aromatic amino acids in a physiological solution in the presence of phosphorus Y0.95Hr0.05VO4 or Y0.95Er0.05YO3Cl. The complex contains donor-acceptor pairs.

EFFECT: use of the invention increases luminescence of the investigated protein and reduces labour input of the protein labelling procedure, widens the range of proteins analysed using a luminescence technique, and lowers the technological level of the process of obtaining donor-acceptor pairs in the protein luminescence-activating hybrid complex.

2 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of obtaining and purifying human inhibin-A. Pieces of placenta are crushed and homogenised. A physiological solution is added in ratio of 1:2. Butyl alcohol is added to the obtained mass in ratio of 1:10 and then left for a day in a refrigerator at t +4°C. The mixture is then centrifuged with diethyl ether in ratio of 1:3 for 10-12 minutes at 5000 rpm in a cold centrifuge twice. Supernatant fluid containing inhibin is put into a flask which is then put into a refrigerator at t +4°C for a day. The supernatant fluid is centrifuged once more and over protein content therein is determined. The obtained solution is mixed with an equal volume of saturated ammonium sulphate solution and centrifuged in a cold centrifuge for 45-50 min at 10000 rpm. The obtained residue is dissolved in a tris-HCl buffer with pH 7.8, and then deposited on a lectin-sepharose column balanced with tris-HCl buffer. The entire volume of the dissolved residue obtained at the previous step is passed through. The column is washed with 1M sodium chloride solution buffered with tris-HCl buffer. The inhibin bound on the column is then eluted with 0.1M lactose solution in a borate buffer at pH 9.0. The eluate undergoes dialysis and concentration.

EFFECT: method enables to obtain inhibin-A with high output, high specific activity and high degree purification.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: Staphylococcus hominis KLP-1 strain is recovered from a clinical material in screening of coagulase-negative staphylococci strains for ability to produce low-molecular antibacterial peptides. The strain is deposited in Tarasevich State Research Institution of Standardisation and Control of Medical Biological Preparations, No. 284. The antibacterial action is shown concerning the bacteria of the following species: Arthrobacter, Bacillus, Enterococcus, Micrococcus, Mycobacterium, Rhodococcus, Staphylococcus, Streptococcus. The low-molecular antibacterial peptide yield makes 1024000 EA in 1 l of the medium.

EFFECT: strain cultured on a nutrient fluid is characterised by higher yield of low-molecular peptide antibacterial compounds inhibiting gram-positive bacteria growth.

1 dwg, 1 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: methods of purifying a target protein involve preparation of a solution containing a mixture of the dissolved target protein and one or more dissolved impurity proteins, contacting said solution with one or more cationic surfactants with increase in the content of the target protein relative proteins remaining in the solution and extraction of the dissolved target protein. The cationic surfactant used can be an amphipathic ammonium compound taken in an amount which is sufficient for preferred precipitation of one or more impurity proteins.

EFFECT: invention increases efficiency of extracting and purifying target proteins, and also increases content of the target protein relative proteins remaining in the solution.

41 cl, 12 dwg, 5 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: position of α-spiral, β-structural fragments and bends of the β-structure in the amino acid sequence of the protein is predicted using a special software PREDICTOR by comparing fragments from five amino acids (penta-fragments) selected in the working file of the protein under analysis successively with shift towards one amino acid, starting with the N-end of the protein, with a special database of penta-fragments of proteins stored in computer memory and obtained using special software based on files with coordinates of atoms of the protein structures from free access Protein Data Bank. Based on information on numbers of folders successively entered into the working file for all penta-fragments selected in the amino acid sequence, the position of α-spiral, β-structural fragments and bends of β-structures in the primary structure of the protein is determined, from which the secondary structure of the analysed protein is determined.

EFFECT: improved method.

18 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a novel biologically active compound such as a peptide which enables production of an antibody which selectively detects the oligomeric form of nucleophosmin protein in preparations containing monomeric or oligomeric protein forms.

EFFECT: invention can be efficiently used to produce an immunodiagnostic preparation for investigating and monitoring cancerous growths.

1 dwg, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a novel biologically active compound such as a peptide which enables production of an antibody which selectively detects the monomeric form of nucleophosmin protein in preparations containing monomeric or oligomeric protein forms.

EFFECT: invention can be efficiently used to produce an immunodiagnostic preparation for investigating and monitoring cancerous growths.

1 dwg

FIELD: biology.

SUBSTANCE: designed is a new recombinant plasmid pETGST-LFmin (7704 nucleotide pairs), containing a catalytically active fragment of the gene of lethal factor anthrax (LF) controlled by a bacteriophage T7 promoter, determinant of resistance to ampicillin and a glutathione-S-transferase sequence for efficient purification of the recombinant protein on a sorbent with immobilised glutathione. The plasmid provided for efficient sythensis of the protein of LF anthrax, chimerised with a sequence of glutathione-S-transferase for purification on immobilised glutathione. Escherichia coli BL-LFminGST strain is obtained from transformation of the said plasmid DNA into a E.coli BL21 (DE3) strain, which provides for output of synthesised LF protein of not less than 90 mg/l g of raw biomass. The active LF protein is obtained using a method which involves culturing the said recombinant strain, destruction of bacterial cells in a buffer solution with pH 7.4 in the presence of Triton X-100 and a protease inhibitor, and purification on a sorbent with immobilised glutathione.

EFFECT: output of proteolytic active recombinant chimeric purified protein of LF anthrax in amount of not less than 70 mg/l g of raw biomass.

3 cl, 3 dwg, 3 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to field of biotechnology, namely to bacteriochlorophyll derivatives and can be used for medical and diagnostic purposes. Anion-containing water-soluble tetracyclic and pentacyclic bacteriochlorophyll (Bchl) derivatives contain one, two or three negatively charged groups and/or acid groups, which transform into negatively charged groups at physiological pH. Obtained derivatives are used for photodynamic therapy and for tumor diagnostics, as well as for killing cells or infectious agents.

EFFECT: increasing selectivity of binding with target in photodynamic therapy and diagnostics.

25 cl, 34 dwg, 5 tbl, 57 ex

FIELD: chemistry, biotechnology.

SUBSTANCE: invention relates to biotechnology. Method includes addition to fermentation broth or homogenate from E. coli of efficient quantity of ethacridinlactate solution for sedimentation of contamination from host-cells in conditions, when greater part of polypeptide remains dissolved, and isolation of heterological polypeptide from broth or homogenate.

EFFECT: simplification of target polypeptide purification and obtaining it with high degree purity.

23 cl, 15 dwg, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: method includes the following stages: conservation of cells in presence of buffered 80-90% glycerine, collapse of cell shells by 3% triton X-100, extraction by increasing concentrations of salts: 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% β-mercaptoethanol, extraction of positively charged proteins from above fractions with the help of ion-exchange chromatography with amberlite IRC-50 in an interrupted gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% on 0.1 M potassium-phosphate buffer pH 6.8 and detection of sites of sensitivity in them to Arg-X proteolysis.

EFFECT: invention may be used in analysis of molecular-genetic mechanisms of procaryote cell structure formation and role of protein components in their organisation, and also when studying features of genome remodelling, which is necessary for opening of paths of regulation mechanisms of macro- and microorganisms.

9 dwg, 1 ex

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