Modified versions of bowman birk protease inhibitors

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to an isolated version of a Bowman Birk protease inhibitor (BBPI), where said BBPI version has a frame containing amino acid sequences 1-40 and 50-66 of SEQ ID NO: 187 and cysteine residues in amino acid positions 41 and 49 of SEQ ID NO: 187, between which is included a binding peptide selected from a binding VEGF peptide, a binding FGF-5 peptide, a binding TGFb peptide and a binding TNFα peptide, where said BBPI version further includes a substituted amino acid in an amino acid position selected from positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55 and 65 of SEQ ID NO: 187, where said substituted amino acid in position 1 is A, in position 4 is V, in position 5 is selected from P and A, in position 11 is 11G, in position 13 is selected from 13Y, 13I, 13F, 13M, 13L, 13V, 13K and 13R, in position 18 is selected from 18I and 18V, in position 25 is selected from 25K, 25N, 25W, 25I, 25A and 25R, in position 27 is selected from 27H, 27K, 27V, 27A and 27Q, in position 29 is selected from 29R, 29K and 29P, in position 31 is selected from 31Q, 31H, 31E, 31A, 31R, 31W, 31K and 31T, in position 38 is selected from 38N, 38K and 38R, in position 40 is selected from 40H, 40K, 40Q, 40R and 40Y, in position 50 is selected from 50R, 50Q, 50K, 50T, 50V, 50M and 50S, in position 52 is selected from 52K, 52T, 52R, 52Q, 52L, 52H, 52A, 52M, 52S and 52E, in position 55 is 55M, in position 65 is selected from 65E and 65D.

EFFECT: invention enables to effectively inhibit trypsin compared with a non-modified variant.

6 cl, 18 dwg, 7 tbl, 18 ex

 

The text descriptions are given in facsimile form.

1. Selected variant of protease inhibitor Bowman Birk (BBPI), where the specified variant BBPI contains the frame containing the amino acid sequence 1-40 and 50-66 sequence SEQ ID NO: 187 and cysteine residues at amino acid positions 41 and 49 of the sequence SEQ ID NO: 187, between which concluded binding peptide, where the specified variant BBPI further comprises a substituted amino acid at amino acid position chosen from positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55 and 65 sequence SEQ ID NO: 187 where a specified substituted amino acid in position 1 represents A, p is the situation 4 is a V, in position 5 is selected from P and A, in position 11 represents 11G in position 13 selected from 13Y, 13I, 13F, 13M, 13L, 13V, 13K and 13R, at position 18 is selected from 18I and 18V in position 25 selected from 25K, 25N, 25W, 25I, 25A and 25R, in position 27 is selected from 27H, 27K, 27V, 27A and 27Q, in position 29 is selected from 29R, 29K and 29P, at position 31 is selected from 31Q, 31H, 31E, 31A, 31R, 31W, 31K and 31T, in position 38 is selected from 38N, 38K and 38R, in position 40 selected from 40H, 40K, 40Q, 40R and 40Y, in position 50 is selected from 50R, 50Q, 50K, 50T, 50V, 50M and 50S, in position 52 is selected from 52K, 52T, 52R, 52Q, 52L, 52H, 52A, 52M, 52S and 52E, in position 55 is a 55M in position 65 is selected from 65E and 65D, where the specified option selected BBPI has higher activity of trypsin inhibitor than the corresponding BBPI, devoid of the specified amino acid substitutions, where the specified binding peptide chosen from a VEGF binding peptide that binds FGF-5 peptide, TGFβ binding peptide and TNFα binding peptide.

2. Selected variant BBPI of claim 1, where the specified modified variant BBPI contains a combination of two amino acid substitutions in positions of amino acids equivalent to positions 50 and 52 of SEQ ID NO: 187.

3. Selected variant BBPI of claim 1, where the specified modified variant BBPI has the amino acid sequence of SEQ ID NO: 595.

4. Selected variant BBPI of claim 1, where the specified modified variant BBPI binds VEGF.

5. Selected variant BBPI of claim 1, where specified vyzyvayushe VEGF peptide selected from SEQ ID NO: 9, 458, 459, 460, 468, 469, 470, 471, 472 and 473.

6. Selected variant BBPI of claim 1, where the specified modified variant BBPI is a VEGF-BBPI chosen from SEQ ID NO: 601, 602, 627, 628, 630, 631, 643, 491, 632, 633, 634, 635 and 636.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

7 cl, 13 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: chicken egg albumin is mixed with an equal volume of a mixture 0.5 M aqueous trichloroacetic acid and an organic solvent in volume ratio 1:1.8-2.3. The formed residue is separated by filtering at 0-5°C. The filtrate is then mixed with an organic solvent in volume ratio of the organic solvent to the filtrate of 3-3.5:1. The supernatant fluid is then removed by decantation at 0-5°C. Low-molecular impurities are removed from ovomucoid through ultrafiltration. The ovomucoid is lyophilically dried. The organic solvent used is a mixture of ethanol and acetone in volume ratio 40-65:60-35.

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1 tbl, 5 ex

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17 cl, 2 ex

FIELD: medicine, biotechnologies.

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20 cl, 2 dwg, 8 ex

FIELD: biotechnology, microbiology, medicine.

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4 cl, 1 dwg, 2 tbl, 12 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to (i) essentially crystalline melagatran in the form of hydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 21.1, 10.5, 7.6, 7,0, 6.7, 6.4, 6.2, 5.7, 5.4, 5.3, 5.22, 5,19, 5.07, 4.90, 4.75, 4,68, 4.35, 4.19, 4.00, 3.94, 3.85, 3.81, 3.73, 3.70, 3.63, 3.52, 3.39, 3.27, 3,23, 3.12, 3.09, 3.06, 2.75, 2.38, and 2.35 Å and/or water content 4.3%; and (ii) essentially crystalline melagatran in the form of anhydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 17.8, 8.9, 8.1, 7.5, 6.9, 6.3, 5.9, 5.6, 5.5, 5.4, 5.3, 5.2, 5.0, 4.71, 4.43, 4.38, 4.33, 4.14, 4.12, 4.05, 3.91, 3.73, 3.61, 3.58, 3.56, 3.47, 3.40, 3.36, 3,28, 3.24, 3.17, 3.09, 3.01, 2.96, 2.83, 2.54, 2.49, 2.41, 2.38, and 2.35 Å. Invention also relates to a method for preparation of indicated form, a method for interconversion of anhydrite form, to use of indicated compounds as pharmaceutical agent, and to preparation of drugs. Pharmaceutical preparation is suitable for treatment of condition, in case of which inhibition of thrombin is needed or desirable. Invention provides a method for treatment of such condition.

EFFECT: increased chemical stability and solid state stability as compared to amorphous forms of melagatran.

14 cl, 4 dwg, 3 tbl, 9 ex

The invention relates to the field of molecular biology and genetic engineering and can be used in medicine

The invention relates to the field of biotechnology and biochemistry, and can be used in medicine

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to (i) essentially crystalline melagatran in the form of hydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 21.1, 10.5, 7.6, 7,0, 6.7, 6.4, 6.2, 5.7, 5.4, 5.3, 5.22, 5,19, 5.07, 4.90, 4.75, 4,68, 4.35, 4.19, 4.00, 3.94, 3.85, 3.81, 3.73, 3.70, 3.63, 3.52, 3.39, 3.27, 3,23, 3.12, 3.09, 3.06, 2.75, 2.38, and 2.35 Å and/or water content 4.3%; and (ii) essentially crystalline melagatran in the form of anhydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 17.8, 8.9, 8.1, 7.5, 6.9, 6.3, 5.9, 5.6, 5.5, 5.4, 5.3, 5.2, 5.0, 4.71, 4.43, 4.38, 4.33, 4.14, 4.12, 4.05, 3.91, 3.73, 3.61, 3.58, 3.56, 3.47, 3.40, 3.36, 3,28, 3.24, 3.17, 3.09, 3.01, 2.96, 2.83, 2.54, 2.49, 2.41, 2.38, and 2.35 Å. Invention also relates to a method for preparation of indicated form, a method for interconversion of anhydrite form, to use of indicated compounds as pharmaceutical agent, and to preparation of drugs. Pharmaceutical preparation is suitable for treatment of condition, in case of which inhibition of thrombin is needed or desirable. Invention provides a method for treatment of such condition.

EFFECT: increased chemical stability and solid state stability as compared to amorphous forms of melagatran.

14 cl, 4 dwg, 3 tbl, 9 ex

FIELD: biotechnology, microbiology, medicine.

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EFFECT: improved preparing method.

4 cl, 1 dwg, 2 tbl, 12 ex

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EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.

20 cl, 2 dwg, 8 ex

FIELD: pharmacology.

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EFFECT: enhanced purity and safety of obtained product.

17 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: aprotinin analogue with a TFFYGGSRGKRNNFKTEEY sequence is obtained, as well as a conjugate based thereon.

EFFECT: invention enables delivery of a compound or medicinal agent through the hematoencephalic barrier in mammals.

6 cl, 9 dwg, 6 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing fused proteins. The fused construct consists of an amino acid sequence of glycosyl phosphatidylinositol anchored tissue inhibitor of metalloproteinase.

EFFECT: cictrisation prevention during skin injures treatment when using the fused construct.

10 cl, 33 dwg, 18 ex

FIELD: chemistry.

SUBSTANCE: chicken egg albumin is mixed with an equal volume of a mixture 0.5 M aqueous trichloroacetic acid and an organic solvent in volume ratio 1:1.8-2.3. The formed residue is separated by filtering at 0-5°C. The filtrate is then mixed with an organic solvent in volume ratio of the organic solvent to the filtrate of 3-3.5:1. The supernatant fluid is then removed by decantation at 0-5°C. Low-molecular impurities are removed from ovomucoid through ultrafiltration. The ovomucoid is lyophilically dried. The organic solvent used is a mixture of ethanol and acetone in volume ratio 40-65:60-35.

EFFECT: high output of ovomucoid and high degree of purity.

1 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

7 cl, 13 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to an isolated version of a Bowman Birk protease inhibitor (BBPI), where said BBPI version has a frame containing amino acid sequences 1-40 and 50-66 of SEQ ID NO: 187 and cysteine residues in amino acid positions 41 and 49 of SEQ ID NO: 187, between which is included a binding peptide selected from a binding VEGF peptide, a binding FGF-5 peptide, a binding TGFb peptide and a binding TNFα peptide, where said BBPI version further includes a substituted amino acid in an amino acid position selected from positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55 and 65 of SEQ ID NO: 187, where said substituted amino acid in position 1 is A, in position 4 is V, in position 5 is selected from P and A, in position 11 is 11G, in position 13 is selected from 13Y, 13I, 13F, 13M, 13L, 13V, 13K and 13R, in position 18 is selected from 18I and 18V, in position 25 is selected from 25K, 25N, 25W, 25I, 25A and 25R, in position 27 is selected from 27H, 27K, 27V, 27A and 27Q, in position 29 is selected from 29R, 29K and 29P, in position 31 is selected from 31Q, 31H, 31E, 31A, 31R, 31W, 31K and 31T, in position 38 is selected from 38N, 38K and 38R, in position 40 is selected from 40H, 40K, 40Q, 40R and 40Y, in position 50 is selected from 50R, 50Q, 50K, 50T, 50V, 50M and 50S, in position 52 is selected from 52K, 52T, 52R, 52Q, 52L, 52H, 52A, 52M, 52S and 52E, in position 55 is 55M, in position 65 is selected from 65E and 65D.

EFFECT: invention enables to effectively inhibit trypsin compared with a non-modified variant.

6 cl, 18 dwg, 7 tbl, 18 ex

FIELD: medicine, pharmaceutics.

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6 cl, 16 dwg, 8 tbl, 6 ex

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