Analogues of insulin-like growth factor-1 (igf-1), containing amino acid substitution in position 59

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

 

The scope of the invention

The present invention relates to novel analogues of insulin-like growth factor-1 (IGF-1), to pharmaceutical compositions containing these analogues, and to the use of such analogs for treating conditions mediated by IGF-1 receptor, such as dwarfism, diabetes, neurodegenerative diseases and to restore cartilage. More specifically, the present invention relates to novel analogs of IGF-1 containing the amino acid substitution at position 59, for example (Asn59)hIGF-1(1-70)-OH and the other(s) substitution(s), as defined in the description.

Prior art

IGF-1 is a polypeptide hormone with a length of 70 amino acids, which is characterized by insulin-like, and mitogenic growth biological activity. This hormone promotes the growth of cells in various tissues, including muscle-skeletal system, liver, kidneys, intestines, tissues, nervous system, heart and lungs.

IGF-1 wild-type and has the following amino acid sequence with three megamachine disulfide bridges, where the side chain of each pair of residues of the A6and A48, A47and A52and A18and A61form disulfide bonds (SEQ ID NO:50):

Although IGF-1 is present in various body tissues, usually it is j is carried out in an inactive form in which it is associated with IGF-binding proteins (IGFBP). Known six IGFBP related, and indicate IGFBP1-IGFBP6. See, for example, Holly and Martin, "Insulin-like Growth Factor Binding Proteins: A Review of Methodological Aspects of Their Purification, Analysis and Regulation,"Growth Regul., 4(Suppl 1):20-30 (1994). IGFBP play an important role in the regulation of IGF-1 due to the presence of inhibitory and/or stimulatory effects on the action of IGF-1. For example, approximately 90% of circulating IGF-1 is present in trimolecular complex containing IGFBP-3 and are sensitive to the acidity of the additional molecule. IGF-1 in the composition of such complexes cannot contact surface receptors, and therefore it is not biologically active. IGF-1 is present in the composition trimolecular complex is also characterized by a significantly longer half-life than not part of the complex of IGF-1.

Violation actions of IGF-1 may contribute in some physiological disorders, including neurodegenerative diseases such as motor neuron disease (i.e. amyotrophic lateral sclerosis (ALS)), muscular dystrophy and multiple sclerosis, diseases of cartilage such as osteoarthritis, bone diseases such as osteoporosis, inflammatory diseases such as rheumatoid arthritis, ischemic damage to organs such as the heart, brain or liver, and so on.

As is well-known specialist is in this area, known and potential uses of IGF-1 are varied and numerous. For example, several studies have reported the use of IGF-1 as a potential therapeutic agent for the treatment of neurodegenerative conditions. Seefor example, Kanjeet al., Brain Res., 486:396-398 (1989); Hantaiet al., J. Neurol. Sci., 129:122-126 (1995); Contreraset al., Pharmac. Exp. Therap., 274:1443-1499 (1995); Di Giulioet al., Society for Neuroscience, 22:1960 (1996); Di Giulioet al., Society for Neuroscience, 23:894 (1997); Hsuet al., Biochem. Mol. Med., 60(2):142-148 (1997); Gorioet al., Neuroscience, 82:1029-1037 (1998). Therapy IGF-1 is shown in many neurological conditions, including ALS, stroke, epilepsy, Parkinson's disease, Alzheimer's disease, acute traumatic injury and other disorders associated with trauma, aging, disease or injury. See, for example, U.S. patent No. 5093137; 5652214; 5703045; international publication WO 90/1483 and WO 93/02695.

Therapy IGF-1 in various other conditions mentioned in several publications. Cm. for example, Schalch et al., "Modern Concepts of Insulin-Like Growth Factors," ed. Spencer (Elsevier, New York), p. 705-714 (1991); Clemmons and Underwood,J. Clin. Endocrinol. Metab., 79(1):4-6 (1994); and Langfordet al., Eur. J. Clin. Invest., 23(9):503-516 (1993) (referring, for example, resistant to insulin conditions and diabetes); and O'sheaet al., Am. J. Physiol., 264:F917-F922 (1993) (referring, for example, to kidney failure). See also U.S. patent No. 7258864 (related to short stature); paten the s U.S. No. 5110604 and 5427778 (related, for example, for wound healing); U.S. patent No. 5126324 (related, for example, to heart disease and stunted growth); U.S. patent No. 5368858 (related, for example, to defects or damage to the cartilage); U.S. patent No. 5543441 and 5550188 (related, for example, to cosmetic transplant tissue); patent No. 5686425 (related, for example, to tissue scarring, localized muscle dysfunction and incontinence); and U.S. patent No. 5656598 (related, for example, to increase bone). Also see international publication WO 91/12018 (related, for example, to the intestine); WO 92/09301 and WO 92/14480 (related, for example, for wound healing); WO 93/08828 (related, for example, to the neuronal damage associated with ischemia, hypoxia or neurodegeneration); WO 94/16722 (related, for example, to resistance to insulin); WO 96/02565A1 (related, for example, to the complex of IGF/IGFBP to stimulate bone formation and regulation of bone restructuring); the publication of the application for the grant of a U.S. patent 2003/0100505 (related, for example for osteoporosis) and the publication of the application for the grant of a U.S. patent 2005/0043240 (related to obesity).

Although therapy IGF-1 use for a number of physiological evidence, the results are sometimes unpredictable. Short-term beneficial effects sometimes are not saved (see,for example, Milleret al., Kidney International, 46:No.201-207 (1994)), and can cause adverse side effect the points, in particular, high doses and/or long-term administration (see,for example, Jabriet al., Diabetes, 43:369-374 (1994); Wilton,Acta Paediatr., 393:137-141 (1992)). It was also reported that high concentrations of IGF-1 increase the risk of prostate cancer (Chanet al., Science, 278:563-566 (1998)).

In accordance with this, in this area there is a need for better ways to treat conditions that respond to IGF-1 and/or other proteins that are associated with proteins that bind insulin-like growth factor. The present invention satisfies these needs and provides other related advantages.

The invention

As found by the authors of the present invention, due to the replacement of the methionine residue at position 59 IGF-1 wild type, which is chemically unstable and can easily be oxidized by another amino acid, as described herein, for example, (Asn59)hIGF-1(1-70)-OH, the resulting analogues of IGF-1 become chemically more stable and as such is less susceptible to oxidation during production, purification, storage, etc.

In one aspect the present invention relates to peptide variants (i.e. peers) IGF-1 following formula (I):

in which:

A-1represents Met, Ser, or demeterova;

A1p is ecstasy a Gly, Ala, Asn, Asp, Gln, Glu or demeterova;

A2is a Pro, Ala, Arg, Asp, Gln, Glu, Lys or demeterova;

A3represents Glu, Ala, Asp, Gln or demeterova;

A4represents Thr, Ala, Asn, Asp, Gln, Glu, Ser;

A5represents Leu, Acc, Ala, Ile or Val;

A6represents Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen or D-Pen;

A7represents Gly, Ala, Asn, Asp, Gln or Glu;

A8represents Ala, Arg, Asn, Asp, Gln, Glu or Lys;

A9represents Glu, Ala, Asp or Gln;

A10represents Leu, Acc, Ala, Ile or Val;

A11represents Val, Ala, Ile or Leu;

A12represents Asp, Ala, Arg, Asn, Gln, Glu or Lys;

A13represents Ala, Asn, Asp, Gln, Glu, Ile, Leu or Val;

A14represents Leu, Acc, Ala, Ile or Val;

A15represents Gln, Ala, Asn, Asp or Glu;

A16represents Phe, Ala, Asn, Asp, Gln, Glu, Trp or Tyr;

A17represents Val, Ala, Ile or Leu;

A18represents Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen or D-Pen;

A19represents Gly, Ala, Asn, Asp, Gln or Glu;

A20represents Asp, Ala, Asn, Gln or Glu;

A21represents Arg, Ala, Asn, Asp, Gln, Glu or Lys;

A22represents Gly, Ala, Asn, Asp, Gln or Glu;

A23represents Phe, Ala, Trp or Tyr;

A24represents Tyr, Ala, Phe or Tp;

A25represents Phe, Ala, Trp or Tyr;

A26represents Asn, Ala, Asp, Gln, Glu, Ser, or Thr;

A27represents Lys, Ala, Arg, Asn, Asp, Gln, Glu or Pro;

A28is a Pro, Ala, Arg or Lys;

A29represents Thr, Ala, Asn, Asp, Gln, Glu or Ser;

A30represents Gly, Ala, Asn, Asp, Gln or Glu;

A31represents Tyr, Ala, Phe or Trp;

A32represents Gly, Ala, Asn, Asp, Gln or Glu;

A33represents Ser, Ala, Thr or Val;

A34represents Ser, Ala, Asn, Asp, Gln, Glu, or Thr;

A35represents Ser, Ala, Asn, Asp, Gln, Glu, or Thr;

A36represents Arg, Ala, Asn, Asp, Gln, Glu or Lys;

A37represents Arg, Ala, Asn, Asp, Gln, Glu or Lys;

A38represents Ala, Asn, Asp, Gln or Glu;

A39is a Pro, Ala, Arg or Glu;

A40represents Gln, Ala, Asn, Asp or Glu;

A41represents Thr, Ala, Asn, Asp, Gln, Glu or Ser;

A42represents Gly, Ala, Arg, Asn, Asp, Gln, Glu or Lys;

A43represents Ile, Ala, Arg, Asn, Asp, Gln, Glu or Lys;

A44represents Val, Ala, Arg, Asn, Asp, Gln, Glu, Ile, Leu, or Lys;

A45represents Asp, Ala, Arg, Asn, Gln, Glu or Lys;

A46represents Glu, Ala, Arg, Asn, Asp, Gln or Lys;

A47represents Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen or D-Pen;

A48represents Cys, D-Cys, hys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen or D-Pen;

A49represents Phe, Ala, Arg, Ile, Leu, Lys, Ser, Thr, Trp, Tyr or Val;

A50represents Arg, Ala, Lys, Ser or Thr;

A51represents Ser, Aib, Ala, Arg, Lys or Thr;

A52represents Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen or D-Pen;

A53represents Asp, Ala, Arg, Asn, Gln, Glu, Lys, Ser or Thr;

A54represents Leu, Acc, Ala, Ile or Val;

A55represents Arg, Ala, Ile, Leu, Lys, Phe, Trp, Tyr or Val;

A56represents Arg, Ala, Asn, Asp, Gln, Glu or Lys;

A57represents Leu, Acc, Ala, Ile or Val;

A58represents Glu, Acc, Ala, Arg, Asn, Asp, Gln or Lys;

A59represents the Acc, Ala, Arg, Asn, Asp, Gln, Glu, Ile, Leu, Lys, Nle, Ser, D-Ser, Thr, Trp, Tyr or Val;

A60represents Tyr, Ala, Phe or Trp;

A61represents Cys, D-Cys, hCys, D-hCys, β-Me-Cys, D-β-Me-Cys, N-Me-Cys, D-N-Me-Cys, Ala, Pen or D-Pen;

A62represents Ala, Asn, Asp, Gln, Glu, Ile, Leu or Val;

A63is a Pro, D-Pro, Ala, Ser, Thr or demeterova;

A64represents Leu, D-Leu, des-Leu, Ala, Ile, Val or demeterova;

A65represents Lys, D-Lys, des-Lys, Ala, Arg, Ile, Leu, Val or demeterova;

A66is a Pro, D-Pro, Ala or demeterova;

A67represents Ala, D-Ala, Aib or demeterova;

A68represents Lys, D-Lys, Ala, Arg, Ile, Leu, Val or deleterow is;

A69represents Ser, D-Ser, Aib, Ala, Thr or demeterova;

A70represents Ala, D-Ala, Asn, Asp, Gln, Glu or demeterova;

A71represents Asn, Ala, Asp, Gln, Glu, Lys, Ser, Thr or demeterova; and

R1represents OH or NH2;

provided that the side chains of each pair of residues of the A6and A48, A47and A52and A18and A61form disulfide bonds; and

also provided that, when A59represents Leu, Ile, Nle, Thr, or Val, analog contains at least one additional amino acid substitution or insertion, defined in this document.

In the formula (I) preferred amino acid substitutions and additions are defined as follows:

A-1represents Met, Ser, or demeterova;

A1represents Gly or demeterova;

A2is a Pro, Lys or demeterova;

A3represents Glu or demeterova;

A4represents Thr;

A5represents Leu;

A6represents Cys, hCys, β-Me-Cys, N-Me-Cys or Pen;

A7represents Gly;

A8represents Ala;

A9represents Glu;

A10represents Leu;

A11represents Val;

A12is an Asp;

A13represents Ala;

14represents Leu;

A15represents Gln;

A16represents Phe;

A17represents Val;

A18represents Cys, hCys, β-Me-Cys, N-Me-Cys or Pen;

A19represents Gly;

A20is an Asp;

A21represents Arg;

A22represents Gly;

A23represents Phe;

A24represents Tyr;

A25represents Phe;

A26represents Asn;

A27represents Lys, Arg or Pro;

A28represents Pro or Lys;

A29represents Thr;

A30represents Gly;

A31represents Tyr;

A32represents Gly;

A33represents Ser;

A34represents Ser;

A35represents Ser;

A36represents Arg;

A37represents Arg;

A38represents Ala;

A39is a Pro;

A40represents Gln;

A41represents Thr;

A42represents Gly;

A43represents Ile;

A44represents Val;

A45is an Asp;

A46represents Glu;

A47represents Cys, hCys, β-Me-Cys, N-Me-Cys or Pen;

A48represents Cys, hCs, β-Me-Cys, N-Me-Cys or Pen;

A49represents Phe, Arg, Leu, or Thr;

A50represents Arg or Ser;

A51represents Ser, Aib, Arg or Thr;

A52represents Cys, hCys, β-Me-Cys, N-Me-Cys or Pen;

A53represents Asp, Arg or Ser;

A54represents Leu or A6c;

A55represents Arg or Tyr;

A56represents Arg or Gln;

A57represents Leu;

A58represents Glu or Arg;

A59represents A6c, Arg, Asn, Asp, Gln, Glu, Ile, Leu, Nle, Ser, D-Ser, Trp or Tyr;

A60represents Tyr or Phe;

A61represents Cys, hCys, β-Me-Cys, N-Me-Cys or Pen;

A62represents Ala or Asn;

A63is a Pro, D-Pro, Thr or demeterova;

A64represents Leu, D-Leu, des-Leu or demeterova;

A65represents Lys, D-Lys, des-Lys, Arg or demeterova;

A66is a Pro, D-Pro or demeterova;

A67represents Ala, D-Ala, Aib or demeterova;

A68represents Lys, D-Lys, Arg or demeterova;

A69represents Ser, D-Ser, Aib, Thr or demeterova;

A70represents Ala, D-Ala, Glu or demeterova; and

A71represents Asp, Glu, Lys, Ser or demeterova.

A subset of the compounds covered by formula (I)includes compounds in which the A 59represents Asn.

Another subset of the compounds covered by formula (I)include compounds in which A59represents Leu, where these compounds contain at least one additional amino acid substitution or insertion selected from the group consisting of Arg27, Arg65, Arg68, Leu49, β-Me-Cys47, β-Me-Cys52, Thr51, Thr69Asp71Glu71, Lys71and Ser71.

Another subset of the compounds covered by formula (I)include compounds in which A59represents Nle, where these compounds contain at least one additional amino acid substitution selected from the group consisting of Aib51, Aib67, Aib69, A6c54N-Me-Cys47N-Me-Cys48, Pen52and Pen61.

Another subset of the compounds covered by formula (I)include compounds in which A59represents Ile, where these compounds contain at least one amino acid substitution selected from the group consisting of Arg58, Arg49, Arg51and Arg53.

Another subset of the compounds covered by formula (I)include compounds in which A59represents Arg, Asp, A6c, Gln, Glu, Ser, Trp or Tyr.

Preferred compounds of formula (I) are:

Example 1: (Asn59)hIGF-1(170)-OH; (SEQ ID NO:1)

Example 2: (Asn59)hIGF-1(1-62)-OH; (SEQ ID NO:2)

Example 3: (Asn59)hIGF-1(4-70)-OH; (SEQ ID NO:3)

Example 4: (Pro27, Lys28, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:4)

Example 5: (Pro27, Lys28, Asn59)hIGF-1(1-62)-OH; (SEQ ID NO:5)

Example 6: (Ser53, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:6)

Example 7: (Ser-Gly1, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:7)

Example 8: (Asn59, Thr63des-Leu64des-Lys65Glu70)hIGF-1(1-70)-OH; (SEQ ID NO:8)

Example 9: (Tyr55, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:9)

Example 10: (Thr49, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:10)

Example 11: (Asn59,62)hIGF-1(1-70)-OH; (SEQ ID NO:11)

Example 12: (Asn59, Phe60)hIGF-1(1-70)-OH; (SEQ ID NO:12)

Example 13: (Ser50, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:13)

Example 14: (Gln56, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:14)

Example 15: (Asn59D-Pro63)hIGF-1(1-70)-OH;

Example 16: (Asn59, D-Leu64)hIGF-1(1-70)-OH;

Example 17: (Asn59, D-Lys65)hIGF-1(1-70)-OH;

Example 18: (Asn59D-Pro66)hIGF-1(1-70)-OH;

Example 19: (Asn59, D-Ala67)hIGF-1(1-70)-OH;

Example 20: (Asn59, D-Lys68)hIGF-1(1-70)-OH;

Example 21: (Asn59, D-Ser69)hIGF-1(1-70)-OH;

Example 22: (Asn59, D-Ala70)hIGF-1(1-70)-OH;

Example 23: (Arg27,65,68, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO:15)

Example 24: (Leu59, Arg65,68)hIGF-1(1-70)-OH; (SEQ ID NO:16)

Example 25: (Leu49,59)hIGF-1(1-70)-OH; (SEQ ID NO:17)

Example 26: (β-Me-Cys52, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO:18)

Example 27: (βMe-Cys 47, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO:19)

Example 28: (Leu59Glu71)hIGF-1(1-71)-OH; (SEQ ID N0:20)

Example 29: (Leu59Asp71)hIGF-1(1-71)-OH; (SEQ ID NO:21)

Example 30: (Leu59, Lys71)hIGF-1(1-71)-OH; (SEQ ID NO:22)

Example 31: (Leu59Ser71)hIGF-1(1-71)-OH; (SEQ ID NO:23)

Example 32: (Leu59, Thr69)hIGF-1(1-70)-OH; (SEQ ID NO:24)

Example 33: (Thr51, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO:25)

Example 34: (N-Me-Cys47, Nle59)hIGF-1(1-70)-OH; (SEQ ID NO:26)

Example 35: (Nle59, Aib69)hIGF-1(1-70)-OH; (SEQ ID NO:27)

Example 36: (N-Me-Cys48, Nle59)hIGF-1(1-70)-OH; (SEQ ID NO:28)

Example 37: (Nle59, Aib67)hIGF-1(1-70)-OH; (SEQ ID NO:29)

Example 38: (hCys52, Nle59)hIGF-1(1-70)-OH; (SEQIDNO:30)

Example 39: (Aib51, Nle59)hIGF-1(1-70)-OH; (SEQ ID N0:31)

Example 40: (Pen52, Nle59)hIGF-1(1-70)-OH; (SEQ ID NO:32)

Example 41: (Nle59, Pen61)hIGF-1(1-70)-OH; (SEQ ID NO:33)

Example 42: (A6c54, Nle59)hIGF-1(1-70)-OH; (SEQ ID NO:34)

Example 43: (Arg53, Ile59)hIGF-1(1-70)-OH; (SEQ ID NO:35)

Example 44: (Arg49, Ile59)hIGF-1(1-70)-OH; (SEQ ID NO:36)

Example 45: (Arg51, Ile59)hIGF-1(1-70)-OH; (SEQ ID NO:37)

Example 46: (Arg58, Ile59)hIGF-1(1-70)-OH; (SEQ ID NO:38)

Example 47: (A6c59)hIGF-1(1-70)-OH; (SEQ ID NO:39)

Example 48: (Asp59)hIGF-1(1-70)-OH; (SEQ ID NO:40)

Example 49: (Trp59)hIGF-1(1-70)-OH; (SEQ ID NO:41)

Example 50: (Ser59)hIGF-1(1-70)-OH; (SEQ ID NO:42)

Example 51: (Tyr59)hIGF-1(1-70)-OH; (SEQ ID NO:43)

Example 52: (Glu59)hIGF-1(1-70)-OH; (SEQ ID NO:44)

Example 53:(Gln 59)hIGF-1(1-70)-OH; (SEQ ID NO:45)

Example 54: (Arg59)hIGF-1(1-70)-OH; (SEQ ID NO:46), and

Example 55: (Met-Gly1, Asn59)hIGF-1(1-70)-OH. (SEQ ID NO:47)

Detailed description of the invention

The application uses the following abbreviations:

Acc: 1-amino-1-cyclo(C3-C9)alkalicarbonate acid

Acc includes:

A3c: 1-amino-1-cyclopropanecarbonyl acid

A4c: 1-amino-1-cyclobutanecarbonyl acid

A5c: 1-amino-1-cyclopentanecarbonyl acid

A6c: 1-amino-1-cyclohexanecarbonyl acid

Aib: α-aminoadamantane acid

Ala or A: alanine

Arg or R: arginine

Asn or N: asparagine

Asp or D: aspartic acid

Cys or C: cysteine

cystine: disulfide dimer of cysteine

hCys: homocysteine

β-Me-Cys: beta-methylcysteine, ie,

(2S,3S)-2-amino-3-mercaptoethane acid

N-Me-Cys: N-methylcysteine

Gln or Q: glutamine

Glu or E: glutamic acid

Gly or G: glycine

Ile or I: isoleucine

Leu or L-leucine

des-Leu: deleteriously Leu

Lys or K: lysine

des-Lys: deleteriously Lys

Met or M: methionine

Nle: norleucine

Pen: penicillamine

Phe or F: phenylalanine

Pro or P: Proline

Ser or S: serine

Thr or T: threonine

Trp or W: tryptophan

Tyr or Y: tyrosine

Val or V: valine

All abbreviations (e.g., Ala) of amino acids in this description to denote the structure-NR-CR'(R”)-CO-, in which each and the R' and R” independently represents hydrogen or a side chain amino acids (for example, R'=H and R”=CH3for alanine) and in which R=H or CH3except Proline, ie,

The peptide according to this invention is also denoted in this document in another format, for example (Asn59)hIGF-1(1-70)-OH (SEQ ID NO:1), amino, substituted compared to the natural sequence placed between the parentheses (i.e. Asn instead of Met at position 59 IGF-1 wild type). The interval, in parentheses, refers to those amino acids that are similar. For example, IGF-1(4-68)-OH” (SEQ ID NO:48) indicates that the analogue contains amino acids from 4 to 68, which correspond to the peptide sequence of the IGF-1 wild type. “NH2” in IGF-1(1-70)-NH2” (SEQ ID NO:49) indicates that the C-end of the peptide amitirova. “IGF-1(1-70)or IGF-1(1-70)-OH” indicates that the C-end is a free acid (SEQ ID NO:50).

Some other abbreviations used herein are defined as follows:

Act: acetonitrile

Boc:tert-butyloxycarbonyl

BSA: bovine serum albumin

DCM: dichloromethane

DIPEA: diisopropylethylamine

DMEM: modified by way of Dulbecco Wednesday Needle

DMF: dimethylformamide

DTT: dithiothreitol

ESI: electrospray ionization

FCS: fetal calf serum

Fmoc: 9-fluorenylmethoxycarbonyl

HBTU: hexaphosphate 2-(1-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium

HOBt, N-hydroxybenzotriazole

HPLC: high performance liquid chromatography

LC-MS: liquid chromatography-mass spectrometry

MPAA: 4-mercaptophenylacetic acid

NMP: N-methylpyrrolidinone

OtBu: O-tert-butyl ester

Pbf: 2,2,4,6,7-pentamethylcyclopentadiene-5-sulfonyl

QC: quality control

tBu:tert-butyl

TCA: trichloroacetic acid

TCEP: Tris-2-carboxyhemoglobin

TIS: triisopropylsilane

TFUCK: triperoxonane acid

Tris: 2-amino-2-(hydroxymethyl)-1,3-propandiol

Trt: trail

UV spectroscopy: ultraviolet spectroscopy

"Alkyl" refers to a hydrocarbon group containing one or more carbon atoms, where multiple carbon atoms, if present, are connected by single bonds. Examples of this group include, but are not limited to, methyl, ethyl, propyl and butyl. Alkyl hydrocarbon group may be linear or may contain one or more branches or cyclic group, examples of which include, but are not limited to, isopropyl and tert-butyl.

"Substituted alkyl" refers to an alkyl in which one or more hydrogen atoms of the hydrocarbon group substituted by one or more substituents selected from the group consisting of halogen, OH, CN, SH, NH2, NHCH3, NO2, (Csub> 1-2)-alkyl, substituted by 1-6 halogen, CF3, OCH3, OCF3and (CH2)0-4-COOH. In other embodiments, implementation of the present 1, 2, 3, or 4 substituent.

"Aryl" refers to optionally substituted aromatic group, at least one ring having a conjugate system of PI electrons, containing up to three conjugated or condensed ring systems. Aryl includes carbocyclic aryl, heterocyclic aryl and burilnye group. Preferably, the aryl is a 5 - or 6-membered ring. Preferred atoms for heterocyclic aryl represents one or more atoms of sulfur, oxygen and/or nitrogen. Examples of aryl include phenyl, 1-naphthyl, 2-naphthyl, indole, quinoline, 2-imidazole, and 9-anthracene. Aryl substituents are selected from the group consisting of-C1-20-alkyl, -C1-20-alkoxy, halogen, -OH, -CN, -SH, -NH2, -NO2, -C1-20-alkyl substituted by halogen, -CF3, -OCF3and -(CH2)0-20-COOH. In other embodiments, implementation of the aryl contains 0, 1, 2, 3, or 4 substituent.

"Alkylaryl" applies to "alkyl"attached to "aryl".

The procedure of synthesis

Presents as an example, analogs of IGF-1 according to the present invention was obtained by the first stage of the synthesis the peptide fragments, the second stage of stitching (whether the investments) and the third stage of folding. The following procedures illustrate the synthesis, as a specialist chemist can provide any of the presents as an example, analogs of IGF-1 according to the present invention.

A)Synthesis of peptide fragment (Gln56, Asn59)hIGF-1(48-70)-OH,i.e. Cys-Phe-Arg-Ser-Cys-Asp-Leu-Arg-Gln-Leu-Glu-Asn-Tyr-Cys-Ala-Pro-Leu-Lys-Pro-Ala-Lys-Ser-Ala-OH_(SEQ ID NO:51)

Based on Fmoc solid-phase peptide synthesis was used for the Assembly specified in the header of the peptide fragment using auxiliary microwave treatment on a peptide synthesizer Liberty (CEM; Matthews, NC, USA). The first fragment of the 14 residues, such as residues 57-70 hIGF-1, or a peptide from the C-terminal acids synthesized in a 1.0 mmol scale using Fmoc-Ala-Wang resin (0.72 mg-EQ./g). The resulting peptide fragment was then divided into four parties 0.25 mmol for lengthening and separating. A sample of the resin weight of 1.36 g) was placed in a conical tube 50 ml with 15 ml of a 1:1 DMF and DCM, which was loaded into the specified position in the synthesizer. Then the resin was transferred into the reaction vessel through the automatic process synthesizer. Used a standard Protocol Liberty for a 1.0 mmol scale. The Protocol included the removal of N-terminal protective Fmoc group by treatment with 20 ml of 20% piperidine containing 0.1 M HOBt in DMF. The initial stage unprotect using a MIC is wave power (45 watts, the maximum temperature 75°C) and ozonation with nitrogen (3 seconds on, 7 seconds off) lasted for 30 seconds. From the reaction vessel solution was removed and the resin thoroughly washed with DMF several times. Then added the following amino acid (cycle 1), which should be attached to the growing peptide (Fmoc-Ser(tBu)-OH)obtained in the form of a stock solution of 0.2 M in DMF (15 ml, 3 equivalents). Added to 6.0 ml of 0.45 M (3 equivalent), HBTU in DMF, after which contributed 3.0 ml of 2 M (6 equivalents) of DIPEA in NMP. Stage of accession was performed using microwave energy (20 watts, maximum temperature 75°C) with bubbling nitrogen in the same respect, both at the stage of removing protection, within a period of 5 minutes. Then the reaction vessel was removed, the solution draining and repeating stage of accession.

The Protocol of accession for Fmoc-Cys(Trt)-OH was a slightly modified version of the standard Protocol. For Cys residues within the first 2 minutes are not used microwave energy.

Followed by 4-minute period using microwave energy (20 watts, maximum temperature 50°C). All amino acids were introduced in the same way, using the dual strategy of joining throughout the whole sequence. Cycles of synthesis for the specified header peptide fragment after the first Se were as follows: cycle 2, Fmoc-Lys(Boc)-OH; cycle 3, Fmoc-Ala-OH; cycle 4, Fmoc-Pro-OH; cycle 5, Fmoc-Lys(Boc)-OH; cycle 6, Fmoc-Leu-OH; cycle 7, Fmoc-Pro-OH; cycle 8, Fmoc-Ala-OH; cycle 9, Fmoc-Cys(Trt)-OH; cycle 10, Fmoc-Tyr(tBu)-OH; cycle 11, Fmoc-Asn(Trt)-OH; cycle 12, Fmoc-Glu(OtBu)-OH; cycle 13, Fmoc-Leu-OH.

As soon as the original peptide fragment was completed, the resin was transferred back into the conical tube 50 ml using DMF as solvent. The resin manually evenly divided into four pieces, which were placed in four conical tubes 50 ml, which was then installed back into the synthesizer. The remaining part is specified in the title peptide was synthesized in 0.25 mmol scale. The Protocol used was the same as the one used for the synthesis on a larger scale, but used smaller amounts of reagents. Removal of N-terminal protective Fmoc group consisted of processing solution containing 10 ml of 20% piperidine and 0.1 M HOBt in DMF. The initial stage unprotect using microwave energy (45 watts, the maximum temperature 75°C) and ozonation with nitrogen (3 seconds on, 7 seconds off) lasted for 30 seconds. Then from the reaction vessel solution was removed and the resin was thoroughly washed several times DMF. Then typed the following amino acid (cycle 14), resulting in a 0.2 M stock solution in DMF (5.0 ml, 4 equivalents), in the growing peptide (Fmoc-Gln(tBu)-OH). Then we use the and 2.0 ml of 0.45 M solution (4 equivalent), HBTU in DMF, then was brought to 1.0 ml of 2 M solution (8 equivalents) of DIPEA in NMP.

The protocols of accession for Fmoc-Cys(Trt)-OH and Fmoc-Arg(Pbf)-OH was a slightly modified version of the standard Protocol. To attach Cys residues microwave energy was first off in the first 2 minutes, then turn on 4 minutes (20 watts, maximum temperature 50°C). To attach residues Arg microwave energy is not used when you first join, but needed a second stage standard connection. In loops 14, 16 and 21 used the procedure capping, which is followed by a stage of accession, including the addition of 7 ml of 0.5 M acetic anhydride containing 0.015 M HOBt and 2 ml of 2 M DIPEA, both compounds were dissolved in NMP using multistage Protocol microwave treatment (50 watts for 30 seconds with a maximum temperature of 65°C, followed by no treatment for 30 seconds, 50 watts for 30 seconds with a maximum temperature of 65°C, then without treatment within 30 seconds). Cycles of synthesis for the specified header peptide fragment after Gln were as follows: cycle 15, Fmoc-Arg(Pbf)-OH; cycle 16, Leu-OH; cycle 17, Fmoc-Asp(OtBu)-OH; cycle 18, Fmoc-Cys(Trt)-OH; cycle 19, Fmoc-Ser(tBu)-OH; cycle 20, Fmoc-Arg(Pbf)-OH; cycle 21, Fmoc-Phe-OH; cycle 22, Fmoc-Cys(Trt)-OH.

After completion of the peptide backbone deleted N-terminal protective Fmoc group, the resin was again washed with DMF. Then the resin was transferred back into the conical tube 50 ml using DMF as solvent for the transfer.

The resin was transferred into a reaction vessel with a porous glass filter. Remove DMF and the resin was intensively washed DCM. The peptide fragment was tsalala and was released from the protection of the following reagent: 5% TIS : 5% water : 90% TFUCK. The reaction mixture was allowed to incubated for 3 hours at room temperature and constant stirring. Then the solution was filtered in a conical tube 50 ml Level TFOC reduced by evaporation in a stream of nitrogen gas. The peptide fragment was besieged by the addition of 40 ml of cold simple ethyl ether, followed by centrifugation at 3000 rpm for 30 minutes at 4°C in a refrigerated centrifuge (Sorvall Legend RT; Thermo Fisher, San Jose, CA, USA). The resulting precipitate was dissolved in 0.1% TFUK in water before purification by preparative HPLC, equipped with a column with reversed phase C18 (Luna 10 μm, column 250×21,2 mm)using a gradient of 0-60% acetonitrile (0.1% TFUC) for 50 minutes at a flow rate of 10 ml/min of Purified peptide fragment was analyzed by HPLC (Luna C18, 3 µm column of 4.6×100 mm) with a gradient of 5-80% acetonitrile (0,08% TFUC) within 30 minutes, with a flow rate of 1 ml/min and by mass spectrometry (LCQ Advantage; Thermo Fisher, San Jose, CA, USA). Peptide frag the UNT then liofilizirovanny and stored at -50°C for further use.

B)Synthesis of a peptide fragment of hIGF-1(1-47)-tiefer,i.e. Gly-Pro-Glu-Thr-Leu-Cys-Gly-Ala-Glu-Leu-Val-Asp-Ala-Leu-Gln-Phe-Val-Cys-Gly-Asp-Arg-Gly-Phe-Tyr-Phe-Asn-Lys-Pro-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr-Gly-Ile-Val-Asp-Glu-Cys-diafiltration-Leu-NH2(SEQ ID NO:52)

N-terminal peptide fragment, such as residues 1-47 hIGF-1, combined with the use based on Boc-chemistry solid phase peptide synthesis. For synthesis, 0.5 mmol scale used peptide synthesizer ABI 433A (Applied Biosystems; Foster City, CA, USA), modified to run standard Protocol FastBoc. The reaction vessel containing to 0.645 mg of 0.77 mg-EQ./g Tampal resin, was placed on the synthesizer. For swelling resin was injected DMF. The Protocol ABI FastBoc of 0.5 was used to obtain the fragment. Each cycle consisted of the release of the N-terminal protective group Boc using net TFUK followed by abundant rinsing DMF. Pre-Packed cartridges 2.0 mmol (4 equivalents) of each amino acid was then dissolved in 0,40 M HBTU, DMF. After complete dissolution of each amino acid solution is automatically moved in the capacity for activation. A solution of DIPEA (net) was injected into the vessel to activate and were subjected to resin for a long period. The reaction vessel was drained and the resin washed with DMF. For cartridges Arg/Asn required a long activation time, to ensure solubility. In addition to the CSO, any amino acid added directly after joining the Gln residue, washed with DCM before and after implementation of the Protocol release. The connection was 30 minutes. The following amino acids used for the specified header peptide fragment: Boc-Arg(Tos)-OH, Boc-Asp(cHex)-OH, Boc-Glu(cHex)-OH, Boc-Asn(Xan)-OH, Boc-Cys(4Me-Bzl)-OH, Boc-Lys(ClZ)-OH, Boc-Gln-OH, Boc-Ser(OBzl)-OH, Boc-Thr(OBzl)-OH and Boc-Tyr(BrZ)-OH.

After the last cycle of joining the resin washed with DCM and dried. With the peptide fragment is removed protection and tsalala it from the resin using processing 10 ml of hydrogen fluoride and anisole. The reaction was allowed to run for 70 minutes, and at this point, the fluoride was washed by a stream of nitrogen. The residue was washed with a simple ether and then the peptide was dissolved in 10-15 ml TFUCK. The peptide fragment was besieged by filtering TFUK in 40 ml of cold simple ethyl ether, followed by centrifugation at 3000 rpm for 30 minutes at 4°C in a refrigerated centrifuge (Sorvall Legend RT; Thermo Fisher, San Jose, CA, USA). The resulting precipitate was dissolved in 0.1% TFUK in water and purified by preparative HPLC, equipped with a column with reversed phase C18 (Luna 10 μm, column 250×21,2 mm)using a gradient of 20-40% acetonitrile (0.1% TFUC) for 120 minutes, with a flow rate of 10 ml/min of Purified peptide fragment was analyzed by HPLC (Luna C18, 3 µm column of 4.6×100 is m) with a gradient of 5-80% acetonitrile (0,08% TFUC) within 30 minutes, with a flow rate of 1 ml/min and by mass spectrometry (LCQ Advantage; Thermo Fisher, San Jose, CA, USA). Then peptide fragment liofilizirovanny and stored at -50°C for further use.

C)General procedure for stitching

Full-sized counterparts hIGF-1 was designed by way of chemical cross linking that occurs in nature between the N-terminal fragment with complex tieferen, for example hIGF-1(1-47)-S-(CH2)2C(0)-Leu-NH2(SEQ ID NO:52), and a C-terminal fragment, for example (Gln56, Asn59)hIGF-1(48-70)-OH (SEQ ID NO:51), which contains at its N-end balance cysteine.

To begin the method of obtaining specified in the header of the peptide of 5.5 mg C-terminal fragment of hIGF-1 was dissolved in 0.5 ml of buffer for stitching (200 mm sodium phosphate, pH 8.5, 6M guanidine hydrochloride) in an Eppendorf tube of 1.5 ml of this solution was added 100 μl of TCEP solution (40 mg/ml) and the mixture was shaken. The mixture was transferred into a second Eppendorf tube containing 6.5 mg of the N-terminal fragment of hIGF-1 complex tieferen. The reagents were thoroughly mixed. A small sample (5 μl) was removed and analyzed by LC-MS (LCQ Deca XP; Thermo Fisher, San Jose, CA, USA). To the reaction mixture were added 100 μl of a solution MPAA (20 mg/ml), followed by stirring. Samples (5 μl) were periodically removed in order to monitor the reaction using LC-MS. After about 3.5 hours, when the reaction was near completion, the mixture was suppressed and was diluted by the addition of 9.5 ml of 0.1% TFU is in the water. The staple product was purified by prepreparation HPLC (Vydac 218TP101510, C18, 10-15 μm, 10×250 mm) in a gradient of 5-80% acetonitrile (0.1% TFUC) for 40 minutes, with a flow rate of 5 ml/min Peak product liofilizirovanny and kept at -50°C. the Mass is not subjected to the folding of the product of crosslinking was determined by physical measurement.

D)The General procedure of folding (redox pair glutathione) for example 14,ie (Gln56, Asn59)hIGF-1 (1-70)-OH(SEQ ID NO:14)

The protein obtained in the fusion process stage C), as described above, was dissolved in the buffer for stitching (200 mm sodium phosphate, pH 8.5, 6M guanidine hydrochloride) to a concentration of 1 mg/ml was Then added to the buffer for folding (100 mm Tris, pH 8.5, 1 mm oxidized glutathione, 10 mm restored glutathione) to achieve a final protein concentration of 0.25 mg/ml Provided a process of folding for 3 hours. After this reaction was suppressed by adding dropwise TFUK to achieve in the reaction mixture pH ≤3. Then the product was purified by prepreparation HPLC (Vydac 218TP101510, C18, 10-15 μm, column, 10×250 mm) in a gradient of 5-60% acetonitrile (0.1% TFUC) for 40 minutes, with a flow rate of 5 ml/min Product liofilizirovanny. The protein content was determined by re-dissolving the product in 0.1% TFUK in the water, then measuring the optical density at 280 nm (spectropho is Omer NanoDrop ND1000). Then protein was analyzed for QC (HPLC and MS).

E)The procedure of oxidation to obtain (glyoxylyl-Gly1, Asn59)hIGF-1(1-70)-OH (SEO ID NO:53) of example 7,ie (Ser-Gly1, Asn59)hIGF-1(1-70) - OH (SEQ ID NO:7)

The mass is subjected to a folding analog hIGF-1 was determined by optical density at 280 nm in 0.1% TFUK in water (spectrophotometer NanoDrop ND1000). The protein obtained in the process of folding in stage D), as described above, re-dissolved in 50 mm imidazole buffer (pH 7.0) to a final concentration of 2 mg/ml (of 2.66×10-4M). Added periodate sodium (NaIO4) (4 equivalents), dissolved in imidazole buffer, and the resulting solution was carefully mixed. The reaction was allowed to occur at room temperature without further agitation. After 5 minutes, the reaction was suppressed by the addition of 10 equivalents of ethylene glycol. The mixture was allowed to stand for 15 minutes at room temperature. The mixture was diluted with 0.1% TFUK in water to a final volume of 10 ml and Then the product was purified by prepreparation HPLC (Vydac 218TP101510, C18, 10-15 μm, column, 10×250 mm) in a gradient of 5-60% acetonitrile (0.1% TFUC) for 40 minutes, with a flow rate of 5 ml/min Then the product liofilizirovanny and stored at -50°C until required application.

F)Synthesis procedure for example 27,ie (β-Me-Cys47, Leu59)hIGF-1(1-70)-OH (SEQ ID NO:19)

is shown in the header of the protein was joined by native chemical crosslinking using hIGF(1-46)-dipropionyl-Leu-NH 2(SEQ ID NO:54) and C-terminal fragments, i.e. (β-Me-Cys47, Leu59)hIGF-1(47-70) (SEQ ID NO:55). Complex tiefer protein (7.4 mg, 1,45 mmol) and C-terminal fragment (3.8 mg, 1.38 mmol) was dissolved in the buffer for stitching (6 M guanidine hydrochloride in 200 mm sodium phosphate, pH 8.5, 400 mm) and TCEP (80 μl, 40 mg/ml, pH 7). Added catalyst MPAA (80 μl, 20 mg/ml, pH 7). The progression of the reaction was monitored by LC-MS on a LCQ Deca XP (Thermo Finnigan) with column Luna C18(2) (5 μm, a 4.6×100 mm) in a gradient of 5-80% acetonitrile (0.1% TFUC) for 30 minutes. The reaction was suppressed by diluting 1:10 dH2O c 0.1% TFOC (about./vol.). The crude mixture was centrifuged and passed through a glass filter with pores of 1.0 μm to remove any sediment MPAA. Full-size protein was purified using a linear gradient B 5-60% within 40 minutes, with a flow rate of 5 ml/min on a Vydac C18 (10 μm, 10×250 mm). Protein was quantified by UV spectroscopy (NanoDrop spectrophotometer ND1000) and liofilizirovanny for future use.

Stored protein (1.8 mg, 235 nmol) was dissolved in a solution of 200 mm H2PO4-, 6 M guanidine-HCl having pH 8.5, to a concentration of 1.0 mg/ml Buffer for folding (100 mm Tris, 10 mm glutathione, 1 mm oxidized glutathione at pH 8.5) was added to the solution to achieve a final protein concentration of 250 μg/ml and the Mixture was left to incubated at room temperature, creating e is ω monitored by HPLC. As he reached the equilibrium (that was visualized in the form of a stable profile HPLC), the reaction was suppressed by mixing with either acetic acid or with TFUK to bring the pH of the solution to 3. The solution was purified by using a first glass filter with pores of 1.0 μm and then prepreparation column.

Subjected to the folding of the protein was purified using a linear gradient B 5-60% within 40 minutes, with a flow rate of 5 ml/min Protein was quantified by UV spectroscopy (NanoDrop spectrophotometer ND1000) and liofilizirovanny. Received approximately 92 µg of purified product with a representation of the output 5%. The mass of the protein was confirmed on a Finnigan LCQ Advantage MAX MS.

G)The procedure of synthesis example 36,ie (N-Me-Cys48, Nle59)hIGF-1(1-70)-OH (SEQ ID NO:28)

Specified in the header of the protein was joined by native chemical crosslinking using hIGF-1(1-46)-dipropionyl-Leu-NH2(SEQ ID NO:52) and C-terminal fragments, i.e. the (N-Me-Cys48, Nle59)hIGF-1(48-70) (SEQ ID NO:56). Complex tiefer protein (4.3 mg, 824 nmol) and C-terminal fragment (2.1 mg, 790 nmol) was dissolved in the buffer for stitching (400 μl, 6 M guanidine hydrochloride in 200 mm sodium phosphate, pH 8.5) and TCEP (80 μl, 40 mg/ml, pH 7). Added catalyst MPAA (80 μl, 20 mg/ml, pH 7). The progression of the reaction was monitored using LC-MS on a LCQ Deca XP (Thermo Finnigan) with column Luna C18(2) (5 μm, a 4.6×100 mm) gradiente 5-80% acetonitrile (0.1% TFOC), within 30 minutes. The reaction was suppressed by diluting 1:10 dH2O c 0.1% TFOC (about./vol.). The crude mixture was centrifuged and passed through a glass filter with pores of 1.0 μm to remove any sediment MPAA. Full-size protein was purified using a linear gradient B 5-60% within 40 minutes, with a flow rate of 5 ml/min on a Vydac C18 (10 μm, 10×250 mm). Protein was quantified by UV spectroscopy (NanoDrop spectrophotometer ND1000) and liofilizirovanny for future use.

Stored protein was dissolved in a solution of 200 mm H2PO4-, 6 M guanidine-HCl (pH 8.5) to achieve a concentration of 1.0 mg/ml Buffer for folding (100 mm Tris, 10 mm glutathione, 1 mm oxidized glutathione, pH 8.5) was added to the solution to achieve a final protein concentration of 250 μg/ml and the Mixture was left to incubated at room temperature while monitoring by HPLC. As he reached the equilibrium (that was visualized in the form of a stable profile HPLC), the reaction is extinguished or acetic acid, or TFOC to pH 3. The solution was purified by using a first glass filter with pores of 1.0 μm and then prepreparation column.

Subjected to the folding of the protein was purified using a linear gradient B 5-60% within 40 minutes, with a flow rate of 5 ml/min Protein was quantified by UV Spectro is the opium (spectrophotometer NanoDrop ND1000) and liofilizirovanny. Received approximately 0,415 mg of the pure product with the provision of the output of 10.6%. The mass of the protein was confirmed on a Finnigan LCQ Advantage MAX MS.

A specialist in this area can be obtained from other peptides according to the invention using the procedures of synthesis, similar to those disclosed in the above examples. Physical data for compounds, examples of which are shown in this document are given in table 1.

7633,2
Table 1
Number exampleMol. mass (expected)Mol. mass (ESI-MS)% purification (HPLC)
17631,67631,699,9
26838,66839,5for 95.2
37348,37347,993,0
47631,67632,197,7
56838,66839,395,9
67603,67602,599,9
77718,77718,799,9
87452,37454,199,9
97638,67639,799,9
107585,57586,099,9
117674,67675,399,9
127615,67616,999,9
137562,57563,699,9
147603,67605,198,5
157631,67634,196,8
167631,697,2
177631,67632,995,1
187631,67631,696,7
197631,67631,997,9
207631,67631,898,5
217631,67631,797,6
227631,67631,898,1
237714,77713,999,9
247686,77686,799,9
257596,67596,799,9
267644,77645,599,9
277644,77644,999,9
287759,87760,4100
297745,87746,5100
307758,87758,4100
317717,77718,5100
327644,77645,5100
337644,77645,495,5
347644,77643,9100
357628,77628,194,0
367644,77644,899,9
377644,77644,6 99,9
387644,77645,399,9
397628,77628,499,9
407658,77658,899,9
417658,77658,899,9
427642,77641,799,9
437671,87672,499,9
447641,77641,899,9
457699,87701,097,7
467657,77658,796,1
477642,77640,999,9
4 7632,67632,599,9
497703,77704,099,9
507604,67604,799,9
517680,77680,5100
527646,67646,2100
537645,67644,9100
547673,77674,799,9

Functional tests

A)The analysis of bindingwith the receptor for IGF-1in vitro

Received a membrane for studies of binding of the radioactive ligand by homogenization of cells MCF-7 human, expressing the natural receptor for IGF-1 in 20 ml of ice-cold 50 mm Tris-HCl by Brinkman transmitter station (Westbury, NY, USA) (6, 15 sec). The homogenates were washed twice by centrifugation (39000 g/10 minutes) and final precipitation resuspendable in 50 mm Tris-HCl containing 2.5 mm MgCl2è0,1% BSA.

For analysis, aliquots were incubated from 0.05 nm [125I]IGF-1. Sometimes consisted of unlabeled competitive tested peptides. The final volume for analysis was of 0.25 ml After incubation period, equal to 120 minutes (20°C) associated with [125I]IGF-1 (~2000 CI/mmol, Perkin Elmer Life Sciences, Boston, MA, USA) was separated from free radioactive particles by centrifugation at 3000 rpm for 10 minutes. The supernatant decantation and radioactive particles trapped sediment, read by gamma-ray spectrometry (Wallac LKB, Gaithersburg, MD, USA). Specific binding was defined as total associated [125I]IGF-1 minus the one that was linked in the presence of 100 nm IGF-1.

Data on the binding of IGF-1 receptors in vitro (i.e. the values of the EU50for compounds listed as examples in this document are shown in table 2.

B) Analyzing the biological activity of IGF-1 in vitro

Mouse 3T3 cells/R (obtained from Dr. E. Rozengurt from UCLA in Los Angeles, CA, USA) were cultured in 24-hole tablet (DMEM+10% FCS) and incubated for 2 days in culture.

For analysis, the medium was removed and washed once with serum-free DMEM. Then incubated without serum for 24 hours. After serum starvation was added [3H]-thymidine and peptides IGF-1. Then cells were incubated for 24 hours at 37°C.

At the end of incubation, the medium aspirated. The cells are then washed ladany the 0.9% NaCl solution. Then added 5% ice-cold TCA solution to incubate for 30 minutes at 4°C. TCA aspirated and the wells were incubated with 95% ethanol for 4 hours. Then the medium was transferred into a vial for liquid scintillation to assess radioactivity.

Data on the biological activity of IGF-1 in vitro (i.e. the values of the EU50for compounds, examples of which are given in this document are also given in table 2.

C) in vitro Screening of peptides IGF-1 on the cross-reactivity with insulin receptors on the cells U2OS

The U2OS cells (catalog No. 93-S, DiscoveRX Corporation, Fremont, CA, USA) were sown in the amount of 6×105cells/ml in 96-well plates coated with poly-D-lysine tablet for 16 hours before analysis in serum-free medium for analysis. Insulin wild-type (catalog No. 10908, Sigma, St. Louis, MO, USA), IGF-1 wild-type (Increlex®, Tercica, Inc., Brisbane, CA, USA) or test peptide IGF-1, described in this application, was added in doses ranging from 10 μm (micromolar) to 0.15 nm (nanomolar), and incubated for 3 hours at 37°C with 5% CO2. Reagent PathHunter™ (catalog No. 93-001, DiscoveRX) received the manufacturer's instructions and added to each well. The plates were incubated at room temperature for 1 hour. Luminescence was read on a spectrophotometer for megamarketing analysis of tablets Envision 2104 (PerkinElmer, Inc., Waltham, MA, USA). Analyzed the activity of each is about the test peptide and reported as the maximum/minimum (max/min) values. Datain vitroon cross-react with the insulin receptor (i.e. the values of the max/min) for compounds, examples of which are given in this document are also presented in table 2.

Discovered that many of the compounds, examples of which are given in this document are significantly more active than IGF-1 wild type, which is characterized by the value of the IC504,59 nm, the value of EC503,75 nm and value max/min 2,1.

Table 2
Number exampleIC50(nm)EC50(nm)max/min
10,570,512,1
20,502,31N/A
32,417,17N/A
40,881,90N/A
51,141,89N/A
60,660,98N/A
74,117,41N/A
819,0232,00N/A
93,628,31N/A
102,901,09N/A
111,853,34N/A
121,475,11N/A
131,257,02N/A
145,94of 3.56N/A
150,813,70N/A
162,61to 7.59N/a
172,774,59N/A
180,723,33N/A
192,77of 7.69N/A
201,603,13N/A
21of 1.573,95N/A
220,914,22N/A
232,693,86N/A
242,892,60N/A
252,406,72N/A
263,601,28N/A
270,58 N/A
283,252,12N/A
2913,254,86N/A
301,951,87N/A
312,031,18N/A
322,66the 5.45N/A
332,252,30N/A
34N/AN/AN/A
35of 3.643,15N/A
36N/AN/AN/A
3723,253,42N/A
382,13 4,21N/A
3928,100,93N/A
40N/AN/AN/A
41N/AN/AN/A
421,492,01N/A
431,275,08N/A
44of 6.313,69N/A
459,276,24N/A
46N/AN/AN/A
471,461,31N/A
48be 18.491,81N/A
4 4,542,69N/A
5030.63 per2,63N/A
512,181,29N/A
521,292,44N/A
53N/AN/AN/A
543,492,34N/A

Introduction

Analogues of IGF-1 in this invention can be represented in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, salts formed with organic acids (e.g. acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluensulfonate or pambou acid), inorganic acids (e.g. hydrochloric acid, sulfuric acid or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic acid sludge is copolymers of polylactic-glycolic acid).

The usual way to obtain the salt of the peptide of the present invention are well known in this field and can be carried out by standard means of exchange of salt. For example, Sol TFUK peptide of the present invention (salt TFUK results peptide purification using preparative HPLC, in which elution is containing TFUK buffer solutions) were converted into another salt, such as acetate salt, by dissolving the peptide in a small amount of 0.25 N. aqueous solution of acetic acid. The resulting solution was applied on the column for prepreparation HPLC (Bond, SB 300, C-8). Elution from the column hold (1) 0.1 N. aqueous solution of ammonium acetate for 0.5 hours, (2) 0,25 N. aqueous solution of acetic acid for 0.5 hours, and (3) a linear gradient (from 20% to 100% solution B over 30 min) at a flow rate of 4 ml/min (solution A represents 0,25 N. aqueous solution of acetic acid, and the solution B is of 0.25 N. acetic acid in a mixture of acetonitrile/water in the ratio 80:20). The fractions containing the peptide, collect and lyophilizers dry.

The dosage of the active ingredient in the compositions of this invention can vary; however, it is necessary that the amount of active ingredient was so, to get a suitable dosage form. Wybran what I dosage depends upon the desired therapeutic effect, from the route of administration and duration of treatment. Dosage is easily determined by professional, competent therapist.

Compounds according to this invention can be administered orally, parenterally (for example, by intramuscular, intraperitoneally, intravenous or subcutaneous injection or via an implant), nazalnam, vaginal, rectal, sublingual, or local route of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms the active compound is mixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose or starch. Such dosage forms can also include, as a normal practice, additional substances, which differ from such inert diluents, for example, lubricants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage form may also include buffer means. Tablets and pills can additionally be obtained intersolubility coatings.

Liquid dosage forms for oral administration include, but are not limited to the texts, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, containing inert diluents commonly used in this field, such as water. Besides such inert diluents, compositions can also include ancillary tools, such as humectants, emulsifying and suspendresume funds, as well as sweeteners, flavorings and fragrances.

Preparations according to this invention for parenteral administration include, without limitation, sterile aqueous or nonaqueous solutions, suspensions, emulsions, etc. are Examples of non-aqueous solvents or carriers include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin and type by injection of organic esters, such as etiloleat. Such dosage forms may also contain auxiliary agents such as preservatives, moisturizing, emulsifying and dispersing funds. The preparations can be sterilized, for example, by filtration through inhibiting bacteria filter, by introducing into the composition sterilizing means, by irradiating the compositions and/or by heating the compositions. Pharmaceutical compositions containing the new analogues of IGF-1, described herein, can also be obtained in the form of sterile solid composition is th, which can be dissolved in sterile water or some other sterile injectable medium immediately before use.

Compositions for rectal or vaginal injection preferably are suppositories which may contain, in addition to the active substance, excipients such as cocoa butter or wax for suppositories.

Compositions for nasal or sublingual introduction also get with standard excipients, well known in this field.

In addition, the connection according to this invention can be fed into the song with a slow release, such as the compositions described in the following patents and patent applications. In U.S. patent No. 5672659 described compositions with delayed release containing a bioactive agent and a complex polyester. In U.S. patent No. 5595760 described compositions with delayed release containing bioactive agent in gel form. In U.S. patent No. 5821221 described compositions with delayed release containing a bioactive agent and chitosan. In U.S. patent No. 5916883 described compositions with delayed release containing a bioactive agent and a cyclodextrin. In PCT publication WO 99/38536 described absorbable compositions with delayed release containing the bioactive agent. In public, the tion PCT WO 00/04916 described a method of producing microparticles, containing a therapeutic agent, such as a peptide, in the system of the emulsion oil-in-water". In PCT publication WO 00/09166 described complexes containing a therapeutic agent, such as a peptide, phosphorylated polymer. In PCT publication WO 00/25826 described complexes containing a therapeutic agent, such as a peptide, and a polymer bearing polimersomy lactone.

In addition, the invention described in U.S. patent No. 7258864, relates to a method of treatment of a subject with deficiency of insulin-like growth factor-1 (IGFD), including the introduction of the pediatric patient profile effective amount of unmodified IGF-1, where the subject is characterized as follows: (a) at the time of treatment or prior to initial treatment with IGF-1, has or had a height at least about 2 standard deviations (SD) below the normal average growth for the corresponding age and sex, and (b) at the time of treatment or prior to initial treatment with IGF-1, has or had the level of IGF-1 in blood of at least about -1 SD below normal averages, where the subject is not suffering from the syndrome of Larona or syndrome partial insensitivity to growth hormone and where specified the introduction is effective for treatment of IGFD the subject.

Similarly, the invention described in WO 2006/130769, relates to a method of treatment of a subject with idiopathic short stature (ISS), including Osamu introduction to the pediatric patient profile, suffering ISS, characterized by incomplete activity or incomplete signaling of endogenous growth hormone, the amount of IGF-1, effective to accelerate the growth of the subject, where the subject is additionally characterized by the following: a) at the time of treatment or prior to initial treatment with IGF-1, has or had a height at least about 2 standard deviations (SD) below the normal average growth for the corresponding age and sex, and (b) has levels of GH and IGF-1 in the blood, which are, at least, normal for the subject of the same age and sex.

In addition, new analogues described herein can be introduced separately or in combination with another therapeutic agent, as determined by a professional therapist.

If not defined otherwise, all technical and scientific terms used herein have the same meaning, which is usually given by a specialist in the field that applies the present invention. Also, all publications, patent applications, patents, and other references mentioned herein are included, therefore, in it by reference, each in full.

1. Similar agonist IGF-1 formula (I),

in which:
A-1represents Ser or demeterova;
A1represents Gly Il is demeterova;
A2represents Pro or demeterova;
A3represents Glu or demeterova;
A4represents Thr;
A5represents Leu;
A6represents Cys;
A7represents Gly;
A8represents Ala;
A9represents Glu;
A10represents Leu;
A11represents Val;
A12is an Asp;
A13represents Ala;
A14represents Leu;
A15represents Gln;
A16represents Phe;
A17represents Val;
A18represents Cys;
A19represents Gly;
A20is an Asp;
A21represents Arg;
A22represents Gly;
A23represents Phe;
A24represents Tyr;
A25represents Phe;
A26represents Asn;
A27represents Lys, Arg or Pro;
A28represents Pro or Lys;
A29represents Thr;
A30represents Gly;
A31represents Tyr;
A32represents Gly;
A33represents Ser;
A34represents Ser;
A35represents Ser;
A36represents Arg;
A37represents Arg;
A38is the Wallpaper Ala;
A39is a Pro;
A40represents Gln;
A41represents Thr;
A42represents Gly;
A43represents Ile;
A44represents Val;
A45is an Asp;
A46represents Glu;
A47represents Cys, β-Me-Cys;
A48represents Cys;
A49represents Phe, Arg, Leu, or Thr;
A50represents Arg or Ser;
A51represents Ser or Thr;
A52is a Cys, or β-Me-Cys;
A53represents Asp, Arg or Ser;
A54represents Leu or A6c;
A55represents Arg or Tyr;
A56represents Arg or Gln;
A57represents Leu;
A58represents Glu;
A59represents Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp, Tyr;
A60represents Tyr or Phe;
A61represents Cys;
A62represents Ala or Asn;
A63is a Pro, D-Pro or demeterova;
A64represents Leu, D-Leu or demeterova;
A65represents Lys, D-Lys, Arg or demeterova;
A66is a Pro, D-Pro or demeterova;
A67represents Ala, D-Ala or demeterova;
A68represents Lys, D-Lys, Arg or demeterova;
A69represents Ser, D-Ser, Aib, Thr Il is demeterova;
A70represents Ala, D-Ala or demeterova; and
A71represents Glu, Lys, Ser or demeterova;
R1represents OH or NH2;
provided that the side chains of each pair of residues of the A6and A48, A47and A52and A18and A61form disulfide bonds; and
also provided that, when A59represents Leu, Ile or Nle, analog contains at least one additional substitution or insertion of amino acids according to Formula I;
or its pharmaceutically acceptable salt.

2. Similar agonist IGF-1 according to claim 1, where the specified analog is:
(Asn59)hIGF-1(1-70) -; (SEQ ID NO: 1)
(Asn59)hIGF-1(1-62)-OH; (SEQ ID NO: 2)
(Asn59)hIGF-1(4-70)-OH; (SEQ ID NO: 3)
(Pro27, Lys28, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 4)
(Pro27, Lys28, Asn59)hIGF-1(1-62)-OH; (SEQ ID NO: 5)
(Ser53, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 6)
(Ser-1-Gly1, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 7)
(Tyr55, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 9)
(Thr49, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 10)
(Asn59,62)hIGF-1(1-70)-OH; (SEQ ID NO: 11)
(Asn59, Phe60)hIGF-1(1-70)-OH; (SEQ ID NO: 12)
(Ser50, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 13)
(Gln56, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 14)
(Asn59D-Pro63)hIGF-1(1-70)-OH;
(Asn59, D-Leu64)hIGF-1(1-70)-OH;
(Asn59, D-Lys65)hIGF-1(1-70)-OH;
(Asn59D-Pro66)hIGF-1(1-70)-OH;
(Asn59, D-Ala67)hIGF-11-70)-OH;
(Asn59, D-Lys68)hIGF-1(1-70)-OH;
(Asn59, D-Ser69)hIGF-1(1-70)-OH;
(Asn59, D-Ala70)hIGF-1(1-70)-OH;
(Arg27,65,68, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO: 15)
(Leu59, Arg65, 68)hIGF-1(1-70)-OH; (SEQ ID NO: 16)
(Leu49, 59)hIGF-1(1-70)-OH; (SEQ ID NO: 17)
(β-Me-Cys52, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO: 18)
(β-Me-Cys47, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO: 19)
(Leu59Glu71)hIGF-1(1-71)-OH; (SEQ ID NO: 20)
(Leu59, Lys71)hIGF-1(1-71)-OH; (SEQ ID NO: 22)
(Leu59Ser71)hIGF-1(1-71)-OH; (SEQ ID NO: 23)
(Leu59, Thr69)hIGF-1(1-70)-OH; (SEQ ID NO: 24)
(Thr51, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO: 25)
(Nle59, Aib69)hIGF-1(1-70)-OH; (SEQ ID NO: 27)
(A6c54, Nle59)hIGF-1(1-70)-OH; (SEQ ID NO: 34)
(Arg53, Ile59)hIGF-1(1-70)-OH; (SEQ ID NO: 35)
(Arg49, Ile59)hIGF-1(1-70)-OH; (SEQ ID NO: 36)
(A6c59)hIGF-1(1-70)-OH; (SEQ ID NO: 39)
(Trp59)hIGF-1(1-70)-OH; (SEQ ID NO: 41)
(Tyr59)hIGF-1(1-70)-OH; (SEQ ID NO: 43)
(Glu59)hIGF-1(1-70)-OH; (SEQ ID NO: 44) or
(Arg59)hIGF-1(1-70)-OH; (SEQ ID NO: 46)
or its pharmaceutically acceptable salt.

3. Similar agonist IGF-1 according to claim 1, in which And59represents Asn; or its pharmaceutically acceptable salt.

4. Similar agonist IGF-1 according to claim 3, where the specified analog is:
(Asn59)hIGF-1(1-70) -; (SEQ ID NO: 1)
(Asn59)hIGF-1(1-62) -; (SEQ ID NO: 2)
(Asn59)hIGF-1(4-70)-OH; (SEQ ID NO: 3)
(Pro27, Lys28, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 4)
(Pro27, Lys28, Asn59)hIGF-1(1-62)-OH; (SEQ ID NO: 5)
(Ser53 , Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 6)
(Ser-1-Gly1, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 7)
(Tyr55, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 9)
(Thr49, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO: 10)
(Asn59, 62)hIGF-1(1-70)-OH; (SEQ ID NO: 11)
(Asn59, Phe60)hIGF-1(1-70)-OH; (SEQ ID NO: 12)
(Ser50, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:13)
(Gln56, Asn59)hIGF-1(1-70)-OH; (SEQ ID NO:14)
(Asn59D-Pro63)hIGF-1(1-70)-OH;
(Asn59, D-Leu64)hIGF-1(1-70)-OH;
(Asn59, D-Lys65)hIGF-1(1-70)-OH;
(Asn59D-Pro66)hIGF-1(1-70)-OH;
(Asn59, D-Ala67)hIGF-1(1-70)-OH;
(Asn59, D-Lys68)hIGF-1(1-70)-OH;
(Asn59, D-Ser69)hIGF-1(1-70)-HE; or
(Asn59, D-Ala70)hIGF-1(1-70)-OH;
or its pharmaceutically acceptable salt.

5. Similar agonist IGF-1 according to claim 1, in which And59represents Leu; or its pharmaceutically acceptable salt.

6. Similar agonist IGF-1 according to claim 5, in which indicated at least one additional substitution or insertion of amino acids selected from the group consisting of Arg27, Arg65, Arg68, Leu49, β-Me-Cys47, β-Me-Cys52, Thr51, Thr69Glu71, Lys71and Ser71; or its pharmaceutically acceptable salt.

7. Similar agonist IGF-1 according to claim 5 or 6, where the specified analog is:
(Arg27, 65, 68, Leu59)hIGF-1(1-70) -; (SEQ ID NO: 15)
(Leu59, Arg65, 68)hIGF-1(1-70) -; (SEQ ID NO: 16)
(Leu49, 59)hIGF-1(1-70)-OH; (SEQ ID NO: 17)
(β-Me-Cys52, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO: 18)
(β-Me-Cys47, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO: 19)
(Leu59Glu71)hIGF-1(1-71)-OH; (SEQ ID NO: 20)
(Leu59, Lys71)hIGF-1(1-71)-OH; (SEQ ID NO: 22)
(Leu59Ser71)hIGF-1(1-71)-OH; (SEQ ID NO: 23)
(Leu59, Thr69)hIGF-1(1-70)-OH; (SEQ ID NO: 24) or
(Thr51, Leu59)hIGF-1(1-70)-OH; (SEQ ID NO: 25)
or its pharmaceutically acceptable salt.

8. Similar agonist IGF-1 according to claim 1, in which And59represents Nle; or its pharmaceutically acceptable salt.

9. Similar agonist IGF-1 according to claim 8, in which indicated at least one additional substitution of amino acids represents Aib69or A6c54; or its pharmaceutically acceptable salt.

10. Similar agonist IGF-1 to item 8 or 9, where the specified analog is:
(Nle59, Aib69)hIGF-1(1-70) -; (SEQ ID NO: 27) or
(A6c54, Nle59)hIGF-1(1-70) -; (SEQ ID NO:34)
or its pharmaceutically acceptable salt.

11. Similar agonist IGF-1 according to claim 1, where a59represents Ile; or its pharmaceutically acceptable salt.

12. Similar agonist IGF-1 according to claim 11, in which indicated at least one more replacement amino acids represents Arg49or Arg53; or its pharmaceutically acceptable salt.

13. Similar agonist IGF-1 according to claim 11 or 12, where the specified analog is:
(Arg53, Ile59)hIGF-1(1-70) -; (SEQ ID NO: 35) or
(Arg49, Ile59)hIGF-1(1-70)-HE; (SQ ID NO:36)
or its pharmaceutically acceptable salt.

14. Similar agonist IGF-1 according to claim 1, in which And59represents Arg, A6c, Glu, Trp or Tyr; or its pharmaceutically acceptable salt.

15. Similar agonist IGF-1 to 14, where the specified analog is:
(A6c59)hIGF-1(1-70) -; (SEQ ID NO: 39)
(Trp59)hIGF-1(1-70) -; (SEQ ID NO: 41)
(Tyr59)hIGF-1(1-70) -; (SEQ ID NO: 43)
(Glu59)hIGF-1(1-70)-OH; (SEQ ID NO: 44) or
(Arg59)hIGF-1(1-70)-OH; (SEQ ID NO: 46)
or its pharmaceutically acceptable salt.

16. Pharmaceutical composition for use in the treatment of IGF-1-mediated conditions or diseases, containing an effective amount of similar-agonist according to any one of claims 1 to 15, where the specified condition or disease selected from the group consisting of short stature, obesity, weight loss, cachexia, anorexia, neurodegenerative disorders associated with fibrosis conditions, disorders of cartilage, bone diseases, inflammatory disorders, intestinal disorders, insulin resistance, diabetes, diabetic ketoacidosis, syndrome Rabson-Mendenhall, retinopathy, acromegaly, fibromuscular hyperplasia and cardiac disorders; and where the specified treatment includes a step of introducing the needy in this subject a therapeutically effective amount of the specified analog or pharmaceutical compositions.

17. The method of treatment Miscrosoft is, including the stage of introduction to a subject in need, a therapeutically effective analog-agonist according to any one of claims 1 to 15 or a pharmaceutical composition according to item 16.



 

Same patents:

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

Anti-mif antibodies // 2509777

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

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2 cl, 1 dwg, 4 ex

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FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

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23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: biotechnologies.

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EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: biotechnologies.

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EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

Organic compounds // 2502802

FIELD: biotechnologies.

SUBSTANCE: invention refers to eucariotic vector for expression of target recombinant product in a mammal cell and to its use, to a mammal cell for production of target recombinant product and to a method for its production, a method of a mammal cell selection and a method for obtaining a target recombinant product. Vector includes the first polynucleotide coding a functional folate receptor bound to a membrane as a selective marker and the second polynucleotide coding the target product that is expressed in a recombinant manner. Target product represents a pharmaceutically active, therapeutically active or diagnostic polypeptide. Functional folate receptor bound to the membrane and target product are expressed from the above expression vector. Sampling system is based on introduction of a gene of exogenic functional folate receptor bound to the membrane to a mammal cell.

EFFECT: invention allows effective selection of transformed cells and high yield of target product.

26 cl, 3 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: expression vector includes: (a) replication origin OriP obtained from Epstein-Barr virus (EBV), where replication origin contains: 1) symmetry element of the second order (DS); and 2) duplication section (FR) that contains fixation point EBNA; (b) replication origin SV40; (c) insertion section for inserting a gene of concern; (d) promoter EF-1b functionally bound to the insertion section; (e) poly-A signal; (f) bacterial replication origin; (g) selected marker; and unnecessarily containing (h) sequence of nucleic acid, which codes constant area of heavy or light chain of antibody, which is functionally bound to the insertion section. With that, replication origin OriP is bound to an initiation factor of replication EBNA 1, which acts from outside and is not coded with an expression vector.

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26 cl, 25 dwg, 3 tbl, 4 ex

FIELD: biotechnologies.

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EFFECT: improvement of the method.

4 cl, 5 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

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5 cl, 5 dwg, 3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and represents a method for preparing virus-like particles containing hepatitis C virus core, E1 and E2 structural antigens, and a method for particle purification. The method for preparing the virus-like particles involves Hansenula polymorpha yeast cell transformation with a recombinant plasmid containing one open reading frame carrying hepatitis C virus core-E1-E2 genes a direct expression of which leads to the formation of core, E1 and E2 antigens. That is followed by culturing and recovering the prepared particles. A method for virus-like particle purification involves bleaching of a damaged cell homogenate, diafiltration, concentration, precipitation with low pH, ion-exchange chromatography, sedimentation centrifugation in a density gradient, hydrophobic chromatography and gel chromatography.

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2 cl, 5 dwg, 5 ex

Anti-mif antibodies // 2509777

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and immunology. Invention discloses a monoclonal antibody and its antigen-binding parts which specifically bind the C-end or central part of the macrophage migration inhibitory factor (MIF). The anti-MIF antibody and its antigen-binding part further inhibit biological function of the human MIF. The invention also describes an isolated heavy and light chain of immunoglobulins obtained from anti-MIF antibodies, and molecules of nucleic acids which encode such immunoglobulins.

EFFECT: disclosed is a method of identifying anti-MIF antibodies, pharmaceutical compositions containing said antibodies and a method of using said antibodies and compositions for treating diseases associated with MIF.

22 cl, 14 dwg, 16 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to creation of recombinant plasmids providing expression of poly-epitopic tumour-associated antigens in dendritic cells capable of stimulation of specific cytocidal cells, and it may be used in medicine. Recombinant plasmid DNA pCI-UB-POLYEPI contains 11 epitopes of tumour-associated antigens of colorectal cancer, its size is 6 355 n. p. and it expresses the following amino acid sequence: DYKDDDDK-LLGVGTFVV-ADRIW-GLKAGVIAV-AAYARY-VLAFGLLLA-ADRIW-YQLDPKFITSI-AAYARY-IMIGVLVGV-ADRIW-YLSGADLNL-AAYARY-CGIQNSVSA-AAYARY-LLLLTVLTV-ADRIW-QYIKANSKFIGlTEL-ANIY-SIINFEKL-ARY-SASFDGWATVSVIAL-ARY-SERVRTYWIIIELKHKARE-ARY-IQNDTGFYTLHVIKSDLVNEE. Mature dendritic cells obtained by adding to immature dendritic cells of pro-inflammatory TNF-α (tumour necrosis factor) cytokine are transfected by constructed plasmid DNA pCl-UB-POLYEPI thus activating them. Then activated dendritic cells are cultured together with peripheral mononuclear blood cells of people sick with colorectal cancer for generation of antigen-specific antitumour cytocidal cells.

EFFECT: invention allows efficient generation of antigen-specific cytocidal cell with antitumour activity in vitro, required for immune response by the 1-st type T-helper to colorectal cancer antigens.

2 cl, 1 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: created is recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype containing expressing cassette with haemagglutinin gene of influenza virus being included. As a haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain haemagglutinin gene with pre-optimised for expression in human being cells nucleotide sequence was used providing overexpression of haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain. Haemagglutinin gene of influenza virus of B/Brisbane/60/2008 strain with optimised nucleotide sequence was cloned in expressing cassette under promoter control and contains polyadenylation signal. Promoter is cytomegalovirus promoter, and polyadenylation signal is SV40. Expressing cassette is located in zone of E1 deletion of human being adenovirus genome of the 5-th serotype. Also method of use of recombinant pseudo adenoviral particle based on human being adenovirus genome of the 5-th serotype for induction of specific immunity to influenza virus B.

EFFECT: possibility of use in pharmaceutical industry for production of vaccine preparations.

6 cl, 9 dwg, 1 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to genetically modified bacteria Salmonella enterica, which contain at least one operon pgI from Campylobacter jejuni or its functional derivative and refers to presentation of at least one N-glycan from Campylobacter jejuni or derivative of this N-glycan on their cell surface. In bacteria one or several genes for biosynthesis of bacillozamine are inactivated by mutation and/or partial or complete deletion of genes pgID, E, F, G. Invention provides method for obtainment of genetically modified bacteria Salmonella enterica. Invention is directed to application of modified bacteria in pharmacological compositions of medical and veterinary purpose and methods for treatment and/or prevention of infections Campylobacter and, not obligatory, Salmonella.

EFFECT: invention provides safety and efficiency of treatment and prevention of infections, which affect people and animals, namely livestock and fowl.

18 cl, 4 dwg, 1 tbl, 6 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to recombinant fused protein dimers intended to inhibit or suppress immune response in a mammal, which bind human CD80 or human CD86 or the extracellular domain of any thereof, and has higher capacity for suppressing immune response than a dimer of the fused protein LEA29Y-Ig. Also disclosed are nucleic acids which code said dimers, expression vectors containing said nucleic acids, as well as recombinant host cells containing said nucleic acids and/or said vectors. Disclosed are pharmaceutical compositions for inhibiting or suppressing immune response in a mammal, which contain said fused protein dimers, as well as use of said dimers to produce drugs for inhibiting or suppressing immune response in a mammal, treating diseases or disorders of the immune system or treating organ or tissue transplant rejection in a mammal. Methods of producing said fused protein dimers are also disclosed.

EFFECT: invention provides effective inhibition or suppression of immune response in a mammal.

9 cl, 15 dwg, 11 tbl, 12 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically binds heparin-binding EGF-like growth factor (HB-EGF) and its antigen-binding fragment. Invention describes a nucleic acid molecule, an expressing vector, a host cell and a method for obtaining an antibody or its antigen-binding fragment, as well as use of antibody or its antigen-binding fragment for obtaining pharmaceutical composition for diagnostics, prevention or treatment of hyperproliferation disease, methods and sets for diagnostics and prevention or treatment of the state associated with HB-EGF expression. This invention can be further found in therapy of diseases determined with or related to HB-EGF expression.

EFFECT: improving efficiency of composition and treatment method.

34 cl, 43 dwg, 28 ex, 12 tbl

FIELD: biotechnologies.

SUBSTANCE: invention describes polynucleotide, expression vector, host cell and production method of humanised antibody together with their use, as well as medical preparation against rheumatoid arthritis, prophylaxis or treatment method of rheumatoid arthritis and use of humanised antibody at production of pharmaceutical preparation for prophylaxis or treatment of rheumatoid arthritis. This invention can be used in therapy of human diseases associated with α9 integrin.

EFFECT: improved activity and thermal stability.

14 cl, 6 dwg, 6 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining modified IGF-1 proteins and can be used in medicine. Constructed is a polypeptide which contains a human IGF-1 precursor protein, wherein amino acids G1, P2 and E3 are removed as a result of deletion or amino acid E3 is removed as a result of deletion, and wherein amino acid R37 is replaced with alanine and amino acids R71 and R72 are removed as a result of deletion. The cleavage of the E-peptide from IGF-1 by a protease is reduced as a result of said modifications. The obtained polypeptide is used to treat a musculoskeletal disease, diabetes, conditions associated with neuron death, anaemia, chronic obstructive pulmonary disease and burn injury.

EFFECT: invention enables to obtain stabilised polypeptides containing a modified sequence of an IGF-1 precursor, in which the cleavage of the E-peptide from IGF1 which occurs in natural physiological conditions is reduced.

21 cl, 12 dwg, 1 tbl, 82 ex

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