Additive cytoprotective effects of two bioactive regions of hormone pro-opiomelanocortine

FIELD: chemistry of peptides, medicine, pharmacology.

SUBSTANCE: invention relates to using the combination of bioactive regions SYSMEHFRWGKPV and YGGFM in pro-opiomelanocortine in manufacturing a medicine used in treatment of inflammatory, degenerative and autoimmune diseases, traumas, infections and burns. Also, invention relates to using polypeptide chosen from group: YGGFMSYSMEHFRWGKPVYGGEM, YGGFMSYSMEHFRWGKPV, SYSMEHFRWGKPVYGGFM and compositions eliciting cytoprotective properties. Invention provides enhancing in peptides effect.

EFFECT: valuable medicinal properties of peptides.

9 cl, 1 tbl, 1 dwg

 

1) the Technical scope of the invention.

This invention is a combination of two bioactive peptides isolated from hormone Pro-opiomelanocortin that contribute to the expression of cytoprotective effects and modulation of the inflammatory response and tone/wound healing. The combination of the two peptides of Pro-opiomelanocortin helps to bring out the best of the pharmacological effects and healing damaged tissue.

2) a Technical problem.

The bioactive part of the protein and gene sequences often contain from 5 to 15 amino acids and repetitive peptides, separated by large blocks of amino acids with uncertain function (1-5).

To select such areas and further manipulation of bioactive effects plots (1-5) can be used for computer analysis methods. This invention is a combination of two repetitive and bioactive sites of hormone Pro-opiomelanocortin, which manifests additive cytoprotective and pharmacological effects.

3) a Technical description.

Repeating peptide fragments within the same protein or family of proteins represent, together with appropriate complementary thereto sequences, sites that are associated with bioactivity larger molecular structures (1-5).

Modern methods the programme is financing, the development of applications and databases, protein and gene structures contribute to computer modeling repetitive, bioactive and complementary structures larger number of different proteins (1-5). Thus, the selection of short and repetitive fragments can be produced on a personal computer quickly and easily (1-5). The inventors have analyzed repetitive and bioactive peptides molecules Pro-opiomelanocortin using the SCAN (3-5) to obtain the possible combinations of elements with additive pharmacological and cytoprotective effects.

4) the Purpose of this invention.

The purpose of this invention to provide an additive cytoprotective pharmacological effects by combining two recurring and bioactive peptides molecules Pro-opiomelanocortin.

Innovation is the fact that instead of independent pharmacological screening of various fragments of Pro-opiomelanocortin used the SCAN to highlight two recurring and bioactive sequence SYSMEHFRWGKPV and YGGFM). Two selected sequences are present in the following molecules: the precursor Pro-opiomelanocortin; Pro-opiomelanocortin, corticotropin, melanotropin, beta-lipotropin, proenkephalin, preproenkephalin, beta-endorphin, adrenalin (table 1)./p>

Fragments of bioactive peptides and SYSMEHFRWGKPV YGGFM were tested separately and in combination, using the standard model of cell protection from the damage of the stomach of male Wistar rats induced by 96% ethanol (6, 7). In the control group of 8 animals that were injected with saline, the area of damage of the stomach was 255,5±78,1 mm2(average, standard deviation, see drawing) (7). In the group of 8 animals that were administered a combination of peptides SYSMEHFRWGKPV (1 mg/kg) and YGGFM (10 mg/kg) area of damage to the stomach was 0.3±07 mm2(p<0.05 compared with controls, see drawing) (7). In the group of 8 animals that were injected SYSMEHFRWGKPV (1 mg/kg) area of damage to the stomach amounted to 5.9±8 mm2(p<0.05 compared with controls, see drawing) (7). In the group of 8 animals that were injected YGGFM (10 mg/kg) area of damage to the stomach was 139,4±36,1 mm2(p<0.05 compared with controls, see drawing) (7). The combination of peptides and SYSMEHFRWGKPV YGGFM, which provided the best cytoprotective effect, patented under the name KOMET. The peptides or their combination did not show any irritants in normal gastric mucosa (7).

5) Drawing and tables.

Table 1 shows the combination of bioactive sequences Pro-opiomelanocortin called KOMET, and three combinations of chemical structural Faure the street KOMET-1, KOMET-2 and KOMET-3 (obtained by combining the individual structural formulas).

The drawing shows cytoprotective effects of bioactive fragments of peptides and SYSMEHFRWGKPV YGGFM molecule Pro-opiomelanocortin and combinations thereof KOMET model lesions of the stomach in rats induced by ethanol.

6) a Description of possible applications of the present invention.

Bioactive sequence KOMET and the combination of their structural formulas KOMET-1, KOMET-2, KOMET-3 defined in table 1, are used as bioactive peptides for the modulation of inflammation and wound healing. Determine structural formulas KOMET-1, KOMET-2 and KOMET-3 bioactive regions (oblasts) hormone Pro-opiomelanocortin (6-9) is to obtain the structural formula (sequences) of potential drugs fast and effective way.

The aim of the invention KOMET-1, KOMET-2 and KOMET-3 is receiving medication that prevents and stops the lesions as in inflammation and autoimmune diseases, and trauma, infections and injuries:

1. connective tissue, joints and bones,

2. Central and peripheral nervous system, optic nerve, eye and ear,

3. skin, hair and nails,

4. the digestive system, liver, pancreas, oral cavity, teeth and sinuses,

5. the immune system, bone marrow, lymphatics the x nodes and spleen,

6. cardiovascular system,

7. respiratory system

8. the reproductive system.

7) Industrial applications of the invention

Bioactive sequence Pro-opiomelanocortin COMETS, COMET-1 and COMET-2 and COMETS-3 injected subcutaneously, intramuscularly, intraperitoneally and intravenously. Dosage forms include ointments, creams, gels, suppositories, vaginal suppositories, eye drops and ear drops.

Links

1. L.Baranyi, W.Campbell, K.Ohishima et al., Nature Medicine 1 (1995) 894-901.

2. J.E.Blalock, Nature Medicine, 1 (1995) 876-878.

3.On the Optimization of Complementary Protein Coding, in: S. Ohno, K. Aoki, M. Usui, E.Uchio (Eds.), Uveitis Today, Elsevier, Amsterdam, 1998, pp 315-318.

4.and P.Konjevoda, Period. Biol. 101 (1999) 363-368.

5.N.Gotovac, M.Martinis et al. Simple Three-step Method for the Analysis and Design of Repetitive and Bioactive protein Motifs, in:(Ed.), Advances in Systems, Signals, Control and Computers, vol. II, IAAMSAD, and ANS, Durban, 1998, pp 310-311.

6. P.Konjevoda,et al. Croat. Chem. Acta 73 (2000) 1111-1121.

7. P.Konjevoda,G.Aralica andJ.Physiol. Paris 95 (1-6) (2001) 277-281.

8. J.M.Lipton and A.Catania, Immunol. Today 18 (1997) 140-145.

9.N.Kopjar,et al. Croat. Chem. Acta 71 (1998) 591-605.

Tables and drawing

Table 1. The combination of two bioactive and repetitive frag the clients proteins (peptide YGGFM and peptide SYSMEHFRWGKPV) was calculated using the SCAN (3-5) of molecules of the precursor Pro-opiomelanocortin and Pro-opiomelanocortin.

Repeating peptide YGGFM also present in the molecules preproenkephalin, proenkephalin, lipotropina-beta endorphin, beta and adrenalinoman (a). Peptide SYSMEHFRWGKPV, also present in the molecules of melanotropin and corticotropin (b).

The combination of bioactive peptides YGGFM and SYSMEHFRWGKPV molecule Pro-opiomelanocortin patented under the name of COMETS.

Other combinations of two sequences of both peptides patented under the following names and structural formulas:

COMET-1 is characterized by the structural formula: YGGFMSYSMEHFRWGKPVYGGFM,

COMETS-2 is characterized by the structural formula YGGFMSYSMEHFRWGKPV and

COMETS-3 is characterized by the structural formula SYSMEHFRWGKPVYGGFM.

(a)

No.The sequence YGGFMBalance
1Adrenalin1-5
2Endorphin beta1-5
3Preproenkephalin54-58, 61-65, 90-94, 140-144, 164-168, 215-219
4Proenkephalin100-104, 107-111, 136-140, 186 to 190, 210-214, 261-265
5Lipotropin-beta59-63, 61-65
6The precursor Pro-opiomelanocortin232-236
7About-about eumelanosomes 3-7, 237-241

(b)

No.The sequence SYSMEHFRWGKPVBalance
1Melanotropin1-13
2The corticotropin1-13
3The precursor Pro-opiomelanocortin133-145
4Pro-opiomelanocortin138-150

A drawing. Cytoprotective effects of bioactive fragments of peptides and SYSMEHFRWGKPV YGGFM molecule Pro-opiomelanocortin in the model damage to the stomach in rats induced by ethanol. The combination of sequences of COMETS (SYSMEHFRWGKPV+YGGFM) demonstrates a strong cytoprotective effects than individual sequences of peptides.

LIST of AMINO acid SEQUENCES

<110> Ademovic, Zlatko and other

<120> Additive cytoprotective effects of two bioactive areas hormone Pro-opiomelanocortin.

<140> PCT/VA 01/00005

<141> 2001-11-29

<160> 5

<170> Version 3.1 patent

<210> 1

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial amino acid sequence with a gap, numbered as a set (combination) of two separate sequence is, have a separate identifiers of sequences 1 and 2 (or Vice versa). Combination (set) of sequence identifiers 1 and 2, called COMETS, contributes to the development of better and more strong cytoprotective and pharmacological effects than those obtained using the individual identity of amino acid sequence.

<400> 1

Ser 1TyrSerMetGlu

5
HisPheArgTrpGly

10
LysProVal

<210> 2

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial amino acid sequence with a gap, numbered as a set (combination) of two separate sequences, determined by the individual identifiers of sequences 1 and 2 (or Vice versa). Combination (set) of sequence identifiers 1 and 2, called COMETS, contributes to the development of better and more strong cytoprotective and pharmacological effects than those obtained using the individual identity of amino acid sequence.

<400> 2

Tyr

1
GlyGlyPheMet

5

<210> 3

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Constructed amino acid sequence, which consists of two non-contiguous segments of a larger sequence or of segments of different sequences, numbered as a separate sequence, with the identifier in a sequence (SEQ ID NO: 3). This artificial sequence named COMET-1.

<400>3

Tyr

1
GlyGlyPheMet

5
SerTyrSerMetGlu

10
HisPheArgTrpGly

15
Lys

ProValTyrGly

20
GlyPheMet

<210> 4

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Constructed amino acid sequence, which consists of two non-contiguous segments of greater posledovatelnostei of segments of different sequences, numbered as a separate sequence, with the identifier in a sequence (SEQ ID NO: 4). This artificial sequence named COMET-2.

<400>4

Tyr

1
GlyGlyPheMet

5
SerTyrSerMetGlu

10
HisPheArgTrpGly

15
Lys

Pro Val

<210> 5

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Constructed amino acid sequence, which consists of two non-contiguous segments from different sequences are numbered as a separate sequence, with the identifier in a sequence (SEQ ID NO: 5). This artificial sequence named COMETS-3.

<400> 5

Ser

1
TyrSerMetGlu

5
HisPheArgTrpGly

10
LysProValTyrGly

15
GlyPhe Met

000

p>

1. Applying a combination of two bioactive peptides derived from Pro-opiomelanocortin, SEQ ID No.1: SYSMEHFRWGKPV and SEQ ID No.2: YGGFM, in the manufacture of medicaments for the treatment of inflammatory, degenerative and autoimmune diseases as well as injuries, infections and burns.

2. The use according to claim 1, characterized in that the content of the peptide SYSMEHFRWGKPV in medicine is from 9.1 to 16.7 wt.%.

3. The use according to claim 1, characterized in that the content of the peptide YGGFM in medicine is 83,3 to 90.9 wt.%.

4. The use according to claims 1 to 3, characterized in that the peptides used in the manufacture of medicaments for the treatment of inflammatory bowel disease.

5. The use according to claims 1 to 3, characterized in that the peptides used in the manufacture of medicaments for the treatment of syndrome of inflamed bowel (IBD).

6. A polypeptide containing an amino acid sequence selected from the group including

SEQ ID No.3.: YGGFMSYSMEHFRWGKPVYGGFM,

SEQ ID No.4.: YGGFMSYSMEHFRWGKPV and

SEQ ID No.5.: SYSMEHFRWGKPVYGGFM.

7. Composition, characterized in that it exhibits cytoprotective properties and contains the polypeptide of claim 6.

8. Composition, characterized in that it exhibits cytoprotective properties and contains a polypeptide that includes the amino acid sequence of SEQ ID No. 1: SYSMEHFRWGKPV, and a polypeptide that includes the amino acid sequence of SEQ ID No.3.: YGGFMSYSMEHFRWGKPVYGGF.

9. The composition according to claim 7 or 8, characterized in that it is a pharmaceutical composition for the treatment or prevention of injury, infection, burns, inflammatory diseases or autoimmune diseases.



 

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