Anti-aging and wound-healing compositions

FIELD: biotechnology.

SUBSTANCE: disclosed are peptides derived from proenzyme forms of matrix proteinases which represent inhibitors of matrix proteinases. Amino acid sequence is disclosed in description. Described are composition for stimulation of healthy skin formation, containing therapeutically effective amount of peptides. Also disclosed are dressing for wounds, method for stimulation of healthy skin formation and wound healing. Disclosed is using of composition in production of drug for wound healing.

EFFECT: new anti-aging and wound-healing agents.

15 cl, 28 dwg, 6 tbl, 7 ex

 

The scope of the invention

This invention relates to compositions containing inhibitors of matrix metalloproteinases, which are suitable for wound healing and elimination of the effects of aging on the skin. These inhibitors are peptides having sequences corresponding to the cut section prefermented forms of matrix metalloproteinases.

Prior art

The skin is always affected by factors such as humidity, ultraviolet radiation, cosmetics, aging, disease, stress and eating habits. As a result, can cause various skin problems. With age, the skin also becomes less elastic, resulting in the formation of wrinkles. Aging is usually accompanied by refinement and overall degradation of the skin. As the natural aging of the skin occurs a decrease in the number of cells and blood vessels that feed the skin. There is also a flattening of the dermal-epidermal connections, thereby weakening its mechanical properties. As a result, older people are more susceptible to blisters mechanical injuries or painful processes (see Oikarinen (1990) "The Aging of Skin: Chronoaging Versus Photoaging", Photodermatal. Photoimmunol. Photomed., Vol.7, pp 3-4).

The skin contains a complex network of elastin fibers, which are responsible C is maintaining its elastic properties. Excessive exposure to sun radiation system of elastic fibers becomes hyperplastic, disorganized and, ultimately, destroyed. This process is known as the actinic elastosis, which is the main cause of wrinkles, discoloration and looseness of the skin on the exposed parts of the body. In the formation of new fibroblasts, endothelial cells and keratinocytes, the skin can regenerate itself. However, as we age, the skin gradually loses this ability. So for prematurely aged skin requires agents that can accelerate the processes of growth and repair.

Wound healing is also accelerated by increasing cell proliferation and migration of certain cell types. The mechanisms involved in the healing process, often divided into four phases: hemostasis, inflammation, proliferation and maturation. During inflammation is the accumulation of white cells to fight bacteria, and increases the permeability of blood vessels, which leads to swelling. If the infection does not develop, the number of cells decreases. Leukocytes are replaced by monocytes. Macrophages and lymphocytes release growth factors (cytokines), as well as a number of chemicals, such as histamine, serotonin and prostaglandins. These substances help regulate the process is healing. In the phase of proliferation of new fibroblasts, endothelial cells and keratinocytes, is formed connective tissue, new blood vessels and restores the damaged tissue. Fibroblasts begin to dominate after about a week, the inflammation subsides and the strength of the fabric around the damaged area increases rapidly. During the ripening phase is delayed collagen, and scar tissue is formed. This maturation phase may continue for a long time, during which restores tissues of different types. For optimal recovery of the skin and associated tissues must have a sufficient supply of various vitamins and minerals and also nutrients and oxygen.

Chronic wounds or painless, non-healing wounds can occur for various reasons, including infection, the presence of foreign bodies or toxic irritants, burns, prolonged mechanical effects on the skin and insufficient blood supply due to impaired blood circulation. In chronic wounds the wound environment affects tissue homeostasis so that healing does not occur, or it starts, but then stops. Factors impeding the healing process in chronic wounds, are necrosis of the tissue and, dehydration, edema chronic wounds, fibrous seal and disease of small blood vessels.

One of the main reasons for messagevine chronic wounds is a special class of proteases called matrix-metalloproteinases (MMP), which destroy the new formed the bed of the wound (Vaalamo et al., 1997; Weckroth et al., 1996; DiColandrea et al., 1998; Moses et al., 1996). Usually the protection Lodge wounds from the destruction of these matrix-metalloproteinases perform four tissue inhibitor of metalloproteinases (TIMP 1-4), which form a very specific inhibitor complexes with the matrix metalloproteinases (Olson et al., 1997; Taylor et al., 1996; Howard et al., 1991). This means that each TIMP inhibits only a certain subclass of matrix metalloproteinases. In chronic wounds, there is a high correlation matrix metalloproteinases to TIMP, resulting in the majority of matrix metalloproteinases not inhibited (Vaalamo et al., 1996; Saarialho-Kere, 1998). In fact, elevated levels of proteases molecules TIMP themselves can be subjected to hydrolysis. Among the natural tissue inhibitors of metalloproteinases none who alone were able to inhibit all types of matrix metalloproteinases.

To control the activity of matrix metalloproteinases was proposed several approaches, including the use of small molecules (Levy et l., 1998; Wojtowicz-Praga et al., 1997; Duivenvoorden, et al., 1997), peptide inhibitors (Odake et al., 1994) and antibodies to the matrix metalloproteinases (Su et al., 1995). However, the perfect remedy for healing wounds and slowing the aging process should not only ensure optimal inhibition of metalloproteinases, but also to promote growth and repair of damaged tissues.

Summary of invention

The present invention provides compositions containing the peptides, which can be used as anti-aging and wound healing agents. The peptides according to the present invention can not only inhibit metalloproteinases, but also to stimulate cell proliferation and migration in several cell types, including fibroblasts, endothelial cells and keratinocytes. In the framework of the present invention also covers a variety of topical lotions actions, headbands and compositions, and methods of using the peptides to slow down the aging process and for healing wounds.

The presented invention relates, therefore, to peptide inhibitors of matrix metalloproteinases. These peptide inhibitors have amino acid sequences identical to or related to the binding site, connecting the two globular domain of matrix metalloproteinases. There are several types of matrix the x metalloproteinases and their sequences, in particular, matrix metalloproteinase-1, matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-4, matrix metalloproteinase-5, matrix metalloproteinase-6, matrix metalloproteinase-7, matrix metalloproteinase-8, matrix metalloproteinase-9, matrix metalloproteinase-10, matrix metalloproteinase-11, matrix metalloproteinase-12 and matrix metalloproteinase-13. The invention includes inhibitors having the amino acid sequence of the binding site of any of matrix metalloproteinases. For example, peptide inhibitors, as claimed in this invention can have an amino acid sequence corresponding to any area of approximately 70 to 120 amino acid sequence of matrix metalloproteinase-2 (SEQ ID NO: 14) and similar areas all other matrix metalloproteinases.

The present invention is peptides, characterized by one of the following formulas (I), (II), (III):

where

XAA1, XAA4and XAA6independently of one another denote non-polar amino acids;

XAA2is a basic amino acid;

XAA3is cysteinate the amino acid;

XAA5is a polar or aliphatic amino acid;

XAA7is an acidic amino acid,

XAA8is an aliphatic or polar amino acid;

XAA9is an aliphatic, non-polar or basic amino acid; and

XAA10is a polar, acidic, basic, or non-polar amino acid;

XAA11is a polar or aromatic amino acid;

XAA12is a polar, basic, aliphatic or nonpolar amino acid;

XAA13is an aromatic, aliphatic, polar, or acidic amino acid;

Xaa14is an aromatic, non-polar or polar amino acid;

XAA15is non-polar, or acidic amino acid;

XAA16is a basic, polar or non-polar amino acid;

Xaa17is a basic, polar, aliphatic, non-polar or acidic amino acid;

XAA18is non-polar or aliphatic amino acid;

XAA19is the main or aliphatic amino acid; and

the specified peptide is able to inhibit the activity of matrix metalloproteinase-1, matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-4, matrix metalloproteinase-5, matrix metalloproteinase-6, full color the UNC metalloproteinase-7, matrix metalloproteinase-8 and matrix metalloproteinase-9, matrix metalloproteinase-10, matrix metalloproteinase-11, matrix metalloproteinase-12 and matrix metalloproteinase-13. In some embodiments of this invention, the peptide can inhibit the activity of matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-7, matrix metalloproteinase-8 and matrix metalloproteinase-9.

As a non-polar amino acids may be, for example, methionine, glycine or Proline. As the basic amino acid can be, in particular, histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine or 2,4-diaminopentane acid. Containerizable amino acids include, in particular, cysteine, homocysteine, penicillamine or β-methylcysteine.

To aliphatic amino acids include, in particular, alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid. To the acidic amino acids include, in particular, aspartic acid or glutamic acid. To polar amino acids include, in particular, asparagine, glutamine, serine, threonine, tyrosine, zither, is Lin, N-acetyl lysine, methionine sulfoxide, or homoserine, and to non-polar amino acids such as methionine, glycine or Proline. In the framework of the present invention to aromatic amino acids include, in particular, phenylalanine, tyrosine, tryptophan, phenylglycine, nafcillin, β-2-titillans, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-forfinally, 3-forfinally, 4-forfinally, pyridinoline or 3-benzothiadiazin.

The present invention relates to peptides of formulas (IV) (SEQ ID NO: 18):

where:

Xaaadenotes Proline;

XAAbdenotes glutamine or glutamic acid;

XAAwithdenotes threonine;

Xaaddenotes glycine;

XAAedenotes aspartic acid or glutamic acid;

XAAfdenotes leucine;

Xaagdenotes aspartic acid;

XAAhdenotes glutamine or serine;

XAAidenotes asparagine or alanine;

Xaajdenotes threonine;

XAAkdenotes isoleucine or leucine;

XAALdenotes glutamic acid or lysine;

XAAmdenotes threonine or alanine;

XAAndenotes methionine;

XAAaboutdenotes arginine;

XAApdenotes lysine or threonine;

Xaa denotes Proline;

XAA2denotes arginine;

XAA3denotes cysteine;

XAA4denotes glycine;

XAA5denotes valine or asparagine

XAA6denotes Proline;

XAA7denotes aspartic acid;

Xaa8denotes valine or leucine;

XAA9denotes alanine or glycine;

XAA10denotes asparagine or arginine;

Xaa11indicates tyrosine or phenylalanine;

Xaa12denotes asparagine or glutamine;

Xaa13represents phenylalanine, or threonine;

Xaa14indicates phenylalanine;

Xaa15denotes Proline or glutamic acid;

Xaa16denotes arginine or glycine;

Xaa17denotes lysine or aspartic acid;

Xaa18denotes Proline or leucine;

Xaa19denotes lysine; and

the specified peptide is able to inhibit the activity of metalloproteinases. Example of matrix metalloproteinases can serve as matrix metalloproteinase-1, matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-4, matrix metalloproteinase-5, matrix metalloproteinase-6, matrix metalloproteinase-7, matrix metalloproteinase-8, matrix metalloproteinase-9, matrix metalloproteinase-10, matrix, metallopro einza-11, matrix metalloproteinase-12 and matrix metalloproteinase-13. Preferred peptides inhibit the matrix metalloproteinase-2 or the matrix metalloproteinase-9.

Sites linking from which the peptide inhibitors according to the present invention may be derived are, for example, amino acid sequence ranging from about 70 to about 120 provisions of SEQ ID NO: 14 and similar other matrix metalloproteinases. In some embodiments of this invention the peptide inhibitors have the amino acid sequence ranging from about 77 to about 110 the provisions of SEQ ID NO: 14, and similar areas of other matrix metalloproteinases. Some examples of peptide inhibitors include inhibitors containing the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

The peptides of the present invention may have different affinity to different the matrix metalloproteinases. For example, in one of the embodiments of the present invention, these peptide inhibitors can inhibit the matrix metalloproteinase-2 with a ki value in the range of about 1.0 μm to 500,0 μm. In another embodiment of this invention these peptide inhibitors can inhibit the Mat is Ixnay the metalloproteinase-2 with a ki value in the range of about 1.0 μm to 400.0Hz in units of microns. In yet another embodiment of the present invention, these peptide inhibitors can inhibit the matrix metalloproteinase-2 with a ki value in the range of about 1.0 μm to 50.0 μm.

The present invention also provides compositions containing a therapeutically effective amount of a peptide according to the present invention and a pharmaceutically acceptable carrier. Compositions for the treatment of wounds and skin lotions are also addressed in this invention.

The object of this invention is also a method of treating wounds or eliminate the effects of aging, involving the application of a therapeutically effective amount of a peptide corresponding to one of the following formulas I, II, III or IV:

(SEQ ID NO: 21)

where:

Xaa1, Xaa4and XAA6independently of one another denote non-polar amino acids;

XAA2is a basic amino acid;

XAA3is cysteinate amino acid;

XAA5is a polar or aliphatic amino acid;

XAA7is an acidic amino acid,

Xaa8is an aliphatic or polar amino acid;

XAA9is Ali eticheskoi, non-polar or basic amino acid; and

XAA10is a polar, acidic, basic, or non-polar amino acid;

Xaa11is a polar or aromatic amino acid;

Xaa12is a polar, basic, aliphatic or nonpolar amino acid;

XAA13is an aromatic, aliphatic, polar, or acidic amino acid;

Xaa14is an aromatic, non-polar or polar amino acid;

Xaa15is non-polar, or acidic amino acid;

Xaa16is a basic, polar or non-polar amino acid;

Xaa17is a basic, polar, aliphatic, non-polar or acidic amino acid;

Xaa18is non-polar or aliphatic amino acid;

Xaa19is the main or aliphatic amino acid;

XAAadenotes Proline;

XAAbdenotes glutamine or glutamic acid;

XAAwithdenotes threonine;

XAAddenotes glycine;

XAAedenotes aspartic acid or glutamic acid;

XAAfdenotes leucine;

Xaagdenotes aspartic acid;

XAAhdenotes glutamine or serine;

XAAidenotes asparagine or alanine;

Xaajdenotes threonine;

Xaakmeans from Azin or leucine;

XaaLdenotes glutamic acid or lysine;

Xaamdenotes threonine or alanine;

Xaandenotes methionine;

Xaaaboutdenotes arginine; and

XAApdenotes lysine or threonine;

moreover, the specified peptide is able to inhibit the activity of matrix metalloproteinases.

As a non-polar amino acids in the stated in this invention, the peptides may be, for example, methionine, glycine or Proline. As the basic amino acid may be, for example, histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine and 2,4-diaminopentane acid. As cysteinate amino acids may be, for example, cysteine, homocysteine, penicillamine or β-methyl cysteine. As the aliphatic amino acids may be, for example, alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid. As acidic amino acids may be, for example, aspartic acid or glutamic acid. As polar amino acids can act as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetylized, methionine sulfoxide, or homoserine, and as depolar the first amino acid, such as methionine, glycine or Proline. As the aromatic amino acids can act as phenylalanine, tyrosine, tryptophan, phenylglycine, nafcillin, β-2-titillans, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-forfinally, 3-forfinally, 4-forfinally, pyridinoline or 3-benzothiadiazin.

In another embodiment, the present invention proposes a method of treatment of wounds or eliminate the effects of aging, involving the application of a therapeutically effective amount of a peptide having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.

Description of the drawings

Figure 1 shows according to CLUSTAL X (version 1.8) multiple sequence alignment cut binding sites selected prefermented forms of MMP protein. On Figa shows the alignment, which clearly highlighted the conservative residues, where the symbol '*' indicates complete identity between sequences, the symbol ':' denotes a position for which the degree of conservatism is 7/9, and the symbol '.' denotes a position for which the degree of identity is more than 80% with a predominantly conservative substitutions. On FIGU heterogeneous provisions bold W is the IFU.

Figure 2 shows the structure preferments forms of MMP-1 (data Bank for proteins the file 1FBL.ENT). The layout area of the sequences of SEQ ID nos: 2-10, are shown in table 1, covers the short stretch between the two major domains. When activating this area is cut out.

Figure 3 shows a three-dimensional model of matrix metalloproteinase-9. Cut out section forming the N-end of active proteases are indicated by hatching. Two zinc ions are depicted as spheres. Cut domain peptide can be contacted with the matrix metalloproteinases near the place of its normal location in the proferment. This link (next to the catalytic zinc) steric blocking part of the active site. This lock prevents linking with the substrate.

Figure 4 shows the graph of the inhibition activity of MMP-9 cut domain peptide 19-mer (SEQ ID NO: 11). MMP-9 was mixed with peptide 19-mer (SEQ ID NO: 11) before RPA analysis. We used the following concentrations of peptide 19-mer (SEQ ID NO: 11): 0 mm (black circles), 0.01 mm (white circles), 0.03 mm (black squares), 0.06 mm (white squares), 0.125 mm (black triangles), 0.25 mm (white triangles), 0.5 mm (crosses), 1 mm (inverted black triangles), 2 mm (inverted white triangles).

Figure 5 shows the graph of the inhibition activity of MMP-9 peptide virtue the CSOs domain 10-mer (SEQ ID NO: 13). MMP-9 was mixed with peptide 10-mer (SEQ ID NO: 13) before RPA analysis. We used the following concentrations of peptide 10-mer (SEQ ID NO: 13): 0 mm (black triangles), 0.25 mm (white triangles), 0.5 mm (inverted white triangles), 1.0 mm (inverted black triangles), 2.0 mm (crosses).

Figure 6 shows the inhibition of the activity of MMP-9 peptide cut domain 9-mer (SEQ ID NO: 12). MMP-9 was mixed with peptide 9-mer (SEQ ID NO: 12) before RPA analysis. We used the following concentrations of peptide 9-mer (SEQ ID NO: 12): 0 mm (black triangles), 0.25 mm (white triangles), 0.5 mm (inverted white triangles), 1.0 mm (inverted black triangles), 2.0 mm (crosses).

7 shows the inhibition of the activity of MMP-9 peptide cut domain of the 19-mer (SEQ ID NO: 11). MMP-9 was mixed with peptide 19-mer (SEQ ID NO: 11) before fluorescence analysis of collagen. We used the following concentrations of peptide 19-mer (SEQ ID NO: 11): 0 mm (black circles), 0.06 mm (white diamonds), 0.1 mm (white squares), 0.25 mm (white circles), 0.5 mm (crosses).

On Fig shown prolonged inhibition of the activity of MMP-9 peptide cut domain of the 19-mer (SEQ ID NO: 11). MMP-9 was mixed with peptide 19-mer (SEQ ID NO: 11) before fluorescence analysis of collagen. We used the following concentrations of peptide 19-mer (SEQ ID NO: 11): 0 mm (black circles), 0.06 mm (white rhombus is key), 0.1 mm (white squares), 0.25 mm (white circles), 0.5 mm (crosses).

Figure 9 shows the long-lasting inhibition of the activity of MMP-9 peptide cut domain 10-mer (SEQ ID NO: 13). MMP-9 was mixed with peptide 10-mer (SEQ ID NO: 13) before fluorescence analysis of collagen. We used the following concentrations of peptide 10-mer (SEQ ID NO: 13): 0 mm (white circles), 0.1 mm (white diamonds), 0.2 mm (white squares), 0.4 mm (crosses).

Figure 10 shows the long-lasting inhibition of the activity of MMP-9 peptide cut domain 9-mer (SEQ ID NO: 12). MMP-9 was mixed with peptide 9-mer (SEQ ID NO: 12) before fluorescence analysis of collagen. We used the following concentrations of peptide 9-mer (SEQ ID NO: 12): 0 mm (black circles), 0.06 mm (white diamonds), 0.1 mm (white squares), 0.25 mm (white circles), 0.5 mm (crosses).

Figure 11 shows the profiles of elution HPLC for typical reactions of splicing. The arrows indicate that the area of the first peak is reduced in the reaction (peak preferments forms of MMP-9), while the area of the two following peaks (Mature MMP-9 and N-terminal cleavage product, respectively) increase.

On Fig shows the transformation preferments forms of MMP-9 in the N-terminal and C-terminal domains under the action of stromelysin. Pro-MMP-9 was reacted with stromelysin in the presence of peptide 19-mer (SEQ ID NO: 11) in the following concentrations: 0 μm (black is rwiki), 0.5 μm (white squares) or 1.0 mm (black squares). At these time points were collected and an aliquot was subjected to HPLC. The peak area preferments forms of matrix metalloproteinases were summed up and was taken as 100% for samples taken at zero time. White circles indicate preferment the form of matrix metalloproteinases, inkubiruemykh in the buffer solution not containing stromelysin or peptide 19-mer (SEQ ID NO: 11).

On Figa the data isothermal titrimetricheskogo calorimetric analysis of the interaction between inhibitory peptide 19-mer (SEQ ID NO: 11) with the enzyme MMP-9. Each peak represents the amount of heat released after the introduction and subsequent binding assays. On FIGU depicts the binding isotherm obtained by time integration value of the peaks for each injection of Figa.

On Fig the data isothermal titrimetrically calorimetric analysis of the interaction between inhibitory peptide 19-mer (SEQ ID NO: 11) with the enzyme MMP-2. On Figa shows the original data of isothermal calorimetric assay for titration of peptide 19-mer (SEQ ID NO: 11) (1 mm) in a solution containing MMP-2 (20 μm), 20 mm cacodylate (pH 6.8) and 10 mm NaCl at a temperature of 25°C. Each peak represents the amount of heat released after the introduction and subsequent reactions swazilan who I am. On FIGU depicts the binding isotherm obtained by time integration value of the peaks for each injection of Figa.

On Fig depicted isotherm surface plasma resonance binding by passing the solution containing the peptide of the 19-mer (SEQ ID NO: 11), on the surface of the immobilized MMP-9.

On Fig histogram that shows the percentage of live cells compared to the positive control skin model after treatment with peptide in two different concentrations. The first sample treated with saline phosphate buffer solution (PBS)served as a positive control to determine the degree of cell viability, taken as 100%. The second sample served as a negative control in which cells were exposed to 1% Triton-X100, demonstrating that this analysis allows you to record cell death. The following three samples were processed peptides 9-mer (SEQ ID NO: 12), 10-mer (SEQ ID NO: 13) and 19-mer (SEQ ID NO: 11) at a concentration of 500 μm. The last three samples were processed peptides 9-mer (SEQ ID NO: 12), 10-mer (SEQ ID NO: 13) and 19-mer (SEQ ID NO: 11) at a concentration of 2 mm. The data are mean values of three experiments.

On Fig graphically displays the dynamics of wound healing in diabetic mice db/db. The graph shows the relative cf the days square wounds in mice depending on the number of days since its application in the processing of either normal saline (white circles) or 20 μg/ml of peptide 19-mer (SEQ ID NO: 11) (black circles). The data are given for the average of the relative diameter of wounds received on the measurement results for the ten experimental animals.

On Fig histogram proliferation of normal human dermal fibroblasts (Clonetics, CC-2509) in the presence of peptide 19-mer (SEQ ID NO: 11) and without it. Cell growth was determined by optical density (OD) at a wavelength of 490 nm for three different concentrations of peptide. The column "19mer6" describes the growth of cells in the presence of peptide 19-mer (SEQ ID NO: 11) at a concentration of 1×10-6Refer to the Column "19mer5" describes the growth of cells in the presence of peptide 19-mer (SEQ ID NO: 11) at a concentration of 1×105Refer to the Column "19mer4" describes the growth of cells in the presence of peptide 19-mer (SEQ ID NO: 11) at a concentration of 1×10-4M Control cells were grown without added peptide.

On Fig histogram proliferation of normal human keratinocytes in the presence of peptide 19-mer (SEQ ID NO: 11) and without it. As can be seen from Fig, adding peptide 19-mer (SEQ ID NO: 11) leads to increased keratinocyte growth depending on the applied dose. Control cells without addition of peptide 19-mer had the lowest cell density. The cage floor is were only 1× 10-5M peptide 19-mer (SEQ ID NO: 11, marked "19mer5" Fig), had significantly greater cell density (P>0.01)than cells not treated with peptide 19-mer. Cells treated with 1×10-4M peptide 19-mer (marked "19mer4" Fig), showed even more intense cell growth (P>0.001). However, cells treated with 1×10-6M peptide 19-mer (marked "19mer6" Fig), showed a weak cell proliferation (P>0.05), which was considered statistically insignificant.

On Fig depicts a 48-hole chemotactic chamber (Neuroprobe, Inc.), used to measure the migration of normal human dermal fibroblasts (NCF).

On Figa and shows the migration of normal human dermal fibroblasts (NCF). On Figa depicted membrane for measuring migration with 8 μm pores (looking like mugs) without cells NCCF (300-fold increase). On FIGU shows the same membrane after migration NCCF adding drug positive control (plasma fibronectin 1.25 µg/ml). This photo was taken with a 300-fold increase. Cell nuclei NCCF painted in purple color. Some cells NCCF migrated through the membrane, while the other is stuck in the 8 μm pores.

On Fig histogram that shows the percentage of migrating normal human is their dermal fibroblasts (NCF) in relation to migratory NCCF in experiments with positive control (plasma fibronectin) at various concentrations of peptide 19-mer (SEQ ID NO: 11). Because some chemotactic substances have a very narrow range of active concentrations in the initial experiments we used ten-fold dilution of peptide 19-mer. Histogram shows mean values for three separate experiments (data for the concentration of 100 µg/ml represent the average of six experiments). Concentrations of 1000 and 100 μg/ml of peptide 19-mer (SEQ ID NO: 11) was chemotactic for fibroblasts (55±3% and 46±3% in positive control, respectively). The number of migrating fibroblasts using concentrations of peptide 19-mer (SEQ ID NO: 11) less than 100 µg/ml was only about two times higher than in the experiment with a negative control, and that was deemed statistically insignificant.

On Fig histogram that shows the percentage of migrating normal human dermal fibroblasts (NCF) in relation to migratory NCCF in experiments with positive control (fibronectin) at various concentrations of peptide 19-mer (SEQ ID NO: 11) and its derivatives. At a concentration of 100 μg/ml acetylated peptide 19-mer (SEQ ID NO: 11) (Ac-19-mer) initiated the migration NCCF to about the same extent as deacetylating peptide 19-mer, but at higher concentrations, As-19-mer operated less efficiently. Peptides 9-mer (SEQ ID NO: 12) and 10-mer (SEQ ID NO: 13) intensified the migration NCCF only at a concentration of 1000 µg/ml Interestingly, peptides 14-mer TMRKPRCGNPDVAN (SEQ ID NO: 19) and 17-mer TLKAMRKPRCGNPDVAN (SEQ ID NO: 20) do not possess chemotactic activity for NCCF in no concentrations (data not shown). The beginning sequences of the peptides of the 14-mer TMRKPRCGNPDVAN (SEQ ID NO: 19) and 17-mer TLKAMRKPRCGNPDVAN (SEQ ID NO: 20)derived from enzymes matrix metalloproteinases, only slightly shifted to the N-end. Therefore, the amino acid sequence of the peptide is essential for the induction of migration NCCF.

On Fig histogram, indicating that the addition of peptide 19-mer (SEQ ID NO: 11) leads to increased synthesis of collagen in the cells of human skin fibroblasts. Control human skin fibroblasts, not treated with peptide 19-mer (SEQ ID NO: 11), produced only a small amount of collagen. In contrast, cells treated with only 1×10-6M peptide 19-mer (SEQ ID NO: 11, marked "19mer6" Fig) or 1×10-5M peptide 19-mer (SEQ ID NO: 11, marked "19mer5" Fig), had significantly greater cell density (P>0.001)than cells not treated with peptide 19-mer.

Detailed description of the invention

The object of the present invention are inhibitors of matrix metalloproteinases, which are suitable to counter the effects of aging on the skin and to stimulate the healing process. In General, the presented inhibitors and compositions contribute to the t the healing of wounds, prevent scarring, improve skin tone, reduce wrinkles and promote smooth, healthy skin.

Matrix metalloproteinases are synthesized in vivo in the form of inactive proenzymes. Proteolytic cleavage of proferment leads to activation and the formation of Mature matrix metalloproteinases. Tsepliaeva peptide sequence is a leader sequence proferment length of about 100 to 110 amino acids, which is located on aminocore protein. According to this invention, these leader peptides proferment can block the active site of matrix metalloproteinases and to inhibit the activity of matrix metalloproteinases. The use of leader peptides proferment matrix metalloproteinase reduces the rate of destruction of the extracellular matrix and accelerates the healing process.

Most of the strategies of inhibition is the inhibition of the enzymatic activity of matrix metalloproteinases using small organic molecules. These compounds are often toxic to the body and contain artificial molecules not found in nature. The use of natural peptides to the activated matrix metalloproteinases provides a high degree of control over the level of proteinase activity without t xeonsaga side effects. In contrast to the strategies of inhibition using small molecules, peptides, claimed in this invention can be used for inhibiting the activation of individual or all classes of matrix metalloproteinases at the same time. These peptides can be directly applied to the skin, injected into the wound, or you can link them or apply through the skin coatings or dressings for wounds.

This invention provides a high degree of control over the level of proteinase activity for chronic wounds and improve the effects of aging. For example, as in the healing of chronic wounds require some level proteinase activity (Agren et al., 1999), the specialist in this area may require only partial inhibition proteinase activity. By adjusting the type and amount used inhibitory peptide, it is possible to control the degree of inhibition of matrix metalloproteinases.

Peptides

According to the present invention are peptides having sequence related leader sequence preferments forms of matrix metalloproteinases in the area of crop land suitable for wound healing and to stimulate the development of healthy skin. These peptides inhibit the activity of various types of matrix metalloproteinases which promote cell growth and migration of fibroblasts and keratinocytes.

The position, which is cleaved leader sequence preferments forms of matrix metalloproteinases, is located about 110 amino acid position in the amino acid sequence of proferment. Peptide inhibitors of the present invention have a sequence, related to any area inside proferment in the range of about 70 to 120 amino acid. These peptides inhibit the activity of many types of matrix metalloproteinases. These peptides can also prevent activation preferments form of matrix metalloproteinases, as well as to inhibit the enzymatic activity of Mature matrix metalloproteinases. Peptides containing the sequence with a higher conservatism among the various matrix metalloproteinases, such as sequences that are closer to the N-terminal region of the cut section can be used to create inhibitors, in General, has efficacy against a broad spectrum of matrix metalloproteinases. At the same time, the peptides containing less conservative sequences, such as sequences that are closer to the C-terminal region of the cut section can be used to create inhibitors having specificity for certain matrix of metallopro eines.

Therefore, peptides with sequences from any leader region preferments forms of matrix metalloproteinases are treated according to the present invention as inhibitors of matrix metalloproteinases, as well as variants of these peptides that have one or more amino acids are replaced by amino acids present in natural matrix-metalloproteinases. A mixture of peptides with different sequences are also considered in this invention.

In General, peptide sequence, variants of peptides and mixtures of peptides are prepared and applied in a way to optimize wound healing, skin regeneration, prevention of scarring or eliminate wrinkles and prevent their formation. Consequently, the composition of these peptides can be adjusted by increasing or decreasing the degree of inhibition, in order to stimulate the healing process and anti-aging.

The size of the peptide inhibitor may vary. Typically, a peptide containing only about five amino acids, may be too short for optimal inhibition. However, peptides consisting of more than eight or nine amino acids are long enough to provide inhibition. Therefore, although the overall length is not critical parameters is ω, in the framework of this invention are often used peptides longer than eight amino acids. Also within the scope of this invention are peptides longer than nine amino acids. In addition, in the framework of the present invention are peptides with a length more than ten amino acids. Moreover, in the framework of this invention are also used peptides with a length of more than about fifteen amino acids. The maximum size of the peptide is no certain restrictions. However, it is usually cheaper to synthesize shorter than longer peptides. Therefore, the length of peptide inhibitors according to the present invention is usually not more than one hundred amino acids. The length of most peptide inhibitors used in this invention does not exceed about fifty amino acids. Also within the scope of this invention are peptide inhibitors containing not more than about thirty amino acids. Can also be used peptides with a length of less than about twenty-five amino acids. Similarly, in the framework of the present invention can be used peptides, the length of which is not more than twenty-three amino acids. An example of a peptide used in the present invention, can serve as SEQ ID NO: 11 with nineteen amino acids.

The sequence about armentia a few typical forms of matrix metalloproteinases approximately 70 to 120 amino acid are shown in table 1.

Table 1:
Sequence cut sections of matrix metalloproteinases
MLMSequenceSEQ ID
mmp2MQKFFGLPQTGDLDQNTIETMRKPRCGNPDVANYNFFPRKPKWDNO: 2
mmp13MQSFFGLEVTGKLDDNTLDVMKKPRCGVPDVGEYNVFPRTLKWSKMNLTYNO: 3
mmp7MQKFFGLPETGKLSPRVMEIMQKPRCGVPDVAEFSLMPNSPKWHSRTVTYRIVSYTNO: 4
mmp3MQKFLGLEVTGKLDSDTLEVMRKPRCGVPDVGHFRTFPGIPKWRKTHLTYRIVNNO: 5
mmp10MQKFLGLEVTGKLDTDTLEVMRKPRCGVPDVGHFSSFPGMPKWRKTHLTYRIVNYNO: 6
mmp12MQHFLGLKVTGQLDTSTLEMMHAPRCGVPDVHHFREMPGGPVWRKHYITYRINNNO: 7
mmp9LQKQLSLPETGELDSATLKAMRTPRCGVPDLGRFQTFEGDLKWHHHNNO: 8
mmp1MQEFFGLKVTGKPDAETLKVMKQPRCGVPDVAQFVLTEGNPRWEQTHLTYRIENNO: 9
mmp8MQRFFGLNVTGKPNEETLDMMKKPRCGVPDSGGFMLTPGNPKWERTNLTYRIRNYNO: 10

Each of the peptides listed in table 1, as well as peptides with SEQ ID NO: 1, 11, 12 and 13, are considered as peptide inhibitors of the present invention. Moreover, variants and derivatives of the peptides having any of the sequences SEQ ID NO: 1-13, and can also what to use as peptide inhibitors. Such variants and derivatives of the peptides can have one or more amino acid substitutions, deletions, insertions or other modifications, provided that these variants or derivatives of the peptides are able to inhibit the matrix metalloproteinase.

As individual amino acid residues of the peptides can be genetically encoded L-amino acids, natural not genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of any of the above groups. In the framework of the present invention makes use of the traditional legend of the twenty genetically encoded L-amino acids and conventional non-coding amino acids, which are shown in table 2.

Table 2
Amino acidA single-letter designationThe usual reduction
AlanineAndAla
ArginineRArg
AsparagineNAsn
Aspartic acidDAsp
CysteineCys
GlutamineQGln
Glutamic acidE Glu
GlycineGGly
HistidineNHis
IsoleucineIIle
LeucineLLeu
LysineToLys
MethionineMMet
PhenylalanineFPhe
ProlinePPro
SerineSSer
ThreonineTThr
TryptophanWTip
TyrosineYTyr
ValineVVal
β-AlanineBAla
2,3-Diaminopropionic acidDpr
α-Aminoadamantane acidAib
N-Methylglycine (sarcosine)MeGly
OrnithineOrn
The citrullineCit
t-butylenet-BuA
t-butylglycolt-BuG

Amino acidA single-letter designationThe usual reduction
N-methylisoleucineMeIle
PhenylglycinePhg
CyclohexylaminCha
NorleucineNIe
NafcillinNal
Pyridylamine
3-Benzothiazol alanine
4-ChlorophenylalaninePhe (4-Cl)
2-ForfinallyPhe (2-F)
3-ForfinallyPhe (3-F)
4-ForfinallyPhe (4-F)
PenicillaminePen
1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acidTic
β-2-titillansThi
Methionine sulfoxideMSO
Homo is rginin HArg
N-acetyl lysineAcLys
2,4-Diaminopentane acidDbu
ρ-aminophenylalaninePhe (pNH2)
N-methylvalineMeVal
HomocysteinehCys
HomoserinehSer
ε-Aminohexanoic acidAha
δ-Aminosalicilova acidAva
2,3-Diaminobutane acidDab

The peptides included in the scope of the present invention, one or more amino acids can be substituted by other amino acids having similar chemical and/or physical properties, provided that these variants or derivatives of the peptides will retain the ability to inhibit the activity of matrix metalloproteinases, to stimulate the cell growth of fibroblasts or keratinocytes or to stimulate the migration of fibroblast cells.

Amino acids that can substitute each other, usually belong to the same class or subclass. As is well known experts who am in this field, amino acids can be divided into three main classes: hydrophilic amino acids, hydrophobic amino acids and containerizable amino acids, depending mainly on the characteristics of the side chain of amino acids. These base classes can be further divided into subclasses. Hydrophilic amino acids include amino acids having acidic, basic, or polar side chains and hydrophobic amino acids include amino acids having aromatic or non-polar side chains. Nonpolar amino acids, in turn, can be subdivided into subclasses, including, in particular, aliphatic amino acids. Below is the definition of classes of amino acids used in this invention is:

"Hydrophobic amino acid" is an amino acid with uncharged at physiological pH values and reject water solution side chain. Examples of genetically encoded hydrophobic amino acids are isoleucine, leucine and valine. The example is not genetically encoded hydrophobic amino acid is t-butylene.

"Aromatic amino acid" refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated π-electron system (aromatic group). The aromatic group can is to be replaced with substitute groups, such as alkyl, alkenyl, quinil, hydroxyl, sulfonyl, nitro and amino groups, and others. Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan. Examples of the most common not genetically encoded aromatic amino acids include phenylglycine, 2-nafcillin, β-2-titillans, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-forfinally, 3-forfinally and 4-forfinally.

"Polar amino acid" refers to a hydrophobic amino acid having a side chain, usually uncharged at physiological pH values and non-polar. Examples of genetically encoded polar amino acids are glycine, Proline and methionine. The example is not genetically encoded polar amino acids can serve as cyclohexylamine.

"Aliphatic amino acid" is a nonpolar amino acid having a saturated or unsaturated unbranched chain, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include alanine, leucine, valine and isoleucine. The example is not genetically encoded aliphatic amino acids is cyclohexylamine.

"Hydrophilic amino acid" is an amino acid having a side chain bound water is the first solution. Examples of genetically encoded hydrophilic amino acids include serine and lysine. The example is not genetically encoded hydrophilic amino acids are citrulline and homocysteine.

"Acidic amino acid" refers to a hydrophilic amino acid having a side chain with a value of RK is less than 7. Acidic amino acids at physiological pH values typically have negatively charged due to the loss of the hydrogen ion side chains. Examples of genetically encoded acidic amino acids are aspartic acid (aspartate) and glutamic acid (glutamate).

"Basic amino acid" refers to a hydrophilic amino acid having a side chain with a value of RK over 7. Basic amino acids at physiological pH values typically have positively charged as a result of merger ion hydronium side chains. Examples of genetically encoded basic amino acids include arginine, lysine and histidine. The example is not genetically encoded basic amino acids are acyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminopentane acid and homoarginine.

"Polar amino acid" refers to a hydrophilic amino acid with uncharged at physiological pH values, a side chain, but containing interatomic bonding, in which a pair of socialized electronicmedia to one of the atoms. Examples of genetically encoded polar amino acids include asparagine and glutamine. Examples of genetically encoded polar amino acids include, citrulline, N-acetyl lysine and methionine sulfoxide.

"Cysteinate amino acid" is an amino acid having a side chain capable of forming covalent bonds with the side chain of another amino acid residue, for example a disulfide bridge. Usually the side chain containedby amino acids contain at least one Tilney (SH) group. An example of a genetically encoded cysteinate amino acid is cysteine. No examples of genetically encoded containedby amino acids are homocysteine and penicillamine.

As can be noted specialists in this field, the above classification is not absolute. Some amino acids have several characteristic properties, and therefore can be included in more than one category. So, for example, tyrosine has both an aromatic ring and a polar hydroxyl group. Thus, tyrosine has two characteristic properties and can be considered as aromatic and polar amino acids. Similarly, cysteine, in addition to the ability to form disulfide bridges also has characteristics of non-polar amine is acid. Thus, although not strictly classified as hydrophobic or nonpolar amino acid, in many cases, the cysteine can be used to make the peptide hydrophobic properties.

Some commonly encountered amino acids that are not encoded genetically, but can be present or to substitute other amino acids in peptides and their analogs described in this invention, include, but are not limited to this list) β-alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; α-aminoadamantane acid (Aib); ε-aminohexanoic acid (Alia); δ-aminosalicilova acid (Ava); methylglycine (MeGly); ornithine (Orn); citrulline (Cit); t-butylene (t-BuA), t-butylglycol (t-BuG), N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylamine (Cha); norleucine (Nle); 2-nafcillin (2-Nal); 4-chlorophenylalanine (Phe (4-Cl)); 2-forfinally (Phe (2-F)); 3-forfinally (Phe (3-F)); 4-forfinally (Phe (4-F)); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-titillans (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutane acid (Dab); 2,3-diaminobutane acid (Dbu); ρ-aminophenylalanine (Phe (pNH2)); N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer). These amino acids also pop is given in the above specified category.

The classification above is encoded and not genetically encoded amino acids are shown in table 3 below. It should be noted that table 3 is for explanatory purposes only and does not constitute an exhaustive list of amino acid residues that can be incorporated into peptides or their analogues, described in this invention. Other amino acid residues, which are suitable for the production of peptides and their analogs described in this invention can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and there in the links. Not shown here amino acids can easily be classified under the above categories based on their known characteristics and/or specific chemical and/or physical properties and comparing them with specific amino acids.

Table 3
ClassificationEncoded geneticallyNot genetically encoded
Hydrophobic
AromaticF, Y, WPhg, Nal, Thi, Tic, Phe (4-Cl), Phe (2-F), Phe (3-F), Phe (4-F), pyridyl alanine, benzothiazyl alanine
NonpolarM, G, P
AliphaticA, V,L, It-BuA, t-BuG, MeIle, Nle, MeVal, Cha, bAla, MeGly, Aib
Hydrophilic
SourD, E
MainH, K, RDpr, Orn, hArg, Phe (p-NH2), DBU, A2BU
PolarQ, N, S, T, YCit, AcLys, MSO, hSer
ContainedbyPen, hCys, β-methylcysteine

The peptides according to the present invention, any amino acid may be replaced by another amino acid of the same class with the formation of variant or derivative of the corresponding peptide, provided that such variants or derivatives of the peptides retain the ability to inhibit the activity of matrix metalloproteinases.

In one of the embodiments of the present invention the peptide inhibitors, as claimed in this invention include any of the peptides of formulas I, II or III.

where

Xaa1, Xaa4and XAA6independently of one another denote non-polar amino acids such as methionine, glycine or Proline;

XAA2is a basic amino acid such as histidine, lysine, arginine, 2,3-diaminopropane the OIC acid, ornithine, homoarginine, ρ-aminophenylalanine, and 2,4-diaminopentane acid;

XAA3is cysteinate amino acid such as cysteine, homocysteine, penicillamine or β-methyl cysteine;

XAA5is a polar or aliphatic amino acid, for example a polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetylized, methionine sulfoxide, or homoserine or aliphatic amino acid such as alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid;

XAA7is an acidic amino acid such as aspartic acid or glutamic acid;

Xaa8is an aliphatic or polar amino acid, for example aliphatic amino acid such as alanine, valine, leucine, isoleucine, t-butylene, t-butylene, methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid, or a polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide, or homoserine;

XAA9is an aliphatic, non-polar or basic amino acid, for example, limfaticheskoi amino acid, such as alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid, non-polar amino acid such as methionine, glycine or Proline, or basic amino acid such as histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine and 2,4-diaminopentane acid;

XAA10is a polar, acidic, basic, or non-polar amino acid, such as polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide, or homoserine, acidic amino acid such as aspartic acid or glutamic acid, basic amino acid such as histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine and 2,4-diaminopentane acid, or non-polar amino acid such as methionine, glycine or Proline;

Xaa11is a polar or aromatic amino acid, for example a polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide, or homoserine, or an aromatic amino acid such as phenylalanine, tyrosine, tryptophan, phenylglycine, nafcillin, β-2-Tieni is alanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-forfinally, 3-forfinally, 4-forfinally, pyridinoline or 3-benzothiadiazin;

Xaa12is non-polar, basic, aliphatic or nonpolar amino acid, for example non-polar amino acid, such asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide, or homoserine, or basic amino acid such as histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine, and 2,4-diaminopentane acid, or aliphatic amino acid such as alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid, or non-polar amino acid such as methionine, glycine or Proline;

XAA13is an aromatic, aliphatic, polar, or acidic amino acid, for example an aromatic amino acid such as phenylalanine, tyrosine, tryptophan, phenylglycine, nafcillin, β-2-titillans, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-forfinally, 3-forfinally, 4-forfinally, pyridinoline or 3-benzothiazol alanine, or an aliphatic amino acid such as alanine, Vali is, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid, or a polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide, or homoserine, or acidic amino acid such as aspartic acid or glutamic acid;

Xaa14is an aromatic, non-polar or polar amino acid, for example an aromatic amino acid such as phenylalanine, tyrosine, tryptophan, phenylglycine, nafcillin, β-2-titillans, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-forfinally, 3-forfinally, 4-forfinally, pyridinoline or 3-benzothiazol alanine, or non-polar amino acid such as methionine, glycine or Proline, or a polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide or homoserine;

Xaa15is non-polar, or acidic amino acid, for example non-polar amino acid such as methionine, glycine or Proline, or acidic amino acid such as aspartic acid or glutamic acid;

Xaa16is a basic, polar or non-polar amino acid, such as the basic amino acid is Oh, such as histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine and 2,4-diaminopentane acid; or a polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide, or homoserine, or non-polar amino acid such as methionine, glycine or Proline;

Xaa17is a basic, polar, aliphatic, non-polar or acidic amino acid, for example, basic amino acid such as histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine, and 2,4-diaminopentane acid, or a polar amino acid such as asparagine, glutamine, serine, threonine, tyrosine, citrulline, N-acetyl lysine, methionine sulfoxide, or homoserine, or aliphatic amino acid such as alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid or non-polar amino acid such as methionine, glycine or Proline and acidic amino acid such as aspartic acid or glutamic acid;

Xaa18is non-polar or aliphatic amino acid, such as non-polar amino acid such as methionine, glycine or Proline, or aliphatic what iminocyclitol, such as alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid; and

Xaa19is the main or aliphatic amino acid, such as basic amino acid such as histidine, lysine, arginine, 2,3-diaminopropionic acid, ornithine, homoarginine, ρ-aminophenylalanine and 2,4-diaminopentane acid, or aliphatic amino acid such as alanine, valine, leucine, isoleucine, t-butylene, t-butylene, N-methylisoleucine, norleucine, N-methylvaline, cyclohexylamine, β-alanine, N-methylglycine or α-aminoadamantane acid.

In some embodiments, the implementation of:

Xaa1denotes Proline,

XAA2denotes arginine,

XAA3denotes cysteine,

XAA4denotes glycine,

Xaa5denotes valine or asparagine,

XAA6denotes Proline,

XAA7denotes aspartic acid,

Xaa8denotes valine, leucine or serine,

XAA9denotes alanine, glycine or histidine,

Xaa10represents asparagine, aspartic acid, histidine, arginine, glutamine, or glycine,

Xaa11indicates tyrosine or phenylalanine,

Xaa12denotes asparagine, serine, arginine, glutamine, valine or ationen,

Xaa13represents phenylalanine, valine, leucine, threonine, serine, or glutamic acid,

Xaa14represents phenylalanine, methionine or threonine,

Xaa15denotes Proline or glutamic acid,

Xaa16denotes arginine, asparagine or glycine,

Xaa17denotes lysine, threonine, serine, isoleucine, methionine, glycine, aspartic acid or asparagine,

Xaa18denotes Proline or leucine, and

Xaa19denotes lysine, valine or arginine.

In the framework of the present invention are also desirable peptides comprising the sequence of SEQ ID NO: 1-13. One example of the desired peptide may be a peptide consisting of nineteen amino acids having SEQ ID NO: 11 (PRCGNPDVANYNFFPRKPK). This peptide (SEQ ID NO: 11) cover crop plot of MMP-2. Two smaller peptide (PRCGNPDVA (SEQ ID NO: 12) and NYNFFPRKPK (SEQ ID NO: 13)), which represents approximately half of the peptide SEQ ID NO: 11, are also desirable peptides. All three peptide in varying degrees, inhibit the activity of MMP-9 and other matrix metalloproteinases.

A single peptide with a sequence identical to the sequence cut plot matrix metalloproteinases, can be used for inhibiting the activity of only one or only a small number of matrix metalloproteinases. The compositions on the basis of which such single peptides are able to inhibit one or more, but usually not all, of matrix metalloproteinases. Such partial inhibition of the activity of matrix metalloproteinases may contribute to wound healing. Alternatively, two or more peptide can be combined to impact on two or more matrix metalloproteinases, which may provide a more complete inhibition of the activity of matrix metalloproteinases.

The person skilled in the art can construct a suitable peptide inhibitor, or a combination of peptide inhibitors to achieve the required quality and the degree of inhibition, using known methods in combination with the techniques described in this invention. "Quality" inhibition means inhibiting type matrix metalloproteinases. Various matrix metalloproteinases may have several different substrates and areas they impact. Degree of inhibition indicates the General degree of inhibition for all of matrix metalloproteinases. By varying the type and amount of the peptide inhibitor, it is possible to vary the quality and the degree of inhibition. The specialist in this area will not be difficult to modify peptides described in this invention, and to achieve the desired type and degree of inhibition of this matrix metalloproteinases.

For example the EP, the person skilled in the art can compare and make the alignment of the peptide sequences shown in figure 1, and construct the corresponding peptide inhibitor of the required quality and the degree of inhibition. In one of the embodiments shown in the example, we compare the amino acid sequences for the three matrix metalloproteinases, typical of the area of damaged tissue, mmp2, mmp9 and mmp1 are compared in order to identify regions of homology and divergence in the sequence.

This alignment of the sequences in bold are marked amino acids present at this location in the protein MMP-1 and absent in proteins MMP-2 or MMP-9, and underlined the indicated amino acids present at this location in the protein MMP-1 and in only one of the proteins MMP-2 or MMP-9.

In one of the embodiments it may be desirable to inhibit MMP-2 and MMP-9, but keep the protein activity of MMP-1 approximately at the same level for the inhibition of tumor development or the healing of chronic wounds. Based on the above sequence alignment, the person skilled in the art can construct a peptide containing amino acids present in the sequence preferential MMP-2 and MMP-9, but missing in the sequence preferments forms of MMP-1, to obtain a peptide capable of inhibiting MMP-2 and MMP-9, but not to inhibit MMP-1. This peptide has the formula IV.

(SEQ ID NO: 18)

where:

XAAanddenotes Proline;

XAAbdenotes glutamine or glutamic acid;

XAAwithdenotes threonine;

Xaaddenotes glycine;

XAAedenotes aspartic acid or glutamic acid;

XAAfdenotes leucine;

Xaagdenotes aspartic acid;

XAAhdenotes glutamine or serine;

Xaaidenotes asparagine or alanine;

Xaajdenotes threonine;

Xaakdenotes isoleucine or leucine, preferably isoleucine;

XaaLdenotes glutamic acid or lysine, preferably glutamic acid;

Xaamdenotes threonine or alanine;

XAAndenotes methionine;

XAAaboutdenotes arginine;

XAApdenotes lysine or threonine;

XAA1denotes Proline;

Xaa2denotes arginine;

XAA3denotes cysteine;

XAA4denotes glycine;

XAA5denotes valine or asparagine, preferably asparagine;

XAA6denotes Proline;

XAA7oboznachaet is aspartic acid;

Xaa8denotes valine or leucine, preferably leucine;

XAA9denotes alanine or glycine, preferably glycine;

XAA10denotes asparagine or arginine;

Xaa11indicates tyrosine or phenylalanine, preferably tyrosine;

Xaa12denotes asparagine or glutamine;

Xaa13represents phenylalanine, or threonine;

Xaa14indicates phenylalanine;

Xaa15denotes Proline or glutamic acid, preferably Proline;

Xaa16denotes arginine or glycine, preferably arginine,

Xaa17denotes lysine or aspartic acid;

Xaa18denotes Proline or leucine, preferably leucine; and

Xaa19denotes lysine.

Modification of peptides

The invention also provides for the modification of peptide inhibitors to stabilize them, facilitate their absorption and absorption, as well as improving other characteristics or properties of peptides, known to specialists in this field. In particular, the peptide inhibitors can be cyklinowanie, the charges on the peptide inhibitors can be neutralized, and the peptides can be linked with other chemical components.

The peptides can be cyklinowanie by any method known to specialists in this field. For example, N - and C-ends of the can is to be condensed with the formation of the peptide bond according to known procedures. Functional groups present in side chains of the amino acids in the peptides may be involved in the cyclization of peptides, as claimed in this invention. Examples of functional groups capable of forming covalent bonds, are-COOH and-HE; -COOH and-NH2; and-COOH and-SH. Pairs of amino acids that can be used for cyclization of the peptide, include Asp and Lys; Glu and Lys; Asp and Arg; Glu and Arg; Asp and Ser; Glu and Ser; Asp and Thr; Glu and Thr; Asp and Cys; and Glu, and Cys. Another example of the amino acid residues capable of forming covalent bonds with each other, are containerizable amino acids such as Cys, hCys, β-methyl-Cys and Pen that can form disulfide bridges with one another. Examples containedby amino acid residues can serve as Cys and Pen. Other pairs of amino acids that can be used for cyclization of the peptide will be obvious to experts in this field.

Group used for cyclization of the peptide does not have to be amino acids. Functional groups capable of forming covalent bonds with aminocom.com peptide, include carboxylic acids and esters. Functional groups capable of forming covalent bonds with carboxyl end of the peptide, are-OH, -SH, -NH2and other, where R is a (C1-C6) alkyl, (C1-C6 1-C6) alkylamino group.

All sorts of reactions between two side chains with functional groups suitable for the formation of such relationships, as well as reaction conditions suitable for the formation of such relationships, is well known to specialists in this field. Reaction conditions used for the cyclization of peptides, are usually chosen is soft enough to avoid destruction or damage to the peptide. Suitable groups that are used when necessary to protect a variety of functional groups are well known in the art (see, for example, Greene &Wuts, 1991, 2nd, John Wiley & Sons, NY), as well as various schemes of reactions to obtain such protected molecules.

In one embodiment, the implementation of the charges on the N - and C-ends are effectively removed. This can be accomplished in any way known to specialists in this field, for example, by acetylation of the N-end or amidation of the C-end.

Methods of obtaining cyclic or other modifications of peptides are well known in the art (see, for example, Spatola, 1983, Vega Data 1(3) for a General review); Spatola, 1983, "Peptide Backbone Modifications" (Modification of the peptide skeleton) in: Chemistry and Biochemistry of Amino Acids, Peptides and Proteins (Chemistry and biochemistry of amino acids, peptides and proteins (Weinstein, ed.), Marcel Dekker, New York, str (General review); Morley, 1980, Trends Pharm. Sci. 1:463-468; Hudson et al., 1979, Int. J. Prot. Res. 14:177-185 (-CH2/sub> NH-, -CH2CH2-); Spatola et al., 1986, Life Sci. 38:1243-1249 (-CH2S); Harm, 1982, J. Chem. Soc. Perkin Trans. I. 1:307-314 (-CH=CH-, CIS and TRANS); Almquist et al., 1980, J. Med. Chem. 23:1392-1398 (-COCH2-); Jennings-White et al., Tetrahedron. Lett. 23:2533 (-COCH2-); European patent application EP 45665 (1982) CA:97:39405 (-CH(OH)CH2-); Holladay et al., 1983, Tetrahedron Lett. 24:4401-4404 (-C(OH)CH2-); and Hruby, 1982, Life Sci. 31:189-199 (-CH2-S-).

Wound healing and anti-aging

The peptides claimed in this invention can be used for healing of wounds, eliminating the effects of aging on the skin and in particular for chronic wounds. Individual peptides, variants of peptides derived peptides and their mixtures (i.e. a mixture of peptides with different sequences) can be combined in the preparation of a composition for promoting the healing of wounds and the prevention or treatment of skin problems associated with aging. For optimal wound healing and skin regeneration may require some activity of matrix metalloproteinases. Thus, compositions and formulations of the present invention does not necessarily require the maximum inhibition of matrix metalloproteinases. In contrast, the activity of the peptide inhibitor vary to optimize the healing process and promote healthy skin. Varying the type, the content and number of the inhibitory Pat the Dov is possible to provide a greater or lesser degree of inhibition, thereby stimulating the healing process and the development of healthy skin.

To encourage the development of healthy skin and/or wound healing peptides, as claimed in this invention can be applied to the skin or injected into the wound in any way chosen by the person skilled in the art. In particular, these peptides can be prepared in the form of a therapeutic composition comprising a therapeutically effective amount of one or more peptides and a pharmaceutical carrier. Such a composition may be applied to the skin or injected into the wound in the form of creams, aerosols, foam, gel, or in any other form. In another embodiment, the peptides according to the present invention can be included in the composition of skin coatings or dressings containing a therapeutically effective amount of one or more peptides, applied by impregnation, covalent binding or otherwise on the coating material or dressings. In one of the embodiments the outer layer of skin or dressings allow the release of the peptide inhibitor. The release of the peptide inhibitor may be uncontrolled or controlled way. Thus, skin patches or dressings for wounds, described in this invention allow the honey is i.i.d. or timed release of the peptide inhibitor in the wound. As skin coatings and dressings can be used any material used in this area, including bandages, gauze, sterile wrapper (sterile wrapping), hydrogels, hydrocolloids, and similar materials.

A therapeutically effective amount of the peptide, as claimed in this invention, is defined as the amount of peptide that inhibits the matrix metalloproteinase to the extent necessary to encourage the development of healthy skin and/or wound healing. In particular, in the preparation of therapeutic or pharmaceutical compositions of the number of peptides according to the present invention can vary from about 0.001% to 35% by weight of the composition. The peptides can be from about 0.5% to 20% by weight of the preparation. Alternatively, the peptides can comprise from about 1.0% to 10% by weight of the preparation. A therapeutically effective amount of the peptide inhibitor depends on the method of administration of the drug. For example, for intravenous administration of a therapeutically effective amount of the peptide can be from 30 to 112,000 micrograms per kilogram of body weight. However, the number of peptide inhibitor that is required for the development of healthy skin or the treatment of wounds, may depend not only on the method of administration, but also from diagnosis, age and condition of the patient and, ultimately, determine aetsa treating physician.

Dosage and method of application can vary depending on the location of the area of skin or tissue to be treated, and/or the severity of the wound. Used dosages of the peptides and peptide conjugates can be determined by comparing their activities in vitro and in vivo in animal models, described below. This connection it is convenient to introduce in the form of single doses of the drug; containing, for example, from about 0.001 μg to 10 mg, preferably from about 0.01 μg to 5 mg, more preferably from about 0.10 μg to 1 mg, and more preferably from about 1.0 μg to 500 μg of peptide in a single dose of the drug. The desired dose may be entered in one of several techniques or by continuous infusion. The desired dose may also be prescribed for use at certain intervals of time, for example two, three, four or more times a day. The specialist in this area will not be difficult to prepare and administer an effective composition on the basis of the information presented here.

Peptide inhibitors, as claimed in this invention can be incorporated into pharmaceutical compositions and administered to mammals, particularly humans, in different dosage forms adapted to the chosen route of administration, i.e. oral or parenteral, nutrion is about, intramuscularly, tapicerki or subcutaneously.

Thus, the peptide inhibitors can be administered systemically, for example, intravenously or intraperitoneally by infusion or injection. Solutions of peptide inhibitor can be prepared in water, in which, if necessary, add non-toxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetine, their mixtures, as well as in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection, infusion or topical administration may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient and suitable for preparation for the immediate reception of sterile injectable or infusion solutions or dispersions, optionally prisoners in liposomes. In all cases, a dosage form in the final form should be sterile, fluid and stable under conditions of manufacture and storage. As the liquid carrier or vehicle can be a solvent or liquid dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils, non-toxic esters of glycerol and their respective mixtures. The desired fluidity can be maintained, for example, due to the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or through the use of surfactants. Protection from exposure to microorganisms can be ensured through the use of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some cases, the person skilled in the art may find it necessary to incorporate means isotonic agents such as sugars, buffers or sodium chloride. Prolonged absorption of the injectable preparations may be achieved through the inclusion of means of agents that slow the absorption such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by introducing a peptide or peptide conjugate in the required amount in the appropriate solvent with the addition if necessary of other above ingredients and followed by sterilization by filtration. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation should include the HAC is smart drying and sublimation technique that allows to obtain a powder containing the active ingredient together with additional ingredients present in pre-sterilized and filtered solutions.

In some cases, the peptide inhibitors can also be administered orally, in combination with a pharmaceutically acceptable delivery vehicle, such as an inert thinner or assimilable edible carrier. They may be enclosed in hard or soft gelatin capsules, compressed into tablets, or directly added to the food of the patient. For oral therapeutic applications of the peptide inhibitor may be combined with one or more excipients and used in the form taken orally tablets, sucking tablets, pastilles, capsules, elixirs, suspensions, syrups, wafers and the like. Such compositions and preparations should contain at least 0.1% of active substance. The percentage of active substance in the compositions and preparations may, of course, vary and is preferably from about 2 to 60% by weight of a given unit dose of the drug. The amount of active substance in such therapeutically suitable compositions are selected in such a way as to obtain the effective dosage.

Tablets, lozenges, pills, capsules and the like may also sod is neigh binder, such as tragacanth gum, gum Arabic, corn starch or gelatin; excipients such as calcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid and the like; lubricants such as magnesium stearate; sweeteners such as sucrose, fructose, lactose or aspartame, as well as flavorings, such as mint oil, wintergrove oil or cherry flavoring. If a single dose of the drug is a capsule, it may contain, in addition to materials of the above type, a liquid media, such as vegetable oil or polyethylene glycol. Various other materials may be present as coatings or membranes, or otherwise modifying the physical form of a solid unit dosage. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, sugar and other similar substances. A syrup or elixir can contain the active compound, sucrose or fructose as sweeteners, methyl and propylparaben as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage should be pharmaceutically acceptable and substantially non-toxic in the amounts two. In addition, the peptide inhibitor may be included in the drugs and devices extended release.

Used solid carriers include finely dispersed solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Used liquid carriers include water, alcohols or glycols or a mixture of water, alcohol and glycol, in which the present compounds can be dissolved or dispersed at effective concentrations, with the addition, if necessary, non-toxic surfactants. To improve properties of the composition in a particular application it may be added excipients such as flavoring agents and additional antimicrobial agents.

Thickeners such as synthetic polymers, fatty acids, salts and esters of fatty acids, fatty alcohols, modified cellulose or modified mineral substances, may also be used together with the liquid media to obtain pastes, gels, ointments, Soaps, and other products for direct application to the skin of the patient.

Typically, the peptides according to the present invention are applied topically to heal wounds and to encourage the development of healthy skin. Active peptides in the local application can apply the change to the selected fabric in any way, direct or indirect, in the form of aerosols, foams, powders, creams, jellies, pastes, suppositories or solutions. The term pasta used in this document, includes creams and other viscous compositions for glazing, which can be applied directly to the skin or used for lubrication of bandages or dressings. The peptides claimed in this invention can be covalently linked, strongly adsorbed or otherwise deposited on the outer layer of skin or dressings for wounds. To facilitate healing after surgery active peptides, as claimed in this invention, can be applied directly to the damaged tissue or be entered in implantable prosthetic devices. These compositions can be applied directly to the skin or the wound through the spray, such as foam or fog, along with other agents.

Compositions on the basis of these peptides can include emulsion peptides in wax, oil, emulsifier, water and/or substantially water-insoluble material that forms in the presence of water gel. Such a composition of the emulsion gives a number of useful qualities, while maintaining its viscous consistency it does not disintegrate if subjected to the usual procedures of sterilization, such as steam sterilization, because the gel stabilizes the emulsion. is also better able to retain water, than conventional gel because the water is held and emulsion and gel.

The composition may also contain a humectant to reduce the partial pressure of water vapor in the cream or lotion to reduce the speed of the drying cream or lotion. Appropriate moisturizers mixed with water in a wide range of concentrations and, as a rule, are suitable for use on the skin. For these purposes, are particularly suitable polyols, and examples of suitable polyols can be monopropellant or glycerin. The polyol may be about 20-50% by weight of the total composition; the concentration range may also be of the order of 30-40%. Such a relatively high content of polyol allows the pasta with any degree of drying to remain soft and pliable as glycerol can act as a plasticizer for the polymer. Thus, for example, when applying the paste on the bandage, after the paste will lose water, it can be easily removed from the skin without cutting bandages. The advantage of polyol is in the ability to suppress the proliferation of bacteria in the pasta when it is contact with the skin or wound, especially infected wounds.

The composition of the mixture may include other ingredients. The ingredients that can be used in the preparation of the mixture include: zinc oxide, at who mmol, calamine, sulfadiazine, chlorhexidine acetate, coal tar, chlorhexidine gluconate, salicylic acid, metronidazole, or other antibacterial agents or their combinations. Other ingredients may also be included in the cream.

These ingredients can be included in appropriate concentrations, for example, the content of zinc oxide can be up to 15 wt.%; commonly used concentration of zinc oxide is 6-10%, possibly in combination with other ingredients, such as ichthammol (0-3 wt.%) and/or calamine (0-15 wt.%). Ichthammol or calamine can also be used separately. Chlorhexidine acetate can be used in concentrations up to 1 wt.%; typical is the concentration of 0.5 wt.%.

One of the examples of the waxes used to obtain emulsions is glyceril the monostearate, or a combination of glyceryl of monostearate and PEG 100 stearate, commercially produced by CITHROL GMS/AS/NA Corporation Croda Universal Ltd. This combination contains both the wax and emulsifier (PEG 100 stearate), is particularly well compatible with the wax, for the formation of aqueous emulsions. To improve the stability of the emulsion in the composition of the funds may be included a second emulsifier, for example, PEG20 stearate, for example CITHROL 1OMS produced by the Corporation Croda Universal Ltd. Total concentration of the emulsifier in a cream typically comprises about 3-15%. P. and the use of two emulsifiers, one of them may be present in higher concentrations than the other.

The water-insoluble material must form a gel upon contact with water contained in the composition. This material must therefore be hydrophilic, but to dissolve in water only to a small extent. Such material may, for example, be a polymeric material that can absorb water, but not soluble in water. Can also be used polimernye materials, capable of forming a gel upon contact with water and remain stable at elevated temperatures, for example, clays such as kaolin or bentonite. Some polymers used in the present invention are superabsorbents, such as the polymers described in the publication WO 92/16245 that contain hydrophilic cellulose derivatives, partially crosslinked with the formation of three-dimensional structures. Suitable crosslinked cellulose derivatives include hydroxyethylcellulose, in which the alkyl group contains from 1 to 6 carbon atoms, such as hydroxyethyl cellulose or hydroxypropylcellulose and carboxycellulose, such as karboksimetiltselljuloza or carboxymethylcellulose. An example of a partially crosslinked polymer is carboxymethylcellulose sodium, marketed under the name AKUCELL X181 firm Akzo Chemicals B.V. is that the polymer is superabsorbent, because it can absorb water in an amount at least ten times its own weight. Crosslinked structure of the polymer prevents it from dissolving in the water, but the water is readily absorbed and retained within the three-dimensional structure of the polymer with the formation of the gel. Water slowly away from such a gel than from a solution, and it allows you to slow down or prevent the drying cream. The content of polymer in the composition tools are typically less than 10%, in particular it is in the range from about 0.5 to about 5.0 wt.% or from about 1.0% to about 2%.

The resulting composition can be sterilized, and its components should be selected by varying the content of the polymer, in order to provide the desired rheological properties of the final product. That is, if the product you want to sterilize, its composition should be selected so that to provide the product with a relatively high viscosity/elasticity before sterilization. If certain components of the structure shall not be subjected to sterilization, the composition may be sterilized before adding these components, or each component may be sterilized separately. This composition can then be prepared by mixing the sterilized ingredients in sterile conditions. When sterilization of the components separately, and then have them mix the m content of the polymer should be chosen in such a way to provide the required rheological properties of the final product. Content of the emulsion determines the processing properties of the composition and its perception by touch, a higher content of the emulsion leads to increased ability to spread and remoortel.

The composition can be packaged in tubes, jars or other suitable form of storage containers, or distributed on the substrate, which is subsequently packaged. A suitable substrate are dressing materials, including film dressings, and bandages.

The following examples serve to illustrate but not limit the invention.

EXAMPLE 1: a Peptide inhibitor

Total materials

All peptides were synthesized by the company Sigma-Genosys, Inc. The resulting peptides were purified in the company to the extent of homogeneity more than 95% by HPLC. Material United buervenich peaks were obessolivanija and liofilizirovanny. Method of mass spectroscopy confirmed the molecular mass and purity of the peptide. Unless otherwise stated, all chemicals were purchased from the companies Sigma Chemical Corp. or Fluka Chemical Co. The active enzyme MMP-9 were purchased from a company Calbiochem.

Molecular modeling

When the molecular modeling was used two programs visualization, Swiss PDB Viewer (Guex and Peitsch, 1997) and Rasmol (Sayle and Milner-White, 1995). The simulation was carried out by the ü on a personal computer Compaq with Windows 95, and workstation Silicon Graphics, Inc. Octane UNIX. In addition, at the station Octane was used a special package for molecular simulation Cerius 2 provided by Molecular Simulations, Inc. Files of three-dimensional structures were taken from the data Bank for proteins in the following format (file name, url): MMP-1 (1FBL, Li et al., 1995), MMP-2 (1GEN, Libson et al., 1995), MMP-8 (1JAO, 1JAN, Grams, et al., 1995; Reinemer et al., 1994), MMP-9 (1MMQ, Browner et al., 1995), TIMP-2/MT-1 MMP complex (1BUV, Femandez-Catalan et al., 1998), TIMP-2 (1BR9, Tuuttila et al., 1998) and TIMP-1/MMP complex (1UEA, Gomis-Ruth et al., 1997; Huang et al., 1996; Becker et al., 1995). These files were used to analyze the three-dimensional structure of proteins, and also served as a source of data on the primary sequence.

The study of inhibition

There were two enzymatic analysis. When the first analysis was measured by the enzymatic hydrolysis fluoresceinlabeled collagen under the action of MMP-9 as a function of time. Fluoresceinlabeled collagen (Molecular Probes, Inc.) with a concentration of 5 μm was added in the reaction buffer solution (50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 5 mm CaCl2, 0.1 mm NaN3) and was placed in a quartz fluorometric cuvette Spectrosil. Matrix metalloproteinase with a concentration of 0.1 μm was mixed with various amounts of peptides at various concentrations and incubated at a temperature of 25°C for 10 minutes to effect the binding. The mixture of proteins was added to the collagen substrate and quickly mixed. The intensity of fluorescence emission at a wavelength of 520 nm was measured as a function of time (wavelength excitation 495 nm) on fluorometry Shimadzu RF5301 (Lakowicz, 1983). Analysis of the release of fluorescein was used to determine the inhibition constants (Ki) peptide inhibitor ([I]) according to Segel (1993) using the method of Dixon (1/v versus [I]), according to the following formula:

where Kmthe Michaelis constant, Vmax- the maximum reaction rate, and [S] is the substrate concentration.

In the second analysis used the technique of fluorescence resonance energy transfer (FRPA). Substrate peptide (Calbiochem) of the seven amino acids was associated with carboxykinase dinitroaniline acceptor, and aminocentesis 2 aminobenzo-anthranilamide (Abz) donor. Cleavage of this substrate under the action of MMP-9 resulted in the release of the fluorescent product (365 nm excitation, 450 nm emission). The peptide concentration of 5 μm was added in the reaction buffer solution (50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 5 mm CaCl2, 0.1 mm NaN3) and was placed in a cell of a black 96-well plates for micrometrology, pre-blocked with 1% BSA solution. Matrix metalloproteinase with a concentration of 0.1 μm was mixed with peptide 9-mer (SEQ ID NO: 12), 10-mer (SEQ ID NO: 13) or 19-mer (SEQ ID NO: 11) in various concentrations and in what was uberalles at a temperature of 25° C for 10 minutes to effect the binding. The mixture of proteins was added to the fluorescent peptide substrate and quickly mixed. The intensity of fluorescence as a function of time was measured using a device Dynex MFX fluorescence reading dice for micrometrology. Revealed the dependence of the fluorescence intensity from moles cleaved peptide by constructing a standard curve Abz containing not FRPA-peptide. The inhibition constants were determined by curve, as described above. Other matrix metalloproteinases were studied in a similar way using the specific substrate of FRPA peptides (all obtained from Calbiochem).

The study suppress activation

This analysis measures the degree of transformation of proferment Mature the matrix metalloproteinases. Preferently form of MMP-9 (100 μg) was mixed with 0.5 μg stromelysin in PBS solution. The reaction solution was incubated at a temperature of 35°C. Aliquots were taken from the reaction solution after every 80 minutes.

Each aliquot was diluted in EDTA to a final concentration of 1 mm, was injected into HPLC column BioSelect 125 and chromatographically in PBS. In zero time (injection) there is a single peak that eluted from the column in approximately 750 seconds. The magnitude of this peak decreases as a function of time is EIW, and there are two new peak. The first peak eluted after approximately 800 seconds and corresponds to the Mature form of MMP-9. The second peak eluted after approximately 1100 seconds and corresponds to the N-terminal fragment preferments form. The peak areas were determined by integrating the profile of elution, and the graph was observed values change square in percent.

Isothermal tetrazona calorimetry

Isothermal tetrazona calorimetry (ITC) was carried out using ITK-tool production MicroCal, Inc. Titration was performed by injecting 5 μl of the solution inhibitory peptide (in the concentration range from 0.5 mm to 2.0 mm) stir in the reaction cell with a volume of 1.4 ml Concentration of MMP-9 was varied in the range from 50 to 80 μm on the cell. As the inhibitor and the enzyme was in the solution 20 mm cacodylate sodium (pH 5.5-7.0), 40 mm NaCl and 20 mm Tris-HCl (pH 7.0-7.5), 40 mm NaCl. Titration was carried out at a temperature of from 20°C to 40°C. Typical conditions titration included a 10-second period of injection, followed 240-second delay before the next injection when the total number of injections is equal to 40. To account for the heat released upon dilution and mixing was carried out a blank titration of inhibitory peptide in buffer solution.

An independent set is a set is TBA binding sites is the most common model for experimental determination of the binding. The formula for the analytical determination of the total amount of heat is given by the following equation (Freire et al., 1990):

where Q is the total heat, V is the volume of the cell ΔH - enthalpy, M is the concentration of macromolecules (associate partner in the cell), n is the stoichiometry of binding, L is the ligand concentration (associate partner in the syringe) and K is a constant of the Association. Data were selected for this model using the fifth edition of Origin (MicroCal, Inc.).

Surface plasmon resonance

The device BiaCore-X for surface plasmon resonance (SPR) (BiaCore, Inc.) was used to measure the interaction between the peptide (P) 19-mer (SEQ ID NO: 11) and MMP-9. In these experiments karboksimetilcellyulozy a sensor chip (CM-5, Lofas et al., 1993) was activated with 50 mm N-hydroxysuccinimide, 0.2 M N-ethyl-N'-(dimethylaminopropyl)-carbodiimide with a flow rate of 10 μl per minute for ten minutes. MMP-9 at a concentration of 75 ng/μl was associated with the activated surface with a flow rate of 10 μl per minute for ten minutes. In conclusion, the surface inaktivirovanie transmission of 1 M ethanolamine-HCl at a rate of 10 μl per minute for five minutes on the surface of the sensor. Peptide 19-mer (SEQ ID NO: 11) in a concentration of from 10 to 50 nm was passed through the sensor at a rate of 20 μl / minute. Phase Association and dissociation from the EPM binding was smoothed out automatically performing fast Fourier transform before modeling of rate constants. Isotherm binding was determined taking into account at the same time direct (ka) and reverse (karate constants:

(Karlsson and Fait, 1997) where [P], [MMP-9] and [R˜MMP-9] the concentration of free peptide, free MMP-9, and complex, respectively. The equilibrium constant of the binding (KA) is defined as follows:

Equation 3 in terms of the SPR signal (Morion et al., 1995) is expressed as follows:

where R is the SPR signal (in units of the response, SW) at time t, Rmax- maximum binding ability of MMP-9 in SW, and the concentration of the chelating peptide. Kinetic analysis (O Shannessy et al., 1993) was carried out using the program Origin (MicroCal, Inc.).

The definition of viability

Relative toxicity of peptides 9-mer (SEQ ID NO: 12), 10-mer (SEQ ID NO: 13) and 19-mer (SEQ ID NO: 11) was determined on a skin model Epiderm(MatTek Corp.). Before adding peptides individual containers with skin samples was pre-incubated for two hours in culture medium at 37 ° °C and 5% CO2. Then the specimen container was transferred to a 6-hole plates containing fresh medium. All peptides were dissolved in PBS to a final concentration of 10 mm, and 100 μl of each peptide is astora has pietravalle on the surface of the container with the sample Epiderm. Incubation was carried out for 12 hours at a temperature of 37°C and 5% CO2. After completion of the incubation period the containers with the samples three times rinsed with PBS solution and transferred to the 24-hole plate containing 300 ál analytical environment MTT one cell (MTT concentration was 1 mg/ml). Colorimetric analysis was performed for three hours (incubation at 37 ° °C and 5% CO2). The containers with the samples then were transferred to 24-hole culture plate containing 2 ml of isopropanol per cell. Extraction colored sediment was carried out for four hours at room temperature. The optical density was measured at a wavelength of 570 nm and 650 nm for each sample. The percent viability for each sample relative to the PBS control was calculated as follows:

For each peptide was carried out three experiments.

Results

The sequence of matrix metalloproteinase-2 (SEQ ID NO: 14) below to help identify the different domains and regions in the matrix metalloproteinases.

1MEALMARGALTGPLRALCLLGCLLSHAAAAPSPIIKFPGD
41VAPKTDKELA VQYLNTFYGCPKESCNLFVLKDTLKKMQKF
81FGLPQTGDLDQNTIETMRKPRCGNPDVANYNFFPRKPKWD
121KNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLRFS
161RIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTG
201VGGDSHFDDDELWTLGEGQVVRVKYGNADGEYCKFPFLFN
241GKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALF
281TMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTT
321EDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKY
361ESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLV
401AAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKG
441IQELYGASPDIDLGTGPTPTLGPVTPEICKQDIVFDGIAQ
481IRGEIFFFKDRFIWRTVTPRDKPMGPLLVATFWPELPEKI
521DAVYEAPQEEKAVFFAGNEYWIYSASTLERGYPKPLTSLG
541LPPDVQRVDAAFNWSKNKK YIFAGDKFWRYNEVKKKMDP
601GFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKL
641ENQSLKSVKFGSIKSDWLGC

Strict pairwise alignment of amino acid sequences of nine matrix metalloproteinases was performed using the program CLUSTAL(Higgins et al., 1992). This alignment allowed us to determine the position of both conservative and nonconservative amino acids flanking the site of activation cleavage proteases. For comparison were taken arbitrary number of amino acids as amino-and carboxylic from the site of activation cleavage. Alignment of sequences of matrix metalloproteinases (table 1), shown in figure 1, indicates that all sections of the activation of matrix metalloproteinases can be aligned in a statistically meaningful way. These areas are selected to match approximately correspond to the sequence of amino acids 70 to 120, with the average signal sequence matrix metalloproteinase comprises amino acids 1-20, propeptide domain corresponds to amino acids 21-100, and the Mature active enzyme comprises an area containing 101 amino acids to the end. Posledovatelno the ü 19-mer (SEQ ID NO: 11), selected for the study, contained within the alignment. In particular, the protein of MMP-2 peptide 19-mer (SEQ ID NO: 11) corresponds to amino acids 100-118.

Alignment of sequences of matrix metalloproteinases indicates that the Central region of activation domain, PRCGVPDV (SEQ ID NO: 1), is highly conserved, whereas the sequences flanking this region have a higher variability. The heterogeneity of the sequence can be used to construct sequences of peptides that can inhibit specific MMP enzymes or combinations of matrix metalloproteinases by simply selecting the appropriate amino acids (based on this alignment). In addition, the length of a particular peptide can be varied in order to modulate its effectiveness.

Three-dimensional structure preferments forms of MMP-1 are presented in figure 2, which shows that the area of activation are shown in table 1 and figure 1, form a bridge connecting the two large globular domain. This cut out area is defined as a short unstructured domain, connecting propeptide domain with the active domain of the enzyme. At one stage the activation of this sequence is cut into two. It is also an area sensitive to activation-mediated chloride mouth and in vitro.

Activation eliminates steric blocking (ongoing propeptide domain) and opens the access to the active site of the Mature enzyme. N-end is located near the catalytic zinc ion, which is indispensable for enzyme activity. The structure of the active enzyme MMP-9 is shown in Figure 3, where the zinc ions are depicted as solid balls. The second zinc ion is structural, that is, it affects the stability of the protein, but not catalysis. C-terminal half of the peptide 19-mer (SEQ ID NO: 11) is now located on aminocore enzyme, she is depicted in the left part of figure 3 in the form of a rising part of the last loop (shaded). Modeling the interaction between the activation domain of the peptide with the surface of the activated matrix metalloproteinases indicates that this peptide (especially with the inclusion of a longer N-terminal region) may interact with the region of the active site, while blocking the access of substrate to the active site. Thus, it can act as a small predomina or enzymatic cap.

It is known that enzymes can be split into fragments and these fragments can be reunited with the formation of active enzyme. Various peptide domains reunited and held together by non-covalent bimolecular the YMI forces. A classic example of such a peptide-protein interactions is the interaction between the S-peptide and S-protein ribonuclease (Levit and Berger, 1976). S-peptide is associated with S-protein ribonuclease in the appropriate position with the formation of the complex, reducing the enzymatic activity of RNase-S.

According to the present invention, the peptides of the activation domain can re-contact with the activated matrix by metalloproteinases in the same area, where they are present in profermance the form of matrix metalloproteinases, forming an inactive complex. The effectiveness of such binding can be measured (see below). Moreover, the peptide of the 19-mer (SEQ ID NO: 11) can bind zinc due to its cysteine residue, again preventing the catalysis.

Inhibition of the enzymatic activity of matrix metalloproteinases

The first studies on the inhibition was performed with the peptide of 19 amino acids (SEQ ID NO: 11)derived from the region of the cut domain of MMP-2. This peptide was selected from the area CLUSTAL alignment, showing the greatest degree of conservatism. The peptide sequence of the 19-mer (SEQ ID NO: 11) is strictly conservative on the N-terminal, but has a high degree of variability at the C-terminal site. Two smaller peptide representing the N-terminal and C-terminal halves of this PE is Chida, were also investigated. These two halves, roughly speaking, divided peptide on a conservative N-terminal part (9-mer (SEQ ID NO: 12)) and non-conservative C-terminal part (10-mer (SEQ ID NO: 13)). This allows to investigate not only the overall efficiency of inhibition, but also its selectivity.

19-mer:PRCGNPDVANYNFFPRKPK(SEQ ID NO: 11)
9-mer:PRCGNPDVA(SEQ ID NO: 12)
10-mer:NYNFFPRKPK(SEQ ID NO: 13)

All three peptide in all fluorescent assays demonstrated the ability to inhibit the enzyme MMP-9. In all investigated cases, the peptide 19-mer (SEQ ID NO: 11) was a more effective inhibitor of the enzyme than the two halves of the peptide. Peptide 9-mer (SEQ ID NO: 12) was a more effective inhibitor than the C-terminal peptide 10-mer (SEQ ID NO: 13). These results indicate that cysteine may be needed as a ligand zinc, or that the N-terminal region is required for the implementation of the steric blocking of the active site of the enzyme. This hypothesis can be tested in the relevant experiments on inhibitory peptides containing a longer N-terminal sequence (amino acids located before the remainder 100). A typical curve of inhibition during the titration the MP-9 peptide 19-mer (SEQ ID NO: 11) shown in Figure 4.

Similar inhibition studies performed with peptides 10-mer (SEQ ID NO: 13) and 9-mer (SEQ ID NO: 12), shown in Figure 5 and 6 respectively. RPA analyses all peptides showed the ability to inhibit the enzyme MMP-9 inhibition constant (Ki) within 45,2 to RUR 327.7 μm (see table 4). The choice of substrate (FRPA peptide or fluoresceinlabeled collagen) has little effect on the relative efficiency of inhibition for the three peptides in all cases was observed following a steady trend: 19-mer (SEQ ID NO: 11)>9-mer (SEQ ID NO: 12)>10-mer (SEQ ID NO: 13). Typical curves of reaction for the titration of MMP-9 relevant peptides shown in Fig.7-9.

In General, the obtained inhibition constants were slightly less for collagen substrate, ranging from 30,3 to 221,3 μm for collagen and 45,2 to RUR 327.7 μm for FRPA-peptide. These data indicate that peptide inhibitors somewhat more effective when used as a substrate to form collagen, which presumably is due to the fact that the inhibitory peptide blocks the active site and, because collagen is much larger than FRPA-peptide, more effectively prevents access to the active site of the enzyme. Less big FRPA-peptide can easily find access to the active site, even in the presence of the inhibitory peptide.

Typical fer entative analyses (shown in Figure 4-7) were usually carried out within 30-40 minutes. With increasing time of the experiment, it was shown that the peptide 19-mer (SEQ ID NO: 11) effectively inhibits catalyzed MMP-9 hydrolysis of collagen over 1000 minutes (Fig). Peptide 10-mer (SEQ ID NO: 13) less effectively prevents collagen breakdown at large times (Fig.9) than peptide 9-mer (SEQ ID NO: 12) (Figure 10). When this peptide 19-mer (SEQ ID NO: 11) again demonstrates the highest degree of inhibition.

Similar enzymatic analyses were performed with other MMP enzymes to test the effectiveness of peptide 19-mer (SEQ ID NO: 11). In these analyses were used FRPA peptides containing its sequence specific crop sections of matrix metalloproteinases. Peptide 19-mer (SEQ ID NO: 11) is able to inhibit various matrix metalloproteinases. The efficiency of peptide 19-mer (SEQ ID NO: 11) in respect of various matrix metalloproteinases as follows: MMP-2>MMP-3>MMP-8>MMP-7>MMP-9>MMP-1, with an inhibition constant in the range from 3.1 μm (MMP-2) to 41.1 μm (MMP-1). These data are shown in table 4.

Table 4.
Summary data on the effectiveness of inhibition
PeptideEnzymeSubstrateToi(µm)
The 19-mer (No. 11)MMP-9To login 30.3
9-mer (No. 12)MMP-9Collagen185.9
10-mer (No. 13)MMP-9Collagen221.3
The 19-mer (No. 11)MMP-9FRPA peptide45.2
9-mer (No. 12)MMP-9FRPA peptide232.8
10-mer (No. 13)MMP-9FRPA peptide327.7
The 19-mer (No. 11)MMP-1FRPA peptide41.1
The 19-mer (No. 11)MMP-2FRPA peptide3.1
The 19-mer (No. 11)MMP-3FRPA peptide6.4
The 19-mer (No. 11)MMP-7FRPA peptide22.8
The 19-mer (No. 11)MMP-8FRPA peptide12.5

Antiplasma activity of peptide 19-mer (SEQ ID NO: 11)

Matrix metalloproteinases biologically synthesized in an inactive preferments form. Proteolytic cleavage of proferment, often through a separate class of membrane-bound matrix metalloproteinases, leading to activation of matrix metalloproteinases. The length of the leader sequence proferment is bring the flax 100 amino acids (it is somewhat varies with different matrix metalloproteinases) and is aminocore protein. Inhibition of activation of proferment can be an effective method of reducing the enzymatic activity of matrix metalloproteinases in chronic wounds. If these enzymes are unable to function, the rate of destruction of the matrix will be reduced, which in turn can lead to more rapid healing of chronic wounds.

Obviously, the peptides activation domain (peptide 19-mer (SEQ ID NO: 11), 9-mer (SEQ ID NO: 12) and 10-mer (SEQ ID NO: 13)) are able to inhibit the enzymatic activity of various matrix metalloproteinases. In addition to this activity, peptide 19-mer (SEQ ID NO: 11) prevents the activation of inactive preferments forms of MMP-9. Thus, the peptide of the 19-mer (SEQ ID NO: 11) may reduce the overall level of activity of matrix metalloproteinases in skin and inside of exudate from chronic wounds by inhibiting the existing activated matrix metalloproteinases or preventing activation of newly synthesized prefermented forms of matrix metalloproteinases.

Figure 11 shows the results of a typical analysis of splicing. The first peak, eluruumis approximately 700 seconds, corresponds preferments form of MMP-9. As the reaction of splicing the intensity of this peak decreases (as indicated by the down arrow) and there are two new peak. The first new peak, eluruumis approximately 800 seconds, which corresponds to the Mature and active MMP-9. The second new peak, eluruumis approximately 1050 seconds, corresponds to pradamano. As the reaction of splicing the intensity of these two peaks increases (as indicated by the arrows up). After completion of the reaction preferently form of MMP-9 ceases to register. The standard titration reaction splicing peptide 19-mer (SEQ ID NO: 11) prevents the conversion of preferments forms of MMP-9 in prodomain and active enzyme. On Fig shows the results of this titration. Splicing can also be inhibited by peptide 19-mer (SEQ ID NO: 11) at micromolar concentrations, the degree of inhibition depends on the dose.

Isothermal tetrazona calorimetry

Calorimetric analysis was used to determine the forms whether or not the peptide 19-mer (SEQ ID NO: 11) stable non-covalent complex with active MMP-9. These data allow us to understand the mechanism of enzyme inhibition and antivaccination properties of peptide 19-mer (SEQ ID NO: 11). On Pig data shown isothermal calorimetric experiment for determining the interaction between the peptide of the 19-mer (SEQ ID NO: 11) inhibitor of matrix metalloproteinases and enzyme MMP-9. The peptide was dissolved in 20 mm cacodylate (pH 6.8), 20 mm NaCl to a final concentration of 1 mm. MMP-9 was diluted in the same buffer solution to a final concentration of 20 μm. The sequence of standard inyecciones so, as explained above. The results of the interaction between the enzyme MMP-9 and peptide 19-mer (SEQ ID NO: 11) shown below:

Stoichiometry: 0,975±0,02

ΔH (kcal/mol): -26,1±1,45

ΔS (cal mol-1K-1): -11,6±2,2

KA(M-1): 1,65×106±4,5×104

These results indicate that the interaction between the peptide of the 19-mer (SEQ ID NO: 11) and the enzyme MMP-9 is determined by the enthalpic factor, that is ΔN is negative. This reaction entropy disadvantageous, as evidenced by the negative value ΔS. However enthalpy member exceeds the member of TΔS therefore, the total free energy (ΔG) is negative.

The reaction of peptide 19-mer (SEQ ID NO: 11) with the enzyme MMP-2 was observed was defined enthalpic factor and entropy disadvantageous. The results of isothermal calorimetric analysis presented on Fig were obtained by titration of peptide 19-mer (SEQ ID NO: 11) by the enzyme MMP-2. In these experiments, there were obtained the following values.

Stoichiometry: 0,99±0,03

ΔH (kcal/mol): -15,4±2,05

ΔS (cal mol-1K-1): -21,1±1,8

KA(M-1): 2,40×106±3,7×104

Thus, binding assays entropic disadvantage. This is presumably due to a loss of configurational entropy in the binding. Should Uch is here, completely flexible peptide has a large number of degrees of freedom. In all cases, the binding stoichiometry of interaction of the peptide with MSE is 1:1, which indicates that one molecule of peptide 19-mer (SEQ ID NO: 11) interacts with one molecule of matrix metalloproteinases.

Surface plasmon resonance

The binding of peptide 19-mer (SEQ ID NO: 11) with the enzyme MMP-9 was kinetically studied using the method of surface plasmon resonance (SPR). A sensor chip is made by linking the active enzyme MMP-9 with the surface of the chip CM-5 (BIACore, Inc.) using standard chemical reactions recommended by the manufacturer. A solution of peptide 19-mer (SEQ ID NO: 11) was passed over the surface of MMP-9 in the instrument BIACore-Xand the binding and dissociation were recorded in real-time. A typical binding isotherm shown in Fig. Phase Association (30-430 seconds) best match the model of a single binding site and led to the rate constant of Association (ka) 2.2×104M-1with-1. Phase dissociation (440-700 seconds) was also consistent with the model of a single binding site and led to the rate constant of dissociation (kd) 4.1×10-3s-1. The calculated equilibrium constant of binding (KA=ka/kd) 5.3×106 good agreement with thermodynamic data. In the experiment it was observed mass transport effects on the order of 100 units of the response in the early phase of dissociation, which was not reflected in the model. Thus, the binding of peptide 19-mer (SEQ ID NO: 11) with MMP-9 is kinetically and thermodynamically favorable.

The definition of viability

Unlike many low molecular weight inhibitors of matrix metalloproteinases, three selected for this study, the peptides are not toxic to cells in experiments on skin model EpiDerm. On Fig shows that the peptide was applied in two concentrations (500 μm and 2 mm) resulted in only a slight decrease in viability compared to the PBS control. Total average viability of the cells after the application of the peptides were $ 97.6% (peptide 19-mer (SEQ ID NO: 11)), 89,6% (peptide 10-mer (SEQ ID NO: 13)) and 95.8% (peptide 9-mer (SEQ ID NO: 12)). These results indicate that these peptides are used for chronic wounds, are not toxic to mammalian cells. Data on Fig, give an average value for the three samples. The standard deviation for the viability ranged from 2.2 to 3.7 in this study and did not show correlation with dose or a particular type of peptide. Viability was slightly reduced at the improving the concentration of the peptide.

These results indicate that these peptides are not toxic in experiments on skin model EpiDermthat kinetic and entropic factors favor the formation of the bound complexes between them and the matrix metalloproteinases, and that they are able to inhibit the enzymatic activity and prevented the activation of matrix metalloproteinases.

EXAMPLE 2: Use of peptide inhibitors for wound healing

Methods

The wound was applied to mice C57BL6/KsJ db/db using a 4 mm biopsy punch. Mice were obtained from Jackson laboratory Laboratories, the age of the animals at the beginning of the experiment was 3-7 months. All mice before applying the wounds were subjected to anesthesia. Two wounds were placed at the upper back of each animal by removing the skin from the underlying structures and push the punch through the isolated skin. A typical wound had an average depth of 1.7 mm, and the size from 1.3 to 2.2 mm When applying a wound muscle tissue was not affected. Immediately after applying the wounds were treated with either saline (animals of the control group)or 5 μl of peptide 19-mer (SEQ ID NO: 11) at a concentration of 20 µg/ml

Every day the site of the wound was photographed by the digital device, and size of the wound was determined by computer integration of the photos. All the% is URS the treatment of wounds and subsequent data analysis was carried out blind (see, for example, Brown et al., 1994). Size of the wound at the time of application (zero-day) for all RAS were arbitrarily taken equal to 1 in relative units; so that in the future space wounds were transformed into relative area of RAS by dividing the absolute value of the square of the wound at day n on the size of the wound in the zero day.

The results:

As you can see in Fig, a single application of peptide 19-mer (at the time of application wounds, zero day) significantly speeds up the process fully tighten the wounds on the tested model of diabetic mice. On average, wounds treated with peptide 19-mer, was delayed on the ninth day after application compared with 14 days in control animals subjected to treatment with saline. In addition, in the processing of wounds peptide 19-mer (SEQ ID NO: 11) observed a reduction in the inflammation of the damaged area on the first day after application of the wound. It should also be noted that the treatment of wounds peptide 19-mer (SEQ ID NO: 11) tighten began earlier than in the treatment of the wound with saline (5 days vs. 8 days).

EXAMPLE 3: Stimulation of fibroblast growth by peptide inhibitors

This example provides data showing that the peptides according to the present invention stimulate the proliferation of fibroblasts.

Materials and methods

Cell lines the human skin is of fibroblasts (Clonetics, Walkersville, MD, normal human neonatal dermal fibroblasts, catalog number CC-2509) studied the ability claimed in this invention the peptide to stimulate cell proliferation. The intensity of the proliferative response of human dermal fibroblasts on peptide 19-mer (SEQ ID NO: 11) was measured in 96-well system analysis using does not contain serum medium as a control. The mother solution containing 0.5 g/l of peptide 19-mer (SEQ ID NO: 11)was prepared in water and then was divorced not contain serum medium Eagle, modified, Dulbecco (DMEM, Sigma Chemical Co., St. Louis, MO) to obtain solutions containing the peptide at a concentration of 1×10-4M, 1×10-5M and 1×10-6M Cells were inoculated in 96-well plates at a concentration of 1×103cells in 100 μl of DMEM medium containing 10% fetal bovine serum (FBS, Sigma Chemical Co., St. Louis, MO). Plates were incubated for 24 hours at a temperature of 37°C in humidified atmosphere with 5% CO2. After incubation the medium was aspirated and cells washed twice with 100 μl containing no serum medium DMEM. Then the medium was aspirated and 100 μl of a solution containing 1×10-4M, 1×10-5M or 1×10-6M peptide 19-mer (SEQ ID NO: 11), was added to the cells (20 cells for each concentration). In addition, 100 μl of media (not contain Asa serum DMEM medium) was added to 10 cells as a control. All cells were incubated for 28 hours at a temperature of 37°C in humidified atmosphere with 5% CO2. After incubation, all cells were added to 20 μl of the solution Cell Titer 96 Aqueous One Solution. Dice was carefully mixed and placed back in the incubator for 45 minutes, and the spectrophotometric absorbance was measured for each cell at 490 nm. The results were analyzed statistically with one-sided variance analysis.

The results:

As you can see in Fig, adding peptide 19-mer (SEQ ID NO: 11) leads to increased fibroblast growth depending on the applied dose. In the control experiment without addition of peptide 19-mer, the cells had the lowest density. Cells treated with only 1×10-5M peptide 19-mer (SEQ ID NO: 11, marked "19mer5" Fig), had significantly greater cell density (P>0.01)than cells not treated with peptide 19-mer. Cells treated with 1×10-4M peptide 19-mer (marked "19mer4" Fig), showed even more intense cell growth (P>0.001). However, cells treated with 1×10-6M peptide 19-mer (marked "19mer6" Fig), showed a weak cell proliferation (P>0.05), which was considered statistically insignificant.

Thus, between control cells and cells fibroblasts treated with peptide 19-mer (SEQ ID NO: 11)observed the Xia statistically significant difference in the intensity of cell growth. On the basis of these results, the peptide 19-mer (SEQ ID NO: 11) seems to be a good proliferation agent for fibroblasts.

EXAMPLE 4: Stimulation of keratinocyte growth peptide inhibitors

This example provides data showing that the peptides of the present invention stimulate the proliferation of keratinocytes.

Materials and methods

The cell line of human skin keratinocytes (Clonetics Walkersville, MD, normal human neonatal epidermal keratinocytes, catalog number SS-2503) was affected by the peptide 19-mer (SEQ ID NO: 11), to determine whether this peptide to stimulate the proliferation of keratinocytes. The proliferative response of human skin keratinocytes to peptide 19-mer (SEQ ID NO: 11) was measured in 96-well system analysis using the basic nutrient medium for keratinocytes (KVM, Clonetics, catalog number SS-3103) as a control. The mother solution containing 0.5 g/l of peptide 19-mer was prepared in water and then was divorced KVM environment to obtain solutions containing the peptide at a concentration of 1×10-4M, 1×10-5M and 1×10-6M Cells were inoculated on a 96-well plate at a concentration of 2.5×103cells in 100 μl medium KVM. Plates were incubated for 24 hours at a temperature of 37°C in humidified atmosphere with 5% CO2. After the Incubus is AI 100 μl of solution, contains 1×10-4M, 1×10-5M or 1×10-6M peptide 19-mer (SEQ ID NO: 11), was added to the cells (10 cells for each concentration). In addition, 100 μl of media KVM was added in 10 cells as a control. The plates were incubated for 48 hours at a temperature of 37°C in humidified atmosphere with 5% CO2. After incubation, all cells were added to 20 μl of the solution Cell Titer 96 Aqueous One Solution. The plate was carefully mixed and placed back in the incubator for 3 hours. Spectrophotometric absorbance was measured for each cell at 490 nm. The results were analyzed statistically with one-sided variance analysis.

Results

As you can see in Fig, adding peptide 19-mer (SEQ ID NO: 11) leads to increased keratinocyte growth depending on the applied dose. Control cells without addition of peptide 19-mer had the lowest cell density. Cells treated with only 1×10-5M peptide 19-mer (SEQ ID NO: 11, marked "19mer5" Fig), had significantly greater cell density (P>0.01)than cells not treated with peptide 19-mer. Cells treated with 1×10-4M peptide 19-mer (marked "19mer4" Fig), showed even more intense cell growth (P>0.001). However, cells treated with 1×10-6M peptide 19-mer (marked "19mer6" Fig), demonstrated the sufficiency of the part of the weak cell proliferation (P> 0,05), which was considered statistically insignificant.

Thus, between control cells and cells keratinocytes treated with peptide 19-mer (SEQ ID NO: 11), there is a statistically significant difference in the intensity of cell growth. Therefore, the peptide of the 19-mer (SEQ ID NO: 11) is, apparently, a good proliferation agent for keratinocytes.

EXAMPLE 5: Stimulation of migration of fibroblasts using peptide inhibitors

This example provides data showing that the peptides claimed in the present invention, can stimulate the migration of fibroblasts.

Materials and methods

Normal human dermal fibroblasts (NCF, Biowhittaker, Walkersville, MD) were inoculated into vials Kzt75 environment FBM (500 ml (Biowhittaker) containing insulin, hFGF-b, GA-1000 and fetal bovine serum (10 ml) and in one case replanted up to 12 times. For analysis of cell migration NCCF washed once with 10 ml of saline buffer solution Hank (HBSS). Then to remove cells NCCF from the vial into the vial Kzt75 added no more than five minutes to three milliliters of trypsin (0.25 per cent) in EDTA. Cells NCCF in the trypsin solution was added to 7 ml of medium FBM, do not contain additional additives. The cells are then NCCF centrifuged for 5 minutes and remove the supernatant. Cells NCCF resuspendable in 10 ml of medium FBM to count. The cells are then NCCF centrifuged the school times for 5 minutes and remove the supernatant of Cells NCCF then resuspendable concentration of 1× 106cells per ml in medium FBM, do not contain additional additives. In the measurement of migration it is important to use the environment FBM, since the full environment contains fibroblast growth factors and serum, which is able to activate the migration of fibroblasts.

Synthesis and purification of peptides was performed on microchemical equipment at Emory University (Emory) or company SigmaGenesis. After chromatography was carried out by HPLC all peptides were subjected to mass spectrometric analysis to determine their purity. Approximately 0.5 to 2.0 milligrams of peptide was used for the preparation of fresh mother solutions (5 mg/ml in PBS) for each experiment by measuring the migration ability. Royal solutions of peptide in PBS was used to prepare more dilute solutions (1 mg/ml, 100 μg/ml, 10 μg/ml, 1 μg/ml, 100 ng/ml, 10 ng/ml) in the environment FBM (without further additives) for measuring migration. Membrane not containing polyvinylpyrrolidone polycarbonate with a pore size of 8 μm (Neuroprobe, Inc., Gaithersburg, MD) were washed with 90% ethanol for 15 minutes and then four times with deionized water. The membrane was then placed in a glass dish containing an aqueous solution of gelatin with a concentration of 5 µg/ml. Glass plate was placed in a water bath at a temperature of about 90°With one hour. The membrane was removed from the solution W is latina and dried for one hour in an incubator at a temperature of 37° C.

For migration analysis was used 48-hole chemotactic chamber (Neuroprobe, Inc., Gaithersburg, MD) (see Fig). Twenty-eight microlitres each of the investigated solution was added at the bottom of the cell (containing four cells). Processed gelatin membrane was carefully placed on top of the bottom cell. The gasket and the upper part of the chamber is then carefully placed on top of the membrane. The device was fixed with six thumbscrews. The solution NCCF containing a given cell density (2×105cells per ml)was prepared by adding 320 μl of solution with a concentration of 1×106cells per ml to 1,28 ml of medium FBM (without further additives). Fifty microlitres solution NCCF with a concentration of 2×105cells per ml were added in the upper part of each cell. The camera was then placed in an incubator at a temperature of 37°and 5% of the content of CO2for three hours. After the camera was removed, Unscrew the thumbscrews and put the camera upside down on a paper towel. The lower part of the chamber was removed, and the membrane through which migrated cells NCCF, on top. The membrane was carefully removed. Cells that were not migrated through the membrane were scraped from the membrane using a cell scraper, washed one side of the membrane with a solution of PBS (Neuroprobe, Inc.). The membrane was fixed with the methanol and stained coloring solutions Diff-Quik I and II. The cell nucleus NCCF was painted in purple color. The membrane was placed on a glass slide, plunging in cedar oil, and closed the cover glass. Cells in three separate fields corresponding to the different cells were counted using an optical microscope Zeiss (25x lens × 10x eyepiece × h). The average number of migrated cells NCCF in negative control subtracted from the values obtained for the experimental solution (peptide), and data of the positive control. These results were then expressed in percentage of migrated cells NCCF with respect to the data of the positive control (plasma fibronectin to 1.25 μg/ml), according to the following formula.

Results

The membrane used to determine the extent of migration of fibroblasts (NCF), had 8 μm pores, shown in Figa (top). The solution chemoattractant initiates the migration of fibroblasts through the membrane. When migrating fibroblasts through the membrane, they are attached to the membrane side chemoattractant. As shown in Figv (bottom), the nuclei of fibroblasts painted in purple color for visual counting. Cells trapped inside the pores (pore looks dark purple), were included in the total number of counted cells. When measuring migration NCCF in positive control STA is in the cells in the field when the count ranged from 27 to 60. As a positive control in the analysis of migration NCCF was used a solution of plasma fibronectin (1,25 µg/ml in the medium FBM). Fibronectin is a molecule that promotes the formation of extracellular matrix during wound healing. Fibronectin binding to the receptor of fibroblasts α4β1the integrin is chemotactic in a very narrow concentration range (0,8-1,6 µg/ml). Postlethwaite, A.E.; Kang, A.H. "Fibroblast Chemoattractants" in Methods of Enzymology, vol.163, Academic Press: New York, 1988, 694-707. In negative control (medium FBM without additives) number of cells in the field when the count ranged from 1 to 7 NCF (less than 10% of positive control) (see Fig).

In the analysis of migration NCCF used several concentrations of peptide 19-mer. Due to the narrowness of the range of concentrations in which plasma fibronectin is chemotactic for NCCF, was used ten-fold dilution of the peptide to study a wide range of concentrations. As shown in Fig, at concentrations of peptide 19-mer above 0.1 mg/ml, a significant number of cells NCCF migrated through the membrane (55%±3% and 46%±3% for 1000 µg/ml and 100 µg/ml, respectively). When the concentration of peptide 19-mer below 100 µg/ml, it has only a minor chemotactically. Because such concentrations of peptide 19-mer induced mi is the radio only 20% NCF, only about two times greater than that for the negative control, these data were considered insignificant. No differences in the data for migration under the action of peptides 19-mer obtained from two different commercial sources, were not detected (data not shown).

To determine whether changes in amino acid sequence to the migration of fibroblasts were synthesized and purified different variations of the sequences of the 19-mer. Were studied the following peptides: 9-mer PRCGNPDVA (SEQ ID NO: 12), 10-mer NYNFFPKKPK (SEQ ID NO: 13), 14-mer TMRKPRCGNPDVAN (SEQ ID NO: 19) and 17-mer TLKAMRKPRCGNPDVAN (SEQ ID NO: 20). The sequence of the peptides of the 14-mer and 17-mer are sequences, taken, respectively, MMP-2 and MMP-9 from the field, shifted closer to the N-end. Peptides 9-mer (SEQ ID NO: 12) and 10-mer (SEQ ID NO: 13) inhibit the activity of matrix metalloproteinases. Peptide 19-mer (SEQ ID NO: 11) was also synthesized with a protective acetyl and amide groups (As-19-mer) at the N - and C-ends, respectively, to determine whether the addition of these groups to influence the intensity of migration of fibroblasts. AC-19-mer was chemotactic for fibroblasts as at a concentration of 100 (44±3%), and 1000 (40±6%) µg/ml, as shown in Fig. Peptide 10-mer (SEQ ID NO: 13) was chemotactic at a concentration of 1000 µg/ml (30±2%), but not at a concentration of 100 µg/ml (11±0%, the same order as negative the control, as shown in Fig). Peptide 9-mer (SEQ ID NO: 12) was chemotactic at a concentration of 1000 µg/ml (32±2%), but not at a concentration of 100 µg/ml (16±0%). Interestingly, the addition of amino acids at the N-end peptide 9-mer resulted in the absence of migration NCCF. Peptides 14-mer TMRKPRCGNPDVAN (SEQ ID NO: 19) and 17-mer TLKAMRKPRCGNPDVAN (SEQ ID NO: 20) do not cause migration NCCF at concentrations ranging from 1000 μg/ml to 10 ng/ml (data not shown). Because of the wide variations in the degree of migration NCCF for different peptides amino acid sequence may be significant to stimulate cells NCCF for migration.

Migration of fibroblasts to the affected area is an important process for proper wound healing. In the experiment on the measurement of migration NCCF were demonstrated chemotactic properties of peptide 19-mer (>100 μg/ml) and its derivatives Ac-19-mer, 9-mer and 10-mer. Since the peptides of 9-mer and 10-mer induce migration NCCF about the same degree, it is difficult to establish how individual amino acids important for activation. Interestingly, peptides 14-mer TMRKPRCGNPDVAN (SEQ ID NO: 19) and 17-mer TLKAMRKPRCGNPDVAN (SEQ ID NO: 20) had no effect on the motility of fibroblasts. Although the exact mechanism of chemoattractive peptide 19-mer is unknown, it is established that the amino acid sequence of the peptide is essential for the activation of fibroblasts.

EXAMPLE 6: Peptide inhibi the ora does not stimulate migration of neutrophils

This example provides data showing that the peptide, as claimed in this invention, does not stimulate migration of neutrophils.

Materials and methods

Synthesis and purification of peptides was performed on microchemical equipment at Emory University or company SigmaGenesis. After chromatography was carried out by HPLC all peptides were subjected to mass spectrometric analysis to determine their purity. Approximately 0.5 to 2.0 milligrams of the obtained peptide was used for the preparation of fresh mother solutions (5 mg/ml in HBSS/HSA) for each migration analysis. Royal peptide solutions were used to prepare more dilute solutions (1 mg/ml, 100 μg/ml, 10 μg/ml, 1 μg/ml, 100 ng/ml and 10 ng/ml) in HBSS/HSA for analysis.

Neutrophils isolated from human blood collected in Vacutainer tubescontaining EDTA) by applying a mixture of blood/EDTA (about 20 ml) over two layers Histopaque1119 (bottom layer 18 ml) and 1077 (top layer, 7 ml) in 50 ml Falcon tubes. The tubes were centrifuged at a speed of 1800 rpm for 30 minutes (without stopping). After centrifugation the tubes were observed four layers (top to bottom): plasma, 1077, 1119 and red blood cells. The intermediate layer between layers of plasma and 1077 contained lymphocytes, monocytes and some platelets. The plasma layer, prom the mediate layer and a large part of the layer 1077 were selected and discarded. Layer 1119 containing neutrophils, were selected and placed in a clean 50 ml tube. The addition of saline phosphate buffer solution, Dulbecco (DPBS), the volume was brought to 50 ml of Cells were centrifuged for 15 minutes at a speed of 2100 rpm Then cells resuspendable in 10 ml DPBS, was transferred to a 15 ml tube and centrifuged again for 10 minutes at a speed of 2000 rpm, This process was repeated for the secondary washing of the cells. After removal of the supernatant liquid red blood cells were literally by adding 6 ml of cold sterile water to each 15 ml portion of the supernatant liquid. The cells were mixed with water only for 30 seconds. Then added 6 ml of cold 2% sterile saline. Cells were centrifuged again for 10 minutes at a speed of 2000 rpm Red supernatant (containing the hemoglobin of red blood cells) were removed. This process lizirovania red blood cells was repeated two more times to remove all of the red blood cells. After removal of the greater part of the red blood cells neutrophils resuspendable in 10 ml HBSS/HSA to count with Trifanova blue. Cells were centrifuged and resuspendable in HBSS/HSA at a concentration of 1.25×106cells/ml

Analysis of neutrophil migration was carried out in two variants on a 24-hole plate with R zelennymi cells (the size of the pores in the separating membrane 3 μm). Separating the membrane was pre-specialise 20-40 μl of a solution of HBSS/HSA (0.04-0.4%). In the lower part of each cell was added chemotactic solutions (500 μl). Neutrophils (200 μl of a solution of 1.25×106cells/ml) were added on top of the membrane in each cell. The cells are then dipped in a chemotactic solutions. The plate for one hour were placed in an incubator at a temperature of 37°and 5% of the content of CO2). The number of cells that migrated through the membrane were counted by staining Trifanova blue.

Results

In experiments on the migration of neutrophils in the allocation of the number of viable neutrophils was approximately 95%. For this experiment, neutrophils resuspendable in solution HBSS/a 0.4% HSA. As a positive control for the study of neutrophil migration was used IL-8 (10 nm in buffer solution HBSS/0.4% HSA). In the experiment we used the following concentrations of peptide 19-mer of 1 mg/ml, 100 μg/ml, 10 μg/ml and 1 μg/ml

In the second column in table 5 shows the results of the first experiment. As negative and positive controls were used, respectively HBSS/HSA and IL-8.

Table 5.
The percentage of migrated neutrophils for each chemotactic substrate.
Chemotactic substrateThe percentage of migrated neutrophils in the first experimentThe percentage of migrated neutrophils in the second experiment, the
HBSS/HSA*29%0%
10 nm IL-881%51%
1 mg/ml of 19-mer23%4%
100 µg/ml of 19-mer29%0%
10 µg/ml of 19-mer22%-
1 µg/ml of 19-mer32%-
*Concentration of HSA was 0.4% in the first experiment and 0.04% in the second experiment.

Peptide 19-mer (SEQ ID NO: 11) induced migration of neutrophils to the same extent as the negative control. Adding IL-8 caused an 81% increase in the migration of neutrophils through the membrane. While the results of the positive control was satisfactory, the results of the negative control (29%) seemed too high. Therefore, in the second experiment, the number of HSA was reduced tenfold to 0.04%. As can be seen from the third column of table 5, in the second experiment, a negative control, the number of migrated neutrophils dropped to 0%. The number of migrating neutrophils in the positive control IL-8 t is the train decreased, but was still quite large (51%). As in the first experiment, the peptide 19-mer had no effect on migration of neutrophils.

Migration of fibroblasts to the affected area is an important process for proper wound healing. In the experiment to measure the degree of migration NCCF were demonstrated chemotactic properties of peptide 19-mer (SEQ ID NO: 11). However, peptide 19-mer had no effect on migration of neutrophils. Apparently, the fibroblasts have a receptor, is absent in the neutrophils, which leads to the recognition of peptide 19-mer binding and chemotactic signaling.

EXAMPLE 7: Stimulation of collagen production

Stimulation of collagen synthesis in response to the addition of a peptide 19-mer (SEQ ID NO: 11) was investigated in cell lines of human dermal fibroblasts (Clonetics, Walkersville, MD, normal human neonatal dermal fibroblasts, catalog number CC-2509) using analytical set Takara Biomedicals EIA (MC)was purchased from Panvera (Madison, WI). Cells were first grown on 96-hole dies in the environment Needles, modified, Dulbecco (DMEM)with the addition of 10% fetal bovine serum (FBS), while the medium and serum were purchased from Sigma Chemical Co, St. Louis, MO. As control was used containing no serum DMEM. The mother solution containing 0.5 g/l of peptide 9-mer (SEQ ID NO: 11), was prepared in water and then was divorced not containing serum-DMEM to obtain solutions containing the peptide at a concentration of 1×10-5M and 1×10-6M Cells were inoculated in 96-well plates at a concentration of 5×103cells in 100 μl of DMEM medium containing 10% fetal bovine serum (FBS, Sigma Chemical Co., St. Louis, MO). Plates were incubated for 24 hours at a temperature of 37°C in humidified atmosphere with 5% CO2. After incubation the medium was aspirated and cells washed twice with 100 μl containing no serum medium DMEM. Then the medium was aspirated and 100 μl of a solution containing 1×10-5M or 1×10-6M peptide 19-mer (SEQ ID NO: 11), was added to the cells (n=2 for each concentration). In addition, 100 μl of media containing no serum medium DMEM) was added in 4 cells as a control. All cells were incubated for 48 hours at a temperature of 37°C in humidified atmosphere with 5% CO2.

For analysis was collected in 20 μl of supernatant from each cell in 96-well plates. Standard buffer solution and stopping the solutions were prepared immediately prior to analysis. 100 μl of the conjugate solution of the antibody-POD (included in the analytical kit) was added to the cells in 96-well plates pre-coated with antibodies (included in the analyte is ical set). 20 ál of the standard and test solution (from another 96-well plates containing fibroblasts) were added to appropriate wells. The plate was gently mixed, tightly closed and incubated for three hours at a temperature of 37°C.

After incubation, each cell four times gently washed with PBS buffer solution (400 µl). After washing of all cells were completely removed all the liquid. In each cell was added 100 μl of substrate solution (hydrogen peroxide and tetramethylbenzidine in buffer solution, supplied with analytic set) and the plate incubated for 15 minutes. At this point in each cell in the same order as the substrate, was added to 100 ál of stopping solution (freshly prepared 1 h H2SO4). The plate was carefully mixed and the absorbance was measured at 450 nm. The results were analyzed statistically with one-sided variance analysis.

The results:

As can be seen from Fig and Table 6, the addition of a peptide 19-mer (SEQ ID NO: 11) leads to increased synthesis of collagen at both concentrations used. Control cells without addition of peptide 19-mer (SEQ ID NO: 11) produced the least amount of collagen. At concentrations of peptide 19-mer (SEQ ID NO: 11) 10-5and 10-6M cells produced a statistically significant (P<0.001) the amount of collagen what. These results indicate that peptide 19-mer (SEQ ID NO: 11) stimulates the production of collagen.

Table 6:
Summary data
GroupThe number of pointsAverageThe standard deviationStandard error of the meanMedian
19mer520,83650,020510,014500,8365
19mer620,83150,012020,0085000,8315
Control40,73880,014310,0071570,7330

LINKS:

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1. The peptide consisting of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, and this peptide inhibits any of the matrix metalloproteinase-1, matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-4, matrix metalloproteinase-5, matrix metalloproteinase-6, matrix metalloproteinase-7, matrix metalloproteinase-8, matrix metalloproteinase-9, matrix metalloproteinase-10, matrix metalloproteinase-11, matrix metalloproteinase-12, or matrix metalloproteinase-13.

2. The peptide according to claim 1, with the indicated peptide is a chemoattractant for fibroblasts or keratinocytes.

3. The peptide according to claim 1, with the indicated peptide stimulates collagen production in fibroblasts.

4. Composition for stimulating the development of healthy skin, containing a therapeutically effective amount of a peptide consisting of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13, and a pharmaceutically acceptable carrier.

5. The composition according to claim 4, with the indicated peptide inhibits proteinase any activity of matrix metalloproteinase-1, matrix metalloproteinase-2, matrix metalloproteinase-3, matrix metalloproteinase-4, matrix metalloproteinase-5, matrix metalloproteinase-6, matrix metalloproteinase-7, matrix metalloproteinase-8, m is texnai metalloproteinase-9, matrix metalloproteinase-10, matrix metalloproteinase-11, matrix metalloproteinase-12, or matrix metalloproteinase-13.

6. The composition according to claim 4, with the indicated peptide stimulates cell growth of fibroblasts or keratinocytes.

7. The composition according to claim 4, with the indicated peptide is a chemoattractant for fibroblasts or keratinocytes.

8. The composition according to claim 4, with the indicated peptide stimulates collagen production in fibroblasts.

9. Dressings for wounds containing composition according to claim 4.

10. Dressings for wounds according to claim 9, which promotes healing, prevents scarring, and improves skin tone, reduces the formation of wrinkles and stimulates the development of smooth, healthy skin.

11. Lotion for skin treatment to reduce the effects of aging, containing the composition according to claim 4.

12. The lotion according to claim 11, which can improve the color of skin, reduce wrinkles or to encourage the development of smooth, healthy skin.

13. The use of a composition according to claim 4 for the manufacture of a medicine for promoting wound healing, prevent scarring, reduce wrinkles, reduce the effects of aging, and improve skin color.

14. Way to stimulate the development of healthy skin, which comprises applying a therapeutically EF the objective amount of the composition according to claim 4.

15. The method according to 14, in which the stimulation of the development of healthy skin includes improving the color of the skin, reducing wrinkles and fostering smooth healthy skin.

Priority items:

16.08.2001 according to claims 1, 4, 5, 9, 13;

21.12.2001 according to claims 1, 4, 5, 9, 13;

21.05.2002 according to claim 2, 3, 6, 7, 8, 10, 11, 12, 14, 15.



 

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