Tetrapeptide, composition (variants), and the method of collagen production stimulation by fibroblast

FIELD: biotechnologies.

SUBSTANCE: invention refers to the area of biotechnology, namely to production of short peptides - stimulators of production of extracellular matrix protein in the skin, and can be used in the medicine. The peptides produced consist of four amino-acid residues that can be used separately or in combination in the method of stimulation of collagen production using fibroblast.

EFFECT: invention provides for effective collagenesis stimulation in fibroblast cells.

25 cl, 3 dwg, 7 tbl, 6 ex

 

This application claims the priority of provisional application U.S. serial number 60/813284, filed June 13, 2006, which is incorporated into this description by reference in full.

The technical FIELD TO WHICH the INVENTION RELATES

The present invention relates to tetrapeptides with amino acid GxxG motif or PxxP, where G (glycine) and P (Proline) are stored, and x denotes a variable amino acid. The present invention relates also to an active tetrapeptide shift frames, which are tetrapeptide sequences joined to one frame is read from tetrapeptide GxxG or PxxP in the protein ESM. In particular, the present invention relates to GxxG, PxxP or active peptides of the shift frame, which stimulate production of extracellular matrix proteins and enhance wound closure monolayer of epithelial cells with excoriation (abrasion, scratches) of human skin. These peptide compositions can be used in compositions for healing damaged skin and maintain healthy skin.

The LEVEL of TECHNOLOGY

Skin aging is seen as the formation of wrinkles and impaired wound healing. A wound is defined as a break in the integrity of the epithelium of the skin. Normal wound healing involves a complex and dynamic, but a well-ordered series of events, PR is spent to heal damaged tissues. The predominant component of normal skin is the extracellular matrix (ECM), gel-like matrix produced by cells, which it surrounds. The ECM consists of two main classes, including fibrous structural proteins and proteoglycans. It is known that changes in the composition and condition of the stitching ECM associated with aging and a range of acquired and inherited skin disorders. It is well described that the ECM not only provides structural support, but also affects cell behavior, such as differentiation and proliferation. In addition, more and more studies suggest that the components of the matrix can be a source of cellular signals to facilitate proliferation and migration of epithelial cells and strengthen the thus wound healing.

The largest class of fibrous ECM molecules is the family of collagens, which consists of at least 16 different types of collagen. The collagen in the dermal matrix consists primarily of collagens type I (80-85%) and type III (8-11%), both of which are fibrillar or plochodrazni collagen. The tensile strength of the skin tensile primarily due to these fibrillar collagen molecules, which samsobeats in microfibers with the placement head to tail and lateral speed. is olekuly collagen be sewn with neighboring collagen molecules, creating additional strength and stability to the collagen fibers. Damage to the collagen network (e.g., enzymes or physical destruction) or total collapse causes healing through reparations.

Various biologically active peptides, which stimulate the production of ECM proteins described in the scientific literature and in patents granted. Peptides were historically isolated from natural sources and have recently been the subject of studies on the relationship of structure and function. Natural peptides served as starting points for konstruirovanie synthetic peptide analogues.

Specific sequences in the proteins of the ECM can stimulate useful elements in the skin, such as collagen type I, collagen type III and fibronectin (Katayama et. al., J. Biol. Chem. 288:9941-9944 (1983)). Katayama et al. identified Pentapeptide, KTTKS (SEQ ID NO:17), in carboxykinase propeptide (residues 197-241) collagen type I. This propeptide cleaved during the production of the Mature protein collagen. This split propeptide may participate in the regulation of collagen production by biochemical feedback mechanism, and the segment KTTKS plays an active role. Maquart et al. (J Soc Biol. 193:423-28 (1999)) reported that the peptide GHK and CNYYSNS also stimulate the synthesis of ECM. These sequences can be released during metabol the ZMA ECM, passing through this signal about the need to repair the ECM. Short peptide sequences released by any mechanism, often called the “matriciana” (Maquart et al., J. Soc. Biol. 193:423-28 (1999)).

Although there are a number of natural and synthetic peptides, there is a need for improved biologically active peptides and methods of use thereof.

The INVENTION

Described tetrapeptide, which are characterized by the amino acid motif sequence GxxG or PxxP, where the remains of G (glycine) and P (Proline) are stored, and x is a variable amino acid. These tetrapeptide originate from sequences that occur multiple times throughout the primary sequence of the protein of the ECM, collagen type IV. The described sequence induce the production of all forms of collagen to a greater extent than previously known peptide sequence, including KTTKS, sold under the trade name MATRIXYL™ SEDERMA SAS (France). In addition, the composition comprising a combination of different repetitive sequences, causes even higher producing collagen reaction. Additional benefits can be expected from combinations of peptides present in various ECM proteins.

Obtaining the specific combination of tetrapeptides to restore the ESM can be commercially excessively costly. Described is relatively simple and economical means of obtaining different combinations of biologically active tetrapeptide. By obtaining a combinatorial library tetrapeptides with the GxxG motif or PxxP can be generated many biologically active tetrapeptide in the same cycle of production (for example, GEPG, GPEG, GPPG and GEEG). The combination of tetrapeptides can induce higher education ECM proteins than the individual peptides. Compositions containing the described tetrapeptide, separately or in combination, are applicable in the markets means for skin care, including, but not limited to, funds that are intended to influence the appearance of wrinkles, skin tone, skin density or sagging skin. Stimulation of collagen described by tetrapeptide can significantly improve the health and appearance of damaged and aged skin.

BRIEF DESCRIPTION of FIGURES

Figure 1 represents SEQ ID NO:45, which is the amino acid sequence of collagen IV, illustrating the location of tetrapeptides GxxG. All sequences are printed in bold, underlined, and overlapping sequences are underlined twice.

Figure 2 represents SEQ ID NO:46, which is the amino acid sequence of collagen III, illustrating the location of the active segments with the shift of the framework PGPR and GAGP. All active sequences with the shift of the frame is printed in bold and are underlined, and the sequence GxxG, with one shift frame, underlined twice.

Figure 3 also represents SEQ ID NO:45, amino acid sequence of collagen IV, illustrating the location of tetrapeptide PGPP.

DETAILED description of the INVENTION

This invention relates in General to tetrapeptides that stimulate the production of ECM proteins and modulate wound healing, and applications such tetrapeptides.

Peptides

One way of carrying out the invention relates to selected tetrapeptide containing the GxxG motif or PxxP. In this embodiment, G (glycine) or P (Proline) are stored, and x denotes a variable amino acid. This peptide can usually be any peptide that fall into the above description and, more preferably, it represents SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

Another variant embodiment of the invention relates to selected tetrapeptide containing motif GxPG, where x denotes P in any variable position, or both. In this embodiment, G (glycine) and P (Proline) are stored, and x denotes a variable amino acid. This peptide can usually be any peptide that fall into the above description and, more preferably, it represents SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

Another variant embodiment of the invention relates to selected tetrapeptide containing motif GxG. In this embodiment, G (glycine) and E (glutamic acid) are stored, and x denotes a variable amino acid. This peptide can usually be any peptide that fall into the above description and, more preferably, it represents SEQ ID NO:5 or SEQ ID NO:8.

Another variant embodiment of the invention relates to selected tetrapeptide containing motif PGxP. In this embodiment, P (Proline) and G (glycine) are stored, and x denotes a variable amino acid. This peptide can usually be any peptide that fall into the above description and, more preferably, it represents SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16.

Another variant embodiment of the invention relates to selected tetrapeptide containing motif PExP. In this embodiment, P (Proline) and E (glutamic acid) are stored, and x denotes a variable amino acid. This peptide can usually be any peptide that fall into the above description and, more preferably, it represents SEQ ID NO:1 or SEQ ID NO:9.

Another variant embodiment of the invention relates to active ones is ropeptide with the shift frame. In this embodiment, this tetrapeptide is one shift range or from tetrapeptide GxxG, or from tetrapeptide Rhhr in ECM protein. This peptide can usually be any peptide that fall into the above description and, more preferably, it represents SEQ ID NO:4 or SEQ ID NO:6.

Each of the above peptides may contain D - or L-amino acids. These peptides can contain all D-amino acids or L-amino acids. These peptides can be acidic C-end (-CO2H) or, preferably, an amide C-end (-CNH2, -CONHR or-CNR2). These peptides can be further enhanced or modified, either chemically or enzymatically. For example, these peptides can be lidirovali (-NH2With the end of that can do this tetrapeptide less sensitive by degradation and to increase their solubility in comparison with the form of free acids. These peptides can also be limitirovany that can provide improved penetration into the skin.

The above peptides can contain the following amino acids: R (arginine), L (leucine), P (Proline), F (phenylalanine), Q (glutamine), E (glutamic acid), I (isoleucine), K (lysine), S (serine), V (valine), A (alanine), N (asparagine), D (aspartic acid), T (threonine), Y (tyrosine) and G (glycine). The above peptides do not include the following AMI is ocelote: M (methionine), C (cysteine), H (histidine) or W (tryptophan). Thus, in one embodiment, x is selected from M (methionine), C (cysteine), H (histidine) or W (tryptophan).

Applications

Additional option of implementing the present invention relates to a method of applying the above-described peptides. These applications may include the use of a single peptide, or may include two or more peptides in combination.

One embodiment of the present invention is a method for activating the healing of damaged skin and maintain healthy skin using tetrapeptide that stimulate the production of ECM proteins. This method usually refers to communication dermal (skin) cells with a composition containing this peptide. These compositions can be spray, emulsion, liquid, lotion, cream, paste, ointment, foam or other pharmaceutically acceptable composition. Typically pharmaceutically acceptable composition may include any acceptable carrier suitable for application to human skin, such as cosmetically acceptable carrier and a dermatologically acceptable carrier. The compositions may contain other active agents such as retinoids or other peptides. These compositions may contain pharmaceutically acceptable carriers or adjus the options. Stage contacting can be performed in vivo, in situ, in vitro or in any way known to a skilled in this area specialists. Most preferably, the phase of the probe must be performed locally in sufficient concentration for the induction of stimulating reactions. The concentration of this peptide in the composition may be equal to about 0.01 μg/ml to approximately 100 μg/ml, approximately 0.1 μg/ml - approximately 50 μg/ml and 0.1 μg/ml - approximately 1 µg/ml stage contacting can be performed on a mammal, cat, dog, cow, horse, pig, or human. The preferred composition for stimulating the production of ECM protein includes SEQ ID NO:8; more preferably, this composition contains SEQ ID NO:8 in a heterogeneous mixture of at least one other tetrapeptides. In the most preferred embodiment, a separate tetrapeptide in this composition could cause sustained production of collagen over a period of at least 48 hours.

Additional option of implementing the present invention relates to a method of activating the healing of skin damaged by natural aging, illness, damage, injury or surgery or other medical procedures. This method may provide an introduction to the Anu animal song where the composition comprises any of the above peptides, alone or in combination. These compositions can be liquid, lotion, cream, paste, ointment, foam or any other pharmaceutically acceptable composition. These compositions may contain pharmaceutically acceptable carriers or adjuvants. These compositions may also contain other biologically active agents, such as antimicrobial agents or growth factors. These compositions can also be used in combination with other therapeutic agents, such as tissue grafts, tissue culture, oxygen or dressings. The concentration of peptide in the composition may be equal to about 0.01 μg/ml to approximately 100 μg/ml, approximately 0.1 μg/ml - approximately 50 μg/ml and 0.1 μg/ml - approximately 1 μg/ml of the Composition may be injected into the wound topically. The animal can usually be animals of any type and preferably is a mammal and more preferably a human, cow, horse, cat, dog, pig, goat or sheep. The preferred composition for applications in wound healing, which stimulates the production of protein ESM, contains SEQ ID NO:8; more preferably the composition comprises SEQ ID NO:8 in a heterogeneous mixture of at least one other tetrapeptides. In most ol doctitle embodiment, a separate tetrapeptide in this composition could cause long-term collagen production for a period of at least 48 hours.

Additional option of implementing the present invention relates to a method for reducing scarring of the skin damaged by normal aging, disease, damage, injury or surgery or other medical procedures. This method may provide an introduction to wound the animal the composition, and this composition contains any of the above peptides, individually or in combination. These compositions can be liquid, lotion, cream, paste, ointment, foam or any other pharmaceutically acceptable composition. These compositions may contain pharmaceutically acceptable carriers or adjuvants. These compositions may also contain other biologically active agents, such as antimicrobial agents or growth factors. These compositions can also be used in combination with other therapeutic agents, such as tissue grafts, tissue culture, oxygen or dressings. The concentration of peptide in the composition may be equal to about 0.01 μg/ml to approximately 100 μg/ml, approximately 0.1 μg/ml - approximately 50 μg/ml and 0.1 μg/ml - approximately 1 μg/ml of the Composition may be injected into the wound topically. The animal can usually be animals of any type and preferably is a mammal, and more pre is respectfully man, cow, horse, cat, dog, pig, goat or sheep. The preferred composition for applications in wound healing, which stimulates the production of protein ESM, contains SEQ ID NO:8; more preferably the composition comprises SEQ ID NO:8 in a heterogeneous mixture of at least one other tetrapeptides. In the most preferred embodiment, a separate tetrapeptide in this composition could cause long-term collagen production for a period of at least 48 hours.

The following variant of implementation of the present invention relates to a method for obtaining the described tetrapeptides in combination. These peptides can be obtained using any method known to a skilled in this area specialists, such as the methods described in Merrifield, R.B., Solid Phase Peptide Synthesis I., J. AM. CHEM. SOC. 85:2149-2154 (1963); Carpino, L.A. et al., [(9-Fluorenylmethyl)Qxy] Carbonyl (Fmoc) Amino Acid Chlorides: Synthesis, Characterization and Application to the Rapid Synthesis of Short Peptides, J. Org. Chem. 37:51:3732-3734; Merrifield, R.B. et al. Instrument for the Automated Synthesis of Peptides, Anal. Chem. 38:1905-1914 (1966) or Kent, S.B.H. et al. High Yield Chemical Synthesis of Biologically Active Peptides on an Automated Peptide Synthesizer of Novel Design, In: PEPTIDES 1984 (Ragnarsson, U., ed.) Almqvist and Wiksell Int., Stockholm (Sweden), pp. 185-188, all of which are included in the present description by reference in full. Preferably, these peptides will be obtained in the device capable of serial Pris the unity of amino acids to a growing peptide chain. However, these peptides can be obtained using standard solid phase methodology.

Observed that the addition of a mixture of free amino acids instead of the homogeneous peptide mixtures during the synthesis of the peptide chains leads to differing inclusion of free amino acids, such as a combination of peptides, resulting in reactions of synthesis. The relative frequency of inclusion of specific amino acids that are included in a mixture of two or more amino acids added during the synthesis, can be adjusted. This correction becomes possible by modifying the relationship of free amino acids made available during the synthesis process, relative to other amino acids in the mixture (called isokinetic mixture).

The following examples are included to illustrate preferred embodiments of the present invention. Specialists with expertise in this area should be clear that the methods described in these examples are ways found by the inventors as the methods that work well in the practice of the present invention, and therefore, can be considered as the preferred methods for practical application. However, qualified in this area specialists should be, in the light of this description of the Oia, it is clear that can be made numerous changes in the specific implementation options are described, and can still be obtained from the same or similar result without deviating from the idea and scope of the present invention.

EXAMPLES

Example 1

Identification of duplicate tetrapeptide sequences in collagen

The relatively high proportion of repetitive sequences tetrapeptide collagen IV is the GxxG motif (where x denotes any amino acid). A number of them are shown in situ as part of a full sequence of collagen IV, shown in figure 1 as SEQ ID NO:45. Collagen IV was investigated first in relation to its role in cooperation with other specialized components of the ECM (see Gregory Schultz et al., 2005). There are eleven sequences with the GxxG motif in collagen IV, which appear more than ten times (GxxG, where xx presents: vp, ek, fp, lp, pp, sp, ep, ip, pk, qp and tp). Among these tetrapeptide sequences of eight of the eleven sequences contain a Proline at position 3, two of the eleven sequences contain R in position 2, one of the eleven sequences contains a Proline at positions 2 and 3 and one of the eleven sequences do not contain Proline. The above sequence is called replacename (REPLIKIN™). "Packing services : THE KIN" is defined as a short sequence in the proteins of the ECM, which occurs many times (i.e. replicated). This sequence may be present in one protein of the ECM (e.g., collagen IV). Preferably, this sequence is present in many proteins of the ECM (e.g., all collagens, elastin, laminin, and so on). The presence of this sequence in multiple ECM proteins increases the likelihood that this fragment is capable of stimulating the synthesis or repair the ECM.

These eleven sequences GxxG found in collagen IV, above, selected in sequence IV collagen person, shown in figure 1. In this figure, all printed in bold sequences are underlined, and the overlapping sequences are underlined twice. All but one of these sequences are found in collagens I, II, III and V. This fact contributes to the ability of the described peptides to stimulate production of all types of collagens, in particular, when these peptides are used in combination. Table 1 shows the occurrence of several tetrapeptide repeats in proteins of the ECM. Bold sequences in table 1 are the sequences found in collagen IV ten or more times.

29
Table 1
The occurrence of tetrapeptides proteins in the ECM
SEQ. ID NOSequenceCollagen ICollagen IICollagen IIICollagen IVCollagen VElastinThe precursor of elastin
19GAAG1057245
20GAKG34355
21GAPG13212569
22GDKG 22493
23GDRG25241
8GEKG3542215
5GEPG111510114
24GERG10111467
2 GFPG48622511
25GIPG22614655
26GKDG14522
27GPG23341
28GLKG21154
GLPG15109421511
30GNPG35321
31GPAG16202036
32GPKG3114129

7GPPG3340404643
33GPQG711975
34GPRG11131047
35GPSG1011515
36GPTG43226
37GPVG93325
38GQPG346127
39GRDG4233
40GRPG33425
3GSPG4621163
41GTPG342112
42GVKG13231
43GVPG131011415
44GYPG11142

As is also apparent from a consideration of the sequence of collagen IV, SEQ ID NO:45, there are also many cases of occurrence of sequences with the motif PxxP. For example, the sequence PGPP found no less than fifteen times, as shown in figure 3. Thus, this sequence is described also called replikins (REPLIKINE™). Preferably, this sequence is present in multiple ECM proteins (e.g., all collagens, elastin, laminin, and so on), because the presence of this series is STI in multiple ECM proteins increases the likelihood that this fragment may be capable of stimulating the synthesis or repair the ECM. These fifteen sequences PGPP found in collagen IV listed above in bold and are underlined in the sequence of collagen, illustrated in figure 3.

Example 2

Identification of the active segments of the shift frame

In addition to the relatively high proportion of tetrapeptide repeated sequences of collagen IV with GxxG motif, identified other tetrapeptide sequences with the shift frame by one amino acid from tetrapeptide sequence or GxxG, or PxxP. These sequences may be repeated or occur only once in the protein ESM and can be located with a shift of one position of amino acids from tetrapeptide sequence or GxxG, or PxxP, as described here. These tetrapeptide sequence known as the active sequence of the shift frame. Such active sequence shift frames, respectively, to contain either G or R, or in the second or in the third position depending on the shift direction of the frame. In addition, it was found that these active sequence of the shift frame can be combined with other tetrapeptide sequences described in this application, formed with the eat bombycina. An example of such CombiLine is N and n.

One example of an active sequence of the shift frame is GAGP or H12 (SEQ ID NO:6). H12 (GAGP) appears as a shift of one residue (or one frame) from the GxxG-tetrapeptide GGAG in collagen III (SEQ ID NO:46), as shown in figure 2. In this figure all active sequence of the shift frame are shown in bold and underlined, and GxxG sequence having one shift frame, underlined twice. In addition, as shown in table 5, this tetrapeptide (GAGP) achieves good results in relation to the production of collagen within 48 hours. Another example is the sequence of PGPR, which is N10 (SEQ ID NO:4), which occurs eleven times in collagens I-IV. Since it appears plural times in individual protein ESM, this tetrapeptide may be additionally considered as replikin (REPLIKINE). Figure 2 (SEQ ID NO:46) shows several cases of this tetrapeptide, each of which meets one of the shift frame from GxxG-tetrapeptide GPRG. This particular active sequence of the shift frame is available in multiple ECM proteins and, therefore, increases the likelihood that this fragment may be capable of stimulating the synthesis or repair the ECM.

Example 3

Identification of repetitive sequences, which stimulate produktionsanlagen

Several sequences identified in examples 1 and 2 were synthesized using the standard method for the synthesis of peptides and analyzed for the stimulation of collagen dermal fibroblasts. These synthesized peptides were amedieval to-end, making these tetrapeptide less sensitive by degradation and increasing their solubility in comparison with the form of the free acid. Dermal fibroblasts were incubated in 96-well tablets at 37°C and 5% CO2within 24 and 48 hours in 150 μl of complete medium for cell culture (Cascade Biologies, Portland, OR; Cat. No. M-106-500), supplemented by a Supplement for growth in low serum (Cascade Biologies, Portland, OR; Cat. No. S-003-10)containing peptides samples at a final concentration of 50 μg/ml In each well were seeded at 10,000 cells. After incubation of each well was extracted samples 100 ál of medium and analyzed for collagen production.

These analyses were performed Tebu-bio Laboratories (France) using a test kit SIRCOL collagen™ (Biocolor Assays, UK) according to the manufacturer's Protocol. Analysis of the SIRCOL collagen™ is a quantitative method with binding dyes designed for the analysis of soluble collagens secreted into the culture medium mammalian cells during in vitro cultivation. Collagen test samples associated with nonnum dye SIRCOL™. Complexes of collagen-dye precipitate from solution and precipitates are collected by centrifugation. The extracted precipitate the collagen-dye dissolved in an alkaline solution prior to measurement of optical density. Measurements in two replications were performed after 24 and 48 hours for two separate samples. The average was calculated from these four measurements for each sample. The optical density of the blind samples (reagents), standards of collagen and samples were measured at 560 nm. The optical density of a blind sample (reagent) subtracted from the optical density for each sample after 24 and 48 hours.

Two separate data set used to obtain the two calibration curves of the standards. First a calibration curve was obtained for the purposes of calculating the amount of collagen in the samples N6 (combination of SEQ ID nos:1-4), H7 H14 (SEQ ID NO:1-8, respectively) and H15 (combination of SEQ ID NO:5-8). The second calibration curve was obtained by calculating the quantity of collagen in the samples H16 (SEQ ID NO:9), H21-23 (SEQ ID NO:10-12, respectively), H25-26 (SEQ ID NO:13 and 14, respectively) or H29-30 (SEQ ID NO:15-16, respectively), H32 (SEQ ID NO:17), H33 (combination of SEQ ID nos:9-12), H34 (combination of SEQ ID nos:11-14), H35 (combination of SEQ ID nos:13-16), H36 (combination of SEQ ID NO:l, 6, 5, 8), H37 (SEQ ID NO:17) and H38 (SEQ ID NO:8) from measurements of optical density and building curve Abs560 nmfrom the respective concentrations of the standards of collagen (in micrograms) every time the implementation of the Yali series analyses. For each data series the same calibration curve was used for samples taken after 24 and 48 hours (table 2A and 2B). Thus, the different curves of the standards received immediately prior to each series of tests.

Table 2A
The calibration curve for analysis of products of collagen peptide H6-H15
Standards of collagen (µg)24 h And560 nmtest48 h And560 nmtest
00,000,00
50,080,10
100,110,15
250,320,35
500,660,65

Table 2B
The calibration curve for analysis of products of collagen peptides H16, H21-23, H25-26 and H29-3
Standards of collagen (µg)And560 nmtest day 1And560 nmtest day 2
00,000,00
50,120,09
100,140,15
250,480,42
500,880,80

Linear regression was performed by building a values AbsNmdepending on the concentrations of the relevant standards collagens using MICROSOFT EXCEL™. The regression resulted in lines described by the formula y=0,013x for both incubation periods specified in table 2A. Because these results were identical for the second series of calibration curves used only the 24-hour period. The formula for the line obtained on the day of analysis 1 and day analysis 2 the second series of samples was y=0,0178x and y=0,0162x respectively. Peptide LL-37 (SEQ ID NO:18) was used as positive control, as was widely reported that he has an effect in the healing of wounds in humans (Heilborn et a. The Cathelicidin Anti-Microbial Peptide LL-37 is Involved in The Re-Epithelialization of Human Skin Wounds and is Lacking in Chronic Ulcer Epithelium, J. Invest. Dermato. 120:379-89 (2003)). The limit of detection of this assay, a specific manufacturer, equal to 2.5 mg.

The total amount of collagen produced in samples containing peptides was calculated from the mean values of optical density obtained after 24 hours (table 3A) and 48 hours (table 3B), using the linear equation generated from the standard curve. The total amount of collagen produced in samples containing peptides H16 (SEQ ID NO:9), H21-23 (SEQ ID NO:10-12, respectively), H25-26 (SEQ ID NO:13 and 14, respectively) or H29-30 (SEQ ID NO:15-16, respectively), H32 (SEQ ID NO:17), H33 (combination of SEQ ID nos:9-12), H34 (combination of SEQ ID nos:11-14), H35 (combination of SEQ ID nos:13-16), H36 (combination SEQ ID NO:1, 6, 5, 8), H37 (SEQ ID NO:17) and H38 (SEQ ID NO:8), was calculated from the values of optical density, measured after 24 hours (table 4A) and 48 hours (table 4B), using the linear equation obtained from the standard curve. These values were compared with values of peptide LL37 (SEQ ID NO:18)peptide, which is known to promote collagen. In each table, the samples marked with an asterisk (*)may not be meaningful, because the limit of detection of this assay is 2.5 mg.

Table 3A
Optical density measurement and quantitative measurement of collagen in the test samples H6-H15 24 hours
SEQ ID NOPeptidesAndNmAverageAverage minus blind experienceCollagen (µg)
18LL37is 0.1020,1360,120,043,0
-H60,0840,1400,110,032,5
1H70,0980,0630,080,000,0*
2H80,1220,0780,100,021,5*
3H90,1470,130,053,5
4H100,1030,1460,120,043,4
5H116,110has 0.1680,140,064,5
6H120,0630,1010,080,000,2*
7H130,1140,0930,100,021,8*
8H140,1150,1220,120,043,0
-H150,1320,0930,112,5
-Blind experience0,0740,0760,080,000,0

Table 3B
Optical density measurement and quantitative measurement of collagen in the test samples H6-H15 after 48 hours
SEQID NOPeptidesAndNmAverageAverage minus blind experienceCollagen (µg)
18LL370,2620,1130,190,075,2
-H60,0860,1890,140,021,3*
1H70,1920,189/td> 0,190,07of 5.4
2H80,1370,1260,130,010,9*
3H90,1170,0610,090,000,0*
4H100,1360,0850,110,000,0*
5H110,1130,1810,150,032,1*
6H120,1060,2310,170,053,7
7H130,1000,1450,12 0,000,2*
8H140,1320,1760,150,032,6
-H150,1770,1740,180,064,3
-Blind experience0,1200,1150,120,000,0

2,1*
Table 4A
Optical density measurement and quantitative measurement of collagen in the test samples W16, n-23, N-26 or N-38 in 24 hours
SEQ ID NOPeptidesAndNmAverageAverage minus blind experienceCollagen (µg)
9H16of 0.133 0,1370,140,063,1
10H210,1290,1190,120,042,5
11H220,1920,0850,140,063,3
12H230,0900,0730,080,000,1*
13H250,1290,0760,000,021,3*
14H260,1140,1490,130,052,9
15H290,1110,0630,09 0,010,4*
16H300,0990,0920,100,020,9*
17H32 (crystals and cellular toxicity)0,0870,0550,07-0,01-0,5*
-H330,0860,1250,110,031,4*
-H340,1170,1200,120,042,2*
-H350,1030,0900,100,020,9*
-H360,1050,1280,120,04
17H370,0990,1000,100,021,1*
8H380,103strength of 0.1590,130,052,9
-Blind experience0,0720,0860,080,000,0

Table 4B
Optical density measurement and quantitative measurement of collagen in the test samples W16, n-23, N-26 or N-38 through 48 hours
SEQ ID NOPeptidesAndNmAverageAverage minus blind experienceBlind experience
9H160,0650,064 0,060,000,3*
10H210,0890,1260,110,052,9
11H22is 0.1020,0870,090,032,1*
12H230,0930,0820,090,031,7*
13H250,0590,0840,070,010,7*
14H26of 0.0810,1530,120,063,5
15H290,0860,0940,090,03 1,9*
16H300,0830,1010,090,032,0*
17H32 (crystals and cellular toxicity)0,0880,0720,080,021,2*
-H330,0960,0920,090,032,1*
-H340,0760,1550,120,063,4
-H350,1200,0740,100,042,3*
-H360,1540,0820,120,063,6
17H370,0780,1140,100,042,2*
8H380,1230,0890,110,052,8
-Blind experience0,1060,01060,060,000,0

Because sample sizes were equal to 100 ál, the concentration of collagen produced in each sample, in micrograms per milliliter was determined by multiplying detektirovanie the amount of collagen ten. The results of all tested samples are summarized in table 5.

Table 5
Collagen synthesis induced by peptides
Produced collagen (µg/ml)
SEQ ID NONameThe primary sequence[Peptide] (µg/ml)24 hours48 h
1H07PEGP50054
2H08GFPG50159
3H09GSPG50350
4H10PGPR50340
-H06H7, H8, H9, H10 (SEQ ID NO:1, 2, 3, 4)502513
5H11GEPG504521
6 H12GAGP50237
7H13GPPG50182
8H14GEKG503026
8H38GEKG0,32928
-H15H11, H12, H13, H14 (SEQ ID NO:5, 6, 7, 8)502543
9H16PEKP50313
10H21PKGP502529
11H22PGQP 503321
12H23PGTP50117
13H25PMGP50137
14H26PGPP502935
15H29PQGP50419
16H30PGNP50920
17H32KTTKS (peptide SEDERMA™)50na12
17H37KTTKS (peptide SEDERMA™)0,322
-H33H16, H21, H22, H23 (SEQ ID NO:9, 10, 11, 12)501421
-H34H22, H23, H25, H26 (SEQ ID NO: 11, 12, 13, 14)502234
-H35H25, H26, H29, H30 (SEQ ID NO: 13, 14, 15, 16)50923
-H36H7, H12, H11, H14 (SEQ ID NO: 1, 6, 5, 8)502136
18LL37LLGDFFRKSKEKIGKEFKRIVQRIDFLRNLVPRTES503052

All tested tetrapeptide stimulated the production of soluble collagen. Of the tested sequences tetrapeptide GxxG with glutamic acid in position 2 best stimulate collagen as at 24-hour and 48-hour time points. These what posledovatelnostei are H11 (GEPG; SEQ ID NO:5), H14 (GEKG; SEQ ID NO:8) and H38 (GEKG; SEQ ID NO:8). These peptides were first subjected to screening using a concentration of peptides 50 µg/ml For finding concentration effective to stimulate collagen production, H14 (SEQ ID NO:8) was also tested at 0.3 µg/ml, as N. As shown in table 5, H38-induced stimulation of collagen was reduced at a lower concentration, which indicates that the maximum stimulatory concentration of SEQ ID NO:8 is equal to or below 0.3 ág/ml

To test the effectiveness of SEQ ID NO:8 (H14 and H38) compared with peptide LL37 (SEQ ID NO:18), which is known that it stimulates the production of collagen. On the basis of the amount of collagen produced by fibroblasts in response to LL37, 25 µg/ml was considered significant amount of collagen released due to contact with tetrapeptides. SEQ ID NO:8 induced approximately the same amount of collagen, and LL37 (SEQ ID NO:18), after 24 hours. It is important that the collagen produced by contact with SEQ ID NO:8, essentially persisted for at least 48 hours. SEQ ID NO:8 was also compared with the basic peptide for skin care, which is known that it stimulates the production of collagen, KTTKS (SEQ ID NO:17) (Katayama et al., J. Biol. Chem. 288:9941-9944 (1983)). KTTKS is an ingredient in the product MATRIXYL™ (SEDERMA SAS, France). SEQ ID NO:8 more stimulated collagen production than this KTTKS peptide (EQ ID NO:17) (table 5) after 24 and 48 hours.

Example 4

Identification of combinations of peptides, which synergistically enhance the stimulation of collagen - COMBILINE

Heterogeneous population of active tetrapeptide can stimulate the production of collagen at a higher level than homogeneous samples tetrapeptides. The components of this heterogeneous composition called kabikinase (COMBIKINE™). CombiLine are a group of replikins combined to obtain a higher or broader actions on one or more types of target cells. Peptides H11 (SEQ ID NO:5), H12 (SEQ ID NO:6), H13 (SEQ ID NO:7) and H14 (SEQ ID NO:8) were combined to a final concentration of 50 μg/ml and analyzed using the same Protocol that was used to separate peptides. As expected, the result in the time point of 24 hours was equal to the average of the individual estimates induction. However, this combination of peptides for 48 hours induced collagen up to 43 µg/ml Unexpectedly this amount was recognized above average (21 μg/ml) of these four separate peptides (see table 5). Thus, specific combinations of peptides can stimulate collagen production to a much greater extent than these individual peptides at the same concentration. In addition, tetrapeptide from various sources ECM, such as collagen, laminin and elastin, can produce uvelichenie the induction of various ECM proteins (see tables 1 and 5).

Example 5

Cheap obtaining BOMBYCINA for increased stimulation of collagen production

The high cost of peptide synthesis limits the possibility of obtaining a heterogeneous compositions of biologically active peptides. This invention significantly reduces this limitation. As now described sequence have a common characteristic (for example, glycine or Proline at both ends), a range of tetrapeptides, varying in positions 2 and 3 can be synthesized in a single cycle of receipt. These synthetic peptides can be obtained by any method known in this field (Benoiton, N., Chemistry of Peptide Synthesis, CRC (2005)). While obtaining these peptides instead of homogeneous samples add a mixture of amino acids. Chemical method of determining the correct ratios of the concentrations of amino acids added to the mixed positions, was described previously (Greenbaum et al. Molecular and Cellular Proteomics 1:60-68, 2002; Krstenansky et al. Letters in Drug Design and Discovery 1:6-13, 2004; both of these references are incorporated herein in their entirety). Using this methodology can be obtained from a library of heterogeneous peptides with almost the same cost, which is necessary for the synthesis of a single peptide.

The application of this manufacturing process makes possible the economical production of biologically active kabikenov. This hundred is ulitsa possible due to the unique composition described tetrapeptides. A mixture of tetrapeptides are more appropriate for inclusion in the preparations for local use than longer peptides. Due to their length tetrapeptide have practical and chemical advantages over a longer peptides comprising the following: an easier on and dissolution of drugs, the higher the permeability of the skin and pores and higher yields more easy ways of making combinations of peptides. Although not required, the ideal drugs tetrapeptides, individually or in combination, are the drugs that support significant production of collagen within 24 hours and up to 48 hours. More preferably, these drugs can induce the synthesis of ECM during the whole period of 48 hours so that more collagen is produced in 48 hours, within 24 hours. Although the scope of this invention tetrapeptide that stimulate the production of ECM proteins within 24 hours, but find reduced products within 48 hours, are less preferred. In this regard, table 6 shows the results described in the present peptides. Preferred peptides are shown in bold.

Table 6
OPI is these peptides
SEQ ID NOPeptidesReleased collagen (μg/ml) 24 hReleased collagen (μg/ml) 48 hA significant release of collagen after 24 h and 48 hThe enhanced release of collagen after 48 h compared to 24 hThe lower release of collagen after 48 h compared to 24 h
18LL373052
-H62513
1H7054
2H8159
3 H9350
4H10340
5H114521
6H12237
7H13182
8H143026
8H382928
-H152543
9H16313
10H212529
11H223321
12H23117
13H25137
14H262935
15H29419
16H30920
17H32 (crystals and cellular toxicity)NA12
17H371122
-H331421
-H3434
-H35923
-H362136

Example 6

Stimulators of collagen also serve as multifactor molecules, amplifying the closure of wounds of epithelial cells

The collagens are key components of all phases of wound healing. Stimulation of collagen production reflects the fact that the damage occurs in the collagen network (for example, under the action of enzymes or due to physical destruction). Indeed, the General collapse of the collagen network actually causes healing. Thus, collagen stimulator may also serve as multifactorial molecules that organize specific remodeling of the matrix and enhancing wound healing.

Experiments on wound healing was performed on monolayers of epithelial cells of human skin (CRL-2592), seeded on 2-hole Cup. Cells were starved in relation to serum for 24 hours before experimentation. On confluent monolayers CRL-2592 inflicted the injuries a P200 pipette tip (200-ál). These wounds were washed and documented in the form of a figure before treatment with peptides. The peptides were added to a final concentration of 20-40 µg/ml of Cells was kept in a thermostat at 37°C, 5% CO2and humidity 92%, except for a short time acquire images at room temperature. Closure of wounds was observed after 6-hour and 10-hour time points. SFR-treated wounds was used as a negative control for comparison purposes.

Table 7
Effect of peptides on closing wounds epithelium of human skin in vitro
0 h6 h10 h
ConnectionSize W*Size W% closureSize W% closure
SR-1362919,40% 2141,70%
SFR-2524219,20%3042,30%
SEQ ID NO:14251252%2,7589%
SEQ ID NO:5483919%3037,50%
*Size W: the size of the wound (relative)

Wound closure monolayer in vitro is the result of cell migration, which is important in many biological processes such as embryogenesis, angiogenesis, inflammatory response and repair (healing) wounds. It is believed that these processes are regulated by interaction with other cells, cytokines and ECM proteins. As shown in table 7, SEQ ID NO:14 significantly induces the closure of wounds in comparison with the actions of one SFR. This activity is a peptide-specific, but also specific regarding the type of cells as SEQ ID NO:14, does not induce closure of wounds in the monolayer of fibroblasts of human skin (data is not shown). SEQ ID NO:5 is also an inducer of collagen, but does not increase closing wounds or migration of epithelial cells to any great extent in comparison with the actions of one SFR. The fact that SEQ ID NO:14 induced migration of cells or closure of wounds of specific in relation to epithelial skin cells way (i.e. without recruitment of fibroblasts), may be an additional advantage of using this peptide for skin care, as it is believed that the recruitment of a large number of active fibroblasts to the site of damage leads to excessive deposition and shrinkage of the tissue, leading to scarring.

All of the described and claimed here compositions or methods may be prepared or executed without undue experimentation in light of this description. Although the compositions and methods of the present invention have been described in the preferred embodiments, a person skilled in this field will be obvious that can be used variation in respect of these compositions and/or methods and in stages or sequence of stages described here ways without deviating from the concepts, ideas and scope of the present invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically similar, can be replaced described here Agay what you're obtaining the same or similar results. It is assumed that all such similar substitutes and modifications apparent to the skilled in this field specialists, both within ideas, scope and concept of the present invention.

1. Tetrapeptide characterized by the formula SEQ ID NO: 1, 3, 4, 5, 8, 9, 10, 11, 12, 13, 15 or 16, where the specified tetrapeptide stimulates the production of collagen by fibroblasts.

2. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 5.

3. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 3.

4. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 8.

5. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 12 or SEQ ID NO: 16.

6. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 1.

7. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 9.

8. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 4.

9. Tetrapeptide according to claim 1, where tetrapeptide characterized by the formula SEQ ID NO: 11.

10. Tetrapeptide according to any one of claims 1 to 9, where tetrapeptide amitirova on carboxykinase.

11. Composition containing tetrapeptide according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier, where the specified composition stimulates the production of collagen by fibroblasts.

12. The composition according to claim 11, where tetrapeptide is present in effective concentrations in d is apatone from about 0.01 μg/ml to approximately 100 μg/ml.

13. The composition according to claim 11, where tetrapeptide is present in an effective concentration in the range from about 0.1 μg/ml to about 1 μg/ml.

14. The composition according to claim 11, where the composition is in the form of an aerosol, emulsion, liquid, lotion, cream, paste, ointment or foam.

15. A composition comprising a mixture of tetrapeptides SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 and a pharmaceutically acceptable carrier, where the specified composition stimulates the production of collagen by fibroblasts.

16. The composition according to claim 11, in which the specified tetrapeptide characterized by the formula SEQ ID NO: 5.

17. The composition according to claim 11, in which the specified tetrapeptide characterized by the formula SEQ ID NO: 8.

18. Composition according to any one of § § 11-17 for use in the tool for skin care.

19. Composition according to any one of § § 11-17 for use in the treatment of wounds on the skin.

20. Way to stimulate the production of collagen by fibroblast, providing the impact on the fibroblast-tetrapeptide characterized by any of the formula SEQ ID NO: 1-16, from induction through this production of collagen by fibroblast.

21. The method according to claim 20, where tetrapeptide characterized by the formula SEQ ID NO: 5.

22. The method according to claim 20, where tetrapeptide characterized by the formula SEQ ID NO: 7.

23. The method according to claim 20, where tetrapeptide characterized by the formula SEQ ID NO: 6.

24. The method according to claim 20, where tetrapeptide across the tsya formula SEQ ID NO: 8.

25. The method according to claim 20, where tetrapeptide characterized by the formula SEQ ID NO: 14.



 

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