PL237242B1 - Composition containing peptide IM, the set and application for stimulation of regeneration of a complex tissue or treatment of wounds - Google Patents

Description

TECHNICAL FIELD

The invention relates to the RDKVYR peptide or a pharmaceutically acceptable salt thereof for use as an agent for promoting the regeneration of composite tissue or the healing of wounds caused by mechanical and / or chemical and / or thermal and / or radiation damage and / or surgery and / or other pathological conditions.

STATE OF THE ART

Regeneration is the process of living organisms recreating lost or damaged parts of the body, organs, tissues or cells. The ability to regenerate is due to the fact that the cells of an organism contain almost the same genetic material, so they have the potential to give rise to any other cell / tissue / organ. In fact, regeneration is only possible in lower organisms. For example, in sponges, the entire body can be restored from a fragment made up of just a few cells. The higher the organism is in the hierarchy of evolution, the worse it has the ability to regenerate, e.g. lizards have the ability to regenerate the tail, which is no longer observed in mammals. Two types of regeneration are most often distinguished. The first type is morphallaxis, which is the reconstruction of the missing body part by changing the specification of the remaining tissue. The second type is epimorphosis, which is the restoration of a missing part of the body by cell proliferation and the growth of new tissue. Epimorphosis can potentially be potentiated by pharmacological stimulation.

When the regenerative capacity of the body is low, connective tissue (scar) is formed at the site of damage or loss of tissue. This tissue fills in the damage so that the damaged tissue or organ maintains its integrity. However, the process of creating connective tissue does not restore the full functionality of the tissue or organ. The formation of scar tissue is a process that is usually irreversible and permanently impairing the functions of the damaged organ. Understanding the basic mechanisms accompanying the regeneration / repair of tissues may enable the use of the natural possibilities of their regeneration and prevent the formation of scar tissue.

The ability to restore the functions and architecture of damaged organs and tissues is particularly important in the case of chronic skin wounds, as they are difficult to treat. Currently, there is a great need to develop new methods of treating skin injuries / defects requiring medical intervention, such as large-area thermal, chemical or radiation burns, chronic wounds caused by civilization diseases such as diabetes or atherosclerosis. Such a method can be the stimulation of regeneration and scar-free wound healing.

The skin is the largest organ of the human body, which plays an extremely important role as protection against physical, chemical and biological factors. It is also a mechanical shield in the body's defense against pathogenic microorganisms. The skin's ability to repair and regenerate is essential to maintaining proper body homeostasis. The natural response to skin trauma is primarily the activation of keratinocytes and fibroblasts and the initiation of the wound healing process. Fibroblasts have the ability to fill large defects in the skin more quickly, usually present at the edge of the wound. Scars usually arise at the expense of the correct stage of skin tissue regeneration, which is a complex process of skin tissue remodeling and growth. Pathological wound healing (chronic wounds, hypertrophic / keloid scars) is a serious medical, economic and social problem that affects millions of people worldwide.

The human immune system, in addition to providing resistance to various types of pathogens and other external and internal threats, can also affect the processes of reconstruction, repair and regeneration of tissues. In the process of regeneration and wound healing, both innate immunity (epidermal barrier, antibacterial peptides, NK cells) and acquired immunity (T and B lymphocytes) and other mechanisms linking both types of immunity (dendritic cells, macrophages) play an important role. It is well known that chronic inflammation (e.g. chronic bacterial infection) mediated, inter alia, by by T lymphocytes it leads to inhibition of regeneration and wound healing processes. On the other hand, a short (up to 3-5 days) physiological stimulation of the immune system in the first days of wound healing can stimulate wound healing by e.g. accelerated migration and proliferation of skin cells. Therefore, it seems that modulating the immune response may be an effective strategy for stimulating the healing of chronic wounds.

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One class of compounds with the potential to stimulate the immune system are peptides, which can also support regenerative processes. Peptides stimulate fibroblasts, stimulating them to proliferate and to produce elastin and collagen. The thrombin peptide, TP508, also known as Chrysalin (OrthoLogic, Tempe, Arizona) is a fragment of the receptor binding domain of the native thrombin molecule. This domain binds to a specific class of receptors found on fibroblasts. The TP508 peptide, which is a fragment of the thrombin domain, accelerates wound healing. Another example is the GHK tripeptide. It occurs naturally in the human body in the extracellular matrix, but its concentration decreases with age. In the human body, it participates in the activation of processes related to skin reconstruction by modulating the activity of metalloproteinases I and II from the intercellular matrix (degradation of collagen and elastin). It also influences the activation of fibroblasts leading to the synthesis of collagen and elastin. Another example of a peptide with pro-regenerative properties is tylothin (KCVRQNNKRVCK), isolated from the skin of the salamander Tylototriton verrucosus, peptide AH90, (ATAWDFGPHGLLPIRPIRIRPLCG) isolated from the skin of the frog Odorran with grahami or the peptide Tiger-NHK 17 (cPRKP2P) [WCCHP2P]. Peptides containing the RGD sequence in their composition have particularly strong pro-regenerative properties. These peptides bind to integrins or growth factor receptors, thus activating the cascade of cellular signals supporting regenerative processes.

Unfortunately, peptides are a class of compounds that are characterized by low stability and durability. The shelf life of a pharmaceutical preparation is defined as the period from its manufacture until it does not comply with the requirements of the Pharmacopoeia, standard or when the concentration of the active substance is reduced by more than 10%. In addition, the preparation must have no change in appearance, taste, bioavailability or toxicity. Pharmaceutical / drug product stability studies are performed at specific time points and conditions (temperature, humidity, pH, etc.) to demonstrate that it remains effective and safe for patients. The obtained results are the basis for determining the durability of the product and its expiry date.

The stability of the active substance is tested in the following form: solid, e.g. with fillers (tablets), emulsions (creams), or dissolved, i.e. in water or buffering systems. Extremely valuable information on the stability of the API (active substance) is provided by tests in plasma and / or whole blood. These studies allow to assess the susceptibility of the compound to enzymatic proteolysis and to select the appropriate dose of the drug to obtain the desired therapeutic effect. Compounds that are characterized by a short half-life usually require further structural modifications (except for pro-drugs) to improve the half-life parameters. Occasionally, compounds have a short plasma half-life due to their binding to plasma proteins, i.e. albumin. Albumin belongs to the group of soluble proteins with MW. 66-69 kDa. In plasma, they account for about 55-65% of all proteins. Their basic physiological function is to bind water, thanks to which the appropriate colloidosmotic (oncotic) pressure of the plasma and its correct volume are maintained. These proteins are also the main transporters of free fatty acids, hormones, vitamins, heavy metals and some drugs (e.g. acetylsalicylic acid). Most drugs bind to proteins reversibly by hydrogen bonding, hydrophobic interactions or van der Waals forces, creating the so-called depot, from which drug molecules gradually release to replace those that have already been eliminated. Currently, more and more attention is paid to albumin as exogenous drug carriers due to the fact that they are not toxic or immunogenic, and additionally improve the pharmacokinetic profile (t1 / 2 of albumin is about 15-29 days) and the stability of drugs that are rapidly degraded in the plasma . Additionally, these proteins are stable in the pH range 4-9, can be heated to 60 ° C for about 10 hours, and are resistant to denaturation by most denaturing reagents, which may be useful for technological reasons in the manufacture of drugs.

There are a number of models for assessing the pro-regenerative activity of chemical compounds in animal models. One of the models that allows for the study of complex tissue regeneration is the experiment of auricle injury in mice, which consists in cutting holes 2 mm in diameter in the center of the auricle of mice, which was a widely used procedure for permanent labeling of laboratory animals. Normal mice heal allows a certain reduction in the surface of the opening, but it never fully overgrows. One exception is the MRL / MpJ mouse, which closes such openings 30 days after injury, restoring the architecture of tissue, including not only skin, but also blood vessels, muscles, cartilage and peripheral nerves. Moreover, in this strain, a stronger regenerative response was observed in others

PL 237 242 B1 in organs, incl. in the heart, the stumps of the amputated fingers, the retina and the cornea, the spinal cord, the tendons and the cartilage of the joints. The regenerative response in the auricle can be considered an indicator of the body's regenerative capacity. This process can be triggered by chemicals in laboratory mice that do not do so spontaneously.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a peptide having the sequence RDKVYR (IM) in the process of complex tissue regeneration and wound healing in mammals. The method of effective wound healing presented in the application is based on the subcutaneous administration of the peptide IM to a mammal.

Both an in vitro cell model as well as an animal model were used in the research of the present invention. The cell model mainly uses human keratinocytes and fibroblasts - cells crucial for wound healing and skin regeneration. The presence of these cells, and especially their proliferation (divisions), is essential for the proper wound healing process. In the animal model, the analysis of tissue loss regeneration in the turbinate of mice was used. This model is widely accepted in the research of new compounds - potential drugs with a pro-regenerative effect. Due to the similar physiology and biochemistry of wound healing in mice and humans, the animal model can be the basis for assessing the effectiveness of new compounds. In addition, animal models provide greater reproducibility of results, control of conditions and standardization of experiments compared to studies involving humans.

The IM peptide is a hydrophilic hexapeptide with the sequence RDKVYR and is fragment 32-37 of the hormone thymopeotin. Thymopoietin is one of the hormones produced by the thymus gland. Its task is to stimulate the formation, differentiation and maturation of T lymphocytes. It controls reactions related to the possibility of transplant rejection and defense mechanisms against neoplastic cells. However, the most important function of this hormone is to enhance lymphopoiesis. This means that the body produces more T cells, which in turn are responsible for the body's immune response. IM peptide is known and sold in Russia and other Eastern European countries as a drug that detoxifies the liver, inactivates free radicals and hydrogen peroxides. It is used in supportive therapies in the treatment of neoplastic diseases, has liver detoxifying properties, is used in the treatment of hepatitis B and C, HIV, AIDS, and human papillomavirus. It is also used in the treatment of psoriasis, brucellosis, rheumatoid arthritis, diphtheria, and bacterial endocarditis. It has been proven that the administration of the IM peptide to patients suffering from ulcerative colitis and inflammation of the digestive system results in a significant reduction of undesirable symptoms, i.e. reduction of ulcers.

Unfortunately, the mechanism of action of the IM peptide is not known so far. The IM peptide alone has been shown to be less effective than lansoprazole, a popular gastric ulcer treatment. However, taking into account the therapeutic efficacy with the simultaneous use of low concentrations and the absence of side effects, unlike lansopral, IM peptide as a drug has greater prospects in the treatment of gastrointestinal diseases. The IM peptide is known as an immunomodulating agent.

The object of the present invention is to show another use of the IM peptide in the multi-tissue regeneration process. Unexpectedly, the IM compound accelerates the regenerative processes.

The invention relates to the RDKVYR peptide or a pharmaceutically acceptable salt thereof for use as an agent for promoting the regeneration of composite tissue or the healing of wounds caused by mechanical and / or chemical and / or thermal and / or radiation damage and / or surgery and / or other pathological conditions.

The RDKVYR peptide is in the form of an injection where a dose concentration of 0.1-50 pg / ml is used.

Terms used in this specification and claims have the following meanings:

The term "wound" includes any type of injury to living tissue induced by impact or pressure and other mechanical factors, burn, frostbite, radiation, chemical agents, ischemia, and other biological agents. In particular, the term "wound" includes damage to the skin but also to other tissues and organs.

The term "wound healing" refers to a post-injury repair process that includes the cleansing of the wound area of foreign matter and dead tissue, and cell proliferation to restore tissue continuity, with or without scar formation.

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The term "regeneration" means the process of restoring lost tissues, structures and appendages, either spontaneously or under the action of pharmacological stimulation.

The term "chronic wound" or "non-healing wound" refers to wounds that show no apparent healing time beyond normal healing; typically wounds that do not heal within 3 months are termed chronic. Chronic wounds or non-healing wounds are often associated with ischemia or ulceration in diabetic patients, but are not limited to this group.

Where a compound is administered in the early phase of wound healing, it is understood that the term "early phase" includes the immune response immediately following the wound.

The terms used above and in the specification and claims have the following meanings:

List of shortcuts:

THEM - a peptide with the sequence RDKVYR DMF - dimethylformamide DCM - dichloromethane MeOH - methanol KI - potassium iodide DIPEA - N, N-diisopropylethylamine Fmoc - 9-fluorenylmethoxycarbonyl group Boc - a tert-butyloxycarbonyl group Oxyma pure - ethyl (hydroxyimino) cyanoacetate DIC - N, N'-Diisopropylcarbodiimide Et2O - diethyl ether Pbf - 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group OtBu - tert-butyl ester tBu - tert-butyl group TIPSI - triisopropylsilane TFA - trifloroacetic acid API - active pharmaceutical substances 46BR.1N - immortalized line of human dermal fibroblasts HaCaT - immortalized line of human keratinocytes FBS - Fetal Bovine Serum PBS - Phospahte-Buffered saline Saline) PBMC - peripheral blood mononuclear cells fMLP - N-formyl-methionyl-leucyl phenylalanine P / S - penicillin / streptomycin ASA - acetylsalicylic acid LPS - lipopolysaccharide PHA - phytohaemagglutinin WHIP - Basophil Activation Test ASC - adipose tissue stem cells (ASCs) (Adipose Derived Stem Cells) Balb / C - a commercially available inbred mouse strain C57BL6J - a commercially available inbred mouse strain

Description of the figures:

Figure 1 - Shows studies of IM compound stability in human plasma (top) and water (bottom). The stability was tested during 24 hours. The presence of the IM compound was monitored using an HPLC technique with a PDA detector.

Fig. 2 - Shows the mass spectrum recorded for the last milliliter of buffer (last wash top) for the IM peptide. The spectrum indicates that the column has been properly washed from excess peptide applied as indicated by no IM mass. Mass spectrum recorded for the elution fraction (bottom) for the IM peptide. The spectrum shows the m / z 836.887 peak, corresponding to the apparent proton ion from this peptide.

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Figure 3 - Shows the effect of IM on the proliferation of 46BR.1N fibroblasts (top) and HaCaT keratinocytes (bottom). The graphs show the results of 4 cycles of experiments for HaCaT (n = 16) and 3 cycles of experiments for 46BR.1N (n = 12). * - statistically significant differences, Mann-Whitney U test (p <0.05).

Fig. 4 - is a graph (top) showing the effect of IM at 0.01 and 0.1 gg / ml on migration of HaCaT keratinocytes and 46BR.1N fibroblasts after 24 h of incubation with compound and pictures of the scratch surface (bottom) in control and after IM stimulation of 46BR.1N fibroblasts from a single experiment. * - statistically significant differences (Mann-Whitney U-test p <0.05).

Figure 5. - Shows immunogenicity studies for IM compound. The graph shows the expression levels of surface activation markers (CD25, CD69, CD71, HLA-DR) on cytotoxic T lymphocytes (CTL). The following experimental conditions were used: control - cells without stimulation, growth medium only; LPS / PHA - positive control, cells stimulated with lipopolysaccharide and phytohaemagglutinin; IM 0.1 - cells stimulated with IM at a concentration of 0.1 mg / ml; IM 1.0 - cells stimulated with IM at a concentration of 1.0 mg / ml. The results are presented as the median (min-max).

Figure 6. - Shows the immunogenicity studies for IM compound. The chart shows the expression levels of surface activation markers (CD25, CD69, CD71, HLA-DR) on T helper lymphocytes (Th). The following experimental conditions were used: control - cells without stimulation, growth medium only; LPS / PHA - positive control, cells stimulated with lipopolysaccharide and phytohaemagglutinin; IM 0.1 - cells stimulated with IM at a concentration of 0.1 mg / ml; IM 1.0 - cells stimulated with IM at a concentration of 1.0 mg / ml. The results are presented as the median (min-max).

Figure 7. - Shows the immunogenicity studies for IM compound. The chart shows the expression levels of surface activation markers (CD25, CD69, CD71, HLA-DR) on cytotoxic cells - Natural Killers (NK). The following experimental conditions were used: control - cells without stimulation, culture medium alone; LPS / PHA - positive control, cells stimulated with lipopolysaccharide and phytohaemagglutinin; IM 0.1 - cells stimulated with IM at a concentration of 0.1 mg / ml; IM 1.0 cells stimulated with IM at 1.0 mg / ml. The results are presented as the median (min-max).

Figure 8. - It shows the result of the basophil activation assay in terms of% stimulated cells; negative control (neg), positive control (fMLP - bacterial derived tripeptide), additional control (ASA acetylsalicylic acid), IM peptide (0.1 gg / mL).

Fig. 9 - shows the regulatory network with the highest consistency score (29.69) for ASCs cells obtained from patient PS9 (top), PS1 (center) and PS8 (bottom) stimulated with IM peptide (0.1 gg / ml ). The numerical values below the individual molecules represent the log2 value of expression change and the p-value (test probability), respectively. Positive and negative values indicate an increase or decrease in expression, respectively. In the bottom panel, an arrow pointing up indicates that the phenomenon is activated.

Figure 10 - Shows the effect of IM on the expression of pluripotency and cell cycle regulation genes (CDKN1A MYC, KIT, POU5F1, TGFB3, TP53) in human ASC cells isolated from three different patients. The reference genes are AKTB and TBP. Patients respond to relationships individually. There are no observable correlations and trends in changes in expression under the influence of the tested compounds.

Figure 11 - Shows the effect of IM on the expression of the pluripotency and cell cycle regulation genes (CDKN1A, MYC, KIT, POU5F1, TGFB3, TP53) in human primary keratinocytes isolated from three different patients. The reference genes are AKTB and TBP. Patients respond to relationships individually. There are no observable correlations and trends in changes in expression under the influence of the tested compounds.

Fig. 12 - Shows a representative photo of the C57BL6 / J mouse study group receiving IM peptide (right column) compared to the representative photos of the solvent alone (saline, NaCl) control group.

Figure 13 - Shows the effect of IM peptide on multi-tissue regeneration in C57BL6 / J mice. The mean surface area of the holes in the auricle and standard deviations (SD) are given. Statistically significant differences (t test * - p <0.05; ** - 001 <p <0.01).

Fig. 14 - Shows a representative photo for the study group of Balb / C mice receiving IM peptide (right column) compared to the representative photos for the solvent alone control group (saline, NaCl).

Figure 15 - Shows the effect of IM peptide on multi-tissue regeneration in Balb / C mice. The mean surface area of the holes in the auricle and standard deviations (SD) are given. Statistically significant differences (ttest * - p <0.05; ** - 0.001 <p <0.01).

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Fig. 16 - Shows histopathological images of the auricle in mice 42 days after the start of the IM dosing procedure (5 µg / g body weight) (panel A) versus vehicle only treated mice (panel B) (saline buffered saline). Mice receiving IM show greater stimulation of the epidermal layers (arrows - panel A2), while in controls the epidermis is in a more differentiated phase (arrows - panel B2). Images from panels A1 and B1 show only those tissue fragments that have been regenerated. Images from panels A3 and B3 show the entire tissue obtained in the preparation.

The invention is illustrated by the following non-limiting examples: CHEMICAL SYNTHESIS

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Chemical synthesis of the IM peptide

Synthesis of the IM peptide with the RDKVYR sequence was performed on a solid support according to the methodology of Fmoc chemistry using a Liberty Blue ™ microwave synthesizer (CEM Corporation). The synthesis was carried out using Cl-TCP (Cl) ProTide resin (CEM Corporation) with a degree of embedding of 0.4 mmol / g.

Reagents used for the synthesis:

• DMF dried over A3 and A4 molecular sieves • 0.2 M solutions of N “-Fmoc-derivative amino acids in DMF:

• Fmoc-Arg (Pbf) -OH;

• Fmoc-Asp (OtBu) -OH;

• Fmoc-Lys (Boc) -OH;

• Fmoc-Val-OH;

• Fmoc-Tyr (tBu) -OH;

• 20% solution of piperidine in DMF;

• 1 M DIPEA + 0.125 M KI in DMF;

• 1 M Oxyma pure + 0.1 M DIPEA in DMF;

• 0.5 M DIC in DMF.

The first N-protected amino acid (Fmoc-Arg (Pbf) -OH) was attached to the resin using a 0.2M solution of N-Fmoc-amino acid in 1M DIPEA + 0.125M KI in DMF under microwave irradiation at 75 W for 60 seconds and at a temperature of 80 ° C, then 20 W for 540 seconds at 90 ° C. A cycle of washes with DMF was performed successively. The Fmoc amino group protection was then removed using a 20% solution of piperidine in DMF by subjecting to microwave irradiation of 125 W for 25 seconds at 70 ° C and 30 W for 65 seconds at 90 ° C. The next 5 amino acid residues were attached at 170 W for 15 seconds at 75 ° C and 30 W for 110 seconds at 89 ° C, each time previously removing the amino shield as above. 0.5M DIC in DMF as coupling reagent and 1M Oxyma pure as racemization suppressor and 0.1 M DIPEA in DMF as free radical scavenger were used each time to join the amino acids. Only the N-terminal arginine residue was double coupled according to the described procedure. After each acylation, the peptidyl-resin was washed four times with DMF.

Cleavage of the peptide from the carrier:

A mixture of 88% TFA (SigmaAldrich) + 5% H2O + 5% phenol + 2% TIPSI (SigmaAldrich) (v / v / v / v) was used to detach the peptide from the support while removing the side covers. The reaction was carried out for three hours using a laboratory shaker, then the resin was filtered off under reduced pressure and the filtrate was evaporated to dryness in a vacuum evaporator. The resulting precipitate was quenched with cold diethyl ether, triturated and then centrifuged (20 min). The precipitate was washed three times with Et2O to remove impurities (swirling after each wash) and then allowed to dry in a vacuum desiccator. The thus obtained crude product was dissolved in water and subjected to the lyophilization process.

Characteristics of the obtained product - HPLC and LC-MS:

The obtained crude product was subjected to chromatographic analysis using reversed phase high performance liquid chromatography (RP-HPLC). Chromatography column: Luna 5 μm C18, 100 A, 250x4.6 mm. Phase system A 0.1% TFA in water, B: 0.1% TFA in 80% acetonitrile in water, flow 1 ml / min, UV detection at 223 nm (tR = 14.093 min). LC-MS ESI TOF on Kromasil C8 column, 5 µm 100 A (250 mm x 1 mm). Determined mass: 835.401 Calculated mass (monoisotopic, average): 835.9618.

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Replacement of the trifluoroacetic counterion by the acetate ion:

The exchange of the trifluoroacetate counter-ion with the acetate ion was performed on a Strata C18-E column (55 μm, 70A) according to the procedure:

- the peptide dissolved in water was applied to the equilibrated column,

- the progress of peptide binding to the column bed was monitored by means of reversed-phase high-performance liquid chromatography,

- after the peptide has bound on the column, it is washed with 0.1 M aqueous ammonium acetate solution,

- the peptide was eluted with 0.1 M ammonium acetate solution in MeOH.

The resulting eluate was evaporated using a vacuum evaporator, and then the excess ammonium acetate was sublimated on a freeze dryer. In order to remove residual ammonium, the lyophilisate was redissolved in water and lyophilized again.

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Investigation of the IM peptide stability in plasma and in water

Blood was collected from healthy donors and centrifuged on tubes coated with lithium heparin. EDTA was used as an anticoagulant. The IM peptide was incubated in plasma at 37 ° C (final peptide concentration: 144 µΜ) and its distribution analyzed at the time points: 1,2, 3, 6 and 24 h. Samples were prepared under sterile conditions. After an appropriate time, a 4-fold excess of ethanol was added, and then the sample was kept on ice for another 15 min. It was then centrifuged (32,000 rcf, 4 ° C, 20 min) on a centrifuge. The supernatant was collected and evaporated and the residue was dissolved in 100 µL of a 0.01% TFA solution in H 2 O. Analyzes were performed by HPLC on a Phenomenex Luna C18 (2) column (5 µm, 100 A 4.6 x 250 mm) with a PDA detector. A 0.01% solution of TFA in H 2 O (A) and 0.01% TFA in 80% MeCN in water (B) was used as the buffer. A linear gradient of 5-100% in 60 min with a flow of 1 mL / min was used for the analysis. The calculations were made using the Shimadzu LCsolution software. After one hour of incubation at 37 ° C, no signal from the IM peptide can be seen (Fig. 1 above).

To stabilize the IM peptide in water, the 0.5 mg / ml peptide was incubated for 24 hours at 37 ° C with continuous shaking. The peptide stability was tested at the following time points 1, 2, 3, 6 and 24 h. Analyzes were performed by HPLC on a Phenomenex Luna C18 (2) column (5 µm, 100 A 4.6 x 250 mm) with a PDA detector. A 0.01% solution of TFA in H 2 O (A) and 0.01% TFA in 80% MeCN in water (B) was used as the buffer. A linear gradient of 5-100% in 60 min with a flow of 1 mL / min was used for the analysis. The calculations were made using the Shimadzu LCsolution software. After 2 hours of incubation at 37 ° C, slight decay of the signal from the IM peptide can be seen (Fig. 1 bottom).

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Determination of the affinity of the IM peptide for bovine albumin

The following buffers were used to prepare the column and perform affinity tests: 1. coupling buffer (CB) - 0.2 M NaHCOs and 0.5 M NaCl (pH 8.3),

2. phosphate buffer saline (PBS) - 5 mM Na2HPO4 and 150 mM NaCl (pH 7.4),

3.blocking buffer (BB) - 0.1 M ethanolamine and 0.5 M NaCl (pH 8.3),

4.washing buffer (WB) - 0.2 M CHsCOONa and 0.5 M NaCl (pH 4),

5. ammonium buffer - 50 mM NH4HCO3 (pH 7.4).

Preparation of the microcolumn for affinity chromatography:

A Mobicol "F" microcolumn from Abo (# M105035F) was used to perform the IM affinity test for albumin. 100 µg of albumin protein was weighed and dissolved in 100 µl of 0.2 mM coupling buffer. 0.06 g of Sepharose (Activated CH-Sepharose 4B, A9019 Sigma-Aldrich) was weighed into a microcolumn and soaked in coupling buffer for 5 minutes to swell the resin. The dissolved albumin was transferred to a microcolumn and incubated for two hours at 25 ° C with continuous shaking. The microcolumn was then washed four times with blocking and washing buffer. The column was again incubated for an hour by shaking at 25 ° C in blocking solution. After the incubation was complete, the column was washed four times with the blocking solution and the washing solution alternately.

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Performing an affinity test:

The column was equilibrated with 20 ml of 50 mM ammonium buffer, then 20 μl of peptide dissolved in 20 μl of ammonium buffer was added and incubated at 25 ° C for 2 hours with constant agitation. After 2 hours, the column was washed with 100 ml of ammonium buffer, collecting the first and last ml of solution (supernatant and last wash fraction). The column bound complex was dissociated after 15 min incubation with 0.1% TFA in water with continuous shaking. The procedure was repeated twice, each time collecting the eluate fractions. The column was washed with PBS solution to renature the immobilized protein and stored at 4 ° C. All fractions were lyophilized and analyzed using mass spectrometry (SHIMADZU ESI-IT-TOF).

Analysis of the spectra obtained:

The spectrum of the last milliliter fraction of the buffers of the IM peptide indicates that the column was properly washed from excess peptide loaded as indicated by the absence of IM mass (Fig. 2 - top). The spectrum of the eluate fraction (Fig. 2 - bottom) shows the m / z peak 836.887 which is consistent with the mass of the IM peptide. On the basis of the obtained spectrum it can be concluded that the tested peptide interacts with albumin, because the m / z signal corresponding to the apparent proton ion derived from this peptide is present in the elution fraction. The affinity test was performed twice with a reproducible result.

BIOLOGICAL RESEARCH

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Cytotoxicity and proliferation tests of human fibroblasts and keratinocytes after stimulation with IM peptide

The IM peptide was tested in in vitro models using:

1) human skin cells and

2) peripheral blood mononuclear cells (PBMC).

The study using skin cells was designed to assess the effect of the IM peptide on their proliferation (multiplication). This process is crucial for wound healing (skin reconstruction). The effect of the IM peptide on the proliferation of immortalized human dermal fibroblast cells (46BR.1N) and dermal keratinocytes (HaCaT) and its cytotoxicity to these cells were investigated. Proliferation tests (XTT) were performed for concentrations: 0.01; 0.1; 1; 10; 25; 50; 100; 150 μg / ml, and cytotoxicity (FDH) for concentrations: 50, 100, 150 μg / ml.

The obtained results show that the IM peptide is not cytotoxic to the cells tested. Moreover, this compound stimulates the proliferation of 46BR.1N fibroblasts - an increase in the number of cells in culture relative to the control by 20-40% after 48 h and 72 h at the following concentrations: 0.1; 1; 10; 25; 50 μg / ml and HaCaT keratinocytes - increase in the number of cells by 10-40% after 48 h and 72 h at the following concentrations: 0.1; 1; 10 µg / ml (Fig. 3). The IM peptide did not inhibit the proliferation of fibroblasts and keratinocytes at all concentrations tested.

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Cell migration assays of human fibroblasts and keratinocytes after stimulation with IM peptide

The effect of IM peptide at concentrations of 0.01 and 0.1 µg / ml on the migration of immortalized cells of the HaCaT human keratinocyte line and 46BR.1N fibroblasts was investigated using the scratch assay. The obtained results show that the IM peptide at a concentration of 0.01 μg / ml stimulates the migration of 46BR.1N cells (fibroblasts) compared to control cells (Fig. 4), cultured in a serum-free medium (statistically significant differences, Mann-Whitney U test) , p <0.05). No pro-migratory effect was observed for HaCaT cells.

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Assessment of immunogenicity for IM peptides

The second type of analysis was to evaluate the immunogenicity of the IM peptide. This aspect is important due to the possible side effects of peptides, which are related to, inter alia, with activation of the immune system and induction of inflammation (lymphocytes) and possible allergic reactions (basophils / mast cells).

The research material was the so-called buffy coat from which peripheral blood mononuclear cells (PBMC) were isolated using density gradient centrifugation (histopaque, Sigma). After washing twice with PBMC in PBS, lysing erythrocytes and counting (Bio-Rad cell counter), cells were seeded in a 24-well plate at 1 million kk / 1 ml RPMI 1640 (P / S, 10% FBS) / well. Then the cells adapted to new conditions for 24 hours (the so-called resting). After this time it was added

PL 237 242 Β1 test compound - IM peptide at two final concentrations: 0.1 μg / ml and 1.0 μg / ml and incubated in an incubator for 48 h. The negative control was unstimulated cells, while the positive control was LPS stimulated cells (1 μg / ml) and PHA (2.5 μg / ml). Cells were harvested from the wells at the end of incubation, washed in PBS, counted and prepared for cytometric analysis: 100,000 kk / 100 μΙ were stained with antibodies, and after 30 minutes incubation at room temperature in the dark, analyzed using a flow cytometer - LSRFortessa, BD.

Immunogenicity studies:

The study of the effect of the IM peptide on the cells of the immune system was performed with the use of isolated peripheral blood mononuclear cells (PBMC). These cells were incubated with the test compound - IM (0.1 µg / ml and 1.0 µg / ml) for 48 hours, then their effects were analyzed by flow cytometry. The degree of stimulation of T lymphocytes (CD3 / CD4 / CD8) and NK cells (CD16 / CD56) was determined by evaluating the expression of activation markers: CD69, CD71, CD25, HLA-DR.

The analyzes performed indicate the lack of immunogenic properties of the IM peptide at both tested concentrations. The levels of selected subpopulations of cells of the immune system (CTL - cytotoxic cells, Th, NK cells) maintained the values similar to the control samples (Fig. 5, Fig. 6 and Fig. 7). These results demonstrate that the IM peptide does not induce inflammation and has a high immune safety profile.

Example 7:

Assessment of the allergenic potential of the IM peptide

The commercial kit Flow CAST® highsens (Buhlmann Laboratories, Switzerland) was used to evaluate basophil activation under the influence of the compound - IM peptide. This test (BAT, Basophil Activation Test) is used to assess the allergic potential of various types of chemical compounds, including drugs. The study was performed on blood samples collected from healthy volunteers and was performed within 24 hours of collection, according to the manufacturer's instructions. 100 μί of whole blood was incubated with the test compound - IM peptide at a final concentration of 0.1 μg / ml. Then the samples were stained with an antibody solution (CD63, CD203c, CCR3) and incubated for 15 min in a water bath at 37 ° C. After incubation, erythrocyte lysis and washing, the samples were analyzed cytometrically (BD FACSCanto II). Negative and positive controls (anti-FceRI mAb and fMLP) were also prepared for each sample. The obtained results indicate no activation of granulocytes in the presence of IM (0.1 µg / ml) - values lower than in the negative control (graph below). This demonstrates a very low probability of an allergic reaction to be triggered by the IM peptide in humans (Fig. 8).

Example 8:

Assessment of the transcriptomic profile of ASCs under the influence of the IM peptide

The effect of the addition of the IM peptide on the transcriptomic profile of primary cell lines - mesenchymal stem cells derived from adipose tissue (ASCs Adipose Derived Stern Cells) was tested. Subcutaneous adipose tissue was obtained from three healthy patients of the MUG's Plastic Surgery Clinic (consent no. NKBBN / 387/2014 of the MUG's Independent Bioethical Research Committee). The isolation of ASCs was performed according to the standard protocol (Gerlach et al. 2012) in DMEM Iow glucose (Dulbecco's modified Eagle medium) with the addition of 10% bovine serum (FBS - Fetal Bovine Serum). The effect of the IM peptide was tested after the second passage, in such a way that after changing the medium, the cells were cultured for 24 h with the addition of 10% FBS, another 24 h with 5% FBS, and then for 48 h only with the addition of the IM peptide at a concentration of 0.1 μg / ml. ASCs cells that had been cultured for the last 48 h without any stimulating factors (control without FBS addition) were used as controls. After the stimulation step, the cells were trypsinized, washed, and RNA was isolated from the frozen cell pellets for transcriptomic studies (for samples with RNA Integrity Number> 7). High-throughput RNA sequencing was performed in order to profile the entire transcriptome for samples obtained from three patients (marked with the symbols PS, PS8 and PS9, respectively). After sequencing, the obtained fragments were mapped to the human reference genome (hg19 edition) using TopHat software. Comparative analyzes of the transcriptomic profiles of ASCs treated with the IM peptide were performed in relation to the FBS Min control (ASCs cells cultured without the addition of stimulants) using the Cufflinks package according to the procedure described by the software developers (workflow for Cufflinks version 2.2.0 and higher - http: // cole -trapnell-lab.qithub.io/cufflinks/manual/). The results of comparative tests for individual samples and for the triplicate in the form of DGE matrices (ang.

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Differential Gene Expression was subjected to functional analysis in the Ingenuity Pathway Analysis (IPA) package.

IPA allows the analysis of transcript changes in the investigated data set and linking them to changes in upstream regulators and biological phenomena occurring as a result of these changes (downstream effects). The coefficient that describes the fit of this data is "consistency score". The regulatory network with the highest consistency score (29.69) for the patient with the symbol PS9 showed significant cell activation, increased migration and chemotaxis, activation of the immune response and activation of nitric oxide synthesis (Fig. 9 - top). Similar biological effects were observed for the PS1 patient, with clearly higher p-values (Fig. 9 - middle), and for the PS8 patient the changes were of the opposite nature (Fig. 9 - bottom).

Additionally, for the PS9 patient, whose ASCs responded most strongly to stimulation with the IM peptide, a whole list of activated biological phenomena was observed (Table 1).

Table 1. List of biological phenomena activated in ASCs cells derived from the PS9 patient upon stimulation with IM peptide (0.1 gg / ml). Z-score is an algorithm in IPA that allows the identification of phenomena and functions activated (z-score> 2) or inhibited (z-score <-2) as a result of changes in expression observed in the tested data set.

Phenomenon z-score Direction of change p-value Quantity of the involved transcripts Cell activation 3.84E-03 Activation 2.685 21 Inflammatory response L33E-04 Activation 2.628 25 Chemotaxis 3.57E-07 Activation 2.555 29 Cell movements L48E-06 Activation 2.455 68 Synthesis of nitric oxide 6.62E-04 Activation 2.391 9 RNA transactivation 1.22E-03 Activation 2.318 twenty Cell movements granulocytes L67E-03 Activation 2.316 12 Endothelial cell permeability 2J5E-03 Activation 2.193 5 Binding of neutrophils 2.82E-03 Activation 2,168 6 Endothelial cell migration 1.21E-05 Activation 2,149 22 Lipid secretion 3.45E-03 Activation 2,122 7 Cellular movements of neutrophils 4.48E-04 Activation 2.099 11 Cell migration l, 78E-04 Activation 2.063 55 Fatty acid synthesis l, 12E-03 Activation 2.021 13

Example 9:

Assessment of the transcriptomic profile of ASCs and keratinocytes under the influence of the IM peptide

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In order to determine the effect of IM derivatives on the expression of pluripotency and cell cycle regulation genes, a number of cultures of human ASC cell lines and keratinocytes from patients were performed. Cultures were stimulated with IM derivatives at 2 different concentrations (0.1 µg / ml and 1.0 µg / ml). As a control, culture of ASC cell lines without the addition of FBS (designated as Control) was taken. Transcript levels for a panel of genes including CDKN1A, MYC, KIT, POU5F1, TGFB3, TP53 were examined. RNA isolation was performed using the RNeasy kit (Qiagen). The levels of selected transcripts were determined using quantitative real-time PCR using TBP and ACTB as reference transcripts.

In almost all tested cases, no significant transcriptional response to the stimulation of ASCs with the IM peptide was observed. Only in the case of treatment with the IM peptide of 0.1 pg / ml of the ASC line derived from patient 1 (Fig. 10), a very strong, over one order of magnitude increase in the expression of the POU5F1 transcript - responsible for embryonic development and stem cell pluripotency was shown.

No significant transcriptional response to the stimulation of keratinocytes with the IM peptide was observed in almost all investigated cases. Only in the case of treatment with the IM peptide 0.1 pg / ml on the keratinocyte line derived from patient 4, a large increase in the expression of POU5F1 transcripts (responsible for embryonic development and stem cell pluripotency) and CDKN1A (responsible for the regulation of the cell cycle) can be seen (Fig. 11). Additionally, for the same culture, a decrease in the expression level of the MYC transcript (responsible for the activation of transcriptional genes related to cell growth) can be noticed.

The lack of a significant transcriptional response of genes encoding the key factors for the regulation of proliferation (CDKN1A, TP53), including oncogenes (MYC, KIT), can be interpreted as a beneficial property of IM, as such results suggest that the pro-regenerative effect of IM would not imply cell cycle disturbance. or activation of oncogenes (Fig. 10 and Fig. 11).

ANIMAL MODEL

P r z v k ł a d 10:

Effect of IM peptide on regeneration of complex tissue in C57BL6 / J mice

Female mice of the C57BL6 strain, 7-9 weeks of age at the start of the experiment, were selected as the experimental model. On the day of starting the experiment, one hole was made in the left and right auricles of mice using a sterile biopsy punch with a diameter of 2 mm. The IM peptide was administered at 5 µg / g body weight of mice by subcutaneous injection between the scapulae. The injection solution was made up in saline and administered once daily on the day of injury induction for four consecutive days and then weekly from days 7 to 42 when animals were euthanized and tissue samples were collected. The condition of the mice was documented every 7 days by collecting photographs. Animals receiving saline in an identical system and with an identical frequency constituted the control for the conducted experiments. The ears were photographed every 7 days for 42 days, and the course of the hole growth was followed by delimiting its surface by computer analysis of the photos taken (ImageJ). A total of 8 mice in the research group, 18 mice in the control group were analyzed. The area of the openings in the ears of the mice from the research and control groups on individual shooting days was counted and compared.

The protocol of animal experiments was approved by the Local Ethical Committee No. 1 at the Institute of Experimental Biology M. Nencki in Warsaw (consent no. 491/2013, taking into account the resolution no. 799/2015).

The multi-tissue regeneration was studied on the basis of the overgrowth of the hole in the auricle of mice. Mean pinhole areas of the ear at day 42 post-injury were 1.67 ± 0.37 mm ² and 3.14 ± 0.43 mm ² for C57BL6J mice treated with IM and saline controls, respectively (Fig. 12 and Fig. 13).

P r z v k ł a d 11:

Effect of IM peptide on regeneration of composite tissue in Balb / C mice

Female mice of the BALBc strain, 7-9 weeks of age at the start of the experiment, were selected as the experimental model. On the day of starting the experiment, one hole was made in the left and right auricles of mice using a sterile biopsy punch with a diameter of 2 mm. The IM peptide was administered at 5 µg / g body weight of mice by subcutaneous injection between the scapulae. The injection solution was made up in physiological saline and administered once daily on the day of induction

The lesions were treated for the following four days, and then weekly from day 7 to 42, when animals were euthanized and tissue samples were collected. The condition of the mice was documented every 7 days by collecting photographs. Animals receiving saline in an identical system and with an identical frequency constituted the control for the conducted experiments. The ears were photographed every 7 days for 42 days, and the course of the hole growth was followed by delimiting its surface by computer analysis of the photos taken (ImageJ). A total of 12 mice in the research group and 12 mice in the control group were analyzed. The area of the openings in the ears of the mice from the research and control groups on individual shooting days was counted and compared.

The protocol of animal experiments was approved by the Local Ethical Committee No. 1 at the Institute of Experimental Biology M. Nencki in Warsaw (consent no. 491/2013, taking into account the resolution no. 799/2015).

The multi-tissue regeneration was studied on the basis of the overgrowth of the hole in the auricle of mice. The mean pinhole areas of the ear at Day 42 post-injury were 1.65 ± 0.29 and 1.95 ± 0.39 mm ² for BALB / c mice treated with IM and saline controls (Fig. 14 and Fig. 15). ).

Histopathological preparations:

Histopathological images of the auricle in mice 42 days after the start of the dosing procedure: (A1 and A2 magnification) IM (5 pg / g body weight) versus (B1 and B2 magnification) vehicle only mice (buffered saline). IM-treated mice (A1 and A2) show the epidermis in a less differentiated phase (Fig. 16 - left side), which may be related to the reorganization / remodeling processes taking place, while in controls (B1 and B2) (Fig. 16 - right side) the epidermis is in the more mature / differentiated phase. In controls, the skin is compact and the basal keratinocytes are mostly flattened. The epidermis of IM-administered mice is thickened with enlarged columnar basal cells. The granular layer and keratohialin grains are abundant in mice treated with vehicle alone as opposed to the skin of IM administered mice. In IM-administered mice, the epithelium shows extensive parakeratosis (the presence of the nuclei of the stratum corneum). This may be related to the shortening of the keratinocyte turnover time from the basal layer of the epidermis to the stratum corneum. Mice with IM administration have a dense stratum corneum. Additionally, the intercellular spaces of the spinous layer in the tissue after the IM are more widened than in the control, where the cells are already tightly packed. In the inner part of the auricle, after IM treatment, a slightly higher density of fibroblasts is observed than in the control, additionally, these fibroblasts are less mature, although in both cases the overall picture of the fibroblast mixture (granulation tissue) is quite similar.

Conclusions:

The use of the IM peptide accelerates the process of complex tissue regeneration. This is confirmed by proliferation and migration studies on fibroblast and keratinocyte cell lines. The IM peptide is non-cytotoxic over a wide concentration range. It is also immunologically and oncologically safe. Acceleration of regeneration after the application of the IM peptide was confirmed by in vivo studies in mice, according to the auricle damage model. Statistically better regeneration in the auricle was achieved in the mice receiving IM in both strains of mice compared to the mice receiving saline. There was a clear orifice reduction effect in mice receiving IM on day 14 of dosing. This effect persisted until the end of the experiment. The maximum overgrowth was visible on the 28th day of the experiment. The results obtained in the Balb / C strain confirm the participation of the IM peptide in the multi-tissue regeneration, also observed in the C57BL6 / J strain. The obtained results indicate that in the reconstituted tissues stimulated with the IM peptide, the process of filling the hole with the regenerated tissue starts earlier compared to the control. The use of the IM peptide affects the quality of the reconstituted tissue. The resulting tissue has the characteristics of a younger tissue with a greater regenerative potential. In summary, IM inhibits keratinocyte differentiation and delays epithelial barrier closure, which may promote blastema formation and accelerate skin regeneration. The blastema is formed by undifferentiated pluripotent cells. Blastema is observed in the early stages of the body's development or during regeneration.

The mechanism of action of the IM peptide is unknown. It is most likely related to some extent to the regulation of the immune system of the mammalian organism, as shown in previous studies with the IM peptide. In the above tests, no changes in the activation of the immune system were observed, which most likely results from the fact that studies were conducted on isolated

And not the whole organism / immune system. The activation of mechanisms promoting regeneration under the influence of IM can also be seen from the example of the increased number of pro-regenerative transcripts under the influence of the IM peptide against ASC cells in transcriptomic analysis.

LITERATURE:

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Claims (3)

1. RDKVYR peptide or a pharmaceutically acceptable salt thereof for use as an agent for promoting the regeneration of composite tissue or the healing of wounds caused by mechanical and / or chemical and / or thermal and / or radiation damage and / or surgery and / or other pathological conditions. 2. The RDKVYR peptide according to claim 1, The method of claim 1, which is in the form of an injection. 3. The RDKVYR peptide according to claim 1, 1-2, characterized in that the dose of RDKVYR is used at a concentration of 0.1-50 µg / ml.