HK1217661B - Use of the cathelicidin ll-37 and derivatives thereof for wound healing - Google Patents

Description

CATHELICIDIN LL-37 and its derivatives for use in wound healing

The application is a divisional application of Chinese patent application 201010150582.3, which has application date of 2004, 01, 28 and has the title of CATHELICIDIN LL-37 and application of derivatives thereof in wound healing.

Technical Field

The present invention relates to peptides LL-37 and N-terminal fragments, and functional derivatives thereof, wherein said peptides are useful for cell proliferation, epithelial repair and wound healing. The invention also relates to pharmaceutical compositions comprising one or more of the peptides.

Background

The epithelium constitutes the first barrier between the host and the potentially harmful environment, and therefore protection of this interface is of paramount importance. Wounds indicate a breakdown of the barrier and immediately initiate a series of closely coordinated events with the aim of rapidly restoring the integrity of the barrier. Emergency wound closure has evolved in higher organisms, unlike the time-consuming process of complete tissue regeneration seen in lower species. Impaired wound healing is a major problem in clinical medicine, ranging from relatively slow "normal" healing seen with age to pathological non-healing (non-healing) ulcers.

Chronic ulcers are a major clinical problem, and although our understanding of the physiological processes of injury has increased over the past decades, only minor therapeutic advances have been achieved. The unique etiology may lead to the development of ulcers in different clinical situations, but, regardless of the cause, non-healing ulcers are characterized by a defect in the inability of epithelial cells to migrate, proliferate and close the barrier. The most common type of chronic skin ulcer is a leg ulcer due to venous insufficiency. These patients develop peripheral venous edema followed by skin ulceration, but the arterial circulation is intact. Leg and foot ulcers due to arteriosclerotic defects are less common.

In addition, the development of skin ulcers is associated with immune diseases such as pyoderma gangrenosum (pyoderma gangrenosum) and vasculitis (vasculitis). Current treatments include long-term systemic immunosuppression, which is not always effective. Epithelial defects and ulcers in the oral, genital and gastrointestinal mucosa are common and cause much pain. The underlying pathological mechanisms are not well understood, such as aphtha (aphtae) and erosive lichen (erosive lichen), and treatment methods are poor.

Traditional wound care involves the formation of granulation tissue by mechanical or enzymatic removal of necrotic debris. Wounds with a dense colonisation of bacteria may require antimicrobial therapy to avoid invasive infection (invasive infection). A variety of topical antimicrobial agents are used, such as iodine (iododine), chlorhexidine (chlorohexidine), hydrogen peroxide (hydrogen peroxide), silver and antibiotics, but the risk of toxic effects of these agents on the matrix and neoepidermises (neoepidemics) must be considered. Once the wound is cleared of necrotic tissue, a dressing is applied to promote granulation tissue formation. A wide variety of such dressings are available and a number of animal studies and clinical trials have demonstrated their beneficial effects on wound healing.

Some parts of the wound remain therapy resistant, so additional therapy is required. The potential use of growth factors in accelerating wound repair has attracted much attention over the past decade. Growth factors are molecules that control cellular processes (cellular processes) critical to tissue repair, including cell migration, proliferation, angiogenesis and extracellular matrix synthesis from the head (denovo). The beneficial effects of these growth factors have been shown in a number of experiments (Schaffetter-Kochanek et al, Basic Res Cardiol 93:1-3,1999). However, the use of growth factors in clinical practice to date has been disappointing in the treatment of chronic ulcers. Now it isBecapplermin licensed in the United states and Europe (except Sweden)Is the only growth factor preferably used in diabetic foot ulcers. The reason for clinical failure of growth factors to treat chronic ulcers is believed to involve delivery problems and rapid degradation.

In parallel, tissue therapy using autologous and allogeneic materials in bioengineered human skin analogs has been developed. Cultured epithelial keratinocytes constitute a functional treatment for covering large areas of damaged skin (e.g. of burn patients), but this treatment is expensive, time consuming and requires laboratory equipment. Various strategies have been used to provide the epidermal substrate (dermallssulate), such as acellular human cadavers and bovine collagen, either with or without cells. All available methods have considerable disadvantages, such as the possibility of disease transmission, high costs, and little suitability for basic wound care.

Antimicrobial peptides are effector molecules of the innate immune system that function to protect a host against potentially harmful microorganisms. They are conserved in evolution and widely distributed in nature. Human antimicrobial peptides, only a few of which have been identified; among them, defensins (defensins) and the human cathelicidin antimicrobial peptide hCAP18 have been implicated in functioning in epithelial defense (Selsted et al, J Biol Chem258:14485-14489, 1983).

WO 96/08508 relates to the human polypeptide FALL-39, and pharmaceutical compositions comprising said peptide and having antibacterial antimicrobial activity. The peptide is designated FALL-39 by the first four amino acid residues and consists of the 39 amino acid C-terminal portion of a precursor protein that is simultaneously identified by three separate groups (Cowland et al, FEBS, 1995; Agerberth et al, Proc Natl Acad Sci USA 1995; Larrick et al, FEBS Letters 1996). The peptides are shown to have potent antimicrobial activity against gram-positive and gram-negative bacteria. Further characterization of the C-terminal peptide showed that the first two (FAs) were removed to give LL-37, a shorter sequence of 37 amino acids, which is now an accepted name (Gudmundsson et al, Eur J Biochem 238:325-332, 1996).

The precursor protein is designated hCAP18, which is a human cationic antimicrobial protein, or one of the members of the cathelicidin protein family comprising cathelins which are conserved in evolution and the C-terminal part is variable in different species in humans hCAP18 is the only member of this protein family, whereas in other species, such as mice and pigs, there are several members of this protein family, the C-terminal peptide LL-37 is believed to act extracellularly and there is no evidence that this precursor protein is cleaved intracellularly hCAP18/LL-37 is present in leukocytes and in barrier organs (barrier organs) such as skin, mucosa, respiratory epithelium and reproductive organs hCAP18/LL-37 is located in the barrier epithelium (hCAP epithelia) and seems to be consistent with the protective effect of this peptide in preventing local infections and systemic microbial invasion, the peptide is described as a cysteine-free peptide which can accommodate the amphipathic peptide (cysteine) and seems to be involved in the amphipathic helix-mediated effects on the skin, i.a gram-positive amphipathic peptide (antisense) and the amphipathic helix-mediated effects on gram-positive bacterial growth, e.a gram-positive protein, a gram-positive protein responsible for the amphipathic peptide (gram-positive protein, a) and gram-positive amphipathic peptide (gram-positive protein responsible for the amphipathic helix-positive protein responsible for the immune response of gram-positive protein responsible for the immune response of fungi, e.g. antibiotic, the immune response of gram-positive protein, antibiotic, Escherichia, EP.

Prior Art

Dorschner et al, J Invest Dermatol 117:91-97,2001, showed that the amount of cathelicidin expression after cleavage was increased in human and mouse skin, and the absence of the murine homolog cathelicidin gene failed to protect these mice against Group A (Group) S.streptococci invasion.

WO 96/09322, Children's Medical Center Corporation, discloses that antimicrobial peptide PR-39 has the induction activity of syndecan-1 and-4, thus simultaneously reducing infection, and as synducin, affects the effects of growth factors, matrix components and other cellular effectors (effectors) involved in tissue repair. The synducin may be administered in a pharmaceutical carrier such as a conventional liposome.

EP 0935965 a1, Toray Industries, inc, relates to anti-helicobacter pylori agents (antipyloriagents) comprising as active agent an antimicrobial peptide, such as the porcine peptide PR-39. It was concluded that exogenous (exogeneous) administration of PR-39 has antimicrobial activity against Helicobacter pylori (Helicobacter pylori) and accelerates the healing of gastric ulcers in mice. FALL39 is mentioned as one of the members of the cathelin family.

US 6,255,282, Helix Biomedix, inc, discloses a newly synthesized lytic peptide (lytic peptide) that shares the structural and functional properties of different known lytic peptides.

Frohm Nilsson, Thesis, Karolinska institute, Stockholm 2001, also states that the human cathelicidin antimicrobial protein hCAP18 is induced at high levels in human skin wounds and releases the active C-terminal peptide LL-37 in physiological healing rather than in chronic non-healing ulcers. hCAP18 was detectable in the wound bed (bed) and epithelium during normal wound healing, but it was not present in the epithelium of chronic leg ulcers, it was only detected in the wound bed and stroma. It is speculated that low levels of hCAP18 and its lack in the epithelium of chronic ulcers contribute to impaired healing.

Zasloff, Nature 415: 389-.

EP 1358888 a1, balss et al, whose publication is 11/5/2003, relates to the use of the peptide LL-37 in the prevention or treatment of diseases caused by reduced blood flow and arteriosclerosis and in the treatment of wounds caused by reduced arterial blood supply. The ability of LL-37 to induce neovascularization and stimulate endothelial cell proliferation is shown. The present invention is fully concerned with angiogenic effects and does not mention epithelia.

Although antimicrobial peptides, specifically LL-37, have shown therapeutic utility, they have not been realized to date. The peptide LL-37 showed cytotoxic effects at high concentrations. However, the potential cytotoxic effects exhibited by LL-37 in the presence of serum are suppressed, but pharmaceutical formulations should avoid the inclusion of serum because of the risk of disease transmission, limited access to and high cost of the drug.

Summary of The Invention

The human antimicrobial peptide hCAP18 was positively regulated in the skin dermis as a normal response to injury. But low levels of hCAP18 were found in chronic non-healing leg ulcers. Clearly, in chronic leg ulcers hCAP18 and LL-37 were completely absent from the epithelium but were present in the inflammatory exudate (infilterte) and stroma of the wound bed. We have now found that hCAP18 is induced in the re-epithelialization of organ-cultured skin wounds, and that this re-epithelialization is inhibited in a concentration-dependent manner by antibodies against LL-37. These findings suggest that LL-37 functions like a growth factor and plays a crucial role in wound closure. The present invention relates to the use of LL-37 or a novel synthetic peptide derived from LL-37 or a functional derivative thereof to compensate for the lack of native LL-37 produced in vivo.

It was also shown that up-regulation of hCAP18 and/or addition of LL-37 peptide stimulates proliferation of normal epithelial and stromal cells, indicating that healing and epithelial regeneration of normal wounds can also be enhanced.

It has also been found that the cytotoxicity of LL-37 can be reduced in compositions comprising certain lipids.

Brief Description of Drawings

Figure 1 is a schematic representation of the 18kDa hCAP18 protein, which contains the signal peptide s.p., a conserved cathelin moiety, and the antimicrobial peptide LL-37 (which is truncated in vivo by the enzyme).

FIG. 2 is a schematic representation of the cathelicidin protein family, illustrating the diversity of C-terminal peptides in different species.

FIGS. 3A, 3B and 3C show the cDNA sequence of pIRES2-EGFP vector including the coding sequence of hCAP18 for transgenic expression of hCAP 18.

Disclosure of Invention

The present invention relates to a peptide having a sequence of at least 20 amino acids of the N-terminal fragment of LL-37, with the proviso that the peptide does not comprise LL-37, and pharmaceutically acceptable salts and derivatives thereof. LL-37 has the amino acid sequence SEQ ID NO: 1:

H-Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser-OH。

the N-terminal sequence of LL-37 refers to the sequence starting from leucine (Leu) at amino acid residue number 1.

Pharmaceutically acceptable salts include, for example, the acetate, carbonate, phosphate, sulfate, trifluoroacetate and chloride salts of the counter ion. The preferred salt is an acetate salt. Esters and amides are examples of pharmaceutically acceptable derivatives.

The peptides of the invention should have an amino acid chain of not more than 40 amino acids. The present invention relates to a peptide having a sequence to which 1 to 3 amino acids have been added to the C-terminus of LL-37. Any amino acid selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, and derivatives thereof may be added. Examples of peptides having 38 amino acids LL-38, SEQ ID NO: 19 having a sequence to which a serine has been added at the C-terminus of LL-37.

The invention relates in particular to a peptide having a sequence of at least 20 amino acids selected from the group consisting of LL-36, LL-35, LL-34, LL-33, LL-32, LL-31, LL-30, LL-29, LL-28, LL-27, LL-26, LL-25, LL-24, LL-23, LL-22, LL-21 and LL-20, said peptide having the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ id no: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

preferred peptides are selected from the group consisting of LL-36, LL-35, LL-34, LL-33, LL-32, LL-31, LL-30, LL-29, LL-28, LL-27, LL-26 or LL-25.

The amino acid sequences of the peptides of the invention are shown in the following table.

The novel peptides can be used as medicaments for cell proliferation, epithelial regeneration, normal or chronic wound healing, and as antimicrobial agents.

It is believed that the novel peptides have the potential to form α -helix structures under physiological conditions.

Other aspects of the invention relate to the use of a peptide having an amino acid sequence selected from one of the following sequences:

a)SEQ ID NO：1；

b) comprises the amino acid sequence of SEQ ID NO: 1 of at least 20 amino acids of the N-terminal fragment;

wherein the peptide enhances proliferation of epithelial and/or stromal cells by a non-lytic mechanism.

The invention specifically relates to a polypeptide having an amino acid sequence of SEQ ID NO: 1 in the form of a salt, preferably in the form of an acetate.

The invention also relates to the use of a peptide selected from LL-20 to LL-36 as described above.

Since the cationic amino acid residues lysine and arginine in the primary structure, LL-37 and LL-25 to LL-36 have a net positive charge at neutral pH (+5 to +7), in particular LL-34 and LL-35 have 7 net positive charges, the other amino acid residues are non-polar/hydrophobic or polar and neutral, or to a lesser extent polar and negatively charged, which makes the entire peptide molecule amphiphilic.

For rabbit CAP18(Cap 18) with 37 amino acids in length_106-142) Studies of C-terminal peptides have shown that broad spectrum antibacterial activity is retained in the highly basic N-terminal sequence of 20 residues, but not if the N-terminus is truncated (Larrick et al, anti Agents Chemother 37:2534-2539, 1993).

LL-37 and the novel LL-20 to LL-36 peptides can be synthesized using an automated peptide synthesizer using standard methods for peptide synthesis.

The invention particularly relates to the use of the LL-37 peptide or any of the peptides LL-20 to LL-36 for the preparation of a medicament for the treatment of chronic ulcers. The chronic ulcers may be caused by venous insufficiency, such as leg ulcers, metabolic dysfunction, such as diabetes, or immune disorders, such as vasculitis and pyoderma gangrenosum. The peptides of the invention may also be used to treat wounds resulting from trauma or burns. The peptides are particularly useful for epithelial tissue regeneration and enhance epithelial regeneration after microdermabrasion.

In addition to being cytotoxic, LL-37 degrades rapidly in the wound environment. Recently it was shown that serine protease 3 is responsible for the extracellular cleavage of hCAP18 (ii)Etc., Blood 97: 3951-.

To prevent deposition of the peptide and also to reduce internal cytotoxicity, the peptide may be formulated with a polar lipid carrier. The formulation should facilitate administration of the peptide to the wound and additionally provide for delayed release of the peptide following administration. This will improve the stability of the peptide in vivo and in vitro.

Another object of the present invention is a pharmaceutical composition comprising an antimicrobial cathelicidin peptide in the form of a pharmaceutically acceptable salt or derivative thereof, together with a carrier consisting of a bilayer-forming polar lipid and an aqueous solution.

The cathelicidin peptides can be derived from different animal species besides human LL-37, such as sheep SC5, cow Bac5, pig PR-39, mouse CRAMP, and rabbit p15, see FIG. 2.

Bilayers generally refer to a lamellar arrangement of polar lipids in water. The acyl chain forms the internal hydrophobic part and the polar head-group forms the hydrophilic part of the bilayer. Examples of polar bilayer-forming lipids, whether natural or synthetic in origin, may be mentioned phosphatidylcholine, phosphatidylglycerol, digalactosyldiacylglycerol, sphingomyelin, and the like. Depending on the concentration of the polar lipid in a polar solvent such as water, viscous gels of the liposome or lamellar liquid crystal type may be formed.

The pharmaceutical composition specifically comprises an amino acid sequence having one of the following sequences selected from:

a)SEQ ID NO：1；

b) comprises the amino acid sequence of SEQ ID NO: 1 of at least 20 amino acids of the N-terminal fragment;

a peptide in the form of a pharmaceutically acceptable salt or derivative thereof, and a carrier consisting of a bilayer-forming polar lipid and an aqueous solution.

Preferred bilayer-forming polar lipids mixed or formulated with the peptides are those that are charge neutral. Particularly useful are digalactosyldiglyceride, and other glycolipids, such as glycosylceramide (glycoceramide), whether natural or synthetic, in which the nonionic sugar moiety (motif) constitutes a polar head-group. Less preferred but still useful are those polar lipids that are zwitterionic and neutral in their physiological environment, such as phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Most preferred are those polar lipids that are negatively charged, and therefore form strong complexes with positively charged peptides.

The bilayer-forming polar lipid carrier according to the present invention is preferably selected from the group consisting of phospholipids, galactolipids and sphingolipids.

Particularly preferred bilayering polar lipids are digalactosyldiglyceride or a mixture of digalactosyldiglyceride-rich polar lipids, due to the excellent skin tolerance of such polar lipids. Digalactosyldiglyceride is a lipid belonging to the glycolipid family, a well-known component of plant cell membranes. One of the most abundant classes contains two galactose units, and its common nomenclature and abbreviation is digalactosyldiglyceride, DGDG, sometimes referred to as galactolipid. Galactolipids, first DGDG and DGDG-rich substances, have been investigated and found to be useful surface-active substances in industrial applications such as food, cosmetics and pharmaceuticals. WO 95/20944 describes the use of the DGDG rich substance "galactolipid substance" as a bilayer-forming substance for pharmaceutical, nutritional and cosmetic use in polar solvents. These applications do not disclose the use of galactolipids and peptides and proteins in general, and specifically the use of the peptides of the present invention.

A preferred aspect of the invention relates to a pharmaceutical composition wherein the bilayer-forming polar lipid carrier is a mixture of polar lipids enriched in digalactosyldiglycerides.

Another preferred aspect of the invention is a pharmaceutical composition, wherein the peptide is in the form of acetate. A preferred peptide is LL-37 in the form of acetate. It is particularly preferred that the pharmaceutical composition comprises the acetate salt of LL-37 and CPL-galactolipids as a bilayer-forming lipid carrier. CPL-galactolipids are a trademark of galactolipid components comprising 50-70% by weight of digalactosyldiglyceride and 30-50% or other polar lipids.

The ratio of the peptide in salt form to the glycolipid carrier in the pharmaceutical composition should preferably be in the weight ratio 1:5 to 1:50, particularly preferably 1: 10-1: 25.

In addition to the bilayer-forming lipid, the carrier also comprises an aqueous solution. Aqueous solution refers to a solution having physiological or pharmaceutically acceptable properties with respect to pH, ionic strength, isotonicity, and the like. As examples, isotonic solutions of water and other biocompatible solvents may be mentioned aqueous solutions, such as physiological saline and dextrose solutions, and hydrogel-forming substances. The aqueous solution may be buffered, such as phosphate buffered saline PBS.

The pharmaceutical composition may further comprise pharmaceutically acceptable excipients such as preservatives to prevent the growth of microorganisms in the composition, antioxidants, isotonicity agents, colorants and the like. In aqueous suspensions the compositions may be combined with suspending agents and stabilizers.

The colloidal nature of the composition allows it to be prepared in sterile form by using a final filter sterilization step.

To form a gel, the peptide is preferably formulated with a hydrogel-forming material. Examples of hydrogel-forming materials are synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, polyethylene glycol, poloxamer (poloxamer) block copolymers, and the like; semi-synthetic polymers such as cellulose esters including carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, methylhydroxypropyl cellulose, and ethylhydroxyethyl cellulose, and the like; natural gums such as gum arabic (acacia), carrageenan (carragenan), chitosan (chitosan), pectin, starch, xanthan gum and the like.

It is advantageous to use mucoadhesive (muco-adhesive) hydrogels. Particularly useful in this regard are the use of hyaluronic acid and its derivatives, cross-linked polyacrylic acids of the carbomer and polycarbophil classes, polymers readily available from gels which are known to adhere strongly to mucous membranes.

Block copolymers of the poloxamer type, i.e. polymers comprising polyethylene glycol and polypropylene glycol blocks, are also advantageous. Certain poloxamers dispersed in water are thermoreversible: they are low viscous at room temperature but show a significant viscosity increase with increasing temperature, resulting in the formation of a gel at body temperature. Thus, the contact time for administering a pharmaceutical formulation to a relatively warm wound may be extended, thus increasing the effectiveness of the incorporated peptide.

The pharmaceutical compositions of the present invention may be formulated for topical or enteral, i.e., buccal, sublingual, mucosal, nasal, bronchial, rectal, and vaginal administration.

Non-limiting examples of pharmaceutical compositions for topical administration are solutions, sprays, suspensions, emulsions, gels and films. If desired, a bandage or band aid (band aid) or plaster (plaster) with the pharmaceutical composition added thereto may be used. Tablets, capsules, solutions or suspensions may be used for enteral administration.

Another aspect of the invention relates to the use of a peptide having an amino acid sequence selected from one of the following sequences, in the form of a pharmaceutically acceptable salt or a derivative thereof, for proliferating epithelial and/or stromal cells in vitro by a non-lytic mechanism:

a)SEQ ID NO：1；

b) comprises the amino acid sequence of SEQ ID NO: 1 of at least 20 amino acids of the N-terminal fragment;

the proliferation can be used for in vitro proliferation of human autologous epithelial cells and stromal cells.

The invention also relates to a growth medium for culturing eukaryotic cells, such as epithelial cells and/or stromal cells, comprising LL-37 or a combination of said peptide and a basal medium. A cytotoxicity reducing agent such as serum may be added. Apolipoprotein A-I (apoA-I) has been found to be the major LL-37-binding protein in human plasma and to act as a scavenger of LL-37 (scuvenger) (Wang et al, J Biol Chem273:33115-33118, 1998;et al, J Biol Chem 274:22445-22451,1999), which suggests that the mechanism involved in the regulation of cathelicidin peptides is involved. The cytotoxicity reducing agent may also be a bilayer-forming polar lipid, such as a lipid selected from the above-mentioned phospholipids, galactolipids or sphingolipids.

The basal medium of the growth medium of the invention is based on double-distilled water (double-distilled water) and several of the following components: inorganic salts, phenol red, glucose, thymidine (thymidine), inosinic acid (hypoxanthinine), HEPES, sodium pyruvate, aminopterin, amino acids and vitamins. For culturing epithelial cells in vitro, such as for example keratinocytes, the growth medium may comprise a basal medium and a kit for promoting growth comprising a) LL-37 peptide in a salt solution, b) penicillin + streptomycin, c) insulin, d) transferrin, e) triiodothyronine (triiodothyronine), f) hydrocortisone, g) cholera toxin, optionally a cytotoxicity reducing agent, such as serum or polar lipids. For culturing stromal cells, such as fibroblasts, in vitro, the growth medium may comprise a basal medium and a kit for promoting growth comprising a) LL-37 peptide in a salt solution, b) penicillin + streptomycin, and optionally a cytotoxicity reducing agent, such as serum or polar lipids.

Other objects of the invention are methods for enhancing the in vitro expansion (expansion) of human autologous epithelial and stromal cells for in vivo cell transplantation, wherein the cells are isolated from sections of healthy skin (expanded pieces), the isolated cells are cultured in vitro in the growth medium of the invention, after which the cultured cells are harvested and used for wound therapy, such as burns and ulcers.

The invention also relates to a kit for promoting growth comprising the peptide LL-37 or said peptide, and bilayer-forming polar lipids with reduced cytotoxicity, and optionally antibiotics, basal medium, and other usual additives in separate containers.

Yet another aspect of the invention relates to the transfection of the full length hCAP18cDNA construct into autologous human keratinocytes for cell transplantation for ulcers and burns. The cDNA construct was designed to regulate the expression of hCAP18 gene by a switching mechanism (Resnitzky et al, Mol Cell Biol 14:1669-1679, 1994). Autologous human keratinocytes were obtained from healthy skin sections of the patients. The keratinocytes are isolated and expanded in cell culture as described. The cDNA constructs were transfected into keratinocytes. The transfected keratinocytes are then further expanded in vitro and returned to the patient.

The invention specifically relates to a polypeptide comprising a polypeptide having the sequence SEQ ID NO: use of a genetic construct of the complete cDNA sequence of hCAP18 of 20 to transfect epithelial and/or stromal cells to enhance proliferation of said cells.

1. A peptide having the sequence of SEQ ID NO: 1, provided that the peptide does not comprise LL-37.

2. The peptide of item 1, wherein LL-37 sequence SEQ ID NO: 1 has 1-3 amino acids added to the C-terminus.

3. A peptide according to claim 1 selected from the group consisting of LL-36, LL-35, LL-34, LL-33, LL-32, LL-31, LL-30, LL-29, LL-28, LL-27, LL-26, LL-25, LL-24, LL-23, LL-22, LL-21 and LL-20, said peptide having the sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

4. a peptide according to any one of items 1 to 3 for use as a medicament.

5. Use of a peptide according to any one of claims 1 to 3 in the manufacture of a medicament for promoting wound healing, cell proliferation, epithelial regeneration and for use as an antimicrobial agent.

6. Use of a peptide having an amino acid sequence selected from one of the following sequences:

a)SEQ ID NO：1；

b) comprises the amino acid sequence of SEQ ID NO: 1 of at least 20 amino acids of the N-terminal fragment;

wherein the peptide enhances proliferation of epithelial and/or stromal cells by a non-lytic mechanism.

7. The use of item 6, the peptide being LL-37 in acetate form, SEQ ID NO: 1.

8. the use of item 6, said peptide being selected from the group consisting of: LL-36, LL-35, LL-34, LL-33, LL-32, LL-31, LL-30, LL-29, LL-28, LL-27, LL-26, LL-25, LL-24, LL-23, LL-22, LL-21, or LL-20, said peptides having the sequences SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, seq id NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

9. the use of a peptide according to any one of claims 6 to 8 in the preparation of a medicament for the treatment of chronic ulcers caused by venous insufficiency, metabolic dysfunction, or immunoregulatory dysfunction.

10. Use of a peptide according to any one of claims 6 to 8 in the manufacture of a medicament for the treatment of wounds due to trauma or burns.

11. A pharmaceutical composition comprising an antimicrobial cathelicidin peptide in the form of a pharmaceutically acceptable salt or derivative thereof, and a carrier consisting of a bilayer-forming polar lipid and an aqueous solution.

12. A pharmaceutical composition comprising a peptide in the form of a pharmaceutically acceptable salt or derivative thereof and a carrier consisting of a bilayer-forming polar lipid and an aqueous solution, wherein the peptide has an amino acid sequence selected from one of the following sequences:

a)SEQ ID NO：1；

b) comprises the amino acid sequence of SEQ ID NO: 1 of at least 20 amino acids of the N-terminal fragment.

13. The pharmaceutical composition of item 11 or 12, wherein the bilayer-forming polar lipid is selected from the group consisting of a phospholipid, a galactolipid, and a sphingolipid.

14. The pharmaceutical composition of any of claims 11-13, wherein the peptide is in the form of acetate.

15. The pharmaceutical composition of any of claims 11-14, wherein the bilayer-forming polar lipid comprises at least 50% (w/w) digalactosyldiglyceride.

16. The pharmaceutical composition of any of claims 12-15, wherein the peptide is LL-37 in the form of acetate.

17. The pharmaceutical composition of any one of items 11-16, comprising a complex of an acetate salt of LL-37 and CPL-galactose ester.

18. The pharmaceutical composition of any one of claims 11-17, wherein the weight ratio of salified peptide to galactolipid carrier is 1: 10-1: 50.

19. use of a peptide having an amino acid sequence selected from one of the following sequences for the in vitro proliferation of epithelial and/or stromal cells by a non-lytic mechanism:

a)SEQ ID NO：1；

b) comprises the amino acid sequence of SEQ ID NO: 1 of at least 20 amino acids of the N-terminal fragment;

wherein the peptide is in the form of a pharmaceutically acceptable salt or derivative thereof.

20. The use of item 19 to proliferate human autologous epithelial and stromal cells in vitro.

21. A growth medium for culturing epithelial and/or stromal cells, comprising LL-37 or a peptide of any of items 1-3, and a basal medium.

22. A growth medium for culturing epithelial and/or stromal cells, comprising LL-37 or a peptide of any of items 1-3 and a bilayer-forming polar lipid in a basal medium.

23. The medium of item 22, wherein the polar lipid is selected from the group consisting of a phospholipid, a galactolipid, and a sphingolipid.

24. A method of enhancing in vitro the expansion of human autologous epithelial and stromal cells for in vivo cell transplantation, wherein the cells are isolated from a cut piece of healthy skin, the isolated cells are cultured in vitro in the growth medium of any one of items 21-23, after which the cultured cells are harvested and used for the treatment of wounds such as burn injuries and ulcers.

25. A kit for culturing epithelial and/or stromal cells, the kit comprising LL-37 or a peptide according to any of items 1-3 and a bilayer-forming polar lipid that reduces cytotoxicity, and optionally antibiotics, basal media, and other common additives.

26. cDNA sequence comprising hCAP18 SEQ ID NO: 20 for the purpose of transfection and transgene expression of epithelial and/or stromal cells to enhance proliferation of said cells.

Examples

EXAMPLE 1 preparation of synthetic peptides

LL-37 peptide was synthesized according to solid phase synthesis using a 9-fluorenylmethoxycarbonyl/tert-butyl strategy (9-fluoronylmethylcarbonyl/tert-butyl strand). Is prepared fromThe processed peptide, as trifluoroacetate salt, was purified by HPLC and finally isolated by freeze-drying (batch No. 971/26, from Polypeptide Laboratories A/S,denmark). Purity was determined by HPLC and integration and found to be 99%. The molecular weight was analyzed by mass spectrometry and corresponded to the theoretical value of 4493g/mol for the free base (free base) form. Analysis of the amino acid composition showed that the relative amount of each amino acid corresponded to the theoretical value of LL-37. The peptide content was calculated from the amino acid analysis results and found to be 73%, with the remainder being the counterion and residual solvent.

Lots of LL-37 were synthesized, and the LL-37 peptides used in examples 2 and 5 below were in the acetate form.

Peptides LL-36 and LL-38 were synthesized as acetates, respectively.

The different peptides used in the following examples and experiments are shown in the following table.

Example 2 preparation of a pharmaceutical composition comprising a mixture of LL-37 peptide and lipid Carrier

A pharmaceutical composition was prepared with the following ingredients:

composition (I)	Concentration of
		LL-37	100ppm*

CPL-galactose esters	0.20％
		2.6% glycerol in sterile water	ad 100％

Ppm is parts per million (by weight)

Peptide LL-37 (batch No. 990/37/A) as acetate, and the lipid carrier CPL-galactose ester from Lipocore Holding AB, which is a lipid material rich in digalactosyldiglyceride and prepared from oats, were weighed into a 50ml glass flask. The two components were mixed gently and then added to the glycerol solution. The mixture was mixed vigorously for 120 minutes and then allowed to stand for 1 hour. The resulting composition is a fine, homogeneous dispersion. It was refrigerated until use.

Example 3 preparation of an aqueous mixture comprising the peptide LL-37 and a lipid Carrier

A mixture of the trifluoroacetate salt of LL-37 (lot 971/26) and a polar bilayer-forming lipid carrier was prepared with the following ingredients (in weight percent):

table 1.

The CPL-Galactolipid ester (CPL-Galactolid) from Lipocore Holding AB is a chromatographically purified Galactolipid fraction from oats, Epikuron 200 from Lucas Meyer GmbH is phosphatidylcholine from soybeans, and CPL-Sphingomyelin (CPL-Sphingomyelin) from Lipocore Holding AB is chromatographically purified Sphingomyelin from milk. DMEM from Invitrogen corp, Dulbecco's modified Eagle medium is an aqueous solution containing inorganic salts, glucose, phenol red, amino acids and vitamins.

Peptide LL-37 and lipid carrier were weighed into a glass flask and then DMEM was added. The resulting dispersion was shaken vigorously with a Heidolph Promax mixer at a frequency of 200/min for 1.5 hours, then allowed to equilibrate and stand at room temperature for about 3 hours. Visual estimation was performed to obtain the following results: all samples were turbid dispersions with no difference in turbidity between any of samples B1, B2, C1 and C2. The only visible difference is between samples a1 and a 2: a1 comprising the peptide was significantly less turbid than a2 not comprising the peptide. The turbidity of sample a2 was lower than the turbidity of samples B1, B2, C1 and C2 in that order. These observations indicate that there is a strong interaction between the two components of sample a1, so that the average particle size of the dispersion is smaller compared to sample a2 without the peptide and the rest of the corresponding samples. After one day of storage at room temperature, samples a1 and a2 did not change, i.e., both were homogeneous dispersions and a1 was less hazy than a2, while the other four samples all had appreciable precipitate at the bottom of the glass flask.

All three mixtures of peptides and polar lipid carriers can be used for various purposes, e.g. as delivery systems, and for experiments in cell culture; however, because the shelf life (shelf-life) of the mixture of peptide and galactolipid is relatively longer (no precipitation) than those of the others, the mixture is most preferred for practical use.

Example 4 preparation of an aqueous mixture comprising a mixture of LL-37 peptide and lipid Carriers

Samples of trifluoroacetate salt of LL-37 (run No. 971/26) and a polar bilayer-forming lipid carrier were prepared with the following ingredients (in weight percent):

table 2.

CPL-galactolipid esters produced by LTP Lipid Technologies Provider AB are chromatographically purified galactolipid fractions from oats. A number of phospholipids used are Phosphatidylcholine (PC) from soybean, about 40% (Sigma; P-3644); PC from egg yolk, approximately 60% (Sigma; P-5394); synthetic Dioleylphosphatidylcholine (DOPC), approximately 99% (Sigma; P-6354); PC from soybean, about 70% (Lipoid S75); and PC from soybean, approximately 94% (Lipoid S100). PBS was phosphate buffered saline from Invitrogen Corp. (Dulbecco's; batch No. 14190-094).

All polar lipids studied had a chain melting transition temperature just below (well below)0 ℃, i.e. in the range of-10 to-15 ℃ when fully hydrated.

The peptide LL-37 and lipid carrier were weighed into a 100ml glass flask and then PBS was added in a total volume of about 30ml the sample was shaken vigorously with an ST mixer (model B1, E.B ü chler, T ü bingen) set to 5.5 (corresponding to a frequency of about 150/min) for 2 hours and then allowed to equilibrate and stand at room temperature for about 30 minutes the turbidity of the resulting dispersion was recorded at 400 and 800nm using Shimadzu UV-VIS Spectrophotometer UV-160A. the turbidity was measured at room temperature using a 10mm cuvette (cuvette cell) against pure water. the turbidity data in Table 3 are expressed in% transmission at 600 nm. the dispersions were also estimated visually and the turbidity measurements were repeated after storing the dispersions at room temperature for one to two days.

TABLE 3 turbidity data

From visual evaluation it was concluded that all mixtures formed more or less turbid dispersions; samples D, E, H and I formed the least turbid dispersions, as seen by the highest transmission in table 3, while samples F, G and J formed the most turbid dispersions, resulting in the lowest transmission detected by the spectrophotometer. After one day of storage at room temperature, samples F, G and J with an initial high turbidity (low transmission) all precipitated and were undetectable. Samples D, E, H and I are stable dispersions so that the turbidity data can be repeated one to two days after preparation.

Samples D and H were in duplicate and both contained CPL-galactolipid, but sample H had a slightly higher weight of peptide compared to the weight of galactolipid. This resulted in slightly lower turbidity (higher transmission) in sample H, suggesting that the peptide and lipid interactions in this sample were stronger than in sample D, resulting in smaller complexes/aggregates that resulted in lower turbidity.

Samples D, E, H and I were further monitored for colloidal stability for 2 months at 2-8 ℃.

TABLE 4 stability data

Sample(s)	Appearance of the product	Stability of
			D	Fine turbid dispersion, little precipitation, easy redispersion and precipitation	Can accept
E	Turbid dispersion, little precipitation; growth of microorganisms	Is not acceptable
			H	Fine turbid dispersion, little precipitation, easy redispersion and precipitation	Can accept
I	Turbid dispersion, little precipitation; growth of microorganisms	Is not acceptable

These data and observations show that the two mixtures of peptide and polar lipid carrier are superior to the rest of the mixtures tested. The carrier comprising CPL-galactose esters (samples D and H) and PC from soy, ca 40% (sample E) makes the dispersion the finest and the longest in colloidal stability; however, only CPL-galactose esters are accepted for pharmaceutical applications, since only phospholipid substances with only 40% phosphatidylcholine can be used for technical applications. These data again indicate the usefulness of galactolipid materials in a variety of pharmaceutical applications, for example as a carrier system for peptides.

Example 5 preparation of an aqueous mixture comprising different amounts of LL-37 peptide and different amounts of galactolipids

Preparation of 995ppm stock solution (acetate; lot No. 990/37/A) of LL-37 peptide in PBS and 1.00% stock solution of CPL-galactose ester in PBS the components of the mixture are shown in Table 5. after equilibrating at room temperature for 1 hour, the ST mixer (model B1, E.B ü chler, T ü bingen) was set to 7.5 (corresponding to a frequency of about 190/min), the vial was shaken in horizontal position for 1 hour, then equilibrated at room temperature overnight and the mixture was left to stand, the mixture was evaluated for appearance of clear colloid, light turbidity, milky at 4 deg.C, one day and five days.

Table 5.

It is clear that the ratio of certain LL-37 peptide and galactolipids results in a solution appearance, indicating the presence of small complexes of a size smaller than the particle size of the corresponding sample without LL-37. Clear solutions indicate excellent colloidal stability.

Example 6 conformational measurements

Measurement of the Circular Dichroism (CD) of LL-37 in solution reveals information about conformational changes LL-37's antibacterial activity is dependent on conformation: high helix content makes the antibacterial action strong and cytotoxic activity high (Johansson et al, J Biol Chem273:3718-3724, 1998). it has been found that the α -helix conformation of LL-37 is dependent on counterion, pH and peptide concentration (Johansson et al, J Biol Chem273:3718-3724, 1998). it is also known that certain fractions of the peptide have a α -helix structure in aqueous solution and due to the presence of additives such as lipids promote this structure, converting it from a random coil to a α -helix (Turner et al, Antimicrob Agents Chemothers 42:2206-2214, 1998).

Samples prepared for Circular Dichroism (CD) measurements were in 10mM phosphate buffered aqueous solution, pH 7.0, containing 200ppm LL-37 (as trifluoroacetate, lot number 971/26), with or without 0.40% CPL-galactose ester ST mixer (model B1, E.B ü chler, T ü bingen) set at 7.5 (corresponding to a frequency of approximately 220/min) was shaken vigorously for 2 hours with 20ml of sample in a 50ml glass flask, the sample was equilibrated at 2-8 ℃ overnight and left to stand.

The CD spectra were recorded on a Jasco J-720(Jasco Inc.) spectrophotometer. A cuvette (1mm diameter length) was placed adjacent to the photomultiplier to reduce the effect of light scattering from the dispersion. The samples were measured at room temperature and scanned from 280 to 200nm at a rate of 20nm/min with a resolution of 1nm, 3 accumulations per round (accumulations per run). The results are expressed as mean residue ellipticity (θ [ ]. theta. ]]And the percentage of the α -helix conformation at 222nm was estimated using the following equation ([ theta ])]₂₂₂+3900)·100/41900。

For CD determination of 200ppm LL-37 in 10mM phosphate buffer, pH 7.0, the dichromatic minimums at 208 and 222nm (double dichromatic minima) show α -helical secondary structure the percentage of α -helical structure was calculated using the minimum at 222nm and was found to be about 63%, when galactolipid concentration added in the same buffer solution was 0.40% (w/w), the α -helical structure of LL-37 was practically unaffected and the α -helical structure was about 64%.

It is speculated that secondary structure is also associated with the wound healing promoting ability of LL-37, where a high percentage of α -helical structure indicates increased activity.

An experiment was performed using the same experimental conditions as described above using the anionic synthetic phospholipid palmitoyl oleoyl phosphatidylglycerol (POPG; Sigma-Aldrich, P6956) as a reference (reference). when this lipid was present, the percentage of α -helix structure was found to be lower, at 58%, indicating the conformation of LL-37 such that the activity was more affected by negatively charged phospholipids than by neutral galactolipids.

Example 7 cytotoxic assay

In vitro cytotoxicity assays are valuable for assessing the cytotoxicity of substances in close contact with living tissue.

Selected preparations were subjected to in vitro cytotoxicity experiments in cultured mammalian cells (L929 mouse fibroblasts). The experimental design is based on US Pharmacopeia 26^thedition,Method<87>And ISO 10993-5 standard.

Formulations D and E (see example 4, table 2) were mixed with complete cell culture medium (HAM F12 medium, 10% fetal bovine serum) at concentrations of 10, 2, 0.4 and 0.08% (v/v). Cell cultures were treated with these test solutions in triplicate for 24 hours. Triplicate untreated cultures, negative control (treated with polypropylene extract) and positive control (treated with tin-stabilized polyvinyl chloride extract) were also included.

The two formulations showed no to slight toxicity (cytotoxicity rating 0-1) tested at 10% (v/v) and no toxicity (cytotoxicity rating 0) at 2%, 0.4% and 0.08% (v/v).

Cytotoxicity experiments were performed with a positive control solution containing 100ppm LL-37 in PBS, resulting in moderate toxicity (cytotoxicity grade 2) at all four concentrations tested (10, 2, 0.4 and 0.08% mixture of the solution with cell culture medium). This level of toxicity is defined as 20-50% cell death or morphological symptoms showing toxicity. The scale ranged from 0 to 4, and when tested on the test extract of the medical device, the scales 3 and 4 failed the test. This positive control solution was considered to be much more toxic than formulations D and E which showed no or only slight toxicity.

Biological experiments

Based on our present findings

hCAP18/LL-37 is induced in the skin and mucous membranes associated with inflammation and wounds, and

hCAP18/LL-37 is deficient in chronic ulcer epithelium despite a wide range of inflammation (major),

we hypothesized that hCAP18/LL-37 is involved in the regenerative capacity of the skin epithelium. To verify this hypothesis, the following experiment was performed.

Experiment 1 study of the type of expression of hCAP18/LL-37 in non-inflammatory human wound healing

Tissue sample

The skin of a person is obtained from a conventional abdominal or breast repair surgery (reduction surgery). Full-thickness wounds were made on the epithelial side with a 3-mm biopsy punch (punch) under sterile conditions. These ex vivo wounds were cut with a 6-mm biopsy punch and then transferred to 24-well plates and covered with 2ml of culture medium. These wounds re-epithelialize repeatedly within 4-7 days (Kratz et al Scand J plant Reconsr Surg Hand Surg 28: 107-. The medium was changed every three days, which was DMEM (Dulbecco's modified Eagle medium, GIBCO) containing 10% Fetal Calf Serum (FCS) and antibiotics (PEST ═ penicillin 50U/ml and streptomycin 50 mg/ml). At different time points, wounds were harvested and snap frozen 2,4 and 7 days after wounding. The test was repeated four times in total. Four different donors were used and three wounds were made in each case in each trial. Only skin from one donor was used for each test.

Preparation of RNA Probe

For detection of hCAP18 gene mRNA and immunoreactivity to hCAP18/LL-37, we performed in situ hybridization and immunohistochemistry on wound specimens representing sequential re-epithelialization at all time points. We use in situ hybridization³⁵S-labeled antisense and sense RNA probes were tested as described (Frohm Nilsson et al, infection Immun67:2561-2566, 1999).

Preparation of LL-37 antibody

For immunohistochemistry, we generated and prepared polyclonal LL-37 antibody as follows: LL-37 peptide (batch No. YS 5253, EuroDiagnostica AB,sweden) was trifluoroacetate and purified by HPLC to 98% purity. The biological activity of the peptide was confirmed in an antibacterial assay. The peptides were used to immunize three rabbits using standard protocols (AgriSera,sweden). Polyclonal antiserum was obtained by affinity purification using synthetic LL-37 peptide and the purified antiserum was evaluated by ELISA. IgG concentration of immune serum was diluted to 0.5 mg/ml. Preimmune serum was collected from each rabbit at an IgG concentration of 2 mg/ml.

Immunohistochemistry

Quick frozen and all biopsies were treated equally. Briefly, 6-7 μm thick frozen sections were incubated with LL-37 antibody at dilutions 1:1000 and 1:2000 and stained with the Vectastain kit (Vectorlaboratories, Burlingame, USA) using the indirect peroxidase method according to the manufacturer's instructions. Sections were counterstained with Mayer hematoxylin solution. All experiments were repeated a minimum of three times to ensure reproducibility. As a control, serial tissue sections were processed simultaneously without primary antibody and pre-immunized rabbit IgG (DAKO, Glostrup, Denmark) was used as primary antibody.

Results

At the 0 hour time point there was moderate expression of hCAP18mRNA, and LL-37 protein in the basal layer of epithelium throughout the tissue was consistent with our previous findings of constitutive expression of hCAP18 in the basal epithelium. Wounds harvested at different time points in re-epithelialization showed unique signals for hCAP18mRNA, and LL-37 protein in the epithelium migrated to cover the wounded surface. None of the cells in the underlying dermal matrix were positive for hCAP 18/LL-37. These results indicate that resynthesis of hCAP18 occurs in keratinocytes during infection-free re-epithelialization, and also support our hypothesis that hCAP18 may be involved in epithelial regeneration.

Experiment 2. inhibition of re-epithelialization of human skin wounds in vitro with LL-37 antibody.

LL-37 antibody prepared in experiment 1 was added to 2ml of medium per well (DMEM, + 10% FCS and PEST) to final antibody dilutions 1:10, 1:100 and 1: 1000. We used the corresponding preimmune serum with a final IgG concentration equal to a 1:10 dilution of LL-37 antiserum as a control, and one group of wounds was treated with medium only. Each experimental condition was done in triplicate and repeated twice. The medium was changed every three days and LL-37 antibody or preimmune serum was added as described above. Isolated wounds were harvested 2,4 and 7 days after wounding. All specimens were snap frozen, sectioned intact and fixed onto Superfrost Plus slides before staining with hematoxylin-eosin. Sections representing the greatest re-epithelialization in the center of the wound were selected for evaluation. The proliferative capacity of keratinocytes was studied by immunohistochemistry on wounds representing all the treatment conditions, using the proliferation marker Ki67 (mouse monoclonal Ki67 antiserum (DAKO, Glostrup, Denmark) at a dilution of 1: 25).

Results

Treatment with LL-37 antibody produced concentration-dependent inhibition of re-epithelialization. All wounds treated with the highest LL-37 antibody concentration (1:10) were non-re-epithelialized. In these wounds only a single keratinocyte with a fragile flat appearance migrates from the edge of each wound. Wounds treated with medium concentrations (1:100) of LL-37 showed delayed re-epithelialization, with most of these wounds healing up to day 7 rather than day 4. And the epithelium is thinner and the keratinocytes appear more fragile. Wounds treated with the lowest concentration (1:1000) of LL-37 antibody did not differ from control wounds, which healed by day 4 and had 2-3 layers of firm epithelium. Control wounds treated with only the medium and control IgG antibodies healed equally. The majority of cells in the re-epithelialized tongue in the control wound were positive for the proliferation marker Ki67, while no Ki67 positive cells were present in the wound treated with LL-37 at 1: 10. We conclude from this experiment: LL-37 may be involved in skin re-epithelialization and proliferative capacity seems to be preferably affected, since blocking with LL-37 antibody would allow migration of the original single cells from the wound margin but would be effective in preventing further proliferation of keratinocytes.

Experiment 3. proliferation of HaCat cells treated by combining synthetic bioactive LL-37 peptide and polar lipid carrier.

HaCat cells were used for three experiments. HaCat cells are an immortalized human keratinocyte cell line (Boukamp et al, JCell Biol 106:761-771,1988), which is suitable for the study of experimental keratinocytes. HaCat cells were cultured in medium (DMEM + 10% FCS and PEST). Both cell cultures were treated with synthetic bioactive LL-37 (batch YS 5253). In addition, a mixture of LL-37 (114. mu.g/ml) and CPL-galactose ester (0.2%) in medium containing 2 or 10% serum was added to evaluate its ability to increase proliferation and inhibit cytotoxicity. Cells were harvested at different time points (24 hours, 48 hours, 72 hours and 96 hours) and counted using a flow cytometer (Becton-Dickinson), and viability was assessed using trypan blue staining. Trypan blue positive indicates that the cell membrane has been disrupted. Proliferation and viability were also determined by measuring mitochondrial activity (WST-1, Roche, Cook et al Anal Biochem 179:1-7,1989).

TABLE 6 HaCat cell proliferation at 96 hours was estimated using flow cytometry.

An increase in cell proliferation was calculated compared to baseline (-EGF). The mean of triplicate samples/conditions in three separate experiments is given.

TABLE 7 proliferation and viability of HaCat cells at 48 hours were determined by mitochondrial activity (WST-1).

An increase in cell proliferation was calculated compared to baseline (-EGF). The mean of 6 samples/condition in one experiment is given.

TABLE 8 HaCat cell proliferation at 72 hours was estimated by flow cytometry.

Proliferation was calculated as compared to baseline (-EGF). The mean of triplicate samples/conditions in one experiment is given.

Results

Treatment of HaCat cells with LL-37 peptide resulted in an increase in the concentration-dependent manner of proliferation. This indicates that the LL-37 peptide has the ability to stimulate keratinocyte proliferation to a level equal to or exceeding the proliferation level of EGF (gold standard for epithelial cell proliferation). We used 1.7nM EGF, since this is already the optimal condition for stimulating the proliferation of cultured keratinocytes and is already the standard culture condition (Cohen et al, Dev Biol 12:394-407, 1965). HaCat cells are highly proliferating epithelial cells, and interestingly LL-37 increased proliferation of these cells even further. The cytotoxic effect induced by LL-37 at 100. mu.g/ml in 2% serum was completely abolished when lipids were added to the mixture, indicating that the lipids were able to replace the serum under this experimental condition.

This experiment has shown that: synthetic bioactivity of LL-37 (25-100. mu.g/ml) added to HaCat cell cultures in medium containing 10% Fetal Calf Serum (FCS) increased proliferation in a concentration-dependent manner. However, if the peptide (100. mu.g/ml) was added to the keratinocyte cultures in a medium containing 2% FCS, all keratinocytes became positive for Trypan blue staining, indicating a cytotoxic effect on these cells.

The presence of serum inhibits the cytotoxic activity of cathelicidins, a mechanism which is thought to protect host cells from potentially harmful effects. Our data demonstrated that the presence of serum (10%) inhibited the cytotoxic effect of LL-37. In addition, the mixture of LL-37 (25. mu.M) and polar lipid carrier (0.2%) inhibited cytotoxic effects and promoted proliferation in medium containing lower serum concentrations (2% FCS). These data suggest that the polar lipid carrier has similar protective capacity as serum, and does not interfere with the biological activity of LL-37.

The raw data show that human keratinocytes proliferate in the same manner as HaCat cells.

Experiment 4 HaCat cells were propagated by treatment with synthetic peptides LL-36, LL-37 and LL-38

HaCat cells were cultured in medium (DMEM + 10% FCS and PEST). HaCat cells were seeded in 96-well plates (Falcon, USA) at a concentration of 2000 cells per well. Cells were plated at-48 hours and stimulated with different concentrations of synthetic peptides LL-37, LL-36 and LL-38 at 0 hours and after 48 hours.

In one experiment, 6 wells were tested under each condition. Adding 1Ci/mmol of³H-thymine (THYMIDINE, [ METHYL-³H]740.0GBq/mmol (20.00Ci/mmol), 1.0ml of ethanol: water, 7:3, PerkinElmer Life Sciences inc. boston ma., USA) were added to each well and incubated for 12-17 hours. After 72 and 96 hours, by³H-Thymidine incorporation proliferation (MicroBet) was assessed by a liquid scintillation counter (liquid scintillator)aPerkin Elmer Life Sciences Inc.Boston MA.,USA)。

TABLE 9.72 and 96 hours later by³H-thymidine incorporation was used to estimate cell proliferation of HaCat due to LL-37 at 96 hours.

The increase in cell proliferation (proliferation coefficient) was calculated compared to baseline (control ═ 0 μ g/ml). The average of four samples for each condition in one experiment is given.

TABLE 10 LL-36 peptide-stimulated HaCat cells. After 96 hours, is prepared from³H-Thymidine incorporation to assess proliferation.

The increase in cell proliferation (proliferation coefficient) was calculated compared to baseline (control ═ 0 μ g/ml). The average of four samples for each condition in one experiment is given.

TABLE 11 LL-38 peptide-stimulated HaCat cells. After 96 hours, is prepared from³H-Thymidine incorporation to assess proliferation.

The increase in cell proliferation (proliferation coefficient) was calculated compared to baseline (control ═ 0 μ g/ml). The average of four samples in each case in one experiment is given.

Experiment 5 proliferation of human fibroblasts treated with LL-37 peptide

The peptide LL-37 used in this and the following experiments was as described in example 1 (batch No. 971/26). Fibroblasts are stromal cells obtained from injured and uninjured skin of patients with chronic leg ulcers caused by venous insufficiency. Punch-biopsies (4-mm) were taken from the wound periphery, which included a 50% epithelialized area, and uninjured skin was taken from the knee area. Individuals with a history of diabetes, arterial insufficiency or chronic inflammatory disease were excluded. Patients with eczema symptoms around the ulcer, with clinical symptoms of infection, or undergoing systemic or local antibiotic treatment at the time of biopsy are also excluded. The included patients were treated with inert topical dressings and standard compression bandages.

Fibroblasts were placed in culture using the explant technique (explant technique) (Hehenberger et al, Cell Biochem Funct 15:197-201, 1997). Fibroblasts were seeded in 96-well plates (Falcon, USA) at a concentration of 2000 cells per well. Cells were seeded at-48 hours and stimulated with varying concentrations of synthetic LL-37 peptide to direct the 0 hour. Experiments were performed on 6 wells per condition in one experiment. Proliferation and viability were determined by measuring mitochondrial activity (WST-1, Roche) after 24 hours and 48 hours. See tables 12 and 13 below. The increase in cell proliferation (proliferation coefficient) was calculated compared to baseline (control ═ 0 μ g/ml). See the mean of the six samples for each condition in one experiment.

Table 12 LL-37 stimulated human wound fibroblasts. Proliferation and viability of human fibroblasts at 48 hours as determined by mitochondrial activity (WST-1).

Table 13 LL-37 peptide-stimulated human normal fibroblasts. Proliferation and viability of human fibroblasts at 48 hours as determined by mitochondrial activity (WST-1).

Experiment 6 transgene expression of hCAP18 in HEK293 cells and proliferation of HEK293-hCAP18 cells

A fragment of Bfa1 of Image clone 3057931(ref), containing the complete coding sequence of hCAP18, comprising 16bp of the 5' -untranslated region, was subcloned into the Sma 1-site of the bicistronic (bicistronic) vector pIRES2-EGFP (BD Biosciences, Bedford, MA). Human embryonic kidney cells HEK293 were transfected with Fugene (Roche Diagnostics, Indianapolis, IN) under standard conditions and selected for two weeks with 400ng/ml G418 antisticum (Invitrogen, Paisley, UK). By usingHigh speed cell sorting flow cytometer (DakoCytomation, FortCollins, CO) sorting EGFP-expressing cells using Summit^TMThe software was used for data analysis and CAP18 expression of the cells was quantified by immunoblotting. Control cell lines were similarly established by transfection with EGFP-only expression vectors.

For proliferation analysis, cells were harvested at 70% confluence and plated in 24-well plates. After 24 hours the medium was changed and the cells were cultured in 2ml of medium supplemented with 5% FCS and PEST (OPTIMEM, Gibco BRL, Life Technologies, Scotland). All conditions were performed in triplicate. The medium was changed every other day. Cell lines were then harvested on day 6 and counted by flow cytometry. Cell viability was determined with trypan blue; cells < 5% were trypan blue positive under all conditions. The increase in cell proliferation (proliferation coefficient) was calculated compared to baseline (HEK 293-EGFP). See the average of triplicate samples for each condition in one experiment.

TABLE 14 proliferation of HEK293-hCAP-18 cells at 144 hours as estimated by flow cytometry.

The proliferation of the HEK293-hCAP18 cell is also used³The results obtained, evaluated for H-thymidine incorporation, are shown in Table 15 below. The increase in cell proliferation (proliferation coefficient) was calculated compared to baseline (HEK 293-EGFP). See each in one experimentAverage of four samples under conditions.

TABLE 15.96 hours later³H-Thymidine incorporation was used to assess proliferation of HEK293-hCAP-18 cells at 144 hours.

Experiment 7 culturing human cells for transplantation in different growth media

Culturing epithelial cells

A1 x 1cm piece of skin was cut from a patient's healthy skin. The skin is minced and treated with trypsin/EDTA (0.05/0.01%), and 2-5X 10⁶The recruited (recruited) keratinocytes of (1) are added at 75cm²1.5X 10 in culture flasks⁶Mitomycin-pretreated (4. mu.g/ml, 2 hours) 3T3 cells. Growth medium A containing LL-37 peptide was added. Lamellar cells were harvested by trypsinization and transplanted into patients.

Growth medium a is used for the in vitro culture of epithelial cells, such as, for example, keratinocytes, and consists of a basal medium and a kit for promoting Growth (GPK) comprising a) LL-37 peptide in a salt solution, b) penicillin + streptomycin, c) insulin, d) transferrin (transferring), e) triiodothyronine (triiodothyronine), f) hydrocortisone, g) cholera toxin and selected cytotoxicity-reducing agents, such as serum or polar lipids.

Stromal cell culture

Stromal cells were obtained from a 4mm skin biopsy, cleaned of subcutaneous tissue, and plated on cell culture dishes using an explantation technique to obtain primary fibroblasts. Growth medium B was used for the culture of biopsies. Cells were harvested by trypsinization and returned to the patient.

Growth medium B is used for the in vitro culture of stromal cells such as, for example, fibroblasts, and consists of a basal medium and a kit for promoting growth, which comprises a) LL-37 peptide in a salt solution, B) penicillin + streptomycin and selected cytotoxicity reducing agents, such as serum or polar lipids.

The basal medium is based on double distilled water containing inorganic salts, phenol red, glucose, thymine, hypoxanthine, HEPES, sodium pyruvate, aminopterin, amino acids and vitamins.

Summary of the experiments

In summary, it has been described that LL-37 is produced in the skin epithelium during normal wound healing, and that LL-37 is required for re-epithelialization to occur. It has been shown that chronic ulcer epithelia lack endogenous LL-37. We therefore believe that treatment with LL-37, and N-terminal fragments of the peptide, and functional derivatives thereof, provides a rational strategy for promoting healing of the ulcer. Additional transgenic expression of LL-37 and hCAP18/LL-37 also stimulated proliferation of healthy skin, indicating that LL-37 could be used to enhance in vivo repair of normal and defective epithelium and in vitro proliferation of epithelial cells for autologous cell transplantation. We have also identified suitable vectors and delivery systems which reduce cytotoxicity and have the potential to protect LL-37 and other cathelicidin peptides from rapid degradation in vivo.

Claims (4)

1. Use of the peptide LL-37 of SEQ ID NO 1 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of chronic ulcers caused by venous insufficiency.
2. 2. The use of claim 1, wherein the chronic ulcer is a leg ulcer.
3. 3. The use of claim 1, wherein the peptide LL-37 is in the form of acetate.
4. 4. The use of claim 2, wherein the peptide LL-37 is in the form of acetate.