WO2016170382A1 - Pharmaceutical compositions comprising a bradykinin 2 receptor antagonist for prevention or treatment of impaired skin wound healing - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for the prevention or treatment of impaired skin wound healing.

Description

PHARMACEUTICAL COMPOSITIONS COMPRISING A BRADYKININ

2 RECEPTOR ANTAGONIST FOR PREVENTION OR TREATMENT

OF IMPAIRED SKIN WOUND HEALING FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for the prevention or treatment of impaired skin wound healing.

BACKGROUND OF THE INVENTION:

Delayed skin wound healing is a source of morbidity, disability, loss of quality of life and mortality in subjects affected by diabetes. Foot ulcers affect at least once in their life about 15% of patients with diabetes and the amputation rate in affected patients is high (1-3). Treatment options for wound healing are currently limited. Improving wound healing in patients with diabetes and shortening time to full recovery is a major therapeutic challenge.

The pathogenesis of chronic foot ulcers in diabetes is complex, multifactorial and still incompletely understood. Loss of insulin action, hyperglycaemia, ischemia secondary to micro- and macro-angiopathy and neuropathy all contribute to delaying skin repair and favor necrosis and infection.

Skin healing after wounding normally comprises an acute response phase with haemostasis and inflammation, a granulation phase with cell migration, angiogenesis and production of extracellular matrix and a late re-epithelisation and scar maturation phase. Various cell types are involved in sequential cooperation in the process, endothelial cells, keratinocytes and fibroblasts (1). In diabetes, cellular infiltration and granulation tissue formation are delayed, together with impaired collagen organization and reduced angiogenesis (4). While impairment of the different components of the healing process has been documented in diabetes, the temporal and local pattern of association of the defects resulting in delaying wound repair is less understood.

Accordingly, there is a need to develop an alternative therapeutic approaches and new drugs that will be suitable for preventing or treating diabetic ulcers and to improve wound healing in diabetic subject. In this way, it has been suggested that characterization of new compounds for treatment of diabetic ulcers may be highly desirable. In this context, kinins could play a role in cutaneous responses to injury. Indeed, kinins are potent vascular endothelium activators and trigger release of a number of endothelial mediators promoting smooth muscle relaxation, inhibition of platelet aggregation and fibrinolysis (5). Kinins have other cellular targets, including keratinocytes and fibroblasts. Studies have documented the presence of all the components of kallikrein-kinin system (KKS) in human skin (6-10). Kinin receptors, bradykinin receptor type 1 (B1R) or bradykinin receptor type 2 (B2R), have been shown to participate in hyperproliferative process in chronic skin inflammation model (11) and to increase skin microvascular blood flow in rabbits and humans (12). Moreover, B1R contributes to differentiation of cultured keratinocytes by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family (7; 13). Overall, these studies suggest a role for the KKS in control of skin homeostasis. However, the involvement of KKS in skin repair, especially in the setting of diabetes is unknown.

The aim of the present invention was to investigate the role of kinin receptors in wound healing in diabetic mice, and how it differs from their role in non-diabetic animals. Effect of potent selective B1R or B2R agonists and/or of B2R antagonist was studied in vivo in mice and in vitro in proliferation/migration of cultured fibroblasts and keratinocytes. The inventors also studied the signalling pathway induced by modulation of kinins receptor activity. These studies demonstrate that B2R activity is deleterious in diabetic wounds and that its blockade is a promising therapeutic approach for diabetic patients.

Targeting the neutralization of a B2R is a radically new strategy for preventing or treating diabetic ulcers and to improve wound healing in diabetic subject. There is no disclosure in the art of the role of B2R in wound healing in diabetic subject, and the use of B2R antagonists in the prevention or treatment of diabetic ulcers and to improve wound healing in diabetic subject.

SUMMARY OF THE INVENTION:

The present invention relates to a compound selected from the group consisting of B2 receptor antagonists, B2 receptor expression inhibitors, kinin expression inihibitors, kininogen expression inhibitors, kallikreins expression inhibitors, kininase expression activators or kininase activators for use in the prevention or treatment of diabetic ulcers in a subject in need thereof. DETAILED DESCRIPTION OF THE INVENTION:

The role of B2 receptors in impaired skin wound healing was investigated by the inventors using streptozotocin-induced model of diabetes or 12-week-old db/db mice compared with non-diabetic mice. The inventors assessed effect of chronic treatment with selective pharmacological Bl or B2 receptor agonists and/or B2 receptor antagonist on healing of experimental skin wound. The inventors also studied effects of Bl or B2 receptor agonists on proliferation and migration of cultured fibroblasts and keratinocytes.

The inventors investigated, through pharmacological manipulation of kinin B l and B2 receptors, the role of kinins in wound healing in diabetic and non-diabetic mice. Diabetes delayed wound healing in mice. Bl and B2 receptor mRNAs increased 2 to 3 fold in diabetic skin. Bl receptor agonist had no effect on wound healing. In contrast, B2 receptor agonist impaired wound repair in both diabetic and non-diabetic mice and induced skin disorganization and epidermis thickening. This effect was abolished by co-administration of B2 receptor antagonist. B2 receptor antagonist alone had no effect in non-diabetic mice. However, it normalized wound healing in streptozotocin or db/db diabetic mice. In vitro, B2 receptor activation unbalanced fibroblast/keratinocyte proliferation and increased keratinocyte migration, through ERK and EGFR signalling.

Accordingly, Kinins, through B2 receptor activation, play a deleterious role in skin wound healing in diabetic mice. B2 receptor blockade improves wound healing in these mice and is a potential therapeutic approach to diabetic ulcers.

Therapeutic methods and uses

Accordingly, the present invention relates to a compound selected from the group consisting of B2 receptor antagonists, B2 receptor expression inhibitors, kinin expression inihibitors, kininogen expression inhibitors, kallikreins expression inhibitors, kininase expression activators or kininase activators for use in the prevention or treatment of diabetic ulcers in a subject in need thereof. As used herein, the term "subject" denotes a mammal. Typically, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with diabetic ulcers. Typically a subject according to the invention is a subject afflicted or susceptible to be afflicted with an impaired skin wound healing. Typically a subject according to the invention is a diabetic subject. The method of the invention may be performed for any type of diabetic ulcers, diabetic wound healing defect and impaired skin wound healing in a diabetic subject. As used herein, the term "diabetic ulcers" has its general meaning in the art and refers to diabetic ulcers such as revised in the World Health Organisation Classification E10-E14 (Diabetes mellitus in ICD-10) such as diabetic foot ulcers.

The term "kinin" has its general meaning in the art and refers to bradykinin and lysil- bradykinin. Kinin, bradykinin and lysil-bradykinin refer to endogenous nona- and deca- peptide that are generated by cleavage of the precursor polypeptide (kininogen) by specific proteases (kallikreins) within numerous tissues of the body (Regoli, D. and Barabe, J. Pharmacol. Rev., 1980, 32, 1-46; Hall, J. M, Pharmacol. Ther., 1992, 56, 131-190; Leeb- Lundberg et al., Pharmacol. Rev. 2005, 57: 27-77). Certain enzymes of the kininase family degrade bradykinin and related peptides and thus inactivate these peptides. Kinins exert their actions through two different G protein-coupled seven transmembrane domains receptors, called Bl and B2.

The term "kininogen" has its general meaning in the art and refers to polypeptide, precursor for the kinin.

The term "Kallikreins" has its general meaning in the art and refers to specific protease responsible of the generation of kinin by the cleavage of the precursor polypeptide (kininogen). The term "kininase" has its general meaning in the art and refers to enzymes responsible of kinin and related peptides degradation and thus their inactivation.

The term "B2-receptor" has its general meaning in the art and refers to kinin receptor type B2 or bradykinin receptor type B2.

The term "expression" when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., phosphatidylserine receptor) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.

An "expression inhibitor" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. An "activator of expression" refers to a natural or synthetic compound that has a biological effect to activate the expression of a gene.

The term "B2 receptor antagonist" refers to a compound that selectively blocks or inactivates B2 receptor. As used herein, the term "selectively blocks or inactivates" refers to a compound that preferentially binds to and blocks or inactivates B2 receptor with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the sub-types or isoforms of the bradykinin receptor family (Bl -receptor). Compounds that prefer B2 receptor, but that may also block or inactivate other bradykinin receptor sub-types, as partial or full antagonists, are contemplated. The term "B2-receptor antagonist" refers to any compound that can directly or indirectly blocks the signal transduction cascade related to the B2-receptor. The "B2-receptor antagonist" may also consist in compounds that inhibit the binding of the kinin to B2-receptor such as compounds having the ability to bind kinin with high affinity and specificity or compounds that compete with kinin. Typically, a B2-receptor antagonist is a small organic molecule, a peptide, a polypeptide, an aptamer or an antibody.

Tests and assays for determining whether a compound is a B2-receptor antagonist are well known by the skilled person in the art such as described in Stewart et al, 1999; Whalley et al., 2012; Regoli et al, 1998; Heitsch, 2003; Howl and Payne, 2003; Figueroa et al., 2012; Charignon et al, 2012; Blaes and Girolami, 2013; Altamura et al, 1999.

The B2 receptor antagonists are well-known in the art as illustrated by Stewart et al, 1999; Whalley et al, 2012; Regoli et al, 1998; Heitsch, 2003; Howl and Payne, 2003; Figueroa et al., 2012; Charignon et al., 2012; Blaes and Girolami, 2013; Altamura et al., 1999. In one embodiment of the invention, the B2 receptor antagonist is a peptide such as HOE140 (also known as icatibant and Firazyr®), NPC-349 (DPhe7-BK with p-(2-thienyl)- alanine), PC-17731, CP-0127 (also known as Bradycor™ and deltibant), B9340, B-9430, B9870 (CU201, breceptin), B-9858, B-10056 and compounds described for example in Stewart et al., 1999; Whalley et al, 2012; Regoli et al., 1998; Charignon et al, 2012; Blaes and Girolami, 2013; Altamura et al., 1999 ; WO 2004/068928; CA2514152.

In one embodiment of the invention, the B2 receptor antagonist is a non-peptide compound, such as the acetamidopyridine-methylquinoline structural class represented by FR173657 ((2E)-3-[6-(acetylamino)-3-pyridinyl]-N-[2-[[2,4-dichloro-3-[[(2-methyl-8- quinolinyl)oxy]methyl]phenyl]methylamino]-2-oxoethyl]-2-propenamide CAS: 167838-64- 4)) (Figure 4) and related molecules; LF 16-0687 ((2S)-N-[3-[[4- (aminoiminomethyl)benzoyl]amino]propyl]-l-[[2,4-dichloro-3-[[(2,4-dimethyl-8- quinolinyl)oxy]methyl]phenyl]sulfonyl]-2-pyrrolidinecarboxamide (CAS: 209733-45-9)) (also known as anatibant; XY2405); Bradyzide ((2S)-N-[2-[[2-(dimethylamino) ethyl]methylamino]ethyl]-l-[[4-[2-[[(diphenylmethyl)amino]thioxomethyl]hydrazinyl]-3- nitrophenyl]sulfonyl]-2-pyrrolidinecarboxamide (CAS: 263011-13-8); MEN16132 ((Fastibant); (5S)-5-amino-4-[[4-[[[2,4-dichloro-3-[[(2,4-dimethyl-8- quinolinyl)oxy]methyl]phenyl]sulfonyl]amino]tetrahydro-2H-pyran-4-yl]carbonyl]-N,N,N- trimethyl-8-oxo-l-piperazinepentanaminium chloride (CAS: 869880-33-1)); BKM-570 ((aS)- 4-[(2,6-dichlorophenyl)methoxy]-a-[[l-oxo-3-(2,3,4,5,6-pentafluorophenyl)-2-propen-l- yl]amino]-N-(2,2,6,6-tetramethyl-4-piperidinyl)benzenepropanamide (CAS: 259885-54-6)); WIN 64338; FR 167344; FR 165649; FR 184280 ; quinoline and imidazo[l,2-a]pyridine derivatives and compounds described, for example, in Stewart et al., 1999; Whalley et al, 2012; Regoli et al, 1998; Altamura et al, 1999 ; U.S. Pat. No. 6,211, 181; U.S. Pat. No. 6,500,831.

In another embodiment, the B2 receptor antagonist of the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). Then after raising aptamers directed against B2 receptor of the invention as above described, the skilled man in the art can easily select those inhibiting B2 receptor.

In one embodiment, the compound of the invention is a B2 receptor expression inhibitor.

B2 receptor expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of B2 receptor mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of B2 receptor proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding B2 receptor can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566, 131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as B2 receptor expression inhibitors for use in the present invention. B2 receptor gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that B2 receptor expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as B2 receptor expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of B2 receptor mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful a B2 receptor expression inhibitors can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing B2 receptor. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma vims, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KREGLER (A Laboratory Manual," W.H. Freeman CO., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

Typically the compound according to the invention as described above are administered to the subject in a therapeutically effective amount.

By a "therapeutically effective amount" of the compound of the present invention as above described is meant a sufficient amount of the compound for preventing or treating diabetic ulcers at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the compound of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the compound of the present invention, preferably from 1 mg to about 100 mg of the compound of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In a particular embodiment, the compound according to the invention may be used in a concentration between 0.01 μΜ and 20 μΜ, particularly, the compound of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 μΜ. In a further aspect, the present invention relates to the compound according to the invention in combination with one or more wound healing agents for use in the prevention or treatment of diabetic ulcers in a subject in need thereof.

The term "wound healing agents" has its general meaning in the art and refers to mineralocorticoid receptor antagonists such as spironolactone, eplerenone and drospirenone; and glucocorticoids such as clobetasol, 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25- diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, anecortave acetate and any of their derivatives. According to the present invention, the compound of the invention is administered sequentially or concomitantly with one or more wound healing agent.

The present invention also relates to a method for preventing or treating diabetic ulcers in a subject in need thereof, comprising the step of administering to said subject the compound of the invention.

Pharmaceutical composition and kits of the invention The compound of the invention may be used or prepared in a pharmaceutical composition.

In one embodiment, the invention relates to a pharmaceutical composition comprising the compound of the invention and a pharmaceutical acceptable carrier for use in the prevention or treatment of diabetic ulcers in a subject in need thereof.

Typically, the compound of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The compound of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used. When the compound of the invention is intended for the prevention or treatment of cutaneous wounds and lesions, it may be desirable to it in admixture with a topical pharmaceutically or cosmetically acceptable carrier. The topical pharmaceutically acceptable carrier is any substantially nontoxic carrier conventionally usable for topical administration of pharmaceuticals in which the active ingredient of the invention will remain stable and bioavailable when applied directly to skin surfaces. For example, carriers such as those known in the art effective for penetrating the keratin layer of the skin into the stratum comeum may be useful in delivering the active ingredient of the invention to the area of interest. Such carriers include liposomes. Active ingredient of the invention can be dispersed or emulsified in a medium in a conventional manner to form a liquid preparation or mixed with a semi-solid (gel) or solid carrier to form a paste, powder, ointment, cream, lotion or the like.

Because dermatologic conditions to be treated may be visible, the topical carrier can also be a topical cosmetically acceptable carrier. The topical cosmetically acceptable carrier will be any substantially non-toxic carrier conventionally usable for topical administration of cosmetics in which the compound of the invention will remain stable and bioavailable when applied directly to the skin surface. Suitable cosmetically acceptable carriers are known to those of skill in the art and include, but are not limited to, cosmetically acceptable liquids, creams, oils, lotions, ointments, gels, or solids, such as conventional cosmetic night creams, foundation creams, suntan lotions, sunscreens, hand lotions, make-up and make-up bases, masks and the like. Topical cosmetically acceptable carriers may be similar or identical in nature to the above described topical pharmaceutically acceptable carriers. The compositions can contain other ingredients conventional in cosmetics including perfumes, estrogen, vitamins A, C or E, alpha-hydroxy or alpha-keto acids such as pyruvic, lactic or glycolic acids, lanolin, vaseline, aloe vera, methyl or propyl paraben, pigments and the like.

It may be desirable to have a delivery system that controls the release of the compound of the invention to the skin and adheres to or maintains itself on the skin for an extended period of time to increase the contact time of the compound of the invention on the skin. Sustained or delayed release of compound of the invention provides a more efficient administration resulting in less frequent and/or decreased dosage of the compound of the invention and better patient compliance. Examples of suitable carriers for sustained or delayed release in a moist environment include gelatin, gum arabic, xanthane polymers. Pharmaceutical carriers capable of releasing the compound of the invention when exposed to any oily, fatty, waxy, or moist environment on the area being treated, include thermoplastic or flexible thermoset resin or elastomer including thermoplastic resins such as polyvinyl halides, polyvinyl esters, polyvinylidene halides and halogenated polyolefins, elastomers such as brasiliensis, polydienes, and halogenated natural and synthetic rubbers, and flexible thermoset resins such as polyurethanes, epoxy resins and the like. Controlled delivery systems are described, for example, in U.S. Pat. No. 5,427,778 which provides gel formulations and viscous solutions for delivery of the compound of the invention to a skin site. Gels have the advantages of having a high water content to keep the skin moist, the ability to absorb skin exudate, easy application and easy removal by washing. Preferably, the sustained or delayed release carrier is a gel, liposome, microsponge or microsphere.

Pharmaceutical compositions of the invention may also include one or more wound healing agent.

In one embodiment, said additional active agents may be contained in the same composition or administrated separately.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the prevention and treatment of diabetic ulcers. The invention also provides kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.

Screenin2 method In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for the prevention or treatment of diabetic ulcers in a subject in need thereof, wherein the method comprises the steps of: i) providing candidate compounds and ii) selecting candidate compounds that blocks or inactivates B2 receptor. The present invention also relates to the candidate compound according to the invention for use in the prevention or treatment of diabetic ulcers in a subject in need thereof.

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for the prevention or treatment of diabetic ulcers in a subject in need thereof, wherein the method comprises the steps of:

providing a B2-receptor, providing a cell, tissue sample or organism expressing a B2-receptor,

providing a candidate compound such as small organic molecule, a peptide, a polypeptide, an aptamer or an antibody, measuring the B2-receptor activity,

and selecting positively candidate compounds that inhibit B2-receptor activity.

Methods for measuring B2-receptor activity are well known in the art. For example, measuring the B2-receptor activity involves determining a Ki on the B2-receptor cloned and transfected in a stable manner into a CHO cell line or measuring one or more of the second messengers of the B2-receptor (inositol phosphates (IPs), intracellular Ca²⁺ concentration [Ca²⁺]i, cGMP, cAMP) in the present or absence of the candidate compound (Stewart et al, 1999; Whalley et al, 2012; Regoli et al, 1998; Heitsch, 2003; Howl and Payne, 2003; Figueroa et al, 2012; Charignon et al., 2012; Blaes and Girolami, 2013; Altamura et al., 1999). The B2-receptor activity resulting from the binding of the candidate compound to B2- receptor may be lower than the biological effect resulting from the binding of the B2-receptor to its natural ligands Tests and assays for screening and determining whether a candidate compound is a

B2-receptor antagonist are well known in the art (Stewart et al, 1999; Whalley et al., 2012; Regoli et al, 1998; Heitsch, 2003; Howl and Payne, 2003; Figueroa et al, 2012; Charignon et al, 2012; Blaes and Girolami, 2013; Altamura et al, 1999). In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to inhibit B2- receptor activity.

Activities of the candidate compounds, their ability to bind B2-receptor and their ability to inhibit B2-receptor activity may be tested using isolated keratinocytes, fibroblasts and endothelial cells expressing B2-receptor, CHO cell line cloned and transfected in a stable manner by the human B2-receptor.

Activities of the candidate compounds and their ability to bind to the B2-receptor may be assessed by the determination of a Ki on the B2-receptor cloned and transfected in a stable manner into a CHO cell line and measuring one or more of the second messengers of the B2- receptor (inositol phosphates (IPs), intracellular Ca²⁺ concentration [Ca²⁺]i, cGMP, cAMP) in the present or absence of the candidate compound. The ability of the candidate compounds to inhibit B2-receptor activity may be assessed by the determination of the pD₂, the concentration causing the B2-receptor-dependent contraction of the human umbilical vein, keratinocyte migration, keratinocyte proliferation and fibroblast proliferation.

Cells expressing another receptor than B2-receptor may be used to assess selectivity of the candidate compounds. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: B1R and B2R mRNA levels are increased in diabetic skin.

Kinin receptor mRNA level in non-diabetic and diabetic mouse skin (wounded or not), measured by RT-qPCR. Data are mean±SEM, n=7/group. **:p<0.01 Diab vs NonDiab

Figure 2: B2R but not B1R activation influences wound healing in NonDiab mice and in Diab mice.

Representative photographs of wound area at day 0 and 1 1 and time course of wound closure (A: NonDiab mice; B: STZ-Diab mice; C: db/db-Diab mice). Results are expressed in percentage of initial wound area at day 0. Data are mean±SEM, n=7-30/group. *:p<0.05 and **:p<0.01 vs PBS-control in each series; $:p<0.05 and $$:p<0.01 Diab vs NonDiab.

Figure 3: B2R but not B1R activation increases epidermal thickness and worsens skin disorganization in wounded skin in NonDiab and STZ-Diab mice.

(A) Representative photographs of Massons's trichrome- stained sections of wounds at day 11 (2x; lOx; 25xmagnification) documenting change in the skin organization. Note thickness of epidermis and vascularisation and cellularisation of dermis. Bar = 1mm. (B) Quantification of epidermal thickness of the scar in NonDiab mice and STZ-Diab mice. Data are mean±SEM, n=7/group.

Figure 4: B2R activation unbalances keratinocytes/fibroblasts proliferation.

Cell proliferation after 48h of treatment with B IR-ag, B2R-ag and/or B2R-ant (A: keratinocytes; B: fibroblasts) at indicated doses. Data are mean±SEM, n=6 wells/group.

Figure 5: B2R but not B1R activation stimulates keratinocyte migration.

(A) Representative images of keratinocytes migration (lOxmagnification) at 0, 15 and 30h post scratch. Scratch area is delimited by black box. (B) Scratch closure after 24h of treatment. Data are mean±SEM and expressed as percentage of scratch width measured at Oh. Each data point represents n=8 wells. *:p<0.05; **:p<0.01 and ***:p<0.001 vs CTL. Figure 6: B2R activation induces ERK phosphorylation in keratinocytes.

Phosphorylation of ERK- 1/2 on keratinocyte lysates assessed by Western blotting after 0, 2, 5, 15 and 40 minutes of tretament with B2R-ag (1x10-6 mol.L-1) and/or B2R-ant (1x10- 5 mol.L-1). Results are representative of 3-5 independent experiments. *:p<0.05 vs t=0min.

EXAMPLE:

Material & Methods

Animals, experimental protocols and treatments

Effects of B1R or B2R agonists or B2R antagonist on wound healing were studied, in both non-diabetic (NonDiab) and diabetic (Diab) mice. All mice were housed with a 12 h light/dark cycle and had free access to standard mice chow and water. All experimental procedures were approved by the local Ethics Committee for Animal Experiment Charles Darwin and performed in accordance with European legislation for the care and use of laboratory animals (L 358-86/609/EEC).

Murine model of type 1. diabetes Diabetes was induced in 10 weeks-old mice (C57BL/6J strain, male, Charles River Laboratories, France) by 5 daily i.p. injections of streptozotocin (STZ) (Sigma-Aldrich, France) (50 mg/kg body weight in 0.05 mol/L-1 sodium citrate, pH 4.5)[10,18]. Diabetes was confirmed by assessing fasting blood glucose level at 0, 7 and 30 days post STZ injection. After 5 weeks of confirmed diabetes (fasting blood glucose>300 mg/dl), excisional wounds were created on the back of the mice as described below. Control NonDiab animals were treated with vehicle only.

Murine model of type 2 diabetes. Diabetic db/db mice were obtained from Janvier Labs (Saint-Berthevin, France). Experiments were performed on male mice, 12 week-old. Only mice with 4 weeks of fasting blood glucose>300 mg/dl were used.

Murine model of skin wound healing. Animals were anesthetized by isoflurane inhalation (1.5% in 02). A dorsal 8 mm diameter full-thickness wound was made on dorsal depilated and cleaned (povidone-iodine solution) skin using a sterile biopsy punch (Kai medical, Japan)[ 19,20]. Buprenorphine (0.05 mg/kg; Buprecare, Axience, France) was administered as analgesic agent ten minutes prior and 24 hours after biopsy. Wounds were harvested by taking high-resolution photos every 2-3 days and the area was quantified relative to a millimetre reference using Image Analyzer Software (ImageJ, NIH) and expressed as percentage of wound area measured at day 0. At day 11 after injury, corresponding to wound closure of NonDiab mice, animals were sacrificed by pentobarbital overdose (120 mg/kg) and a skin sample including wound and surrounding tissues was collected. One half of the sample was immediately fixed in 10% formalin for histological analysis, the other part being kept at - 80°C for molecular analyses.

Treatments administration. Immediately after wounding, mice were implanted i.p. with osmotic mini pumps (Alzet, model 1002, Charles River, France) delivering the selective B1R agonist (SarLys[Hyp3,Igl5,DPhe8]desArg9-bradykinin, B IR-ag) or the selective B2R agonist ([Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin, B2R-ag)[21,22], both at non- hypotensive dose of 720 nmol/kg.day-1 (B IR-ag: 796 mg/kg. day-1 ; B2R-ag: 813 mg/kg. day- 1) [10], associated or not with the specific B2R antagonist (HOE140, Icatibant, B2R-ant)[23] at a dose of 380 nmol/kg.day-1 (500 mg/kg. day-1) [24]. B2R-ant was also administered alone at the same dosage. Control mice received only vehicle (Phosphate Buffered Saline, PBS). Treatments were pursued until sacrifice (n=7-30/group).

Histological analysis. Skin biopsies fixed in 10% formalin and embedded in paraffin were cut into 7-μπι section and stained with Masson's trichrome according to the manufacturer's instructions (Sigma- Aldrich, France). Photomicrographs were obtained using digital camera attached to light microscope (Leica DM 4000B and LAS v3.8 software). Epidermal thickness was measured in wounded skin and healthy surroundings area by using Image Analyzer Software (ImageJ, NIH) (3 measurements for each compartment/section, 4 sections/animal, n=7-13/group). Structure and organisation of the granulation tissue and/or the scar were observed. Wound closure and histological studies were performed blindly with regard to treatment.

Real-time PCR. Total RNA was isolated from the skin, collected at day 11, using TRIzol (Invitrogen, France) and reverse transcribed with superscript II reverse transcriptase as previously described[25]. The cDNAs were amplified and quantified using TaqMan Universal Master Mix and Assays-on-Demand Gene Expression Probes for gene of B1R and B2R (Applied Biosystems, France) in an ABI PRISM-7000 Sequence Detection System (Applied Biosystems, France). Each sample was tested in triplicate. Data were normalized to 18S rRNA. Changes in the target gene were calculated by the 2-ΔΔΟΤ comparative method for each sample[26].

Keratinocytes and fibroblasts cell culture

Keratinocytes. (human keratinocyte cell line HaCaT; ATCC, Virginia) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, LifeTechnologies, UK) supplemented with 10% Foetal Bovine Serum (FBS, Gibco, LifeTechnologies, UK) and antibiotics (50 U/mL Penicillin, 50 U/mL Streptomycin, Gibco, LifeTechnologies, UK). Keratinocytes were used between passages 68 and 75. Fibroblasts. (NIH-3T3 mouse fibroblasts; Sigma- Aldrich, Missouri) were cultured in

DMEM supplemented with 5% New Born Calf Serum (NBCS, Gibco, LifeTechnologies, UK), 5% FBS and antibiotics according to manufacturer's guidelines. Fibroblasts were used between passages 162 and 171. Keratinocytes and fibroblasts were kept at 37°C in a 5% C02 environment. Culture media were changed every two-day. When the cells reached subconfluence, they were harvested using 0.05% trypsin-EDTA (Gibco, LifeTechnologies, UK), and fresh culture medium was added for obtaining single cell suspensions used for further study. Cell proliferation assay. Cell proliferation was measured using Quick Cell

Proliferation Assay kit (Abeam, Cambridge, MA) according to the manufacturer's instructions[27]. This assay is based on cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Briefly, keratinocytes or fibroblasts were seeded on 96-well plates (1x104 cells/well) and incubated with two different concentrations of B1R- ag or B2R-ag (1x10-6 mol.L-1 and 1x10-7 mol.L-1) and/or with B2R-ant (1x10-5 mol.L-1) in DMEM supplemented with 2% FBS. Cell proliferation was measured after 24, 48, 72 and 96 hours of incubation. Optical density was measured at 440 nm with a reference wavelength of 650 nm using a precision microplate reader (iMark Microplate Absorbance Reader, BIORAD). Results were expressed as percentage of control (DMEM supplemented with 2% FBS). Each experiment was performed in duplicate per condition and repeated three times.

Wound closure assay. Spreading and migration capabilities of cells were assessed using a scratch assay[28]. Keratinocytes or fibroblasts were seeded on 6-well plates (3x105 cells/well). After reaching confluence, cultures were scratch-wounded with a 0.2 ml sterile pipette tip at the centre of each well and cell debris were removed by PBS washing. For keratinocytes, plates were pre-coated with collagen I (BD Biosciences, California). BIR-ag or B2R-ag (1x10-6 mol.L-1 and 1x10-7 mol.L-1) and/or B2R-ant (1x10-5 mol.L-1) were added in DMEM supplemented with 2% FBS. Mitomycin C (Sigma-Aldrich, Missouri) (l(^g/mL) was also added in medium to inhibit cellular proliferation. After 15 hours, photographs of the scratch width were taken every 3 hours until closure and scratch closure was quantified by monitoring the front of migration. Results were expressed in percentage of scratch width measured at hour 0. Each experiment was realized in duplicate per condition and repeated four times.

Western blotting. Cells were deprived in FBS during 24 hours and then treated 0, 2, 5, 15 and 40 minutes with B2R-ag (1x10-6 mol.L-1) and/or B2R-ant (1x10-5 mol.L-1). Proteins were extracted from treated-cells using standard protein extraction protocols and were dosed using BCA protein assay (Pierce Protein Biology Products, USA). Fifteen^g proteins were separated on a Mini-PROTEAN TGX gel (Biorad, USA) and membranes were blotted with phospho- and total p44/42 MAPK (Erk-1/2) (Thr202/Tyr204) rabbit antibody (dilution 1/1000; Cell signalling technologies, Danvers, USA). They were exposed to ECL plus western blotting reagents (Biorad, USA) and reactive bands were detected in ImageQuant LAS 4000 (GE Healthcare, France) and quantified using Multi Gauge software 2.0 (FujiFilm, Japan). Each blot was then stripped and re-probed with anti-P-actin antibodies for data normalization. Results were confirmed by repeating the experiment 3-5 times.

Statistical analysis

Data are presented as mean±SEM. Statistical analysis was performed using one-way or two-way ANOVA for comparing effect of diabetes and/or treatments in mice. One-way ANOVA was used for comparing effect of treatment on cell migration or proliferation and effect of diabetes on receptor mRNA level. ANOVA was followed by ad-hoc multiple comparison tests. Statistical significance was accepted at p<0.05. Results

Treatments did not affect metabolic parameters. Body weight and plasma glucose level of the different groups of mice at the beginning and end of the experiment are presented in Tablel . Treatment with BIR-ag, B2R-ag and/or B2R-ant did not affect these parameters (Table 1).

TABLE 1: Weight and blood glucose level of different experimental groups at time of wounding (beginning of the experiment) and 11 days after wounding (end of the experiment).

** ><0.001 : glucose level in all Diab groups vs NonDiab. NS among Diab groups.

BIR and B2R mRNA levels are increased in diabetic skin. Analysis of bradykinin receptor levels in healthy skin and wounded area at day 11 demonstrated that BIR mRNA level were increased roughly 3 times in STZ-Diab mice compared to NonDiab mice (p<0.01). Similarly, B2R mRNA level were also increased 2 times compared to Non-Diab mice (p<0.01) (Figure 1). BIR agonist treatment had no effect on wound closure. BIR-ag treatment had no effect on wound closure in NonDiab mice (Figure 1A) and in STZ-Diab mice (Figure IB). Moreover, histological analysis showed that B IR activation did not alter skin organisation and had no effect on epidermal thickness, neither in intact surrounding skin nor in wounded skin (data not shown).

B2R activation delays wound closure in mice. In NonDiab mice, B2R-ag treatment significantly delayed wound healing from the 1 st day of treatment onward (Figure 2A). Co- treatment with the B2R-ant totally abrogated the wound healing delaying effect of B2R-ag. Kinetic profile of wound closure in mice co-treated with B2R-ag and B2R-ant was indistinguishable from PBS-treated mice (Figure 2A). In STZ-Diab mice, wound closure was significantly delayed when compared with NonDiab mice (Figure 2B). On day 11, wounds were healed in NonDiab mice whereas they remained open in STZ-Diab mice (12.7 ± 1.2 %, p<0.05). In STZ-Diab mice, B2R-ag treatment resulted in a further significant delay in wound healing from the 1st day of treatment onward (Figure 2B). On day 11, wound area was 2.3 times larger in B2R-ag-treated STZ-Diab mice compared to PBS-treated STZ-Diab mice (p<0.05).

B2R blockade improves skin wound healing in two models of diabetic mice. B2R-ant alone had no significant effect on wound healing in NonDiab mice (Figure 2A) but significantly improved skin wound healing in diabetic mice. In STZ-Diab mice, wound area was reduced on the 1st day of B2R-ant treatment and was indistinguishable from NonDiab mice during the 11 days follow-up period (Figure 2B). The beneficial effect of B2R-ant on wound healing was also observed in another diabetic model, db/db-Diab mice. In these mice, B2R-ant significantly reduced wound area during the 11th days of treatment (Figure 2C). On day 11, wound area was 3.2 times smaller in B2R-ant-treated db/db-Diab mice compared to PBS-treated db/db-Diab mice (p<0.05).

B2R activation induces epidermal thickening and skin layer disorganization in wounded skin. B2R activation induced no alteration in intact part of skin in NonDiab and STZ-Diab mice (data not shown). But, in the wound of NonDiab and Diab mice, B2R-ag treatment induced important skin layer disorganisation, with a defect in skin and epidermal maturation, hypervascularisation and hypercellularisation of granulation tissue (Figure 3 A). In B2R-ag-treated NonDiab mice, epidermal thickness of wound area was increased compared to PBS-treated mice (11 1.1±8.7 μπι vs 69.9±5.7 μπι; p<0.05). This effect was abrogated by co- treatment with B2R-ant (56.3±8.2 μιη; p<0.05) (Figure 3B). In Diab mice, treatment with B2R-ag had no further effect on epidermal thickness (Figure 3C).

B2R antagonist treatment restores wounded skin histology in diabetic mice. After B2R-ant treatment alone, the epidermis of Diab-mice was histologically indistinguishable from NonDiab mice (Figure 3A). B2R-ant decreased by 45% the epidermal thickness of diabetic wounds (112.4±21.9 μιη vs 206.3±21.6 μιη; p<0.05) (Figure 3C).

B2R activation unbalances keratinocyte and fibroblast proliferation in vitro. Treatment with BIR-ag or B2R-ag significantly increased keratinocyte proliferation, assayed using Quick Cell Proliferation Assay kit (+36% to 46% at 48h, p<0.05) (Figure 4A). Moreover, fibroblast proliferation was significantly decreased by the B2R-ag (-33%) at 48h, p<0.05) but was not altered by the BIR-ag (Figure 4B). These B2R-ag effects on cell proliferation were abolished by co-treatment with B2R-ant (Figure 4). B2R-ant alone had no effect on cell proliferation.

B2R activation stimulates keratinocyte but not fibroblast migration. Figure 5 shows that keratinocyte migration, assessed using scratch assay, was significantly stimulated with B2R-ag during the 30 hours scratch closure process and independently of the dose used (wound closure +32%> compared to medium alone after 24h, p<0.05). This effect was abolished in presence of the B2R-ant (Figure 5B). B2R-ant alone or B IR-ag had no effect on cell migration (Figure 5B). B2R-ag treatment had no effect on fibroblast migration (data not shown). B2R activation induces ERK phosphorylation in keratinocytes. B2R-ag induced phosphorylation of ERK-1/2 in keratinocytes. Peak phosphorylation occurred 5 to 40 min after stimulation (p<0.05) and thereafter, the phosphorylation level gradually decreased (60- 120 min). This effect was blocked by B2R-ant co-treatment (Figure 6). Discussion

The main findings of the present invention are that kinin B2R activation delays healing in non-diabetic and diabetic mouse skin wound models. Treatment with a selective B2R antagonist accelerates skin wound repair in diabetic mice. The healing effect of B2R antagonist was consistent in two different murine models of diabetes, close to either type 1 or type 2 human diabetes. The deleterious effects of B2R activity in the wounded skin may involve unbalancing of keratinocyte and fibroblast proliferation during the re-epithelialisation and scar remodelling phases after injury. Previous reports indicated that B1R and B2R are present in healthy human skin and in skin of patients with cutaneous wound or proliferative skin diseases[l 1-13]. The inventors show that both receptors are present in murine skin and are upregulated by diabetes mellitus.

Kinins can exert their biological actions through these two types of G protein-coupled membrane receptors. While the B2R is constitutively present in most tissues and mediates the main effect of kinins, the B1R is considered as being mainly inducible and its functions are less well understood. Interestingly, the inventors recently showed that B 1R activation protects the heart against ischemia damage [29] and has pro-angiogenic effects in a diabetic mouse model of hindlimb ischemia [10]. However in the skin, B 1R activation had no effect on wound healing in mice, diabetic or non-diabetic. This receptor does not seem to be importantly involved in control of skin trophicity in diabetes, despite its strong induction by chronic hyperglycaemia.

By contrast, B2R activation disorganized the architecture of the wound, increased epidermis thickness and significantly delayed wound healing in mice. The deleterious effect of B2R activation on wound repair was observed in both non-diabetic and diabetic mice and was additive to the effect of diabetes. Co-treatment with a specific B2R antagonist abrogated the effects of B2R agonist administration indicating that these effects were truly due to B2R activation.

Wound healing involves tightly regulated humoral and cellular processes. Alteration of these timely-controlled processes by chronic hyperglycaemia expands tissue damage and delays tissue repair [30]. After a cutaneous injury, the healing process can be divided into four successive, overlapping phases: coagulation, inflammation, cell migration and proliferation, and remodelling. Kinins, through B2R activation, could likely influence several of these phases.

The first steps of wound healing are haemostasis and inflammation [30]. Kinins, through vascular endothelium activation, have anti-clotting and profibrinolytic effects [31,32], increase blood flow and trigger leucocyte migration [33]. In the present invention, pharmacological manipulation of B2R activity influenced wound repair already on the first day of follow-up after surgery. This observation demonstrates that kinins influence the initial phase of wound repair.

But kinins can also influence late phases. The histological results show that B2R agonist induced skin disorganisation and increased epidermal thickness and hypercellular granulation tissue. Moreover, previous studies suggested that bradykinin enhances keratinocyte motility [34] and differentiation even if effect on cell proliferation is unclear [12,14]. All these findings demonstrate that kinins influence proliferative and remodelling phases of healing. In this context, the inventors studied in vitro effect of B2R activation on proliferation and migration of the two major cell types involved in scar formation: keratinocytes and fibroblasts. The results show that B2R activation inhibited fibroblast proliferation but increased keratinocyte proliferation and migration in a cell layer injury model. These in vitro effects of B2R agonist are consistent with the wound repair delaying effect observed in vivo. Indeed, accumulation of fibroblasts, formation of granulation tissue, reconstitution of the dermis and reformation of an intact epidermis via keratinocyte migration and proliferation are important features of the wound healing process [30]. Skin layer disorganization, involving stratum corneum, epidermis and dermis, observed in mice treated with B2R agonist, especially in diabetic mice, may thus reflect unbalance between hypo- proliferation of fibroblasts and hyper-proliferation of keratinocytes, impairing healing. Stimulation of keratinocyte proliferation and migration in vitro by B2R activation is in line with the thickening of epidermis observed in B2R agonist treated mice.

B2R activation effects on keratinocytes were associated with ERK-1/2 phosphorylation. This result is in agreement with previous observations showing that keratinocyte migration, proliferation and wound re-epithelialization involve ERK activation [35-37] and confirms the importance of this pathway in keratinocyte functionality.

The deleterious effect of B2R stimulation on wound healing was additive to the effect of diabetes. Abnormal keratinocyte and fibroblast migration, proliferation and differentiation, and decreased vascularisation contribute to delayed wound healing in diabetic patients [4,38]. In these patients, keratinocytes in epidermis of chronic ulcers are highly proliferative [39]. B2R agonist treatment probably worsened keratinocyte hyper-proliferation due to diabetes and stimulated migration, resulting in abnormal scar remodelling.

Interestingly, whereas B2R antagonist alone had no effect on the wounded skin in non- diabetic mice, it significantly accelerated wound repair in diabetic mice. Indeed, B2R blockade restored the normal proliferation and maturation pattern of the wounded epidermis in a murine model of type 1 diabetes (STZ). The beneficial effect on wound healing of B2R antagonist was extended to a second diabetic mouse model, closer to type 2 human diabetes (db/db mice). These observations indicate that endogenous kinins exert deleterious effects opposing wound repair in diabetic skin. The lack of effect of B2R antagonist in non-diabetic mice may be explained by low kinins bioavailability and/or reduced B2R synthesis in the skin in normoglycaemic condition. By contrast, in diabetes, B2R synthesis is upregulated in the skin and kinins production may also be enhanced. In conclusion, B2R but not B1R activation exerts deleterious effects in the wounded mouse skin resulting in delayed healing, especially in diabetic animals. Bl and B2 receptors are upregulated by diabetes in skin. Treatment with a B2R antagonist accelerates wound repair in two different mouse models of diabetes. Improving skin healing is still a therapeutic issue in diabetic patients, especially for treatment of foot ulcers. A kinin B2R antagonist, already approved clinically in an unrelated indication [40], is a new treatment for diabetic cutaneous injury.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Ramsey S.D., Newton K., Blough D., McCulloch D.K., Sandhu N., et al. (1999) Incidence, outcomes, and cost of foot ulcers in patients with diabetes. Diabetes Care 22: 382-387

2. Bartus C.L., Margolis D.J. (2004) Reducing the incidence of foot ulceration and amputation in diabetes. Curr. Diab. Rep. 4: 413-418

3. Boulton A.J., Vileikyte L., Ragnarson-Tennvall G., Apelqvist J. (2005) The global burden of diabetic foot disease. Lancet 366: 1719-1724

4. Brem H., Tomic-Canic M. (2007) Cellular and molecular basis of wound healing in diabetes. J Clin Invest 117: 1219-1222

5. Falanga V. (2005) Wound healing and its impairment in the diabetic foot. Lancet 366: 1736-1743 6. Couture R., Blaes N., Girolami J.P. (2014) Kinin receptors in vascular biology and pathology. Curr. Vase. Pharmacol. 12: 223-248

7. Waeckel L., Potier L., Richer C, Roussel R., Bouby N., et al. (2013) Pathophysiology of genetic deficiency in tissue kallikrein activity in mouse and man. Thromb. Haemost. 110: 476-483

8. Furchgott R.F., Vanhoutte P.M. (1989) Endothelium-derived relaxing and contracting factors. FASEB J. 3 : 2007-2018

9. Couture R., Harrisson M., Vianna R.M., Cloutier F. (2001) Kinin receptors in pain and inflammation. Eur. J. Pharmacol. 429: 161-176

10. Desposito D., Potier L., Chollet C, Gobeil F. Jr., Roussel R., et al. (2015) Kinin receptor agonism restores hindlimb postischemic neovascularization capacity in diabetic mice. J. Pharmacol. Exp .Ther. 352: 218-226

11. Komatsu N., Takata M., Otsuki N., Toyama T., Ohka R., et al. (2003) Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages. J. Invest. Dermatol. 121 : 542-549

12. Vidal M.A., Astroza A., Matus C.E., Ehrenfeld P., Pavicic F., et al. (2005) Kinin B2 receptor-coupled signal transduction in human cultured keratinocytes. J. Invest. Dermatol. 124: 178-186

13. Schremmer-Danninger E., Naidoo S., Neuhof C, Valeske K., Snyman C, et al. (2004) Visualisation of tissue kallikrein, kininogen and kinin receptors in human skin following trauma and in dermal diseases. Biol. Chem. 385: 1069-1076

14. Poblete M.T., Reynolds N.J., Figueroa CD., Burton J.L., Muller-Esterl W., et al. (1991) Tissue kallikrein and kininogen in human sweat glands and psoriatic skin. Br. J. Dermatol. 124: 236-241

15. Milia A.F., Del Rosso A., Pacini A., Manetti M., Marrelli A., et al. (2005) Differential expression of tissue kallikrein in the skin of systemic sclerosis. Histol. Histopathol. 20: 415- 422

16. Pietrovski E.F., Paludo K.S., Mendes D.A., Guimaraes F.de S., Veiga S.S., et al. (2011) Bl and B2 kinin receptor participation in hyperproliferative and inflammatory skin processes in mice. J. Dermatol. Sci. 64: 23-30

17. Warren J.B., Loi R.K. (1995) Captopril increases skin microvascular blood flow secondary to bradykinin, nitric oxide, and prostaglandins. FASEB J. 9: 411-418 18. Johnson M.S., Ryals J.M., Wright D.E. (2008) Early loss of peptidergic intraepidermal nerve fibers in an STZ-induced mouse model of insensate diabetic neuropathy. Pain 140: 35- 47

19. Ebrahimian T.G., Squiban C, Roque T., Lugo-Martinez H., Hneino M., et al. (2012) Plasminogen activator inhibitor- 1 controls bone marrow-derived cells therapeutic effect through MMP9 signaling: role in physiological and pathological wound healing. Stem Cells 30: 1436-1446

20. Balaji S., Lesaint M., Bhattacharya S.S., Moles C, Dhamija Y., et al. (2014) Adenoviral- mediated gene transfer of insulin-like growth factor 1 enhances wound healing and induces angiogenesis. J. Surg. Res.

21. Cote J., Savard M., Bovenzi V., Belanger S., Morin J., et al. (2009) Novel kinin B l receptor agonists with improved pharmacological profiles. Peptides 30: 788-795

22. Belanger S., Bovenzi V., Cote J., Neugebauer W., Amblard M., et al. (2009) Structure- activity relationships of novel peptide agonists of the human bradykinin B2 receptor. Peptides 30: 777-787

23. Wirth K., Hock F.J., Albus U., Linz W., Alpermann H.G., et al. (1991) Hoe 140 a new potent and long acting bradykinin-antagonist: in vivo studies. Br. J. Pharmacol. 102: 774-777

24. Tschope C, Seidl U., Reinecke A., Riester U., Graf K., et al. (2003) Kinins are involved in the antiproteinuric effect of angiotensin-converting enzyme inhibition in experimental diabetic nephropathy. Int. Immunopharmacol. 3 : 335-344

25. Bodin S., Chollet C, Goncalves-Mendes N., Gardes J., Pean F., et al. (2009) Kallikrein protects against microalbuminuria in experimental type I diabetes. Kidney Int. 76: 395-403

26. Livak K.J., Schmittgen T.D. (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25: 402-408

27. Guo S., Al-Sadi R., Said H.M., Ma T.Y. (2013) Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14. Am. J. Pathol. 182: 375-387

28. Fronza M., Heinzmann B., Hamburger M., Laufer S., Merfort I. (2009) Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts. J. Ethnopharmacol. 126: 463-467

29. Potier L., Waeckel L., Vincent M.P., Chollet C, Gobeil F. Jr., et al. (2013) Selective kinin receptor agonists as cardioprotective agents in myocardial ischemia and diabetes. J. Pharmacol. Exp. Ther. 346: 23-30 30. Li J., Chen J., Kirsner R. (2007) Pathophysiology of acute wound healing. Clin. Dermatol. 25: 9-18

31. Brown N.J., Gainer J.V., Murphey L.J., Vaughan D.E. (2000) Bradykinin stimulates tissue plasminogen activator release from human forearm vasculature through B(2) receptor- dependent, NO synthase-independent, and cyclooxygenase-independent pathway. Circulation 102: 2190-2196

32. Murphey L.J., Malave H.A., Petro J., Biaggioni L, Byrne D.W., et al. (2006) Bradykinin and its metabolite bradykinin 1-5 inhibit thrombin-induced platelet aggregation in humans. J. Pharmacol. Exp. Ther. 318: 1287-1292

33. Blaes N., Girolami J.P. (2013) Targeting the 'Janus face' of the B2-bradykinin receptor. Expert. Opin. Ther. Targets 17: 1145-1166

34. Coutant K.D., Corvaia N., Ryder N.S. (1997) Bradykinin induces actin reorganization and enhances cell motility in HaCaT keratinocytes. Biochem. Biophys. Res. Commun 237: 257- 261

35. Gao L., Chao L., Chao J. (2010) A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: activation of proteinase-activated receptor 1 and epidermal growth factor receptor. Exp. Cell. Res. 316: 376-389

36. Li W., Henry G., Fan J., Bandyopadhyay B., Pang K., et al. (2004) Signals that initiate, augment, and provide directionality for human keratinocyte motility. J. Invest. Dermatol. 123 : 622-633

37. Pastore S., Mascia F., Mariani V., Girolomoni G. (2008) The epidermal growth factor receptor system in skin repair and inflammation. J. Invest. Dermatol. 128: 1365-1374

38. Albiero M., Menegazzo L., Boscaro E., Agostini C, Avogaro A., et al. (2011) Defective recruitment, survival and proliferation of bone marrow-derived progenitor cells at sites of delayed diabetic wound healing in mice. Diabetologia 54: 945-953

39. Usui M.L., Mansbridge J.N., Carter W.G., Fujita M., Olerud J.E. (2008) Keratinocyte migration, proliferation, and differentiation in chronic ulcers from patients with diabetes and normal wounds. J. Histochem. Cytochem. 56: 687-696

40. Cicardi M., Banerji A., Bracho F., Malbran A., Rosenkranz B., et al. (2010) Icatibant, a new bradykinin-receptor antagonist, in hereditary angioedema. N. Engl. J. Med. 363 : 532-541

Claims

CLAIMS:

1. A method for preventing or treating diabetic ulcer in a subject in need thereof, comprising the step of administering to said subject a compound selected from the group consisting of B2 receptor antagonists, B2 receptor expression inhibitors, kinin expression inihibitors, kininogen expression inhibitors, kallikreins expression inhibitors, kininase expression activators or kininase activators.

2. The method according to claim 1 wherein said diabetic ulcer is diabetic foot ulcer.

3. The method according to claim 1 wherein said B2 receptor antagonist is selected from the group consisting of small organic molecules, peptides, polypeptides, aptamers, and antibodies.

4. The method according to claim 3 wherein said peptide is selected from the group consisting of HOE 140, PC-349, PC-17731, CP-0127, B9340, B-9430, B9870, B-9858, and B-10056.

5. A pharmaceutical composition for use in the prevention or treatment of diabetic ulcers in a subject in need thereof comprising a compound selected from the group consisting of B2 receptor antagonists, B2 receptor expression inhibitors, kinin expression inihibitors, kininogen expression inhibitors, kallikreins expression inhibitors, kininase expression activators or kininase activators and a pharmaceutical acceptable carrier.

6. A method of screening a candidate compound for use as a drug for the prevention or treatment of diabetic ulcers in a subject in need thereof, wherein the method comprises the steps of:

providing a B2-receptor, providing a cell, tissue sample or organism expressing a B2-receptor,

- providing a candidate compound such as small organic molecule, a peptide, a polypeptide, an aptamer or an antibody,

measuring the B2-receptor activity,

and selecting positively candidate compounds that inhibit B2-receptor activity.