FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular in dermatology.

BACKGROUND OF THE INVENTION:

Impaired cutaneous wound healing, responsible for chronic ulcers, represents one of the most important complications of diabetes. Indeed, the incidence of diabetic foot ulcers is increasing because of the high prevalence of diabetes mellitus worldwide and the longer life expectancy of patients (Boulton et al., 2005). The prevalence of global diabetic foot ulcers was recently reported to be 6.3% and up to 13.0% in North America (Zhang et al., 2017). Ulcers remain in a chronic inflammation state that prevents them from healing, resulting in other severe complications, such as pain, infection, and eventually amputation (Greenhalgh, 2003, Noor et al, 2017, Peters and Lipsky, 2013). Previous studies showed that most diabetic amputations are preceded by a foot ulceration, which subsequently results in serious gangrene or infection (Boulton et al, 2005, Lepantalo et al., 2011). Diabetic foot ulcers are thus a major health issue that significantly affects the lives of patients, resulting in a high financial burden in many countries (Boulton et al., 2005). The prevention and appropriate care of diabetic ulcers are thus of major importance.

Several therapies have been proposed for diabetic ulcers but effective treatment is still needed (Clokie et al, 2017, Dinh et al, 2012, Galiano et al., 2004). The lack of therapeutic tools likely results from the complex mechanisms involved in the development of unhealed wounds. Many pathogenic factors, such as vascular defects and neuropathy, are responsible for diabetic ulcers (Ahmed and Antonsen, 2016, Boulton, 2014, Brem and Tomic-Canic, 2007, Dinh et al, 2012). A chronic inflammatory environment is also a common feature observed in unhealed wounds and is mainly associated with the uncontrolled recruitment and activation of inflammatory cells, in particular monocytes/macrophages (Boniakowski et al., 2017, Leal et al., 2015, Okizaki et ak, 2015).

During the wound-repair process, macrophages present various phenotypes and functions, depending on the stage of the healing response and how they are activated. During the early phase of wound healing, classically activated macrophages, known as Ml macrophages, are recruited and secrete pro-inflammatory cytokines to kill pathogens and clear away the damaged tissue. In contrast, alternatively activated M2 macrophages secrete anti- inflammatory factors to resolve inflammation and produce factors required for later regenerative phases. The balance between these macrophage subpopulations is pivotal for maintaining a physiological healing process (Gordon, 2003, Mahdavian Delavary et al, 2011, Mantovani et al, 2004). However, in unhealed wounds, such as diabetic ulcers, macrophages are chronically activated and restrained to the Ml phenotype, heavily contributing to the chronic inflammatory microenvironment observed in these wounds. Moreover, such prolonged inflammation delays the process of tissue regeneration, including re-epithelialization, granulation tissue formation, and vascularization (Boniakowski et al., 2017, He et al., 2017, Maruyama et al, 2007, Okizaki et al, 2015). Enhancing macrophage polarization towards M2 phenotype may help to promote cellular proliferation and angiogenesis and to accelerate diabetic wound closure (He et al., 2017, Leal et al., 2015, Okizaki et al., 2015).

It was recently reported the involvement of MR activation in delayed wound closure in type-1 diabetes and the improvement of impaired wound re-epithelialization following local application of a MR antagonist to the wounds (Nguyen et al., 2016). It was also recently shown that NGAL is a primary target of aldosterone/mineralocorticoid receptor signaling in many organs and that NGAL plays a key role in the action of mineralocorticoids in the cardiovascular system (Buonafme et al, 2018). However, its role in chronic wounds has never been investigated.

SUMMARY OF THE INVENTION:

As defined by the invention, the present invention relates to the use of NGAL inhibitor for the treatment of chronic wound.

DETAILED DESCRIPTION OF THE INVENTION:

Chronic wounds and in particular diabetic ulcers are a serious complication of diabetes. Unresolved inflammation, associated with the dysregulation of both the phenotype and function of macrophages, is involved in the poor healing of diabetic wounds. Here, the inventor report that topical pharmacological inhibition of the mineralocorticoid receptor (MR) by canrenoate or MR siRNA can resolve inflammation to improve delayed skin wound healing in diabetic mouse models; importantly, wounds from normal mice are unaffected. Furthermore, they show that MR blockade leads to downregulation of the MR target, lipocalin 2 (Lcn or NGAL), which may facilitate macrophage polarization towards the M2 phenotype and improve impaired angiogenesis in diabetic wounds. Indeed, diabetic Lcn2-deficient mice showed improved wound healing, associated with macrophage M2 polarization and angiogenesis. In addition, recombinant Lcn2 protein prevented IL4-induced macrophages switch from Ml to M2 phenotype. In conclusion, inhibiting the activity or expression of NGAL would very suitable for the treatment of chronic wound.

Accordingly, the present invention relates to a method of treating chronic wound in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a NGAL inhibitor.

In some embodiments, the method herein disclosed is particularly suitable for the treatment of chronic wound. As used herein, the term“chronic wound” or“delayed wound closure” refers to those wounds that do not heal in an orderly set of stages and in a predictable amount of time. Typically, wounds that do not heal within three months are considered chronic.

Examples of chronic wounds include, but are not limited to, venous stasis ulcers, diabetic foot ulcers, and the like. Chronic, wounds may also include those relating to trauma (or repeated trauma), thermal injury (e.g., burns) and radiation damage. In some embodiments, treatment of chronic wound in subject suffering from sickle-cell disease is also encompassed. In some embodiments, treatment of chronic wound in elderly is also encompassed.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term“NGAL” or“Lcn2” has its general meaning in the art and refers to the Neutrophil Gelatinase- Associated Lipocalin as described in Schmidt-Ott KM. et al. (2007) (Schmidt-Ott KM, Mori K, Li JY, Kalandadze A, Cohen DJ, Devarajan P, Barasch J. Dual action of neutrophil gelatinase-associated lipocalin. J Am Soc Nephrol. 2007 Feb; 18(2):407-13. Epub 2007 Jan 17. Review.). An exemplary human amino acid sequence is represented by SEQ ID NO: 1.

SEQ ID NO: l>sp I P80188 I NGAL HUMAN Neutrophil gelatinase-associated lipocalin OS=Homo sapiens OX=9606 GN=LCN2 PE=1 SV=2

MPLGLLWLGLALLGALHAQAQDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYWGLAGNA

ILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNI KSY

PGLTSYLVRWSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKS LG

LPENHIVFPVPIDQCIDG

As used herein, the term“NGAL inhibitor” refers to a molecule that partially or fully blocks, inhibits, or neutralizes a biological activity or expression of NGAL. Suitable inhibitor molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, recombinant proteins or peptides, etc. A NGAL inhibitor can be a molecule of any type that interferes with the signaling associated with NGAL, for example, either by decreasing transcription or translation of NGAL encoding nucleic acid, or by inhibiting or blocking NGAL activity, or both. In some examples, a NGAL inhibitor is an agent that interferes with the signaling associated with NGAL. Examples of NGAL inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, NGAL -specific aptamers, anti-NGAL antibodies, NGAL-binding fragments of anti- NGAL antibodies, NGAL-binding small molecules, NGAL-binding peptides, and other polypeptides that specifically bind NGAL (including, but not limited to, NGAL-binding fragments of one or more NGAL ligands, optionally fused to one or more additional domains), such that the interaction between the NGAL inhibitor and NGAL results in a reduction or cessation ofNGAL activity or expression. It will be appreciated that NGAL inhibitors described herein may be strong inhibitors ofNGAL.

In particular, the NGAL inhibitor is a small molecule, such as a small organic molecule, which typically has a molecular weight less than 5,000 kDa.

In some embodiments, the NGAL inhibitor is an anti-NGAL antibody. As used herein, the term “antibody” as includes but is not limited to polyclonal, monoclonal, humanized, chimeric, Fab fragments, Fv fragments, F(ab’) fragments and F(ab’)2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. Antibodies can be made by the skilled person using methods and commercially available services and kits known in the art. Methods of preparation of monoclonal antibodies are well known in the art and include hybridoma technology and phage display technology. Further antibodies suitable for use in the present disclosure are described, for example, in the following publications: Antibodies A Laboratory Manual, Second edition. Edward A. Greenfield. Cold Spring Harbor Laboratory Press (Sep. 30, 2013); Making and Using Antibodies: A Practical Handbook, Second Edition. Eds. Gary C. Howard and Matthew R. Kaser. CRC Press (Jul. 29, 2013); Antibody Engineering: Methods and Protocols, Second Edition (Methods in Molecular Biology). Patrick Chames. Humana Press (Aug. 21, 2012); Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Eds. Vincent Ossipow and Nicolas Fischer. Humana Press (Feb. 12, 2014); and Human Monoclonal Antibodies: Methods and Protocols (Methods in Molecular Biology). Michael Steinitz. Humana Press (Sep. 30, 2013)).

In some embodiments, the NGAL inhibitor is an inhibitor of NGAL expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of NGAL mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of NGAL, 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 NGAL can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting 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 inhibitors of expression for use in the present invention. NGAL gene expression can be reduced by contacting a patient 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 NGAL gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs 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, shRNA or ribozyme nucleic acid to the cells and typically cells expressing NGAL. Typically, 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, shRNA 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 virus, murine mammary tumor virus, and rous 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. In some embodiments, the inhibitor of expression is an endonuclease. In a particular embodiment, the endonuclease is CRISPR-cas. In some embodiment, the endonuclease is CRISPR-cas9, which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 B1 and US 2014/0068797. In some embodiment, the endonuclease is CRISPR-Cpfl, which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA- guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of drug may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of drug to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active ingredient (i.e. the NGAL inhibitor) are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for drug depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of drug employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of drug is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target.

Typically, the drug of the present invention is administered to the subject in the form of a pharmaceutical composition, which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a subject, the composition will be formulated for administration to the subject. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

In some embodiments, it may be desirable to administer the active ingredient (i.e. the NGAL inhibitor) of the invention 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 (i.e. the NGAL inhibitor) will remain stable and bioavailable when applied directly to skin or corneal 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 (i.e. the NGAL inhibitor) to the area of interest. Such carriers include liposomes wherein the active ingredient of the invention (i.e. the NGAL inhibitor) 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.

Suitable topical pharmaceutically acceptable carriers include water, buffered saline, petroleum jelly (vaseline), petrolatum, mineral oil, vegetable oil, animal oil, organic and inorganic waxes, such as microcrystalline, paraffin and ozocerite wax, natural polymers, such as xanthanes, gelatin, cellulose, collagen, starch, or gum arabic, synthetic polymers, alcohols, polyols, and the like. The carrier can be a water miscible carrier composition. Such water miscible, topical pharmaceutically acceptable carrier composition can include those made with one or more appropriate ingredients outset of therapy.

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 active ingredient of the invention (i.e. the NGAL inhibitor) 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 active ingredient of the invention (i.e. the NGAL inhibitor) to the wound and adheres to or maintains itself on the wound for an extended period of time to increase the contact time of the active ingredient of the invention (i.e. the NGAL inhibitor) on the wound. Sustained or delayed release of active ingredient of the invention (i.e. the NGAL inhibitor) provides a more efficient administration resulting in less frequent and/or decreased dosage of active ingredient of the invention (i.e. the NGAL inhibitor) 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 active ingredient of the invention (i.e. the NGAL inhibitor) 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 active ingredient of the invention (i.e. the NGAL inhibitor) to a wound site. Gels have the advantages of having a high water content to keep the wound moist, the ability to absorb wound exudate, easy application and easy removal by washing. Preferably, the sustained or delayed release carrier is a gel, liposome, microsponge or microsphere.

The active ingredient of the invention (i.e. the NGAL inhibitor) can also be administered in combination with other pharmaceutically effective agents including, but not limited to, antibiotics, other wound healing agents, and antioxidants.

The route of administration of the active ingredient of the invention (i.e. the NGAL inhibitor) will depend on the site of the wound and the type and extent of the injury. Any suitable application method can be used as long as an effective amount of the active ingredient of the invention (i.e. the NGAL inhibitor) is able to reach the areas which require reepithelialisation to occur. Routes of administration include, but are not limited to, topical, transdermal and parenteral. Typically, the ingredient of the invention will be administered by topical or transdermal application.

Topical administration for cutaneous treatment is accomplished via a topically applied solution, cream, ointment, gel or other suitable formulation healing bandage which can then be applied to the wound such that the active ingredient of the invention (i.e. the NGAL inhibitor) composition contacts the wound. Examples of suitable transdermal devices are described, for instance, in U.S. Pat. No. 4,818,540. The active ingredient of the invention (i.e. the NGAL inhibitor) can be mixed with a pharmaceutically acceptable cream, applied to the wound, and covered with an occlusive dressing. Alternatively, the wound area can be irrigated or soaked with a solution of the active ingredient of the invention (i.e. the NGAL inhibitor). The solution will be applied two to twelve times per day. For transdermal application, the active ingredient of the invention (i.e. the NGAL inhibitor) is formulated in a composition capable of allowing the active ingredient of the invention (i.e. the NGAL inhibitor) to penetrate the skin and site of the wound. Such compositions are applied directly to the skin or incorporated into a protective carrier such as a transdermal or "patch" device. The active ingredient of the invention (i.e. the NGAL inhibitor) formulations for transdermal administration can be used to coat the fibers of an absorbent gauze dressing.

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. Lcn2 deficiency prevents the diabetes-induced delay of wound healing

Lcn2mRNA (A) and protein (B) levels in wounds at day 5 were analyzed by real-time PCR and ELISA. C-G: Diabetes was induced in wildtype (CT) and Lcn2 KO mice by STZ injections before wounding. Quantification of the wound area (C) from CT and Lcn2 KO mice with or without STZ treatment at the indicated times post-wounding. Ml (D) and M2 (E) macrophages and endothelial cells (F) of wounds at day 5 were quantified by FACS analysis. Total macrophages were sorted from wounded skin at day 5 and angiogenic factors mRNA levels were analyzed by real-time PCR (G). Data represent mean ± SEM; n= number of mice per group, from 2 experimental series. Statistics: a, e, f, g: one-way ANOVA followed by the Newman-Keuls Multiple Comparison test; d: 2-way ANOVA followed by the Newman-Keuls Multiple Comparison test. *E<0.05; ** <0.01; ***/ ^> <o.001; **** ^> <().0001 CT + STZ vs CT. ^§§ E<0.01 CT + STZ vs KO Lcn2 + STZ (d).

EXAMPLE:

Methods

Wound healing in vivo

Two mouse models of diabetes were used for studying wound healing in vivo: streptozotocin-induced type I diabetes ad type II diabetes (db-db mice), with their appropriate controls. Wounds were generated with 6 mm biopsy punch. For the db-db groups, wounds were done at 10 weeks of age. At that time all the dorsum skin displayed a homogenous pink color. For the streptozotocin groups, diabetes was induced at 10 weeks and we performed wounds at 15 weeks of age. We have given a special attention to select dorsum areas at a telogen stage, featured by pink skin color after hair clipping. The histology of the lateral skin surrounding the healing wounds in both models confirmed our clinical evaluation since we could not find any evidence of anagen hair follicles. MR blockade over the wound was achieved by local application of the MR antagonist potassium canrenoate (Canre) or PBS and wounds were photographed to evaluate wound closure. At day 5 (STZ groups) or 7 (db-db groups), mice were euthanized and wound and skin specimens were collected for RNA and protein extraction, immunolabelling, or FACS analysis.

Diabetes was also induced by STZ injections in 10 week-old female Lipocalin 2 knockout mice (Berger et ah, 2006) and their appropriate controls before wounding.

Local MR silencing by siRNA in wounds of diabetic type 1 mouse model

Mouse MR Stealth siRNAs (Set of 3, MSS201383, MSS201384, MSS272869) were purchased from ThermoFisher Scientific (Renfrew, UK). Stock solution was made by adding lml H20 to 20 nMol MR siRNA set. The siRNA working solution was prepared by 1 : 10 dilution of siRNA stock solution with Mirus transfection reagent. After wounding, 100 pi of siRNA solution (0.2 nMol) was locally injected with needle into each wound at day 0 and day 2. The scrambled siRNA (Stealth™ RNAi negative control, medium GC duplex, ThermoFischer Scientific) was prepared according to manufacturer’s instruction and used as negative control. Photographs of wounds at day 3 and day 5 were used to evaluate the degree of wound closure. Mice were sacrificed at day 5, and wounded skin samples were collected for histological and molecular analysis.

Animal study approval statement

All animal experiments were approved by the Darwin ethics committee of Pierre et Marie Curie University and the French Ministry of Research (APAFIS#4438- 2015092514508030 v7) and in accordance with the INSERM guidelines and European Community directives for the care and use of laboratory animals.

Results

MR blockade rescues delayed cutaneous wound closure in diabetic mice

We have shown previously that local MR blockade with Canrenoate (Canre) improves the delayed wound re-epithelialization of type-1 diabetic mice (STZ mice) (Nguyen et al., 2016). To exclude MR-independent effects of Canrenoate, we designed an in vivo experimental strategy to interfere specifically with the MR. Topical treatment with MR siRNA blocked MR upregulation in STZ wounds (data not shown) and restored the defective re-epithelialization of STZ mice, as compared to those of diabetic mice treated by scrambled siRNA (data not shown). These results are quite comparable to those previously obtained using Canrenoate, indicating that MR antagonism/silencing is beneficial for wounding in type I diabetic mice.

Next we questioned the effect of Canrenoate on the delayed wound healing of type 2 diabetes using db-db mice, a genetic mouse model of type 2 diabetes. MR mRNA expression was higher in wounded skin and the surrounding normal skin of db-db mice than that of normal db/+ control (CT) mice (data not shown), raising the question of the role of MR overexpression in abnormal wound repair in type 2 diabetes. Wound healing was strongly impaired in db-db mice relative to that of CT mice (data not shown). Consistent with our observations in STZ mice, local treatment with Canre improved the wound healing delay in db-db mice while it did not modify wound closure in normal CT mice (data not shown). Moreover, keratin- 14 staining showed that impaired re-epithelialization of db-db wounds was rescued by local Canre treatment, as illustrated by the longer neo-epidermis at the edges of the wound sections (data not shown) and the shorter residual wound length (the distance between two edges) in Canre- treated db-db mice than in PBS-treated db-db mice (data not shown). This reduction in wound surface is indicative of re-epithelialization rather than wound contraction. The improvement of re-epithelialization was accompanied by a higher number of proliferating Ki67-positive keratinocytes in the neo-epidermis of the wounds of canre-treated than PBS-treated db-db mice (data not shown). These observations suggest that the effects of topical Canre treament on diabetic wound healing are mediated by MR signaling. Thus, topical MR blockade restored the impaired proliferation of epidermal keratinocytes and blunted the delayed re-epithelialization of wounds in 2 diabetes mouse models. In contrast, these phenomena were not found in wounds of normal CT mice.

Impaired wound angiogenesis in diabetic mice is improved by MR antagonism

Impaired dermal wound angiogenesis is a key contributor to the wound healing defect of diabetes. Herein, we explored the role of MR in the defective wound angiogenesis of diabetic mouse models. The density of CD31 ⁺ blood vessels was lower in the wounds of STZ-induced (data not shown) and db-db diabetic mice than those of control mice (data not shown), consistent with previous reports. Decreased vessel density of diabetic wounds was partly rescued by topical treatment of these wounds with Canre (data not shown). MR inactivation by local MR siRNA treatment of STZ wounds also showed an beneficial effect on wound angiogenesis (data not shown). The improvement of vessel density in Canre-treated diabetic wounds was associated with an increased number of CD45 CD31 ⁺ endothelial cells in the wound beds, as quantified by FACS analysis of wound specimens (data not shown). Moreover, by quantification of CD31 ⁺ Ki67 ⁺ double positive cells on wound sections of STZ mice (data not shown), we found less proliferating endothelial cells in STZ wounds as compared to those of controls (2.87% ± 1.08, n=7 vs 7.18% ± 0.71, n=7, respectively), and this decrease was rescued by Canre treatment (STZ + PBS: 2.87% ± 1.08, n=7 vs STZ + Canre: 6.37% ± 1.2, n=7). These observations suggest that the activation of MR in skin of diabetic mice is involved in the impaired wound angiogenesis of these animals and that MR blockade could prevent such defects through increased proliferation of endothelial cells forming novel microvessels in wounds.

MR blockade ameliorates inflammation of diabetic wounds

Chronic inflammation is a common feature of diabetic ulcers. We examined whether MR plays a role in maintaining inflammation in the diabetic wounds by studying the impact of MR blockade on the expression of some pro/anti-inflammatory markers in wounds (Ashcroft et ah, 2012, Barrientos et al, 2008, Ramalho et ah, 2018). Local treatment with Canre attenuated the overexpression of some pro-inflammatory genes, such as TNF-a, MCP-1, IL-12, and IL-23 in diabetic wounds (data not shown). Consistent with this effect, the repression of anti-inflammatory factors in diabetic wounds was rescued by Canre treatment (data not shown). Thus, MR blockade blunted the inflammation observed in diabetic wounds and resulted in an anti-inflammatory status that could improve the impaired cellular proliferation and poor angiogenesis.

MR antagonism induces the switch of unrestrained Ml towards M2 macrophages in diabetic wounds

A failure to switch from activated Ml macrophages to alternative M2 macrophages leads to chronic inflammation and impaired angiogenesis in various situations of delayed wound healing, including venous leg ulcers and diabetic foot ulcers (Guo et al, 2016, Okizaki et al., 2015, Sindrilaru et al., 2011). We investigated whether the beneficial effect of canrenoate treatment was associated with a switch of activated Ml macrophages towards the alternative M2 macrophage population. Wounds from both STZ and db-db mice showed more Ml macrophages (Ly6C ^l ) associated with fewer M2 macrophages (Ly6C ^low ) than those of control mice, whereas the total number of macrophages in diabetic wounds was not different from that in control wounds (data not shown). These observations are consistent with a number of previous reports showing the accumulation and persistence of activated Ml macrophages in diabetic wounds (Guo et al., 2016, Leal et al., 2015, Okizaki et al., 2015). Importantly, the polarization of diabetic-wound macrophages was nearly restored to the levels of control mice by topical canrenoate treatment: Canre blunted the increase in Ml macrophages in STZ and db- db wounds (data not shown) and the associated decrease in M2 macrophages (data not shown). MR blockade also blunted over-expression of the pro-inflammatory marker Ly6C by diabetic- wound macrophages (data not shown). These results suggest that topical MR blockade promotes a shift of pro-inflammatory Ml macrophages towards the anti-inflammatory M2 phenotype to resolve inflammation, promote angiogenesis, and accelerate wound healing.

MR blockade rescues the impaired macrophage expression of angiogenic factors in diabetic wounds

In addition to their pathogen-killing activity, macrophages in wounds also promote cellular proliferation and tissue regeneration, including re-epithelialization and dermal vascularization (Gordon, 2003, Lucas et al, 2010). We tested whether canrenoate treatment improves delayed diabetic wound angiogenesis through the modulation of macrophage polarization by assessing a panel of pro-angiogenic factors in wound tissue. Five days after wounding, gene expression of pro-angiogenic factors was impaired in STZ wounds with a significant decrease of FGF-2, PLGF, Tie-2, and angiopoietin-2 mRNA levels (data not shown). Importantly, topical canrenoate application restored the expression of these factors in diabetic wounds (data not shown). We then isolated macrophages (CD1 lb ⁺ F4.80 ⁺ Ly6G ) from wounded skin by FACS to analyze the expression of pro-angiogenic genes. VEGF-a, FGF-2, PLGF, and Tie2 mRNA levels were significantly lower in macrophages isolated from STZ wounds than those from control wounds, whereas topical application of Canre restored the inadequate expression of these angiogenic factors (data not shown).

The MR target Lcn2 promotes macrophage phenotypic polarization/switching and angiogenesis in diabetic wounds

We recently showed that Lcn2 is a primary target of aldosterone/mineralocorticoid receptor signaling in many organs and that Lcn2 plays a key role in the action of mineralocorticoids in the cardiovascular system (Buonafme et al., 2018). Lcn2 mRNA and protein expression was much higher in diabetic wounds than in control, non-diabetic wounds (Figures 1A-1B). Local canre or MR siRNA treatment lowered the upregulated expression of Lcn2 in diabetic wounds to near the level found in control wounds, indicating that Lcn2 is also an MR target in diabetic skin (Figures 1A-1B). We assessed whether Lcn2 is involved in the impaired healing of diabetic wounds using a global Lcn2-knockout mouse model. Wound closure in diabetic STZ mice was significantly impaired relative to that of CT nondiabetic mice, but Lcn2 inactivation prevented the delay in wound healing in Lcn2-knockout diabetic mice (Figure 1C). Of note, Lcn2 deficiency did not affect wound healing in normal non-diabetic mice (Figure 1C). FACS analysis of wound specimens demonstrated that Lcn2 inactivation resulted in Ml to M2 macrophage polarization: the increase of pro-inflammatory Ml macrophages in CT diabetic wounds was blunted in Lcn2-deficient diabetic mice (Figure ID) while the decrease of M2 macrophages in diabetic wounds was prevented (Figure IE). Importantly, Lcn2 deficiency prevented the impaired angiogenesis associated with diabetes, with the presence of more endothelial cells in the wounds of diabetic Lcn2 KO mice than those of CT diabetic mice (Figure IF). Moreover, expression of the pro-angiogenic genes VEGF-a, FGF-2, PLGF and Tie2 was higher in STZ Lcn2 KO macrophages than in STZ control mice (Figure 1G).

Lcn2 protein induces an unrestrained pro-inflammatory Ml macrophage phenotype in vitro

We hypothesized that the MR target Lcn2 participates in the deleterious effect of MR activation in diabetic wound healing by tipping macrophage functional/phenotypic polarization from proangiogenic M2 macrophages towards proinflammatory Ml . Macrophages isolated from the peritoneum of wild-type mice were first pretreated with LPS to induce an Ml phenotype and then with IL4 to switch them towards an M2 phenotype (data not shown). FACS analysis showed that treatment with recombinant Lcn2 resulted in a higher percentage of Ly6C ^M Ml macrophages (data not shown), together with fewer Ly6C ^low M2 macrophages (data not shown), indicating that recombinant Lcn2 inhibited the ability of LPS -pretreated macrophages to switch to the M2 phenotype in response to IL4. This was associated with decreased expression of the angiogenic genes VEGFa and PLGF (data not shown).

Overall, these data show that MR blockade controls the phenotypic polarization of macrophages towards a repair M2 phenotype and promotes dermal angiogenesis through modulation of the expression and activity of Lcn2, thereby improving the delayed wound healing in diabetes (data not shown).

Discussion: By using two mouse models of diabetes (STZ-induced type 1 and db-db type 2 diabetic mice), we demonstrate that inflammation and impaired healing of diabetic wounds are associated with the activation of MR signaling. Topical inhibition of this pathway provided a clear benefit to improve healing of these pathological wounds. This effect acts via inducing the polarization of macrophages toward the M2 phenotype, helping to resolve inflammation and rescue the angiogenesis defect of diabetic wounds. Moreover, we identified the MR target lipocalin 2 as one of the underlying signaling pathways. In contrast, MR blockade do not modify wound closure from normal mice. We propose that inadequate MR occupancy by exogenous GC or locally produced GC, as well as enhanced MR expression, may explain the benefit of MR blockade in a variety of pathological situations such as dermocorticoid treatments, UV irradiation, diabetic delayed wound healing and perhaps psoriatic skin or glucocorticoid-treated psoriasis (Hannen et ah, 2017, Nguyen et ah, 2016, Stojadinovic et ah, 2016). In addition, local steroidogenesis and modulation of I ΐb-Hydroxysteroid dehydrogenase type 1 may interfere with MR/GR signaling balance in skin diseases (Sevilla and Perez, 2018, Slominski et ah, 2014, 2015, Tiganescu et ah, 2013). Efforts towards better integrations of these factors should help to propose novel therapeutic improvements of delayed wound healing.

During wound healing, macrophages are significantly involved in the repair process (Brancato and Albina, 2011, Lucas et ah, 2010, Rahmani et ah, 2018). Importantly, they show heterogeneous phenotypes and functions and can switch/polarize between phenotypes to adapt to the wound stage (Boniakowski et al, 2017, Novak and Koh, 2013). The balance of such macrophage populations is pivotal for the progression of wound healing. However, most diabetic wounds do not progress, but remain in a chronic state of inflammation. This mainly results from a defect in the phenotypic switch of macrophages, leading to the sustained presence of pro-inflammatory Ml macrophages and over-production of pro-inflammatory cytokines in wounds (Boniakowski et al, 2017, Leal et al, 2015, Okizaki et al, 2015). Here, we demonstrate that topical inhibition of the MR promotes the polarization of activated Ml macrophages (CDl lb ⁺ /F4.80 ⁺ /Ly6C ^low ) in diabetic wounds toward an anti-inflammatory M2 phenotype (CD1 lb ⁺ /F4.80 ⁺ /Ly6C ^low ). Accordingly, MR antagonism up-regulated the expression of some anti-inflammatory factors, while downregulating the expression of pro-inflammatory cytokines in wound tissues. This is consistent with previous findings that showed the role of MR activation in various diseases involving chronic inflammation, including diabetes (Guo et al., 2008, Jaisser and Farman, 2016, Marzolla et al., 2014). Previous studies reported a central role of MR over-activation in sustained macrophage polarization after renal injury induced by ischemia reperfusion (Barrera-Chimal et al, 2018). In this setting, pharmacological MR antagonism also promoted the polarization of activated Ml macrophages toward an anti inflammatory M2 phenotype, suggesting that this may be a common mode of action of MR antagonists in wound healing in cardiovascular, renal, or metabolic injury (Barrera-Chimal et al, 2018).

Defective angiogenesis often occurs in diabetic wounds. The defect of wound angiogenesis may be due to inadequate mobilization and abnormal activation of endothelial progenitor cells (Loomans et al., 2004). Several therapies based on promoting the functional activity of endothelial cells and their progenitors have been proposed to improve healing (Liu et al, 2014, Marrotte et al, 2010, Nishimura et al, 2012). Impaired angiogenesis may also be associated with chronic inflammation, inhibiting the production of pro-angiogenic factors and limiting endothelial cell activity. Macrophages are important sources of pro-angiogenic factors. These inflammatory cells have been shown to be involved in many disease settings, including the complications of diabetes (Maruyama et al., 2007, Okizaki et al., 2015). Here, we found that M2 polarized macrophages, induced by the inhibition of MR signaling, expressed higher levels of pro-angiogenic factors than those from non-treated diabetic wounds, contributing to angiogenesis and wound healing improvement.

Overall, our results suggest an important role for MR signaling in the sustained polarization of macrophages and impaired wound angiogenesis in diabetes. These findings provide further insight concerning the impact of MR activation in various skin diseases. Indeed, we previously reported the role of cutaneous MR over-activation in the deleterious effects caused by dermo-glucocorticoid treatment in both mice and humans, including skin atrophy and impaired wound healing: glucocorticoid-induced MR activation impaired the proliferation and activation of epidermal keratinocytes linked to over-activation of epithelial sodium channels. Topical cutaneous treatment with MR antagonists provided significant benefits, limiting atrophy and improving wound healing (Maubec et al., 2015, Nguyen et al., 2016). MR overexpression in keratinocyte was shown to induce epidermal atrophy in mice (Sainte Marie et al., 2007). Boix et al. demonstrated that epidermal deletion of MRs leads to increased keratinocyte proliferation and differentiation (Boix et al., 2016). Other studies proposed that epidermal MR cooperates with glucocorticoid receptor acting as an anti-inflammatory factor to counteract skin inflammation and regulate epidermal development in inflamed skin (Bigas et al, 2018, Sevilla and Perez, 2018).

The mechanism underlying the modulation of inflammation and angiogenesis by MR in diabetic wounds may be complex and multifactorial. We identified lipocalin 2, a primary target of aldosterone/mineralocorticoid receptor signaling (Buonafme et al., 2018), as a promising candidate in diabetes-associated delayed wound healing. Lipocalin 2 secretion is increased in the wound lysates of diabetic foot ulcers due to the release of neutrophil extracellular traps (Fadini et al., 2019). Lcn2 activation is also considered to be a biomarker for chronic inflammatory diseases of skin, such as psoriasis and venous ulcers (Serra et al, 2013, Shao et al, 2016). In the present study, wounds from diabetic Lcn2 knockout mice had lower inflammation scores, with a lower ratio of M1/M2 macrophages, with increased angiogenesis, and better wound healing than those from wild-type mice with STZ injection. The role of Lcn2 in the regulation of inflammation is complex. Cheng et al. demonstrated that Lcn2 promotes Ml macrophage polarization after cardiac ischemia-reperfusion injury (Cheng et al, 2015). In contrast Guo et al. reported that Lcn2-deficient mice displayed up-regulation of Ml macrophage markers and down-regulation of M2 markers in the adipose tissue and liver of mice fed a high-fat diet (Guo et al, 2014). In another setting, Warszawska et al. identified Lcn2 as both a marker of deactivated macrophages and a macrophage deactivator in the lungs of mice with bacterial pneumonia (Warszawska et al, 2013). In the present study, Lcn2 inactivation had a positive effect on diabetic wounds whereas it did not affect the healing of non-diabetic normal mice. Our in vitro study on the function/polarization of macrophages showed a direct effect of recombinant Lcn2 protein to prevent the switching of pre-activated Ml macrophages to M2 the phenotype (although a limit of our approach is the use of macrophages isolated from peritoneum rather than those of the skin).

In summary, this study indicates the efficacy of NGAL blockade to restore impaired reepithelialization, angiogenesis and to reduce inflammation after an acute wound in diabetic mice; whether these benefits extend to human chronic diabetic wounds as leg ulcers remain to be demonstrated.

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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.

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