ES2665019B1 - USE OF CALPROTECTIN FOR THE TREATMENT OF INTESTINAL INFLAMMATORY DISEASES - Google Patents

Description

d e s c r ip c io n

USE OF CALPROTECTIN FOR THE TREATMENT OF INTESTINAL INFLAMMATORY DISEASES

TECHNICAL FIELD OF THE INVENTION

The invention relates to the treatment and prevention of inflammatory bowel disease using calprotectin, preferably administered intrarectally. The invention is also related to compositions suitable for intrarectal administration comprising calprotectin (CP), as well as its use in the treatment and prevention of said disease.

STATE OF THE ART

Inflammatory bowel disease

Inflammatory Bowel Disease (IBD ) represents a group of idiopathic chronic conditions associated with various areas of the gastrointestinal tract, basically comprising ulcerative colitis (UC ) and the disease de Crohn (CD, from English "Crohn's disease"). The incidence of IBD is increasing slowly among the population of developed countries, which is a significant problem since the quality of life of patients is seriously compromised. The underlying triggering cause of IBD remains unclear, but a multifactorial origin that involves environmental and genetic contributions is generally accepted. The last decade has been critical to understanding the pathology of IBD, particularly the importance of maintaining an adequate interaction between the commensal microbiota and the intestinal mucosa of the host [Rubin DC, Shaker A, Levin MS. Chronic intestinal inflammation: inflammatory bowel disease and colitis-associated colon cancer. Front Immunol. 2012; 3: 107]. The epithelium is in continuous contact with microorganisms and antigens of the diet, creating a crucial site for the regulation of innate and adaptive immunity.

Nowadays, there is a growing interest in research into the regulation of mechanisms that modulate the intestinal barrier function, focusing on both physical and immunological aspects. As it is now accepted that the development of IBD depends on a dysregulated immune response of the host's mucosa towards the intestinal microbiota, it seems feasible that an impaired intestinal barrier function leading to a greater intestinal permeability and / or translocation exposes the mucosal immune system to bacteria and unprocessed antigens, which could result in a complete inflammatory response [Sánchez de Medina F, Romero-Calvo I, Mascaraque C, et al. Intestinal inflammation and mucosal barrier function. Inflammatory Bowel Diseases. 2014; 20: 2394-2404].

Calprotectin

Calprotectin (CP) is a heterocomplex of proteins formed by two subunits of 14 and 8 kD, encoded by the genes of mice S100a8 (also known as MRP8 or CP-10) and S100a9 (MRP14). S100A8 and S100A9 belong to the S100 protein family with EF hand domain ( EF-hand) for calcium binding. S100A8 and S100A9 are ligands capable of at least partially activating TLR4 [Vogl T, Tenbrock K, Ludwig S, et al. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxininduced shock. Nat Med. 2007; 13: 1042-1049] or the receptor for advanced glycation end products (RAGE ) [Hofmann MA, Drury S, Fu C, et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100 / calgranulin polypeptides. Cell. 1999; 97: 889-901]. CP is expressed in neutrophils, macrophages, epithelial cells (including perhaps intestinal epithelial cells [Lee MJ, Lee JK, Choi JW, et al., Interleukin-6 induces S100A9 expression in colonic epithelial cells through STAT3 activation in experimental ulcerative colitis, PLoS One. 2012; 7: e38801]) and other cell types. In neutrophils, the CP comprises 20-45% of the cytosolic proteins. In many inflammatory processes, PC is substantially elevated and has been widely studied as a biomarker of disease activity, especially in IBD, where CP is measured in faeces for diagnosis [Tibble JA, Bjarnason I. Non-invasive investigation of inflammatory bowel disease. World J Gastroenterol. 2001; 7: 460-465]. CP has generally been considered a proinflammatory protein, mainly via TLR4 ligation, acting as an alarmin in inflammatory sites [Loser K, Vogl T, Voskort M, et al. The Toll-like receptor4 ligands Mrp8 and Mrp14 are crucial in the development of autoreactive CD8 + T cells. Nat Med. 2010; 16: 713-717]. S100A8 is a leukocyte chemoattractant, although it presents an atypical regulation [Cornish CJ, Devery JM, Poronnik P, et al. S100protein CP-10 stimulates myeloid cell chemotaxis without activation. J Cell Physiol. 1996; 166: 427-437], since it is induced in monocytes / macrophages by the anti-inflammatory cytokine IL-10 and is potentiated by glucocorticoids [Hsu K, Passey RJ, Endoh Y, et al. Regulation of S100A8 by glucocorticoids. J Immunol. 2005; 174: 2318-2326], [Xu K, Yen T, Geczy CL. II-10 upregulates macrophage expression of the S100 protein S100A8. J Immunol. 2001; 166: 6358-6366]. Inhibits oxidative metabolism in neutrophils [Sroussi HY, Lu Y, Villines D, et al. The down regulation of neutrophil oxidative metabolism by S100A8 and S100A9: implication of the protease-activated receptor-2. Molecular immunology. 2012; 50: 42-48] and is protective against sepsis in mice, although contradictory evidence has been published in this regard [Sun Y, Lu Y, Engeland CG, et al. The anti-oxidative, anti-inflammatory, and protective effect of S100A8 in endotoxemicmice. Molecular immunology.2013; 53: 443-449]. Calprotectin also shows antibacterial properties due, at least partially, to its ability to tightly bind and sequester transition metals [Kehl-Fie TE, Chitayat S, Hood MI, et al. Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus. Cell host & microbe. 2011; 10: 158-164]. PC also plays an important role in the biology of the skin [Kerkhoff C, Voss A, Scholzen TE, et al. Novel insights into the role of S100A8 / A9 in skin biology. Exp Dermatol. 2012; 21: 822-826], lung [Hiroshima Y, Hsu K, Tedla N, et al. S100A8 induces IL-10 and protects against acute lung injury. J Immunol. 2014; 192: 2800-2811], in cancer [Markowitz J, Carson WE, 3rd. Review of S100A9 biology and its role in cancer. Biochim Biophys Acta. 2013; 1835: 100-109] and in fungal infections [Mehra T, Koberle M, Braunsdorf C, et al. Alternative approaches to antifungal therapies. Exp Dermatol. 2012; 21: 778-782].

To the extent that PC is clearly upregulated in the inflamed bowel and has proinflammatory actions as described above, it could be expected to contribute to the inflammatory response in inflammatory bowel disease and intestinal inflammation in general. However, there is no direct evidence available on the role of PC in intestinal inflammation.

With respect to the uses of calprotectin as an anti-inflammatory agent, [US20090305985] describes the use of S100A8 and / or S100A9 as immunomodulatory agents for the treatment of inflammatory diseases. However, this patent does not disclose specific details such as the administration of calprotectin by the intrarectal route, nor the disadvantages of its administration intraperitoneally, and would not induce a person skilled in the art to use CP for the treatment of diseases such as inflammatory bowel disease (IBD). Rather, it mostly presents in vitro data that describe the chemotactic modulatory effects of S100A8 and S100A9 separately (ie, not as calprotectin but as monomers, either in a native form or as modified sequences), together with some other data about effects on microbial growth that have little or no relevance with the treatment of inflammatory bowel disease. Said document indicates that the monomers S100A8 and S100A9 had a fugactic (anti-chemotactic) effect at concentrations in the range 1 nM-1 pM in human monocytes and neutrophils in assays in agarose and transwell, and chemotactic effects at 0.1 -1 nM in lymphocytes in transwell assays. No trials with calprotectin are presented. There is a single in vivo test using the acute inflammatory air bag model of rat and a variant of S100A8, not calprotectin, administered intraperitoneally. It should be noted that this model, although useful for screening purposes, has very little validity in terms of correlation with the therapeutic activity in intestinal inflammation. For example, non-steroidal anti-inflammatory agents such as ibuprofen and related drugs are active in the air bag model [Goindi S, Narula M, Kalra A. "Microemulsion-Based Topical Hydrogels of Tenoxicam for Treatment of Arthritis". AAPS PharmSciTech. 2015 Aug 19], [Jukanti R1, Devaraj G, Devaraj R, Apte S. "Drug targeting to inflammation: studies on antioxidant surface loaded diclofenac liposomes '' Int J Pharm. 2011 Jul 29; 414 (1-2): 179-85] but they actually exacerbate inflammatory bowel disease (IBD) [Long MD1, Kappelman MD, Martin CF, Chen W, Anton K, Sandler RS. "Role of Nonsteroidal Anti-Inflammatory Drugs in Exacerbations of Inflammatory Bowel Disease." J Clin Gastroenterol, 2016 Feb; 50 (2): 152-6].

The complete sequence of S100A8 is known and can be accessed, for example, at [https://www.ncbi.nlm.nih.gov/gene/6279#reference-sequences]. The complete sequence of S100A9 is also known and can be accessed, for example, at [http://www.ncbi.nlm.nih.gov/gene/6280#reference-sequences].

SEQS100A8:

MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIRKKGADVWFKELDI NTDGAVNFQEFLILVIKMGVAAHKKSHEESHKE

SEQS100A9:

MTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEKVIE HIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTP

OBJECT OF THE INVENTION

The object of this invention is related to the use of calprotectin (CP) in the treatment of damage or degeneration of the intestinal epithelium, particularly in diseases or conditions related to intestinal inflammation.

The inventors aimed to establish the role of CP in intestinal inflammation and its possible therapeutic use using the Dextran Sulfate Sodium (DSS) model of colitis in mice. Their results have shown that CP has dose-dependent protective effects when administered intrarectally, but not intraperitoneally. In addition, CP shows immunostimulatory effects in intestinal epithelial cells, which indicates that it can act on the epithelium from the luminal side (reachable directly using the intrarectal route). These results also suggest a possible mechanism of action.

This invention provides significant new results in the following aspects: (1) it provides data on the therapeutic value of calprotectin instead of its individual components; (2) provides valuable data on the usefulness of the intrarectal versus intraperitoneal route; (3) presents information on active doses; and (4) shows the therapeutic efficacy of CP in a model of real inflammatory bowel disease.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, the present invention is related to the use of calprotectin for the treatment of intestinal epithelial damage or degeneration, preferably inflammatory bowel disease or disease.

A further aspect of the invention is the use of a calprotectin as a medicament or in the preparation of a medicament for the treatment of intestinal epithelial damage or degeneration, preferably inflammatory bowel disease or disease.

Another aspect is a pharmaceutical composition, preferably an oral composition, more preferably for topical administration and even more preferably a composition intrarectal, which comprises a therapeutically effective amount of calprotectin and at least one pharmaceutically acceptable carrier, adjuvant or vehicle, useful for the treatment of damage or degeneration of the intestinal epithelium, preferably inflammatory bowel disease or disease.

A further aspect is a method for treating a mammal, preferably a human being, against intestinal epithelial damage or degeneration, preferably inflammatory bowel disease or condition, comprising local and / or topical administration, preferably oral administration, more preferably intrarectal administration, of a therapeutically effective amount of calprotectin.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Effect of intrarectal CP on body weight loss and macroscopic colon parameters in colitis DSS mice. Colitis was induced with 2% DSS, as described in materials and methods, and treated with 50 or 100 ^ g / day of CP (n = 10 per group). (A) Evolution of body weight, expressed as a percentage of initial weight. (B) Weight / length ratio of colon. (C) Representative images of the large intestine of the different groups. (D) Macroscopic result. (E) Spleen weight. p <0.05 against the DSS group. All groups are significantly different from the control from day 5 onwards in panel A (not shown).

Figure 2. Colonic histology of cols DSS mice treated with CP intrarectally. Histological sections representative of hematoxylin and eosin are shown. Colitis was induced with 2% DSS and treated with 50 or 100 ^ g / day of CP intrarectally (n = 10 per group). (A) Control; (B) DSS; (C) CP 50 ^ g / day; (D) CP 100 ^ g / day.

Figure 3. Effect of intrarectal CP on the activity of colonic alkaline fostatase (AP) in colitis DSS mice. (A) Alkaline phosphate (AP). (B) Inhibition of colonic AP activity by levamisole in vitro. Colitis was induced with 2% DSS and treated with 50 or 100 ^ g / day of CP intrarectally (n = 10 per group). The activity of AP was measured by spectrophotometry and its sensitivity to the specific inhibitor levamisole was determined. The values are means ± SEM. p <0.05 vs. control group; * p <0.05 vs. DSS group.

Figure 4. Effect of intrarectal CP on the expression of colon myeloperoxidase (MPO) and S100A8 in eolithic DSS mice. (A) Colonic MPO activity. (B) mRNA levels of S100A8 (C) Immunostaining of MRP8 (S100A8). Colitis was induced with 2% DSS and treated with 50 or 100 jg / day of CP intrarectally (n = 10 per group). The activity of MPO was measured by spectrophotometry. S100A8 was determined by RT-PCR and immunohistochemistry. The values are means ± SEM with the standard errors represented by vertical bars. p <0.05 vs. control group; * p <0.05 vs. DSS group.

Figure 5. Colonic expression of different genes in DSS mice with colitis treated with CP 50 or 100 jg / day or vehicle intrarectally. Colitis was induced with 2% DSS and treated with 50 or 100 μg / day of CP intrarectally (n = 10 per group). The mRNA levels were measured by T-PCR. (A) Levels of IL-10 expression by FOXP3. (B) IL-22. (C) TLR4. The values are means ± SEM. p <0.05 vs. control group; * p <0.05 vs. DSS group.

Figure 6. Secretion of cytokines by the spleen (A, B) or mesenteric lymph node cells (C, D) ex vivo in mice with DSS colitis treated with CP intrarectally. Colitis was induced with 2% DSS and treated with 50 or 100 jg / day of CP intrarectally (n = 10 per group). Cells were isolated and cultured with or without concanavalin A, and the levels of cytokines in the supernatant were measured by ELISA (A, C) production of IL-17 and (B, D) production of IL-10. The values are means ± SEM. p <0.05 vs. control group; * p <0.05 vs. DSS group.

Figure 7. Analysis of leukocyte populations in the spleen of mice with DSS colitis treated with CP intrarectally. Colitis was induced with 2% DSS and treated with 50 or 100 jg / day of CP intrarectally (n = 10 per group). The cells were isolated and analyzed by flow cytometry. Relative amount of (A) neutrophils, CD4 lymphocytes, CD8 lymphocytes and (B) IFN- and IL-4 expression by CD4 T lymphocytes (EC) Ly6G positive histograms in peripheral blood in control (C), DSS groups (D) and CP50 (E). The values are means ± SEM. p <0.05 vs. control group; * p <0.05 vs. DSS group.

Figure 8. Effect of CP on intestinal epithelial cells EC18. (A, B) Effect on the secretion of GROa and MCP-1. (C, D) Impact of TLR4 knockdown on the effect of CP on the secretion of GRO a and MCP-1. (E, F) Impact of pharmacological inhibition of NF ^k B (Bay11-7082, 10 ^ M) and RAGE (FPS-ZM1, 10 ^ M) on the effect of CP in the secretion of MCP-1 and GRO. Different concentrations of CP were added to the culture medium and the concentration of cytokines in the culture medium was measured using ELISA after a 24 h incubation. The results are expressed as mean ± SEM of three different experiments (n = 3 in each experiment). p <0.05 vs. control; * p <0.05 vs. stimulated group.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

In the context of the present invention, the following terms are defined:

Calprotectin is defined as a dimer of the S100A8 protein or any polypeptide whose nucleic acid sequence possesses an identity greater than 50%, 60%, 70%, 80%, 90%, 95%, 99% or more with the sequence of S100A8; and the S100A9 protein or any polypeptide whose nucleic acid sequence possesses an identity greater than 50%, 60%, 70%, 80%, 90%, 95%, 99% or more with the sequence of S100A9.

The term "dimer", as used in this document, refers to the association of these subunits.

The term "S100A8" refers to the human S100A8 gene (eg Homo sapiens-GENBANK Accession No. NM.sub.-002964.4) and its gene product (GENBANK Accession No. NP.sub .-- 002955.2), including products wild ( wild type) and mutants. S100A8 is also known as MRP-8, "S100 calcium-binding protein A8", calgranulin A, calgranulin-A, calprotectin subunit L1L, cystic fibrosis antigen, L1 leukocyte complex light chain, protein related to the migration inhibitory factor 8 or "urinary stone protein band A")

The S100A8 protein has the following sequence:

SEQ S100A8:

MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETECPQYIRKKGADVWFKELDI NTDGAVNFQEFLILVIKMGVAAHKKSHEESHKE

The term "S100A9" refers to the human S100A9 gene (eg, Homo sapiens - GENBANK Accession No. NM.sub.-002965.3) and its gene product (GENBANK Accession No. NP.sub .-- 002956.1), including wild and mutant products (wild - type). S100A9 is also known as MRP-14, "S100 calcium-binding protein A9", calgranulin B, calgranulin-B, calprotectin subunit L1H, heavy chain complex of leukocytes L1 or protein related to the migration inhibitory factor 14 in humans. There are genes and equivalent proteins (orthologs) in different species of mammals such as rats and mice.

The S100A9 protein has the following sequence:

SEQ S100A9:

MTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEKVIE HIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTP

In each case, the S100A8 and S100A9 sequences preferably encode a protein that overregulates the fugetaxis. Moreover, the terms "gene S100A8 / S100A9". "S100A8 / S100A9 nucleotide sequence" and "S100A8 / S100A9 polynucleotide sequence" encompass the DNA, complementary DNA (cDNA) and RNA sequences.

The terms "' fugetaxis " (or fugetaxia) and "anti-guimiotaxis" (or anti-chemotaxis or reverse chemotaxis) refer to the response of mobile cells in which the direction of movement moves them away from the gradient of a disseminable substance or agent chemokinetic

The term "amino acid sequence" to refer to an amino acid sequence of a naturally occurring protein molecule, and similar terms such as "polypeptide" or "protein", is not intended to limit the amino acid sequence to the native amino acid sequence complete, associated with the mentioned protein molecule.

As used herein, the term "amino acid substitution" refers to an act, process or result of amino acid substitution.

The term "plasmid", as used herein, refers to a small DNA fragment that reproduces independently.

The term " purified " refers to molecules (polynucleotides or polypeptides) that are removed from their natural environment, isolated or separated. "Substantially purified" molecules are free at less than 50%, preferably at least 75%, and more preferably at least 90% other components with which they are naturally associated.

The term " wild-type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a natural source. A wild-type gene is the one most frequently observed in a population and therefore the "normal" or "wild-type" form of the gene is arbitrarily employed.

The terms "mutant". "polymorphism"' and "variant", in reference to a gene or gene product, refer to alterations in the sequence and / or functional properties (ie, different characteristics) when compared to the wild type gene or to a product of the parental gene. In some preferred embodiments, the term "mutant" refers to a gene or gene product that differs from a gene or parental gene product as a result of mutation. It is observed that the natural and induced mutants can be isolated; these are identified by the fact that they have altered characteristics compared to the wild type gene or parental gene product. In addition, mutant genes can be produced artificially (for example, site-directed mutagenesis) or synthetically in the laboratory. The term mutation refers to a change in the number, arrangement or molecular sequence of nucleotides in a genetic sequence. In other embodiments, "mutation" refers to a change in the number, arrangement or a specified amino acid sequence of a peptide or a protein.

As used herein in reference to a protein, the term "modified" refers to proteins with structural changes including primary, secondary, tertiary, etc. changes. Therefore, the term "modified" encompasses, but is not limited to deletions, insertions and amino acid substitutions (eg, as a result of mutation), as well as post-translational modifications such as glycosylation, acylation, limited proteolysis, phosphorylation, isoprenylation. and oxidation.

In addition, the term "modified" encompasses the substitution of a native amino acid for a non-standard residue including, but not limited to, acetamidomethyl, aminohexanoic acid, aminoisobutyric acid, beta-alanine, cyclohexylalanine, D-cyclohexylalanine, e-acetyl lysine, gamma aminobutyric acid, Hydroxyproline , nitro-arginine, nitro-phenylalanine, nitro-tyrosine, norleucine, norvaline, octahydroindole carboxylate, ornithine, penicillamine, phenylglycine, phosphoserine, phosphothreonine, phosphotyrosine, pyroglutamate and tetrahydroisoquinoline.

The term "antibody" refers to polyclonal and monoclonal antibodies. Polyclonal antibodies that are formed in the animal as a result of an immunological reaction against a protein of interest or a fragment thereof, can be easily isolated from the blood using well-known and purified methods, for example, by column chromatography. Monoclonal antibodies can also be prepared using known methods (see, for example, Winter and Milstein, Nature, 349, 293-299, 1991). As used herein, the term "antibody" includes antibodies prepared recombinantly and antibodies modified as well as antigen-binding fragments, such as chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligo-specific antibodies, single-chain antibodies. and F (ab) or F (Ab). sub.2 fragments. The term "reagent" used in reference to an antibody indicates that the antibody is capable of binding to an antigen of interest. For example, an antibody reactive with S100A8 is an antibody, which binds S100A8 or a fragment of S100A8.

The terms "transporter", "adjuvant" and / or "vehicle" refer to entities or molecular substances with which the active ingredients are administered. Such carriers, adjuvants or pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, excipients , shelling, wetting agents or diluents. Pharmaceutical carriers, adjuvants and / or suitable pharmaceutical vehicles are described, for example, in "Remington's Pharmaceutical Sciences" by EW Martin.

The terms "localized" and "local" refer to the participation of a limited area, therefore, in contrast to the "systemic" treatment , in which the whole body is involved, usually through the vascular and vascular systems. / or lymphatic, localized treatment implies the treatment of a specific and limited area. Thus, in some embodiments, the affected areas are treated locally using the methods and compositions of the present invention.

The term "topically" means application to the surface of the skin, mucosa, viscera, etc. Similarly, the terms "topically active agent" refer to a substance or composition that elicits a pharmacological response at the site of application (eg, skin or intestinal epithelium).

The terms "systemically active agent" are used broadly to mean a substance or composition that will produce a pharmacological response at a site remote from the point of application.

^{The term "anima} r ^{" refers to any member of the Animalia kingdom , which includes} living ^beings that have cells that differ from plant cells with respect to the absence of a cell wall and chlorophyll and the capacity for spontaneous movement. Preferred embodiments of the present invention are directed primarily to mammals of the animal kingdom.

The terms "patient" and "subject" refer to an animal, preferably mammal, more preferably human, that is a candidate for medical treatment.

The terms "sample" and "specimen" are used in their broadest sense. On the one hand, they are intended to include a specimen or a crop. On the other hand, they are intended to include both biological and environmental samples. These terms encompass all types of samples obtained from humans and other animals, including, but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid, semen, saliva and wound exudates, as well as solid tissue. However, these examples should not be construed as limiting the types of samples applicable to the present invention.

The term " inflammation ", as used herein, refers to the tissue response to trauma, characterized by increased blood flow and the entrance of leukocytes into the tissues, resulting in swelling, redness, elevated temperature and pain.

The term "symptom" refers to any subjective evidence of a patient's illness or condition (for example, a change in a patient's condition indicative of some bodily or mental state). For example, the phrase "symptoms of inflammation" in the context of inflammatory bowel disease (IBD) is defined herein to include, but is not limited to, symptoms such as abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, and other more serious complications, such as dehydration, anemia and malnutrition. Several of these symptoms are subject to a quantitative analysis (eg, weight loss, fever, anemia, etc.). Some symptoms are easily determined from a blood test (for example, anemia) or a test that detects the presence of blood (for example, rectal bleeding).

The term "treatment" as used herein refers to combating the effects caused as a result of a disease or pathology of interest in a subject (preferably mammal, more preferably a human being). The "treatment" also includes the prevention, improvement or elimination of the physiological sequelae of the disease. More specifically, the treatment includes:

(I) preventing the disease or pathological condition in a mammal, particularly when the mammal has a predisposition for the pathological condition to occur, but has not yet been diagnosed to have it.

(II) inhibit the disease or pathological state, that is, stop its development;

(III) alleviating the disease or pathological state, that is, causing the regression of the disease or pathological state or its symptoms;

(IV) stabilize the disease or pathological state.

Similarly, the phrase "in conditions such that symptoms are reduced" in the context of IBD refers to any degree of qualitative or quantitative reduction of the detectable symptoms of IBD, including but not limited to, a detectable impact on the recovery rate of diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, bloating, fibrosis, inflammation of the intestines and malnutrition, or reduction of at least one of the following symptoms: abdominal pain , diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, bloating, fibrosis, intestinal inflammation and malnutrition.

Use of CP for the treatment of intestinal inflammatory diseases or conditions

In a first aspect, the present invention is related to the use of calprotectin, as previously defined, for the treatment of diseases or conditions mediated by intestinal epithelial damage or degeneration, preferably inflammatory disease or intestinal disease.

In a particular embodiment, the disease or condition is colitis.

Medications for the treatment of inflammatory bowel diseases or conditions

Another aspect of the invention is the use of a calprotectin as a medicament, or in the preparation of a medicament, for the treatment of diseases or conditions mediated by intestinal epithelial damage or degeneration, preferably inflammatory bowel disease or disease.

Another aspect is a pharmaceutical composition comprising a therapeutically effective amount of calprotectin and at least one pharmaceutically acceptable carrier, adjuvant or vehicle, useful for the treatment of diseases or conditions mediated by intestinal epithelial damage or degeneration, preferably inflammatory bowel disease or disease.

In a particular aspect, the only active ingredient comprised in the composition is calprotectin.

In a particular aspect, the composition is suitable to be administered locally. In another particular aspect, the composition is suitable to be administered topically.

In another particular aspect, the composition is suitable for oral administration.

In a preferred aspect, the composition should be administered intrarectally, ie, the composition comprises at least one pharmaceutically acceptable carrier useful for intrarectal administration.

In a particular aspect, the vehicle used in the intrarectal composition is 10 mM pmercaptoethanol in distilled water.

In another particular aspect, a vehicle which prevents or limits the destruction or denaturation of CP in gastric secretions can be used by using enteric coating such as coating based on eudragit L.

In a particular embodiment, the disease or condition is colitis.

Method of treatment of intestinal inflammatory diseases or conditions

A further aspect of the invention is a method, hereinafter the "method of the invention". for treating a mammal, preferably human, against diseases or conditions mediated by intestinal epithelial damage or degeneration, preferably inflammatory bowel disease or disease, comprising administration, preferably oral administration, more preferably intrarectal administration, of a therapeutically effective amount of calprotectin to said mammal ..

In a particular aspect, the method of the invention comprises the local administration of a composition comprising a therapeutically effective amount of calprotectin. In another particular aspect, the method comprises a topical administration of a composition comprising a therapeutically effective amount of calprotectin.

In a particular embodiment, the method of the invention comprises:

A) provide a composition comprising calprotectin; Y

B) contacting the intestinal epithelium with said composition

In a more particular embodiment, the composition comprising calprotectin is administered intrarectally.

In a particular embodiment, the disease or condition is colitis.

Contrary to previous evidence, such as those mentioned in the US patent [US20090305985], CP is not effective when administered systemically (for example, using the intraperitoneal route). Rather, protection against experimental colitis is achieved only when the same amount of CP is administered intrarectally, so that it exerts primarily a local action on the inflamed mucosa. This mode of action is consistent with the mechanisms described for this protein, and it has also been specifically shown that CP can modulate epithelial biology by acting from the apical (luminal) domain, although alternative sites of action can not be excluded. As indicated, CP as such was not tested in vivo in the US patent [US20090305985], nor was it tested in vitro in chemotactic experiments.

A local mechanism is also compatible with oral dosing, provided that CP is administered in sufficiently large doses and / or in suitable galenic forms.

Based on the mechanisms described for CP, it has traditionally been attributed a proinflammatory role. However, the role of CP in inflammation is controversial. Thus, while intramucosal CP can act as alarmin and chemoattractant, recent studies suggest that the predominant role of CP or its heterodimeric subunits may ultimately be antiinflammatory [Hiroshima Y, Hsu K, Tedla N, et al. S100A8 induces IL-10 and protects against acute lung injury. J Immunol. 2014; 192: 2800-2811] [Averill MM, Barnhart S, Becker L, et al. S100A9 differentially modifies phenotypic states of neutrophils, macrophages, and dendritic cells: implications for atherosclerosis and adipose tissue inflammation. Circulation. 2011; 123: 1216-1226], [Geczy CL, Chung YM, Hiroshima Y. Calgranulins may contribute vascular protection in atherogenesis. Circulation journal: official journal of the Japanese Circulation Society. 2014; 78: 271-280]. The situation is complicated by the particular characteristics of the intestine, where immune activation can actually result in protection against inflammation. Thus, although ligature of TLR4 causes cellular activation in immune and epithelial cells, it also modulates the epithelial dynamics to face the challenge imposed by mucosal inflammation, in which epithelial proliferation and restitution are considered important for healing and protection against ulceration, one of the main characteristics of IBD. In fact, TLR4 KO mice show elevated colitis. Similarly, blocking RAGE (another a CP receptor) attenuates colitis, but RAGE KO mice are no less sensitive to the induction of colitis.

MODES OF REALIZATION

Materials and methods

Reagents

Except where indicated, all reagents and primers were obtained from Sigma. Reverse transcription was carried out using the iScript cDNA Synthesis Kit, and for the amplification (Bio-Rad) iTM SYBR Green Supermix was used. Mouse ELISA kits were obtained from eBioscience. The DSS was obtained from MP Biomedicals.

Calprotectin

The expression and purification of CP was performed using a modified version of the original protocol described in Kehl-Fie et al (2011). Briefly, the S100A8 and S100A9 proteins are overexpressed in BL21 rosetta cells of E. coli in inclusion bodies. The protein is split into guanidinium hydrochloride and redoubled by dialysis at 4 ° C in a buffer containing 20 mM phosphate at pH 7.5. The purification involves a series of chromatographic steps involving exchange of hydroxylapatite anions, Source Q and Superdex 75 columns. The lipopolysaccharides were removed using the ToxinEraser ™ Endotoxin Removal Kit (Genscript) following the manufacturer's instructions.

Animals

The C57BL / 6 female mice were obtained from the Jackson Laboratory and maintained at the Biomedical Research Center of the University of Granada (Granada, Spain), in air-conditioned premises with a 12-hour cycle of light and dark. The animals were housed in groups under specific conditions without pathogens ( Specific Pathogen-Free, SPF), in individual cages ventilated with a system of air insufflation and exhalation with double filter (pre-filter and HEPA filter) and were given free access to the tap water and food in autoclave (Harlan-Teklad 2014, Harlan Ibérica, Barcelona, Spain). All the animal procedures carried out in the present study were in accordance with the Directive for the Protection of Vertebrate Animals Used for Experimental Purposes and Other Scientific Purposes of the European Union (86/609 / EEC) and were approved by the Welfare Committee Animal of the University of Granada.

Induction of DSS colitis and experimental design.

A total of 67 female C57BL / 6 mice were used. Colitis was induced by the addition of DSS to drinking water for 8 days. The experimental conditions were selected to achieve a mild to moderate degree of colitis using 2% w / v of DSS. The state of the mice was monitored individually by a general examination and measuring the loss of body weight. In addition, the presence of diarrhea and hematochezia was observed. Food intake and water consumption were controlled every day. In the first experiment, the mice were randomly assigned to four different groups. The control group (C) (n = 10) did not receive DSS at any time. The remaining mice drank water supplemented with DSS and additionally received intrarectally 50 μg / d of calprotectin (group CP50, n = 10), 100 ^ g / d (group CP100, n = 10) or vehicle (P-mercaptoethanol 10 mM In distilled water, group DSS, n = 10). The administration of calprotectin began at the same time as the DSS supplementation (day 0) and continued until the mice were sacrificed on day 8 by cervical dislocation.

In a separate experiment, 27 mice were randomly assigned to a control group and a DSS group as described above (n = 9), plus one group, CP, that received 50 ^ g / d intraperitoneally. The DSS group was injected daily with vehicle. The animals were followed for 8 days and sacrificed under the same conditions.

Evaluation of the damage in the colon.

The entire colon was removed, rinsed gently with saline and placed on an ice plate, cleaned of fat and mesentery and dried on filter paper. Each specimen was weighed and its length was measured with a constant load (2 g). A small segment of the intestine was dissected and used to isolate the RNA. The colon was subsequently cut longitudinally into several pieces for the determination of biochemical parameters. The fragments were immediately frozen in liquid N ² and kept at -80 ° C until use. Colonic tissue fixed with formalin was cut and stained with hematoxylin and eosin. Histology was scored on a scale of 0-8 according to the following criteria: erosion (0-2); Infiltration (0-2); Loss of the structure of the crypts (0-2); Submucosal thickening (0 2). The activities of colon myeloperoxidase (MPO) and alkaline phosphatase (AP) were measured spectrophotometrically as described. In addition, the sensitivity of AP to the specific inhibitor levamisole in vitro was measured .

Immunohistochemistry

The formalin-fixed colonic tissue was deparaffinized in xylene and rehydrated through a series of alcohol treatments. Recovery of the antigen was achieved by boiling the matrices in sodium citrate buffer (pH 6.0). The endogenous HRP activity was blocked with 0.5% H ² O ² . After blocking in 10% normal horse serum, the MRP-8 antibody (ab92331, Abcam) was applied and the signal was detected using the VECTASTAIN Elite ABC kit (Vectashield, PK-6100). AEC (3-amino-9-ethylcarbazole) was added and the samples were developed for three to five minutes. The reaction was terminated by placing the slides in distilled water. The sheets were then countercolored with Harris Hematoxylin. The tissues were then assembled using aqueous mounting medium.

Secretion of cytokines by mesenteric lymph node cells and splenocytes.

The mesenteric lymph nodes (MLN) and the spleen of the mice were extracted using a sterile technique and mechanically dissected. The cells were washed once with fresh medium and filtered using a 70 ^ M filter (Falcon BD cell strainer, Reference 352350, Becton Dickinson) to obtain a mononuclear suspension, mostly of T cells. The cells were incubated in RPMI 1640 medium containing fetal bovine serum (10%), 2 mM L-glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 2.5 mg / ml amphotericin B and 0.05 mM of p-mercaptoethanol. The cells were cultured at 106 cells / ml and stimulated with concanavalin A (ConA) to a final concentration of 5 mg / ml. ConA is a polyclonal stimulant of T cells that evokes an increase in cytokine secretion, which is typically improved in colitic animals. The cell culture medium was collected after 48 h and the cytokine content was assayed by commercial ELISA. The cytokines tested were IL-6, IL-10, IL-17, IFN- y and TNF-a. The plates (Nunc ™ Immuno plate) were read at 450 nm using a plate reader (Tecan, model Sunrise-basic). ELISA measurement of cytokines in cell supernatants did not show a consistent trend in the case of IFN- y , TNFa and IL6 (data not shown).

Flow cytometry.

Single cell suspensions of freshly isolated splenocytes were washed in staining buffer (5% FBS / 0.02% NaN ³ in PBS), blocked with anti-CD16 / 32 antibody for 5 min at room temperature (RT) and then stained for 30 min at 4 ° C with the following surface antibodies: anti-CD3 APC, anti-CD4 PerCP, anti-CD8 FITC, anti-Ly6G PE-Cy7 (BD Pharmingen). Cell suspensions were washed and analyzed using a FACsCalibur flow cytometer (BD Biosciences) and FlowJo software (Version 9.3.1, Treestar).

For in vitro stimulation of isolated splenocytes, cells were cultured in RPMI 1640 complete medium supplemented with 10% (v / v) fetal bovine serum (FBS), 2.0 mM L-glutamine, 50 ^ M 2-mercaptoethanol, penicillin 100 U / ml and streptomycin 100 ^ g / ml, plus phorbol myristate acetate (0.5 ^ g / ml), ionomycin (2.5 ^ g / ml) and monensin 2 ^ M (GolgiStop®), which were added simultaneously at the beginning of cell culture. The samples were incubated for 4 h at 37 ° C in a 5% CO ² atmosphere, then the cells were stained overnight at 4 ° C with the following antibodies: anti-IFN- and PE and anti-IL-4 APC . The cell suspensions were then washed and the standard extracellular procedure was followed for anti-CD3 FITC and anti-CD4 PerCP.

Culture of intestinal epithelial cells

Non-modified rat small intestine epithelia IEC18 (ECACC 88011801) (passages 25-50) were obtained from the Cell Culture Service of the University of Granada and cultured in DMEM containing fetal bovine serum (10%), 100 U / ml of penicillin, 0.1 Mg / ml of streptomycin and 2.5 mg / ml of amphotericin B. All experiments were performed with monolayers 2-5 days after reaching confluence. The IEC18 cells were pretreated in some experiments with shRNA specific for TLR4 for knockdown genes. All the reagents used in these experiments were obtained from Santa Cruz Biotechnologies (Heidelberg, Germany) and the manufacturer's instructions were followed. Briefly, IEC18 cells were seeded in six-well plates and cultured for 24 h until 50% confluency. Before infection, the IEC18 medium was supplemented with polybrene 5 ^ g / mL and the cells were incubated for 10 h. the control particles and TLR4 lentiviral shRNA were also pretreated separately with polybrene 5 ^ g / mL for 30 min, added to the culture medium and incubated overnight. On the third day, the fresh medium was replaced by lentiviral particles containing medium and the cells were cultured until confluence for 24 h. Finally, the IEC18 cells were divided (1: 5), cultured again for 24 h and selected with 10 ^ g / ml puromycin dihydrochloride.

To explore signaling pathways, confluent monolayers were exposed to IEC18 Bay 11-7082 (10 M), a selective inhibitor of phosphorylation of IkBa that blocks signaling via NF ^k B, or FPS-ZM1 (10 ^ M) (Merck Millipore, Billerica, MA), a RAGE antagonist. All inhibitors were dissolved in DMSO and added to the culture medium 2 h before exposure.

Statistic analysis.

In all the experiments, the samples were taken in triplicate, and the results are expressed as means with their standard errors (SEM). Differences between the means were tested using one-way ANOVA to see their statistical significance and tests of minimum significant difference of Fishera posteriori were performed on preselected pairs. All analyzes were performed with the SigmaStat 3.5 program (Jandel Corporation). The differences were considered significant with P <0.05.

Results

Mice treated with DSS (DSS mice) exhibited classic signs of colitis including loss of body weight from day 5 onwards, diarrhea and rectal bleeding, in addition to a general "diseased" appearance (curling behavior, reduced movements, erect hair ) (Figure 1A, Data not shown) The large intestine was hyperemic, thickened and rigid, with loss of the vascular pattern of the mucosa, resulting in a significant increase in the colonic damage score (Figure 1B / C / D). At the histological level, inflammation of the colon was characterized by an abnormal crypt structure and loss of crypts, infiltration of leukocytes (ie, neutrophils), enlargement of the submucosa, etc. (Figure 2), resulting in a score significantly higher (Table 1) Mice with colitis showed an increase in colonic AP activity, an indicator of epithelial stress and inflammation, associated with an in vitro sensitivity increased to the specific inhibitor levamisole (Figure 3). DSS-induced colitis was also associated with an increase in neutrophil markers MPO and S100A8 (Figure 4). Immunohistochemical analysis revealed that the S100A8 carrier cells were infiltrating the mucosa and submucosa, without expression at the epithelial level (Figure 4C). Colitis was also associated with splenomegaly (Figure 1E).

The mice treated with CP showed a reduced body weight loss compared to the DSS control, which was significant only for the C50 group on the last day (Figure 1a). The CP had no effect on the weight of the colon: length ratio (Figure 1B). The macroscopic damage score was reduced by half with both CP doses compared to the DSS group, but this did not reach statistical significance (Figure 1D). A dose-dependent improvement of the microscopic score was also observed (significant only with the highest dose, Table 1), corresponding to the preservation of the mucosal structure, particularly at the epithelial level (Figure 2). The activity of MPO was completely normalized with the dose of 50 jg / day and was actually below the non-inflamed control when CP was administered at 100 | jg / day (Figure 4A). Comparable results were observed with S100A8 (Figure 4B / C). Similarly, the activity of colonic AP and sensitivity to levamisole were generally lower in both treated groups, although only with the lowest CP dose was significance achieved (Figure 3). Spleen size was significantly reduced with the highest dose of CP (Figure 1E).

Table 1. Histological score of DSS mice treated with Calprotectin at 50 jg / d, 100 jg / d intrarectally; or vehicle. p <0.05 vs. control group; * p <0.05 vs. DSS group.

In order to elucidate in depth the beneficial effect of the CP, the colonic expression of a wide range of genes known to be differentially expressed in acute phase of DSS-induced colitis and at least in part associated with the disease was analyzed. inflammatory bowel Strong regulation of colonic cytokine expression was observed, including IL-10, TNF, IL-4 and IFN- and, (Figure 5). In addition, the colon mRNA levels of the Foxp3 regulatory T cell marker and the antibacterial peptide Reg3 and also markedly increased (p> 0.05 for the latter). It should be noted that the high variability in the DSS group prevented achieving statistical significance compared to the control group in some cases (IL-22). The CP caused a reduction of the expression of these inflammatory markers, which was significant for TNF, IL-10, Foxp3, and Reg3 and IFN and in one or both doses. IL-22 mRNA levels also were lower than in the DSS group, but without reaching statistical significance. Since CP has been described as a TLR4 ligand, its expression was also measured (Figure 5C). There was no effect of DSS colitis on this parameter, but both doses of CP treatment significantly reduced their mRNA levels.

On the other hand, in order to evaluate the immunological state within the intestinal environment, mouse splenocytes and MLNCs were cultured ex vivo, either in the presence or absence of the ConA T cell stimulator. The level of IL-17A was significantly reduced in the groups that received CP when the splenocytes (Figure 6A) and MLNCs (Figure 6C) were exposed to ConA. In contrast, the production of IL-10 was not altered neither in splenocytes nor in MLNC under stimulation with ConA (Figure 6B / D). In turn, the basal release of IL-10 was reduced in splenocytes from mice treated with CP, whereas that of MLNC remained unchanged (Figure 6C). ELISA measurement of cytokines in cell supernatants did not show a consistent trend in the case of IFN- y , TNFa and IL6 (data not shown).

Finally, splenocytes and whole peripheral blood cells were analyzed by flow cytometry. DSS-induced colitis was associated with a marked increase in blood neutrophil levels, which was reduced in the CP groups (Figure 7A). There were no significant changes in the number of CD4 and CD8 lymphocytes. In the spleen, CP increased, in a dose-dependent manner, the number of both CD4 + cells IL-4 + and IFN- and +, although this was not affected by DSS colitis (only significant for the 100 pg dose). , Figure 7B).

Tests carried out to demonstrate a systemic effect

In order to verify the possible systemic effects of the CP, a second experiment was carried out in which calprotectin was administered intraperitoneally to mice with DSS-induced colitis (DSS mice) following a protocol identical to the previous one. As shown in Table 2, there was no effect of CP on these experimental conditions, except for a slightly higher spleen weight.

Table 2. Effect of calprotectin in mice with DSS-induced colitis. Colitis was induced in mice with 2.5% DSS in drinking water. The DSS mice were treated with calprotectin at 50 ^ g / d intraperitoneally (i.p.) or vehicle.

Effect of CP on epithelial cells in vitro

Additionally, the effect of CP on intestinal epithelial cells was examined in vitro. IEC18 cells, a non-tumor cell line, were used. As expected, these cells showed minimal cytokine production under basal conditions, but released markedly increased amounts of GRO a and MCP1 when stimulated with LPS (Figure 8). The CP produced a substantial secretion of cytokines at 10 ^ M (equivalent to 360 ^ g / mL). Since it has been described that the CP acts via ligature of TLR4 and RAGE, the impact of the receptor interference was evaluated using knockdown genes and a specific antagonist, respectively. As expected, silencing TLR4 resulted in significant inhibition of cytokine secretion induced by LPS but had no effect on the evocation of secretion GRO, while MCP-1 (Figure 8 C is enhanced / D). In turn, the RAGE antagonist, FPS-ZM1, almost completely eliminated the secretion of GRO a / MCP-1 caused by CP or LPS (which is also a RAGE ligand). Similar effects were obtained with the inhibitor of NF ^k B, Bay11-7082 (Figure 8E / F).

Summary of trial results

The results presented demonstrate that exogenous CP exerts protective effects in DSS-induced colitis in mice. In order to obtain therapeutic benefits, the CP it must be administered intrarectally, since the activity was completely lost when it was administered intraperitoneally. Although pharmacokinetic data are not available, proteins, including large ones, such as antibodies, are usually efficiently absorbed when injected subcutaneously and are expected to follow similar behavior with the intraperitoneal route.

The main source of PC in DSS-induced colitis are presumably infiltrated neutrophils, which are avidly recruited to activate active mucosal lesions in both experimental colitis and patients with IBD, since they express large amounts of the protein. This is consistent with immunohistochemical analysis and elevated levels of MPO in the tissue, another predominant neutrophil protein, universally used as a marker of intestinal inflammation, as in the present study. Continuous intestinal inflammation is associated with increased fecal CP levels, as mentioned above, but it is not clear exactly how the protein accesses the luminal side. The neutrophils are able to perform a transepithelial migration, giving rise to crypt abscesses and, finally, also to the presence of neutrophils in the intestinal lumen. These are, therefore, a plausible source of fecal CP. In fact, the feces of patients with active IBD show increased levels of several neutrophil proteins. Activated macrophages can also contribute to the increase in luminal CP. It is also possible that mucosal PC can be filtered through the epithelium into the lumen, although this mechanism has not been established. Furthermore, it should be noted that although intestinal epithelium was traditionally considered not to express CP, it has recently been shown to produce S100A8 and / or S100A9. Therefore, this may be a possible additional source of fecal CP. However, Holgersen et al. We did not observe epithelial staining with anti-S100A8 in IL-10 KO colicy mice, and our own data do not support the epithelial expression of S100A8 in DSS-induced colitis.

Therefore, it is expected that the intrarectally administered CP acts mainly, or even exclusively, in the colonic lumen. Since the present study was designed as a proof-of-concept experiment, we have little direct evidence of the mechanisms that explain the beneficial effect observed. However, based on the known properties of the CP, we can speculate about several possibilities. First, CP can exert antibacterial actions, possibly in areas near the epithelium or in sites of epithelial injury with bacterial translocation. This has been identified as a relevant pathogenic factor in IBD and experimental colitis, so that protection against bacterial penetration and / or damage to the epithelial barrier is expected to be beneficial for the host. A possible impact on the composition of the microbiota is also conceivable, but this possibility has not been explored so far. Second, CP can act as an antioxidant in damaged mucosal areas. Third, CP can modulate the function of epithelial cells, for example by activating apical TLR4 or RAGE receptors.

In vitro results indicate that CP evokes the secretion of cytokines by IEC18 cells, a line of rat intestinal epithelial cells. This was also observed in mouse intestinal epithelial cells (IEC4.1 cells, data not shown). The effect was reached at relatively high concentrations (10 ^ M). However, this concentration is probably reached in vivo with the doses tested. Since a completely distended mouse colon contains ~ 0.3 ml, an intrarectal dose of 100 ^ g / day would result in a concentration of at least 333 ^ g / ml. Our data further indicate that CP evokes cytokine secretion through RAGE ligation and activation of NF ^k B. TLR4 was not involved in the release of GRO a , but appears to exert an inhibitory action on the production / secretion of MCP- one. Although the consequences of the secretion of cytokines evoked by increased epithelial cells are debatable, it is important to note that GRO to KO mice are more sensitive to DSS colitis. Our data also indicate that the CP activates in vitro macrophages (data not shown), consistent with previous reports. This can not be the only mechanism of the CP at the epithelial level, since the preliminary evidence obtained by analyzing microarrays of Caco 2 cells exposed to CP suggests that a set of genes related to the tight junction and epithelial junction is up-regulated, a effect that is consistent with the protection against inflammation (data not shown).

It is also interesting to note that the spleen and mesentery nodule mononuclear cells isolated from mice treated with CP exhibited a negatively regulated IL-17A production, while the levels of IL-10 remained stable, ie, similar to those in the group of DSS control. IL-10 is an important anti-inflammatory cytokine in different organs including the intestine, and IL-10 KO mice develop chronic colitis spontaneously. IL-10 is positively regulated in chronic colitis and tends to decrease as the inflammatory reaction decreases. In addition, IL-10 will correlates with the levels of FOXP3 colon, which is a marker of Treg cells, one of the main producers of IL-10. The use of CP to protect against pulmonary inflammation by induction of IL-10 has recently been described. Therefore, it is plausible that this is also an operative mechanism in DSS colitis.

It is interesting to consider the biological importance of the doses tested. Assuming a CP fecal level of ~ 50 ^ g / g as the normal upper limit in humans, the amount of CP in the colonic lumen in non-colitic mice would be approximately 20 ^ g, since normal fecal production in mice is approximately 0 4g / day Therefore, the doses tested in our study correspond to luminal levels higher than normal, and are probably in the range of those measured in colitis in humans. This raises the possibility that "physiological" CP may have a limiting role in the colic response. In this regard, it is interesting to consider the available evidence with KO S1008 / S100a9 mice. K0 S100a8 mice are not viable and die before birth. S100a9 mice are, on the contrary, viable and apparently normal, although they lack S100A8 / S100A9 heterodimers (ie, CP). The impact of the absence of CP on neutrophil biology is not resolved, with almost any effect detected in one study and the reduction of the chemoattractant response and the expression of CD11b in another. The effects in vivo are also controversial, with no differences observed in the models of peritonitis, inflammation of the skin and urinary tract infection, while a greater susceptibility of K0 S100a9 mice to the formation of edema after infection of the paw with Leishmania and impaired tissue repair after renal ischemic-reperfusion injury. Unfortunately, there are no reports on the effects on experimental colitis.

In conclusion, our data indicate that calprotectin exerts protective effects in experimental colitis when taken intrarectally, suggesting a possible similar beneficial role in patients with inflammatory bowel disease. Further experimentation is required to explore the mechanical and therapeutic aspects of this effect.

Claims (7)

A composition comprising a therapeutically effective amount of calprotectin, a dimer of the S100A8 protein or any polypeptide whose nucleic acid sequence possesses an identity greater than 50%, 60%, 70%, 80%, 90%, 95%, 99% or more with the sequence of S100A8; and the S100A9 protein or any polypeptide whose nucleic acid sequence possesses an identity greater than 50%, 60%, 70%, 80%, 90%, 95%, 99% or more with the sequence of S100A9, for use in the treatment of a disease or condition measured by the damage or degeneration of the intestinal epithelium. 2. A composition according to previous claim, suitable to be administered topically. 3. A composition according to any of the preceding claims, suitable to be administered intrarectally. 4. A composition according to any of the preceding claims, wherein the calprotectin is a dimer of the human proteins S100A8 and S100A9. 5. A composition according to any of the preceding claims wherein the sole active principle of the composition is calprotectin. 6. A composition according to any of the preceding claims, for the treatment of intestinal inflammatory diseases or conditions. 7. A composition according to any of the preceding claims, for the treatment of colitis.