WO2014170712A1 - Rac-1 inhibitors or pi3k inhibitors for preventing intestinal barrier dysfunction - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for intestinal barrier dysfunction in particular in patients suffering from inflammatory bowel diseases. In particular, the present invention relates to a compound selected from the group consisting of Rac-1 inhibitors or PI3K inhibitors for use in a method for preventing intestinal barrier dysfunction in a patient in need thereof.

Description

RAC-1 INHIBITORS OR PI3K INHIBITORS FOR PREVENTING INTESTINAL

BARRIER DYSFUNCTION

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for intestinal barrier dysfunction in particular in patients suffering from inflammatory bowel diseases.

BACKGROUND OF THE INVENTION:

Crohn's Disease (CD) is supposed to result from an alteration of the intestinal barrier associated with an over-reactivity of the mucosal immune system toward luminal stimuli in genetically predisposed individuals¹. Altered expression and distribution of tight junction (TJ) proteins have been reported in the colonic mucosa of CD patients². This breach in the intestinal barrier can be the cause or the consequence of a chronic inflammation. Enhance production of TNF-a and IFN-γ increases the myosin light chain kinase (MLCK, a key regulator of tight junction permeability) expression and activity³ and increases both the epithelial cells death⁴'⁵ and endocytosis of TJ proteins⁶ all contributing to deregulate intestinal permeability. The intestinal barrier dysfunction may also precede the onset of CD as suggested by intestinal barrier dysfunction reported in healthy first degree relatives of CD patients⁷'⁸. To explain this finding, a genetic predisposition to intestinal barrier dysfunction has been proposed^9"11. Among the identified CD susceptibility genes, NOD2, ATG16L1 and IRGM are those with the highest impact¹². NOD2^1007fs, ATG16L1^300A and IRGM variants have been associated with a deficiency of autophagy bulk degradation process that enables degradation and recycling of proteins and damaged organelles¹³.

Today, little is known about the relationship between autophagy and intestinal permeability. CD patients and their healthy relatives carrying NOD2 risk alleles have an increased paracellular permeability (PP)^{9 11}. Paneth cells from CD patients are characterized by the presence of crinophagy, an autophagy process directed against the secretory granules¹⁴. However, this effect does not seem related to CD genetic risk factors. Finally, the activation of autophagy triggers Paneth cell abnormalities and increases bacterial translocation in mouse¹⁵. The aim of the inventors was to unravel the molecular mechanisms which relate autophagy and intestinal permeability with special attention on the role of NOD2. SUMMARY OF THE INVENTION:

The present invention relates to a compound selected from the group consisting of Rac-1 inhibitors or PI3K inhibitors for use in a method for preventing intestinal barrier dysfunction in a patient in need thereof.

DETAILED DESCRIPTION OF THE INVENTION:

NOD2, the main Crohn's Disease (CD) susceptibility gene, plays a role in the regulation of autophagy. NOD2 mutated CD patients are characterized by an increased intestinal permeability. To better understand the relationship between autophagy and intestinal paracellular permeability (PP), the inventors induced autophagy by nutrient starvation or rapamycin treatment in mice and Caco-2 monolayers. Activation of autophagy increased the PP through tight junction (TJ) disassembling. Occludin and Zonula-occludens- 1 were redistributed within cell vesicles due to macropinocytosis through PI3K and the subsequent Racl activation. ΝΟϋ2^ (but not the NOD2^1007fs CD variant) was able to interact with Rac-1 in order to block TJ macropinocytosis and the subsequent increased PP. Finally, biopsies from CD patients showed autophagy vacuoles together with increased PP. These results contribute to explain intestinal barrier dysfunction associated with CD and other disorders especially when associated with NOD2 dysfunction.

The present invention relates to a compound selected from the group consisting of Rac-1 inhibitors or PI3K inhibitors for use in a method for preventing intestinal barrier dysfunction in a patient in need thereof. According to the invention, the patient suffers from a disease that leads to an intestinal barrier dysfunction. Typically, the patient suffers from an inflammatory bowel disease. However, intestinal barrier dysfunction (e.g. gut permeability) is also an essential component of many disorders. For most of them, an association with NOD2 mutations or IBD lesions has been documented including Graft Versus Host Disease (Holler, E., et al. Both donor and recipient NOD2/CARD15 mutations associate with transplant-related mortality and GvHD following allogeneic stem cell transplantation. Blood 104, 889-894 (2004)); sepsis related mortality (Brenmoehl, J., et al. Genetic variants in the NOD2/CARD15 gene are associated with early mortality in sepsis patients. Intensive Care Med 33, 1541-1548 (2007)); spontaneous bacterial peritonitis and survival in liver cirrhosis (Appenrodt, B., et al. Nucleo tide-binding oligomerization domain containing 2 (NOD2) variants are genetic risk factors for death and spontaneous bacterial peritonitis in liver cirrhosis. Hepatology 51, 1327- 1333); intestinal failure in patients with short bowel syndrome (Ningappa, M., et al. NOD2 gene polymorphism rs2066844 associates with need for combined liver-intestine transplantation in children with short-gut syndrome. Am J Gastroenterol 106, 157-165.); arthritis (De Keyser F, Elewaut D, De Vos M, De Vlam K, Cuvelier C, Mielants H, Veys EM. Bowel inflammation and the spondyloarthropathies. Rheum Dis Clin North Am. 1998 Nov;24(4):785-813); irritable bowel syndrome and graft rejection after small bowel transplantation (Fishbein, T., et al. NOD2-expressing bone marrow-derived cells appear to regulate epithelial innate immunity of the transplanted human small intestine. Gut 57, 323- 330 (2008).).

In some embodiments, the patient has at least one NOD2 mutation as above exemplified.

As used herein the term "inflammatory bowel disease" has its general meaning in the art and refers to any inflammatory disease that affects the bowel. The term includes but is not limited to ulcerative colitis, Crohn's disease, microscopic colitis (lymphocytic colitis and collagenous colitis), infectious colitis caused by bacteria or by virus, radiation colitis, ischemic colitis, pediatric colitis, undetermined colitis, and functional bowel disorders (described symptoms without evident anatomical abnormalities).

As used the term "intestinal barrier dysfunction" refer to the intestinal barrier dysfunction typically observed in the patients suffering from an inflammatory bowel disease in particular from Crohn's disease, but that may occur in other diseases as above exemplified. Typically, the intestinal barrier dysfunction is characterized by intestinal paracellular permeability and bacterial translocation.

In some embodiments, the compounds of the present invention are thus particularly suitable for preventing intestinal paracellular permeability and bacterial translocation.

In some embodiments, the compounds of the invention are thus useful in the treatment of inflammatory bowel diseases, especially for maintaining the patient in a long term remission after a standard treatment selected from the group consisting of corticosteroids, immunosuppressive drugs, aminosalicylates sulfasalazine, such as Mesalazine (also known as 5-aminosalicylic acid, mesalamine, or 5-ASA. Brand name formulations include Apriso, Asacol, Pentasa, Mezavant, Lialda, Fivasa, Rovasa and Salofalk.), Sulfasalazine (also known as Azulfidine), Balsalazide (also known as Colazal or Colazide (UK)), Olsalazine (also known as Dipentum), immuno suppressors (azathioprine, 6-mercaptopurine, methotrexate, rapamycine, cyclosporine and tacrolimus) or biological treatments such as Infliximab, Visilizumab, Adalimumab, or Vedolizumab or a combination thereof.

As used herein the term "Racl" has its general meaning in the art and refers to ras- related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Racl). Racl is a member of the Rho family of low molecular weight GTPases and are related to each other based on sequence homology and function (Vojtek, A. B., and Cooper, J. A., Cell 1995, 82, 527-529). In an active state, they bind to GTP and transduce signals of other proteins in signal transduction pathways. In their inactive state, they are bound to GDP. Members of the Rho family are typically involved in regulation of the actin cytoskeleton.

As used herein the term "Rac-1 inhibitor" designates any compound or treatment that reduces or blocks the activity of Rac-1. More preferred Rac-1 inhibitors are compounds that inhibit Rac-1 activation by its GEFs (guanine nucleotide exchange factor) in an exchange assay, and/or that inhibit Rac-1 -dependent cytoskeleton rearrangements. The term also includes inhibitors of Rac-1 expression.

Rac-1 inhibitors are well known in the art. A typical example of Racl inhibitors includes NSC 23766 described in international patent application WO 2007/016539. The compound is a cell- permeable pyrimidine compound that specifically and reversibly inhibits Racl GDP/GTP exchange activity by interfering Racl interaction with the Rac-specific GEFs. A typical example of Racl inhibitors includes EHT 1864 described in international patent application WO 2004/076445. EHT 1864 is a small molecule that blocks the Racl signaling pathways. A typical example of Racl inhibitors includes Berberine which is a member of the protoberberine class of isoquinoline alkaloids. It is probably the most widely distributed of all alkaloids, having been found in the roots, rhizomes, and stem bark of the plants of nine botanical families, Berberidaceae, Papaveraceae, Rununculaceae, Rutaceae, Menispermaceae, Rubiaceae, Rhamnaceae, Magnoliaceae, and Annonaceae.

Other examples of Racl inhibitors include those described in EP2433636, WO2007031878, WO2007016539, WO2009007457 and WO2005051392. Examples of Racl inhibitors which are activity inhibitors include Racl Inhibitor W56, sold by Tocris Biosciences (Ellisville, Mo.), NSC23760 sold by EMD Biosciences (San Diego, Calif.) or the inhibitors described in Yuan Gao, et al. PNAS, May 18, 2004, vol. 101, 7618-7623. As used herein the term "PI3K" has its general meaning in the art and refers to any phosphoinositide 3-kinase. Phosphoinositide 3-kinases (also called Phosphatidylinositol 3- kinases or PI3Ks) constitute a family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways. PI3Ks are divided into three different classes: Class I, II and III. They are further divided between class IA and IB. Class IA PI3K is composed of a heterodimer between a pi 10 catalytic (α, β and δ isoforms) subunit and a p85 regulatory subunit. Class IB PI3K heterodimers contain a plOl regulatory subunit and a pi 10 catalytic subunit (γ isoform). Class I PI3Ks are intracellular signal transducer enzymes capable of phosphorylating the phosphatidylinositol-4,5- diphosphate (PIP2) to form the phosphatidylinositol-3,4,5-triphosphate (PIP3). The formation of PIP3 plays a role in the PI3K-dependent activation of the PI3K/AKT pathway. Indeed, PIP3 allows the recruitment of AKT and PDK1 (PDK1 or Phosphoinositide-Dependent Protein Kinase 1). Since PIP3 is restricted to the plasma membrane, this results in recruitment of AKT and PDK1 to the plasma membrane. The colocalization of activated PDK1 and AKT allows AKT to become phosphorylated by PDK1 on threonine 308, leading to partial activation of AKT. Full activation of AKT occurs upon phosphorylation of serine 473 by the TORC2 complex comprising the mTOR protein kinase. AKT (also known as PKB, Protein Kinase B) is a serine/threonine protein kinase that regulates cellular survival and metabolism by binding and regulating many downstream effectors. As use herein the term 'PI3K inhibitor" designates any compound or treatment that reduces or blocks the activity of PI3K. When the PI3K enzyme is inhibited, PI3K is unable to exert its enzymatic, biological and/or pharmacological effects. In one embodiment, the activity of PI3K alpha is inhibited. In another embodiment, the activity of PI3K beta is inhibited. In another embodiment, the activity of PI3K gamma is inhibited. In yet another embodiment, the activity of PI3K delta is inhibited. In a preferred embodiment the activity of PI3K gamma is inhibited. Such inhibitory activity can be determined by assays or animal models well-known in the art. The term also includes inhibitors of PI3K expression. PI3K inhibitors are well known in the art (Current Medicinal Chemistry, 201 1 , 18,

2686-2714; Macias-Perez IM, Flinn IW. GS-1101 : A Delta-Specific PI3K Inhibitor in Chronic Lymphocytic Leukemia. Curr Hematol Malig Rep. 2013 Mar;8(l):22-7. doi: 10.1007/s 11899-012-0142-1.; Kong DX, Yamori T. ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor identified using the JFCR39 drug discovery system. Acta Pharmacol Sin. 2010 Sep;31(9): l 189-97. doi: 10.1038/aps.2010.150. Epub 2010 Aug 23. Review.).

Two well-known PI3K inhibitors are LY294002, a morpholine derivative of quercetin, and wortmannin, a furanosteroid metabolite of the fungi Penicillium funiculosum or Talaromyces (Penicillium) wortmannii. LY294002 is a reversible inhibitor of PI3Ks whereas wortmannin acts irreversibly. LY294002 and wortmannin are both pan- inhibitors of PI3Ks.

An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.

Inhibitors of gene expression for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein (i.e. PI3K or Rac-1), 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 the target protein can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically 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 (siR As) can also function as inhibitors of gene expression for use in the present invention. Gene expression can be reduced by contacting the tumor, subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschi, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

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

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

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

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

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

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

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

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

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Galenic adaptations may be done for specific delivery in the small intestine or colon.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

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

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

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

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

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

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

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The Compound of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations ; time release capsules ; and any other form currently used.

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. EXAMPLE: Nod2-Racl interactions control the paracellular permeability related to macropinocytosis of enterocyte tight junctions induced by autophagy

Material & Methods Patients: ileal biopsies from nn non inflammatory controls and nn CD and nn

Ulcerative colitive patients were obtained from non inflamed areas of the ileum during routine endoscopy. All participants signed an informed written consent.

Animals. All the animals were on the C57BL/6 background. Wild type (WT), Nodi^1'

²² , Nod2^2939iC47 and ATG16L1^HP48 mice were housed in animal facility with free access to food (UAR pellets) and water. Free pathogen conditions were checked according to FELASA recommendations.

Cell lines. Caco-2 cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen collection. Enterocytic Caco-2/TC7 were previously reported⁴⁹. Cells were grown at 37°C in a 5% C0₂ water saturated atmosphere in Glutamax DMEM (Gibco) supplemented with 20% of heat inactivated fetal bovin serum (Biowest), 1% non-essential amino acids and 1% antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, Gibco). Cells were seeded in Transwell inserts (Costar) and grown for 14 days.

Reagents. MDP, 3-methyl adenine (3-MA), Wortmanin, cytochalasin D, 5-(N-ethyl- N-isopropyl)-amiloride (EIPA), methyl-B-cyclodextrin (MBCD), Nystatin were obtained from Sigma. NSC23766 and W56 were obtained from Tocris Biosciences. Latrunculin B was obtained from Enzo Life Sciences. and rapamycin from Calbiochem.

Expression Vectors and cell transfections. All cloning experiments were performed using the Gateway™ system (Invitrogen). All cDNAs were cloned and sequenced into pDONR™/Zeo and subsequently shuttled into pEGFP-GW as described in Ref⁵⁰. Cells transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection, three cycles of selection were performed using G418 (sigma, 400-800μg/mL, four days/cycle). Then, the most efficiency transfected Caco- 2/TC7 cells were selected by FACS. NOD2 expression, cellular growth, cell differentiation (evidenced by mRNA expression of sucrase-isomaltase and alkaline phosphatase as well as alkaline phosphatase activity) were checked in the three cell lines. mRNA expressions of MLCK, occludin and ZO-1 were not modified by NOD2 transfection. Caco-2/TC7 cells were similarly transfected with the Rac-1N17 and Rac-1 V12 expression vectors.

Induction of autophagy. For in vivo and ex vivo experiments, mice were housed in metabolic cages to prevent coprophagia. They had free access to water. They were fasted for 24 hours⁵¹ or let with free access to food (for the control group). All animals survived 24 hours of food restriction. Induction of autophagy was also induced by an i.p. injection of 100μg of rapamycin 24 hours before experiments. When indicated, mice were pre-treated i.p. with 3-MA (3mg/mice, 3 hours before NS) or MDP (100μg/mice, one day before NS). For in vitro experiments, the cells were incubated in a medium without amino acids (NS), washed with PBS and cultured in Earle's Balanced Salt Solution (EBSS, Sigma-Aldrich) for the indicated times. Autophagy was alternatively induced by adding rapamycin into the medium (0.5μΜ).

Measures of permeabilities. For in vivo experiments, mice were gavaged with fluorescein-isothiocyanate labelled dextran 4kDa (FD4; 15η¾/100μΕ per mice; Sigma) 5 hours prior the sacrifice. Whole serum FD4 level was determined with a fluorometer (PerkinElmer). For ex vivo experiments, mice were sacrified 24h after NS and biopsies from their ileal mucosa were mounted in Ussing Chambers and maintained in circulating oxygenated Ringer solution at 37°C. PP was assessed by measuring the mucosal-to-serosal flux of FD4 with a fluorometer (PerkinElmer). For in vitro experiments, the cell lines were cultivated for 14 days in a TC system⁵². Then, FD4 (10^"5M) was added into the apical compartment and the permeability was monitored by sampling 400μί from the basolateral compartment 2, 4, 6 and 8 hours following autophagy induction. When indicated Caco-2 cells were pre-incubated with wortmaninn (115nM), NSC23766 (250μΜ), W56 (200μΜ), cytochalasin D (^g/ml), latrunculin B (O^mol/L), EIPA (50μΜ), MB CD (2.5mM/ml)), Nystatin (300ng/mL) or ML-7 (20μg/mL).

RT-PCR. After extraction by the NucleoSpin RNA II Kit (Macherey-Nagel), total RNAs were converted to cDNAs using random hexonucleotides. PCR was performed using QuantiTect SYBR Green PCR Kit (Applied), sense and antisense primers specific for ZO-1 , ZO-2, ZO-3, Occludin, JAM-A, Claudin 5, Claudin 8, the long isoform of MLCK, Sucrase isomaltase, Phosphatase alkaline and G3PDH (table 1). After amplification, the cycle threshold (Ct) was determined and expressed as 2^~AACt.

Biological assays. Total protein concentrations were determined using a commercial kit (Biorad). MLCK concentrations were measured by ELISA (Cusabio) ⁵². Enzymatic activities were measured using commercial kits for Alkaline phosphatase (Biovision), PI3K (Millipore) and LDH (Cytotox 96^R , Promega). For LDH assays, Caco-2/TC7 cells exposed to Triton X100 (0.9%) were used as a control of total release (100% LDH release) while the background level was determined on the culture medium (0%> LDH release).

Transmission electron microscopy. Caco-2/TC7 filters or fresh biopsies were flushed with cold PBS and treated with a cold glutaraldhehyde/tannic acid in cacodylate buffer. Fine samples were processed as previously described⁵². Observations were made using a JEOL CXI 00 equipped with a Gatan Digital camera and the micrographs were processed with Gatan software. Measurements of tight junctions were performed with Image J software.

Immunofluorescence studies. Caco-2/TC7 filters were washed in PBS, fixed with cold methanol and washed again in PBS. After incubation in PBS/1%BSA at room temperature, Caco-2 filters were incubated with anti-rabbit primary antibodies (occluding or ZO-1, Invitrogen) and washed in PBS. Filters were then incubated with the Alexa 488 secondary antibody (Invitrogen). Fluoprep reagent solution (Biomerieux) was used to mount the filters on the cover slips.

Caspase-3 staining. Caco-2/TC7 filters were washed in PBS, fixed in 4% phosphate buffered formalin and embedded in paraffin blocks and cut into 5μιη sections. Sections were deparafmised and subjected to a heat-induced antigen recovery in sodium citrate buffer (pH6). Endogen peroxydases were blocked with 3% H₂0₂ (DAKO) and slides were incubated (30 min) with the primary rabbit polyclonal antibodies against Cleaved Caspase-3 (Cell Signaling Technology, Inc Ozyme). A biotin- labeled secondary antibody was then applied for 30 min, followed by avidin-biotin-per oxydase conjugate for 30 min (Vector Laboratories, Burlingame, CA, USA). Peroxidase enzyme substrat 3,3'-diaminobenzidine was finally added to yield a brown reaction product. Western blot analyzes. Caco-2 monolayers or ileal biopsies from mice were lysed in RIPA buffer and centrifuged. Soluble proteins (lOOug) were boiled, separated on a SDS- PAGE gel and transferred to a membrane (iBlot gel transfer stacks, Nitrocellulose regular; Invitrogen) overnight. The membranes were incubated overnight in a blocking solution containing anti-LC3 (Sigma), anti-Lamp-1, anti-Occludin (Zymed) or anti-ZO-1 (Cell Signaling). After being washed, the membranes were incubated with HRP-conjugated antibody (GE-Healthc are) and develop ed using the Sup er S ignal West Pico Chemiluminescent Substrate (Thermo Scientific) on the Amersham Hyperfilm™ film ECL.

Co-immunoprecipitation experiments. Cells were lysed with a 1% Digitonin- containing buffer and processed as previously described in ref⁵³. Cell lysates were immunoprecipitated over 2 hours at 4°C with either Rac-1 or NOD-2 antibodies (Santa Cruz Biotechnology) and incubated with 75 μ\ protein G Sepharose overnight at 4°C, as reported⁵². Proteins were resolved by sodium dodecylsulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE), transferred on to nitrocellulose membranes (Invitrogen) and immunob lotted with primary antibodies (rabbit anti-RAC-1 (Santa Cruz Biotechnology), and rabbit anti-PAKl (Cell Signaling Technology)) followed by goat anti-rabbit IgG (Jackson Immuno Research Laboratories) coupled to horseradish peroxidase (HRP). Membranes were developed by enhanced chemical luminescence (ECL) treatment (Amersham Biosciences).

Cell fractionation. Caco-2/TC7 cells s were rinsed, scraped off, and collected in ice- cold PBS containing 1% protease/phosphatase I and II inhibitor cocktail (Sigma). Cells were then disrupted by nitrogen cavitation (650 Psi, 30min, 4°C). Centrifugation at 10,000 x g for l h at 4°C was used to sediment cell debris, mitochondria, and nuclei. The resulting supernatant was centrifuged at 100,000 x g for lh to sediment plasma membranes. The 100,000 x g pellet was washed and resuspended in RIPA buffer [50 mM Tris-Cl (pH 8.0), 320 mM sucro s e , 0 . 1 mM EDTA, 1 mM DTT, 1% Nonidet P-40 , 0. 1 % SD S , 1 % protease/phosphatase I and II inhibitor cocktail]. The 100,000 x g supernatant represent cytosol. The membrane and cytosol proteins (50-100 μg) were separated on 10% SDS-PAGE, transferred using iBlotGel Transfer Device (Invitrogen) and probed with primary antibodies: Occludin, Lamp-1 (Zymed) or Actin (Sigma). HRP-conjugated secondary antibodies were detected using ECL reagents (Pierce).

Statistical analysis. Two groups' comparisons were performed using unpaired t-test (Mann- Whitney) with a two-tailed P value. Multiple groups comparisons were performed using an one-way analysis variance test (Bonferroni). Statistical analyzes were performed using GraphPad Prism 4.00 (GraphPad Software). A P-value less than 0.05 was considered as statistically significant (two sided tests). Values were expressed as mean±SEM.

Study approvals. Patient studies were approved by the French ethic committee (Comite de Protection des Personnes, He de France IV n°2008/53). Housing and experiments were led following institutional animal healthcare guidelines and were approved by the local ethical committee for animal experimentation (Comite Regional d'Ethique en matiere d'Experimentation Animale n°4).

Results

Autophagy increases the permeability by a direct effect on the enterocyte functions.

Autophagy was induced in C57B6 wild type (WT) mice by fasting or by i.p. injection of rapamycin. Transmission electron microscopy (TEM) analyses showed a strong accumulation of autophagic vacuoles associated with an increased number of lysosomal structures in enterocytes of the ileal mucosa. Consistently, an enhanced conversion of LC3-I to LC3-II was also noted. No apoptosis was observed.

Biopsies from the ileal mucosa were mounted in Ussing chambers and PP was investigated ex vivo by monitoring the flux of 4kD-dextran FITC (FD4). A threefold increase of the PP was detected after fasting or rapamycin treatment. An increased translocation of Escherichia, coli was also observed after fastin. Treatment with 3-methyladenine (3 -MA, a canonical inhibitor of autophagy) significantly reduced fasting-induced PP and E. coli translocation. Moreover, fasting or rapamycin treatment did not alter the PP in ATG16L1 hypomorphic (ATG16L1^HP) mice which exhibit impaired autophag. We next assessed FD4 serum concentrations in both WT and ATG16L1^HP mice after fasting or rapamycin treatment. Increased PP was only detected in WT mice demonstrating that a functional autophagy increases the PP in vivo.

As autophagy modulates the expression of pro -inflammatory cytokines (TNF-a and IFN-γ), which are known to increase the gut permeability³, PP was further assessed in polarized Caco-2 or Caco-2/TC7 monolayers after nutrient starvation (NS) or rapamycin treatment. The induction of autophagy was checked by LC3 Western blotting and TEM. An increased PP was demonstrated by the accumulation of FD4 into the baso lateral compartment of Transwell chambers (TC) and by a partial drop of the transepithelial electrical resistance.

An increased PP could result from epithelial cell death (necrosis or apoptosis), or from the modulation of TJ¹⁶'¹⁷. Cell death was thus monitored by the release of the cytosolic lactate deshydrogenase (LDH) into the medium and by Dapi, Tryptan blue and Caspase 3 stainings. No effect of autophagy on cell vitality was observed. As a whole, we thus concluded that the autophagy directly affects the enterocyte function. Activation of the autophagy process induces TJ disassembling.

We assessed TJ opening both in mouse ileal mucosa and Caco-2/TC7 cells by TEM after fasting or rapamycin treatment. TJ intercellular spaces were significantly enlarged under autophagy activation in both in vitro and in vivo models. Cytoskeletal reorganization and more specifically myosin light chain (MLC) phosphorylation via MLCK is a mediator of physiological and pathophysiological TJ regulation³. We thus measured MLCK mRNA and protein expression levels in Caco2/TC7 cells. NS and rapamycin treatment did not affect MLCK expression. In addition, the MLCK inhibitor ML-7 had no effect on the autophagy- dependent increased PP in Caco-2/TC7 cells. Altogether, these results showed that MLCK was not involved in TJ disassembling.

In contrast, NS was associated with reduced expressions of occludin and zonula- occludens 1 (ZO-1), two constitutive TJ proteins, both in the mouse ileal mucosa and Caco- 2/TC-7 cells while no modification of the expressions of occludin, ZO-1 or others TJ proteins were observed at the mRNA level. Confocal analyses show an altered apical localization of occludin and ZO-1 in Caco-2/TC7: the proteins were heterogeneously spread on the cell membrane but also visible within the cytosol. These alterations were reversible by the addition of serum, glucose and amino acids in the culture medium. Altogether, these data suggested that autophagy accounted for the redistribution and degradation of both occludin and ZO-1 through a dynamic process. Autophagy induced TJ disassembling is mediated by macropinocytosis.

Because endocytosis and autophagy are intertwined pathways¹⁸, the hypothesis of TJ endocytosis was further explored. TEM analyses showed that fasting triggered the formation of endosomal vacuoles and amphisomes (autophagic vacuoles formed by fusion between autophago somes and endosomes), along the lateral cell membrane. Subcellular fractionation by gradient density evidenced a redistribution of occludin into the cytosolic compartment in Caco-2/TC7 after 8 hours of NS. Consistent with an increased endocytosis along the membrane, an enrichment of actin in the membrane sub-fraction was also observed. Lamp-1 (a marker of lysosomes) was overexpressed after NS confirming the induction of autophagy. Its strict intracellular distribution confirmed the absence of contamination during subcellular fractionation. Finally, co-localization analyses with EEA1, LC3 and Lamp-1 (three vesicular markers) further demonstrated that occludin was internalized through the endosomal and autophago-lysosomal pathways.

To decipher the endocytosis pathways involved in the occludin internalization, PP was monitored in Caco-2/TC7 monolayers in the presence of inhibitors of endocytosis. Chloropromazine, Methyl-P-cyclodextrin (Μβ-CD) or Nystatin did not affect the NS induced PP, excluding the involvement of clathrin- or lipid raft/caveolae-mediated endocytosis. In contrast, EIPA (an inhibitor of the sodium-proton exchange); Cytochalasin D (which blocks actin polymerization) and Latrunculin (which binds to monomeric actin preventing its incorporation into filaments) strongly reduced autophagy-dependent PP. As a result, we concluded that TJ proteins were internalized by macropinocytosis and degraded into the autophago-lysosomal machinery.

PHK and Rac-1 activation are involved in T J macropinocytosis.

Phosphoinositide 3-kinases (PI3K) controls essential cellular functions like cytoskeletal dynamics, signal transduction and membrane trafficking and therefore mediates endocytic membrane traffic, macropinocytosis and autophagy. We thus assessed whether the PI3K signaling pathway was involved in the autophagy-dependent PP and TJ macropinocytosis. The activity of PI3K was increased in Caco-2/TC7 cells after NS. Furthermore, wortmanin (a PI3K specific inhibitor), abrogated the increased PP and prevented the activation of PI3K by inhibiting its phosphorylation.

Given the ro le o f Rac-1 (a key downstream target/effector of PI3K), in

19 20 21

macropinocytosis ' and in cell junction remodeling , autophagy-induced PP was investigated in the presence of W56 and NSC23766, two Rac-1 inhibitors. These drugs also abrogated or partially thwarted the PP induced by NS. As Rac-1 regulates the actin cytoskeleton, through its binding which PAKs, we investigated the interaction of Rac-1 with PAK-1. Co-immunoprecipitation experiments showed that Rac-1 was associated with PAK-1 after NS, consistent with the activation of Rac-1 during NS. This association was alleviated in the presence of Wortmanin.

To further explore the role of Rac-1, the impact of Rac-1 mutations on occludin expression in Caco-2/TC7 was studied in stably transfected cells. Under basal conditions, the expression of occludin was reduced in cells transfected with a constitutively active Rac-1 variant (VI 2), while no modifications were seen in cells transfected with a Rac-1 dominant negative cDNA (N17). After induction of autophagy, a decreased expression of occludin was observed in control and V12 cells while no effect was seen in N17 cells. As a whole, we concluded that autophagy triggers TJ macropinocytosis via PI3K and the actin-myosin cytoskeleton regulator Rac-1. NOD2 abrogates the autophagy dependent PP.

Since NOD2 plays a pivotal role in the regulation of intestinal PP 9 ' 10 ' 22 ' 23 and in the induction of the autophagic process²⁴'²⁵, we tested its putative role on the autophagy dependent PP. NOD2 activation by MDP (a NOD2 ligand) inhibited the autophagy-dependent PP in the ileum of starved mice. This effect was not observed in Nod2 knock-out (Nod2^_/~) and Nod2^2939lC mice carrying a mutation homologous to the NOD2^1007fs human CD associated varian. Treatment of Caco-2/TC7 cells with MDP blunted the degradation of occludin and ZO-1 proteins induced after NS. Altogether, these data indicated the inhibitory role of NOD2 on the autophagy-dependent PP.

Caco-2/TC7 cells were next stably transfected with NOD2™¹ or NOD2^1007fs expression plasmids. NOD2™¹ and NOD2^1007fs mRNA levels were 800 fold higher than in control cells transfected with an empty vector. Although NS decreased the LC3I/LC3II ratio and increased Lamp-1 expression at the protein level in the three cell lines, activation of autophagy did not influence PP in NOD2^wt-overexpressing Caco-2/TC7 cells. In contrast, NS-induced PP was significantly increased in the NOD2^1007fs-overexpressing cell line when compared to controls. As expected, PP was correlated with loss of occludin and ZO-1. Altogether, we concluded that NOD2^wt but not the CD associated 1007fs variant inhibited TJ disassembling without modifying the autophagic flux.

NOD2 blocks Rac-1 activation and macropinocytosis.

To explain how NOD2 reverses the autophagy-induced PP, we checked the level of

PI3K activity in the panel of stably transfected Caco-2/TC7 cell lines. PI3K activity was similarly increased after NS in all cell lines, suggesting an action of NOD2 downstream of PI3K. A recent study reported a molecular interaction between NOD2 and Rac-1 in intestinal epithelial cells after F-actin disruption²⁶. We thus investigated whether NOD2 physically interacts with Rac-1. In the control cell line, only a weak interaction was visible following NS. Although no interaction was observed in NOD2™¹ overexpressing Caco-2/TC7 cells under basal conditions, NOD2/Racl strongly interacted after the induction of autophagy. In contrast, a very weak interaction was detected following NS in Caco-2/TC7 cells overexpressing NOD2^1007fs. PAK-1 immunoblotting experiments showed that PAK-1 did not belong to the NOD2 /Rac-1 complex, suggesting that NOD2 interacts only with the GDP-bound inactive form of Rac-1 in these conditions. Following autophagy induction, an absence of Rac- l/PAKl interaction was observed in Caco-2/TC7 over-expressing ΝΟϋ2^. In contrast, a strong interaction between Rac-1 and PAK1 (577% of empty vector cells) was observed in cells overexpressing NOD2^1007fs, suggesting that Rac-1 is activated after NS in presence of _NOD2ioo7fs _{s imi l arly ?} in Caco-2/Tc7 non transfected cells, NS strongly increased the interaction between NOD2 and Rac-1. NOD2 activation by MDP did not modify the interaction between NOD2 and Rac-1 but strongly decreased Rac-l/PAKl interactions after NS. As a whole, we concluded that Nod2 inhibits Rac-1 by a direct physical interaction and that MDP reinforces this effect. Considering that Rac-1 inhibition line up NOD2/Rac-l interactions, this effect might be due to the sequestration of the inactive form of Rac-1 by NOD2.

Autophagy and PP are increased in the epithelium of CD patients.

To explore the relevance of these findings in CD, we studied fresh ileal biopsies obtained from non inflamed areas in CD patients. Autophagy vacuoles were easily observed in the patients by TEM while they were very rarely found in controls. Consistently, Ussing Chambers experiments using the FD4 marker demonstrated an increased PP. As a whole, these observations suggested that the above reported experimental findings may contribute to explain the intestinal barrier dysfunction associated with CD.

Discussion:

This study demonstrates that the induction of autophagy alters the PP of the intestinal mucosa and highlights the cellular and molecular mechanisms by which it dismantles the epithelial barrier. Autophagy triggers both PI3K/Rac-1 and PBK/mTOR signalling pathways which are responsible of the macropinocytosis and autophago-lysosomal TJ degradation. NOD2^wt (but not the CD associated variant NOD2^1007fs) maintains the epithelial barrier homeostasis by inhibiting Rac-1 and subsequently the macropinocytosis of TJ.

The deleterious role of autophagy on intestinal barrier reported here converges with previous data⁶'¹⁸. An abnormal autophagic degradation of endothelial TJ proteins was also reported to disrupt the brain-blood barrier after exposure to nanoaluminia particles²⁷. In endothelial cells, the nitrosative stress leads to the induction of autophagy and to the degradation of ZO-1 by lysosomes²⁸. As a result, autophagy causes epithelial or endothelial barrier disruption by abnormal elimination of TJ proteins²⁸. Here we further show that, at least in enterocytes, this process involves Rac-1 which participates to the formation of macropinocytosis vacuoles and cell junction architecture²⁹.

NOD2 was previously reported to interact with Rac-1 but the impact of NOD2-Rac-l interaction remained unclear²⁶'³⁰. This work demonstrates that NOD2^wt (but not the NOD2¹⁰⁰⁷^ variant) interacts with the inactive form of Rac-1 and inhibits its activation. The final result is the inhibition of TJ macropinocytosis. Interestingly, Nod2 also decreases MLCK dependent macromolecule transcellular permeability and bacterial translocation²³. As a result, Nod2 may be seen as a general inhibitor of the epithelial permeability for small molecules, antigens and bacteria³¹. Its role in endothelial cells has now to be explored.

Epithelial barrier functions with its fencing properties against small and large molecules are impaired in CD⁸'^32"37. Nevertheless, the causes and consequences of the "leaky gut" is a subject of debate. A defect in barrier function may increase antigen uptake into the gut mucosa with a subsequent inflammatory and adaptive immune response³⁸ but in turn, inflammation is known to increase the intestinal permeability especially through the production of Thl cytokines like TNF-a and INF-γ³⁹. Indeed, TNF -a is able to alter the integrity of the epithelial barrier by modulating MLCK expression and activity³, epithelial cell death⁴'⁵ and endocytosis of TJ proteins⁶. IFN-γ also alters the epithelial barrier function by macropinocytosis of TJ proteins¹⁸. Finally, autophagy is able to modulate the expression of TNF-a or IFN-γ³. In the other hand, the increased permeability observed in CD patients in remission and in their healthy relatives (especially in case of NOD2^l007is mutation), suggests that the inflammation is not the cause of the leaky gut. Recently, the inflammatory consequences of a constitutive gut barrier dysfunction have been demonstrated in mice overexpressing MLCK³⁸. The data presented here further support the opinion that the gut barrier defect may precede mucosal inflammation.

In addition to CD, several Human diseases have been associated with a defect of the epithelial barrier function. Among them, an association with NOD2 mutations has been documented in Graft Versus Host Disease⁴⁰; sepsis related mortality⁴¹; spontaneous bacterial peritonitis and survival in liver cirrhosis⁴²; intestinal failure in patients with short bowel syndrome⁴³ and graft rejection after small bowel transplantation⁴⁴. This work suggests to further explore the autophagic process in the gut of these critically sick patients.

In these disorders, the present data suggest that Racl inhibitors may provide new therapeutic options to treat the intestinal barrier dysfunction. Interestingly, azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are widely used in inflammatory bowel diseases. They induce a specific blockade of Racl activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Racl instead of GTP⁴⁵'⁴⁶. Altogether, these observations argue for further studying Racl inhibitors in diseases associated with a gut barrier defect. REFERENCES:

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Claims

CLAIMS:

A compound selected from the group consisting of Rac-1 inhibitors or PI3K inhibitors for use in a method for preventing intestinal barrier dysfunction in a patient in need thereof.

The compound for use according to claim 1 , wherein the patient suffers from a disease selected from the group consisting of inflammatory bowel diseases, graft host disease, sepsis related mortality, spontaneous bacterial peritonitis and survival in liver cirrhosis, intestinal failure in patients with short bowel syndrome, arthritis, irritable bowel syndrome and graft rejection after small bowel transplantation.

The compound for use according to claim 2, wherein the patient suffers from Crohn's disease.