WO2013121034A1

WO2013121034A1 - Methods and pharmaceutical compositions for reducing adipose tissue inflammation

Info

Publication number: WO2013121034A1
Application number: PCT/EP2013/053148
Authority: WO
Inventors: Nicolas VENTECLEF; Karine Clement; Elise DALMAS
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2012-02-17
Filing date: 2013-02-18
Publication date: 2013-08-22

Abstract

The present invention relates to an inhibitor of interferon regulatory factor 5 (IRF5) activity or expression for use in a method for reducing adipose tissue inflammation in a subject. The present invention also relates to a method for screening a plurality of candidate compounds useful for reducing adipose tissue inflammation comprising the steps consisting of (a) testing each of the candidate compounds for its ability to inhibit interferon regulatory factor 5 activity or expression and (b) and positively selecting the candidate compounds capable of inhibiting said interferon regulatory factor 5 activity or expression.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR REDUCING

ADIPOSE TISSUE INFLAMMATION

FIELD OF THE INVENTION:

The present invention relates methods and pharmaceutical compositions for reducing adipose tissue inflammation.

BACKGROUND OF THE INVENTION:

Given the increasing prevalence of human obesity worldwide, there is an urgent need for a better understanding of the molecular mechanisms linking obesity to metabolic and cardiovascular co -morbidities. This knowledge is a prerequisite to the development of effective pharmacological means of preventing these pathologies and/or acting on them at early stages. Amongst the obesity devastating complications is the increased incidence of type-2 diabetes, of which 60 % can be directly attributed to body fat gain.

For a decade now, obesity is seen as an inflammatory disease characterized by a chronic low-grade inflammatory state. Obesity associates with increased circulating concentrations of inflammatory cytokines and acute phase proteins. Local up-regulation in genes encoding inflammatory factors has been described, associated with a marked accumulation of macrophages in adipose tissue. It is generally considered that the inflammation and alterations in white adipose tissue (WAT) function lead to changes in the release of adipokines as well as non-esterified fatty acids³. Such alterations will eventually alter glucose homeostasis, as manifested by reduced insulin-stimulated glucose uptake in skeletal muscle, reduced insulin secretion from pancreatic β-cells and increased glucose output from the liver³'⁴. Moreover, visceral adipose tissue in the obese state is characterized by higher expression of pro -inflammatory mediators and increased accumulation of macrophages, when compared to the subcutaneous depot^5"7. Since the visceral fat drains directly to the liver through the portal vein, inflammation of the visceral adipose tissue may have detrimental consequences on glucose and lipid hepatic metabolism, i.e. increased glycogen and lipid storage as well as enhanced glucose output.

Although the primary reason for adipose inflammation is still unclear, adipose tissue macrophages (ATMs) are viewed as important actors of this process. In WAT from lean, insulin-sensitive subjects, ATMs are eventually involved in normal tissue turnover by clearing the tissue from apoptotic cells, lipids and cellular debris. ATMs, primarily serving as scavenger cells, secrete cytokines with anti- inflammatory properties (e.g. IL-10) and have been termed M2-ATMs¹⁰. In contrast, in the obese state, monocytes are recruited from the circulation and accumulate in the expanding WAT where they, through unclear mechanisms, can undergo polarization into a class of macrophages with pro -inflammatory properties termed Ml -ATMs⁸'⁹. These cells release high levels of pro -inflammatory factors like ILlb, TNFa, IL-6 and monocyte chemoattractant protein-1 (MCP-1). The later may attract more monocytes from the circulation and at the same time stimulates lipolysis and inhibit insulin signaling in the adipocytes at least in different cellular and rodent models.

Recent studies have reported that members of the interferon regulatory factor (IRF) family have been implicated in macrophage polarization during acute inflammation. IRFs family members are transcription factors that possess a helix-turn-helix DNA-binding motif and mediate virus- and interferon (Interferon)-induced signaling pathways. IRF4 has been reported to control the commitment of dendritic cells (DCs) and to regulate the polarization of M2 macrophages¹³'¹⁴. Recently, IRF5 was shown to be an important player in promoting polarization of macrophages to a Ml phenotype (Krausgruber T, Blazek K, Smallie T, Alzabin S, Lockstone H, Sahgal N, Hussell T, Feldmann M, Udalova IA. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses. Nat Immunol. 2011 Mar;12(3):231-8. Epub 2011 Jan 16.). Indeed, the overexpression of IRF5 was shown to induce polarization of macrophages to an Ml phenotype, whereas silencing of IRF5 promoted a switch towards an M2 phenotype¹⁵. In addition, polymorphisms in the human IRF5 gene have been associated with several autoimmune diseases and IRF5 directly controls the expression of inflammatory mediators, including type I interferon, TNFa, IL-6, IL-12 and IL- 23^15"20. Altogether, IRF5 is an important transcription factor controlling macrophage polarization and thereby a regulator of the inflammatory responses. Hitherto, the role of IRF5 in adipose tissue inflammation and in relation to obesity as well as obesity-associated disorders has not been investigated.

SUMMARY OF THE INVENTION:

The present invention relates to an inhibitor of interferon regulatory factor 5 (IRF5) activity or expression for use in a method for reducing adipose tissue inflammation in a subject.

The present invention also relates to a method for screening a plurality of candidate compounds useful for reducing adipose tissue inflammation comprising the steps consisting of (a) testing each of the candidate compounds for its ability to inhibit interferon regulatory factor 5 activity or expression and (b) and positively selecting the candidate compounds capable of inhibiting said interferon regulatory factor 5 activity or expression.

DETAILED DESCRIPTION OF THE INVENTION:

For the present invention, the inventors demonstrated the role for IRF5 in the control of macrophage Ml polarisation and and adipose tissue inflammation in human obesity. As such, IRF5 could be a driving factor behind the development of life threatening obesity comorbidities such as insulin resistance or liver steatosis.

Accordingly, the present invention relates to an inhibitor of interferon regulatory factor 5 (IRF5) activity or expression for use in a method for reducing adipose tissue inflammation in a subject.

The terms "subject," and "patient," used interchangeably herein, refer to a mammal, particularly a human who has been previously diagnosed. In a particular embodiment, the subject is a subject at risk of obesity or an obese subject. The term "obesity" refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in meter squared (kg/m²). Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg/m², or a condition whereby a subject with at least one comorbidity has a BMI greater than or equal to 27 kg/m². An "obese subject" is an otherwise healthy subject with a BMI greater than or equal to 30 kg/m² or a subject with at least one comorbidity with a BMI greater than or equal 27 kg/m². A "subject at risk of obesity" is an otherwise healthy subject with a BMI of 25 kg/m² to less than 30 kg/m² or a subject with at least one co-morbidity with a BMI of 25 kg/m² to less than 27 kg/m². The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian-Pacific countries, including Japan, "obesity" refers to a condition whereby a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m². An "obese subject" in these countries refers to a subject with at least one obesity- induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m². In these countries, a "subject at risk of obesity" is a person with a BMI of greater than 23 kg/m2 to less than 25 kg/m². As used herein the term 'interferon regulatory factor 5" or "IRF5" has its general meaning in the art. IRF5 is a member of the interferon regulatory factor (IRF) family, a group of transcription factors with diverse roles, including virus-mediated activation of interferon, and modulation of cell growth, differentiation, apoptosis, and immune system activity. Interferon regulatory factor 5 (IRF-5) is a member of a family of transcription factors that controls inflammatory and immune responses (Honda et al. (2005) Int. Immunol. 17: 1367- 1378). IRF-5 has a critical role in the production of the pro-inflammatory cytokines tumor necrosis factor-a (TNF-a), interleukin-12 (IL-12), and IL-6 following Toll Like Receptor (TLR) signaling as determined by knockout mouse studies (Takaoka et al. (2005) Nature 434:243-249), and is also important for transactivation of type I Interferon and Interferon- responsive genes (Barnes et al. (2001) J. Biol. Chem. 276:23382-23390; Barnes et al. (2004) J. Biol. Chem. 279:45194-45207). An "inhibitor of IRF5 activity" has its general meaning in the art, and refers to a compound (natural or not) which has the capability of reducing or suppressing the activity of IRF5. Typically, said compound inhibits or reduces the transcription from promoters containing IRF5 binding sites. For example the compound may block the interaction of IRF 5 with the IRF5 binding sequences, or may bind to IRF5 in manner that IRF is not able to bind to the IRF5 binding sites. In a particular embodiment said inhibitor is a selective inhibitor of IRF5 activity. As used herein the term "selective inhibitor of IRF5 activity" refers to such compound which inhibits the IRF5 activity more strongly than for the other members of the IRF family (IRF1 , IRF2, IRF5, IRF4...) in a manner that the inhibitor is at least 10 times, more preferably at least 100 times and most preferably at least 1000 times over the other IRF family members. Typically, said inhibitor is a small organic molecule or a biological molecule (peptides, lipid, aptamer).

In a particular embodiment, the activity of IRF5 can be reduced using a "dominant negative." To this end, constructs which encode, for example, defective IRF5 polypeptide, such as, for example, mutants lacking all or a portion of the DNA binding domain, can be used in gene therapy approaches to diminish the activity of IRF5 on appropriate target cells. For example, nucleotide sequences that direct host cell expression of IRF5 in which all or a portion of the DNA binding domain is altered or missing can be introduced into monocytes or macrophages, or even adipose tissue in general (either by in vivo or ex vivo gene therapy methods described herein or otherwise known in the art). Alternatively, targeted homologous recombination can be utilized to introduce such deletions or mutations into the subject's endogenous IRF5 gene in monocytes or macrophages. The engineered cells will express nonfunctional IRF5 polypeptides.

An "inhibitor of IRF5 expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for IRF5. Inhibitors of expression for use in the present invention may be based on anti-sense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of IRF5 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of IRF5, 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 IRF5 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 (siRNAs) can also function as inhibitors of expression for use in the present invention. IRF5 gene expression can be reduced by contacting a 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 IRF5 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 Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). All or part of the phosphodiester bonds of the siRNAs of the invention are advantageously protected. This protection is generally implemented via the chemical route using methods that are known by art. The phosphodiester bonds can be protected, for example, by a thiol or amine functional group or by a phenyl group. The 5'- and/or 3'- ends of the siRNAs of the invention are also advantageously protected, for example, using the technique described above for protecting the phosphodiester bonds. The siR As sequences advantageously comprises at least twelve contiguous dinucleotides or their derivatives.

As used herein, the term "siR A derivatives" with respect to the present nucleic acid sequences refers to a nucleic acid having a percentage of identity of at least 90% with erythropoietin or fragment thereof, preferably of at least 95%, as an example of at least 98%, and more preferably of at least 98%.

As used herein, "percentage of identity" between two nucleic acid sequences, means the percentage of identical nucleic acid, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the nucleic acid acids sequences. As used herein, "best alignment" or "optimal alignment", means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two nucleic acids sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity. The best sequences alignment to perform comparison can be realized, beside by a manual way, by using the global homology algorithm developed by SMITH and WATERMAN (Ad. App. Math., vol.2, p:482, 1981), by using the local homology algorithm developped by NEDDLEMAN and WUNSCH (J. Mol. Biol, vol.48, p:443, 1970), by using the method of similarities developed by PEARSON and LIPMAN (Proc. Natl. Acd. Sci. USA, vol.85, p:2444, 1988), by using computer software using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), by using the MUSCLE multiple alignment algorithms (Edgar, Robert C, Nucleic Acids Research, vol. 32, p: 1792, 2004 ). To get the best local alignment, one can preferably used BLAST software. The identity percentage between two sequences of nucleic acids is determined by comparing these two sequences optimally aligned, the nucleic acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.

shRNAs (short hairpin RNA) can also function as inhibitors of expression for use in the present invention. Ribozymes can also function as inhibitors of 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 endonucleo lytic cleavage of IRF5 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.

Both antisense oligonucleotides and ribozymes useful as inhibitors of 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, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and preferably cells expressing IRF5. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A 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, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adenoviruses and adeno-associated (AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. Actually 12 different AAV serotypes (AAVl to 12) are known, each with different tissue tropisms (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAV are derived from the dependent parvovirus AAV2 (Choi, VW J Virol 2005; 79:6801-07). The adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316- 27). It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion. Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 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 micro encap sulation.

In a preferred embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for the monocyte or macrophage.

Accordingly, the inhibitors of interferon regulatory factor 5 (IRF5) activity or expression are thus particularly suitable for preventing life threatening obesity co-morbidities such as type II diabetes, insulin resistance, dyslipidemia, liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), syndrome X, cardiovascular disorders such as hypertension, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, angina pectoris, cerebral infarction, cerebral thrombosis and transient ischemic attack. The inhibitor of IRF5 activity or expression may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

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

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

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

The Inhibitor of IRF5 activity or expression of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active 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 inhibitor of IRF5 activity or expression 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 present invention also relates to a method for screening a plurality of candidate compounds useful for reducing adipose tissue inflammation comprising the steps consisting of (a) testing each of the candidate compounds for its ability to inhibit interferon regulatory factor 5 activity or expression and (b) and positively selecting the candidate compounds capable of inhibiting said interferon regulatory factor 5 activity or expression.

Testing whether a candidate compound can inhibit interferon regulatory factor 5 activity or expression can be determined using or routinely modifying reporter assays known in the art.

For example, the method may involve contacting cells expressing IRF5 with the candidate compound, and measuring the IRF5 mediated transcription (e.g., activation of promoters containing IRF5 binding sites), and comparing the cellular response to a standard cellular response. Typically, the standard cellular response is measured in absence of the candidate compound. A decrease cellular response over the standard indicates that the candidate compound is an inhibitor of IRF5 activity.

In another embodiment the invention provides a method for identifying a ligand which binds specifically to IRF5. For example, a cellular compartment, such as a membrane or a preparation thereof, may be prepared from a cell that expresses a molecule that binds IRF5. The preparation is incubated with labelled IRF5 and complexes of ligand bound to IRF5 are isolated and characterized according to routine methods known in the art. Alternatively, the IRF5 interacting polypeptide may be bound to a solid support so that binding molecules solubilized from cells are bound to the column and then eluted and characterized according to routine methods. In another embodiment, a cellular compartment, such as a membrane or a preparation thereof, may be prepared from a cell that expresses a molecule that binds IRF5 such as a molecule of a signalling or regulatory pathway modulated by IRF5. The preparation is incubated with labelled IRF5 in the absence or the presence of a candidate compound. The ability of the candidate compound to bind the binding molecule is reflected in decreased binding of the labelled ligand. Molecules which bind gratuitously, i.e., without inducing the effects of IRF5 on binding the IRF5 binding molecule, are most likely to be good inhibitor of IRF5 activity.

Another method involves screening for compounds which inhibit IRF5activity by determining, for example, the amount of transcription from promoters containing IRF5 binding sites in a cell that expresses IRF5. Such a method may involve transfecting a eukaryotic cell with DNA encoding IRF5 such that the cell expresses IRF5, contacting the cell with a candidate compound, and determining the amount of transcription from promoters containing IRF5 binding sites. A reporter gene (.e.g, GFP) linked to a promoter containing an IRF5 binding site may be used in such a method, in which case, the amount of transcription from the reporter gene may be measured by assaying the level of reporter gene product, or the level of activity of the reporter gene product in the case where the reporter gene is an enzyme. A decrease in the amount of transcription from promoters containing IRF5 binding sites in a cell expressing IRF5, compared to a cell that is not expressing IRF5, would indicate that the candidate compound is an inhibitor of IRF5 activity.

In a particular embodiment, the candidate compound is selected from the group consisting of small organic molecules, peptides, polypeptides or oligonucleotides. Other potential candidate compounds include antisense molecules.

The candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties in adipose tissue macrophages from obese subjects. For example, the candidate compounds that have been positively selected with the screening method as above described may be further selected for their ability to inhibit ILlbeta expression in adipose tissue macrophages from obese subjects. Typically, the screening method may further comprise the steps of i) bringing into contact an adipose tissue macrophage from obese patients with a positively selected candidate compound ii) determining the expression of ILlbeta by said macrophage and iii) comparing the expression determined at step ii) with the expression determined when step i) is performed in the absence of the positively selected candidate compound. Step i) as above described may be performed by adding an amount of the candidate compound to be tested to the culture medium of the macrophages. Usually, a plurality of culture samples are prepared, so as to add increasing amounts of the candidate compound to be tested in distinct culture samples. Generally, at least one culture sample without candidate compound is also prepared as a negative control for further comparison.

Finally, the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on animal models for obesity. Typically, C57/BL6 wild-type mice may be fed with a high fat diet (calories from fat 60 % of total calories versus 10 % in standard laboratory chow). This nutritional challenge is widely used to induce adipose tissue inflammation by increasing macrophage infiltration, mimicking aspects of western-type diet in humans. Then the positively selected candidate compound may be administered to the animal model and finally adipose tissue inflammation and/or metabolic status (including improved insulin sensitivity and glucose tolerance and reduced liver steatosis) may be evaluated. Said inflammation or status may be then compared with a negative control (e.g. an animal that was not administered with the candidate compound) or compared with a positive control (e.g. an IRF5 knockout animal fed with the same nutritional regiment as described in Example 3). Thus in one embodiment, the screening method may further comprise the steps of i) administered a C57/BL6 wild-type mouse fed with a high fat diet with a positively selected candidate compound ii) measuring the level of adipose tissue inflammation and/or at least one metabolic parameter selected from the group consisting of insulin sensitivity, glucose tolerance and liver steatosis and iii) comparing said level and/or saids parameter measured at step ii) with the level and/or parameters measured when step i) is performed in the absence of the positively selected candidate compound and iv) finally selected the candidate compound that provides a reduced adipose tissue inflammation and/or an increased glucose tolerance or insulin sensitivity and/or a reduced liver steatosis.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1. Messenger RNA expression of IRF5, -3 and -4 was measured using RT-

PCR in adipose tissue samples from non obese and obese subjects. ADD stars the figures for significance. Figure 2. Messenger RNA expression of IRF5, CD68 and IL6 was measured using RT- PCR in the stroma vascular fraction (SVF) of adipose tissue samples from non obese (N.Ob) and obese (Ob) subjects. Figure 3. Messenger RNA expression of IRF5, CD68 and IL6 was measured using

RT- PCR in subcutaneous (Sc) and visceral (Vis) adipose tissue samples.

Figure 4: IRF5 is more recruited onto ILlbeta promoter of SVF from obese subjected. IRF5 recruitment is associated with an increased recruitment of RNA polymerase 2 (Pol2) which indicates an increased of ILlbeta transcription.

EXAMPLE 1: IRF5 REGULATION IN HUMAN OBESITY (ADIPOSE TISSUE AND STROMA VASCULAR FRACTION) To investigate the role of IRFs family members in adipose tissue inflammation, we analyzed the gene expression profile of IRF5 in subcutaneous adipose tissue of lean (n=10) and obese (n=60) subjects by RT-PCR. Unexpectedly, preliminary results show that IRF5 mRNA expression was significantly up regulated, in WAT of obese vs. lean subjects. By contrast, IRF3 was expressed at similar levels, while IRF4 was also increased although to a lesser extent than IRF5 (Fig. 1).

To further confirm IRF5 up-regulation in obese adipose tissue is driven by proinflammatory macrophage's infiltration, we analyzed IRF5 expression in stroma vascular fraction of obese compared to lean adipose tissues. Interestingly, the expression of IRF5 was higher in obese SVF, along with the pro-inflammatory cytokine IL-6 and the macrophage marker CD68 (Fig 2). In addition, IRF5, IL-6 and CD68 mRNA expression was also higher in visceral (more inflamed WAT) than in subcutaneous WAT in obese subjects (Fig. 3). In addition, IRF5 mRNA expression in WAT correlated with CD68 expression.

These data strongly support that IRF5 is associated with macrophage accumulation and adipose tissue inflammation.

EXAMPLE 2: IRFS IS MAINLY EXPRESSED IN ADIPOSE TISSUE MACROPHAGE AND CONTROLLED IL1BETA EXPRESSION To identify the stroma vascular cells fraction-expressing IRF5, we immunoselected different immune cells from the SVF fraction. As we expected, IRF5 is mainly expressed in adipose tissue macrophages. The gene expression data are confirmed at the protein levels. Indeed, IRF5 staining revealed a specific staining in macrophages. In addition, IRF5 presented an over-activity in macrophages of obese subjects. Chromatin Immuno- Precipitation revealed that IRF5 is more recruited onto ILlbeta (Pro -inflammatory cytokine) promoter in SVF of obese subjects (n=6) (Figure 6). Collectivity, these results collected in human strongly support a key role of IRF5 in macrophage polarization into a proinflammatory phenotype that contributes to adipose tissue inflammation.

EXAMPLE 3: IRF5 KNOCKOUT MICE ARE RESISTANT TO ADIPOSE TISSUE INFLAMMATION AND PROMOTE ADIPOSITY

IRF5 knockout mice have previously been described and were obtained from Prof. Udalova's team. For each set of experiments, age and sex matched C57/BL6 wild-type mice were used as controls. Mice were fed either a normal chow laboratory diet or a high fat diet (calories from fat 60 % of total calories versus 10 % in standard laboratory chow). This nutritional challenge is widely used to induce adipose tissue inflammation by increasing macrophage infiltration, mimicking aspects of western-type diet in humans. IRF5 knockout mice present a less inflamed adipose tissue than wild-type controls. In turn, this might be associated with the amelioration of their metabolic status, including improved insulin sensitivity and glucose tolerance and reduced liver steatosis compared to wild-type mice. In order to phenotype the consequences of IRF5 depletion on adipose tissue inflammation and metabolic status, wild-type and IRF5 knockout mice (n=5-7) were fed a high fat diet for 16 weeks. IRF5 -/- and WT mice did not present weight difference under chow diet. Unexpectedly, in vivo experiments revealed that IRF5 -/- mice are more obese than WT mice. We also measured the serum levels of glycemia and insulinemia and revealed that both are lower in IRF5 -/- mice and WT suggesting IRF5 -/- mice are protected against HFD-induced sytstemic inflammation (Figures 5 and 6). REFERENCES:

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

Claims

CLAIMS:

1. A method for reducing adipose tissue inflammation in a subject comprising administering the subject with an inhibitor of interferon regulatory factor 5 (IRF5) activity or expression.

2. The method according to claim 1 for preventing life threatening obesity co-morbidities such as type II diabetes, insulin resistance, dyslipidemia, liver steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), syndrome X, cardiovascular disorders such as hypertension, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, angina pectoris, cerebral infarction, cerebral thrombosis and transient ischemic attack.

3. A method for screening a plurality of candidate compounds useful for reducing adipose tissue inflammation comprising the steps consisting of (a) testing each of the candidate compounds for its ability to inhibit interferon regulatory factor 5 activity or expression and (b) and positively selecting the candidate compounds capable of inhibiting said interferon regulatory factor 5 activity or expression.