WO2015007326A1

WO2015007326A1 - Agents comprising a terminal alpha-galactosyl moiety for use in prevention and/or treatment of inflammatory diseases

Info

Publication number: WO2015007326A1
Application number: PCT/EP2013/065243
Authority: WO
Inventors: Rafael Mañez Mendiluce; Cristina COSTA VALLÉS; Magdiel PÉREZ CRUZ
Original assignee: Institut D'investigació Biomèdica De Bellvitge (Idibell)
Priority date: 2013-07-18
Filing date: 2013-07-18
Publication date: 2015-01-22

Abstract

The present invention relates to the field of immunomodulation and, more in particular, to agents and methods for the prevention and/or treatment of inflammatory diseases or of systemic inflammatory response syndrome (SIRS), all of them based on agents comprising an α-Gal epitope, wherein said epitope comprises a terminal α- galactosyl.

Description

AGENTS COMPRISING A TERMINAL ALPHA-GALACTOSYL MOIETY FOR USE IN PREVENTION AND/OR TREATMENT OF INFLAMMATORY

DISEASES

TECHNICAL FIELD

The present invention relates to the field of immunomodulation and, more in particular, to methods for the prevention and/or treatment of inflammatory diseases based on agents comprising a terminal a-galactosyl moiety. BACKGROUND ART

Severe sepsis (acute organ dysfunction secondary to infection) and septic shock (severe sepsis plus hypotension not reversed with fluid resuscitation) are major healthcare problems, affecting millions of individuals around the world each year, killing one in four (and often more), and increasing in incidence.

There is not any specific treatment for severe sepsis and septic shock. Dotrecogin alpha (Xigris®) was considered for several years the unique therapy for these conditions. However, it has been recently retired from the market because a repeated clinical trial asked for European authorities failed to show the efficacy observed in the initial PROWESS trial. The Intensive Care Department at the Bellvitge Biomedical Research Institute has participated in most of the clinical trials performed during the last 10 years that investigated specific therapies for sepsis/septic shock. The outcome has been so far identical: all of them failing to show significant improvements. Therefore, an approach that could help to prevent these conditions may have a major impact in clinical medicine.

Two types of sepsis/septic shock can be considered. The first type is caused by exogenous microorganisms that colonize upper airway such as pneumococcus or meningococcus, which already have specific vaccines to prevent the disease. The impact of vaccination in meningococcal infections is unquestionable. In the case of pneumococcal sepsis or meningitis the benefit is not so unanimous. The second type of sepsis is caused by endogenous enteric bacteria. The "gut origin of sepsis" hypothesis proposes that bacteria, which are normally resident within the lumen of the intestinal tract, translocate across the intestinal epithelial barrier and act as a source of sepsis at distant sites, which has been supported by many studies in animals.

The clinical significance of bacterial translocation is noteworthy in nosocomial and neutropenic patient infections, playing an important role in the causation of sepsis. This may affect up to 12% of patients admitted to Intensive Care Unit (ICU-acquired sepsis) and 1.6% of people with cancer per year. Compared with the overall population, cancer patients are 4 times much more likely to be hospitalized with severe sepsis, with and in hospital mortality of 37.8%. Other patients at risk for enteric bacterial sepsis are those submitted to surgery o medical procedures. After cardiac surgery 1.4% of patients develop sepsis/septic shock with a mortality rate of 32%. Also, a recent study showed that after general surgery sepsis and septic shock occurred in 2.3% and 1.6% of patients, respectively, whilst the incidence of pulmonary embolism and heart attack were 0.3% and 0.2%), respectively. Death rates within 30 days were 5.4% for sepsis, 33.7% for septic shock, 9.1% for pulmonary embolism and 32% for heart attack. These data suggest that sepsis is a common and serious complication in general surgery patients and occurs more frequently than pulmonary embolism or heart attack.

Immune-mediated inflammatory diseases (IMID) present a group of common and highly disabling chronic conditions that share inflammatory pathways. Several incidence and prevalence studies of IMID during the past decades have reported a considerable variation of the disease occurrence among different populations. Overall, the estimated prevalence of IMID in Western society is 5%-7%.

It has been showed that the serum level of anti-Gal antibodies correlated with the inflammatory status in patients initiating renal replacement therapy. Subsequent survival and peritoneal-dialysis-related enteric peritonitis was predicted, with a worse prognosis for those patients with higher level of antibodies and inflammatory status at baseline (Perez-Fontan M et al. 2006 Am. J. Kidney Dis 48:972-982).

All these data suggest that a large population of individuals could benefit from a strategy to reduce inflammation and avoid the clinical complications of sepsis caused by enteric bacteria. In a previous study, this issue has been addressed by using statins in patients admitted to ICU with severe sepsis (Kruger P et al. 2013 Am J Respir Crit Care Med 2013; 187:743-750). The drug could not modify inflammation in these patients, but it was associated with a lower inflammatory status at the time of ICU admission and better outcome in those patients that were previous statins users.

Thus, there is a need in the state of the art to identify agents for use in the treatment of septic and inflammatory processes, which overcome the problems associated to current methodologies.

BRIEF SUMMARY OF THE INVENTION

The authors of the present invention have shown that removal of anti-Gal antibodies mediated by compounds comprising terminal a-galactosyl moieties prevents sepsis and reduces cytokine levels in mice genetically modified so as to express anti-Gal antibodies.

Thus, the present invention is related to a method for the prevention and/or treatment of inflammation in a subject, comprising the administration to a subject in need thereof of a therapeutically effective amount of an agent comprising a terminal a- galactosyl moiety.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the effects of the removal of anti-Gal antibodies in Gal-KO mice. Survival rate (A) and welfare (B) in GAS914-treated mice (n = 17) before CLP and control mice (n = 17). (C) Survival rate in GAS914-treated mice 12h after CLP and control mice. (D) Anti-Gal IgM and IgG levels in GAS914-treated mice before CLP and control mice.

Figure 2 shows cytokines profile after the removal of anti-Gal antibodies in Gal-

KO mice. (A) Cytokine levels in Gal-KO mice at the time of CLP compared to untreated animals. (B) Cytokine levels in Gal-KO mice 24h after CLP compared to untreated animals. (C) Cytokine levels in wild type mice lacking anti-Gal antibodies before CLP compared to untreated animals. DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have shown that the removal of antibodies for galactose al-3 galactose (anti-Gal antibodies) using a compound containing a terminal a-galactosyl moiety and, in particular, GAS914 prevents sepsis mortality (see Example 1) and modifies the inflammatory status by reducing the cytokine levels (see Example 2) in Gal-KO mice which generate natural anti-Gal antibodies without any need of additional immunization. These findings allow the use of compounds containing a terminal α-galactosyl moieties for the treatment of inflammatory diseases or for the inflammatory symptoms of a disease which result in inflammation.

Definitions

The term "a- galactosyl" or "q-galactosyl moiety" or "a- galactosyl residue", as used herein, relates to a terminal galactose residue in a molecule, i.e., to a glycosyl radical derived from a-galactose. (which can be bound/linked to, for example, to a second monosaccharide). In some contexts, the term alfa-galactosyl is related in particular to a unit of alfa-galactose bound to a second monosaccharide.

The term "agent comprising a terminal a-galactosyl moiety", as used herein, also known as "alpha-galactosyl agent", or "anti-Gal binding agent" refers to any molecule, or part of a molecule, with a terminal structure comprising Galal-3Gaipi-4GlcNAc-R, Galal-3Gah31-3GlcNAc-R, or any carbohydrate chain with terminal Galal-3Gal at the non-reducing end, or any molecule with terminal α-galactosyl unit, capable of binding the anti-Gal antibody, wherein said α-galactosyl unit may be bound to a second monosaccharide, such as galactose or glucose. The a-Gal epitope (also known as "alpha-galactosyl epitope", or "anti-Gal binding epitope") is synthesized by the glycosylation enzyme a-l,3-galactosyltransferase (al,3GT) and it is expressed in very large amounts on the cells of non-primate mammals, prosimians and in New World monkeys. The al,3GT gene was inactivated in ancestral Old World primates. Thus humans, apes, and Old World monkeys lack a-gal epitopes and produce high titer anti- Gal antibodies. In particular, the agent of the invention comprises a terminal a- galactosyL wherein said α-galactosyl is selected from the group comprising terminal Galal-3Gal, terminal Galal-2GaL terminal Galal-6GaL terminal Galal-6Glc, and terminal α-galactose sugar unit(s) capable of binding anti-Gal antibodies. In a particular embodiment, the agent comprising a terminal a-galactosyl moiety is part of a glycolipid, a glycoprotein or a liposome. The term "a-Gal glycolipid", refers to any glycolipid that has at least one a-gal moiety on its nonreducing end of the carbohydrate chain. The term "a-Gal liposomes" refers to any liposomes that have an a-gal moiety and are capable of binding the anti-Gal antibody.

The term "anti-Gal antibody", or "anti-Gal", as used herein, relates to an antibody that interacts specifically with the a-Gal epitope (Galal-3Gaipi-4GlcNAc-R or Galal-3Gaipi-3GlcNAc-R) on glycolipids, glycoproteins or other molecules comprising the alpha-galactosyl epitope. This antibody constitutes approximately 1% of circulating IgG in human serum and is produced, upon stimulation, by 1% of circulating B lymphocytes. Anti-Gal antibody is also present as IgA antibodies in body secretions such as saliva, milk and colostrum. It has been proposed that the antigenic source for the constant production of anti-Gal antibodies are the alpha-galactosyl- like epitopes found on many bacteria of the gastrointestinal flora. Whereas anti-Gal is abundant in humans, apes and Old World monkeys, it is absent from New World monkeys, prosimians and nonprimate mammals. The latter group of species produces, however, large amounts of alpha-galactosyl epitopes. It is estimated that anti-Gal appeared in ancestral Old World primates less than 28 million years ago, possibly as a result of an evolutionary event which exerted a selective pressure for the suppression of alpha-galactosyl epitopes expression by inactivation of the gene for the enzyme alpha 1,3 galactosyltransferase. This also resulted in the loss of immune tolerance to the alpha-galactosyl epitope and the production of anti-Gal. Anti-Gal antibodies bind in vivo to a-gal epitopes when administered to humans or Old World monkeys. This is particularly evident in the context of xenotransplantation, where the in vivo binding of anti-Gal to a-gal epitopes on transplanted pig heart or kidney is the main cause for the rapid rejection of such grafts in humans and Old World monkeys. Anti-Gal antibodies are responsible for the hyper-acute rejection of a xenograft.

The terms "antibody", "immunoglobulin" and the like terms refer to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. An exemplary immunoglobulin (antibody) structural unit is composed of two pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N- terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. The C-terminal ends of each heavy chain are disulfide bonded together, and form the constant region of the antibody. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different "classes". There are five-major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 1 10 amino acids at the H2-terminus and a kappa or lambda constant region at the COOH-terminus. Full- length immunoglobulin "heavy chains" (of about 50 kDa or about 446 amino acids) similarly comprise a variable region (of about 1 16 amino acids) and one of the aforementioned heavy chain constant regions or classes, e.g., gamma (of about 330 amino acids). The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

In the context of the invention, the antibody is an anti-Gal antibody, that recognizes and binds to a a-Gal epitope, wherein said epitope comprises a terminal a- galactosyl. More particularly, the anti-Gal antibody recognizes and binds to an agent comprising a terminal a-galactosyl moiety which is selected from the group comprising Galal-3Gal, Galal-2GaL Galal-6GaL and a-galactose sugar unit(s) capable of binding anti-Gal antibodies. More preferably, the anti-Gal antibody recognizes and binds to an a-Gal epitope or to an agent comprising a terminal Galal-3Gal. Even more preferably, the anti-Gal antibody recognizes and binds to GAS914. As used herein, the expression "an antibody targets" is referred to the specific recognition and binding of the antibody to a particular antigen. As it is used herein, the expression "binds specifically to" refers to the capacity of the antibodies for binding specifically to epitopes comprising a terminal a-galactosyl and not to epitopes comprising other carbohydrates.

The term "epitope" or "antigenic determinant", as used herein, includes any region of an antigen which is specifically recognized by an antibody. One and the same antigen can have different epitopes. Each epitope usually consists of clusters of chemically active surfaces of molecules such as amino acids or sugar side chains, which have specific three-dimensional structural characteristics, as well as specific charge characteristics. In the context of the present invention, the epitope is an a-Gal epitope, wherein said epitope comprises a terminal a-galactosyl, and wherein said epitope is recognized and bound to by anti-Gal antibodies.

The term "galactose" or "Gal", as used herein, relates to a C-4 epimer of glucose that exists in both open chain and cyclic form. The open-chain form has a carbonyl at the end of the chain. In the open-chain form D- and L- isomers cannot be separated, but the cyclic forms can be crystallized and isolated.

The term "Gal(al.3)Gal" or "Gal(al,3)" or al,3 galactobiose (in some contexts, also referred to as alpha-Gal) relates to galactose(al,3)galactose (CAS No 13168-24-6). Compounds comprising a galactose-a-l,3-galactose epitope include, without limitation, galactose-a-l,3-galactose and derivatives thereof including oligomers (e.g., dimers, trimers) of galactose-a-l,3-galactose and glycopeptides bearing oligosaccharides with terminal galactose-a-l,3-galactose. Derivatives of galactose-a-l,3-galactose include, for example, amides, esters, ethers, amines, sulfonamides, thioethers, acetals, carbamates, ureas and amidines. Examples of compounds comprising a galactose-a-l,3-galactose epitope, by way of illustration and not limitation, include galactose-a-l,3-galactose (al- 3 galactobiose) (Galal-3Gal) and derivatives thereof such as, for example, glucosamine derivatives, linear B-2 trisaccharide (Galal-3Gah31-4GlcNAc), linear B-6 trisaccharide (Galal-3Gaipi-4Glc) and derivatives thereof, al-3 galactobiosyl β-methyl glycoside, al-3, β1-4 galactotriose (Galal-3Gaipi-4Gal), galactotetraose (Galal-3Gaipi-4Galal- 3-D-Gal), and derivatives of the above such as, e.g., amino acid derivatives (e.g., dodecalysine, glycine) and glycopeptides with galactose-a-l,3-galactose.

Similarly, the terms "Gal(al.2)Gal". "Gal(al.6)Gal" and "Gal(al.6)Glc" relate, respectively, to galactose(al,2)galactose, galactose(al,6)galactose and galactose(a 1 ,6)glucose. The term "GAS914". as used herein, relates to a soluble Gal trisaccharide- polylysine conjugate of approximately 500 kDa that effectively competes for a-Gal binding by a-Gal IgM (IC(50), 43 nM) and IgG (IC(50), 28 nM) antibodies in vitro (Katopodis G et al. 2002 J. Clin. Invest., 110: 1869-1877; Zhong R et al. 2003 Transplantation 75: 10-19). It is an artificial injectable antigen with a linear polylysine backbone (with an average length of 1,000 lysines) and with approximately 25% of side chains conjugated to Linear B trisaccharide (linear B type 2 trisaccharide, Galal- 3Gaipi-4GlcNAc). It is described in W09847915 by Novartis AG (BaseL Switzerland). GAS914 has the following structure (Katopodis G et al. 2002 J Clin Invest 110(12): 1869-1877).

GAS914 is shown herein as the chemical structure of random copolymer, wherein n represents the average degree of polymerization; x represents the fraction of glycosylated monomer; and 1 - x represents the fraction of thioglycerol- capped monomer.

The term "glycoconjugate". as used herein, relates to carbohydrates covalently linked with a second chemical species, wherein said second chemical species is selected from the group comprising proteins (known as glycoproteins), peptides (peptidoglycans, glucopeptides), lipids (glycolipids, lipopolisaccharides), saccharides (glycosaccharides). Glycoconjugates according to the invention include, without limitation, those wherein a carbohydrate comprising a terminal alpha-galactosyl moiety is conjugated to bovine serum albumin (BSA), human serum albumin (HSA), or 1,2-di-O-hexadecyl-sn- glycero- 3 -phosphoethano lamine (HDPE) .

The term "glycolipid" relates to a lipid molecule attached to a carbohydrate. Glycolipids comprise glyceroglycolipids, glycosphingolipids and glycosylphosphatidylinositols. Glycolipids according to the invention comprise at least a carbohydrate residue comprising a terminal alpha-galactosyl residue. In a particular embodiment, glycolipids according to the invention comprise a terminal Galal-3Gal, terminal Galal-2Gal, terminal Galal-6Gal, terminal Galal-6Glc, or terminal a- galactose sugar unit(s) capable of binding an anti-Gal antibody, more preferably a terminal Galal-3Gal.

The term "glycoprotein" relates to any protein which is covalently modified by at least one carbohydrate residue. Glycoproteins which can be studied according to the method of the invention include those which are modified by a monosaccharide or by an oligosaccharide and in which said monosaccharide or oligosaccharide is bound to the polypeptide chain by a side chain comprising a nitrogen atom (N-glycosylation) or an oxygen atom (O-glycosylation). Typically, N-glycosylation comprises the modification of proteins in asparagine residues which form part of a consensus sequence of the Asn- X-Ser or Asn-X-Thr type. O-glycosylation occurs in the side chains of serine and/or threonine residues. Glycoproteins and glycopeptides according to the invention comprise at least a carbohydrate residue comprising a terminal a-galactosyl moiety, in particular selected from the group comprising a terminal Galal-3Gal, terminal Galal- 2Gal, terminal Galal-6Gal, terminal Galal-6Glc, or terminal a-galactose sugar unit(s) capable of binding an anti-Gal antibody, more preferably a terminal Galal-3Gal.

The term "immune disorder" or "immune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunological reaction of the subject. In particular, immune diseases include autoimmune diseases and immunologically mediated diseases. The term "autoimmune disorder" or "autoimmune disease" refers to a condition in a subject characterized by cellular, tissue and/or organ injury caused by an immunological reaction of the subject to its own cells, tissues and/or organs. Illustrative, non- limiting examples of autoimmune diseases which can be treated with the peptide or with the nucleic acid of the invention include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CF1DS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia- fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjogren's syndrome, Good pasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, Wegener's granulomatosis, Anti-Glomerular Basement Membrane Disease, Antiphospholipid Syndrome, Autoimmune Diseases of the Nervous System , Familial Mediterranean Fever, Lambert-Eaton Myasthenic Syndrome, Sympathetic Ophthalmia, polyendocrinopathies, psoriasis, etc.

The term "immune mediated inflammatory disease" shall be taken to mean any disease mediated by the immune system and characterized by chronic or acute inflammation, resulting from, associated with or triggered by, a dysregulation of the normal immune response e.g. Crohn's disease, type 1 diabetes mellitus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, Hashimoto's disease, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome, ulcerative colitis, autoimmune hemolytic anemia, sterility, myasthenia gravis, multiple sclerosis, Basedow's disease, thrombopenia purpura, Guillain-Barre syndrome, allergy, asthma, atopic disease, arteriosclerosis, myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia, and rejection after organ transplantation. In a particular embodiment, immune mediated inflammatory diseases are selected from the group comprising rheumatoid arthritis and ischemia/reperfusion injury.

The term "inflammation", as used herein, is intended to encompass both acute responses (i.e., a response in which the inflammatory processes are active) and chronic responses (i.e., a response marked by slow progress and formation of new connective tissue). Chronic and acute inflammation may be distinguished by the cell types involved. Acute inflammation often involves polymorphonuclear neutrophils; whereas chronic inflammation is normally characterized by a lymphohistiocytic and/or granulomatous response. Examples of specific types of inflammation are diffuse inflammation, focal inflammation, croupous inflammation, interstitial inflammation, obliterative inflammation, reactive inflammation, specific inflammation, toxic inflammation, and traumatic inflammation. The term "inflammation" encompasses both internal inflammation and external inflammation. The term "inflammatory condition" refers to a symptom or manifestation associated with inflammation. In particular embodiments, an inflammatory condition is manifested by one or more symptoms selected from the group consisting of redness, heat, swelling (enlargement of any organ or tissue due to accumulation of excessive fluid in said organ or tissue), pain and dysfunction of the tissues or organs involved. All of the five conditions may be observed or associated with an inflammation, but none is necessarily always present. The degree or the severity of the five conditions may vary. For example, the heat, can be localized to the site of inflammation, or it can be systemic, with an increase of several degrees in the body temperature. The dysfunction of the tissues or organs involved can be a partial or a total failure of the tissue or organ. It can range from annoying to life-threatening, depending on severity and on the type of tissue or organ in which the dysfunction occurs. In particular embodiments, the swelling is edema, which refers to the accumulation of an excessive amount of watery fluid in cells or intercellular tissues. Edema can accumulate excessive liquid in almost any location in the body, with the most common sites at the feet and ankles. It can be localized, for example, due to venous or lymphatic obstruction or increased vascular permeability. It can also be systemic, for example, due to heart failure or renal disease. In the context of the present invention, the inflammation in a subject is not due to SIRS or to xenotransplant rejection.

The term "inflammatory disease", as used herein, refers to a condition in a subject characterized by inflammation, e.g. chronic inflammation. Illustrative, non- limiting examples of inflammatory disorders include, but are not limited to, celiac disease, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), asthma, encephalitis, chronic obstructive pulmonary disease (COPD), inflammatory osteolysis, Crohn's disease, ulcerative colitis, allergic disorders, septic shock, pulmonary fibrosis (e.g. , idiopathic pulmonary fibrosis), inflammatory vacultides (e.g. , polyarteritis nodosa, Wegner's granulomatosis, Takayasu's arteritis, temporal arteritis, and lymphomatoid granulomatosus), post-traumatic vascular angioplasty (e.g. restenosis after angioplasty), undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, chronic hepatitis, and chronic inflammation resulting from chronic viral or bacteria infections. In the present invention, this term does not include systemic inflammatory response syndrome (SIRS). A particular group of inflammatory diseases are immune-mediated inflammatory diseases. The term "immune mediated inflammatory disease" shall be taken to mean any disease mediated by the immune system and characterized by chronic or acute inflammation, resulting from, associated with or triggered by, a dysregulation of the normal immune response e.g. Crohn's disease, type 1 diabetes mellitus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, Hashimoto's disease, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison disease, scleroderma, Goodpasture's syndrome, ulcerative colitis, autoimmune hemolytic anemia, sterility, myasthenia gravis, multiple sclerosis, Basedow's disease, thrombopenia purpura, Guillain-Barre syndrome, allergy, asthma, atopic disease, arteriosclerosis, myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia, and rejection after organ transplantation. In a particular embodiment of the present invention, the inflammatory disease is selected from the group comprising rheumatoid arthritis and ischemia/reperfusion injury.

The expression "disease associated with inflammation" relates to all those diseases where pathogenic inflammation occurs i.e. when said process is harmful or undesirable, whether cancerous or not. Diseases associated with inflammation include inflammatory diseases, where there is an excessive or altered inflammatory response that leads to inflammatory symptoms. Said inflammatory diseases which may be treated by the agent of the invention include, without limitation, Addison's disease, acne vulgaris, alopecia areata, amyloidosis, ankylosing spondylitis, ulcerations, aphthous stomatitis, arthritis, arteriosclerosis, osteoarthritis, rheumatoid arthritis, bronchial asthma, Bechet's disease, Boeck's disease, intestinal inflammatory disease, Crohn's disease, choroiditis, ulcerative colitis, celiac's disease, cryoglobulinemia, macular degeneration, dermatitis, dermatitis herpetiformis, dermatomyositis, insulin dependent diabetes, juvenile diabetes, inflammatory demyelinating disease, Dupuytren contracture, encephalomyelitis, allergic encephalomyelitis, endophthalmia, allergic enteritis, autoimmune enteropathy syndrome, erythema nodosum leprosum, ankylosing spondylitis, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, cystic fibrosis, gingivitis, glomerulonephritis, Goodpasture syndrome, Graves syndrome, Hashimoto's disease, chronic hepatitis, histiocytosis, regional ileitis, iritis, disseminated lupus erythematous, systemic lupus erythematous, cutaneous lupus erythematous, lymphogranuloma, infectious mononucleosis, miastenia gravis, transverse myelitis, primary idiopathic myxedema, nephrosis, obesity, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, panniculitis, pemphigus vulgaris, periodontitis, polyarteritis nodosa, chronic polyarthritis, polymyositis, acute polyradiculitis, psoriasis, chronic obstructive pulmonary disease, purpura, gangrenous pioderma, Reiter's syndrome, diabetic retinopathy, rosacea, sarcoidosis, ataxic sclerosis, progressive systemic sclerosis, scleritis, sclerodermia, multiple sclerosis, disseminated sclerosis, acute anterior uveitis, vitiligo, Whipple's disease, diseases associated with AIDS, severe combined immunodeficiency and Epstein Barr's virus such as Sjogren's syndrome, osteoarticular tuberculosis and parasitic diseases such as leishmaniasis. Preferred inflammatory diseases are immune mediated inflammatory diseases.

The term "ischemia/reperfusion injury", as used herein, refers to tissue damage caused when blood supply returns to the tissue after a period of ischemia. The absence of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function. Oxidative stress associated with reperfusion may cause damage to the affected tissues or organs. Ischemia-reperfusion injury is characterized biochemically by a depletion of oxygen during an ischemic event followed by reoxygenation and the concomitant generation of reactive oxygen species during reperfusion.

The term "moiety", as used herein, relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures.

The term "recipient", as used herein in the context of transplantation, relates to a human or animal subject from which a organ for transplantation originates.

The term "rejection" or "transplant rejection" is used in the present invention in a context of cell/tissue/organ transplant, and is related to the process by which a transplantated cell, tissue and/or organ is rejected by the immune system of the recipient, which destroys the transplanted cell, tissue and/or organ. Different categories of rejection can be distinguised: hyperacute rejection (HAR, also known as accelerated humoral rejection or ACHR, with onset within minutes of anastomosis of blood supply, and caused by circulating antibodies), acute rejection (also known as cellular rejection, with onset 2-60 days after transplantation, characterized by interstitial vascular endothelial cell swelling, interstitial accumulation of lymphocytes, plasma cells, immunoblasts, macrophages, neutrophils; tubular separation with edema/necrosis of tubular epithelium; swelling and vacuolization of the endothelial cells, vascular edema, bleeding and inflammation, renal tubular necrosis, sclerosed glomeruli, tubular 'thyroidization', creatinine clearance, malaise, fever, HTN, oliguria) and chronic rejection (with a late onset, often more than 60 days after transplantation, and frequently accompanied by acute changes superimposed, increased mesangial cells with myointimal proliferation and crescent formation; mesangioproliferative glomerulonephritis, and interstitial fibrosis; there is in general a poor response to corticosteroids). In a particular embodiment, the transplant rejection is xenotransplant rejection. In a particular embodiment, the transplant rejection is xenotransplant (also known as xenograft, xenogeneic transplant or heterograft) wherein a transplantated cell, tissue and/or organ from a donor to a recipient is rejected by the immune system of the recipient, which destroys the transplanted cell, tissue and/or organ, wherein the donor and the recipient are members of different species. In a particular embodiment, the xenotransplant rejection is acute humoral rejection. The term "rheumatoid arthritis", as used herein, relates to an autoimmune disease that results in a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks flexible (synovial) joints. In the present invention, rheumatoid arthritis is selected from the group comprising acute arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerativa arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, and vertebral arthritis.

The term "sample" as used herein, relates to a biofluid sample that can be obtained from a subject. In a preferred embodiment of the invention, the biofluid is any fluid of the body comprising antibodies. In a more preferred embodiment of the invention, the biofluid is selected from blood (such as peripheral blood), serum or plasma. In a more preferred embodiment of the methods of the invention, the sample is serum or plasma. The blood sample is typically extracted by means of puncturing an artery or vein, normally a vein from the inner part of the elbow or from the back of the hand, the blood sample being collected in an air-tight vial or syringe. A capillary puncture normally on the heel or on the distal phalanxes of fingers can be performed for analysis by means of a micromethod. Serum can be obtained from the complete blood sample and in the absence of anticoagulant by leaving the sample to settle for 10 minutes so that it coagulates and subsequently centrifuging it at 1,500 rpm for 10 minutes for the purpose of separating the cells (precipitate) from the serum (supernatant). In turn, to obtain the plasma sample the complete blood is contacted with an anticoagulant and is centrifuged at 3,000 rpm for 20 minutes. The precipitate of said centrifugation corresponds to the formed elements, and the supernatant corresponds to the plasma. The serum or the plasma obtained can be transferred to a storage tube for sample analysis by means of the methods of the invention.

The term "subject" or "individual" or "animal" or "patient" is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. In a preferred embodiment of the invention, the subject lacks a-Gal epitopes but has natural anti-Gal antibodies, and includes humans, apes, and Old World monkeys (superfamily Cercopithecoidea, clade Catarrhini, including baboons and macaques). In a more preferred embodiment of the invention, the subject is a human. In a particular embodiment, the subject suffers from inflammation or is a candidate to suffer from inflammation, in particular the subject suffers or is candidate to suffer from an immune mediated inflammatory disease, wherein said inflammation is not due to systemic inflammatory response syndrome (SIRS) or to xenotransplant rejection. In an alternative embodiment, the subject is candidate to suffer from SIRS.

The term "systemic inflammatory response syndrome" or "SIRS", as used herein, is also known as "sepsis" or "septic syndrome", and is related to a condition secondary to infection characterized by a manifested infection induced by microorganisms, preferably bacteria or fungi, by parasites or by viruses or prions. The term comprises different forms of sepsis, e.g. urosepsis, sepsis due to pneumonia, intraabdominal infection, postoperative sepsis, sepsis due to invasion of a foreign body, sepsis due to bone marrow insufficiency or neutropenia, cholangiosepsis, sepsis after skin injury, burn or dermatitis, dentogenic or tonsillogenic sepsis. The more general term SIRS describes a generalized hyper- inflammatory reaction of diverse geneses, e.g. infection, burn and trauma. Thus, the sepsis is a particular form of SIRS, namely a SIRS characterized by infection of normally or physiologically sterile tissue. Particular forms of the condition of sepsis are "severe sepsis" and "septic shock". "Severe sepsis" is defined as "a sepsis associated with (multiple) organ dysfunction, hypoperfusion, or hypotension". "Septic shock" is defined as a sepsis with hypotension, despite fluid resuscitation, along with the presence of perfusion abnormalities.

SIRS manifestations include, without limitation, body temperature less than 36°C or greater than 38°C, heart rate greater than 90 beats per minute, tachypnea (high respiratory rate), with greater than 20 breaths per minute or an arterial partial pressure of carbon dioxide less than 4.3 kPa (32 mmHg), leukocytes less than 4000 cells/mm³ (4 x 10⁹ cells L) or greater than 12,000 cells/mm³ (12 x 10⁹ cells/L); or the presence of greater than 10% immature neutrophils. Methods to determine whether a subject is suffering from SIRS are known by the skilled person and include, without limitation, the method based on determining the levels of pro-hepcidin (pro-HEPC), soluble TNF- receptor 2 (sT FR2), Pentraxin-3 (PTX-3), Macrophage Colony-Stimulating Factor (MCSF), pro-Brain Natriuretic Protein (pro-BNP), Histone proteins, Procalcitonin (PCT) and c-Reactive Protein (CRP) in a biological sample taken from a subject as in EP2223119 Al, or the method based on determining the level of follistatin-like protein- 1 in a serum sample from a subject as in US20130011863.

In one embodiment, the condition associated with sepsis syndrome is selected from the group consisting of an organ dysfunction, preferably a kidney dysfunction or a liver dysfunction, a multiple organ dysfunction syndrome (MODS), an acute respiratory distress syndrome (ARDS), and disseminated intravascular coagulation (DIC). In another embodiment, the sepsis syndrome is induced by a bacterium or more than one bacterium selected from the group consisting of Gram-negative bacteria and Gram- positive bacteria. In yet another embodiment, the Gram-negative bacterium is selected from the group consisting of Escherichia coli, Klebsiella species, Serratia species, Enterobacter species, Proteus species, Pseudomonas aeruginosa, Haemophilus influenzae, Neisseria species, and Listeria species. In another embodiment, the Gram- positive bacterium is selected from the group consisting of Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative Staphylococci, Enterococcus species, Streptococcus pyogenes, and Streptococcus viridans. In one embodiment, the bacterium is a Gram-negative bacterium, preferably E. coli. In one embodiment, the sepsis syndrome is induced by a microorganism or more than one microorganism selected from the group consisting of anaerobic bacteria, fungi, rickettsiae, chlamydiae, mycoplasma, spirochetes, and viruses.

The term "transplantation" refers to a surgical procedure by which a celL tissue or organ is transferred from a donor subject to a recipient subject or from one part of the body to another in the same subject. The "donor subject" is the subject who gives blood, cells, tissues, or an organ for another subject by blood transfusion or an organ transplant. In the context of the invention, the donor subject gives cell(s), tissue(s) and/or organ(s) to a recipient subject. According to the invention, the donor subject is a human or another mammal. The "recipient subject" is the subject who receives blood, cells, tissues, or an organ from another subject by blood transfusion or an organ transplant. In the context of the invention, the recipient subject receives tissue(s) and/or organ(s) from a donor subject subject. According to the invention, the recipient subject is a human or another mammal. Transplanted tissues comprise, but are not limited to, bone tissue, tendons, corneal tissue, heart valves, veins and bone marrow. Transplanted organs comprise, but are not limited to, heart, lung, liver, kidney, pancreas and intestin. The particular surgical procedure of transplantation wherein the donor subject and the recipient subject are genetically non- identical members of the same species is known as allotransplantation. Thus, the term allotransplant (also known as allograft, allogeneic transplant or homograft) is related to the transplantation of cells, tissues or organs sourced from a genetically non-identical member of the same species as the recipient. The term "allotransplantable" refers to organs or tissues that are relatively often or routinely transplanted. Examples of allotransplantable organs include heart, lung, liver, pancreas, kidney and intestine. The particular surgical procedure of transplantation wherein the donor subject and the recipient subject are members of different species is known as xenotransplantation. Thus, the term xenotransplant (also known as xenograft, xenogeneic tranplant or heterograft) is related to the transplantation of cells, tissues or organs sourced from a donor to a recipient, wherein donor and recipient are members of different species. Methods for the prevention and/or treatment of inflammation

In a first aspect, the invention relates to a method for the prevention and/or treatment of inflammation in a subject, comprising the administration to a subject in need thereof of a therapeutically effective amount of an agent comprising a terminal a- galactosyl moiety.

Alternatively, the invention relates to an agent comprising a terminal a- galactosyl moiety for use in the prevention and/or treatment of inflammation in a subject.

Alternatively, the invention relates to the use of an agent comprising a terminal a-galactosyl moiety, for the manufacture of a medicament for the prevention and/or treatment of inflammation in a subject.

Thus, the invention relates to agents comprising a terminal a-galactosyl moiety. In a particular embodiment of the method of the invention, the inflammation is due to an immune mediated inflammatory disease. In a particular preferred embodiment, the immune mediated inflammatory disease is selected from the group comprising rheumatoid arthritis, ischemia/reperfusion injury, systemic inflammatory response syndrome (SIRS) and transplant rejection. The inflammatory symptoms of a disease that causes inflammation comprise those symptoms related to an inflammatory process with are caused by any disease or condition causing inflammation, wherein said disease or condition includes, without limitation, acid reflux/heartburn, acne, allergies and sensitivities, Alzheimer's disease, asthma, aterosclerosis, bronchitis, cancer, carditis, celiac disease, chronic pain, Crohn's disease, cirrosis, colitis, dementia, dermatitis, diabetes, dry eyes, edema, emphysema, eczema, fibromyalgia, gastroenteritis, gingivitis, heart disease, hepatitis, high blood pressure, insulin resistance, interstitial cystitis, joint pain/arthritis/rheumatoid arthritis, metabolic syndrome (syndrome X), myositis, nephritis, obesity, osteopenia, osteoporosis, Parkinson's disease, periodontal disease, polyarteritis, polychondritis, psoriasis, scleroderma, sinusitis, Sjogren's sindrome, spastic colon, systemic candidiasis, tendonitis, UTI's and vaginitis. Symptoms commonly associated with low- grade chronic inflammation include, without limitation, body aches and pain, congestion, frequent infections, diarrhea, dry eyes, indigestion, shortness of breath, skin outbreaks, swelling, stiffness, and weight gain/obesity.

In a particular embodiment, the subject suffering from inflammation, or candidate to suffer from inflammation, has natural endogenous anti-Gal antibodies. In a particular preferred embodiment, the subject is a human.

As previously described, the agent for administration according to the method of the invention comprises a terminal a-galactosyl moiety. In a particular embodiment, the terminal a-galactosyl moiety is selected from the group comprising terminal Galal- 3GaL terminal Galal-2GaL terminal Galal-6GaL terminal Galal-6Glc, and terminal a- galactose sugar unit(s) capable of binding an anti-Gal antibody. In a particular preferred embodiment, the terminal α-galactosyl moiety of the agent for use according to the invention comprises a terminal Galal-3Gal.

Natural or synthetic molecules comprising one or more terminal a-galactosyl moietys according to the present invention include, without limitation, the following: i. galactose-a-l,3-galactose, oligomers (e.g., dimers, trimmers, tetramers, pentasaccharides) of galactose-a-l,3-galactose, and derivatives thereof, including glucosamine derivatives, linear B-2 trisaccharide (Galal-3Gaipi-

4GlcNAc, CAS No. 101627-01-4), linear B-6 trisaccharide (Galal-3Gah31- 4Glc, CAS No. 56038-36-9) and derivatives thereof, al-3 galactobiosyl β- methyl glycoside; al-3, β1-4 galactotriose (Galal-3Gah31-4Gal, CAS No. 56038-36-9), galactotetraose (Galal-3Gaipi-4Galal-3-D-Gal, CAS No. 56038- 38-1), Galili pentasaccharide (L537, Gal-al,3Gal-pi,4GlcNAc-pi,3Gal- pi,4Glc, CAS No. 119502-59-9), and derivatives of the above such as, e.g., amino acid derivatives (e.g., dodecalysine, glycine), amides, esters, ethers, amines, sulfonamides, thioethers, acetals, carbamates, ureas and amidines, glycopeptides comprising oligosaccharides with terminal galactose-a-1,3- galactose,

commercially available glycolipids (Dextra Laboratories, Ltd., United Kingdom) such as Galal-3Gal glycolipids comprising blood group B type 2 linear trisaccharide (GN334, Galal-3GaU31-4GlcNac, CAS No. 101627-01-4); and comprising Galili pentasaccharide (L537, Gal-al,3Gal-pi,4GlcNAc-pi,3Gal- pi,4Glc, CAS No. 119502-59-9),

commercially available glycoconjugates with galactose-a-l,3-galactose epitopes include for instance: Galal-3Gaipi-4Glc-BSA (#NGP0330, 3 atom spacer), Galal-3GaU31-4(3-deoxyGlcNAc)-HSA (#NGP2335), Galal-3Gah31- 4GlcNAcpi-HDPE (#NGL0334), Galal-3Gal-BSA (#NGP0203, 3 atom spacer), Galal-3Gaipi-3GlcNAc-BSA (#NGP0333, 3 atom spacer), Galal- 3Gaipi-3GlcNAc-HSA (#NGP2333, 3 atom spacer), Galal-3GaU31-4(6- deoxyGlcNAc)-HSA (#NGP2336, 3 atom spacer), Galal-3GaU31-4Glc-HSA (#NGP2330, 3 atom spacer), Galal-3Gal-BSA (#NGP1203, 14 atom spacer), Galal-3Gal-HSA (#NGP2203, 3 atom spacer), Galal-3Gal-HSA (#NGP3203, 14 atom spacer), Galal-3Gaipi-4GlcNAc-BSA (#NGP0334, 3 atom spacer), Galal-3GaU31-4GlcNAc-BSA (#NGP1334, 14 atom spacer), Galal-3Gah31- 4GlcNAc-HSA (#NGP2334, 3 atom spacer), Galal-3GaU31-4GlcNAc-HSA (#NGP3334, 14 atom spacer), and Galal-3GaU31-4Glc-BSA (#NGP0330, 3 atom spacer), Galal-3Gaipi-HDPE (#NGL0203),

galactose-a-l,3-galactose oligosaccharides, such as those commercially available from Elicityl, including without limitation Galal-3Gaipi-3(Fucal- 4)GlcNAc (#GLY076), Galal-3Gaipi-4(Fucal-3)GlcNAc (#GLY075), Galal- 3 [Gaip 1 -4GlcNAcP 1 -3]4Gaip 1 -4Glc (#GL Y079), Galal -3 [Gaip 1 -4GlcNA_Cp 1 - 3]3Gaipi-4Glc (#GLY078), Galal -3Gaipi-4Glc (#GLY070), Galal-3[Gaipi- 4GlcNA_Cpi-3]2Gaipi-4Glc (#GLY077), Galal-3Gaipi-4GlcNA_Cpi-3Gaipi- 4Glc (#GLY071), Galal-3Gaipi-4GlcNAc (#GLY74-2, linear B-2 trisaccharide), and Galal-3Gaipi-3GlcNAc (#GLY74-1), and

vi. additional macromolecules with a-Gal epitopes that are suitable for injection and subsequent in situ binding to anti-Gal antibodies include, without limitation:

o mouse laminin with 50-70 a-gal epitopes (Galili U 1993 Springer Seminars in Immunopathology 15, 155), commercially available from Life Technologies (#23017-015),

o multiple synthetic a-gal epitopes linked to BSA (Stone KR et al. 2007 Transplantation 83(2): 211-219),

o GAS914, commercially produced by Novartis (W09847915) and disclosed in Zhong R et al. 2003 Transplantation 75(1): 10-19,

o the a-Gal polyethylene glycol conjugate TPC (Schirmer JM et al. 2004 Xenotransplantation 11(5): 436-443), and

o a-Gal epitope-mhnicking peptides linked to a macromolecule backbone

(Sandrin MS et al. 1997 Glycoconj J 14(1): 97-105), and peptides encoded by mucin genes MUC1, 3 and 4.

These compounds, and similar synthetic and natural glycoconjugates, glycolipids, glycoproteins or oligosaccharides linked to reactive groups comprising a terminal a-galactosyl moiety, preferably a terminal Galal-3GaL may also bind anti-Gal antibodies and thus be considered functional equivalents of the agents for their use according to the invention. In a particular preferred embodiment, the agent comprising a terminal a-galactosyl moiety for use according to the invention is GAS914.

Assays to determine whether a molecule is capable of specifically binding to an antibody, particularly an anti-Gal antibody, are known by the skilled person and include, without limitation, immunoprecipitation, radioimmunoassay (RIA), enzyme- linked immunoabsorbent assay (ELISA) and immunofluorescent techniques such as fluorescence microscopy or flow cytometry.

Appropriate amounts of an agent according to the present invention can be formulated with pharmaceutically acceptable excipients and/or carriers to obtain a pharmaceutical composition to be administered in a method for the prevention and/or treatment of inflammation. A composition that includes an agent according to the invention can be delivered to a subject by a variety of routes including, without limitation, systemically delivery, e.g., by intravenous, subcutaneous or intramuscular injection. Additionally, it is also possible to administer the composition comprising the agent of the invention intranasally which allows systemic administration by a non- aggressive mode of administration. Also, intraventricular administration may be adequate. A preferred route of delivery is intravenous injection.

Those skilled in the art are familiar with the principles and procedures discussed in widely known and available sources as Remington's Pharmaceutical Science (17th Ed., Mack Publishing Co., Easton, Pa., 1985) and Goodman and Gilman's The Pharmaceutical Basis of Therapeutics (8th Ed., Pergamon Press, Elmsford, N.Y., 1990) both of which are incorporated herein by reference.

In a preferred embodiment of the present invention, the agents of the invention are formulated in accordance with standard procedure as a pharmaceutical composition adapted for delivered administration to human beings and other mammals producing natural anti-Gal antibodies. Typically, compositions for intravenous or intraventricular administration are solutions in sterile isotonic aqueous buffer.

Where necessary, the agent of the invention is comprised in a composition also including a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

In cases other than intravenous administration, the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, geL polymer, or sustained release formulation. The composition can be formulated with traditional binders and carriers, as would be known in the art. Formulations can include standard carriers such as pharmaceutical grades of mannitoL lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., inert carriers having well established functionality in the manufacture of pharmaceuticals. Various delivery systems are known and can be used to administer a therapeutic of the present invention including encapsulation in liposomes, microparticles, microcapsules and the like.

In yet another preferred embodiment, therapeutics containing the agents of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, thriethylamine, 2-ethylamino ethanoL histidine, procaine or similar.

The pharmaceutical compositions containing the agent according to the method of the invention can occur at any pharmaceutical form of administration considered appropriate for the selected administration route, for example, by systemic, oraL parenteral or topical administration, for which it will include the pharmaceutically acceptable excipients necessary for formulation of the desired method of administration.

The effective quantity of the agent of the invention can vary within a wide range and, in general, will vary depending on the particular circumstances of application, duration of the exposure and other considerations.

Solid dosage forms for oral administration may include conventional capsules, sustained release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. At these solid dosage forms, the active compounds can be mixed with at least one inert excipient such as sucrose, lactose or starch. Such dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, e.g. lubricating agents such as magnesium stearate. In the case of capsules, tablets, effervescent tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can be prepared with enteric coatings.

Liquid dosage forms for oral administration may include emulsions, solutions, suspensions, syrups and elixirs pharmaceutically acceptable containing inert diluents commonly used in the technique, such as water. Those compositions may also comprise adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening agents, flavoring and perfuming agents.

Injectable preparations, for example, aqueous or oleaginous suspensions, sterile injectable may be formulated according with the technique known using suitable dispersing agents, wetting agents and/or suspending agents. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. Sterile oils are also conventionally used as solvents or suspending media.

For topical administration, compounds of the method of the invention can be formulated as creams, gels, lotions, liquids, pomades, spray solutions, dispersions, solid bars, emulsions, microemulsions and similars which may be formulated according to conventional methods that use suitable excipients, such as, for example, emulsifiers, surfactants, thickening agents, coloring agents and combinations of two or more thereof.

Additionally, the compounds of the method of the invention may be administered in the form of transdermal patches or iontophoresis devices. In one embodiment, the compounds of the invention are administered as a transdermal patch, for example, in the form of sustained-release transdermal patch. Suitable transdermal patches are described in more detail in, for example, US5262165, US5948433, US6010715 and US6071531.

The compositions comprising the agents of the method of the invention can additionally include conventional excipients, ie pharmaceutically acceptable carriers suitable for parenteral application which do not react damaging with the active compounds. Suitable pharmaceutically acceptable vehicles include, for example, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, monoglycerides and diglycerides of fatty acids, fatty acid esters petroetrals, hydroxymethyl cellulose, polyvinylpyrrolidone and similars.

Several drug delivery systems are known and can be used to administer the agents or compositions according to the method of the invention, including, for example, encapsulation in liposomes, microbubbles, emulsions, microparticles, microcapsules and similars. The required dosage can be administered as a single unit or in a sustained release form.

Sustainable-release forms and appropriate materials and methods for their preparation are described in, for example, "Modified-Release Drug Delivery Technology", Rathbone, M. J. Hadgraft, J. and Roberts, M. S. (eds.), Marcel Dekker, Inc., New York (2002), "Handbook of Pharmaceutical Controlled Release Technology", Wise, D. L. (ed.), Marcel Dekker, Inc. New York, (2000). In one embodiment of the invention, the orally administrable form of a compound according to the invention is in a sustained release form further comprises at least one coating or matrix. The coating or sustained release matrix include, without limitation, natural polymers, semisynthetic or synthetic water-insoluble, modified, waxes, fats, fatty alcohols, fatty acids, natural semisynthetic or synthetic plasticizers, or a combination of two or more of the them.

Enteric coatings may be applied using conventional processes known to experts in the art, as described in, for example, Johnson, J. L., "Pharmaceutical tablet coating", Coatings Technology Handbook (Second Edition), Satas, D. and Tracton, A. A. (eds), Marcel Dekker, Inc. New York, (2001), Carstensen, T., "Coating Tablets in Advanced Pharmaceutical Solids", Swarbrick, J. (ed.), Marcel Dekker, Inc. New York (2001), 455- 468.

Even though individual needs vary, determination of optimal ranges for effective amounts of the agent of the invention belongs to the common experience of those experts in the art. In general, the dosage needed to provide an effective amount of such compound, which can be adjusted by one expert in the art will vary depending on age, health, fitness, sex, diet, weight, degree of alteration of the receptor, frequency of treatment and the nature and extent of impairment or illness, medical condition of the patient, route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profile of the particular compound used, if using a system drug delivery, and if the compound is administered as part of a combination of drugs.

The amount of the compound according to the invention that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, in particular an inflammatory disease or of the inflammation symptoms of a disease that causes inflammation, and can be determined by conventional clinical techniques, including reference to Goodman and Gilman, supra; The Physician's Desk Reference, Medical Economics Company, Inc., Oradell, NJ, 1995, and Drug Facts and Comparisons, Inc., St. Louis, MO, 1993. The precise dose to use in the formulation will also depend on the route of administration, and severity of the disease or disorder, and should be decided in the physician's opinion and the patient's circumstances.

The levels of anti-Gal antibodies specific for an agent comprising a terminal a- galactosyl moiety can be determined by any method suitable for determination of antibody levels or antibody titer of antibodies with a desired specificity known by the skilled in the art and include, but are not limited to, immunochemical assays such as ELISA (Enzyme-Linked Immuno Sorbent Assay), immunostaining, immunochemical assays, immunofluorescence, flow cytometry, Western blot, radioimmunoassays, immunohistochemical assays and immunoprecipitations. By way of non-limiting example, the levels of anti-Gal antibodies can be quantified by means of secondary antibodies with a capacity to specifically bind to anti-Gal antibodies (or to fragments thereof) and subsequent quantification of the resulting anti-Gal antibody-secondary antibody complexes. The antibodies to be employed in these assays can be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' y F(ab')2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies. At the same time, the antibodies can be labeled or not. Illustrative, but nonexclusive examples of markers which can be used include radioactive isotopes, enzymes, fluorophores, chemilumine scent reagents, enzymatic substrates or cofactors, enzymatic inhibitors, particles, colorants, etc. There are a wide variety of well-known assays that can be used in the present invention, which use non- labeled antibodies (primary antibody) and labeled antibodies (secondary antibodies); among these techniques are included Western-blot or Western transfer, ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzymatic immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or microarrays including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks. Other ways of detecting and quantifying the anti-Gal antibodies include techniques of affinity chromatography, binding- ligand assays, etc. The invention is described in detail below by means of the following examples which are to be construed as merely illustrative and not limitative of the scope of the invention. EXAMPLES

Example 1. Removal of anti-Gal antibodies prevents sepsis in Gal knockout mice

A knockout mice lacking the a-l,3-galactosyltransferase (al,3GT) gene was used. This mice generates natural anti-Gal antibodies without any need of additional immunization, thereby allowing the inventors to investigate whether the removal of anti-Gal antibodies with GAS914 as unique action might have an impact in the sepsis developed in Gal knockout mice after colon ligation and puncture (CLP). In this model a total of 0.5 cm of cecum was ligated and punctured twice using a 30-gauge needle to allow protrusion of contents of cecum and assure the presence of bacteria in the peritoneum.

To evaluate the clinical status of the mice, the physical activity was analyzed using an established welfare scoring system that evaluates spontaneous activity, food intake and response to exogenous stimuli. A total of 34 animals were included in the study. Seventeen (control group) did not receive any treatment. The other 17 (GAS914 group) were treated with 10 mg/kg of GAS914 intraperitoneally every other day from day -3 of CLP and thereafter. The dosage and interval of GAS914 injections were selected based on previous local studies that showed the complete removal of anti-Gal antibodies on day 0 (CLP day). Essentially, all anti-Gal antibodies are eliminated with the initial GAS914 injection.

As it is shown in Figure 1A, 4 of the 17 (24%) Gal knockout mice treated with GAS914 died after CLP compared with 11 (65%) of untreated animals (p=0.01), with most of deaths occurring within 48h after CLP. Also, welfare of animals was better from day one after CLP in GAS914 treated animals compared to controls (Figure IB). To obtain the benefit of removing anti-Gal antibodies in sepsis, these have to be depleted before CLP. The inventors also investigated in two groups of 10 animals, whether removal of anti-Gal antibodies with GAS 914, 12h after CLP had an impact in animal survival and welfare. In this case, GAS914 did not provide any benefit compared to control animals (Figure 1C).

Treatment with GAS914 before CLP was associated with nearly the depletion of all anti-Gal IgM and IgG antibodies at the time of CLP (Figure ID). These antibodies barely change after CLP, whilst in non-treated animals there was a significant reduction of anti-Gal IgM and IgG antibodies after CLP, suggesting the presence of the antigen after the procedure (Figure ID). Despite of this, no differences were observed 24 h after CLP between animals that did or did not receive GAS914 regarding the number of bacteria isolated in the blood, the type (Escherichia coli in all the cases) and the genotype of these microorganisms. This suggests that the favorable outcome obtained in sepsis with GAS914 was not due to modification of the bacterial infection by the molecule. Example 2. Removal of anti-Gal antibodies reduces cytokine levels in Gal knockout mice

The other capital element for the development of sepsis during bacterial infections is the host inflammatory response. For this reason, the inventors evaluated the impact of GAS914 in the pattern of cytokines before and after CLP, in Gal-KO and wild type mice, using an array of 40 cytokines, including both proinflammatory and antiinflammatory mediators.

The results showed that treatment with GAS914 was associated with a reduction of most cytokines at the time of CLP in Gal-KO mice compared to untreated animals (Figure 2A), which was not observed 24 h after CLP (Figure 2B) or in wild type mice lacking anti-Gal antibodies before CLP (Figure 2C). This suggests that the improved outcome in sepsis mediated by GAS914 requires the presence of anti-Gal antibodies, and results from a reduction of baseline net inflammatory status likely mediated by the soluble immune complexes generated between anti-Gal antibodies and GAS914.

Claims

1. A method for the prevention and/or treatment of inflammation in a subject, comprising the administration to a subject in need thereof of a therapeutically effective amount of an agent comprising a terminal a-galactosyl moiety.

2. The method according to claim 1, wherein the inflammation is due to an immune mediated inflammatory disease.

3. The method according to claim 2, wherein the immune mediated inflammatory disease is selected from the group comprising rheumatoid arthritis, ischemia/reperfusion injury, systemic inflammatory response syndrome (SIRS) and transplant rejection.

4. The method according to claim 1, wherein the terminal α-galactosyl is selected from the group consisting of terminal Galal-3Gal, terminal Galal-2Gal, terminal Galal-6Gal, terminal Galal-6Glc, and terminal a-galactose sugar unit(s) capable of binding an anti-Gal antibody.

5. The method according to claim 1, wherein the terminal α-galactosyl is terminal Galal-3Gal.

6. The method according to claim 1, wherein the agent is selected from the group comprising

- galactose-a-l,3-galactose (Galal-3Gal),

galactose-a-l,3-galactose oligomers and oligosacharides, comprising linear B-2 trisaccharide (Galal-3GaU31-4GlcNAc, CAS No. 101627-01-4), linear B-6 trisaccharide (Galal-3GaU31-4Glc, CAS No. 56038-36-9), al-3 galactobiosyl β-methyl glycoside; al-3, β1-4 galactotriose (Galal-3GaU31- 4Gal, CAS No. 56038-36-9), galactotetraose (Galal-3GaU31-4Galal-3-D-

Gal, CAS No. 56038-38-1), Galili pentasaccharide (L537, Gal-al,3Gal- pi,4GlcNAc-pi,3Gal-pi,4Glc, CAS No. 119502-59-9), Galal-3GaU31- 3(Fucal-4)GlcNAc (#GLY076), Galal-3GaU31-4(Fucal-3)GlcNAc (#GLY075), Galal-3[Gaipi-4GlcNA_Cpi-3]4Gaipi-4Glc (#GLY079), Galal-3[Gaipi-4GlcNA_Cpi-3]3Gaipi-4Glc (#GLY078), Galal-3GaU31- 4Glc (#GLY070), Galal-3[GaU31-4GlcNA_Cpi-3]2GaU31-4Glc (#GLY077), Galal-3Gaipi-4GlcNA_Cpi-3Gaipi-4Glc (#GLY071), Galal-3GaU31- 4GlcNAc, and Galal-3GaU31-3GlcNAc (#GLY74-1), and derivatives thereof,

galactose-a-l,3-galactose derivatives, comprising glucosamine derivatives, amides, esters, ethers, amines, sulfonamides, thioethers, acetals, carbamates, ureas and amidines,

glycopeptides and glycoproteins comprising oligosaccharides with terminal galactose-a- 1,3-galactose,

glycolipids comprising galactose-a- 1,3-galactose, comprising glycolipids comprising blood group B type 2 linear trisaccharide (GN334, Galal- 3Gaipi-4GlcNac, CAS No. 101627-01-4) and glycolipids comprising Galili pentasaccharide (L537, Gal-al,3Gal-pi,4GlcNAc-pi,3Gal-pi,4Glc, CAS No. 119502-59-9),

galactose-α- 1,3-galactose glycoconjugates, comprising Galal-3Gaipi-4Glc- BSA (#NGP0330, 3 atom spacer), Galal-3Gaipi-4(3-deoxyGlcNAc)-HSA (#NGP2335), Galal-3GaU31-4GlcNAcpi-HDPE (#NGL0334), Galal-3Gal- BSA (#NGP0203, 3 atom spacer), Galal-3Gaipi-3GlcNAc-BSA (#NGP0333, 3 atom spacer), Galal-3GaU31-3GlcNAc-HSA (#NGP2333, 3 atom spacer), Galal-3Gaipi-4(6-deoxyGlcNAc)-HSA (#NGP2336, 3 atom spacer), Galal-3Gaipi-4Glc-HAS (#NGP2330, 3 atom spacer), Galal-3Gal- BSA (#NGP1203, 14 atom spacer), Galal-3Gal-HSA (#NGP2203, 3 atom spacer), Galal-3Gal-HSA (#NGP3203, 14 atom spacer), Galal-3Gah31- 4GlcNAc-BSA (#NGP0334, 3 atom spacer), Galal-3Gaipi-4GlcNAc-BSA (#NGP1334, 14 atom spacer), Galal-3Gaipi-4GlcNAc-HSA (#NGP2334, 3 atom spacer), Galal-3Gaipi-4GlcNAc-HSA (#NGP3334, 14 atom spacer), and Galal-3GaU31-4Glc-BSA (#NGP0330, 3 atom spacer), Galal-3Gaipi- HDPE (#NGL0203), and additional macromolecules comprising galactose-a-l,3-galactose, comprising mouse laminin, synthetic a-gal epitopes linked to BSA, GAS914, the a-Gal polyethylene glycol conjugate TPC, and a-Gal epitope-mimicking peptides linked to a macromolecule backbone.

7. The method according to claim 1, wherein the agent is a the random copolymer GAS914 having the following structure:

wherein n represents the average degree of polymerization, x represents the fraction of glycosylated monomer; and 1 - x represents the fraction of thioglycerol- capped monomer.

The method according to claim 1, wherein the subject has endogenous anti-Gal antibodies.

9. The method according to claim 1, wherein the subject is a human.