MXPA01007564A - Methods for preventing graft rejection and ischemia-reperfusion injury - Google Patents

Abstract

A method for inhibiting the rejection of transplanted grafts is disclosed. The method comprising administering an effective amount of an antagonist of CCR1 function to a graft recipient. Also disclosed is a method of inhibiting ischemia/reperfusion injury comprising administering to a subject in need thereof an effective amount of an antagonist of CCR1 function. The disclosed methods can also comprise the co-administration of one or more additional therapeutic agents, for example, immunosuppressive agents and cell adhesion inhibitors.

Description

METHODS OF PREVENTION OF THE REJECTION OF GRAFTING AND THE INJURY FOR ISCHEMIA-REPERFUSION BACKGROUND OF THE INVENTION In many cases, the best and only treatment available for patients suffering from certain degenerative conditions of terminal phase or congenital genetic disorders is the transplantation of a healthy graft (for example, organs, tissues). Advances in surgical techniques and post-operative immunosuppressive therapy have mitigated some of the barriers to the long-term survival of grafts and graft recipients and have previously converted experimental therapy into a broader clinical practice. A major barrier to long-term survival of transplanted grafts is rejection by the recipient's immune system. The rejection of grafts can be classified as hyperacute rejection, which is mediated by preformed antibodies that can bind to the graft and are present in the circulation of the recipient; acute rejection, which is mediated by the recipient's cellular immune response; or chronic rejection, which occurs through a multifactorial process that includes an immune component. The practice of matching the allelic variants of cellular antigens, most notably the major histocompatibility antigens ("MHC") / also referred to as tissue typing, as well as the blood type matching of the donor and the donor. receptor, has reduced the incidence of hyperacute rejection. However, most of the grafts that are transplanted do not exactly correspond to the type of tissue of the recipient (for example, allografts) and do not remain viable without therapeutic intervention. Rejection of allografts can be inhibited by long-term prophylactic immunosuppressive therapy (eg, for life, most notably with agents that inhibit calcineurin (eg, cyclosporin A (CsA), FK-506). not only does it inhibit graft rejection, it can make the recipient susceptible to infection with, for example, viruses, bacteria and fungi (for example, yeasts, molds) and present a higher risk of developing certain malignancies. , therapeutic doses of immunosuppressive agents can produce adverse side effects, such as diabetes melli-tus, neurotoxicity, nephrotoxicity, hyperlipidemia, hypertension, hirsutism and gingival hyperplasia (Spencer, CM et al., Drugs 54 (6): 925-975 ( 1997).) Thus, the degree of immunosuppression must be carefully calculated to avoid rejection of the graft and to preserve the general health of the recipient. Prophylactic ion, acute and chronic rejection of grafts remains a clinical problem. Acute episodes of rejection are characterized by infiltration of the graft by receptacle leukocytes (eg, monocytes, macrophages, T cells) and cell necrosis. These episodes usually occur during the days to months following the transplant. Acute rejection has been treated with high doses of certain immunosuppressive agents, such as glucocorticoids (eg, prednisone) and certain antibodies that bind to leukocytes (eg, OKT3). However, these therapies do not always stop rejection, they are associated with systemic side effects and may lose efficacy in cases of recurrent rejection activity. Chronic rejection is the major cause of graft failure and death of the recipient for those patients who survive the first year. Evidence of chronic rejection can be found in approximately 40-50% of heart and / or lung allograft recipients who survive for five years and most kidney grafts succumb to chronic rejection. The pathogenesis of chronic rejection is complex and involves accelerated arteriosclerosis (eg, atherosclerosis) of the vasculature associated with the graft and leukocyte infiltration. Unlike episodes of acute rejection, chronic rejection does not respond, in general, to more immunosuppressive therapy. Moreover, the accelerated atherosclerosis of the graft characteristic of chronic rejection is generally diffuse and not subject to conventional therapeutic procedures (for example, angioplasty, bypass graft, endarterectomy). In this way, patients who chronically reject their grafts may require a second transplant. (Schroeder J.S., "Cardiac Transplantation", pp. 1298-1300; Maurer, J.R., "Lung Transplantation", pp. 1491-1493; Carpenter, C.B. and Lazaras, J.M., "Dialysis and Transplantation in the Treatment of Renal Failure," pp. 1524-1529; Dienstag, J., "Liver Transplantation", pp. 1721-1725; all in Harrison's Principles of Internal Medicine, 14th ed., Fauci et al., Eds. McGraw Hill (1998)). The flow of oxygenated blood to all trans-planted grafts stops during graft procurement and transplant surgery. The lack of blood leads to hypoxia and necrosis of some or all of the graft cells, depending on the amount of time during which the blood flow stops. The resulting damage or injury is apparent after reinstatement of blood flow (reperfusion). This type of injury, referred to as an ischemia / reperfusion injury, can lead to the death of the endothelial cells lining the blood vessels (eg, arteries) associated with the graft (Gohra, H. et al., Transplantation. 60 (1): 96-102 (1995)). Thus, ischemia / reperfusion injury may be a contributing factor to accelerated graft arteriosclerosis and graft rejection.

Therapeutic methods are needed to prevent graft rejection and ischemia / reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a Kaplan-Meier survival curve illustrating the survival of CCRl - / - (- -) mice and wild type B6 / 129 mice (-? -) after renal ischemia / reperfusion. CCR1 - / - mice survived significantly more than B6 / 129 mice (p <0.001, log rank sum test, survival limited to 100 days). Figure 2 is a histogram illustrating the serum creatinine levels of CCR1 - / - and B6 / 129 mice after renal ischemia / reperfusion or sham surgery (unilateral nephrectomy without ischemia / reperfusion). Creatine levels were measured 2, 7 and 100 days after the ischemia / reperfusion or sham surgery. Only B6 / 129 (control) mice had significantly elevated serum creatinine levels at 2 and 7 days of ischemia / reperfusion (** p <0.005). All groups had elevated serum creatinine levels 100 days after ischemia / reperfusion or sham surgery (* p < 0.01). Figure 3 is a histogram illustrating serum alanine aminotransferase (SGPT) levels, an indicator of hepatic dysfunction, in CCRl - / - ("KO") and B6 / 129 ("WT") mice at predetermined times after ischemia / reperfusion. Serum SGPT levels are also shown in B6 / 129 mice that underwent surgery but did not experience hepatic ischemia / reperfusion (sham). CCRl - / - mice experienced significantly less liver dysfunction than B6 / 129 mice, as determined by serum SGPT levels, over the period from 0.5 to 7 days after ischemia / reperfusion Hepatic (* p < 0.0001). Figure 4A is a histogram illustrating T-cell proliferation induced by concanavalin-A in splenocyte cultures isolated from CCR1 - / - or B6 / 129 mice (CCR1 + / +). T cell proliferative responses induced by concanavalin-A in cell cultures from CCR1 - / - mice or cultures of B6 / 129 mice were approximately equivalent. The data are the mean ± standard deviation of six cultures that were stimulated for 48 hours. The data presented are representative of four trials. Figure 4B is a histogram illustrating mixed lymphocyte responses ("MLR") of cells isolated from CCR1 - / - mice (CCR1 KO) or wild type B6 / 129 (WT) mice stimulated with allogeneic splenocytes (isolated from mice Balb / c). Cells of both CCR1 - / - and wild type B6 / 129 mice showed strong MLR towards Balb / c stimulator cells treated with mitomycin-c (** p <0.001 against unstimulated CCR1 responder cells or B6 / 129 wild-type unstimulated). The CCR1 - / - cells proliferated less than the sal-vaje type cells in the assay (* p <0.01). The data are the mean ± the standard deviation of six 5-day crops. The data presented are representative of four trials. BALB / c es-tim. : splenocytes treated with mitomycin -c isolated from a Balb / c mouse; WT resp .: splenocytes isolated from a B6 / 129 mouse; CCRl KO resp. : splenocytes isolated from a CCR1 - / - mouse; BALB / c-WT: splenocytes isolated from a B6 / 129 mouse stimulated with splenocytes treated with mitomycin-c isolated from a Balb / c mouse; BALB / c-KO: splenocytes isolated from a CCR1 - / - mouse stimulated with splenocytes treated with mito-mycin-c isolated from a Balb / c mouse. COMPENDIUM OF THE INVENTION The invention relates to transplants and the promotion of the viability of transplanted grafts. In one aspect, the invention relates to a method for inhibiting (reducing or preventing) graft rejection (eg, acute rejection, chronic rejection). In one embodiment, the method consists of administering to an graft recipient an effective amount of an antagonist of the CCR1 function. In another realization, the graft is an allograft. In a particular embodiment, the allograft is a heart. In a preferred embodiment, the method consists in administering an antagonist of the CCR1 function and one or more immunosuppressive agents to a graft recipient. In another aspect, the invention relates to a method for inhibiting (reducing or preventing) ischemia / reperfusion injury. In one embodiment, the method consists of administering to a subject in need thereof an effective amount of an antagonist of the CCR1 function. In some embodiments, the ischemia / reperfusion injury may be the result of a trauma or a medical procedure, for example surgery. In other embodiments, the ischemia / reperfusion injury may be the result of a pathological condition, for example arteriosclerosis, myocardial infarction, cerebrovascular accident or transient ischemic attack. In a particular embodiment, the ischemia / reperfusion injury is a consequence of a graft transplant. In another particular embodiment, the graft is a kidney. In another embodiment, the method consists of the administration of an antagonist of the CCR1 function and one or more additional therapeutic agents, for example thrombolytic agents, inhibitors of cell adhesion, anticoagulants, antithrombotic agents and activators or inhibitors of nitric oxide synthase. DETAILED DESCRIPTION OF THE INVENTION The invention relates to the transplantation and viability of transplanted grafts. Specifically, the invention relates to the inhibition of graft rejection (e.g., acute graft rejection, erogenous graft rejection) by administering to an graft recipient an effective amount of a CC chemokine receptor antagonist, CCR1, of mammals (for example, humans). Chemokines are a family of proinflammatory mediators that promote the recruitment and activation of multiple leukocyte lines (eg, lymphocytes and macrophages). They can be released by many types of tissue cells after their activation. The continuous release of chemokines at sites of inflammation can mediate the continuous migration and recruitment of effector cells to sites of chronic inflammation. The chemokines are related in terms of primary structure and share four conserved cysteines, which form disulfide bonds. Based on this conserved cysteine motif, the family can be divided into different branches, including the CXC chemokines (-quimiokines) and the CC chemokines (ß-chemokines), where the first two conserved cysteines are separated by an intermediate residue or they are adjacent residues, respectively (Baggiolini, M. and Dahinden, CA, Immunology To-day, 15: 127-133 (1994)). C-X-C chemokines include a series of potent chemoattractants and neutrophil activators, such as interleukin 8 (IL-8), PF4 and neutrophil activating peptide 2 ("NAP-2"). CC chemokines include "RAN-TES" (Regulated on Activation, Normal T Expressed and Secreted), the inflammatory proteins of macrophages la and lß ("MlP-la" and "MlP-lß"), eotaxin and chemotactic proteins of human monocytes 1-3 ("MCP-1", "MCP-2", "MCP-3"), which have been characterized as chemoattractants and activators of monocytes or lymphocytes. Chemokines, such as RANTES and MlP-la, have been implicated in acute and chronic human inflammatory diseases, including respiratory diseases, such as asthma and allergic disorders. The chemokine receptors are members of a super family of G-protein coupled receptors ("GPCRs"), which share structural features that reflect a common mechanism of signal transduction action (Gerard, C. and Gerard, NP, Annu. Rev. Immunol., 12: 775-808 (1994); Gerard, C. and Gerard, N.P., Curr. Opin. Immunol .. 6: 140-145 (1994)). Conserved features include seven hydrophobic domains that extend across the plasma membrane, which are connected by extracellular and intracellular hydrophilic loops. Most of the primary sequence homology occurs in the transmembrane hydrophobic regions, with the most diverse hydrophilic regions. The receptors for the CC chemokines include: CCR1, which can be linked, for example, to MlP-la, RANTES, MCP-2, MCP-3, MCP-4, CKbetad, CKbeta8-l, leukotactin-1, HCC-1 and MPIF-1; CCR2, which can be linked, for example, to MCP-1, MCP-2, MCP-3 and MCP-4; CCR3, which can be linked, for example, to eotaxin, eo-taxin-2, RA? TES, MCP-2, MCP-3 and MCP-4; CCR4, which can be linked, for example, to TARC, RANTES, MlP-la and MCP-1; CCR5, which can be linked, for example, to MlP-la, RANTES and MlP-lβ; CCR6, which can be linked, for example, to LARC / MIP-3a / exodus; CCR7, which can be linked, for example, to ELC / MIP-3β, and CCR8, which can be linked, for example, to 1-309 (Baggiolini, M., Nature 392: 565-568 (1998); Luster, AD, New England Journal of Medicine, 338 (1): 436-445 (1998), Tsou et al., J. Exp. Med., 188: 603-608 (1998), Nardelli et al., J. Immunol. (1): 435-444 (1999); Youn et al., Blood 91 (9): 3118-3126 (1998); Youn et al., J. Immunol. 159 (11): 5201-5201 (1997)) . CCR1, as well as cellular processes and responses mediated by CCR1, are implicated in the rejection of transplanted grafts. As described here, allograft survival studies were performed using a murine cardiac transplant model. Mice lacking the functional chemokine receptor CCR1 as a result of a white alteration of the CCR1 gene (CCRl KO; Gerard, C. et al., J ".

Clin. Invest. 100: 2022-2027 (1997)) did not reject the transplanted allografts, which did not correspond in the MHC (major histocompatibility complex) class I and MHC class II, as rapidly as the control mice, which have a CCR1 gene and they are, for the rest, genetically identical to CCRl KO mice (Example 1, Table 1, groups 1 and 2). As described herein, administration of low dose immunosuppressive therapy (cyclosporin A) to CCR1 + / + control mice resulted in only a 2-3 day increase in graft viability compared to animals CCR1 + / + not treated (Example 1, Table 1, group 3). Surprisingly, administration of the same low dose of CsA with inhibition of CCR1 function resulted in permanent grafting (> 100 days) in the CCR1 KO mice receiving CsA for a maximum period of only 21 days (Table 1, group 4) . In other studies, allografts that corresponded only in the MHC class II to CCR1 KO mice and CCR1 + / + control were transplanted. As expected, partial tissue matching led to prolonged graft survival in control CCRl + / + mice. However, all control CCRl + / + mice still rejected the graft by day thirty-five (Example 1, Table 1, group 5). Partial correspondence of MHC antigens, such as low dose immunosuppression, with inhibition of CCR1 function resulted in permanent grafting (> 100 days) (Example 1, Table 1, group 6). Histological examination of permanently grafted hearts excised from group 4 and group 6 mice (see Table 1) at 100 days post-transplant revealed only minimal infiltration of mononuclear cells and there was no evidence of accelerated arteriosclerosis of the transplant. Survival of uncorrected allografts in Class I and Class II can be prolonged by administration of anti-CD4 monoclonal antibody (mAb) (Mottram et al., Transplantation 59: 559-565 (1995)). However, the long-term survival of these grafts is complicated by the development of chronic rejection with widespread arteriosclerosis in the graft vasculature (Hancock et al., Nature Medicine 4: 1392-1396 (1998)). As described herein, allografts without correspondence in Class I and Class II survived for 60 days in CCRl KO and control CCRl + / + mice that received anti-CD4 mAb therapy. The morphological examination of grafts extracted from CCR1 + / + control receptors at sixty days revealed severe arteriosclerosis. In contrast, grafts extracted from CCR1 KO receptors showed no evidence of arteriosclerosis (Example 2, Table 2). Therefore, the alteration of the CCR1 function can provide the double benefit of inhibition of both acute and chronic allograft rejection. Accordingly, a first aspect of the invention provides a method of inhibiting the rejection (e.g., acute and / or chronic rejection) of a graft, comprising administering to an graft recipient an effective amount of an antagonist of the CCR1 function. CCR1 Antagonists As used herein, the term "CCR1 function antagonist" refers to an agent (e.g., a molecule, a compound) that can inhibit a (i.e., one or more) function of CCR1. For example, an antagonist of the CCR1 function can inhibit the binding of one or more ligands, for example, MlP-la, RANTES, MCP-2, MCP-3, MCP-4, CKbetad, CKbe-ta8-l, leukotactin- 1, HCC-1, MPIF-1) to CCR1 and / or inhibit signal transduction mediated through CCR1 (eg, exchange of GDP / GTP for G proteins associated with CCR1, intracellular calcium flux). Consequently, processes mediated by CCR1 and cellular responses (eg, pro-life, migration, chemotactic responses, secretion or degranulation) can be inhibited with an antagonist of CCR1 function. Preferably, the antagonist of the CCR1 function is a compound which is, for example, a small organic molecule, a natural product, a protein (for example, antibody, chemokine, cytokine), a peptide or a peptidomimetic. Various molecules are known in the art which can antagonize one or more functions of the chemokine receptors (eg, CCR1), including the small organic molecules described, for example, in International Patent Application WO 97/24325, of Takeda Chemical Industries, Ltd.; WO 98/38167, from Pfizer, Inc .; WO 97/44329, Teijin Limited; WO 98/04554, from Banyu Pharmaceutical Co., Ltd .; WO 98/27815, WO 98/25604, WO 98/25605, WO 98/25617 and WO 98/31364, from Merck & Co., Inc .; WO 98/02151 and WO 99/37617, LeukoSite, Inc .; WO 99/37651 and WO 99/37619, LeukoSite, Inc. et al .; U.S. Provisional Application Number 60 / 021,716, filed July 12, 1996; US Patent Applications Nos. 09 / 146,827 and 09 / 148,236, filed September 4, 1998; Hesselgesser et al., J ". Biol. Chem. 273 (25): 15687-15692 (1998), and Howard et al., J. Medicinal Chem. 41 (13): 2184-2193 (1998); proteins, such as antibodies (eg, polyclonal, monoclonal, qui-maric, humanized sera) and antigen-binding fragments thereof (eg, Fab, Fab ', F (ab') 2, Fv), for example those described by Su et al., J. Leukocyte Biol. 60: 658-656 (nineteen ninety six); mutants and chemokine analogs, for example those described in US Pat. No. 5,739,103, issued to Ro-llins et al., WO 96/38559, Dana Farber Cancer Institute, and WO 98/06751, to Research Corporation Technologies, Inc .; peptides, for example those described in WO 98/09642, of the United States of America. The complete teachings of each of the aforementioned patent applications and references are incorporated herein by reference. The antagonists of the CCR1 function can be identified, for example, by studying libraries or collections of molecules, such as the Chemical Repository of the National Cancer Institute (USA), as described herein or using other suitable methods. Another source of CCR1 antagonists are combinatorial libraries that may contain many structurally distinct molecular species. The combinatorial libraries can be used to identify a line of compounds or to optimize a previously identified line. Said libraries can be manufactured by well-known methods of combinatorial chemistry and studied by suitable methods, such as methods described herein. The term "natural product," as used herein, refers to a compound that can be found in nature, for example natural metabolites of marine organisms (eg, tunicates, algae) and plants and which possess biological activity, for example They can antagonize the CCR1 function, for example, lactacystin, paclitaxel and cyclosporin A are natural products that can be used as antiproliferative or immunosuppressive agents.Natural products can be isolated and identified by suitable means. homogenizing (eg grinding) a suitable biological source (eg, vegetation) in a suitable buffer and clarifying it by centrifugation, thereby producing an extract.The resulting extract can be studied for its ability to antagonize CCR1 function, for example, by the assays described here, extracts containing an activity that antagonizes CCRl function can be r still processed to isolate the CCR1 antagonist by suitable methods, such as fractionation (e.g., column chromatography (e.g., ion exchange, inverted phase, affinity), phase-sharing, fractional crystallization), and studying biological activity ( for example, antagonism of CCR1 activity). Once isolated, the structure of a natural product can be determined (for example, by nuclear magnetic resonance (NMR)) and those skilled in the art can devise a synthetic scheme to synthesize the natural product. Therefore, a natural (for example, substantially purified) product can be isolated from nature or it can be totally or partially synthetic. A natural product can be modified (for example, derivatized) to optimize its therapeutic potential. Thus, the term "natural product", as used herein, includes those compounds that are produced using standard medicinal chemistry techniques to optimize the therapeutic potential of a compound that can be isolated from nature. The term "peptide", as used herein, refers to a compound consisting of about two to about ninety amino acid residues, wherein the amino group of one amino acid is attached to the carboxyl group of another amino acid by a peptide bond. A peptide can, for example, be derived or separated from a native protein by enzymatic or chemical cleavage, or it can be prepared using conventional peptide synthesis techniques (e.g., solid-phase synthesis) or molecular biology techniques (see Sam-brook). , J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). A "peptide" can contain any suitable L- and / or D-amino acid, for example common a-amino acids (e.g., alanine, glycine, valine), non-aa amino acids (eg β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statin) and non-usual amino acids (eg, citrulline, homocitrulline, homoserine, norleucine, norvaline , ornithine). The amino, carboxyl and / or other functional groups on a peptide can be free (for example, unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups and means for adding or removing protecting groups are known in the art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991. The functional groups of a peptide can also be derivatized (eg, alkylated) using methods known in the art. Peptides can be synthesized and assembled in bookstores consisting of a few to many discrete species. Such libraries can be prepared using well-known methods of combinatorial chemistry and can be studied as described herein or using any suitable methods to determine whether the library contains peptides that can antagonize the CCR1 function. Said peptide antagonists can then be isolated by suitable methods. The term "peptidomimetic". as used herein, it refers to molecules that are not polypeptides, but that mimic aspects of their structures. For example, polysaccharides having the same functional groups as the peptides that can antagonize CCR1 can be prepared. Peptidomimetics can be designed, for example, by establishing the three-dimensional structure of a peptide agent in the environment in which it is attached or will bind to CCR1. The peptidomimetic consists of at least two components, the rest or binding moieties and the backbone or support structure. The binding moieties are the atoms or chemical groups that react or form a complex (for example, through hydrophobic or ionic interactions) with CCR1, for example with the amino acid (s) that are (are) in the site of ligand binding or proximate (s) to it. For example, the binding moieties in a peptidomimetic can be the same as those of a CCR1 peptide antagonist. The binding moieties can be an atom or chemical group that reacts with the receptor in a manner equal to or similar to that of the binding moiety of a CCR1 peptide antagonist. Examples of suitable linking moieties for use in the design of a peptidomimetic for a basic amino acid in a peptide are nitrogen-containing groups, such as amines, ammoniums, guanidines and amides or phosphoniums. Examples of suitable linking moieties for use in the design of a peptidomimetic for an acidic amino acid, for example, carboxyl, lower alkyl ester and carboxylic acid, sulfonic acid, a lower alkyl ester and sulfonic acid or a phosphorous acid or ester of it. The support structure is the chemical entity that, when attached to the rest or binding residues, provides the three-dimensional configuration of the peptidomimetic. The support structure can be organic or inorganic. Examples of organic support structures include polysaccharides, polymers or oligomers of organic synthetic polymers (such as polyvinyl alcohol or polylactide). It is preferred that the support structure possess substantially the same size and dimensions as the peptide backbone or support structure. This can be determined by calculating or measuring the size of the peptide and peptidomimetic atoms and bonds. In one embodiment, the nitrogen of the peptide bond can be replaced with oxygen or sulfur, thus forming a polyether backbone. In another embodiment, the carbonyl can be substituted with a sulfonyl group or a sulfinyl group, thereby forming a polyamide (e.g., a polysulfonamide). Inverted amides of the peptide can be made (for example, by substituting one -? HC0 - group with one or more -COHN groups). In yet another realization, the peptide skeleton can be substituted with a polysilane skeleton. These compounds can be manufactured by known methods. For example, a polyester peptidomimetic can be prepared by replacing the corresponding a-amino group on the amino acids with a hydroxyl group, thereby preparing a hydroxy acid and sequentially esterifying the hydroxy acids, optionally blocking the basic and acid side chains to minimize side reactions. The determination of an appropriate chemical synthetic route can be easily identified by determining the chemical structure. Peptidomimetics can be synthesized and assembled in libraries consisting of a few to many discrete molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry and can be studied as described herein to determine whether the library contains one or more peptidomimetics that antagonize the CCR1 function. Said peptidomimetic antagonists can then be isolated by suitable methods. In one embodiment, the CCR1 antagonist is an anti-body or antigen-binding fragment thereof that has specificity for CCR1. The antibody can be polyclonal or monoclonal and the term "antibody" is intended to include both polyclonal and monoclonal antibodies. The terms "polyclonal" and "monoclonal" refer to the degree of homogeneity of an antibody preparation and are not intended to be limited to particular methods of production. The term "antibody", as it is? used herein, also includes functional fragments of antibodies, including fragments of chimeric, humanized, primatized, coated or single chain antibodies. Functional fragments include antigen-binding fragments that bind to CCR1. For example, fragments of antibodies capable of binding to CCR1 or portions thereof, including, without limitation, the Fv, Fab, Fab 'and F (ab') 2 fragments. they are covered by this invention. Said fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, cleavage with papain or pepsin can generate Fab or F (ab ') 2 fragments. respectively. Other proteases with the substrate specificity required to generate Fab or F (ab ') 2 fragments can also be employed. Antibodies can also be produced in a variety of truncated forms using antibody genes where one or more stop codons have been introduced. upstream of the natural stop site. For example, a chimeric gene encoding a heavy chain portion of F (ab ') 2 can be designed to include DNA sequences encoding the CHi domain and the hinge region of the heavy chain. The present invention and the term "antibody" also include single chain antibodies and chimeric, humanized or primatized antibodies (grafted with "CDRs" - complementarity determining regions) or coated, as well as chimeric single chain antibodies, grafted with CDRs or coated which contain portions derived from different species and the like. The various portions of these antibodies can be chemically linked together by conventional techniques or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, for example, Ca-billy et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0125.023 Bl; Boss et al., US Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, M.S. et al., WO 86/01533; Neuberger, M.S. et al., European Patent No. 0,194,276 Bl; Winter, US Patent ? 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al., European Patent No. 0,451,216 Bl, and Padlan, E.A. et al., EP 0 519,596 Al. See also New an, R. et al., Bio-Technology 10: 1455-1460 (1992) for pri- matized antibodies, and Ladner et al., US Pat. . No. 4,946,778, and Bird, R.E. et al., Science 242: 423-426 (1988) for single chain antibodies. Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques. Nucleic acid sequences (eg, cDNAs) encoding humanized variable regions can also be constructed using PCR mutagenesis methods to alter the DNA sequences encoding a human or humanized chain, such as a DNA template of a previously humanized variable region (see, for example, Kamman, M. et al., Nucí Acids Res. 17: 5404 (1989); Sato, K. et al., Cancer Research 53: 851-856 (1993); Daug-herty, BL et al., Nucleic Acids Res. 19 (9): 2471-2476 (1991), and Lewis, AP and JS Crowe, Gene 101: 297-302 (1991)). Using these or other suitable methods, variants can also be easily produced. In one embodiment, cloned variable regions can be mutated and coding sequences of variants with the desired specificity can be selected (eg, from a phage library, see, for example, Krebber et al. 5,514,548; Hoogenboom et al., WO 93/06213, published April 1, 1993). Antibodies specific for mammalian (e.g., human) CCR1 can be raised against an appropriate immunogen, such as isolated and / or recombinant human CCR1 or portions thereof (including synthetic molecules, such as synthetic peptides). Antibodies can also be produced by immunizing a suitable host (e.g., mouse) with cells expressing CCR1, such as activated T cells. (see, for example, US Pat. No. 5,440,020, the teachings of which are hereby incorporated by reference in their entirety). In addition, cells expressing recombinant CCR1, such as transfected cells, can be used as immunogens or in a selection for antibodies that bind to the receptor. (see, for example, Chuntharapai et al., J. Immunol., 152: 1783-1789 (1994), Chuntharapai et al., US Patent No. 5,440,021). The preparation of immunizing antigen and the production of polyclonal and monoclonal antibodies can be carried out using any suitable technique. A variety of methods have been described (see, for example, Kohier et al., Nature 256: 495-497 (1975), and Eur. J. Immunol., 6: 511-519 (1976); Milstein et al., Nature. 266: 550-552 (1977), Ko-prowski et al., US Patent No. 4,172,124, Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Cu_rrertt Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer 94), Ausubel, FM et al., Eds. (John Wiley &Sons:? Ew York, NY), Chapter 11 ( 1991)). In general, a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2 / 0 or P3X63Ag8.653) with antibody producing cells. The antibody-producing cells, preferably from the spleen or lymph nodes, can be obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions and cloned by limiting dilution. Cells that produce antibodies with the desired binding properties can be selected by means of a suitable assay (e.g., ELISA). Other suitable methods of producing or isolating antibodies with the required specificity can be employed, including, for example, human or artificial antibodies, including, for example, methods that select recombinant antibody from a library (e.g., an exhibit library). of phages), or based on the immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see, for example, Jakobovits et al., Proc. Nati. Acad. Sci. USA 90: 2551-2555 (1993), Jakobovits et al., Nature 362: 255-258 (1993), Lonberg et al., U.S. Patent No. 5,545,806, Surani et al., US Pat. No. 5,545,807; Lonberg et al., W097 / 13852). In one embodiment, the antibody or antigen-binding fragment thereof has specificity for a mammalian chemokine CC receptor (CCR1) 1, such as human CCR1. In a preferred embodiment, the antibody or antigen-binding fragment can inhibit the binding of a ligand (i.e., one or more ligands) to CCR1 and / or one or more functions mediated by CCR1 in response to ligand binding. Preferred antagonist antibodies of the CCR1 function are described in our co-pending U.S. Patent Application entitled "Anti-CCRl Antibodies and Methods of Use therefor", by Shixin Qin, Walter Newman and Nasim Kassam, Attorney Docket No. LKS97-13 , in the EE.UTJ. Serial No. 09 / 239,938, filed on January 29, 1999 and in International Application No. PCT / US99 / 04527, the teachings of each of these applications being incorporated herein by reference. Assessment of antagonist activity The ability of an agent (eg, proteins, peptides, natural products, small organic molecules, peptidomimetics) to antagonize CCR1 function can be determined using a suitable selective study (eg, assay of high performance) . For example, an agent can be studied in an extracellular acidification assay, a calcium flux assay, a ligand binding assay or a chemotaxis assay (see, for example, Hesselgesser et al., J. Biol. Chem. 273 (25): 15687-15692 (1998) and WO 98/02151). In a particular assay, membranes can be prepared from cells expressing CCR1, such as THP-1 cells (American Type Culture Collection, Manassas, VA; Accession No. TIB202). The cells can be harvested by centrifugation, washed twice with PBS (phosphate-buffered saline) and the resulting pellets frozen at -70 to -85 ° C.

The frozen pellet can be thawed in ice-cold lysis buffer consisting of 5 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.5, 2 mM EDTA (ethylenediamine-tetraacetic acid), 5 μg / ml of each of aprotinin, leupeptin and chemostatin (protease inhibitors) and 100 μg / ml of PMSF (phenylmethanesulfonyl fluoride - also a protease inhibitor), at a concentration of 1 to 5 x 107 cells / ml, to achieve lysis cell phone. The resulting suspension can be mixed thoroughly to resuspend the entire frozen cell pellet. Nucleic acid and cell debris can be removed by centrifugation 400 x g for 10 minutes at 4 ° C. The resulting supernatant can be transferred to a fresh tube and the membrane fragments can be collected by centrifugation at 25,000 x g for 30 minutes at 4 ° C. The resulting supernatant can be aspirated and the pellet can be resuspended in freezing buffer, consisting of 10 mM HEPES, pH 7.5, 300 mM sucrose, 1 μg / ml of each of aprotinin, leupeptin and chemostatin and 10 μg / ml. ml of PMSF (approximately 0.1 ml per 108 cells). All aggregates can be redissolved using a mini-homogenizer and the total protein concentration can be determined by suitable methods (eg, Brad-Ford assay, Lowery assay). The membrane solution can be divided into aliquots and frozen at -70 to -85 ° C until needed. The membrane preparation described above can be used in a suitable binding assay. For example, the membrane protein (2 to 20 μg of total membrane protein) can be incubated with 0.1 to 0.2 nM of RANTES or MlP-la labeled with 1 5I, with or without unlabeled competitor (RANTES or MlP-la) or various concentrations of the compounds to be studied.

RANTES labeled with 125I and MlP-labeled with 12? I can be prepared by suitable methods or can be purchased from commercial vendors (eg, DuPont-? EN (Boston, MA)). The binding reactions can be carried out in 60 to 100 μl of a binding buffer consisting of 10 mM HEPES, pH 7.2, 1 mM CaCl 2, 5 mM MgCl 2 and 0.5% BSA (bovine serum albumin), during 60 min at room temperature. The binding reactions can be terminated by collecting the membranes by rapid filtration through glass fiber filters (eg, GF / BO GF / C, Packard), which can be pre-packed in 0.3% polyethylenimine. . The filters can be washed with approximately 600 μl of binding buffer, containing 0.5 M NaCl, and dried and the bound radioactivity can be determined by scintillation counting. The CCR1 antagonist activity of the test agents (e.g., compounds) may be given as the concentration of inhibitor required for 50% inhibition (IC50 values) of the specific binding in receptor binding assays (e.g. using 125 I-RANTES or 125 I-MIP-la as ligand and membranes of THP-1 cells). The specific binding is preferably defined as the total binding (e.g., total cpm on the filters) minus non-specific binding. Non-specific binding is defined as the amount of cpm still detected in the presence of an excess of unlabeled competitor (eg, RANTES or MlP-la). If desired, membranes prepared from cells expressing recombinant CCRl can be used in the described assay. The ability of the compounds to antagonize CCR1 function in a leukocyte chemotaxis assay using suitable cells can also be determined. Suitable cells include, for example, cell lines, recombinant cells or isolated cells expressing CCR1 and undergoing chemotaxis induced by CCR1 ligands (eg, MlP-la, RANTES, MCP-2, MCP-3)., MCP-4, HCC-1 or MPIF-1). In one example, recombinant Ll2 cells expressing CCR1 (see Campbell et al., J "Cell Biol. 134: 255-266 (1996)), peripheral blood mononuclear cells or differentiated HL60 cells can be used. butyric acid in a modification of a transendothelial migration assay (Carr, MW et al., TA, Proc. Nati, Acad. Sci., USA (91): 3652 (1994)). Mononuclear cells can be isolated from peripheral blood of whole blood by suitable methods, for example density gradient centrifugation and positive or preferably negative selection with specific antibodies The endothelial cells used in this assay are preferably the ECV 304 endothelial cell line, obtained from the European Collection of Animal Cell Cultures ( Porton Down, Salisbury, UK) Endothelial cells can be cultured in 6.5 mm diameter Transpocillo culture inserts (Costar Corp., Cambridge, MA) with a pore size of 3.0 μm. The culture medium for ECV 304 cells can consist of M199 + 10% FCS, L-glutamine and antibiotics. The test medium may consist of equal parts of RPMI 1640 and M199 with 0.5% BSA. Two hours prior to the assay, 2x005 ECV 304 cells can be plated on each insert of the 24-well Transpocillo chemotaxis plate and incubated at 37 ° C. Chemotactic factors can be added, such as RANTES or MlP-la (Peprotech) (diluted in assay medium) to the 24-well tissue culture plates in a final volume of 600 μl. Endothelial cell-coated Transports can be inserted into each well and 10 6 cells of the leukocyte type being studied are added to the upper chamber in a final volume of 100 μl of assay medium. The plate can then be incubated at 37 ° C in 5% C02 / 95% air for 1-2 hours. Cells that migrate to the bottom chamber during incubation can be counted, for example, using flow cytometry. To count the cells by flow cytometry, 500 μl of the cell suspension of the lower chamber can be placed in a tube and relative counts can be obtained for a set period of time, for example 30 seconds. This method of counting is highly reproducible and allows to open the gate on leukocytes and the exclusion of debris or other cell types of the analysis. Alternatively, the cells can be counted with a microscope. Assays can be performed to evaluate inhibitors of chemotaxis in the same way as the control experiment described above, except for the fact that antagonist solutions can be added, in assay medium containing up to 1% DMSO cosolvent, both to the upper chamber as to the lower one before the addition of the cells. The potency of the antagonist can be determined by comparing the number of cells migrating to the bottom chamber in the antagonist containing wells with the number of cells migrating to the bottom chamber in the control wells. The control wells may contain equivalent amounts of DMSO, but not antagonist. The activity of an antagonist of the CCR1 function can also be assessed by monitoring the cellular responses induced by the active receptor, using appropriate cells expressing the receptor. For example, exo-kyphosis can be monitored (eg, degranulation of the cells that results in the release of one or more enzymes or other components of the granules, such as esterases (eg, serine esterases), perforin and / or granzymes), the release of inflammatory mediators (such as the release of bioactive lipids such as leukotrienes (e.g., leukotriene C4)) and respiratory burst by methods known in the art or by other suitable methods (see, for example. , Taub, DD et al., J. Immunol., 155: 3877-3888 (1995), regarding serine-esterase-derived granule release assays, Loetscher et al., J. Immuno 1. 156: 322-327 (1996), in terms of assays for the release of enzymes and granzymes, Rot, A. et al., "Exp. Med. 176: 1489-1495 (1992), regarding the respiratory explosion: Bischoff, SC et al. ., Eur. J. Immunol., 23: 761-767 (1993), and Bag-gliolini, M. and CA Dahinden, Immunology Today 15: 127-133 ( 1994).) In one embodiment, a CCR1 antagonist is identified by monitoring the release of an enzyme after degranulation or exocytosis by a cell capable of this function. Cells expressing CCR1 can be maintained in a suitable medium under suitable conditions and de-granulation can be induced. The cells are placed in contact with an agent to be studied and the release of enzyme can be assessed. The release of an enzyme to the medium can be detected or measured using a suitable assay, such as in an immunological assay or a biochemical assay for enzymatic activity.

The medium can be studied directly by introducing the test components (eg, substrate, cofactors, antibody) into the medium (eg, before, simultaneously or after combining the cells and the agent). The assay can also be carried out in medium that has been separated from the cells or additionally processed (for example, fractionated) before the assay. For example, convenient assays are available for enzymes, such as serine esterases (see, for example, Taub, D.D. et al., J ". Immunol., 155: 3877-3888 (1995) in regard to the release of serine-esterases derived from granules.) In another embodiment, cells expressing CCRl are combined with a CCR1 ligand or promoter. the CCR1 function, an agent to be studied before, after or at the same time is added and the degranulation is evaluated.The inhibition of the degranulation induced by ligand or promoter is indicative that the agent is an inhibitor of the CCR1 function of In a preferred embodiment, the antagonist of the CCR1 function does not significantly inhibit the function of other chemokine receptors (eg, CCR2, CXCR1, CCR3). Such CCR1-specific antagonists can be identified by suitable methods, such as by a appropriate modification of the methods described here For example, cells that do not express CCR1 (CCR1") can be created or identified, but that they express one or more other chemokine receptors (by axis) mplo, CCR2, CXCR1, CCR3) using suitable methods (e.g., transfection, antibody staining, Western blot, RNase protection). Said cells or cell fractions (e.g., membranes) obtained from said cells may be used in a suitable binding assay. For example, when a cell which is CCR1"and CCR3 + is chosen, the CCR1 antagonist can be studied for the ability to inhibit the binding of a suitable CCR3 ligand (e.g., RANTES, MCP-3) to the cell or Cellular fraction, as described herein In another preferred embodiment, the CCR1-binding agent is an agent that binds to CCRl.These antagonists that bind to CCRl can be identified by suitable methods, for example in binding assays. employing a labeled antagonist (eg, enzymatically labeled (eg, with alkaline phosphatase or horseradish peroxidase), biotinylated or radiolabelled (eg with 3H, 14C or 125D) In another preferred embodiment, the antagonist of The CCR1 function is an agent that can inhibit the binding of a (ie, one or more) ligand from CCR1 to CCR1 (eg, human CCR1.) In a particularly preferred embodiment, the CCR1 function antagonist is an agent that can a go to CCR1 and thus inhibit the binding of a (ie, one or more) ligand from CCR1 to CCR1 (eg, human CCR1). Methods of therapy The term "graft", as used herein, refers to organs and / or tissues that can be obtained from a first mammal or donor and transplanted to a second mammal, preferably a human. The term "graft" embraces, for example, skin, eye or portions of the eye (e.g., cornea, retina, lens), muscle, bone marrow or cellular components of the bone marrow (e.g., stem cells). ), progenitor cells), heart, lung, heart-lung (eg, heart and a single lung, heart and both lungs), liver, kidney, pancreas, parathyroid, intestine (eg, colon, small intestine, duodenum), neuronal tissue, bone and vasculature (eg, artery, vein). A graft of a suitable mammal (eg, human, pig, baboon, chimpanzee) can be obtained or, under certain circumstances, an engraftment can be obtained by culturing cells, for example embryonic, fetal cells, skin cells, cells blood and bone marrow cells, which were obtained from a suitable mammal. The graft of a genetically modified animal can be obtained or can be modified (eg, genetically, chemically or physically) by suitable means. A graft is preferably obtained from a human. An "allograft", as the term is used herein, refers to a graft that contains antigens that are allelic variants of the corresponding antigens that are found in the recipient. For example, a human graft containing an MHC class II antigen encoded by the HLA-DRB1 * 0401 allele is an allograft if it is transplanted into a human receptor whose genome does not contain the HLA-DRB1 * 0401 allele. In an embodiment, the method of inhibition (reduction or prevention) of graft rejection consists in administering an effective amount of a (i.e., one or more) antagonist of the CCR1 function to a recipient of a graft. In another embodiment, the method of inhibiting graft rejection consists in administering an effective amount of an antagonist of CCR1 function to an allograft receptor. In a preferred embodiment, the method consists of administering an effective amount of an antagonist of the CCR1 function to a recipient of a cardiac allograft. In another embodiment, the CCR1 function antagonist is selected from the group consisting of small organic molecules, natural products, peptides, peptidomimetics and proteins, wherein said proteins are not chemokines or mutants or analogs thereof. In a preferred embodiment, the invention provides a method of inhibiting (reducing or preventing) graft rejection, comprising administering to a graft receptor an effective amount of an antagonist of the CCR1 function and an effective amount of a (FIG. that is, one or more) additional therapeutic agent, preferably an immunosuppressive agent. Advantageously, the effects of inhibiting the rejection of CCR1 antagonists and immunosuppressive agents can be additive or synergistic and can lead to permanent grafting. Yet another benefit of the coadministration of a CCR1 antagonist and an immunosuppressive agent is that the dose of the immunosuppressive agent required to inhibit graft rejection can be reduced to subtherapeutic levels (eg, a dose that does not inhibit graft rejection when administered as only therapeutic agent). The ability to reduce the dose of the immunosuppressant agent can greatly benefit the graft recipient, since many immunosuppressive agents have serious and well-known side effects, including, for example, a higher incidence of infection, a higher incidence of certain malignancies, diabetes mellitus, neurotoxicity, nephrotoxicity, hyperlipidemia, hypertension, hirsutism, gingival hyperplasia, alteration in wound healing, lymphopenia, jaundice, anemia, alopecia and thrombocytopenia (Spencer, CM et al., Drugs 54 (6): 925- 975 (1997); Physicians Desk Reference, 53rd Edition, Medical Economics Co., pp. 2081-2082 (1999)). The term "immunosuppressive agent", as used herein, refers to compounds that can inhibit an immune response. The immunosuppressive agent used in the invention may be a new compound or may be selected from compounds that are known in the art, for example calcineurin inhibitors (eg, cyclosporin A, FK-506), signal transduction inhibitors. IL-2 (e.g., rapamycin), glucocorticoids (e.g., prednisone, dexamethasone, methylprednisolone), inhibitors of nucleic acid synthesis (e.g., azathioprine, mercaptopurine, mycophenolic acid) and antibodies to lymphocytes and antigen-binding fragments of them (for example, OKT3, anti-IL2 receptor). The new immunosuppressive agents can be identified by those skilled in the art by suitable methods, for example by selecting compounds for their ability to inhibit antigen-dependent T cell activation. The immunosuppressant agent used for therapeutics (eg, co-administration with an antagonist of CCR1 function) is preferably a calcineurin inhibitor. More preferably, the immunosuppressive agent used for therapeutics is cyclosporin A. When the graft is bone marrow, cells (e.g. leukocytes) derived from the graft can produce an immune response directed to the organs and tissues of the recipient. This condition is referred to in the art as graft versus host disease ("GVHD"). Administration of an antagonist of the CCR1 function with or without an additional therapeutic agent (e.g., immunosuppressive agent, hematopoietic growth factor) can inhibit GVHD. Accordingly, in another embodiment, the invention provides a method of inhibition (reduction or prevention) of GVHD in a bone marrow graft recipient, comprising administering an effective amount of an antagonist of CCR1 function. In a further embodiment, the method of inhibiting GVHD consists in the administration of an antagonist of the CCR1 function and one or more additional therapeutic agents, for example an immunosuppressive agent. In another embodiment, the inhibition method of GVHD consists in the administration of an antagonist of the CCR1 function, which is selected from the group consisting of small organic molecules, natural products, peptides, peptidomimetics and proteins, where said proteins are not chemokines or mutants or analogs thereof. Another aspect of the invention relates to the inhibition of ischemia / reperfusion injury using CCR1 antagonists. Ischemia / reperfusion injury refers to cell death due to necrosis that occurs when the flow of blood to an organ or tissue is restricted or stopped (ischemia), resulting in oxygen deprivation (hypoxia). The lesion maintained by an organ or tissue under ischemic conditions is apparent after restoration of blood flow (reperfusion). The ischemia / reperfusion injury can be the result of a pathological condition in which ischemia can occur, for example in myocardial infarction, in arteriosclerosis, in stroke, in transient ischemic attacks and the like. Ischemia / reperfusion injury can occur as a result of trauma or medical procedures that stop, restrain or redirect (for example, shunt) blood flow. Examples of such trauma include, for example, surface freezing, burns and injuries caused by pinching which restrict the flow of blood to the limbs or limbs. Medical procedures that can result in ischaemia / reperfusion injury include, for example, placement of a tourniquet, angioplasty (eg, balloon angioplasty) and surgery (eg., organ transplant). In the context of organ transplantation, the ischemia / reperfusion injury that occurs during the transplantation of all grafts, including isogenic grafts (for example, when the donor and recipient are identical twins), may be a contributing factor to the rejection of grafts (for example, acute rejection, chronic rejection). We describe here a murine model of renal cold ischemia / reperfusion injury, which mimics the storage conditions of organs (eg, kidneys) that have been excised from a donor in preparation for transplantation (see Example 3). Studies using this model revealed that inhibition of CCR1 function can significantly inhibit ischaemia / reperfusion injury. Specifically, renal ischemia / reperfusion had no measurable effect on the renal function of CCR1 KO mice, with 100% survival of the mice during the 72-hour follow-up period. In contrast, the CCR1 + / + control mice experienced a considerable alteration in renal function and an 80% mortality at 72 hours (Table 3). Histological examination of kidneys excised for CCR1 KO mice and CCR1 + / + control 48 hours after ischemia / reperfusion revealed that inhibition of CCR1 function can inhibit tubular necrosis and neutrophil infiltration in the kidney. Thus, by inhibiting CCR1 function, the ischaemia / reperfusion injury can be inhibited (reduced or prevented). Consequently, the alteration of the CCR1 function can have significant beneficial actions in the prevention of lesions of the transplanted grafts (for example, kidneys). For example, inhibition of CCR1 function can inhibit (reduce or prevent) the initial lesion post-graft transplantation and, thereby, result in a reduction of acute and chronic allograft rejection. Furthermore, inhibition of CCR1 function may increase the pool of donor organs, since many organs (eg, the kidneys) are not transplanted because they are thought to have a high risk of ischemia / reperfusion injury as a result of a prolonged storage. Accordingly, another embodiment of the invention is a method for inhibiting ischemia / reperfusion injury, comprising administering to a subject (e.g., a human) in need thereof an effective amount of an antagonist of CCR1 function. In certain embodiments, the ischemia / reperfusion injury may be a consequence of a medical procedure or trauma or the result of a pathological condition. In a particular embodiment, the invention provides a method for inhibiting ischemia / reperfusion injury that may occur during graft transplantation. In a preferred embodiment, the invention provides a method for inhibiting the ischemia / reperfusion injury that may occur during the transplantation of a kidney. In another embodiment, the method of ischemia / reperfusion injury inhibition consists in the administration of an antagonist of the CCR1 function, which is selected from the group consisting of small organic molecules, natural products, peptides, peptidomimetics and proteins, where said proteins are not chemokines or mutants or analogs thereof. In another embodiment, the method of inhibiting ischemia / reperfusion injury consists in administering to a subject in need thereof an effective amount of a CCR1-function antagonist and an effective amount of one or more additional therapeutic agents that can promote the flow of blood and / or inhibit leukocyte infiltration. For example, the additional agent can be selected from the group consisting of a fibrinolytic agent (e.g., Reta-vase), a thrombolytic agent, such as a plasminogen activator (e.g., tissue plasminogen activator, urokinase, streptokinase). , recombinant tissue plasminogen activator), an anticoagulant (eg, a coumarin anticoagulant (eg, warfarin, etiline dicumarol), heparin, hirulog, hirudin, aspirin), a vasodilator (eg, nitroglycerin, amotriphene, erythritol, prenylamine), an agent that stimulates or inhibit the production of nitric oxide (for example, a stimulator or inhibitor of nitric oxide synthase, for example the compounds described in U.S. Patent Nos. 5,811,437, issued to Singh et al., 5,854,234 , issued to Hansen et al., and 5,854,251, issued to Hallinan et al.), an immunosuppressive agent and an inhibitor of cell adhesion. Inhibitors of cell adhesion suitable for co-administration with antagonists of CCR1 function include, for example, pro-teins, such as cytokines, antibodies that bind to cell adhesion molecules (eg, integrins, selectins) and fragments of antigen binding thereof and soluble adhesion molecules (eg, chimeric adhesion molecules), small organic molecules, peptides and pepti-domimetics, for example the compounds described in U.S. Patent Nos. 5,843,441, issued to Gundel and col., 5,695,760, issued to Faanes et al., 5,843,425, issued to Tedder et al., 5,753,617 and 5,710,123, assigned to Heavner et al., 5,837,689, issued to Anderson et al. , 5,725,802, issued to Barrett et al., 5,510,332, issued to Kogan et al., And 5,707,985 and 5,260,277, issued to McKenzie et al., International Patent Applications WO 97/03094, WO 98. / 04247 and WO 96/22966, by Biogen Inc., WO 97/10839 and WO 96/00581, of Texas Biotechnology Corporation and WO 94/15958, WO 93/08823 and WO 92/08464, from Tanabe Seiyaku Co., Ltd. The complete teachings of each of the aforementioned references are hereby incorporated by reference. In a preferred embodiment, the additional agent that is coadministered with the antagonist of the CCR1 function can be selected from the group consisting of an immunosuppressive agent and a cell adhesion inhibitor. In another preferred embodiment, the additional agent can be selected from the group consisting of a fibrinolytic agent, a thrombolytic agent, an anticoagulant, a vasodilator and an agent that stimulates or inhibits the production of nitric oxide.

The invention further relates to an antagonist of CCR1 function for use in therapy (including prophylaxis), for example, as described herein, and with the use of said antagonist for the manufacture of a medicament for inhibiting graft rejection ( for example, acute rejection, chronic rejection) and / or ischemia / reperfusion injury as described here. The invention also relates to a medicament for inhibiting graft rejection (eg, acute rejection, chronic rejection) and / or ischemia / reperfusion injury, wherein said medicament includes an antagonist of CCR1 function. A "subject" is preferably a human, but may also be a mammal in need of veterinary treatment, for example domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, poultry, pigs, horses and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). An effective amount of the CCR1 antagonist can be administered to a subject to inhibit (reduce or prevent) graft rejection and / or ischemia / reperfusion injury. For example, an effective amount of the CCR1 antagonist may be administered before, during and / or after transplant surgery or other medical procedure that may result in an ischemia / reperfusion injury. When co-administration of an antagonist of CCR1 function and an additional therapeutic agent to inhibit graft rejection and / or ischemia / reperfusion injury is indicated or desired, the antagonist of CCR1 function can be administered before, at the same time or after administration of the additional therapeutic agent. When the antagonist of the CCR1 function and the additional therapeutic agent are administered at different times, they are preferably administered in a suitable period of time to obtain a substantial overlap of the pharmacological activity (eg, inhibition of the CCR1 function, immunosuppression). of the agents. The person skilled in the art will be able to determine the appropriate time for the coadministration of an antagonist of the CCR1 function and an additional therapeutic agent depending on the particular agents selected and other factors. An "effective amount" of a CCR1 antagonist is an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, such as an amount sufficient to inhibit graft rejection and / or ischemia / reperfusion injury. For example, an effective amount is an amount sufficient to inhibit one (i.e., one or more) CCR1 function (e.g., leukocyte migration induced by CCR1 ligands, integrin activation induced by CCR1 ligands, transient increase induced by CCR1 ligands in the concentration of free intracellular calcium [Ca +] i and / or secretion (for example, degranulation) induced by CCR1 ligands of proinflammatory mediators) and thus inhibit graft rejection and / or Ischemia / reperfusion injury. An "effective amount" of an additional therapeutic agent (e.g., immunosuppressive agent) is an amount sufficient to achieve a desired therapeutic and / or prophylactic effect (e.g., immunosuppression).

The amount of agent (eg, CCR1 antagonist, additional therapeutic agent) administered to the individual will depend on the characteristics of the individual, such as general health, age, sex, body weight and drug tolerance, as well as as to the degree, severity and type of rejection and / or ischemia / reperfusion injury. The person skilled in the art will be able to determine the appropriate dosages depending on these and other factors. Typically, an effective amount may vary between about 0.1 mg per day and about 100 mg per day for an adult,. Preferably, the dosage ranges from about 1 mg per day to about 100 mg per day. The agent (for example, CCR1 antagonist, additional therapeutic agent) can be administered by any suitable route, including, for example, orally in capsules, suspensions or tablets or by parenteral administration. Parenteral administration may include, for example, intramuscular, intravenous, subcutaneous or intraperitoneal administration. The agent (eg, CCR1 antagonist, additional therapeutic agent) can also be administered orally (eg, in the diet), transdermal, topical, inhalation (eg, by intrabronchial, intranasal or oral inhalation or intranasal drops). ) or rectal. The administration can be local or systemic, as indicated. The preferred mode of administration may vary depending on the particular agent (e.g., CCRl antagonist, additional therapeutic agent) chosen; however, oral or parenteral administration in general is preferred. The agent (eg, CCR1 antagonist, additional therapeutic agent) can be administered as a neutral compound or as a salt. Salts of compounds containing an amine or other basic group can be obtained, for example, by reaction with an appropriate organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. Compounds with a quaternary ammonium group also contain a counter-anion, such as chloride, bromide, iodide, acetate, perchlorate and the like. Salts of compounds containing a carboxylic acid or other acid functional group can be prepared by reaction with a suitable base, for example a hydroxide base. The salts of acid functional groups contain a countercation, such as sodium, potassium and the like. The CCR1-function antagonist can be administered to the individual as part of a pharmaceutical composition for the inhibition of graft rejection and / or ischemia / reperfusion injury containing a CCR1 antagonist and a pharmaceutically acceptable carrier. Pharmaceutical compositions for therapy may contain an antagonist of CCR1 function and one or more additional therapeutic agents. An antagonist of the CCR1 function and an additional therapeutic agent can be components of separate pharmaceutical compositions, which can be mixed together before administration or administered separately. The formulation will vary according to the selected route of administration (e.g., solution, emulsion, capsule). Suitable pharmaceutical carriers may contain inert ingredients that do not interact with the CCR1-function antagonist and / or the additional therapeutic agent. Standard techniques of pharmaceutical formulation can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Pharmaceutical carriers suitable for parenteral administration include, for example, sterile water. physiological saline solution, bacteriostatic saline solution (saline containing approximately 0.9% mg / ml of benzyl alcohol), phosphate buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions (such as in a hard gelatin or cyclodextran coating) are known in the art (Baker et al., "Controlled Relase of Biological Active Agents," John Wiley and Sons, 1986). The present invention will now be illustrated by the following Examples, which are not intended to be limiting in any way. EXAMPLES Example 1 Target search CCRl and heart transplant Experimental design Three sets of data were generated: (i) The acute rejection rate was determined in mice with CCR1 (KO) suppression against controls. (ii) The effect of immunosuppression with cyclosporin A (CsA) on CCRl KO mice was determined against controls. (iii) The effects of the deletion of CCR1 on the development of chronic rejection were determined. Methods Mice. CCRl KO mice (also referred to as CCRl - / -) (strain B6 / 129, H-2b), which are homozygotes for a white gene disruption of CCRl, were produced by Craig Gerard (Children's Hospital , Boston, MA; Gerard, C. et al., J ". Clin. Invest. 100: 2022-2027 (1997)) and reproduced in LeukoSite (Cambridge, Mass.) All other mice were obtained from Jackson Laboratory (Bar. Harbor, ME.) These included donor strains (BALB / c, H-2d, B6.C-H2 (bml2) / KhEg (bml2), H-2b) and control receptors (B6 / 129). BALB / c and B6 / 129 differ in the two loci of class I and class II of the major histocompatibility complex (MHC), while Bml2 and B6 / 129 differ only in MHC class II, a cardiac allograft was performed in mice ( Mot-tram, PL et al., Transplantation 59: 559-565 (1995); Hancock, WW et al., Proc. Nati, Acad. Sci (USA) 93: 13967-13972 (1996)) with the aid of a microscope. for operations (Nikon, 4x to 38x increase) ) under clean conditions (rigorously sterile conditions were not required for studies with CCRl KO or control mice, while they were necessary for other immunodeficient mice): Preparation of the donor's heart. Donor mice were anesthetized with Nembutal (50 mg / 10 g body weight) and atropine sulfate (0.17 mg / 100 g body weight) i.p .; additional anesthesia was administered with methoxyflurane supplementation through a face mask as needed during the procedure. The mice were shaved and cleaned with 70% alcohol. An abdominal incision was made by the midline in the donor animal and 1 ml of a 10% solution of heparin in the inferior vena cava was injected. The incision was then extended cephalad to open the thorax through a median sternotomy. The thorax opened. The inferior vena cava was ligated with 6-0 silk and divided inferiorly to the ligature. The superior vena cava was then ligated in a similar manner and divided in a position superior to the ligature. The aorta and pulmonary artery were separated and divided as distally as possible. At this point, the blood was evacuated from the heart by applying pressure with applicator rods. A cross section was made in the aorta just proximal to the brachiocephalic artery and a transversal section was made in the main pulmonary artery just proximal to its bifurcation. The pulmonary veins were then ligated and divided into masses and the heart was placed in ice-cold saline. Preparation of the receiver. After being anesthetized in the same way as the donor, the recipient was taken under a microscope, an abdominal incision was made in the midline, and segments of the aorta and vena cava were dissected below the renal vessels to free them, but without separate them from each other, along a path of approximately 2 mm. A clamp was placed in the proximal aorta and vena cava and a distal 6-0 silk ligature was placed around the aorta and the vena cava in preparation for the subsequent occlusion of the vessels. Heart transplant The ligature that had been placed around the distal aorta and the vena cava was secured by a simple knot. An aortotomy and venotomy were performed in the vena cava adjacent to each other. The donor's heart was then extracted from the iced saline and the donor's aorta and pulmonary artery were attached end-to-side to the aorta and the vena cava of the recipient, respectively, with a sliding suture, using 10-0 with a BV-3 needle. As adjacent anastomoses were made to each other, the side of the pulmonary artery-cava suture line near the aortic anastomosis was sutured from the inside with an eversion slide suture. During this period, ice-cold saline was dripped onto the ischemic heart at frequent intervals. After completion of the anastomoses, the inferior vascular occlusive occlusion was first released, filling the inferior vena cava and the pulmonary artery of the donor with venous blood from the recipient. Upon release of the proximal occlusive ligature, the aorta and coronary arteries of the transplant were perfused with oxygenated blood from the recipient. Blood loss was minimized by gradual libration of the proximal ligature. Externally tuned saline was used to warm the heart immediately after establishing coronary perfusion. With heating and coronary perfusion, the heart began to fibrillate and normally reversed in a few minutes spontaneously to a sinus rhythm. Occasionally, a cardiac massage was necessary to restore a normal heartbeat. The intestines were carefully placed back into the abdominal cavity around the auxiliary heart and the abdomen closed with a single sliding suture for all layers (saline with antibiotic was used to wash the peritoneal cavity as necessary). The mouse was placed in a constant temperature at 35 ° C for the recovery of the anesthesia. Immunosuppression. The effect of therapy with cyclosporine A (CsA) (Sigma, St. Louis, MO) (10 mg / kg / day, injected subcutaneously) was studied using our standard BALB / c-B6 / l29 or B6 cardiac allograft model / 129-CCR1 KO and injecting CsA daily until rejection occurs or for a maximum of 21 days, starting on the day of the transplant. Monitoring the survival of allografts. The survival of cardiac allografts was monitored twice a day by palpation of ventricular contractions through the abdominal wall (Mottram, PL et al., Transplantation 59: 559-565 (1995)), rejection was defined as the day of cessation of a palpable heartbeat and was verified by autopsy (Gerard, C. et al., J., Clin.Research 100: 2022-2027 (1997); Mottram, P.L. et al., Transplantation 59: 559-565 (1995)). Once the function of the cardiac graft had ceased, the mice were anesthetized as before, the grafts were surgically cut, subdivided into portions for (a) fixation with formalin, imbibition in paraffin and subsequent examination of optical microscopy, or (b) ) sudden freezing in liquid nitrogen and storage at -70 ° C until processing for immunohistology or for RNAse protection assays. Immunopathology For histology, sections of paraffin were stained with hematoxylin and eosin (HE), in order to evaluate the morphology of the graft, and with Weigert's elastin stain to examine the degree of intimal proliferation in the penetrating branches of the myocardial arteries. -dicas (a key feature of arteriosclerosis in transplants). (Gerard, C. et al., J. "Clin Invest. 100: 2022-2027 (1997); Mot-tram, PL et al., Transplantation 59: 559-565 (1995)). chemokines and mRNA for the chemokine receptor using AR? asa protection assay kits (Pharmingen, San Diego, CA) Results Table 1 summarizes the graft survival data (mean ± SD) (using 6-10 animals / group).

Table 1 - Effect of CCRl KO on survival of cardiac allografts in mice The p values were determined by the Mann-Whitney U test. The key points that emerge from Table 1 are: CCR1 + cells contribute to the pathogenesis of allograft rejection. The alteration of the CCR1 function in a complete lack of MHC correspondence significantly prolongs the rejection of allografts (group 1 vs 2). As anticipated, rejection in control mice was associated with infiltration of the grafts by CCR1 + mononuclear cells (mainly macrophages) and mRNA studies showed that rejection in control mice was associated with intragraft induction of expression of the MRNA for the ligands of CCR1, MlP-la and RANTES. Although rejection in CCRl KO mice was accompanied by dense infiltration of mononuclear cells and further regulation of the mRNA of the CCR1 ligands, the allografts lacked AR? M expression or proteins corresponding to CCR1. The addition of CsA (10 mg / kg / day) produced only a minor prolongation of the survival of the allografts in the control mice (2-3 days) compared to the untreated recipients (group 1 vs 3). However, the same dose of CsA in CCRl KO mice (for a maximum of 21 days) gave rise to a permanent graft in all the receptors (group 4, a higher evaluation revealed that the mice of group 4 survived during > 200 days after the heart transplant). The beneficial actions of some experimental agents may be impaired by concomitant immunosuppression. However, CsA and the inhibition of CCR1 function are synergistic in terms of efficacy. In addition, the grafts collected on day 100 of CCR1 KO receptors showed only a minor cellular infiltrate and no evidence of arteriosclerosis of the transplant. These findings are concomposed to the severe arteriosclerosis observed in control allograft recipients that were treated with high dose CsA (30 mg / kg / day) or with CD4 mAb therapy (Mottram, PL, Han, WR et al., "Increased expression of IL-4 and IL-10 and decreased expression of IL-2 and IFN- in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy", Transplantation 59: 559-565 (1995); Hancock, WW , Buelow, R. et al., "Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes", Nature Medicine 4: 1392-1396 (1998)). The alteration of the CCR1 function in combinations of mismatch of class II is highly effective. While the untreated recipients rejected the allografts around day 35 (group 5), the alteration of the CCR1 function in this combination resulted in a permanent in-jertation (group 6). Furthermore, as with group 4 (Csa at low dose), the allografts collected on day 100 showed no evidence of arteriosclerosis of the transplant or other features of chronic rejection. Example 2 CCR1 and chronic rejection in cardiac allograft recipients The administration of monoclonal antibody (mAb) CD4 can prolong the survival of cardiac allografts in the described murine model (Mottram et al., Transplantation 59: 559-565 (1995)). ). However, prolonged survival of the grafts in animals treated with anti-CD4 is complicated by the development of chronic rejection with florid arteriosclerosis of the transplant (Hancock et al., Nature Medicine 4: 1392-1396 (1998)). Methods Cardiac allografts derived from Balb / c donors were transplanted into control CCR1 + or CCR1 + / + mice as described in Example 1. Immunosuppression. CD4 mAb (GK1.5, American Type Culture Collection, Manassas, VA; Accession No. TIB-207) was administered four times to allograft recipients CCR1 - / - or CCR1 + / + (6 / group), at 250 μg by intraperitoneal injection on day 0 (time of transplant) and days 1, 2 and 3 following. Moni tori zac ion of chronic rejection. The su-pervi gingiva of the cardiac allograft was monitored twice a day by palpation of the ventricular contractions through the abdominal wall. All the grafts survived until day 60 and the reading was the morphological examination, particularly the degree of arteriosclerosis development of the transplant. Accordingly, the formalin grafts were fixed, embedded in paraffin and the sections were counterstained with elastin staining of Weigert. All the intramyocardial arteries were scored as to the degree of intimal proliferation as <5% occlusion (0), > 5-20% (1), > 20-40% (2), > 40-60% (3), > 60-80% (4) or > 80-100% (5) (Murphy et al., Transplantation 64: 14-19 (1997)). Results Table 2 shows the results of the vessel score in cardiac allografts (6 grafts / group) and the statistical evaluation (Mann-Whitney U test). Table 2 - Effect of CCRl KO on the development of arteriosclerosis of transplants The key points that emerge from Table 2 are: CCR1 + cells contribute to the pathogenesis of chronic allograft rejection. The alteration of the CCR1 function blocks the development of the atherosclerosis of the transplant. The alteration of the CCR1 function blocks the development of other characteristics of chronic rejection. Example 3 CCRl and renal ischemia / reperfusion injury The prolonged interruption of blood supply to an organ will result in its complete necrosis even if the circulation is eventually restored. Interruption for shorter periods can lead to death of specific cell types that are highly susceptible to hypoxia, but the organ as a whole can be partially or even completely recovered, depending on the period of lack of blood flow (ischemia) and others. f. The damage, known as ischemia / reperfusion injury, which is apparent after restoration of blood flow to a transiently ischemic kidney or other commonly transplanted organs, is primarily mediated by neutrophils, which are recruited to the organ with revascularization. Although multiple pathways are implicated in such neutrophil recruitment, we hypothesized that the chemokine receptor, CCR1, could play a key role, so blocking this route would reduce the lesion after ischemia / reperfusion. We established a new mouse model of cold kidney injury due to ischemia / reperfusion, in which the maintenance of the organ at 4 ° C during the period of ischemia simulates the maintenance conditions of a kidney that has been collected in preparation for a transplant. . Therefore, these observations are directly related to the ways in which the clinical lesion due to ischemia / reperfusion could be reduced or even eliminated in the context of renal allograft, by blocking the CCR1 route. Experimental design The kidneys of inbred CCRl KO or control B6 / 129 mice were perfused in-situ until they were pale using cold saline and packed on ice for varying periods before revascularization. After the preliminary studies. We caused a cold ischemia of 60 minutes and we followed up for 48-72 hours, with analysis of renal function, histology, immunopathology (with quantitative image analysis) and analysis of MRNA. Methods Mice: CCRl KO mice (strain B6 / 129, H-2b) were produced by Craig Gerard (Children's Hospital, Boston, MA; Gerard, C. et al., "Clin Invest. 100: 2022-2027 (1997)) and reproduced in LeukoSite (Cambridge, Mass.) B6 / 129 control mice were obtained from Jackson Laboratory (Bar Harbor, ME) .

Ischemia / reperfusion model. The ischemia / reperfusion procedure was performed under sterile conditions with the help of a microscope for operations (Zeiss, 2-50x magnification). Mice (CCRl KO or B6 / 129) were anesthetized with Nembutal (50 mg / 100 g body weight); Metoxiflurane supplementation was used, administered through a face mask, to obtain additional anesthesia, as necessary. After shaving and disinfecting the abdomen with 70% alcohol, a laparotomy was performed through an incision through the midline. After accessing the renal artery and vein of the right kidney, the vena cava and the adrenal and infrarenal aorta were separated, the perirenal tissue was cut and the kidney was mobilized. To stop the flow of arterial blood to the kidney, the infra- and supra-rrenal aorta was closed by microclips. An aortotomy was then performed and the kidney was washed with cold saline until pale. To allow retrograde flow of the wash solution in the abdomen, a small incision was made in the renal vein, between the clip and the kidney, after clamping the vein with a microclip. All incisions were sutured with 10-0 proleno sutures. After this procedure, the kidneys were exposed for varying periods of ischemia of up to 75 minutes, during which time the cold kidneys were kept packed in ice. After removing the clips and reperfusing the organ, a contralateral nephrectomy was performed. The abdomen was closed with 6-0 silk. The animals were kept in the post-operative in a heated cage, under observation, until they were seen to recover from the procedure. Moni tori zac ion of the effects of ischemia / reperfusion injury. Serum creatinine levels were measured daily. As the creatinine levels had a peak at 48 hours post-ischemia / reperfusion, the mice (8 / group) were sacrificed at 48 hours for the histological and mRNA studies of the kidneys or were followed for 72 hours. to determine the effects on animal survival. For histology, the kidneys were fixed in formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H-E) and periodic acid / Schiff stain (PAS). The expression of chemokines and chemokine receptor mRNA was determined using AK? Asa protection assay kits (Pharmingen, San Diego, CA). Results In Table 3, the contrasting findings of control mice B6 / 129 vs. CCRl. Table 3 - Effect of CCRl KO on renal injury by ischemia / reperfusion in mice The p values were determined by the Mann-Whitney U test. The key points that emerge from Table 3 are: The alteration of the CCR1 function blocked the recruitment of neutrophils and completely prevented the alteration of renal function after the ischemia / reperfusion injury.

The blocking of CCRl also almost completely abolished tubular cell necrosis. The results of the mRNA analysis are not shown in Table 3. The kidneys of CCRl KO and B6 / 129 mice showed in both cases induction of mRNA expression for the chemokines associated with CCR1, MIP-1 and RANTES. Therefore, the effects of the CCR1 deletion left the proinflammatory cell responses intact, but the absence of CCR1 on the host cells inhibited leukocyte recruitment and subsequent renal damage. The study was extended by increasing the number of animals in each group to 15 and extending the analysis period to 100 days. The results given in Table 3 were included in the extended study analysis. The extended study showed that CCR1 - / - mice survived significantly more after ischemia / reperfusion than B6 / 129 mice (92 + 6.1 vs 45 + 12.4 days, mean survival time ± standard error of the average, limited to 100 days, p <0.001). 86% of the CCRl - / - mice were alive 100 days after the ischemia / reperfusion procedure, while only 26% of the B6 / 129 mice survived for 100 days after the procedure (Figure 1). Serum creatinine measurements were made at 2, 7 and 100 days after the ischemia / reperfusion procedure to assess renal function. The CCR1 - / - mice were protected from renal injury and showed serum creatinine levels similar to those detected in animals subjected to sham treatment (unilateral nephrectomy without ischemia / reperfusion). However, serum creatinine levels were significantly increased, approximately 5-fold, in wild type B6 / 129 animals two days after ischemia / reperfusion (p <0.005). Significantly elevated levels (4 fold) of serum creatinine were also detected in wild type B6 / 129 animals seven days after ischemia / reperfusion (p <0.005). Serum creatinine was elevated in all groups, including mock animals, 100 days after the procedure (p <0.01) (Figure 2). Example 4 CCR1 and hepatic injury by ischemia / reperfusion Methods Male C57B6 / 129 (B6 / 129) mice or male CCR1 - / - mice weighing 22-28 g were used. Partial hepatic ischemia was induced in mice anesthetized with sodium pentobarbital (60 mg / kg i.p.). A midline laparotomy was performed and an atraumatic clip was used to interrupt the blood supply to the middle and left lobes of the liver, thus producing partial hepatic ischemia. At 90 minutes, the clip was removed, starting the hepatic reperfusion. The sham control mice underwent the same surgical procedure without interrupting the blood supply to the middle and left lobes of the liver. Mice were sacrificed at serial intervals and samples of liver and blood tissue were taken for analysis. Hepatic function is measured by measuring serum alanine aminotransferase (SGPT) and / or serum aspartate aminotransferase (SGOT). Results CCRl - / - mice were protected against hepatic dysfunction for 0.5-7 days after hepatic ischemia / reperfusion, as assessed by the serum alanine aminotransferase (SGPT) assays (Figure 3) and Serum aspartate aminotransferase (SGOT). In addition, CCRl - / - mice survived significantly more than wild type B6 / 129 mice after hepatic ischemia / reperfusion (13.4 + 3.6 days compared with 8.7 ± 5.1 days, p < 0.01). Immunohistological analysis of liver tissue demonstrated the expression induced by ischemia / reperfusion of CCR1 ligands in CCR1 - / - and B6 / 129 mice. Extended liver necrosis and neutrophil infiltration were observed in the liver tissue excised from B6 / 129 mice after hepatic ischemia / reperfusion. Nevertheless, only a minor and focal lesion of the hepatocytes was observed and no infiltration of neutrophils into the hepatic tissue of CCR1 - / - mice after hepatic ischemia / reperfusion. Therefore, the absence of CCRl in mice inhibited hepatic injury by ischemia / reperfusion and subsequent liver dysfunction. Example 5 Immunocompetence of CCRl - / - mice T cell proliferative responses in vi tro Mixed lymphocyte responses ("MLR") were assessed by culturing splenocytes responders (isolated from CCR1 - / - or wild type B6 / 129 mice) with splenocytes allogenic stimulators inactivated with mitomycin C (isolated from Balb / c mice) in RPMI 1640 medium containing 5% FBS, 1% penicillin / streptomycin and 2-mercaptoethanoi 5 x 10"5 M, in plates of 96 flat-bottom wells The cultures were incubated at 37 ° C in 5% C02 for 3 to 5 days and pulsed with [3 H] thymidine for 6 hours before collection, the amount of [3H] thymidine was measured Incorporated by the cells cultured by scintillation counting, the average amount of incorporated radioactivity (counts per minute) and standard deviations were calculated using 12 wells per group, and the mitogen-induced proliferation of T cells was measured using Concanavalin-A ( With- A, a mitogen of T cells). Splenocytes isolated from wild-type CCR1 - / - or B6 / 129 mice were cultured in 96-well flat-bottomed plates in RPMI-1640 medium containing 5% FBS, 1% penicillin / streptomycin, 2-mercaptoethanoi 5 x 10 ~ 5 M and 1.25-10 μg / ml of Con-A (Sigma Chemical Co., St. Louis, MO). The cultures were incubated at 37 ° C in 5% C02 for 72 hours and pulsed with [3 H] thymidine for 6 hours before collection. The amount of [3 H] thymidine incorporated by the cultured cells was measured by scintillation counting. The average amount of incorporated radioactivity (counts per minute) and the standard deviation were calculated using 12 potions per group. Results The results of the T cell proliferation studies in vitro are presented in Figures 4A and 4B. The mitogen-induced T cell proliferation detected in the splenocyte cultures of CCR1 - / - mice was identical to that detected in the splenocyte cultures of wild type B6 / 129 mice (Figure 4A). CCRl - / - mice developed a vigorous response to allogeneic stimulator cells in MLR assays (p <; 0.001) (Figure 4B). However, the overall magnitude of the 5-day MLR repeat test response was consistently 20-25% lower in cultures containing responding splenocytes isolated from CCR1 - / - mice compared to cultures containing B6 responding splenocytes / 129 (p < 0.01). These studies of T cell responses in vitro demonstrate that CCR1 - / - mice are immunocompetent. Although this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention, such as it is defined by the appended claims.

Claims (48)

Claims

1. A method of inhibiting graft rejection, comprising administering to a subject in need thereof an effective amount of an antagonist of CCR1 function.

2. The method of Claim 1, wherein said graft is an allograft.

3. The method of Claim 2, wherein said allograft is selected from the group consisting of kidney, liver, lung, heart-lung, pancreas, intestine and heart.

4. The method of Claim 3, wherein said allograft is a heart.

5. The method of Claim 1, wherein said antagonist of the CCR1 function is selected from the group consisting of small organic molecules, natural products, peptide Sj. proteins and peptidomimetics.

6. The method of Claim 5, wherein said antagonist of the CCR1 function is a small organic molecule.

7. The method of Claim 5, wherein said antagonist of the CCR1 function is a natural product.

8. The method of Claim 5, wherein said antagonist of the CCR1 function is a peptide.

9. The method of Claim 5, wherein said antagonist of the CCR1 function is a peptidomimetic.

10. The method of Claim 5, wherein said antagonist of the CCR1 function is a protein.

11. The method of Claim 10, wherein said pro tein is an anti-CCRl antibody or antigen-binding fragment thereof.

12. A method of inhibiting graft rejection, comprising administering to a subject in need thereof an effective amount of an antagonist of CCR1 function and an immunosuppressive agent.

13. The method of Claim 12, wherein said immunosuppressive agent is one or more agents selected from the group consisting of calcineurin inhibitors, glucocorticoids, nucleic acid synthesis inhibitors, and antibodies that bind to lymphocytes or binding fragments. antigen of these.

14. The method of Claim 13, wherein said immunosuppressive agent is an inhibitor of calcineurin.

15. The method of Claim 14, wherein said calcineurin inhibitor is cyclosporin A.

16. The method of Claim 14, wherein said calcineurin inhibitor is FK-506.

17. The method of Claim 13, wherein said immunosuppressant agent is a glucocorticoid.

18. The method of Claim 17, wherein said glucocorticoid is prednisone or methylprednisolone.

19. A method of inhibiting graft versus host disease, comprising administering an effective amount of an antagonist of CCR1 function to a recipient of a transplanted graft.

20. The method of Claim 19, wherein said graft is bone marrow.

21. The method of Claim 20, which further includes the administration of an immunosuppressive agent.

22. The method of Claim 21, wherein the immunosuppressive agent is a calcineurin inhibitor.

23. The method of Claim 22, wherein said calcineurin inhibitor is cyclosporin A or FK-506.

24. A method of ischemia / reperfusion injury inhibition, comprising administering to a subject in need thereof an effective amount of an antagonist of CCR1 function.

25. The method of Claim 24, wherein said antagonist of the CCR1 function is selected from the group consisting of small organic molecules, natural products, peptides, proteins and peptidomimetics.

26. The method of Claim 25, wherein said antagonist of the CCR1 function is a small organic molecule.

27. The method of Claim 25, wherein said antagonist of the CCR1 function is a natural product.

28. The method of Claim 25, wherein said antagonist of the CCR1 function is a peptide.

29. The method of Claim 25, wherein said antagonist of the CCR1 function is a peptidomimetic.

30. The method of Claim 25, wherein said antagonist of the CCR1 function is a protein.

31. The method of Claim 30, wherein said pro tein is an anti-CCR1 antibody or antigen-binding fragment thereof.

32. The method of Claim 25, wherein said ischemia / reperfusion injury is a consequence of a medical procedure that stops, restricts or redirects blood flow.

33. The method of Claim 32, wherein said medical procedure is angioplasty or surgery.

34. The method of Claim 32, wherein said medical procedure is surgery.

35. The method of Claim 34, wherein said surgery is a graft transplant.

36. The method of Claim 35, wherein said graft is selected from the group consisting of kidney, lung, liver, heart-lung, pancreas, intestine and heart.

37. The method of Claim 36, wherein said graft is a kidney.

38. The method of Claim 24, wherein said ischemia / reperfusion injury is the result of a pathological condition selected from the group consisting of arteriosclerosis, myocardial infarction, cerebrovascular accident and transient ischemic attack.

39. The method of Claim 38, wherein said pathological condition is myocardial infarction.

40. The method of Claim 38, wherein said pathological condition is the cerebrovascu lar accident.

41. The method of Claim 38, wherein said pathological condition is arteriosclerosis.

42. The method of Claim 24, further comprising administering to said subject an effective amount of one or more additional therapeutic agents selected from the group consisting of fibrinolytic agents, thrombolytic agents, anti-coagulants, inhibitors of cell adhesion, antithrombotic agents. , stimulators of nitric oxide synthase and inhibitors of nitric oxide synthase.

43. The method of Claim 42, wherein said additional therapeutic agent is a fibrinolytic agent.

44. The method of Claim 42, wherein said additional therapeutic agent is a thrombolytic agent.

45. The method of Claim 42, wherein said additional therapeutic agent is an anticoagulant.

46. The method of Claim 42, wherein said additional therapeutic agent is an inhibitor of cell adhesion.

47. The method of Claim 42, wherein said additional therapeutic agent is an antithrombotic agent.

48. The method of Claim 42, wherein said additional therapeutic agent is an activator or inhibitor of nitric oxide synthase.