MXPA01007565A - Method of treating demyelinating inflammatory disease using ccr1 antagonists - Google Patents

Abstract

The invention relates to a method for treating inflammatory demyelinating diseases. The method comprises administering to a subject in need an effective amount of an antagonist of CCR1 function. In a preferred embodiment, the invention provides a method for treating multiple sclerosis.

Description

METHOD OF TREATMENT OF INFLAMMATORY DISEASE DEMYELINIZING USING CCRl ANTAGONISTS BACKGROUND OF THE INVENTION Demyelinating inflammatory diseases share the common characteristics of inflammation (e.g., leukocyte infiltration) and destruction of myelin of the central nervous system (CNS). The pathophysiology of these diseases involves infiltration of the CNS by leukocytes (for example, T cells, macrophages) and the subsequent development of plaques, which are areas of demyelination. These diseases can be acute (for example, acute disseminated encephalomyelitis, Guillain-Barre syndrome and acute haemorrhagic leukoencephalitis) or chronic (for example, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuropathy). There is no specific diagnostic test for demyelinating diseases and the diagnosis is usually based on the recognition of the distinctive pattern of CNS injury that each disease produces. The most common demyelinating disease is multiple sclerosis (MS), which affects approximately 350,000 Americans and, with the exception of trauma, is the most common cause of neurological disability that begins in the early years of the adult phase to the median. age. The manifestation of MS is highly variable and the disease can be benign or rapidly debilitating. The clinical course of MS can be grouped into four general categories. Recurrent-remitting MS is characterized by recurrent acute attacks of neurological dysfunction followed by periods in which recovery may occur. There is no progression of the neurological alteration between attacks. Secondary progressive MS has a recursive-remitting course at the beginning, but later the neurological dysfunction progresses (ie becomes more severe) during the period between acute attacks. Primary progressive MS is characterized by a progression of disability from the onset of the disease without acute differentiated attacks. Some patients with primary progressive MS may experience periods of apparent clinical stability. The rarer course is referred to as progressive-recurrent MS, which is characterized by a progression of disability from the onset of the disease with recurrent acute attacks. The hypotheses on the pathogenesis of demyelinating diseases are derived from the study of experimental allergic encephalomyelitis (EAE) in mice and rats. EAE is an inflammatory demyelinating disease mediated by Th1 CD4 + cells that serves as a model for MS. EAE, MS, and other demyelinating diseases appear to be mediated by an autoimmune mechanism that involves the activation and recruitment of lymphocytes (eg, T cells) that react with certain autoantigens expressed in the CNS. Furthermore, there is some evidence of a genetic predisposition to the development of demyelinating disease and environmental factors (eg, viral infection) can trigger the onset of the disease. The treatment of demyelinating disease is primarily focused on the inhibition of acute attacks with adrenocorticotropic hormone (ACTH) or immunosuppressive agents (eg, glucocorticoids). These agents can produce serious systemic side effects and, therefore, only moderate and severe attacks are usually treated. Interferons (for example, IFN-la, IFN-Ib) and copolymer 1 have been used for prophylaxis against recurrent acute attacks (recurrence) in MS. However, the efficacy of these therapies is often limited by systemic reactions (eg, neutralizing antibodies) to the drugs. Additionally, chronic immunosuppression (for example with methotrexate, azathioprene, cyclophosphamide, 2-chlorodeoxyadenosine) has been used with only modest efficacy for the treatment of progressive MS. There is, therefore, a need for an effective method of treating demyelinating diseases.

COMPENDIUM OF THE INVENTION The invention relates to a method for treating inflammatory demyelinating diseases. The method consists of administering an effective amount of an antagonist of CCR1 function to a subject in need thereof. In one aspect, the invention provides a method of treating an inflammatory demyelinating disease, comprising administering to an individual in need thereof an effective amount of a (i.e., one or more) antagonist of CCR1 function. In one embodiment, the method is directed to the treatment of an acute inflammatory demyelinating disease, for example acute disseminated encephalomyelitis, Guillain-Barre syndrome or acute haemorrhagic leukoencephalitis. In another embodiment, the method is directed to the treatment of a chronic inflammatory disease, for example EM or chronic inflammatory demyelinating polyradiculo-neuropathy. In a preferred embodiment, the invention provides a method of treating MS. The antagonist of the CCR1 function is a molecule, such as a protein, peptide, peptidomimetic, natural product or small organic molecule, which inhibits (reduces, prevents) one or more functions of CCR1. In another aspect, the invention provides a method of treating an inflammatory demyelinating disease, comprising administering to a subject in need thereof an effective amount of an antagonist of the CCR1 function and an effective amount of one or more additional therapeutic agents, such as antiviral, antibacterial and immunosuppressive agents.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a histogram illustrating the clinical scores of young female mice deleted for CCRl (CCRl KO, n = 10, solid bars) and wild-type control mice with age and sex correspondence (FIG. B6 / 129, n = 10, blank bars) at various times after immunization with MOG peptide (35-55) in complete Freund's adjuvant ("CFA") supplemented with Mycobacterium tuberculosis antigen (4 mg / ml). The clinical scores shown are the average daily score for each group. Figure 2 is a histogram illustrating the clinical scores of young female mice deleted for CCRl (CCRl KO, n = 8, solid bars) and wild-type control mice with age and sex correspondence (B6 / 129 , n = 8, blank bars) at various times after immunization with MOG peptide (35-55) in complete Freund's adjuvant (CFA) supplemented with Mycobacterium tuberculosis antigen (4 mg / ml). The clinical scores shown are the average daily score for each group. Figure 3 is a histogram illustrating the clinical scores of female mice suppressed for CCR1 (CCRl KO, n = 10, solid bars) and wild-type control mice with age and sex correspondence (B6 / 129, n = 10, blank bars) at various times after immunization with MOG peptide (35-55) in complete Freund's adjuvant (CFA) supplemented with Mycobacterium tuberculosis antigen (4 mg / ml). The clinical scores shown are the average daily score for each group. Figure 4 is a histogram illustrating the change in the thickness of the ear associated with a delayed-type hypersensitivity response in young female CCR1 KO mice (n = 8) and wild-type control mice with age and sex correspondence (FIG. n = 8), which were sensitized and inoculated with 2,4-dinitrofluorobenzene (DNFB) 0.5%. Figure 5A is a graph illustrating the dose-dependent proliferation of splenocytes in an in vitro proliferation assay. Splenocytes isolated from CCR1 - / - mice or B6 / 129 wild type mice (CCR1 + / +) were cultured with MOG peptide (35-55). Proliferation was detected by pulsing the cultures with 3H-thymidine and quantifying the amount of radioactivity incorporated into the DNA of the cells. Figure 5B is a graph illustrating the dose-dependent production of IL-2 in splenocyte cultures stimulated with MOG peptide (35-55). Splenocytes isolated from CCR1 - / - mice or wild type B6 / 129 mice (CCR1 + / +) were cultured in serum-free medium containing MOG peptide (35-55) for 40 hours. The culture supernatants were removed and the amount of IL-2 in the supernatants was determined by quantitative ELISA. Figure 5C is a graph illustrating the do-sis-dependent production of IFN-? in cultures of splenocytes stimulated with MOG peptide (35-55). Splenocytes isolated from CCR1 - / - mice or B6 / 129 wild type mice (CCR1 + / +) were cultured in serum-free medium containing MOG peptide (35-55) for 40 hours. The culture supernatants were removed and the amount of IFN? in the supernatants by quantitative ELISA. Figure 5D is a graph illustrating the dose-dependent production of IL-6 in cultures of splenocytes stimulated with MOG peptide (35-55). Isolated splenocytes were cultured from CCR1 - / - mice or wild type B6 / 129 mice (CCR1 + / +) in serum-free medium containing MOG peptide (35-55) for 40 hours. The culture supernatants were removed and the amount of IL-6 in the supernatants was determined by quantitative ELISA.

DETAILED DESCRIPTION OF THE INVENTION Chemokines are a family of proinflammatory mediators that promote the recruitment and activation of multiple leukocyte lines (e.g., 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 may 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 (a-chemokines) and the CC chemokines (β-chemokines), where the first two conserved cysteines are separated by an intermediate residue or they are adjacent residues, respectively (Baggiolini, M. and Dahinden, CA, I munology Today, 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 the "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 human monocyte chemoattractant proteins 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 superfamily 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, NP, 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, CKbeta8, 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, RANTES, 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, RA? TES 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 (7): 436-445 (1998), Tsou et al., J. Exp. Med., 188: 603-608 (1998);? Ardelli et al., J. Immunol. 162 { 1): 435-444 (1999); Youn et al., Blood 91 (9): 3118-3126 (1998); Youn et al., J. Immunol. 259 (11): 5201-5201 (1997)). The human chemokine receptor CCR1 is expressed in a variety of different cells, including neutrophils, monocytes, lymphocytes and eosinophils and CCR1 binds to a variety of chemokine ligands (Gong, X. et al., J *. Biol. Chem. 272: 11682-11685 (1997), Wong, M. and E.? Fish, J. Biol. Chem. 273: 309-341 (1998), and Youn, BS et al., J. Immunol. 159: 5201 -5205 (1997)). To determine if the CCR1 function is involved in the initiation, progression and / or maintenance of demyelinating lesions in the CNS, studies of EAE, autoimmune disease mediated by cellular immunity (for example, mediated by Thl) that is a model of MS, in wild-type and genetically altered mice were conducted. As described herein, neurological dysfunction, CNS inflammation and destruction of CNS myelin, which are all characteristics of EAE and MS, were dramatically inhibited (eg, reduced or prevented) in CCRl mice - / - in comparison with CCRl + / + control mice with age and sex correspondence. The incidence of EAE, as of MS, is influenced by gender and female mice develop the disease more frequently and with greater severity than male mice. Consequently, EAE is usually studied in young female mice (eg, 6-9 weeks of age). As described herein, EAE was induced in CCR1 - / - and CCR1 + / + mice corresponding in age and sex by immunization with an immunodominant epitope of oligodendrocyte myelin glycoprotein (MOG (35-55); Hilton, A.. et al., J ". Neurochem 65: 309-318 (1995)) and the establishment, course and severity of the disease were determined by monitoring neurological dysfunction (eg, tail tone, posterior paresis, loss of the straightening response, tetraparesis.) The young female CCR1 - / - mice developed EAE, which was significantly less severe than the disease in CCR1 + / + control animals. Furthermore, the young female CCR1 - / - mice had a The lower incidence of the disease and the onset of clinical disease (for example, neurological dysfunction) was delayed compared to control CCRl + / + mice (Figures 1 and 2, Example 1, Experiments 1 and 2). In a study using adult male mice, 80% of CCRl + / + control mice developed EAE, whereas CCRl - / - mice had a significantly lower incidence of the disease, with EAE developing only one of ten animals. mouse CCR - / - who developed EAE had only minimal clinical symptoms, which were dramatically less severe than the clinical symptoms of CCRl + / + control mice. (Figure 3, Example 1, Experiment 3). Histological examination of brains and spinal cords excised from mice immunized with MOG (35-55) revealed a significant reduction in the degree of inflammation and destruction of myelin in the CNS of CCRl - / - mice (n = 3) compared to control mice CCR1 + / + (n = 4) (Example 1, Microscopic examination). Therefore, CCR1 - / - mice develop less severe clinical symptoms compared to control CCR1 + / + mice, since impaired CCR1 function inhibits CNS inflammation. Other studies showed that the marked reduction in the severity and incidence of the pathology associated with EAE in CCRl - / - mice is not due to a general defect in the cellular in u-nity of these animals. Certainly, mice CCR1 - / - have the ability to produce a normal cellular immune response, as determined by a mixed lymphocyte reaction (MLR), and non-sensitized CCR1 - / - mice are hyperresponders in a type hypersensitivity assay Standard Delay (HTR) (Example 2, Figure 4). Therefore, the CCR1 function is specifically involved in the pathogenesis of EAE and the alteration of CCR1 function inhibits (reduces or prevents) the neurological dysfunction associated with EAE, CNS inflammation and the destruction of CNS myelin in CCRl mice. - / - Accordingly, a first aspect of the invention provides a method of treating an inflammatory demyelinating disease, comprising administering to a subject in need thereof an effective amount of an antagonist of the CCR1 function. CCR1 Antagonists As used herein, the term "CCR1 function antagonist" refers to an agent (eg, a molecule, a compound) that can inhibit one (i.e., one or more) CCR1 function . 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- l, 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). Accordingly, processes mediated by CCR1 and cellular responses (eg, proliferation, 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 that is, for example, a small organic molecule, a natural product, a protein (eg, antibody, chemokine7 cytokine), a peptide or a peptidomimetic. Various molecules that can antagonize one or more functions of the chemokine receptors (eg, CCR1), including the small organic molecules described, are known in the art, 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; U.S. Patent Applications Nos. 09 / 146,827 and 09 / 148,236, filed September 4, 1998; Hesselgesser et al., J ". Biol. Chem. 273 (25): 15687-15692 * (1958), and Howard et al., J. Medicinal Chem. 41 (13): 2184-2193 (1998); proteins, such as antibodies (eg, polyclonal, monoclonal, chimeric, 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 (1996), mutants and chemokine analogs, for example those described in US Patent No. 5,739,103, issued to Ro-llins. et al., WO 96/38559, Dana Farber Cancer Institute, and WO 98/06751, Research Corporation Technologies, Inc., Peptides, for example those described in WO 98/09642, of the United States of America. The full complement of each of the aforementioned patent applications and references are hereby incorporated by reference.CNR1 antagonists 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 here or using other methods The antagonists thus identified can be used in the therapeutic methods described herein. Another source of CCR1 antagonists are combinatorial libraries that may contain many structurally distinct molecular species. Combined libraries can be used to identify a line of compounds or to optimize a previously identified line. These 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 appropriate means. a suitable biological source (eg, vegetation) can be homogenized (eg, by grinding) in a suitable buffer and clarified 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 may be 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 to study the biological activity ( 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 Sambrook, J ., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, Y (1989)). A "peptide" can contain any suitable L- and / or D-amino acid, for example, common a-amino acids (eg, alanine, glycine, valine), non-a-amino acids (eg, β-alanine, aminobutyric acid, 6-aminocaproic acid, sarcosine, statin) and non-usual amino acids (eg, ci-truline, homocitrulin, homoserin, 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, 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 CCRl, for example with the amino acid (s) that are (are) in the ligand binding site or proximate 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. Organic polysaccharides, polymers or oligomers of organic synthetic polymers (such as polyvinyl alcohol or polylactide) are included as organic carrier structures. 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 substituted with oxygen or sulfur, thus forming a polyether skeleton. 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 -? HCO- group) with one or more -COHN- groups. In yet another embodiment, the peptide backbone can be replaced with a polysilane backbone. 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 reactions. collateral 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 production methods. The term "antibody", as used herein, also includes functional fragments of antibodies, including chimeric, humanized, primatized, coated or single chain anti-body fragments. Functional fragments include antigen-binding fragments that bind to CCR1. For example, fragments of antibodies capable of binding to CCR1 or portions thereof, including, but not limited to, the Fv, Fab, Fab 'and F (ab') 2 fragments are protected 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 wherein 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 antibodies, humanized or primatized (grafted with "CDRs" - complementarity determining regions) or coated, as well as chimeric single chain antibodies, grafted with CDRs or coated containing 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 No. 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 Newman, R. et al., Bio-Technology 10: 1455-1460 (1992) for primatized 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 techno- logy 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, e.g., U.S. Pat. No. 5,440,020, the teachings of which are herein incorporated in its entirety as a reference). 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, e.g., Chuntharapai et al., J. Immunol., 152: 1783-1789 ( 1994); Chuntharapai et al., U.S. 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, Kohler 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), Current Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer 94), Ausubel, FM et al., Eds. (John Wiley &Sons: New 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 production or isolation of antibodies with the required specificity can be used, including, for example, human or artificial antibodies, including, for example, methods that select recombinant antibody from a library (for example, a phage display library), or that are based on the immunization of transgenic animals. (for example, 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., US Pat. 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 to 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 USA 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; Access No.

TIB202). The cells can be collected 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 marked with 125 I and MlP-labeled with 125 I can be prepared by suitable methods or can be purchased from commercial vendors (eg, DuPont-? E? (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 / B or 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 αCl, and dried and the bound radioactivity can be determined by scintillation counting. The CCR1 antagonist activity of the test agents (e.g., compounds) can be given as the inhibitor concentration 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 (e.g., 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 HL60 cells differentiated with butyric acid can be used in a modification of a transendothelial migration assay (Carr, MW et al., TA, Proc. Na ti, Acad. Sci., USA (91): 3652 (1994)). Peripheral blood mononuclear cells can be isolated from 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, U.K.). The 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 may 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, such as RANTES or MlP-la (Peprotech) (diluted in assay medium) can be added 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 the 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, exoxyphosis can be monitored (eg, degranulation of 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 C)) 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 granule-derived serine esterase release assays, Loetscher et al., J. Immunol., 156: 322-327. (1996), in terms of assays for the release of enzymes and granzymes, Rot, A. et al., J ". Exp. Med. 176: 1489-1495 (1992), regarding the respiratory explosion: Bischoff, SC and col., 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 tests for enzymes are available, such as serine esterases (see, for example, Taub, D.D. et al., J. Immunol., 155: 3877-3888 (1995) regarding the release of serine-esterases derived from granules). In another embodiment, the cells expressing CCR1 are combined with a CCR1 ligand or promoter of the CCR1 function, an agent to be studied before, after or at the same time is added and the degranulation is evaluated. Inhibition of ligand or promoter-induced degranulation is indicative that the agent is an inhibitor of mammalian CCR1 function. In a preferred embodiment, the antagonist of the CCR1 function does not significantly inhibit the function of other chemokine receptors (eg, CCR2, CXCR1, CCR3). Said CCR1-specific antagonists can be identified by suitable methods, such as by an appropriate modification of the methods described herein. For example, cells that do not express CCR1 (CCR1"), but which express one or more other chemokine receptors (eg, CCR2, CXCR1, CCR3) can be created or identified using appropriate methods (eg, transfection, staining antibodies, Western blot, RNase protection.) Such cells or cell fractions (eg, membranes) obtained from said cells can be used in a suitable binding assay, eg, when choosing a cell which is CCR1"and CCR3 +, 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 cell fraction, as described herein. In another preferred embodiment, the CCR1 function antagonist is an agent that binds to CCR1. Such antagonists that bind to CCR1 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 radiolabeled (for example, with H, C or 125D.) In another preferred embodiment, the CCR1 function antagonist is an agent that can inhibit the binding of a (i.e., one or more) ligand from CCR1 to CCR1 (e.g. Human CCR1. In a particularly preferred embodiment, the CCR1 function antagonist is an agent that can bind to CCR1 and thus inhibit the binding of a (i.e., one or more) ligand from CCR1 to CCR1 (eg, human CCR1). Methods of therapy In one embodiment, the method of treating an inflammatory demyelinating disease consists in administering an effective amount of a (i.e., one or more) antagonist of CCR1 function to a subject in need thereof. Attachment includes therapeutic or prophylactic treatment. According to the method, the severity of the disease can be prevented or reduced in whole or in part. In particular embodiments, the inflammatory demyelinating disease may be an acute inflammatory demyelinating disease, for example acute disseminated encephalomyelitis, Guillain-Barre syndrome or acute hemorrhagic leukoencephalitis. In other embodiments, the inflammatory demyelinating disease may be a chronic inflammatory demyelinating disease, for example multiple sclerosis or chronic inflammatory demyelinating polyradiculoneuropathy. In a preferred embodiment, the invention provides a method of treatment (including therapeutic or prophylactic treatment) of multiple sclerosis, comprising administering an effective amount of an antagonist of CCR1 function to a subject in need thereof. As discussed here, the manifestation of MS is variable and the clinical course of MS can be grouped into four categories: recurrent-remitting, primary progressive, secondary progressive, and progressive-recurrent. The method of the invention can be used to treat MS that occurs with each of the recognized clinical courses. Accordingly, an antagonist of CCR1 function can be administered to a patient with a progressive course of MS to delay or prevent the progression of the neurological disorder. An antagonist of CCR1 function can also be administered to a subject with relapsing-remitting MS, progressive secondary or progressive-recurrent as prophylaxis against recurrence (for example, acute attack). For example, the CCR1-function antagonist can be administered to a subject with relapsing-remitting MS during the remitting phase of the disease to inhibit (e.g., prevent, delay) relapse. In another embodiment, the CCR1 function antagonist is selected from the group of molecules that can inhibit one or more functions of CCR1, for example certain small organic molecules, natural products, peptides, peptidomimetics and proteins, where said proteins are not chemokines or mutants or analogs thereof. In another embodiment, the invention provides a method of treating (reducing or preventing) inflammatory demyelinating disease, comprising administering an effective amount of an antagonist of the CCR1 function and an effective amount of a (i.e., one or more) agent additional therapy to a subject who needs it. The therapeutic benefit of an antagonist of CCR1 function and certain other therapeutic agents can be additive or synergistic when coadministered, thus providing a highly effective treatment. Suitable therapeutic agents for administration with an antagonist of CCR1 function include, for example, antiviral agents (eg, acyclovir, ganciclovir, famciclovir, penciclovir, valaciclovir, vidarabine, foscarnet, indinavir), antibacterial agents (eg, antibiotics ( for example, erythromycin, penicillin, tetracycline, ciprofloxacin, norfloxacin, flurazo-lidone, azithromycin, chloramphenicol), sulfonamides, quinalo-nas), immunosuppressive agents, such as calcineurin inhibitors (eg, cyclosporin A, FK-506 ), inhibitors of signal transduction IL-2 (eg, rapamycin), glucocorticoids (eg, prednisone, dexamethasone, methylprednisolone), inhibitors of nucleic acid synthesis (eg, azathioprine, mercaptopurine, acid mycophenolic) and antibodies to lymphocytes and antigen-binding fragments thereof (eg, OKT3, anti-IL2 receptor). Additional suitable therapeutic agents for coadministration with an antagonist of CCR1 function include, for example, anti-inflammatory agents (eg, aspirin, ibuprofen, naproxen, lyophilin), hormoins (eg, adrenocorticotropic hormone (ACTH)), cytokines. -nas (e.g., interferons (e.g., IFNβ-la, IFNβ-lb), Th2-promoting cytokines (e.g., IL-4, TGF-β)) and antibodies, such as antibodies that bind to chemokines, cytokines or cell adhesion molecules (eg, anti-CDll / CD18, anti-MIP-la) and copolymer 1. The term "Th2 promoter cytokine," as used herein, refers to cytokines that inhibit development. Differentiation of Thl cells and / or promote the development / differentiation of Th2 cells. Several "Th2 promoter cytokines" are known in the art and additional "Th2 promoter cytokines" can be identified using suitable methods (see, eg, Lingnau et al., J. Immunol. 161 (9): 4709 -4718 (1998).) The particular co-therapeutic agent selected for administration with an antagonist of CCR1 function will depend on the type and severity of the inflammatory demyelinating disease under treatment, as well as the characteristics of the individual, such as the condition of general health, age, sex, body weight and tolerance to drugs.

For example, in one embodiment, an antagonist of CCR1 function may be administered together with methylprednisolone or ACTH to treat a severe relapse of MS. In another embodiment, an antagonist of the CCR1 function can be administered together with an immunosuppressive agent, IFNβ-la, IFNβ-lb or copolymer 1 during the remitting phase of MS as prophylaxis against recurrence. The person skilled in the art will be able to determine the preferred co-therapeutic agent based on these considerations and other factors. The invention is further related to the use of an antagonist of CCR1 function in therapy (including prophylaxis), for example, as described herein, and with the use of said antagonist for the manufacture of a medicament for the treatment of a disease inflammatory demyelinating (eg, MS). The invention also relates to a medicament for the treatment of an inflammatory demyelinating disease (e.g., EM), wherein said medicament contains 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 treat (reduce or prevent) the inflammatory demyelinating disease. For example, an effective amount of the CCR1 antagonist can be administered before, during and / or after a demyelinating event. When co-administration of an antagonist of the CCR1 function and an additional therapeutic agent to treat the inflammatory demyelinating disease is indicated or desired, the antagonist of the CCR1 function can be administered before, at the same time or after the 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 CNS inflammation, CNS demyelination and / or neurological dysfunction. 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 [Ca2 +] i and / or secretion (eg, degranulation) induced by CCR1 ligands of proinflammatory mediators) and thus inhibit CNS inflammation, CNS demyelination and / or neurological dysfunction. 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 the degree, severity and type of inflammatory demyelinating disease. 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 (eg, 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 by intranasal drops). ) or rectal. 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 a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like. The compounds containing a quaternary ammonium group also contain a counter-anion, such as chloride, bromide, iodide, acetate, per-chlorate and the like. Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reaction with a suitable base, for example a base hydroxide. The salts of acid functional groups contain a countercation, such as sodium, potassium and the like. The antagonist of the CCR1 function can be administered to the individual as part of a pharmaceutical composition for the treatment of an inflammatory demyelinating disease containing a CCR1 antagonist and a pharmaceutically acceptable carrier. The pharmaceutical compositions for coteraction may contain an antagonist of the CCR1 function and one or more additional therapeutic agents. Alternatively, 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 (eg, solution, emulsion, capsule). Suitable pharmaceutical carriers may contain inert ingredients that do not interact with the CCR1 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 solution of Hank, 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 Relay of Biologi- cal 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 Materials and Methods Animals B6 / 129 CCRl - / - mice (also referred to as CCRl KO) were obtained, which are homozygous for a target gene alteration of CCRl, from the laboratory of Dr. Craig Gerard and maintained at Charles River Laboratories (Wilmington, MA) (Gerard et al., J. Clin. Invest. 200 (8): 2022-2027 (1997)). Animals Fl B6 / 129 CCRl + / + were obtained from wild-type Taconic labs (Germantown, NY). Both males (> 20 weeks of age) and females (6-8 weeks of age) were used for the experiments. MOG Peptide The MOG peptide (35-55) (MEVGWYRSPFSRWH-LYRNGK, SEQ ID NO: 1) previously described (Hilton, AA et al., J. Neurochem, 65: 309-318 (1995)) was synthesized on a Synthesizer. Peptidic Applied Biosystems 433A (Applied Biosystems, Foster City, CA). The peptide containing free amino and carboxyl ends was stored as desiccated in amounts of 5 mg at -80 ° C. Immunization Protocol Experiment 1 Young female mice (6-8 weeks) were immunized (CCR1 - / -, n = 10; CCR1 + / +, n = 10) as previously described (Bernard, CC et al., J. Mol. Med. 75: 77-88 (1997)). To explain it briefly, the animals were injected subcutaneously into the ventral abdomen with 100 μg of MOG peptide (35-55) emulsified in complete Freund's adjuvant (Sigma Chemical Co., St. Louis, MO) supplemented with 4 mg / ml (final concentration) of Mycobacterium tuberculosis antigen (Difco, Detroit, MI). The total injected volume was 100 1 divided between two sites. The mice were also injected with Pertussis toxin (Sigma, St. Louis, MO) in isotonic saline (150 ng, I.P.) on the day of immunization with MOG peptide (35-55) and two days later. Experiment 2 Young female mice were immunized (6-8 weeks) (CCRl - / -, n = 8; CCR1 + / +, n = 8) as described above in Experiment 1. Experiment 3 Male adult mice (> 20 weeks) were immunized (CCR1 - / -, n = 10; CCR1 + / +, n = 10 ) as described earlier in Experiment 1. Disease scoring system The mice were weighed and scored on a daily basis based on the following scale: 0 = normal, 1 = loss of tail tone, 2 = paresis posterior, 3 = loss of straightening response, 4 = tetraparesis, moribund. All data were analyzed using a two-tailed Student's T test. RESULTS The CCR1 - / - mice are resistant to the development of Experimental Allergic Encephalomyelitis (EAE). In each experiment, the animals of both groups (CCR1 + / + and CCR1 - / -) began to develop symptoms of disease towards 10 days post-immunization, with the exception of adult male CCR1 + / + mice, which developed symptoms of disease on day 15, and the symptoms of disease were more severe towards approximately 16 days post-immunization. The incidence of the disease in the CCR1 - / - group was lower than in the CCR1 + / + group (Experiment 1: 8/10 vs. 10/10, Experiment 2: 5/8 vs. 8/8, Experiment 3: 1/10 versus 8/10). In each experiment, the mean clinical score in the CCR1 - / - group was lower than that of the CCR1 + / + group at all time points. The difference in the mean clinical score reached significance (p <0.05) on days 11-23 and 36 of Experiment 1, on days 10-28 of Experiment 2 and on day 17 of Experiment 3). After completing each experiment, the maximum clinical score of each animal was determined and the mean score SD for each group was derived (ie CCRl KO animals and CCRl + / + controls). The Student's T test was used to determine if the difference between the groups was statistically significant at the level of p < 0.05 (Table 1).

Table 1 - Mean maximum clinical score of EAE of CCRl KO mice and CCRl + / + controls Microscopic examination Methods On day fourteen of Experiment 2, the animals CCR1 + / + (n = 4) and CCR1 - / - (n = 3) were sacrificed and the selected tissues were excised and fixed in neutral buffered formalin, including brain, cerebellum, brainstem and thoracolumbar spinal cord. After routine processing, the tissues were embedded in paraffin, sectioned at 6 μm, stained with hematoxylin and eosin (H + E) and examined microscopically. Results Inflammation of the CNS is less severe in mice with their CCRl-pressure. All (4 of 4) animals CCRl + / + had lesions of varying severity and distribution in the tissues examined (some animals had an injury at one site) as previously described (Slavin et al., Autoimmuni ty 28: 109-120 (1998)). The lesions consisted of focal multifocal perivascular accumulation of lymphocytes in the Virchow-Robin space. In some lesions, the lymphocytes extended to the adjacent neutrophil for a short distance, resulting in swelling and demyelination of the axons. The most serious lesions were observed in the spinal cords of two animals. In contrast, the lesions were not observed in the examined tissues of CCRl - / - animals. Therefore, CNS inflammation in CCR1 - / - mice is less severe than in CCR1 + / + control mice. EXAMPLE 2 Delayed-type hypersensitivity reaction Mice were sensitized on the shaved abdomen with 25 μl of 2, 4-dinitrofluorobenzene (DNFB, Aldrich, Milwaukee, Wl) at 0.5% in an acetone vehicle: 4: 1 olive oil. (Sigma, St. Louis, MO) on days 1 and 2 of the experiment. On day 5 of the experiment, the animals were anesthetized with metaphane (Pitman / Moore, Mundelein, IL), the thickness of the ear was measured (at 0.001 inches) and 20 μl of 0.5% DNFB was applied in a vehicle of acetone: 4: 1 olive oil to the top and bottom of each ear. On day 6, the animals were sacrificed and the ears were re-measured. The results are expressed as the change in the thickness of the ear. Results Non-sensitized B6 / 129 wild-type mice demonstrated a minimal reaction to treatment with DNFB and sensitized B6 / 129 mice demonstrated a strong reaction to treatment with DNFB. In contrast, non-sensitized CCR1 - / - mice had a modest reaction to DNFB, which was increased by pre-sensitization. Therefore, non-sensitized CCR1 - / - mice produce a hyperresponse to DNFB compared to wild-type mice, but sensitized CCR1 - / - mice respond poorly. Example 3 Proliferation / activation in vi tro Fourteen days after immunization with MOG peptide, B6 / 129 CCR1 - / - and wild-type mice were sacrificed and spleens were extracted. Splenocytes were cultured in 96-well plates at a concentration of 5 x 106 cells / ml in Dulbecco's minimal essential medium (DMEM, Gibco) supplemented with 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, L-glutamine 2 mM, penicillin (100 U / ml), streptomycin (100 U / ml), 10% heat-inactivated bovine serum (Biowhittaker, Walkersville, MD) and 2-mercaptoethanol 5 x 10"5 M (Sigma, St. Louis, MO) For the proliferation assays, serial dilutions (final concentration of 100, 50, 10, 5, 1 μg / ml) of the MOG peptide (35-55) were added to the cultures at the time of establishment. control wells did not contain MOG peptide (33-55) .After 72 hours of culture, 3H-thymidine was added to each well and the plates were cultured for an additional sixteen hours.The cultures were harvested and the amount of 3H- was measured. Thymidine incorporated into cell DNA For cytokine assays, splenocytes were cultured in serum-free medium X-Vi or 20 (Biowhit-taker) containing MOG peptide (35-55) at a final concentration of 100 μg / ml, 10 μg / ml or 1 μg / ml. The control wells did not contain MOG peptide (35-55). The cells were cultured for 40 hours, culture supernatants were collected and the amount of IL-2, IL-6 or IFN-α was measured. in the supernatants by ELISA. Cytokine ELISAs Quantitative ELISAs were performed for IL-2, IL-6 and IFN-? mice using paired monoclonal antibodies (mAbs) specific for particular cytokines according to the recommendations of the antibody supplier (Pharmingen, San Die-go, CA). Briefly, 96-well microtiter plates (Dynatech, Chantilly, VA) were coated overnight at 4 ° C with 5 μg of mouse rat an-ti-cytokine capture purified antibody in 100 μl of carbonate buffer, pH 8 ,2. The plates were then washed three times with PBS containing 0.5% Tween 20 (polyethylenesorbatoyl monolaurate, Sigma, St. Louis, MO), blocked with 3% bovine serum albumin (Sigma, St. Louis, MO) in PBS, washed and incubated with culture supernatants or cytokine standards overnight at 4 ° C. The plates were then washed and incubated with a rat biotinylated anti-cytokine detection antibody for 1 hour. The plates were washed, avidin-peroxidase was added and the plates were incubated for 20 minutes at room temperature. Color was developed with a corapo-nent, the TMB reagent (3, 3 ', 5, 5' -tetramethylbenzidine) (KPL, Gaithersburg, MD). Results Splenocytes isolated from both CCR1 - / - and wild type B6 / 129 mice proliferated and produced IL-2, IL-6 and IFN-α. in response to MOG (35-55). There was no difference in the amount of splenocyte antigen-specific proliferation of CCR1 - / - compared to splenocytes from wild type B6 / 129 mice when stimulated with MOG (35-55) (Figure 5A). Moreover, there was no difference in the amount of IL-2 (Figure 5B) or IL-6 (Figure 5D) produced in splenocyte cultures of CCR1 - / - or wild type B6 / 129 mice. In contrast, splenocytes from CCR1 - / - mice produced more IFN-? splenocytes from wild type B6 / 129 mice after MOG stimulation (35-55) (Figure 5C). The results of the in vitro proliferation / activation assays and the HTR assay demonstrate that the CCR1 - / - mice are not immunosuppressed in general as a result of the deletion of CCR1. Having shown and described this invention in particular with reference 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 deviating from the spirit and scope of the invention as it remains. defined by the appended claims.

LIST OF SEQUENCES < 110 > LeukoSite, Inc. Rottman, James B. Hancock, Wayne W. < 120 > Method of treatment of inflammatory demyelinating disease using CCRl antagonists < 130 > 1855.1065002 < 150 > 09 / 240,253 < 151 > 01-29-1999 < 160 > 1 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 21 < 212 > PRT < 213 > Artificial sequence < 220 > < 223 > Synthetic peptide < 400 > 1 Met Glu Val Gly Trp Tyr Arg Ser By Phe Ser Arg Val Val His Leu 1 5 10 15 Tyr Arg Asn Gly Lys 20

Claims (27)

Claims

1. A method of treating a demyelinating inflammatory disease consisting in administering to a subject in need thereof an effective amount of an antagonist of CCR1 function.

2. The method of Claim 1, wherein said disease is an acute inflammatory demyelinating disease.

3. The method of Claim 2, wherein said acute inflammatory demyelinating disease is selected from the group consisting of acute disseminated encephalomyelitis, Guillain-Barre syndrome and acute hemorrhagic leukoencephalitis.

4. The method of Claim 3, wherein said disease is acute disseminated encephalomyelitis.

5. The method of Claim 3, wherein said disease is Guillain-Barre syndrome.

6. The method of Claim 3, wherein said disease is acute hemorrhagic leukoencephalitis.

7. The method of Claim 1, wherein said disease is a chronic inflammatory demyelinating disease.

8. The method of Claim 7, wherein said chronic inflammatory demyelinating disease is selected from the group consisting of multiple sclerosis and chronic inflammatory demyelinating polyrra-diculoneuropathy.

9. The method of Claim 8, wherein said disease is multiple sclerosis.

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

11. The method of Claim 10, wherein said antagonist of the CCR1 function is a small organic molecule.

12. The method of Claim 10, wherein said antagonist of the CCR1 function is a natural product.

13. The method of Claim 10, wherein said antagonist of the CCR1 function is a peptide.

14. The method of Claim 10, wherein said antagonist of the CCR1 function is a peptidomimetic.

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

16. The method of Claim 15, wherein said pro tein is an anti-CCR1 antibody or an antigen-binding fragment thereof.

17. The method of Claim 1, further comprising administering to said subject an effective amount of an additional therapeutic agent selected from the group consisting of antiviral agents, antibacterial agents, immunosuppressive agents, cytokines, and hormones.

18. The method of Claim 17, wherein said additional therapeutic agent is one or more immunosuppressive agents selected from the group consisting of calcineurin inhibitors, glucocorticoids, inhibitors of nucleic acid synthesis, and antibodies that bind to lymphocytes or fragments of antigen binding of these.

19. The method of Claim 18, wherein said immunosuppressive agent is an inhibitor of calcineurin.

20. The method of Claim 19, wherein said immunosuppressive agent is cyclosporin A.

21. The method of Claim 18, wherein said immunosuppressive agent is a glucocorticoid.

22. The method of Claim 21, wherein said glucocorticoid is prednisone or methylprednisolone.

23. The method of Claim 17, wherein said additional therapeutic agent is a cytokine.

24. The method of Claim 23, wherein said cytokine is an interferon.

25. The method of Claim 23, wherein said cytokine is a Th2 promoter cytokine.

26. The method of Claim 17, wherein said additional therapeutic agent is a hormone.

27. The method of Claim 26, wherein said hormone is adrenocorticotropic hormone.