WO2000044408A2 - Methode de traitement des troubles inflammatoires demyelinisants utilisant des agonistes de la fonction ccr1 - Google Patents

Methode de traitement des troubles inflammatoires demyelinisants utilisant des agonistes de la fonction ccr1 Download PDF

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WO2000044408A2
WO2000044408A2 PCT/US2000/002124 US0002124W WO0044408A2 WO 2000044408 A2 WO2000044408 A2 WO 2000044408A2 US 0002124 W US0002124 W US 0002124W WO 0044408 A2 WO0044408 A2 WO 0044408A2
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ccrl
antagonist
disease
function
mice
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PCT/US2000/002124
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WO2000044408A3 (fr
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James B. Rottman
Wayne W. Hancock
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Millennium Pharmaceuticals, Inc.
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Priority to CA002361256A priority Critical patent/CA2361256A1/fr
Priority to AU27409/00A priority patent/AU2740900A/en
Priority to EP00905778A priority patent/EP1148889A2/fr
Priority to JP2000595710A priority patent/JP2002535376A/ja
Publication of WO2000044408A2 publication Critical patent/WO2000044408A2/fr
Publication of WO2000044408A3 publication Critical patent/WO2000044408A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the inflammatory demyelinating diseases share the common features of inflammation (e.g., leukocyte infiltration,) and destruction of the myelin of the central nervous system (CNS).
  • the pathophysiology of these diseases involves infiltration of the CNS by leukocytes (e.g., T cells, macrophages) and the subsequent development of plaques, which are areas of demyelination.
  • leukocytes e.g., T cells, macrophages
  • plaques which are areas of demyelination.
  • These diseases can be acute (e.g., acute disseminated encephalomyelitis, Guillain-Barre syndrome and acute hemorrhagic leukoencephalitis) or chronic (e.g., multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuropathy ).
  • MS multiple sclerosis
  • the most common demyelinating disease is multiple sclerosis (MS) which affects about 350,000 Americans, and with the exception of trauma, is the most frequent cause of neurological disability commencing in early adulthood to middle age.
  • MS multiple sclerosis
  • 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. Relapsing-remitting MS is characterized by recurrent acute attacks of neurological dysfunction followed by periods when recovery may occur. No progression of neurological impairment occurs between attacks. Secondary progressive MS has a relapsing-remitting course at first, but later, neurological dysfunction progresses (i.e.. becomes more severe) during the period between acute attacks.
  • Primary progressive MS is characterized by a progression of disability from the onset of disease with no distinct acure attacks. Some patients with primary progressive MS can experience periods of apparent clinical stability. The most rare course is referred to as progressive-relapsing MS which is characterized by a progression of disability from the onset of disease with recurrent acute attacks. Insights into the pathogenesis of the demyelinating diseases have been derived from studying experimental allergic encephalomyelitis (EAE) in mice and rats. EAE is an inflammatory demyelinating disease mediated by CD4 * Thl cells which serves as a model for MS.
  • EAE allergic encephalomyelitis
  • EAE EAE
  • MS and other demyelinating diseases appear to be mediated by an autoimmune mechanism which involves the activation and recruitment of lymphocytes (e.g., T cells) that are reactive to certain self-antigens expressed in the CNS.
  • lymphocytes e.g., T cells
  • a genetic predisposition for developing demyelinating disease and environmental factors may trigger the onset of disease.
  • Treatment of the demyelinating disease is primarily focused on inhibiting acute attacks with adrenocorticotropic hormone (ACTH) or immunosuppressive agents (e.g., glucocorticoids). These agents can produce severe systemic side effects and, thus, only moderate and severe attacks are usually treated.
  • Interferons e.g., EFN ⁇ -la, LFN ⁇ -lb
  • copolymer 1 have been used for prophylaxis against recurrent acute attacks (relapse) in MS.
  • systemic reactions e.g., neutralizing antibodies
  • chronic immunosuppression e.g., with methotrexate, azathioprene, cyclophosphamide, 2-chlorodeoxyadenosine
  • methotrexate e.g., methotrexate, azathioprene, cyclophosphamide, 2-chlorodeoxyadenosine
  • the invention relates to a method for treating inflammatory demyelinating diseases.
  • the method comprises administering an effective amount of an antagonist of CCRl function to a subject in need thereof.
  • the invention provides a method of treating an inflammatory demyelinating disease comprising administering to a subject in need thereof an effective amount of an (i.e., one or more) antagonist of CCRl function.
  • the method is directed toward treating an acute inflammatory demyelinating disease, for example, acute disseminated encephalomyelitis, Guillain-Barre syndrome or acute hemorrhagic leukoencephalitis.
  • the method is directed toward treating a chronic inflammatory disease, for example, MS or chronic inflammatory demyelinating polyradiculoneuropathy.
  • the invention provides a method of treating MS.
  • the antagonist of CCRl function is a molecule, such as, a protein, peptide, peptidomimetic, natural product or small organic molecule, that inhibits (reduces, prevents) one or more functions of CCRl .
  • 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 CCRl function and an effective amount of one or more additional therapeutic agents, such as, antiviral, antibacterial and immunosuppressive agents.
  • CFA Freunds complete adjuvant
  • DNFB 2,4-dinitrofluorobenzene
  • Figure 5 A is a graph illustrating dose-dependent proliferation of splenocytes in an in vitro proliferation assay.
  • Splenocytes isolated from CCRl -/- mice or wild type B6/129 mice (CCRl +/+) were cultured with MOG(35-55) peptide. Proliferation was detected by pulsing the cultures with 3 H-thymidine and quantifying the amount of radioactivity incorporated into the DNA of cells.
  • Figure 5B is a graph illustrating dose-dependent production of IL-2 in cultures of splenocytes stimulated with MOG(35-55) peptide.
  • Splenocytes isolated from CCRl -/- mice or wild type B6/129 mice (CCRl +/+) were cultured in serum- free media containing MOG(35-55) peptide 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 dose-dependent production of IFN- ⁇ in cultures of splenocytes stimulated with MOG(35-55) peptide.
  • Splenocytes isolated from CCRl -/- mice or wild type B6/129 mice (CCRl +/+) were cultured in serum- free media containing MOG(35-55) peptide for 40 hours. The culture supernatants were removed and the amount of IFN ⁇ in the supernatants was determined by quantitative ELISA.
  • Figure 5D is a graph illustrating dose-dependent production of IL-6 in cultures of splenocytes stimulated with MOG(35-55) peptide.
  • Chemokines are a family of pro inflammatory mediators that promote recruitment and activation of multiple lineages of leukocytes (e.g., lymphocytes, macrophages). They can be released by many kinds of tissue cells after activation. Continuous release of chemokines at sites of inflammation can mediate the ongoing migration and recruitment of effector cells to sites of chronic inflammation.
  • the chemokines are related in primary structure and share four conserved cysteines, which form disulfide bonds.
  • the family can be divided into distinct branches, including the C-X-C chemokines ( ⁇ -chemokines), and the C-C chemokines ( ⁇ -chemokines), in which the first two conserved cysteines are separated by an intervening residue, or are adjacent residues, respectively (Baggiolini, M. and Dahinden, C. A., Immunology Today, 75:127-133 (1994)).
  • the C-X-C chemokines include a number of potent chemoattractants and activators of neutrophils, such as interleukin 8 (IL-8), PF4 and neutrophil-activating peptide-2 (NAP -2).
  • the C-C chemokines include, for example, RANTES (Regulated on Activation, Normal T Expressed and Secreted), the macrophage inflammatory proteins l and l ⁇ (MlP-l ⁇ and MlP-l ⁇ ), eotaxin and human monocyte chemotactic 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-l have been implicated in human acute and chronic inflammatory diseases including respiratory diseases, such as asthma and allergic disorders.
  • the chemokine receptors are members of a superfamily of G protein-coupled receptors (GPCR) which share structural features that reflect a common mechanism of action of signal transduction (Gerard, C. and Gerard, N.P., Annu Rev. Immunol, 72:775-808 (1994); Gerard, C. and Gerard, N. P., Curr. Opin. Immunol., (5:140-145 (1994)).
  • GPCR G protein-coupled receptors
  • conserveed features include seven hydrophobic domains spanning the plasma membrane, which are connected by hydrophilic extracellular and intracellular loops. The majority of the primary sequence homology occurs in the hydrophobic transmembrane regions with the hydrophilic regions being more diverse.
  • the receptors for the C-C chemokines include: CCRl which can bind, for example, MlP-l ⁇ , RANTES, MCP-2, MCP-3, MCP-4, CKbeta8, CKbeta8-l, leukotactin-1, HCC-1 and MPIF-1; CCR2 which can bind, for example, MCP-1, MCP-2, MCP-3 and MCP-4; CCR3 which can bind, for example, eotaxin, eotaxin-2, RANTES, MCP-2, MCP-3 and MCP-4; CCR4 which can bind, for example, TARC, RANTES, MlP-l ⁇ and MCP-1; CCR5 which can bind, for example, MlP-l ⁇ , RANTES, and MlP-l ⁇ ; CCR6 which can bind, for example, LARC/MIP- 3 ⁇ /exodus; CCR7 which can bind, for example, ELC/MIP-3 ⁇ and CCR8 which can bind
  • the human chemokine receptor CCRl is expressed on a variety of different cells including neutrophils, monocytes, lymphocytes, and eosinophils, and CCRl binds a variety of chemokine ligands (Gong, X. et al, J. Biol. Chem., 272: 11682- 11685 (1997); Wong, M. and E.N. Fish, J. Biol. Chem., 273: 309-341 (1998); and Youn, B.S. et al, J. Immunol., 159: 5201-5205 (1997)).
  • EAE EAE
  • MS MS
  • female mice develop disease more frequently and with greater severity than do male mice. Consequently, EAE is usually studied in young (e.g., 6-9 week old) female mice.
  • EAE was induced in age and sex matched CCRl-/- and
  • mice by immunization with an immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG(35-55); Hilton, A.A., et al. J. Neurochem., (55:309-318 (1995)), and the onset, course and severity of disease was assessed by monitoring neurological dysfunction (e.g., tail tone, posterior paresis, loss of righting response, tetraparesis).
  • MOG(35-55) myelin oligodendrocyte glycoprotein
  • CCRl -/- mice develop less severe clinical symptoms, in comparison with CCRl +/+ control mice, because disruption of CCRl function inhibits CNS inflammation. Further studies demonstrated that the remarkable decrease in severity and incidence of EAE-associated pathology in CCRl -/- mice is not due to a general defect in cellular immunity in these animals. In fact, CCRl -/- mice have the capacity to mount a normal cellular immune response as assessed by a mixed lymphocyte reaction (MLR), and unsensitized CCRl -/- mice are hyper-responsive in a standard delayed type hypersensitivity (DTH) assay (Example 2, Figure 4).
  • MLR mixed lymphocyte reaction
  • DTH delayed type hypersensitivity
  • a first aspect of the invention provides a method for treating an inflammatory demyelinating disease comprising administering to a subject in need thereof an effective amount of an antagonist of CCRl function.
  • CCRl antagonists As used herein, the term "antagonist of CCRl function" refers to an agent
  • an antagonist of CCRl function can inhibit the binding of one or more Hgands (e.g., MlP-l ⁇ , RANTES, MCP-2, MCP-3, MCP-4, CKbeta ⁇ , CKbeta8-l, leukotactin-1, HCC-1, MPIF-1) to CCRl and/or inhibit signal transduction mediated through CCRl (e.g., GDP/GTP exchange by CCRl associated G proteins, intracellular calcium flux).
  • Hgands e.g., MlP-l ⁇ , RANTES, MCP-2, MCP-3, MCP-4, CKbeta ⁇ , CKbeta8-l, leukotactin-1, HCC-1, MPIF-1
  • CCRl -mediated processes and cellular responses can be inhibited with an antagonist of CCRl function.
  • the antagonist of CCRl function is a compound which is, for example, a small organic molecule, natural product, protein (e.g., antibody, chemokine, cytokine), peptide or peptidomimetic.
  • chemokine receptors e.g., CCRl
  • CCRl chemokine receptors
  • Antagonists of CCRl function can be identified, for example, by screening libraries or collections of molecules, such as, the Chemical Repository of the National Cancer Institute (U.S.A.), as described herein or using other suitable methods. Antagonists thus identified can be used in the therapeutic methods described herein.
  • combinatorial libraries can comprise many structurally distinct molecular species.
  • Combinatorial libraries can be used to identify lead compounds or to optimize a previously identified lead.
  • Such libraries can be manufactured by well-known methods of combinatorial chemistry and screened by suitable methods, such as the methods described herein.
  • natural product refers to a compound which can be found in nature, for example, naturally occurring metabolites of marine organisms (e.g., tunicates, algae) and plants and which possess biological activity, e.g., can antagonize CCRl function.
  • marine organisms e.g., tunicates, algae
  • biological activity e.g., can antagonize CCRl function.
  • lactacystin, paclitaxel and cyclosporin A are natural products which can be used as anti-proliferative or immunosuppressive agents.
  • Natural products can be isolated and identified by suitable means.
  • a suitable biological source e.g., vegetation
  • a suitable buffer e.g., water
  • the resulting extract can be assayed for the capacity to antagonize CCRl function, for example, by the assays described herein.
  • Extracts which contain an activity that antagonizes CCRl function can be further processed to isolate the CCRl antagonist by suitable methods, such as, fractionation (e.g., column chromatography (e.g., ion exchange, reverse phase, affinity), phase partitioning, fractional crystallization) and assaying for biological activity (e.g., antagonism of CCRl activity).
  • a natural product can be isolated (e.g., substantially purified) from nature or can be fully or partially synthetic.
  • a natural product can be modified (e.g., derivatized) to optimize its therapeutic potential.
  • natural product includes those compounds which are produced using standard medicinal chemistry techniques to optimize the therapeutic potential of a compound which can be isolated from nature.
  • peptide refers to a compound consisting of from about two to about ninety amino acid residues wherein the amino group of one amino acid is linked to the carboxyl group of another amino acid by a peptide bond.
  • a peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or 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, NY (1989)).
  • a "peptide” can comprise any suitable L- and/or D-amino acid, for example, common ⁇ -amino acids (e.g., alanine, glycine, valine), non- ⁇ -amino acids (e.g., ⁇ -alanine, 4-aminobutyric acid, 6- aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine).
  • the amino, carboxyl and/or other functional groups on a peptide can be free (e.g., 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 know in the art and are disclosed in, for example, Green and Wuts, "Protecting Groups in Organic Synthesis ", John Wiley and Sons, 1991.
  • the functional groups of a peptide can also be derivatized (e.g., alkylated) using art-known methods.
  • Peptides can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry, and can be screened as described herein or using other suitable methods to determine if the library comprises peptides which can antagonize CCRl function. Such peptide antagonists can then be isolated by suitable methods.
  • peptidomimetic refers to molecules which are not polypeptides, but which mimic aspects of their structures. For example, polysaccharides can be prepared that have the same functional groups as peptides which can antagonize CCRl .
  • Peptidomimetics can be designed, for example, by establishing the three dimensional structure of a peptide agent in the environment in which it is bound or will bind to CCRl.
  • the peptidomimetic comprises at least two components, the binding moiety or moieties and the backbone or supporting structure.
  • the binding moieties are the chemical atoms or groups which will react or form a complex (e.g., through hydrophobic or ionic interactions) with CCRl, for example, with the amino acid(s) at or near the ligand binding site.
  • the binding moieties in a peptidomimetic can be the same as those in a peptide antagonist of CCRl.
  • the binding moieties can be an atom or chemical group which reacts with the receptor in the same or similar manner as the binding moiety in a peptide antagonist of CCRl.
  • binding moieties suitable for use in designing a peptidomimetic for a basic amino acid in a peptide are nitrogen containing groups, such as amines, ammoniums, guanidines and amides or phosphoniums.
  • binding moieties suitable for use in designing a peptidomimetic for an acidic amino acid can be, for example, carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, a lower alkyl sulfonic acid ester or a phosphorous acid or ester thereof.
  • the supporting structure is the chemical entity that, when bound to the binding moiety or moieties, provides the three dimensional configuration of the peptidomimetic.
  • the supporting structure can be organic or inorganic. Examples of organic supporting structures include polysaccharides, polymers or oligomers of organic synthetic polymers (such as, polyvinyl alcohol or polylactide). It is preferred that the supporting structure possess substantially the same size and dimensions as the peptide backbone or supporting structure. This can be determined by calculating or measuring the size of the atoms and bonds of the peptide and peptidomimetic. In one embodiment, the nitrogen of the peptide bond can be substituted with oxygen or sulfur, thereby forming a polyester backbone.
  • the carbonyl can be substituted with a sulfonyl group or sulfinyl group, thereby forming a polyamide (e.g., a polysulfonamide).
  • Reverse amides of the peptide can be made (e.g., substituting one or more -CONH- groups for a -NHCO- group).
  • the peptide backbone can be substituted with a polysilane backbone.
  • a polyester peptidomimetic can be prepared by substituting a hydroxyl group for the corresponding ⁇ -amino group on amino acids, thereby preparing a hydroxyacid and sequentially esterifying the hydroxyacids, optionally blocking the basic and acidic side chains to minimize side reactions. Determining an appropriate chemical synthesis route can generally be readily identified upon determining the chemical structure.
  • Peptidomimetics can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using well- known methods of combinatorial chemistry, and can be screened as described herein to determine if the library comprises one or more peptidomimetics which antagonize CCRl function. Such peptidomimetic antagonists can then be isolated by suitable methods.
  • the CCRl antagonist is an antibody or antigen-binding fragment thereof having specificity for CCRl .
  • the antibody can be polyclonal or monoclonal, and the term “antibody” is intended to encompass 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 used herein also encompasses functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies. Functional fragments include antigen-binding fragments which bind to CCRl.
  • antibody fragments capable of binding to CCRl or portions thereof including, but not limited to Fv, Fab, Fab' and F(ab') 2 fragments are encompassed by the invention.
  • Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively.
  • Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab') 2 fragments.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.
  • Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody".
  • the various portions of these antibodies can be joined together chemically 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.
  • Humanized antibodies can be produced using synthetic or recombinant DNA technology using standard methods or other suitable techniques.
  • Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., et al, Nucl Acids Res., 17: 5404 (1989)); Sato, K, et al., Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al, Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P.
  • variants can also be readily produced.
  • cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al, U.S. 5,514,548; Hoogenboom et al, WO 93/06213, published April 1, 1993).
  • Antibodies which are specific for mammalian (e.g., human) CCRl can be raised against an appropriate immunogen, such as isolated and/or recombinant human CCRl or portions thereof (including synthetic molecules, such as synthetic peptides). Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express CCRl, such as activated T cells (see e.g., U.S. Pat. No. 5,440,020, the entire teachings of which are incorporated herein by reference). In addition, cells expressing recombinant CCRl such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor (See e.g.,
  • Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique.
  • a variety of methods have been described (see e.g., Kohler et al, Nature, 256: 495-497 (1975) and Eur. J. Immunol 6: 511-519 (1976); Milstein et al, Nature 266: 550-552 (1977); Koprowski et al, U.S. 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, F.M.
  • a hybridoma is 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.
  • a suitable immortal cell line e.g., a myeloma cell line such as SP2/0 or P3X63Ag8.653
  • the antibody producing cells preferably those obtained 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 which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
  • Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, for example, methods which select recombinant antibody from a library (e.g., a phage display library), or which rely upon immunization of transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies (see e.g., Jakobovits et al, Proc. Natl. 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, U.S. Patent No. 5,545,807; Lonberg et al, WO97/13852).
  • a library e.g., a phage display library
  • transgenic animals e.g., mice
  • the antibody or antigen-binding fragment thereof has specificity for a mammalian CC chemokine receptor- 1 (CCRl), such as human CCRl .
  • CCRl mammalian CC chemokine receptor- 1
  • the antibody or antigen-binding fragment can inhibit binding of a ligand (i.e., one or more Hgands) to CCRl and/or one or more functions mediated by CCRl in respond to ligand binding.
  • a ligand i.e., one or more Hgands
  • Preferred antibody antagonists of CCRl function are disclosed in our co-pending United States Patent Application titled "Anti-CCRl Antibodies and Methods of Use Therefor", by Shixin Qin, Walter Newman and Nasim Kassam, Attorney's docket number LKS97-13, U.S. Serial No. 09/239,938, filed January 29, 1999, and in International Application No. PCT/US99/04527, the teachings of each of these applications are incorporated
  • an agent e.g., proteins, peptides, natural products, small organic molecules, peptidomimetics
  • a suitable screen e.g., high through-put assay.
  • an agent can be tested in an extracellular acidification assay, calcium flux assay, ligand binding assay or chemotaxis assay (see, for example, Hesselgesser et al, J. Biol. Chem. 273(25):15687-15692 (1998) and WO 98/02151).
  • membranes can be prepared from cells which express CCRl, such as THP-1 cells (American Type Culture Collection, Manassas, VA; Accession No. TIB202). Cells can be harvested by centrifugation, washed twice with PBS (phosphate-buffered saline), and the resulting cell pellets frozen at -70 to -85°C.
  • CCRl a cell which express CCRl
  • the frozen pellet can be thawed in ice-cold lysis buffer consisting of 5 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.5, 2 mM EDTA (ethylenediaminetetraacetic acid), 5 ⁇ g/ml each aprotinin, leupeptin, and chymostatin (protease inhibitors), and 100 ⁇ g ml PMSF (phenyl methane sulfonyl fluoride - also a protease inhibitor), at a concentration of 1 to 5 x 10 7 cells/ml, to achieve cell lysis.
  • HEPES N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid
  • 2 mM EDTA ethylenediaminetetraacetic acid
  • PMSF phenyl methane sulfonyl fluoride - also a protease inhibitor
  • the resulting suspension can be mixed well to resuspend all of the frozen cell pellet. Nuclei and cell debris can be removed by centrifugation of 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 ⁇ g/ml each aprotinin, leupeptin, and chymostatin, and 10 ⁇ g/ml PMSF (approximately 0.1 ml per each 10 8 cells). All clumps can be resolved using a minihomogenizer, and the total protein concentration can be determined by suitable methods (e.g., Bradford assay, Lowery assay). The membrane solution can be divided into aliquots and frozen at -70 to -85°C until needed.
  • freezing buffer consisting of 10 mM HEPES pH 7.5, 300 mM sucrose, 1 ⁇ g/ml each aprotinin, leupeptin, and chymostatin, and 10 ⁇ g/ml PMSF (approximately 0.1 ml per each 10 8 cells). All clumps can
  • membrane protein (2 to 20 ⁇ g total membrane protein) can be incubated with 0.1 to 0.2 nM 125 I-labeled RANTES or MlP-l ⁇ with or without unlabeled competitor (RANTES or MLP-l ⁇ ) or various concentrations of compounds to be tested.
  • 125 I-labeled RANTES and 125 I-labeled MIP- 1 ⁇ can be prepared by suitable methods or purchased from commercial vendors (e.g., DuPont- NEN (Boston, MA)).
  • the binding reactions can be performed in 60 to 100 ⁇ l of a binding buffer consisting of 10 mM HEPES pH 7.2, 1 mM CaCl2, 5 mM MgCl2, and 0.5% BSA (bovine serum albumin), for 60 min at room temperature.
  • the binding reactions can be terminated by harvesting the membranes by rapid filtration through glass fiber filters (e.g., GF/B or GF/C, Packard) which can be presoaked in 0.3% polyethyleneimine.
  • the filters can be rinsed with approximately 600 ⁇ l of binding buffer containing 0.5 M NaCl, dried, and the amount of bound radioactivity can be determined by scintillation counting.
  • test agents e.g., compounds
  • IC 50 values the inhibitor concentration required for 50% inhibition (IC 50 values) of specific binding in receptor binding assays
  • Specific binding is preferably defined as the total binding (e.g., total cpm on filters) minus the non-specific binding.
  • Non-specific binding is defined as the amount of cpm still detected in the presence of excess unlabeled competitor (e.g., RANTES or MlP-l ⁇ ).
  • membranes prepared from cells which express recombinant CCRl can be used in the described assay.
  • the capacity of compounds to antagonize CCRl function can also be determined in a leukocyte chemotaxis assay using suitable cells.
  • suitable cells include, for example, cell lines, recombinant cells or isolated cells which express CCRl and undergo CCRl ligand-induced (e.g., MLP-l ⁇ , RANTES, MCP-2, MCP- 3, MCP-4, HCC-1 or MPIF-1) chemotaxis.
  • CCRl -expressing recombinant LI .2 cells see Campbell, et al.
  • peripheral blood mononuclear cells or HL60 cells differentiated with butyric acid can be used in a modification of a transendothelial migration assay (Carr, M.W., et al. T.A., Proc. Natl. Acad. Sci., USA, (97):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 endothelial cell line, ECV 304, obtained from the European
  • Endothelial cells can be cultured on 6.5 mm diameter Transwell culture inserts (Costar Corp., Cambridge, MA) with 3.0 ⁇ m pore size.
  • Culture media for the ECV 304 cells can consist of Ml 99+ 10% FCS, L-glutamine, and antibiotics.
  • the assay media can consist of equal parts RPMI 1640 and Ml 99 with 0.5% BSA. Two hours before the assay, 2xl0 5 ECV 304 cells can be plated onto each insert of the 24 well Transwell chemotaxis plate and incubated at 37°C.
  • Chemotactic factors such as RANTES or MlP-l (Peprotech)(diluted in assay medium) can be added to the 24- well tissue culture plates in a final volume of 600 ⁇ L. Endothelial-coated Transwells can be inserted into each well and 10 6 cells of the leukocyte type being studied are added to the top chamber in a final volume of 100 ⁇ L of assay medium. The plate can then be incubated at 37°C in 5% CO 2 /95% air for 1-2 hours. The cells that migrate to the bottom chamber during incubation can be counted, for example using flow cytometry.
  • ⁇ L of the cell suspension from 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 counting method is highly reproducible and allows gating on the leukocytes and the exclusion of debris or other cell types from the analysis.
  • cells can be counted with a microscope.
  • Assays to evaluate chemotaxis inhibitors can be performed in the same way as control experiment described above, except that antagonist solutions, in assay media containing up to 1% of DMSO co-solvent, can be added to both the top and bottom chambers prior to addition of the cells.
  • Antagonist potency can be determined by comparing the number of cell that migrate to the bottom chamber in wells which contain antagonist, to the number of cells which migrate to the bottom chamber in control wells. Control wells can contain equivalent amounts of DMSO, but no antagonist. The activity of an antagonist of CCRl function can also be assessed by monitoring cellular responses induced by active receptor, using suitable cells expressing receptor.
  • exocytosis e.g., degranulation of cells leading to release of one or more enzymes or other granule components, such as esterases (e.g., serine esterases), perform, and/or granzymes
  • esterases e.g., serine esterases
  • inflammatory mediator release such as release of bioactive lipids such as leukotrienes (e.g., leukotriene C 4 )
  • respiratory burst can be monitored by methods known in the art or other suitable methods (see e.g., Taub, D.D. et al, J. Immunol, 155: 3877-3888 (1995), regarding assays for release of granule-derived serine esterases; Loetscher et al, J.
  • an antagonist of CCRl is identified by monitoring the release of an enzyme upon degranulation or exocytosis by a cell capable of this function.
  • Cells expressing CCRl can be maintained in a suitable medium under suitable conditions, and degranulation can be induced.
  • the cells are contacted with an agent to be tested, and enzyme release can be assessed.
  • the release of an enzyme into the medium can be detected or measured using a suitable assay, such as in an immunological assay, or biochemical assay for enzyme activity.
  • the medium can be assayed directly, by introducing components of the assay
  • the assay can also be performed on medium which has been separated from the cells or further processed (e.g., fractionated) prior to assay.
  • convenient assays are available for enzymes, such as serine esterases (see e.g., Taub, D.D. et al, J. Immunol, 155: 3877-3888 (1995) regarding release of granule-derived serine esterases).
  • cells expressing CCRl are combined with a ligand of
  • CCRl or promoter of CCRl function an agent to be tested is added before, after or simultaneous therewith, and degranulation is assessed. Inhibition of ligand- or promoter-induced degranulation is indicative that the agent is an inhibitor of mammalian CCRl function.
  • the antagonist of CCRl function does not significantly inhibit the function of other chemokine receptors (e.g., CCR2, CXCRl, CCR3).
  • CCRl -specific antagonists can be identified by suitable methods, such as by suitable modification of the methods described herein.
  • cells which do not express CCRl can be created or identified using suitable methods (e.g., transfection, antibody staining, western blot, RNAse protection).
  • suitable methods e.g., transfection, antibody staining, western blot, RNAse protection.
  • Such cells or cellular fractions e.g., membranes
  • the CCRl antagonist can be assayed for the capacity to inhibit the binding of a suitable CCR3 ligand (e.g., RANTES, MCP-3) to the cell or cellular fraction, as described herein.
  • the antagonist of CCRl function is an agent which binds to CCRl .
  • CCRl-binding antagonists can be identified by suitable methods, for example, in binding assays employing a labeled (e.g., enzymatically labeled (e.g., alkaline phosphatase, horse radish peroxidase), biotinylated, radio- labeled (e.g., ⁇ , 14 C, 125 I)) antagonist.
  • a labeled e.g., enzymatically labeled (e.g., alkaline phosphatase, horse radish peroxidase), biotinylated, radio- labeled (e.g., ⁇ , 14 C, 125 I)
  • the antagonist of CCRl function is an agent which can inhibit the binding of a (i.e., one or more) CCRl ligand to CCRl (e.g., human CCRl).
  • the antagonist of CCRl function is an agent which can bind to CCRl and thereby inhibit the binding of a (i.e., one or more) CCRl ligand to CCRl (e.g., human CCRl).
  • the method of treating an inflammatory demyelinating disease comprises administering an effective amount of an (i.e., one or more) antagonist of CCRl function to a subject in need thereof.
  • Treatment includes therapeutic or prophylactic treatment.
  • the severity of disease can be prevented or reduced in whole or in part.
  • the inflammatory demyelinating disease can be an acute inflammatory demyelinating disease, for example, acute disseminated encephalomyelitis, Guillain-Barre syndrome or acute hemorrhagic leukoencephalitis.
  • the inflammatory demyelinating disease can be a chronic inflammatory demyelinating disease, for example, multiple sclerosis, chronic inflammatory demyelinating polyradiculoneuropathy.
  • the invention provides a method of treating (including therapeutic or prophylactic treatment) multiple sclerosis, comprising administering an effective amount of an antagonist of CCRl function to a subject in need thereof.
  • an antagonist of CCRl function can be administered to a patient with a progressive course of MS to retard or prevent the progression of neurological impairment.
  • An antagonist of CCRl function can also be administered to a subject with relapsing-remitting, secondary progressive or progressive-relapsing MS as prophylaxis against relapse (e.g., an acute attack).
  • the antagonist of CCRl function can be administered to a subject with relapsing- remitting MS during the remitting phase of the disease to inhibit (e.g., prevent, delay) relapse.
  • the antagonist of CCRl function is selected from the group of molecules which can inhibit one or more functions of CCRl, for example, certain small organic molecules, natural products, peptides, peptidomimetics and proteins, wherein said proteins are not chemokines or mutants or analogues thereof.
  • the invention provides a method for treating (reducing or preventing) inflammatory demyelinating disease comprising administering an effective amount of an antagonist of CCRl function and an effective amount of an (i.e., one or more) additional therapeutic agent to a subject in need thereof.
  • an antagonist of CCRl function and certain other therapeutic agents can be additive or synergistic when co-administered, thereby providing a highly efficacious treatment.
  • Therapeutic agents suitable for administration with an antagonist of CCRl function include, for example, antiviral agents (e.g., acyclovir, ganciclovir, famciclovir, penciclovir, valacyclovir, vidarabine, foscarnet, indinavir), antibacterial agents (e.g., antibiotics (e.g., erythromycin, penicillin, tetracyclin, ciprofloxacin, norfloxacin, flurazolidone, azithromycin, chloramphenicol), sulfonamides, quinalones), immunosuppressive agents, such as, calcineurin inhibitors (e.g., cyclosporin A, FK-506), IL-2 signal transduction inhibitors (e.g., rapamycin), glucocorticoids (e.g., prednisone, dexamethasone, methylprednisolone), nucleic acid synthesis inhibitors (e.g., aza
  • Additional therapeutic agents suitable for co- administration with an antagonist of CCRl function include, for example, anti- inflammatory agents (e.g., aspirin, ibuprofen, naproxen, lysofylline), hormones (e.g., adrenocorticotropic hormone (ACTH)), cytokines (e.g., interferons (e.g., IFN ⁇ -la, IFN ⁇ -lb), Th2-promoting cytokines (e.g., IL-4, TGF- ⁇ )) and antibodies, such as antibodies that bind chemokines, cytokines or cell adhesion molecules (e.g., anti- CD11/CD18, anti-MIP l ⁇ ) and copolymer 1.
  • anti- inflammatory agents e.g., aspirin, ibuprofen, naproxen, lysofylline
  • hormones e.g., adrenocorticotropic hormone (ACTH)
  • cytokines e.
  • Th2 -promoting cytokine refers to cytokines which inhibit the development/differentiation of Thl cells and/or promote the development/differentiation of Th2 cells.
  • Th2 -promoting cytokines are known in the art and additional "Th2 -promoting cytokines” can be identified using suitable methods (see, for example, Lingnau et al, J. Immunol 161(9):4709-4718 (1998)).
  • the particular co-therapeutic agent selected for administration with an antagonist of CCRl function will depend on the type and severity of inflammatory demyelinating disease being treated as well as the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs.
  • an antagonist of CCRl function can be administered together with methylprednisolone or ACTH to treat a severe relapse of MS.
  • an antagonist of CCRl function can be administered together with an immunosuppressive agent, IFN ⁇ -la, LFN ⁇ -lb or copolymer 1 during the remitting phase of MS as prophylaxis against relapse.
  • an immunosuppressive agent IFN ⁇ -la, LFN ⁇ -lb or copolymer 1 during the remitting phase of MS as prophylaxis against relapse.
  • the invention further relates to use of an antagonist of CCRl function in therapy (including prophylaxis), for example, as described herein, and to the use of such an antagonist for the manufacture of a medicament for the treatment of inflammatory demyelinating disease (e.g., MS).
  • the invention also relates to a medicament for the treatment of inflammatory demyelinating disease (e.g., MS) wherein said medicament comprises an antagonist of CCRl function.
  • a "subject” is preferably a human, but can also be a mammal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, fowl, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
  • domestic animals e.g., dogs, cats, and the like
  • farm animals e.g., cows, sheep, fowl, pigs, horses, and the like
  • laboratory animals e.g., rats, mice, guinea pigs, and the like.
  • An effective amount of the antagonist of CCRl function can be administered to a subject to treat (reduce or prevent) inflammatory demyelinating disease.
  • an effective amount of the antagonist of CCRl function can be administered before, during and/or after a demyelinating episode.
  • the antagonist of CCRl function can be administered before, concurrently with or after administration of the additional therapeutic agent.
  • the antagonist of CCRl function and additional therapeutic agent are administered at different times, they are preferably administered within a suitable time period to provide substantial overlap of the pharmacological activity (e.g., inhibition of CCRl function, immunosuppression) of the agents.
  • the skilled artisan will be able to determine the appropriate timing for co-administration of an antagonist of CCRl function and an additional therapeutic agent depending on the particular agents selected and other factors.
  • an "effective amount" of a CCRl 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.
  • an effective amount is an amount sufficient to inhibit a (i.e., one or more) function of CCRl (e.g., CCRl ligand-induced leukocyte migration, CCRl ligand-induced integrin activation, CCRl ligand-induced transient increase in the concentration of intracellular free calcium [Ca 2+ ], and/or CCRl ligand-induced secretion (e.g.
  • an "effective amount" of an additional therapeutic agent is an amount sufficient to achieve a desired therapeutic and/or prophylactic effect (e.g., immunosuppression).
  • agent e.g., CCRl antagonist, additional therapeutic agent
  • amount of agent (e.g., CCRl 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 tolerance to drugs as well as the degree, severity and type of inflammatory demyelinating disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Typically, an effective amount can range from about 0.1 mg per day to 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 e.g., CCRl antagonist, additional therapeutic agent
  • Parenteral administration can include, for example, intramuscular, intravenous, subcutaneous, or intraperitoneal administration.
  • the agent e.g., CCRl antagonist, additional therapeutic agent
  • Administration can be local or systemic as indicated.
  • the preferred mode of administration can vary depending upon the particular agent (e.g., CCRl antagonist, additional therapeutic agent) chosen, however, oral or parenteral administration is generally preferred.
  • the agent e.g., CCRl antagonist, additional therapeutic agent
  • Salts of compounds containing an amine or other basic group can be obtained, for example, by reacting with a suitable 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 counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like.
  • Salts of compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base, for example, a hydroxide base. Salts of acidic functional groups contain a countercation such as sodium, potassium and the like.
  • the antagonist of CCRl function can be administered to the individual as part of a pharmaceutical composition for treating inflammatory demyelinating disease comprising a CCRl antagonist and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions for co-therapy can comprise an antagonist of CCRl function and one or more additional therapeutic agents.
  • an antagonist of CCRl function and an additional therapeutic agent can be components of separate pharmaceutical compositions which can be mixed together prior to administration or administered separately. Formulation will vary according to the route of administration selected (e.g., solution, emulsion, capsule).
  • Suitable pharmaceutical carriers can contain inert ingredients which do not interact with the antagonist of CCRl function and/or additional therapeutic agent. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
  • Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like.
  • Methods for encapsulating compositions(such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al, "Controlled Release 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.
  • CCRl-/- B6/129 mice also referred to as CCRl KO
  • CCRl KO CCRl-/- B6/129 mice
  • Wild type CCR1+/+ B6/129 FI animals were obtained from Taconic labs (Germantown, NY). Both male (>20 weeks of age) and female (6-8 weeks of age) animals were used for experiments.
  • MOG(35-55) peptide (MEVGWYRSPFSRVVHLYRNGK, SEQ ID NO:l) described previously (Hilton, A.A. et al, J. Neurochem., 65: 309-318(1995)) was synthesized on an Applied Biosystems 433A Peptide Synthesizer (Applied Biosystems, Foster City, CA). The peptide which contains free amino and carboxyl termini was stored as a dessicate in 5 mg quantities at -80°C.
  • Mycobacterium tuberculosis antigen (Difco, Detroit, MI). The total injectate volume was 100 ⁇ l divided between two sites. 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(35-55) peptide and two days later.
  • Pertussis toxin Sigma, St. Louis, MO
  • I.P. isotonic saline
  • mice in both groups began to develop disease symptoms by 10 days post-immunization, with the exception of adult male CCR1+/+ mice which developed disease symptoms on day 15, and disease symptoms were most severe by about 16 days post-immunization.
  • the incidence of disease in the CCRl -/- group was less than in the CCRl +/+ group (Experiment 1: 8/10 vs. 10/10, Experiment 2: 5/8 vs. 8/8, Experiment 3: 1/10 vs. 8/10).
  • CNS inflammation is less severe in CCRl knockout mice. All (4 of 4) CCRl +/+ animals had lesions of variable severity and distribution in the examined tissues (some animals had one lesion at one site) as previously described (Slavin, et al, Autoimmunity, 25:109-120 (1998)). Lesions consisted of focal to multifocal perivascular accumulation of lymphocytes in the space of Virchow-Robin. In some lesions, the lymphocytes extended into the adjacent neutropil for a short distance resulting in axonal swelling and demyelination. The most severe lesions were observed in the spinal cords of two animals. In contrast, lesions were not observed in the examined tissues from CCRl -/- animals. Thus, CNS inflammation in CCRl -/- mice is less severe than in CCRl +/+ control mice.
  • mice were sensitized on the shaved abdomen with 25 ⁇ l of 0.5% 2,4-dinitrofluorobenzene (DNFB, Aldrich, Milwaukee, WI) in a vehicle of 4:1 acetone:olive oil (Sigma, St. Louis, MO) on days 1 and 2 of the experiment.
  • DNFB 2,4-dinitrofluorobenzene
  • a vehicle of 4:1 acetone:olive oil Sigma, St. Louis, MO
  • mice were anesthetized with metafane (Pitman/Moore, Mundelein, IL), ear thickness was measured (to 0.001 inch) and 20 ⁇ l of 0.5% DNFB in a vehicle of 4:1 acetone:olive oil was applied to the top and bottom of each ear.
  • animals were euthanized and ears were measured again. Results were expressed as the change in ear thickness.
  • Non-sensitized wild type B6/129 mice demonstrated minimal reaction to DNFB challenge and sensitized B6/129 mice demonstrated a vigorous reaction to DNFB challenge.
  • non-sensitized CCRl -/- mice had a modest reaction to DNFB, which was enhanced by prior sensitization.
  • non-sensitized CCRl -/- mice are hyper-responsive to DNFB in comparison to wild type mice, but sensitized CCRl -/- mice respond normally.
  • Splenocytes were cultured in 96-well plates at a concentration of 5 x 10 6 cell/ml in Dulbecco's minimal essential media (DMEM; Gibco) supplemented with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 U/ml), 10% heat inactivated bovine serum (Biowhittaker, Walkersville, MD) and 5 x 10 "5 M 2-mercaptoethanol (Sigma, St. Louis, MO).
  • DMEM Dulbecco's minimal essential media
  • splenocytes were cultured in X-Vivo 20 serum free media media (Biowhittaker) containing MOG(35-55) peptide at 100 ⁇ g/ml, 10 ⁇ g/ml or 1 ⁇ g/ml final concentration. Control wells did not contain MOG(33-55) peptide. The cells were cultured for 40 hours, the culture supernatants were collected and the quantity of IL-2, IL-6 or IFN- ⁇ in the supernatants was measured by ELISA.
  • Quantitative ELISAs for murine IL-2, IL-6 and LFN- ⁇ were performed using paired monoclonal antibodies (mAbs) specific for particular cytokines as recommended by the antibody supplier (Pharmingen, San Diego, CA). Briefly, 96- well microtiter plates (Dynatech, Chantilly, VA) were coated overnight at 4°C with 5 ⁇ g of purified rat anti-mouse cytokine capture antibody in 100 ⁇ l of carbonate buffer, pH 8.2. The plates were then washed three times with PBS containing 0.5% Tween 20 (polyethylenesorbate monolaurate; Sigma, St Louis, MO), blocked with 3% bovine serum albumin (Sigma, St.

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Abstract

L'invention se rapporte à une méthode de traitement des troubles inflammatoires démyélinisants. Cette méthode consiste à administrer à un sujet nécessitant un tel traitement une quantité efficace d'un antagoniste de la fonction CCR1. Dans une réalisation préférée, l'invention se rapporte à une méthode de traitement de la sclérose en plaques.
PCT/US2000/002124 1999-01-29 2000-01-27 Methode de traitement des troubles inflammatoires demyelinisants utilisant des agonistes de la fonction ccr1 WO2000044408A2 (fr)

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AU27409/00A AU2740900A (en) 1999-01-29 2000-01-27 Method of treating demyelinating inflammatory disease using ccr1 antagonists
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JP2000595710A JP2002535376A (ja) 1999-01-29 2000-01-27 Ｃｃｒ１アンタゴニストを用いた脱髄性炎症性疾患の治療方法

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WO2002028419A3 (fr) * 2000-10-04 2002-06-13 Applied Research Systems Mutants de chimiokines intervenant dans le traitement de la sclerose en plaques
WO2002028419A2 (fr) * 2000-10-04 2002-04-11 Applied Research Systems Ars Holding N.V. Mutants de chimiokines intervenant dans le traitement de la sclerose en plaques
CZ303409B6 (cs) * 2000-10-04 2012-09-05 Merck Serono Sa Zkrácený a mutovaný lidský chemokin
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HRP20030215B1 (en) * 2000-10-04 2011-09-30 Laboratoires Serono Sa Chemokine mutants in the tratment of multiple sclerosis
US11248051B2 (en) 2002-02-25 2022-02-15 Biogen Ma Inc. Administration of agents for the treatment of inflammation
US7335350B2 (en) 2002-04-04 2008-02-26 Laboratoires Serono Sa Chemokines mutants having improved oral bioavailability
EP1534293A4 (fr) * 2002-06-12 2009-09-16 Chemocentryx Inc Compositions anti-inflammatoires et procedes d'utilisation
EP1534293A1 (fr) * 2002-06-12 2005-06-01 Chemocentryx, Inc. Compositions anti-inflammatoires et procedes d'utilisation
EP1592386A2 (fr) * 2003-01-24 2005-11-09 Elan Pharmaceuticals, Inc. Composition et methode destinees a traiter les maladies de demyelinisation et la paralysie par administration d'agents de remyelinisation
US7576101B2 (en) 2003-01-24 2009-08-18 Elan Pharmaceuticals, Inc. Composition for and treatment of demyelinating diseases and paralysis by administration of remyelinating agents
AU2004207536B2 (en) * 2003-01-24 2010-05-20 Elan Pharmaceuticals Inc. Composition for and treatment of demyelinating diseases and paralysis by administration of remyelinating agents
EP1592387A4 (fr) * 2003-01-24 2009-05-06 Elan Pharm Inc Composition permettant de traiter des maladies dues a la demyelinisation et a la paralysie par administration d'agents de remyelinisation
EP2484381A1 (fr) * 2003-01-24 2012-08-08 Elan Pharmaceuticals Inc. Composition permettant de traiter des maladies dues à la démyélinisation et à la paralysie par administration d'agents de remyélinisation
EP1592386A4 (fr) * 2003-01-24 2007-06-13 Elan Pharm Inc Composition et methode destinees a traiter les maladies de demyelinisation et la paralysie par administration d'agents de remyelinisation
EP1592387A2 (fr) * 2003-01-24 2005-11-09 Elan Pharmaceuticals, Inc. Composition permettant de traiter des maladies dues a la demyelinisation et a la paralysie par administration d'agents de remyelinisation

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CA2361256A1 (fr) 2000-08-03
JP2002535376A (ja) 2002-10-22
EP1148889A2 (fr) 2001-10-31
WO2000044408A3 (fr) 2000-12-14
AU2740900A (en) 2000-08-18

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