WO2001083546A1 - Recepteur car en tant que mediateur du chimiotactisme et/ou de la chemocinetique des cellules migrantes - Google Patents

Recepteur car en tant que mediateur du chimiotactisme et/ou de la chemocinetique des cellules migrantes Download PDF

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Publication number
WO2001083546A1
WO2001083546A1 PCT/US2000/015440 US0015440W WO0183546A1 WO 2001083546 A1 WO2001083546 A1 WO 2001083546A1 US 0015440 W US0015440 W US 0015440W WO 0183546 A1 WO0183546 A1 WO 0183546A1
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Prior art keywords
cells
car receptor
car
subject
cell
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PCT/US2000/015440
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English (en)
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David T. Scadden
Mark C. Poznansky
Ivona T. Olszak
Edward M. Brown
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The General Hospital Corporation
The Brigham And Women's Hospital, Inc.
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Priority to AU2000253227A priority Critical patent/AU2000253227A1/en
Priority to US10/002,854 priority patent/US7176243B2/en
Publication of WO2001083546A1 publication Critical patent/WO2001083546A1/fr
Priority to US11/429,902 priority patent/US7951364B2/en
Priority to US13/118,511 priority patent/US20120329153A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • This invention relates to methods and compositions for modulating movement of eukaryotic cells with migratory capacity. More specifically, the invention relates to methods and compositions for modulating movement of CaR receptor expressing cells of hematopoietic, neural, epithelial, endothelial, or mesenchymal origin, in a specific site in a subject.
  • the foregoing are useful, mter alia, in the treatment of conditions characterized by a need to modulate migratory-cell movement associated with specific sites in a subject. Specific sites include sites of inflammation and modulation of migratory-cell movement is movement away from an agent source, or repulsion.
  • Coli chemotaxis has revealed that a chemical which attracts the bacteria at some concentrations and conditions may also act as a negative chemotactic chemical or chemorepellent at others (Tsang N et al., Science, 1973 , 181 :60-69; Repaske D and Adler J., J Bacteriol, 1981, 145:1196-1208; Tisa LS and Adler J., Proc Natl Aca Sci U.S.A., 1995, 92:10777-10781; Taylor BL and Johnson MS., FEES Lett, 1998, 425:377-381).
  • chemokines capable of inducing cell migration.
  • chemokines which can be produced by virtually every cell type in mammals (Wells, T.N., et al., Trends Pharmacol Sci, 1998, 19:376-380; Baggiolini, M., Nature, 1998, 392:565-568; Luster, A.D., N Engl J Med, 1998, 338:436-445).
  • Chemokines mediate their function via seven-transmembrane, G protein-coupled receptors (7-TMR); the absence of either chemokines or their receptors results in marked phenotypic alterations in mice (Luster. A.D., supra; Ma, Q., et al., Proc Natl Acad Sci U S A, 1998, 95:9448-53; Ma, Q.. et al, Immunity, 1999, 10:463-
  • the calcium-sensing receptor (CaR) is a member of the 7-TMR superfamily and is responsive to Ca ++ concentrations within the millimolar range found in extracellular fluids (Brown, E.M., et al., Nature, 1993, 366:575-80).
  • Activation of the receptor is maximal at 5 mM Ca* ' (Brown, E.M., et al., Vitamins and Hormones, 1999, 55:1-71), and selective CaR activators have been developed that efficiently mimic Ca ++ -induced activation through an allosteric mechanism (e.g., ⁇ PS R-467 and its less active stereoisomer, S-467) ( ⁇ emeth, E.F., et al., Proc Natl Acad Sci USA, 1998, 95:4040-5).
  • Calcimimetics are low molecular weight compounds, termed "calcimimetics", that interact with the CaR's transmembrane domains and potentiate the actions of polycationic agonists, such as Ca ++ itself, which bind to the receptor's amino-terminal extracellular domain.
  • Calcimimetics are currently in clinical trials for treating primary hyperparathyroidism, a disorder in which the CaR is underactive, and represent useful pharmacological tools for assessing the CaR's mediatory role in CaR-expressing cells in which high Ca ++ modulates cellular function.
  • CaR signal transduction is mediated via a pertussis toxin (PTX)-inhibitable G oti pathway as well as a PTX-insensitive mechanism, likely involving G ⁇ q /n (Chen, C.J., et al.,
  • compositions and methods relating to the migration of CaR receptor expressing cells are also described in more detail below.
  • the foregoing can be used, inter alia, in the treatment of conditions characterized by a need to modulate migratory-cell movement in specific sites in a subject. Important such sites include inflammation sites.
  • a method of enhancing migration of CaR receptor expressing cells to a specific site in a subject involves, locally administering to a specific site in a subject in need of such treatment a nonCa ++ CaR receptor agonist in an amount effective to enhance migration of CaR receptor expressing cells to the specific site in the subject.
  • the CaR receptor expressing cells can be hematopoietic cells, immune cells (including antigen presenting cells), neural cells, epithelial cells, endothelial cells (including endothelial cell progenitors), and/or mesenchymal cells.
  • the CaR receptor expressing hematopoietic cells are hematopoietic progenitor cells.
  • the nonCa 4"1" CaR receptor agonist can be ⁇ PS R-467 and/or ⁇ PS S-467.
  • a method of inhibiting migration of CaR receptor expressing cells to a specific site in a subject involves locally administering to a specific site in a subject in need of such treatment a CaR receptor antagonist, in an amount effective to inhibit migration of CaR receptor expressing cells to the specific site in the subject.
  • the specific site is a site of inflammation.
  • the method further comprises co-administering an agent that is not a CaR receptor antagonist that inhibits migration of immune cells to the site of inflammation in the subject.
  • the agent includes an anti-inflammatory agent and/or an immunosuppressant.
  • the subject has an autoimmune disease.
  • the autoimmune disease includes rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, Crohn's disease. Guillain-Barre syndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, systemic lupus erythematosus.
  • the subject has multiple sclerosis, an abscess, a transplant, an implant, atherosclerosis, and/or myocarditis.
  • Preferred CaR receptor expressing cells are as described above.
  • the CaR receptor antagonist is NPS-2143.
  • a method of repelling CaR receptor expressing cells from a material surface involves coating a material surface with an amount of a CaR receptor antagonist effective to repel CaR receptor expressing cells from the material surface.
  • the material surface is part of an implant.
  • the material comprising the implant may be synthetic material or organic tissue material.
  • Important CaR receptor antagonist, cell-types, and so on, are as described above.
  • a method of attracting CaR receptor expressing cells to a material surface involves coating a material surface with a nonCa 44" CaR receptor agonist in an effective amount to attract CaR receptor expressing cells to the material surface.
  • Important nonCa ++ CaR receptor agonists, cell-types, material surfaces and so on, are as described above.
  • a method of enhancing an immune response in a subject having a condition that involves a specific site involves locally administering to the specific site in a subject in need of such treatment a nonCa 4- * " CaR receptor agonist, in an amount effective to enhance immune cell migration to the specific site in the subject.
  • the specific site is a site of a pathogenic infection.
  • the specific site is a germ cell-containing site.
  • the specific site is an area immediately surrounding a tumor.
  • a method for enhancing migration of a cell toward a chemokine involves contacting a cell known to migrate toward a chemokine that is not a CaR receptor agonist with the chemokine and a CaR receptor agonist in a combined amount effective to enhance migration of the cell toward the chemokine, wherein the amount of CaR receptor agonist is effective to potentiate the amount of chemokine versus the same amount of the chemokine if administered without the CaR receptor agonist.
  • Important CaR receptor agonists including Ca ++ ), cell-types, and so on. are as described above.
  • the chemokine is selected from the group consisting of MCP- l, MIP-l ⁇ , and SDF-1.
  • a method for enhancing expression of a chemokine receptor in a cell involves contacting a cell expressing a chemokine receptor with a CaR receptor agonist in an effective amount to enhance expression of the chemokine receptor in the cell.
  • a CaR receptor agonist including Ca ++
  • the chemokine receptor is selected from the group consisting of CCR-2, CCR-5, and CXCR-4.
  • a method for enhancing bone marrow engraftment following bone marrow transplantation is provided.
  • the method involves contacting isolated bone marrow cells to be transplanted with a CaR receptor agonist in an effective amount to increase chemokine receptor expression in the isolated bone marrow cells to enhance bone marrow engraftment following bone marrow transplantation of said cells.
  • a CaR receptor agonist including Ca ++ ), chemokine receptors, and so on, are as described above. If Ca ++ is used as the CaR receptor agonist, the Ca 4.41" concentration used to increase chemokine receptor expression in the isolated bone marrow cells is higher than the Ca 4"1" concentration found in the media in which the isolated bone marrow cells are kept/cultured.
  • the isolated bone marrow cells are hematopoietic progenitor cells.
  • a method for enhancing mobilization of hematopoietic cells in a subject involves administering to a subject in need of such treatment a CaR receptor antagonist in an amount effective to enhance mobilization of hematopoietic cells in the subject.
  • the CaR receptor antagonist is NPS-2143.
  • the hematopoietic cells are hematopoietic progenitor cells.
  • the hematopoietic cells are hematopoietic stem cells.
  • the subject is a bone marrow donor.
  • a method for treating a subject to enhance immune reactivity to a specific antigen in the subject involves administering to a subject in need of such treatment an amount of a CaR receptor agonist together with an amount of a specific antigen, wherein the amount of the CaR receptor agonist is sufficient to enhance in the subject immune reactivity to the specific antigen versus the same amount of the specific antigen if administered without the a CaR receptor agonist.
  • Important CaR receptor agonists (including Ca ++ ) are as described above.
  • the method further comprises co-administering a non-CaR receptor agonist adjuvant.
  • the non-CaR receptor agonist adjuvant is Freund's incomplete adjuvant.
  • a method for treating a subject to enhance immune tolerance in the subject involves administering to a subject in need of such treatment an amount of a CaR receptor antagonist, wherein the amount of the
  • CaR receptor antagonist is sufficient to enhance in the subject immune tolerance to a self or a non-self antigen.
  • Important CaR receptor antagonists are as described above.
  • the subject has an autoimmune disease.
  • the autoimmune disease includes rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre syndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, systemic lupus erythematosus.
  • the subject has multiple sclerosis, an abscess, a transplant, an implant, atherosclerosis, and/or myocarditis.
  • a pharmaceutical preparation is provided.
  • the pharmaceutical preparation comprises a CaR receptor related agent of the invention (CaR receptor agonist or CaR receptor antagonist) in an effective amount to modulate migration of a CaR receptor expressing cell, and a pharmaceutically acceptable carrier.
  • a CaR receptor related agent of the invention CaR receptor agonist or CaR receptor antagonist
  • Important CaR receptor related agents, cell-types, and so on, are as described above.
  • Figure 1(a) Scattergram showing CaR positive stain on CD14 + monocytes (upper panel), and inhibition of anti-CaR antibody binding to CaR by preincubating CD14 + monocytes with CaR peptide (lower panel);
  • Figure 1(h) Graphs showing elevation of CD14 + intracellular
  • Figure 2 Various bar graphs showing that monocytes migrate toward Ca 4-1" in a dose dependent manner that is inhibitable by pretreatment with PTX, genistein or herbimycin and is potentiated by the selective CaR activator, NPS R-467, and the chemokine, MCP-1 ;
  • Figure 2(a) - upper panel checkerboard analysis for transmigration assays were used to determine if Ca* 4" was capable of inducing monocyte chemotaxis
  • Figure 2(a) - lower panel bar graph showing a Ca ++ dose dependent effect;
  • Figure 2(b) bar graph showing chemotaxis of monocytes towards a positive gradient of Ca ++ that is inhibitable by pretreatment with the tyrosine kinase inhibitors, genistein (gen) or herbimycin (herb), but not by the PI-3 kinase inhibitor, wortmannin (wort);
  • the invention involves the discovery that the CaR receptor plays an important role in chemotaxis and chemokinesis of cells.
  • Pharmaceutical compositions containing the foregoing CaR receptor related agents, and various therapeutic and diagnostic methods utilizing the foregoing of CaR receptor related agents, are also described in more detail below.
  • the foregoing can be used, mter alia, in the treatment of conditions characterized by a need to modulate migratory-cell movement in specific sites in a subject. Important such sites include inflammation sites.
  • CaR receptor refers to a polypeptide encoded by the nucleic acid having anyone of GenBank Ace. Nos.: U20759, S81755, or NM_000388, or having a nucleic acid sequence as described in WO9418959, all of which are exressly incorporated herein by reference.
  • CaR receptor related agents refer to molecules that can mimic (agonists) or block (antagonists) an effect of extracellular Ca ++ through the CaR receptor (described above), on a cell expressing such a receptor.
  • the effect is chemotactic and/or chemokinetic (collectively “migratory”) in nature.
  • CaR receptor related agents include CaR receptor agonists, for example, Ca ++ , CaR peptide (NPS Pharmaceuticals, Inc., Salt Lake City, UT), NPS R-467 (NPS Pharmaceuticals), NPS S-467 (NPS Pharmaceuticals), and CaR receptor antagonists, for example NPS-2143 (NPS Pharmaceuticals), agonist binding agents as described below, and the molecules described in US Patents 5,858,684, 5,763, 569, and 5,688,938.
  • binding agents which, for example, can be antibodies or fragments of antibodies having the ability to selectively bind to, for example, CaR receptor agonists and inhibit the migratory properties of the CaR receptor agonist (as described above).
  • the binding agents are antibodies.
  • Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology.
  • the pFc' and Fc regions are effectors of the complement cascade but are not involved in antigen binding.
  • An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an if) F(ab') fragment retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDR3 complementarity determining regions
  • non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody.
  • This is most clearly manifested in the development and use of "humanized” antibodies in which non-human CDRs are covalently 0 joined to human FR and/or Fc/pFc' regions to produce a functional antibody.
  • PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin.
  • Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the
  • FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non- human sequences.
  • the present invention also includes so-called single chain antibodies.
  • a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent. In all embodiments, human subjects and cells are preferred.
  • the chemotactic, chemokinetic (collectively "migratory") activity of the foregoing CaR receptor related agents on the different cell-types of the invention can be detected using any of the transmigration systems described herein (see Examples), or a variety of other systems well known in the art (see, e.g., U.S. patent 5,514,555, entitled: “Assays and therapeutic methods based on lymphocyte chemoattractants,” issued May 7, 1996, to Springer, TA, et al.).
  • CaR receptor expressing cells are cells that express the CaR receptor. Expression can be detected using techniques well known to those of ordinary skill in the art and include nucleic acid hybridization (e.g., Southern, Northern). PCR (Polymerase Chain Reaction), and immunohistochemistry.
  • the CaR receptor expressing cells can be, inter alia, hematopoietic cells, immune cells (including antigen presenting cells), neural cells, epithelial cells, endothelial cells (including endothelial cell progenitors), and or mesenchymal cells.
  • the CaR receptor expressing hematopoietic cells are hematopoietic progenitor cells.
  • Hematopoietic cells”and/or cells of "hematopoietic origin” include, but are not limited to, pluripotent stem cells, multipotent progenitor cells and/or progenitor cells committed to specific hematopoietic lineages.
  • the progenitor cells committed to specific hematopoietic lineages may be of T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage and or lymphoid tissue-specific macrophage cell lineage.
  • the hematopoietic cells may be derived from a tissue such as bone marrow, peripheral blood (including mobilized peripheral blood), umbilical cord blood, placental blood, fetal liver, embryonic cells (including embryonic stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue. Lymphoid soft tissue includes the thymus, spleen, liver, lymph node, skin, tonsil and Peyer's patches.
  • the "hematopoietic origin" cells may be derived from in vitro cultures of any of the foregoing cells, and in particular in vitro cultures of progenitor cells.
  • Immune cells refer to cells of hematopoietic origin (see above) that are involved in the specific recognition of antigens. Immune cells also include antigen presenting cells (APCs). such as dendritic cells or macrophages, B cells, T cells, etc.
  • APCs antigen presenting cells
  • Neurotrophic cells include neurons and glia, and/or cells of both central and peripheral nervous tissue that express RR/B (see, U.S. Patent 5,863,744, entitled: “Neural cell protein marker RR/B and DNA encoding same,” issued January 26, 1999, to
  • epithelial cells include cells of a tissue that covers and lines the free surfaces of the body.
  • epithelial tissue includes cells of the skin and sensory organs, as well as the specialized cells lining the blood vessels, gastrointestinal tract, air passages, ducts of the kidneys and endocrine organs.
  • mesenchymal cells include cells that express typical fibroblast markers such as collagen, vimentin and fibronectin.
  • Cells of endothelial origin are cells of the vasculature that are involved in blood vessel formation (angiogenesis).
  • An embryonic stem cell is a cell that can give rise to cells of all lineages; it also has the capacity to self-renew.
  • a germ cell is a cell specialised to produce haploid gametes. It is a cell further differentiated than a stem cell, that can still give rise to more differentiated germ-line cells.
  • the invention in another part relates to the unexpected discovery that the CaR receptor related agents of the invention (and in particular CaR receptor agonists), enhance migration of a cell toward a chemokine.
  • the method involves contacting a cell known to migrate toward a chemokine that is not a CaR receptor agonist with the chemokine and a CaR receptor agonist in a combined amount effective to enhance migration of the cell toward the chemokine, wherein the amount of CaR receptor agonist is effective to potentiate the amount of chemokine versus the same amount of the chemokine if administered without the CaR receptor agonist.
  • Cytokinines refer to a large family of protein cytokines capable of inducing cell migration.
  • Cytokine is a generic term for nonantibody soluble proteins which are released from one cell subpopulation and which act as intercellular mediators, for example, in the generation or regulation of an immune response. See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal, et al. eds., Blackwell Scientific, Boston, Mass. 1991)
  • Cytokines include, e.g., interleukins IL-1 through IL-15, tumor necrosis factors ⁇ & ⁇ , interferons ⁇ , ⁇ , and ⁇ , tumor growth factor beta (TGF- ⁇ ), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GM-CSF).
  • TGF- ⁇ tumor growth factor beta
  • CSF colony stimulating factor
  • GM-CSF granulocyte monocyte colony stimulating factor
  • the action of each cytokine on its target cell is mediated through binding to a cell surface receptor.
  • Cytokines share many properties of hormones, but are distinct from classical hormones in that in vivo, they generally act locally on neighboring cells within a tissue. The activities of cytokines range from promoting cell growth (e.g., IL-2,
  • the cytokine is a cytokine with chemoattractant and/or chemokinetic properties.
  • cytokines include: PAF, N-formylated peptides.
  • C5a, LTB , LXA 4 chemokines: CXC, IL-8, GCP-2, GRO ⁇ , GRO ⁇ , GRO ⁇ , ENA-78, NAP-2, IP- 10, MIG, I-TAC, SDF-l ⁇ , BCA-1, PF4, Bolekine, MlP-l ⁇ , MlP-l ⁇ , RANTES, HCC-1, MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 (mouse only), Leukotactin-1 (HCC-2, MIP-5), Eotaxin, Eotaxin-2 (MPIF2), Eotaxin-3 (TSC), MDC, TARC, SLC (Exodus-2, 6CKine), MIP-3 ⁇ (LARC, Exodus-1), ELC (MIP-3 ⁇ ), 1-309, DC-CK1 (PARC, AMAC-1), TECK, CTAK, MPIF1 (MIP-3), MIP-5 (HCC-2), HCC-4 (NCC- 4), M
  • the cytokine is a member of the Cys-X-Cys family of chemokines (chemokines that bind to the CXCR-4 receptor).
  • Preferred such agents of the invention include SDF-l ⁇ , SDF-1 ⁇ , and met-SDF-1 ⁇ .
  • such fugetactic agents include other CXCR-4 receptor ligands.
  • CXCR-4 ligands include, but are not limited to, HIV-1 1I1B gpl20, small molecules T134 and MD3100, and/or T22 ([Tyr5,12,Lys7]- polyphemusin II) (Heveker et al., Curr Biol, 1998, 8:369-76).
  • the invention involves a method of repelling or attracting immune cells from and to a material surface.
  • "Material surfaces” as used herein include, but are not limited to, dental and orthopedic prosthetic implants, artificial valves, and organic implantable tissue such as a stent, allogeneic and/or xenogeneic tissue, organ and/or vasculature.
  • Implantable prosthetic devices have been used in the surgical repair or replacement of internal tissue for many years.
  • Orthopedic implants include a wide variety of devices, each suited to fulfill particular medical needs. Examples of such devices are hip joint replacement devices, knee joint replacement devices, shoulder joint replacement devices, and pins, braces and plates used to set fractured bones.
  • Some contemporary orthopedic and dental implants use high performance metals such as cobalt-chrome and titanium alloy to achieve high strength. These materials are readily fabricated into the complex shapes typical of these devices using mature metal working techniques including casting and machining.
  • the material surface is coated or impregnated with an amount of a CaR receptor related agent (a CaR receptor agonist or CaR receptor antagonist) effective to modulate the migration of CaR receptor expressing cells (e.g., immune cells) toward or away from the coated material surface.
  • a CaR receptor related agent a CaR receptor agonist or CaR receptor antagonist
  • the material surface is part of an implant.
  • the material surface may also be coated with a cell-growth potentiating agent, an anti-infective agent, and/or an antiinflammatory agent.
  • Coatings or materials which can be impregnated are well known in the art. Some of the same materials used to make microspheres and the like, described below, may be used. Such materials are natural or synthetic, and may be polymers, gels, hydrogels, proteins, peptides, and the like.
  • a cell-growth potentiating agent as used herein is an agent which stimulates growth of a cell and includes growth factors such as PDGF, EGF, FGF, TGF, NGF, CNTF, and GDNF.
  • An anti-infective agent as used herein is an agent which reduces the activity of or kills a microorganism and includes: Aztreonam; Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane; Pirazmonam Sodium; Propionic Acid; Pyrithione Sodium; Sanguinarium Chloride; Tigemonam Dicholine; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin
  • Cephapirin Sodium Cephradine; Cetocycline Hydrochloride; Cetophenicol;
  • Netilmicin Sulfate Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;
  • Nifurdazil Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole; Nitrocycline;
  • Nitrofurantoin Nitromide; Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline;
  • Oxytetracycline Calcium Oxytetracycline Hydrochloride; Paldimycin; Parachlorophenol;
  • Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V
  • Polymyxin B Sulfate; Porfiromycin; Propikacin; Pyrazinamide; Pyrithione Zinc;
  • Repromicin Rifabutin; Rifametane; Rifamexil; Rifamide: Rifampin; Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; Rosaramicin Butyrate; Rosaramicin
  • Sisomicin Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin;
  • Stallimycin Hydrochloride Steffimycin; Streptomycin Sulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide; Sulfacetamide Sodium; Sulfacytine; Sulfadiazine;
  • Sulfadiazine Sodium Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;
  • Sulfamethizole Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc;
  • Temocillin Tetracycline; Tetracycline Hydrochloride; Tetracycline Phosphate Complex;
  • a method of inhibiting migration of CaR receptor expressing cells to a specific site in a subject involves locally administering to a specific site in a subject in need of such treatment a CaR receptor antagonist, in an amount effective to inhibit migration of CaR receptor expressing cells to the specific site in the subject.
  • the invention provides a method of inhibiting migration of immune cells to a site of inflammation in the subject.
  • “Inflammation” as used herein is a localised protective response elicited by a foreign (non-self) antigen, and/or by an injury or destruction of tissue(s), which serves to destroy, dilute or sequester the foreign antigen, the injurious agent, and/or the injured tissue. Inflammation occurs when tissues are injured by viruses, bacteria, trauma, chemicals, heat, cold, or any other harmful stimuli.
  • T cells classic weapons of the immune system
  • B cells macrophages
  • soluble products that are mediators of inflammatory responses (neutrophils, eosinophils, basophils, kinin and coagulation systems, and complement cascade).
  • a typical inflammatory response is characterized by (i) migration of leukocytes at the site of antigen (injury) localization; (ii) specific and nonspecific recognition of "foreign” and other (necro tic/injured tissue) antigens mediated by B and T lymphocytes, macrophages and the alternative complement pathway; (iii) amplification of the inflammatory response with the recruitment of specific and nonspecific effector cells by complement components, lymphokines and monokines, kinins, arachidonic acid metabolites, and mast cell/basophil products; and (iv) macrophage, neutrophil and lymphocyte participation in antigen destruction with ultimate removal of antigen particles (injured tissue) by phagocytosis.
  • Non-self antigens are those antigens on substances entering a subject, or exist in a subject but are detectably different or foreign from the subject ' s own constituents, whereas "self antigens are those which, in the healthy subject, are not detectably different or foreign from its own constituents.
  • an individual's immune system will identify its own constituents as “non-self,” and initiate an immune response against “self-antigens,” at times causing more damage or discomfort as from, for example, an invading microbe or foreign material, and often producing serious illness in a subject.
  • the inflammation is caused by an immune response against "self-antigen," and the subject in need of treatment according to the invention has an autoimmune disease.
  • "Autoimmune disease” as used herein results when a subject's immune system attacks its own organs or tissues, producing a clinical condition associated with the destruction of that tissue, as exemplified by diseases such as rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus. hemolytic anemias, rheumatic fever.
  • Autoimmune disease may be caused by a genetic predisposition alone, by certain exogenous agents (e.g., viruses, bacteria, chemical agents, etc.), or both.
  • Some forms of autoimmunity arise as the result of trauma to an area usually not exposed to lymphocytes, such as neural tissue or the lens of the eye. When the tissues in these areas become exposed to lymphocytes, their surface proteins can act as antigens and trigger the production of antibodies and cellular immune responses which then begin to destroy those tissues.
  • Other autoimmune diseases develop after exposure of a subject to antigens which are antigenically similar to. that is cross-reactive with, the subject ' s own tissue.
  • rheumatic fever for example, an antigen of the streptococcal bacterium, which causes rheumatic fever, is cross-reactive with parts of the human heart.
  • the antibodies cannot differentiate between the bacterial antigens and the heart muscle antigens, consequently cells with either of those antigens can be destroyed.
  • autoimmune diseases for example, insulin-dependent diabetes mellitus (involving the destruction of the insulin producing beta-cells of the islets of Langerhans), multiple sclerosis (involving the destruction of the conducting fibers of the nervous s ⁇ stem) and rheumatoid arthritis (involving the destruction of the joint-lining tissue), are characterized as being the result of a mostly cell-mediated autoimmune response and appear to be due primarily to the action of T cells (See, Sinha et al., Science, 1990, 248:1380). Yet others, such as myesthenia gravis and systemic lupus erythematosus, are characterized as being the result of primarily a humoral autoimmune response.
  • inhibition of migration of immune cells to a specific site of inflammation involved in any of the foregoing conditions according to the invention is beneficial to the subject since it inhibits escalation of the inflammatory' response, protecting the specific site (e.g., tissue) involved, from "self-damage.”
  • the subject has rheumatoid arthritis, multiple sclerosis, or uveitis.
  • the inflammation is caused by an immune response against "non-self-antigens" (including antigens of necrotic self-material), and the subject in need of treatment according to the invention is a transplant recipient, has atherosclerosis, has suffered a myocardial infarction and/or an ischemic stroke, has an abscess, and/or has myocarditis.
  • non-self-antigens including antigens of necrotic self-material
  • a method for enhancing mobilization of hematopoietic cells in a subject involves administering to a subject in need of such treatment a CaR receptor antagonist in an amount effective to enhance mobilization of hematopoietic cells in the subject.
  • the CaR receptor antagonist is NPS-2143.
  • the hematopoietic cells are hematopoietic progenitor cells.
  • the hematopoietic cells are hematopoietic stem cells.
  • the subject is a bone marrow donor. By enhancing mobilization of bone marrow cells, the need for bone marrow isolation may be obviated.
  • bone marrow cells including hematopoietic stem cells
  • the circulating bone marrow cells can then be easily isolated using techniques well known in the art (for example, utilizing bone marrow cell-specific cell surface markers -e.g., CD34), and be transplanted into a different subject in need of bone marrow transplantation.
  • a method of enhancing migration of CaR receptor expressing cells to a specific site in a subject involves, locally administering to a specific site in a subject in need of such treatment a nonCa ++ CaR receptor agonist in an amount effective to enhance migration of CaR receptor expressing cells to the specific site in the subject.
  • the specific site is a site of a pathogenic infection.
  • the specific site is a germ cell-containing site.
  • the specific site is an area immediately surrounding a tumor. Efficient recruitment of immune cells to help eliminate the infection, unwanted germ-cell and/or tumor, is therefore beneficial.
  • the specific site is a germ cell containing site.
  • the recruitment of immune cells to these specific sites will help eliminate unwanted germ cells, and/or implanted and nonimplanted embryos.
  • co-administration of contraceptive agents other than anti-fugetactic agents is also provided.
  • Non-CaR receptor agonist contraceptive agents are well known in the art.
  • the specific site is an area immediately surrounding a tumor.
  • Non-CaR receptor agonist anti-cancer agents include: Acivicin; Aclarubicin; Acodazole Hydrochloride;
  • Aminoglutethimide Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;
  • Carmustine Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;
  • Cisplatin Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; dacarbazine;
  • Dactinomycin Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine;
  • Eflornithine Hydrochloride Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin
  • Epothilones Erbulozole; Esorubicin Hydrochloride; Estramustine;
  • Fadrozole Hydrochloride Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine
  • Interferon Gamma-I b Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;
  • Leuprolide Acetate Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate;
  • Mitomalcin Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic
  • Tirapazamine Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride;
  • a method of inhibiting tumor cell metastasis in a subject involves locally administering to a tumor site in a subject in need of such treatment a CaR receptor agonist in an amount effective to inhibit metastasis of tumor cells from the tumor site in the subject.
  • the method involves co-administering a cytokine binding agent.
  • the cytokine binding agent is an anti-cytokine antibody or a cytokine agonist.
  • Antibodies are as described earlier in relation to CaR receptor agonist binding agents except that they selectively bind a cytokine.
  • Preferred cytokines are as described elsewhere herein.
  • co-administration of anti-cancer agents other than CaR receptor agonists is also provided.
  • Anti-cancer agents are as described above.
  • a method of inhibiting endothelial cell migration to a tumor site in a subject involves locally administering to an area surrounding a tumor site in a subject in need of such treatment a CaR receptor antagonist in an amount effective to inhibit endothelial cell migration to the tumor site in the subject (to prevent angiogenesis and/or metastasis).
  • the area surrounding the tumor site is not immediate to the tumor site.
  • Important fugetactic agents are as described above.
  • the tumor cell may be of a cancer or tumor type thought to escape immune recognition.
  • Such cancers or tumors may be of the folowing origin: biliary tract cancer; brain cancer, including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer (hepatocarcinoma); lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells
  • cancers or tumors escaping immune recognition include glioma, colon carcinoma, colorectal cancer, lymphoid cell-derived leukemia, choriocarcinoma. and melanoma.
  • the CaR receptor expressing cell is a hematopoietic cell.
  • the hematopoietic cell is an immune cell.
  • the hematopoietic cell is hematopoietic progenitor cell.
  • the foregoing methods of therapy may include co-administration of a non-CaR receptor related agent together with a CaR receptor related agent (CaR receptor antagonist - including Ca ++ -, or antagonist) of the invention that can act cooperatively, additively, or synergistically with the CaR receptor related agent of the invention to modulate migration of CaR expressing cells (e.g., immune cells) to or from a site of inflammation in the subject.
  • Co- administering refers to administering simultaneously two or more compounds of the invention (e.g., a CaR receptor related agent and a non-CaR receptor related agent), as an admixture in a single composition, or sequentially, close enough in time so that the compounds may exert an additive or even synergistic effect, i.e., to modulate migration of CaR expressing cells to or from a site of inflammation.
  • the non-CaR receptor related agents are immunosuppressants.
  • Such immunosuppressants include: Azathioprine; Azathioprine Sodium; Cyclosporine; Daltroban; Gusperimus Trihydrochloride; Sirolimus; Tacrolimus.
  • the non-CaR receptor related agents are anti-inflammatory agents.
  • anti-inflammatory agents include: Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclof
  • Etodolac Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide
  • Fluquazone Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen;
  • Halcinonide Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen
  • Ibuprofen Piconol Inol
  • Ibuprofen Piconol Inonidap
  • Indomethacin Indomethacin Sodium
  • Indoprofen Indoxole
  • Intrazole Isoflupredone Acetate
  • Isoxepac Isoxicam
  • Ketoprofen Lofemizole
  • Pirfenidone Piroxicam
  • Piroxicam Cinnamate Piroxicam Olamine
  • Pirprofen Prednazate
  • Salcolex Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam;
  • Sulindac Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate;
  • a method for treating a subject to enhance immune reactivity toward a specific antigen in the subject involves administering to a subject in need of such treatment an amount of a CaR receptor agonist together with an amount of a specific antigen, wherein the amount of the CaR receptor agonist is sufficient to enhance in the subject immune reactivity toward the specific antigen versus the same amount of the specific antigen if administered without the a CaR receptor agonist.
  • administering a CaR receptor agonist "together with" a specific antigen it is meant that the two agents are administered at the same time, preferably admixed together in a single composition (e.g., the CaR receptor agonist is acting as adjuvant).
  • a “specific antigen”, as used herein, falls into four classes: 1) antigens that are characteristic of a pathogen; 2) antigens that are characteristic of an autoimmune disease; 3) antigens that are characteristic of an allergen; and 4) antigens that are characteristic of a tumor.
  • Antigens in general include polysaccharides, glycolipids, glycoproteins, peptides, proteins, carbohydrates and lipids from cell surfaces, cytoplasm, nuclei, mitochondria and the like.
  • Antigens that are characteristic of pathogens include antigens derived from viruses, bacteria, parasites or fungi.
  • pathogens include vibrio choleras, enterotoxigenic Escherichia coli, rotavirus, Clostridium difficile, Shigella species, Salmonella typhi, parainfluenza virus, influenza virus, Streptococcus pneumonias, Borella burgdorferi,
  • HIV HIV, Streptococcus mutans, Plasmodium falciparum. Staphylococcus aureus, rabies virus and
  • Viruses in general include but are not limited to those in the following families: picornaviridae; caliciviridae; togaviridae; flaviviridae; coronaviridae; rhabdoviridae; filoviridae; paramyxoviridae; orthomyxoviridae; bunyaviridae; arenaviridae; reoviridae; retroviridae; hepadnaviridae; parvoviridae; papovaviridae; adenoviridae; he ⁇ esviridae; and poxyviridae; and viruses including, but not limited to. cytomegalovirus; Hepatitis A,B,C, D, E;
  • HTLV-I Human T-lymphotropic
  • HTLV-II Human he ⁇ esvirus-6
  • HHV-8 Guanarito virus
  • Bartonella henselae Sin regularly virus
  • Sabia virus Sabia virus
  • Exemplary cytomegalovirus epitopes include GP 33-43, NP396-404, and GP276-286.
  • An exemplary influenza epitope includes the HA peptide.
  • Bacteria in general include but are not limited to: P. aeruginosa; Bacillus anthracis; E. coli, Enterocytozoon bieneusi; Klebsiella sp.; Klebsiella pneumoniae; Serratia sp.; Pseudomonas sp.; P. cepacia; Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumoniae; S. aureus; Haemophilus sp.; Haemophilus Influenza; Neisseria Sp.; Neisseria gonorheae;
  • Neisseria meningitis Helicobacter pylori; Bacteroides sp.; Citrobacter sp.; Branhamella sp.;
  • Salmonella sp. Salmonella typhi; Shigella sp.; S. pyogenes; Proteus sp.; Clostridium sp.;
  • Mycobacteria sp. Mycobacterium tuberculosis; Ureaplasma sp.; Streptomyces sp.;
  • EF Anthrax toxin
  • EF Adenylate cyclase toxin
  • Cholera enterotoxin E. coli LT toxin
  • Botulinum Neurotoxin Type B heavy and light chains Tetanus toxin; Tetanus toxin C fragment; Diphtheria toxin; Pertussis toxin; Parvovirus B19; Staphylococcus enterotoxins; Toxic shock syndrome toxin (TSST-1); Erythrogenic toxin; and Vibrio cholerae 0139.
  • Parasites include but are not limited to: Ehrlichia chafeensis; Babesia; Encephalitozoon cuniculi; Encephalitozoon hellem; Schistosoms; Toxoplasma gondii; Plasmodium falciparum,
  • microti Isospore belli, L hominis; Dientamoeba fragiles; Onchocerca volvulus; Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale; Strongyloides stercoralis; Capillaria philippinensis; Angiostrongylus cantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcus granulosus, E. multilocularis; Paragonimus westermani, P. caliensis; Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciola hepatica, Sarcoptes scabiei, Pediculus humanus; Phthirius pubis; and Dermatobia hominis.
  • Fungi in general include but are not limited to: Cryptococcus neoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis; Histoplasfria capsulatum; Coccidioides immitis; Candida species, including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii and C. krusei, Aspergillus species, including A. fumigatus, A. flavus and A. niger, Rhizopus species; Rhizomucor species; Cunninghammella species; Apophysomyces species, including A. saksenaea, A. mucor and A. absidia; Sporothrix schenckii, Paracoccidioides brasiliensis; Pseudallescheria boydii, Torulopsis glabrata; and Dermatophyres species.
  • Antigens that are characteristic of autoimmune disease typically will be derived from the cell surface, cytoplasm, nucleus, mitochondria and the like of mammalian tissues. Examples include antigens characteristic of uveitis (e.g. S antigen), diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, primary myxoedema, thyrotoxicosis, rheumatoid arthritis, pernicious anemia, Addison's disease, scleroderma, autoimmune atrophic gastritis, premature menopause (few cases), male infertility (few cases), juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic opthalmia, phacogenic uveitis, autoimmune haemolytic anemia, idiopathic thrombocylopenic pu ⁇ ura, idi
  • Antigens that are allergens are generally proteins or glycoproteins, although allergens may also be low molecular weight allergenic haptens that induce allergy after covalently combining with a protein carrier (Remington's Pharmaceutical Sciences). Allergens include antigens derived from pollens, dust, molds, spores, dander, insects and foods. Specific examples include the urushiols (pentadecylcatechol or heptadecyicatechol) of Toxicodendron species such as poison ivy, poison oak and poison sumac, and the sesquite ⁇ enoid lactones of ragweed and related plants.
  • Antigens that are characteristic of tumor antigens typically will be derived from the cell surface, cytoplasm, nucleus, organelles and the like of cells of tumor tissue. Examples include antigens characteristic of tumor proteins, including proteins encoded by mutated oncogenes; viral proteins associated with tumors; and tumor mucins and glycolipids.
  • Tumors include, but are not limited to, those from the following sites of cancer and types of cancer: biliary tract cancer; brain cancer, including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic and myelogeneous leukemia; multiple myeloma; AIDS associates leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rec
  • Antigens characteristic of tumors may be proteins not usually expressed by a tumor precursor cell, or may be a protein which is normally expressed in a tumor precursor cell, but having a mutation characteristic of a tumor.
  • An antigen characteristic of a tumor may be a mutant variant of the normal protein-having an altered activity or subcellular distribution. Mutations of genes giving rise to tumor antigens, in addition to those specified above, may be in the coding region, 5' or 3' noncoding regions, or introns of a gene, and may be the result of point mutations frameshifts, deletions, additions, duplications, chromosomal rearrangements and the like.
  • tumor antigens include: proteins such as Ig-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4 of melanoma, Pmel-17 (gp 100) of melanoma,
  • MART-1 Melanoma
  • pi 5 protein of melanoma pi 5 protein of melanoma
  • tyrosinase of melanoma MAGE
  • melanoma thyroid medullary, small cell lung cancer, colon and/or bronchial squamous cell cancer, BAGE of bladder, melanoma, breast, and squamous-cell carcinoma, gp75 of melanoma, oncofetal antigen of melanoma; carbohydrate/lipids such as muci mucin of breast, pancreas, and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes such as mutant p53 of carcinoma, mutant ras of colon cancer and HER21neu proto-onco-gene of breast carcinoma; viral products such as human papilloma virus proteins of squamous cell cancers of cervix and esophagus; and antigens (shown in parenthesis) from the following tumors: acute lymphoblastic leukemia (etv6; amll ; cyclophilin b), gliom
  • proteinaceous tumor antigens may be presented by HLA molecules as specific peptides derived from the whole protein. Metabolic processing of proteins to yield antigenic peptides is well known in the art; for example see U.S. patent 5,342,774 (Boon et al.). and the ones on the lists previously.
  • Antigens may also include: C reactive protein; Coxsackie BI, B2, B3, B4, EI5, B6 proteins; Myelin basic protein; pancreatic beta-cell antigens; arthritis associated antigens (cartilage, aggrecan, type II collagen); AP-1 ; NF-kappaB; desmoglein (Dsg 1 or 3); and alzheimer's associated antigens (prions, amyloid-beta protein), and/or any synthetic agent that binds to the T-cell receptor.
  • the method according to this aspect of the invention further comprises co-administering a non-CaR receptor agonist adjuvant.
  • a "non-CaR receptor agonist adjuvant,” as used herein, refers to an agent that augments, stimulates, activates. potentiates, or modulates the immune response at either the cellular or humoral level. As a result, less vaccine (specific antigen) may be used to produce an immune response toward the antigen.
  • Well known immunologic adjuvants include Freund's adjuvant, beg, and corynebacterium parvum.
  • the non-CaR receptor agonist adjuvant is Freund's incomplete adjuvant.
  • Freund's incomplete adjuvant is prepared by mixing 9 parts Marcol 52 (a white mineral oil of national formulary grade with a viscosity of not more than 37 centistokes at 100 degree F, and a specific gravity range of 0.818 to 0.880 at 77 degree F) and 1 part of Arlacel A (mannide monoleate, purest grade for use in human and veterinary adjuvant formulations) and filtering through a 0.45 ⁇ filter.
  • Marcol 52 a white mineral oil of national formulary grade with a viscosity of not more than 37 centistokes at 100 degree F, and a specific gravity range of 0.818 to 0.880 at 77 degree F
  • Arlacel A mannide monoleate, purest grade for use in human and veterinary adjuvant formulations
  • a method for treating a subject to enhance immune tolerance in the subject involves administering to a subject in need of such treatment an amount of a CaR receptor antagonist, wherein the amount of the CaR receptor antagonist is sufficient to enhance in the subject immune tolerance to a self or a non-self antigen.
  • Important CaR receptor antagonists are as described above.
  • the subject has an autoimmune disease.
  • the autoimmune disease includes rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre syndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, systemic lupus erythematosus.
  • the subject has multiple sclerosis, an abscess, a transplant, an implant, atherosclerosis, and/or myocarditis.
  • compositions are administered in effective amounts.
  • the effective amount will depend upon the mode of administration, the particular condition being treated and the desired outcome. It will also depend upon, as discussed above, the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result. In some cases this is a local (site-specific) reduction of inflammation. In other cases, it is inhibition of tumor growth and/or metastasis. In further cases it is mobilization of stem cells. Generally, doses of active compounds of the present invention would be from about
  • parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
  • a desirable route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing peptides are well known to those of skill in the art.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration.
  • CaR receptor related agents (CaR receptor agonists -including Ca ++ -, or antagonists), may be combined, optionally, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the invention in other aspects includes pharmaceutical compositions of CaR receptor related agents.
  • the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptably compositions.
  • Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • CaR receptor related agents that are nucleic acid or peptide molecules (e.g., CaR peptide) preferably are produced recombinantly, although such molecules may be isolated from biological extracts.
  • Recombinantly produced CaR receptor related agents such as CaR peptides (ligands for the CaR receptor), include chimeric proteins comprising a fusion of a CaR peptide with another polypeptide, e.g., a polypeptide capable of providing or enhancing protein-protein binding, sequence specific nucleic acid binding (such as GAL4). enhancing stability of the CaR peptide under assay conditions, or providing a detectable moiety, such as green fluorescent protein.
  • a polypeptide fused to a CaR peptide or fragment may also provide means of readily detecting the fusion protein, e.g., by immunological recognition or by fluorescent labeling.
  • the CaR receptor related agent if a polypeptide
  • nucleic acids of the invention may be employed for introducing nucleic acids of the invention (CaR peptide sense and anti-sense, CaR receptor dominant negative, etc.) into cells, depending on whether the nucleic acids are introduced in vitro or in vivo in a host.
  • Such techniques include transfection of nucleic acid-CaPO precipitates, transfection of nucleic acids associated with DEAE, transfection with a retrovirus including the nucleic acid of interest, liposome mediated transfection, and the like.
  • a vehicle used for delivering a nucleic acid of the invention into a cell can have a targeting molecule attached thereto.
  • a targeting molecule e.g., a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or inco ⁇ orated within the nucleic acid delivery vehicle.
  • proteins which bind to a surface membrane protein associated with endocytosis may be inco ⁇ orated into the liposome formulation for targeting and/or to facilitate uptake.
  • Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like.
  • Polymeric delivery systems also have been used successfully to deliver nucleic acids into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acids.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the CaR related agent, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • sylastic systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • peptide based systems such as fatty acids
  • wax coatings such as those described in U.S. Patent Nos.
  • a preferred delivery system of the invention is a colloidal dispersion system.
  • Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system of the invention is a liposome.
  • Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 ⁇ m can encapsulate large macromolecules. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., (1981) 6:77).
  • LUV large unilamellar vessels
  • a liposome In order for a liposome to be an efficient gene transfer vector, one or more of the following characteristics should be present: (1) encapsulation of the gene of interest at high efficiency with retention of biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information.
  • Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[l-(2, 3 dioleyloxy)-propyl]- N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA dimethyl dioctadecylammonium bromide
  • Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis, G. in Trends in Biotechnology, (1985) 3:235-241.
  • the preferred vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System”).
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient.
  • the CaR receptor related agents described herein are encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere
  • a CaR receptor related agent is dispersed throughout a solid polymeric matrix
  • a microcapsule wherein a CaR receptor related agent is stored in the core of a polymeric shell
  • polymeric matrix for containing a CaR receptor related agent examples include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced.
  • the size of the polymeric matrix further is selected according to the method of delivery which is to be used.
  • the polymeric matrix and CaR receptor related agent are encompassed in a surfactant vehicle.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.
  • the delivery system is a biocompatible microsphere that is suitable for local, site-specific delivery.
  • microspheres are disclosed in Chickering et al., Biotech. And Bioeng, 1996, 52:96-101, and Mathiowitz et al., Nature, 1997, 386:410- 414.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the CaR receptor related agents of the invention to the subject.
  • Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers. Synthetic polymers are preferred. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multi-valent ions or other polymers.
  • CaR receptor related agents can be delivered using a bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,
  • non-biodegradable polymers examples include ethylene vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, et al., in Macromolecules, 1993, 26:581-587, the teachings of which are inco ⁇ orated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • implantable pumps include controlled-release microchips.
  • a preferred controlled-release microchip is described in Santini, JT Jr., et al., Nature, 1999, 397:335-338, the contents of which are expressly inco ⁇ orated herein by reference.
  • Long-term sustained release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • the isolated CaR receptor related agents of the invention are delivered directly to the site at which there is inflammation, e.g., the joints in the case of a subject with rheumatoid arthritis, the blood vessels of an atherosclerotic organ, etc.
  • this can be accomplished by simple injection, or by attaching an isolated CaR receptor related molecule (agonist/antagonist) to the surface of a balloon catheter; inserting the catheter into the subject until the balloon portion is located at the site of inflammation, e.g. an atherosclerotic vessel, and inflating the balloon to contact the balloon surface with the vessel wall at the site of the occlusion.
  • the compositions can be targeted locally to particular inflammatory sites to modulate immune cell migration to these sites.
  • the local administration involves an implantable pump to the site in need of such treatment.
  • Preferred pumps are as described above.
  • the CaR receptor related agent may be delivered topically, e.g., in an ointment/dermal formulation.
  • the CaR receptor related agents of the invention are delivered in combination with a non-CaR receptor related agents (e.g., antiinflammatory, immunosuppressant, etc.).
  • the isolated CaR receptor related agents of the invention are administered to a subject in combination with a balloon angioplasty procedure.
  • a balloon angioplasty procedure involves inserting a catheter having a deflated balloon into an artery.
  • the deflated balloon is positioned in proximity to the atherosclerotic plaque and the site of inflammation, and is inflated such that the plaque is compressed against the arterial wall.
  • the layer of endothelial cells on the surface of the artery is disrupted, thereby exposing the underlying vascular smooth muscle cells.
  • the isolated CaR receptor related molecule is attached to the balloon angioplasty catheter in a manner which permits release of the isolated CaR receptor related molecule at the site of the atherosclerotic plaque and the site of inflammation.
  • the isolated CaR receptor related agent may be attached to the balloon angioplasty catheter in accordance with standard procedures known in the art.
  • the isolated CaR receptor related agent may be stored in a compartment of the balloon angioplasty catheter until the balloon is inflated, at which point it is released into the local environment.
  • the isolated CaR receptor related agent may be impregnated on the balloon surface, such that it contacts the cells of the arterial wall as the balloon is inflated.
  • the CaR receptor related agent also may be delivered in a perforated balloon catheter such as those disclosed in Flugelman, et al., Circulation, 1992, 85:11 10-1 117. See, also, e.g., published PCT Patent Application WO 95/23161, for an exemplary procedure for attaching a therapeutic protein to a balloon angioplasty catheter. This procedure can be modified using no more that routine experimentation to attach a therapeutic nucleic acid to the balloon angioplasty catheter.
  • CD14 + monocytes Low density cells were isolated from human and mouse peripheral blood using Ficoll-Hypaque (Pharmacia, Piscataway, NJ). CD14 + monocytes were purified by sorting on FACSVantage (Becton Dickinson, San Jose, CA) based on CD14 expression. Purified cells were incubated in either calcium-free medium (Hanks' Balanced Salt Solution IX, CellGro, Herndon. VA) or Iscove's Medium IX (Mod)(CellGro, Herndon, VA) which contains 1.5 mM Ca ++ .
  • the medium was subsequently supplemented with 0.5 mM, 1 mM, 2 mM, 3 mM or 5 mM calcium chloride (CaCl 2 ) (Sigma) to achieve the desired level of extracellular calcium, or with 1 ⁇ M of the selective CaR activator, NPS R-467, or its less active stereoisomer, NPS S-467, in 3 mM Ca ++ (NPS Pharmaceuticals, Inc., Salt Lake City, UT). Cells were incubated for up to 24 hours in 5% CO humidified air. Monocytes were then detached from the plate using a cell scraper. All calcium concentrations indicated in the presentation of the data represent the total Ca ++ present in the basal medium plus any added Ca 4 ". Transmigration assays.
  • Transwells (5 ⁇ m pore size polycarbonate membrane, 12 mm diameter) (Costar, Corning, NY) were used to assess cell migration using an established methodology (25).
  • the concentration of Ca ++ in IMDM (CellGro, Herndon, VA) in the upper and lower chambers of the transwell were adjusted according to a checkerboard analysis of chemotaxis. Calcium concentrations varied from OmM to 6.5mM.
  • Purified monocytes (1 x 10 4 ) were then placed in the top chamber with 150 ⁇ l of IMDM (CellGro, Herndon, VA).
  • Cytosolic calcium changes Purified monocytes were loaded with Indo-1/AM (Molecular Probes, Eugene, OR). Cells were collected on FACSVantage for 30 sec to establish a baseline emission value for Indo-1/AM. Chemokine ligands were then added to cells incubated in Ca ++ - free media, or those supplemented with 1.5 mM, or 4.5 mM CaCl 2 , or with the selective calcium receptor activator, NPS R-467. or the less active S-467 (1 ⁇ M) (A gift of E. Nemeth,
  • Chemokine receptor expression and flow cytometric analysis Purified monocytes were incubated in a Ca + -free medium supplemented with 1 mM, 3 mM or 5 mM CaCl or with 1.5 mM CaCl 2 plus 1 ⁇ M NPS R-467 or S-467. Cells were washed once in PBS with 1% FCS and resuspended in 100 ⁇ l of Ca ++ -free PBS. Monoclonal antibody against the CaR was added to the cells and incubated alone or with the CaR peptide for 30 min at room temperature.
  • Flow cytometric analysis was performed using a dual laser FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA) calibrated using 2 ⁇ m Calibrite beads (Becton Dickinson). Data acquisition and analysis were performed using CellQuest software (Becton Dickinson).
  • CaR-/- were bred as previously described (Ho, C, et al., Nat Genet 1995, 1 1 :389-94). To produce CaR-/- mice for this study, heterozygote mice were intercrossed. The mice studied were either 129S6/svev or 129S6/svev /Swiss Webster mixed genetic background. Mice homozygous for the CaR knockout allele do not live longer than three weeks. Mice were genotyped using the following protocol. A l ⁇ l sample of DNA sample was obtained from a ⁇ 5mm section of the tail of each mouse to be genotyped by according to established techniques !9 .
  • mice were obtained.
  • Peripheral blood was obtained from each genotype of mouse at sacrifice at day 6 to 8 post partum.
  • Biologic activity of ionic calcium in vivo C57BL/6 mice (Jackson Laboratories) were injected subcutaneously at a marked, shaved spot on the abdomen with 20 ⁇ l of 5 mM CaCl 2 or 20 ⁇ l of
  • MCP-1 at a concentration of 10 ng/ml, with a combination of MCP-1 and 5 mM CaCl 2 or with
  • mice 10 ⁇ M NPS R-467 in PBS.
  • Control mice were injected subcutaneously with 20 ⁇ l PBS alone. All subcutaneously injected agents contained less than 0.004 ng/ml of LPS by the limulus assay (Sigma) as previously described (Bleul, C.C., et al., J Exp Med. 1996, 184:1 101-1 109).
  • mice were sacrificed by CO asphyxiation 18 hours after injection, and injection sites were excised, snap-frozen and 4 ⁇ m cryosections were obtained and stained with hematoxylin and eosin. Immunocytochemistry was performed on the frozen sections using alkaline phosphatase- conjugated Mac-1 antibody (Pharmingen) as previously described and assessed by light microscopy (Bleul, C.C., et al., supra). Results
  • CD14 + PBMC, and CXCR-4 was present on 87%; co-expression of the CaR with CCR2 was noted in 97% of CD14 ⁇ PBMC and in 83% with CXCR4.
  • Functional responses of monocytes to activation of receptors for chemokines were then measured in the presence or absence of CaR stimulation using transmigration assays.
  • Chemotaxis of CD14 + PBMC to a positive concentration gradient of MCP-1 was dependent upon extracellular calcium, as has been reported by others (Sozzani, S., et al. J Immunol, 1991 , 147:2215-21 ; Sozzani, S..
  • Ionic calcium is highly regulated in vivo, yet can be elevated in specific tissue microenvironments such as the bone marrow and is particularly altered in the context of ongoing cell death and inflammation. Hypothesizing that this phenomenon may serve as a primitive regulator of immune cells, we examined a number of hematopoietic cell types for expression of the calcium sensing receptor. While this receptor is expressed on primitive
  • the calcium sensing receptor is a 7-membrane
  • ionic calcium serves to directly and indirectly influence monocyte migratory response. It can play a role in the tissue localization of primary monocytes and may potentiate protein-mediated induction of chemotaxis.
  • the contribution of this signal to inflammation is dependent upon local calcium concentrations and therefore may be relevant for settings of either extensive injury or in the context of chronic inflammation.
  • the CaR induction of monocyte infiltration provides insight regarding the persistence of inflammation at sites of prior injury subsequent to resolution of the inciting event. The presence of local calcium may further monocytic recruitment, thereby pe ⁇ etuating inflammatory infiltration. Sites of local accumulation of calcium such as atherosclerotic plaques, granulomata, calcific tendonitis or calcium pyrophosphate disease (pseudogout) may provide a reservoir of calcium ions serving to encourage monocyte localization. Interruption of this chemokinetic stimulus through specific inhibition of the CaR therefore provides a therapeutic opportunity in these disease contexts.
  • FIG. 1 CD14 + monocytes stain positively for the CaR, and binding of anti-CaR antibody is inhibitable by preincubation with CaR peptide.
  • Purified peripheral blood CD 14+ monocytes were exposed to anti-CaR antibody (solid area in histogram) or isotype control (open area) and examined by flow cytometry. Monocytes were also preincubated with CaR peptide prior to staining with anti-CaR antibody (dashed area). Data represent one often independent experiments with comparable results.
  • Figure lb Elevating the extracellular Ca ++ concentration or addition of the selective CaR activator, NPS R-467, induces elevation in cytosolic Ca ++ .
  • FIG. 2 Monocytes migrate toward Ca ++ in a dose dependent manner that is inhibitable by pretreatment with PTX, genistein or herbimycin and is potentiated by the selective CaR activator, NPS R-467, and the chemokine, MCP-1. Transmigration assays were used to determine if Ca ++ was capable of inducing monocyte chemotaxis within a checkerboard analysis ( Figure 2a - upper panel). An input of 1 x 10 monocytes was utilized and the mean number of cells migrating in response to each Ca ++ gradient are shown. The results of three independent experiments are shown.
  • Table 1 Exposure of CD14 monocytes to Ca 4 " enhances CCR2 expression in a dose dependent manner. Monocytes were exposed to varying concentrations of Ca”" and to the selective CaR activator, NPS R-467 (1 ⁇ M), or to NPS S-467 (1 ⁇ M) in the presence of 1.5 mM Ca 4 ". The median fluorescence intensity for CCR2 expression was measured for each Ca 4 " concentration and in the presence of NPS R-467 or S-467. Mean values +/- s.e.m. from three independent experiments are shown. Table 1.

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Abstract

L'invention concerne des méthodes et des compositions permettant de moduler le mouvement de cellules eucaryotes ayant une capacité de migration. D'une manière plus spécifique, l'invention concerne des méthodes et des compositions permettant de moduler le mouvement du récepteur CaR exprimant des cellules d'origine hématopoïétique, neurale, épithéliale, endothéliale ou mésenchymateuse, dans un site spécifique chez un sujet. Ces méthodes et ces compositions conviennent notamment pour le traitement de maladies caractérisées par le besoin de moduler le mouvement de migration cellulaire associé à des sites spécifiques chez un sujet. Ces sites spécifiques comprennent des sites d'inflammation et la modulation du mouvement de migration cellulaire consiste en un mouvement d'éloignement d'une source d'agent ou répulsion.
PCT/US2000/015440 2000-05-01 2000-06-02 Recepteur car en tant que mediateur du chimiotactisme et/ou de la chemocinetique des cellules migrantes WO2001083546A1 (fr)

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US11/429,902 US7951364B2 (en) 2000-05-01 2006-05-08 CaR receptor as a mediator of migratory cell chemotaxis and/or chemokinesis
US13/118,511 US20120329153A1 (en) 2000-05-01 2011-05-30 Car receptor as a mediator of migratory cell chemotaxis and/or chemokinesis

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Publication number Priority date Publication date Assignee Title
US7176243B2 (en) 2000-06-02 2007-02-13 The General Hospital Corporation CaR receptor as a mediator of migratory cell chemotaxis and/or chemokinesis
WO2008141268A2 (fr) * 2007-05-10 2008-11-20 Smith & Nephew, Inc. Amélioration du récepteur sensible au calcium
US9775816B2 (en) 2013-11-07 2017-10-03 The General Hospital Corporation Eluting matrix and uses thereof

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US5686574A (en) * 1992-02-26 1997-11-11 The General Hospital Corporation Constitutive activator of retinoid acid response (car) receptor fusion protien

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US5686574A (en) * 1992-02-26 1997-11-11 The General Hospital Corporation Constitutive activator of retinoid acid response (car) receptor fusion protien
US5756448A (en) * 1992-02-26 1998-05-26 The General Hospital Corporation Constitute activator of retinoid (CAR) receptor polypeptides

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7951364B2 (en) 2000-05-01 2011-05-31 The General Hospital Corporation CaR receptor as a mediator of migratory cell chemotaxis and/or chemokinesis
US7176243B2 (en) 2000-06-02 2007-02-13 The General Hospital Corporation CaR receptor as a mediator of migratory cell chemotaxis and/or chemokinesis
WO2008141268A2 (fr) * 2007-05-10 2008-11-20 Smith & Nephew, Inc. Amélioration du récepteur sensible au calcium
WO2008141268A3 (fr) * 2007-05-10 2009-07-02 Smith & Nephew Inc Amélioration du récepteur sensible au calcium
US9775816B2 (en) 2013-11-07 2017-10-03 The General Hospital Corporation Eluting matrix and uses thereof
US9849094B2 (en) 2013-11-07 2017-12-26 The General Hospital Corporation Eluting matrix and uses thereof
US9849159B2 (en) 2013-11-07 2017-12-26 The General Hospital Corporation Eluting matrix and uses thereof
US10580262B2 (en) 2013-11-07 2020-03-03 The General Hospital Corporation Eluting matrix and uses thereof

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