US20120213800A1 - Methods for treating and diagnosing disease - Google Patents

Methods for treating and diagnosing disease Download PDF

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US20120213800A1
US20120213800A1 US13/400,831 US201213400831A US2012213800A1 US 20120213800 A1 US20120213800 A1 US 20120213800A1 US 201213400831 A US201213400831 A US 201213400831A US 2012213800 A1 US2012213800 A1 US 2012213800A1
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gpbp
alkyl
alkoxy
subject
binding
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Juan Saus
Fernando Revert
Ramón Merino
Jesús Merino
Francisco Revert-Ros
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FibroStatin SL
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FibroStatin SL
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Assigned to FIBROSTATIN, S.L. reassignment FIBROSTATIN, S.L. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: REVERT-ROS, FRANCISCO, MERINO, JESUS, MERINO, RAMON, REVERT, FERNANDO, SAUS, JUAN
Publication of US20120213800A1 publication Critical patent/US20120213800A1/en
Priority to US15/485,827 priority patent/US10633455B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11009—Goodpasture-antigen-binding protein kinase (2.7.11.9)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Definitions

  • NC1 domain of the ⁇ 3 chain of the basement membrane collagen IV [ ⁇ 3(IV)NC1] depends in part on phosphorylation.
  • Goodpasture Antigen Binding Protein GPBP, 77 kD GPBP or GPBP-1) (WO 00/50607; WO 02/061430) is a non-conventional protein kinase that catalyzes the conformational isomerization of the ⁇ 3(IV)NC1 domain during its supramolecular assembly, resulting in the production and stabilization of multiple ⁇ 3(IV)NC1 conformers in basement membranes.
  • the present invention provides methods for treating chronic kidney disease (CKD), comprising administering to a subject in need thereof an amount effective of an inhibitor of 77 kD GPBP to treat the CKD.
  • CKD chronic kidney disease
  • the present invention provides methods for treating immune complex-mediated glomerulonephritis (GN), comprising administering to a subject in need thereof an amount effective of an inhibitor of 77 kD GPBP to treat the immune complex-mediated GN.
  • the immune complex-mediated GN is associated with an autoimmune disorder selected from the group consisting of IgA nephropathy, systemic lupus erythematosus (SLE) and Goodpasture disease.
  • the present invention provides methods for treating pulmonary fibrosis (PF), comprising administering to a subject in need thereof an amount effective of an inhibitor of 77 kD GPBP to treat the PF.
  • PF pulmonary fibrosis
  • the PF is non-idiopathic PF.
  • the subject is preferably a human.
  • any suitable 77 kD GPBP inhibitor may be used; in one embodiment, the inhibitor is an antibody that binds to 77 kD GPBP, such as an antibody selective for the 77 kD isoform of GPBP.
  • the GPBP inhibitor comprises a polypeptide comprising an amino acid sequence according to the general formula X1-SHClX2-X3 (SEQ ID NO: 2), wherein:
  • X1 is 0-10 amino acids of the sequence ATTAGILATL (SEQ ID NO: 3);
  • X2 is E or Q
  • X3 is 0-10 amino acids of the sequence LMVKREDSWQ (SEQ ID NO: 4).
  • the inhibitor comprises a compound of formula (I):
  • R is selected from N and CR 5 ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), or (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , (aryl)C 1 -C 6 alkyl, or (heteroaryl)C 1 -C 6 alkyl;
  • R 3 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 1 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5
  • R 4 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), benzyloxy, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy), —O(CH 2 ) 1-5 —C(O)OH, —O(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), (aryl)C 1
  • the present invention provides methods for diagnosing rheumatoid arthritis (RA) or pulmonary fibrosis (PF) comprising
  • the 77 kD GPBP-binding molecule comprises an antibody that binds to native 77 kD GPBP, such as an antibody is selective for the 77 kD isoform of GPBP.
  • the detecting comprises use of a technique selected from the group consisting of ELISA, immunoflourescence, and chromatography.
  • the subject may be suffering from one or more symptoms selected from the group consisting of: one or more swollen joints, one or more stiff joints, one or more warm joints, joint stiffness early in the morning that typically lasts for more than an hour, excess synovial fluid, development of fibrous tissue in the synovium, and impaired range of joint movement.
  • the subject may be suffering from one or more symptoms selected from the group consisting of: chronic dry coughing, fatigue, weakness, chest discomfort, loss of appetite, rapid weight loss, and dyspnea with exertion.
  • the methods may further comprise determining the subject's plasma level of C reactive protein (CRP), comparing the amount of CRP in the subject's plasma to control, and using the CRP comparison in aiding the diagnosis of RA or PF.
  • CRP C reactive protein
  • the invention provides methods for diagnosing CKD or immune complex-mediated GN, comprising
  • the present invention provides methods for identifying compounds to treat CKD, immune complex-mediated GN, and/or PF, comprising contacting a 77 kD GPBP-77 kD GPBP-substrate binding complex with one or more test compounds under binding conditions, wherein those test compounds that displace 77 kD GPBP binding from the binding complex are candidate compounds for treating CKD, immune complex-mediated GN, and/or PF.
  • the present invention provides methods for identifying compounds to treat CKD, immune complex-mediated GN, and/or PF, comprising contacting a 77 kD GPBP-substrate under binding conditions with
  • test compounds that outcompete 77 kD GPBP for binding to the 77 kD GPBP-binding substrate are candidate compounds for treating CKD, immune complex-mediated GN, and/or PF.
  • FIG. 1 Characterization of GPBP-1 blocking mAbs.
  • A The binding between recombinant GPBP-1 and a3(IV)NC1 was measured by ELISA in the absence (Control) or presence of the indicated mAbs. The values are expressed as binding percentage of the control which was set at 100%. Other mAbs to GPBP did not block significantly GPBP-1 binding to ⁇ 3(IV)NC1 (not shown).
  • FIG. 2 Reduction of circulating GPBP-1 (cGPBP-1) levels in NZW mice treated with GPBP-specific antibodies.
  • B Represented are the serum levels of cGPBP-1 in each individual sample collected in A (circles) and the mean value of each series (line).
  • HE Heamatoxylin/Eosin
  • Masson Masson's trichrome
  • Standard indirect immunofluorescence was used to visualize IgG and IgM deposits using conventional microscopy and the distribution of C3c, GPBP-1 and ⁇ 3(IV) using confocal microscopy.
  • Micrographs show electron-dense material in the epithelial side of an enlarged capillary GBM in control mice (red arrows), abnormalities not found in mice treated with GPBP blocking mAbs.
  • Original magnifications ⁇ 400.
  • Confocal scale bars 35 and 54 ⁇ m (top and bottom).
  • Electron microscopy scale bars 2 ⁇ m (top) and 1 ⁇ m (bottom).
  • FIG. 4 Downregulation of GPBP-1 reduces pro-IL-1 ⁇ levels and delays IL-1 ⁇ secretion.
  • A Wild type and GPBP-1-silenced (pSi-GPBP-1) RAW 264.7 cells were primed for 8 hours with LPS and pro-IL-1 ⁇ and GPBP-1 were detected in cell lysates by Western blot with specific antibodies.
  • B and C Wild type and GPBP-1-silenced (pSi-GPBP-1) RAW 264.7 cells were primed overnight with 1 ⁇ g/ml of LPS and NLRP3 was activated with 10 ⁇ M doxorubicin for the indicated times.
  • IL-1 ⁇ (B) and cGPBP-1 (C) were detected in media supernatants by ELISA.
  • FIG. 5 cGPBP-1 and C reactive protein (CRP) levels are inversely related. cGPBP-1 and CRP were measured in sera of patients with proteinuria. The plot represents the mean (bar) and the individual (circles) cGPBP-1 levels of three groups of patients with the indicated CRP levels. Statistically significant differences were found between the group of patients with CRP ⁇ 3 ⁇ g/mL and CRP>4 ⁇ g/mL, according to Kruskal Wallis/Dunn test (***P ⁇ 0.001).
  • FIG. 6 Patients with RA show augmented circulating GPBP-1 levels which are inversely regulated with circulating CRP levels.
  • a circles represent circulating GPBP-1 (levels from individual serum of 113 patients having significant levels of anti-cyclic citrullinated peptide (anti-CCP) (RA) and 100 healthy donors (Control). Bars indicate the mean value within a series. On the right is indicated the result of the Mann-Whittney analysis.
  • each circle represents the respective values of circulating CRP and GPBP-1 found in individual sera from RA patients having significant levels of anti-CCP.
  • FIG. 7 Circulating GPBP-1 levels in mice developing collagen-induced artritis (CIA). Dots represent cGPBP-1 levels in individual serum of the indicated mice before immunization with bovine type II collagen (pre-immunization) or at the indicated times after immunization.
  • FIG. 8 Induction of pulmonary fibrosis (PF) by doxorubicin. Macroscopic aspect (upper panels) and representative histological appearance of the lungs ( ⁇ 10) stained with heamatoxylineosin (middle panels) or Masson's trichrome (lower panels) in control and doxorubicin treated mice 12 days after the i.t. instillation of the drug.
  • PF pulmonary fibrosis
  • FIG. 9 Expression of GPBP-1 during the development of doxorubicin-induced PF.
  • a shown are the levels of cGPBP-1 at the indicated time points after the i.t. instillation of doxorubicin. Results are expressed as the values of individual mice. Bars represent the mean value of each examination.
  • B shown are the levels of expression of GPBP-1 mRNA in the lung of control and doxorubicin treated mice 12 days after drug instillation. Results are expressed as mean ⁇ SD fold change of GPBP-1 expression relative to the ribosomal 18S subunit expression measured in parallel in each sample.
  • C Immunohistochemical examination of GPBP-1 expression using a polyclonal anti-GPBP-1 antibody in lung sections of control and doxorubicin treated mice 12 days after drug instillation ( ⁇ 10).
  • FIG. 10 Effect of pinacidil treatment in the development of doxorubicin-induced PF.
  • Lungs from untreated mice or animals treated with different doses of pinacidil were examined 12 days after doxorubicin instillation. Lungs sections stained with heamatoxylineosin ( ⁇ 10) are shown (left panels).
  • the extension of histological lesions is expressed in individual mice of each experimental group as the percentage of affected parenchyma (right panel).
  • FIG. 11 Effects of pinacidil or myricetin treatment in the induction of collagen I expression during the development of doxorubicin induced PF.
  • the expression of al (I) collagen mRNA was determined in untreated or pinacidil or myricetin treated mice 12 days after doxorubicin instillation using RT-qPCR. Results are expressed as mean ⁇ SD fold change of al (I) collagen expression relative to the ribosomal 18S subunit expression measured in parallel in each sample. Statistic differences are indicated as follow: ns: non-significant, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.005.
  • FIG. 12 Effect of GPBP mAbs on doxorubicin-induced PF.
  • the expression of al (I) and ⁇ 1 (IV) collagens mRNAs was determined in untreated, anti-TGF ⁇ , mAb 14 or mAb N12 treated mice 12 days after doxorubicin instillation using RT-qPCR. Results are expressed as mean ⁇ SD fold change of ⁇ 1(I) collagen expression relative to the ribosomal 18S subunit expression measured in parallel in each sample. Statistic differences are indicated as follow: ns: non-significant, *p ⁇ 0.05, ***p ⁇ 0.005.
  • native protein means the protein naturally produced by the cell, including any post-translational modifications (PTMs), and includes non-denatured protein, or denatured protein (as, for example, naturally produced protein substantially purified and subjected to one or more denaturing agents to, for example, run on a SDS-PAGE gel).
  • PTMs post-translational modifications
  • substantially purified polypeptide means that the polypeptide has been separated from its in vivo cellular environments. It is further preferred that the isolated polypeptides are also substantially free of gel agents, such as polyacrylamide, agarose, and chromatography reagents.
  • the present invention provides methods for treating chronic kidney disease (CKD), comprising administering to a subject in need thereof an amount effective of an inhibitor of 77 kD GPBP to treat the CKD.
  • CKD chronic kidney disease
  • inhibitors of 77 kD GPBP reduce inflammatory infiltrates in kidneys, and thus can be used for treating CKD.
  • the last stage of any inflammation is fibrosis, and renal tubulo-interstitial fibrosis represents the final common pathway for all kidney diseases, leading to gradual expansion of the fibrotic mass which destroys the normal structure of the tissue and results in CKD.
  • the subject can be any subject that might benefit from treatment, such as a mammal.
  • the subject is a human subject.
  • the amino acid sequence of 77 kD GPBP, now also called GPBP or GPBP-1 was disclosed in U.S. Pat. No. 6,579,969 Issued Jun. 17, 2003 and corresponding PCT publication WO 00/50607, published Aug. 31, 2000. As disclosed therein, and in WO2010/009856 and U.S. Pat. No.
  • kidney disease is a progressive loss in renal function over a period of months or years, regardless of underlying pathology.
  • individuals with a glomerular filtration rate (GFR) ⁇ 60 mL/min/1.73 m 2 for 3 months are classified as having CKD, irrespective of the presence or absence of kidney damage.
  • individuals with kidney damage are classified as having CKD, irrespective of the level of GFR.
  • Kidney damage is defined as pathological abnormalities or markers of damage, including abnormalities in blood or urine test or imaging studies
  • the methods can be used to treat non-dialysis dependent CKD, such as:
  • Stage 1 Slightly diminished function; kidney damage with normal or relatively high GFR ( ⁇ 90 mL/min/1.73 m 2 );
  • Stage 2 Mild reduction in GFR (60-89 mL/min/1.73 m 2 ) with kidney damage;
  • Stage 3 Moderate reduction in GFR (30-59 mL/min/1.73 m 2 );
  • Stage 4 Severe reduction in GFR (15-29 mL/min/1.73 m 2 ).
  • treating means (a) slowing progression of the subject to end stage renal disease (ESRD); (b) slowing the reduction in GFR in the subject; (c) limiting the progression of renal disease in the subject; and/or (d) reducing the severity of CKD symptoms.
  • ESRD end stage renal disease
  • limiting the progression of CKD means to reduce or prevent decreases in renal function in those patients receiving treatment relative to patients not receiving the treatment. Such treatment thus reduces the need for kidney dialysis or transplantation in patients.
  • the progression of renal disease can be measured in various ways, including the following:
  • Proteinuria ie: increased loss of protein into the urine; often assessed by measurement of albumin levels (ie: “albuminuria”));
  • Impaired glomerular clearance ie: kidney function to clear substances from blood; can be measured, for example, by creatinine (ie: “impaired creatinine clearance”), inulin, or urea clearance);
  • TGF-(3) urinary transforming growth factor beta
  • the methods of the invention can be used, for example, to limit the increase in one or more of proteinuria, albuminuria, serum creatinine levels, and urinary TGF- ⁇ levels, and/or to limit the impairment of glomerular clearance and/or creatinine clearance in subject being treated by the methods of the invention.
  • the present invention provides methods for treating immune complex-mediated GN, comprising administering to a subject in need thereof an amount effective of an inhibitor of 77 kD GPBP to treat the immune complex-mediated GN.
  • the subject can be any subject that might benefit from treatment, such as a mammal.
  • the subject is a human subject.
  • GN is a renal disease characterized by glomerular damage
  • immune complex-mediated GN means that the GN is characterized by glomerular depositions of immune complexes.
  • the immune complex-mediated GN is associated with an autoimmune disorder selected from the group consisting of IgA nephropathy, systemic lupus erythematosus (SLE) and Goodpasture disease.
  • autoimmune disorder selected from the group consisting of IgA nephropathy, systemic lupus erythematosus (SLE) and Goodpasture disease.
  • “treating” immune complex-mediated GN means (a) slowing progression of the glomerular damage; (b) slowing progression of glomerular immune complex deposits; (c) reducing glomerular inflammation; (d) reducing glomerular immune complex deposits; and/or (d) slowing progression to ESRD.
  • inhibitors of 77 kD GPBP repair glomerular basement membrane collagen-based alterations, reduce the glomerular deposits of immune complexes and attenuate inflammation associated with immune complex-mediated GN.
  • the 77 kD GPBP inhibitors may be co-administered with one or more other therapeutics, such as standard of care therapeutics for the disease being treated.
  • one or more compounds are selected from the group consisting of angiotensin converting enzyme inhibitors and angiotensin receptor blockers, or pharmaceutically acceptable salts thereof in a pharmaceutically acceptable carrier.
  • Non-limiting examples of angiotensin converting enzyme inhibitors for use in the present invention include benazepril, benazeprilat, captopril, delapril, fentiapril, fosinopril, libenzapril, moexipril, pentopril, perindopril, pivopril, quinapril, quinaprilat, ramipril, spirapril, spiraprilat, zofenopril, ceronapril, enalapril, indolapril, lisinopril, alacepril, and cilazapril, or pharmaceutically acceptable salts thereof.
  • Non-limiting examples of angiotensin receptor blockers for use in the present invention include losartan, candesartan, irbesartan, olmesartan, valsartan, telmisartan, eprosartan, and tasosartan.
  • the inhibitor in another embodiment, can be administered in combination with belimumab.
  • the present invention provides methods for treating pulmonary fibrosis (PF), comprising administering to a subject in need thereof an amount effective of an inhibitor of 77 kD GPBP to treat the PF
  • PF pulmonary fibrosis
  • the subject can be any subject that might benefit from treatment, such as a mammal.
  • the subject is a human subject.
  • PF is the formation or development of excess connective tissue (fibrosis) in the lungs.
  • Symptoms of pulmonary fibrosis include, but are not limited to shortness of breath, chronic dry, hacking coughing, fatigue and weakness, chest discomfort, and/or loss of appetite and rapid weight loss.
  • Pulmonary fibrosis is suggested by a history of progressive shortness of breath (dyspnea) with exertion. Sometimes, fine inspiratory crackles can be heard at the lung bases on auscultation. A high resolution CAT scan will generally demonstrate abnormalities.
  • inhibitors of GPBP were effective in treating doxorubicin-induced PF, an animal model for human PF.
  • the PF to be treated by the methods of the invention may be a secondary effect of other diseases (i.e.: “interstitial lung disease”, including but not limited to autoimmune disorders (ex: rheumatoid arthritis-RA-, SLE, scleroderma, etc.), viral infections or other microscopic injuries to the lung (such as exposure to asbestos, silicon, cigarette smoke, etc.), or may be idiopathic (i.e. “idiopathic pulmonary fibrosis.”)
  • diseases i.e.: “interstitial lung disease”
  • autoimmune disorders ex: rheumatoid arthritis-RA-, SLE, scleroderma, etc.
  • viral infections or other microscopic injuries to the lung such as exposure to asbestos, silicon, cigarette smoke, etc.
  • idiopathic pulmonary fibrosis i.e. “idiopathic pulmonary fibrosis.”
  • treating PF means (a) slowing progression of the lung fibrosis; and/or (b) reducing PF symptoms.
  • GPBP inhibitors may be co-administered with one or more other therapeutics, such as standard of care therapeutics for the disease being treated.
  • one or more compounds are selected from the group consisting of corticosteroids, immunosupressants (including but not limited to cyclophosphamide, azathioprine, and methotrexate), anti-inflammatory agents, IFN- ⁇ , mycophenolate mofetil, and pirfenidone.
  • the methods may be carried out on any suitable subject, such as those that have been identified as over-expressing 77 kD GPBP.
  • a normal value of 77 kD GPBP as a reference for a standard curve is less than 10 ng/ml in plasma.
  • a normal 77 kD GPBP range is between ⁇ 1 ng/ml-10 ng/ml in plasma.
  • subjects identified as having more than 10 mg/ml of 77 kD GPBP in their plasma are treated according to the methods of the invention.
  • Methods for determining the amount of circulating 77 kD GPBP are known in the art (see WO 2010/009856 and U.S. Pat. No. 7,935,492), and examples are described below.
  • any suitable inhibitor of 77 kD GPBP can be used.
  • the inhibitor is selected from the group consisting of 77 kD GPBP antisense RNA, 77 kD GPBP siRNA, and 77 kD GPBP antibodies.
  • the inhibitor of 77 kD GPBP is a 77 kD GPBP antibody, or pharmaceutically acceptable salts thereof.
  • Exemplary antibodies are disclosed, for example, in WO 2010/009856 and U.S. Pat. No. 7,935,492.
  • the antibodies recognize native 77 kD GPBP, including but not limited to those antibodies disclosed in WO 2010/009856 and U.S. Pat.
  • antibodies to native 77 kD GPBP means that the antibodies bind to native 77 kD GPBP, and does not require that they not bind to other GPBP species.
  • the antibodies are specific for 77 kD GPBP.
  • the antibody is a monoclonal antibody, such as a humanized monoclonal antibody.
  • antibody as used herein is intended to include antibody fragments thereof which are selectively reactive with the polypeptides of the invention, or fragments thereof.
  • Antibodies can be fragmented using conventional techniques, and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab′) 2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′) 2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments.
  • monoclonal antibody fragments include (i) a Fab fragment, a monovalent fragment consisting essentially of the VL, VH, CL and CH I domains; (ii) F(ab)2 and F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists essentially of a VH domain; and (vi) one or more isolated CDRs or a functional paratope.
  • a Fab fragment a monovalent fragment consisting essentially of the VL, VH, CL and CH I domains
  • F(ab)2 and F(ab′)2 fragments bivalent fragments
  • Monoclonal antibodies can be produced by obtaining spleen cells from the animal. (See Kohler and Milstein, Nature 256, 495-497 (1975)).
  • monoclonal antibodies (mAb) of interest are prepared by immunizing inbred mice with the 77 kD GPBP, or an antigenic fragment thereof. The mice are immunized by the IP or SC route in an amount and at intervals sufficient to elicit an immune response. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of by the intravenous (IV) route.
  • IV intravenous
  • Lymphocytes from antibody positive mice are obtained by removing spleens from immunized mice by standard procedures known in the art.
  • Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner under conditions which will allow the formation of stable hybridomas.
  • the antibody producing cells and fusion partner cells are fused in polyethylene glycol at concentrations from about 30% to about 50%.
  • Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art.
  • DMEM Dulbecco's Modified Eagles Medium
  • Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
  • Humanized antibody refers to antibodies derived from a non-human antibody, such as a mouse monoclonal antibody.
  • humanized antibodies can be derived from chimeric antibodies that retain or substantially retains the antigen-binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans.
  • chimeric antibodies can comprise human and murine antibody fragments, generally human constant and mouse variable regions. Since humanized antibodies are far less immunogenic in humans than the non-human monoclonal antibodies, they are preferred for therapeutic antibody use.
  • Humanized antibodies can be prepared using a variety of methods known in the art, including but not limited to (1) grafting complementarity determining regions from a non-human monoclonal antibody onto a human framework and constant region (“humanizing”), and (2) transplanting the non-human monoclonal antibody variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (“veneering”). These methods are disclosed, for example, in, e.g., Jones et al., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855 (1984); Morrison and 01, Adv.
  • the polypeptides of the present invention are typically formulated with a pharmaceutically acceptable carrier for parenteral administration.
  • acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA.
  • the formulation of such compositions, including the concentration of the polypeptide and the selection of the vehicle and other components, is within the skill of the art.
  • the 77 kD GPBP inhibitor is a peptide inhibitor disclosed in WO 2004/070025 and U.S. Pat. No. 7,326,768, or pharmaceutically acceptable salts thereof.
  • the peptide inhibitor is a polypeptide comprising or consisting of an amino acid sequence according to the general formula X1-SHClX2-X3 (SEQ ID NO: 2), wherein:
  • X1 is 0-10 amino acids of the sequence ATTAGILATL (SEQ ID NO:3);
  • X2 is E or Q
  • X3 is 0-10 amino acids of the sequence LMVKREDSWQ (SEQ ID NO:4).
  • the peptide inhibitor comprises or consists of a sequence selected from the group consisting of SHClE (SEQ ID NO:5), SHClQ (SEQ ID NO:6), ILATLSHCIELMVKR (SEQ ID NO: 7), and ILATLSHCIQLMVKR (SEQ ID NO: 8).
  • the peptide inhibitor comprises or consists of at least six (6, 7, 8, 9, or all) contiguous amino acids EKTAGKPILF (SEQ ID NO: 9).
  • the isolated polypeptide comprises or consists of the sequence EKTAGKPILF (SEQ ID NO: 10).
  • the polypeptides can further be derivatized to provide enhanced half-life, such as by the addition of polyethylene glycol (PEG) or as otherwise known in the art.
  • the polypeptides of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), a combination of D- and L-amino acids, and various “designer” amino acids (e.g., ⁇ -methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids, etc.) to convey special properties.
  • Synthetic amino acids include ornithine for lysine, and norleucine for leucine or isoleucine.
  • polypeptides can have peptidomimetic bonds, such as ester bonds, to prepare polypeptides with novel properties.
  • a polypeptide may be generated that incorporates a reduced peptide bond, i.e., R 1 —CH 2 —NH—R 2 , where R 1 and R 2 are amino acid residues or sequences.
  • a reduced peptide bond may be introduced as a dipeptide subunit.
  • Such a polypeptide would be resistant to protease activity, and would possess an extended half-live in vivo.
  • polypeptide is used in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits are linked by peptide bonds, although the polypeptide can comprise further moieties that are not necessarily linked to the polypeptide by a peptide bond.
  • the polypeptide can further comprise a non-amino acid molecule that contains an aromatic ring.
  • polypeptides described herein may be chemically synthesized or recombinantly expressed. Recombinant expression can be accomplished using standard methods in the art, as disclosed above.
  • expression vectors can comprise bacterial or viral expression vectors, and such host cells can be prokaryotic or eukaryotic.
  • the polypeptides for use in the methods of the present invention are chemically synthesized.
  • Synthetic polypeptides prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected Na-t-butyloxycarbonyl) amino acid resin with standard deprotecting, neutralization, coupling and wash protocols, or standard base-labile N ⁇ -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids.
  • Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, such as by using automated synthesizers.
  • the peptide/antibody inhibitors may be administered together in a pharmaceutical composition with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may comprise in addition to the peptide inhibitor (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer.
  • the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer.
  • the pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose.
  • the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof.
  • the pharmaceutical composition includes a bulking agent, like glycine.
  • the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof.
  • the pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood.
  • Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride.
  • the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form.
  • Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
  • the peptide inhibitor/antibody may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use.
  • the 77 kD GPBP inhibitor is selected from the group consisting of DAB-Am32, pinacidil, and mirycetin, and pharmaceutically acceptable salts thereof, each of which is demonstrated in the examples that follow to be effective in the methods of the present invention.
  • the 77 kD GPBP inhibitor is one disclosed in WO 2011/054530 and US 20110105545.
  • the compounds may be compounds of formula (I), or pharmaceutically acceptable salts thereof:
  • R is selected from N and CR 5 ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), or (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , (aryl)C 1 -C 6 alkyl, or (heteroaryl)C 1 -C 6 alkyl;
  • R 3 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 1 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5
  • R 4 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), benzyloxy, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy), —O(CH 2 ) 1-5 —C(O)OH, —O(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), (aryl)C 1
  • inhibitor compounds of formula (I) may be those of formula (II):
  • the inhibitors are compounds of formulae (I) or (II) wherein:
  • R is selected from N and CR 5 ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), and —(CH 2 )
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), or (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), or —(CH 2 ) 1-5 —C(O)NH 2 ;
  • R 3 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 1 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5
  • R 4 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), benzyloxy, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy), —O(CH 2 ) 1-5 —C(O)OH, or —O(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy).
  • the inhibitors are compounds of formulae (I) or (II) wherein:
  • R is selected from N and CR 5 ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, and amino(C 1 -C 6 alkyl);
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), or (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl);
  • R 3 is C 1 -C 6 alkyl, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy); and
  • R 4 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), benzyloxy, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy), —O(CH 2 ) 1-5 —C(O)OH, or —O(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy).
  • the inhibitors are compounds of formula (II), wherein R is N. These compounds can be represented by formula (III):
  • the inhibitors are compounds of formula (II), wherein R is CR 5 . These compounds can be represented by formula (IV):
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 1 is hydrogen, hydroxy, or C 1 -C 6 alkoxy.
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 1 is hydrogen.
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), or sulfanyl(C 1 -C 6 alkyl).
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), or formyl(C 0 -C 6 alkyl).
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 2 can be C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), or hydroxy(C 1 -C 6 alkyl).
  • R 2 can be C 1 -C 6 alkyl such as methyl, ethyl, or isopropyl.
  • R 2 can be halo(C 1 -C 6 alkyl) such as fluoromethyl, difluoromethyl, or trifluoromethyl.
  • R 2 can, in certain embodiments, be hydroxy(C 1 -C 6 alkyl).
  • the hydroxy(C 1 -C 6 alkyl) can be hydroxymethyl, 1-hydroxyethyl, or 2-hydroxyethyl.
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 2 is C 1 -C 6 alkyl. In certain preferred embodiments R 2 is methyl.
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 3 is C 1 -C 6 alkyl, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —CH ⁇ CH—C(O)OH, or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy).
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 3 is —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), or —(CH 2 ) 1-5 —C(O)NH 2 .
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 3 is —(CH 2 ) 1-2 —C(O)OH, or —(CH 2 ) 1-2 —C(O)(C 1 -C 6 alkoxy).
  • R 3 can be —(CH 2 ) 2 —C(O)OH, —(CH 2 ) 2 —C(O)(OCH 3 ), —(CH 2 ) 2 —C(O)(OCH 2 CH 3 ), or —(CH 2 ) 2 —C(O)(OC(CH 3 ) 3 ).
  • R 3 can be —(CH 2 ) 2 —C(O)OH, or —(CH 2 ) 2 —C(O)(OCH 2 CH 3 ).
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 3 is —(CH 2 ) 1-2 —C(O)OH.
  • R 3 is —(CH 2 ) 2 —C(O)OH.
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 4 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), or benzyloxy.
  • the inhibitors are compounds as described above with reference to any of formulae (I)-(IV), wherein R 4 is hydroxy or C 1 -C 6 alkoxy (e.g., methoxy).
  • R 4 is C 1 -C 6 alkoxy.
  • R 4 is methoxy.
  • the inhibitors are compounds as described above with reference to formulae (I), (II), or (IV), wherein R 5 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy).
  • the inhibitors are compounds as described above with reference to formulae (I), (II), or (IV), wherein R 5 is C 1 -C 6 alkyl, such as methyl.
  • the inhibitors are compounds as described above with reference to formulae (I), (II), or (IV), wherein R 5 is halo(C 1 -C 6 alkyl), such as trifluoromethyl.
  • the inhibitors are compounds of any of formulae (I), (II), or (IV), wherein:
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, and amino(C 1 -C 6 alkyl);
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo (C 1 -C 6 alkoxy);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino (C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), or (C 1 -C 6 alkyl)thio(C 1 -C 6 alkyl);
  • R 3 is —(CH 2 ) 1-2 —C(O)OH, —(CH 2 ) 1-2 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-2 —C(O)NH 2 , —(CH 2 ) 1-2 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-2 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy); and
  • R 4 is hydroxy, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), or benzyloxy.
  • the inhibitors are compounds of any of formula (III), wherein:
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), or (C 1 -C 6 alkyl)thio(C 1 -C 6 alkyl);
  • R 3 is —(CH 2 ) 1-2 —C(O)OH, —(CH 2 ) 1-2 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-2 —C(O)NH 2 , —(CH 2 ) 1-2 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-2 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy); and
  • R 4 is hydroxy, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), or benzyloxy.
  • the inhibitors are compounds of any of formulae (I), (II), or (IV), wherein R 1 is hydrogen; R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), or formyl(C 1 -C 6 alkyl); R 3 is —(CH 2 ) 1-2 —C(O)OH, —(CH 2 ) 1-2 —C(O)(C 1 -C 6 alkoxy), or —(CH 2 ) 1-2 —C(O)NH 2 ; R 4 is hydroxy or C 1 -C 6 alkoxy; and R 5 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy).
  • the inhibitors are compounds of any of formula (III), wherein R 1 is hydrogen; R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), or formyl(C 1 -C 6 alkyl); R 3 is —(CH 2 ) 1-2 —C(O)OH, —(CH 2 ) 1-2 —C(O)(C 1 -C 6 alkoxy), or —(CH 2 ) 1-2 —C(O)NH 2 ; R 4 is hydroxy or C 1 -C 6 alkoxy; and R 5 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy).
  • the inhibitors are compounds of any of formulae (I)-(IV), wherein:
  • R if present, is selected from N and CR S ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, and amino(C 1 -C 6 alkyl);
  • R 1 is hydrogen
  • R 2 is C 1 -C 6 alkyl
  • R 3 is —(CH 2 ) 1-2 —C(O)OH
  • R 4 is C 1 -C 6 alkoxy.
  • the inhibitors are compounds of any of formulae (I)-(IV), wherein:
  • R if present, is selected from N and CR S ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, and amino(C 1 -C 6 alkyl);
  • R 1 is hydrogen
  • R 2 is methyl
  • R 3 is —(CH 2 ) 2 —C(O)OH
  • R 4 is methoxy
  • the inhibitors are compounds of formula (V) are of formula (VI):
  • the inhibitors are compounds of formulae (V) or (VI) wherein:
  • R is selected from N and CR 5 ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), or (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —CH ⁇ CH—C(O)OH, or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy); and
  • R 6 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), benzyloxy, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy), or —OS(O) 2 CF 3 .
  • the disclosure provides compounds of formulae (V) or (VI) wherein:
  • R is selected from N and CR 5 ;
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, and amino(C 1 -C 6 alkyl);
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)sulfanyl(C 1 -C 6 alkyl), —CH ⁇ CH—C(O)OH, or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy); and
  • R 6 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), benzyloxy, —(CH 2 ) 1-5 —C(O)OH, —(CH 2 ) 1-5 —C(O)(C 1 -C 6 alkoxy), —(CH 2 ) 1-5 —C(O)NH 2 , —(CH 2 ) 1-5 —C(O)NH(C 1 -C 6 alkyl), —(CH 2 ) 1-5 —C(O)N(C 1 -C 6 alkyl) 2 , —CH ⁇ CH—C(O)OH, —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy), or —OS(O) 2 CF 3 .
  • the inhibitors are compounds as described above with reference to any of formulae (V)-(VI), wherein R 1 is hydrogen.
  • the inhibitors are compounds as described above with reference to any of formulae (V)-(VI), wherein R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), —CH ⁇ CH—C(O)OH, or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy).
  • the inhibitors are compounds as described above with reference to any of formulae (V)-(VI), wherein R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), formyl(C 0 -C 6 alkyl), —CH ⁇ CH—C(O)OH, or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy).
  • R 2 can be C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy).
  • R 2 can be C 1 -C 6 alkyl such as methyl, ethyl, or isopropyl.
  • R 2 can be halo(C 1 -C 6 alkyl) such as fluoromethyl, difluoromethyl, or trifluoromethyl.
  • R 2 can, in certain embodiments, be hydroxy(C 1 -C 6 alkyl).
  • the hydroxy(C 1 -C 6 alkyl) can be hydroxymethyl, 1-hydroxyethyl, or 2-hydroxyethyl.
  • the inhibitors are compounds as described above with reference to any of formulae (V)-(VI), wherein R 2 is C 1 -C 6 alkyl. In certain preferred embodiments R 2 is methyl.
  • the inhibitors are compounds as described above with reference to any of formulae (V)-(VI), wherein R 6 is hydroxy, halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), benzyloxy, or —OS(O) 2 CF 3 .
  • the inhibitors are compounds as described above with reference to any of formulae (V)-(VI), wherein R 6 is hydroxy or C 1 -C 6 alkoxy (e.g., methoxy).
  • the disclosure provides compounds as described above with reference to formulae (V)-(VI), wherein R 5 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy).
  • the inhibitors are compounds as described above with reference to formulae (V)-(VI), wherein R 5 is C 1 -C 6 alkyl, such as methyl.
  • the inhibitors are compounds as described above with reference to formulae (V)-(VI), wherein R 5 is halo(C 1 -C 6 alkyl), such as trifluoromethyl.
  • the disclosure provides compounds of any of formulae (V)-(VI), wherein:
  • R 5 is selected from the group consisting of hydrogen, halogen, cyano, nitro, hydroxy, C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), amino, (C 1 -C 6 alkyl)amino, di(C 1 -C 6 alkyl)amino, hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, and amino(C 1 -C 6 alkyl);
  • R 1 is hydrogen, halogen, hydroxy, C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy);
  • R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), (C 1 -C 6 alkoxy)C 1 -C 6 alkyl, formyl(C 0 -C 6 alkyl), amino(C 1 -C 6 alkyl), sulfanyl(C 1 -C 6 alkyl), (C 1 -C 6 alkyl)thio(C 1 -C 6 alkyl), or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy); and
  • R 6 is hydroxy, C 1 -C 6 alkoxy, halo(C 1 -C 6 alkoxy), or —OS(O) 2 CF 3 .
  • the inhibitors are compounds of any of formulae (V)-(VI), wherein R 1 is hydrogen; R 2 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), hydroxy(C 1 -C 6 alkyl), formyl(C 0 -C 6 alkyl), or —CH ⁇ CH—C(O)(C 1 -C 6 alkoxy); R 6 is hydroxy, C 1 -C 6 alkoxy, or —OS(O) 2 CF 3 ; and R 5 is C 1 -C 6 alkyl, halo(C 1 -C 6 alkyl), C 1 -C 6 alkoxy, or halo(C 1 -C 6 alkoxy).
  • the GBPB inhibitor compounds include pharmaceutically acceptable salts, esters, amides, and prodrugs thereof, including but not limited to carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention.
  • salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts, and the like.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, and the like
  • non-toxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like See, for example, Berge S. M. et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977; 66:1-19 which is incorporated herein by reference.)
  • esters of the inhibitors include C 1 -C 6 alkyl esters, wherein the alkyl group is straight or branched, substituted or unsubstituted, C 5 -C 7 cycloalkyl esters, as well as arylalkyl esters such as benzyl and triphenylmethyl.
  • C 1 -C 4 alkyl esters are preferred, such as methyl, ethyl, 2,2,2-trichloroethyl, and tert-butyl.
  • Esters of the compounds of the present invention may be prepared according to conventional methods.
  • Examples of pharmaceutically acceptable, non-toxic amides of the inhibitors include amides derived from ammonia, primary C 1 -C 6 alkyl amines and secondary C 1 -C 6 dialkyl amines, wherein the alkyl groups are straight or branched. In the case of secondary amines, the amine may also be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C 1 -C 3 alkyl primary amines and C 1 -C 2 dialkyl secondary amines are preferred. Amides of the compounds of the invention may be prepared according to conventional methods.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood.
  • prodrugs are provided in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference.
  • alkenyl as used herein, means a straight or branched chain hydrocarbon containing from 2 to 10 carbons, unless otherwise specified, and containing at least one carbon-carbon double bond.
  • Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and 3,7-dimethylocta-2,6-dienyl.
  • alkoxy as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
  • Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.
  • alkyl as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms unless otherwise specified.
  • Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.
  • an “alkyl” group is a linking group between two other moieties, then it may also be a straight or branched chain; examples include, but are not limited to —CH 2 —, —CH 2 CH 2 —, —CH 2 CH 2 CHC(CH 3 )—, —CH 2 CH(CH 2 CH 3 )CH 2 —.
  • alkylene refers to a bivalent alkyl group.
  • An “alkylene chain” is a polymethylene group, i.e., —(CH 2 ) n —, wherein n is a positive integer, preferably from one to six, from one to four, from one to three, from one to two, or from two to three.
  • a substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.
  • An alkylene chain also may be substituted at one or more positions with an aliphatic group or a substituted aliphatic group.
  • alkynyl as used herein, means a straight or branched chain hydrocarbon group containing from 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond.
  • Representative examples of alkynyl include, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, and 1-butynyl.
  • aryl means a phenyl (i.e., monocyclic aryl), or a bicyclic ring system containing at least one phenyl ring or an aromatic bicyclic ring containing only carbon atoms in the aromatic bicyclic ring system.
  • the bicyclic aryl can be azulenyl, naphthyl, or a phenyl fused to a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocyclyl.
  • the bicyclic aryl is attached to the parent molecular moiety through any carbon atom contained within the phenyl portion of the bicyclic system, or any carbon atom with the napthyl or azulenyl ring.
  • the fused monocyclic cycloalkyl or monocyclic heterocyclyl portions of the bicyclic aryl are optionally substituted with one or two oxo and/or thia groups.
  • bicyclic aryls include, but are not limited to, azulenyl, naphthyl, dihydroinden-1-yl, dihydroinden-2-yl, dihydroinden-3-yl, dihydroinden-4-yl, 2,3-dihydroindol-4-yl, 2,3-dihydroindol-5-yl, 2,3-dihydroindol-6-yl, 2,3-dihydroindol-7-yl, inden-1-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen-2-yl, 2,3-dihydrobenz
  • the bicyclic aryl is (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • halo or “halogen” as used herein, means —Cl, —Br, —I or —F.
  • haloalkyl refers to an alkyl, alkenyl or alkoxy group, as the case may be, which is substituted with one or more halogen atoms.
  • heteroaryl means a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring.
  • the monocyclic heteroaryl can be a 5 or 6 membered ring.
  • the 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom.
  • the 6 membered ring consists of three double bonds and one, two, three or four nitrogen atoms.
  • the 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl.
  • monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl.
  • the bicyclic heteroaryl consists of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups which are independently oxo or thia.
  • the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl, or heterocyclyl ring
  • the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon or nitrogen atom contained within the monocyclic heteroaryl portion of the bicyclic ring system.
  • the bicyclic heteroaryl is a monocyclic heteroaryl fused to a benzo ring
  • the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon atom or nitrogen atom within the bicyclic ring system.
  • bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, benzothiazolyl, cinnolinyl, 5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl, 5,6,7,8-tetrahydroquinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroisoquinolin-1-yl, thienopyridinyl, 4,5,6,7-tetrahydrobenzo[c]
  • the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the GPBP inhibitors are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the inhibitors may be mixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the inhibitors may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • the inhibitors can be administered as the sole active pharmaceutical agent, or they can be used in combination with one or more other compounds useful for carrying out the methods of the invention.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • the inhibitors may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the inhibitors may be applied in a variety of solutions and may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • the inhibitors may be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
  • the pharmaceutical compositions containing inhibitors may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservative agents in order to provide palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques. In some cases such coatings may be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monoole
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the inhibitors in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the inhibitors in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent exemplified by those already mentioned above.
  • Additional excipients for example sweetening, flavoring and coloring agents, may also be present.
  • Inhibitors may also be administered in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavoring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents.
  • the inhibitor compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the inhibitor-containing compositions may also be administered in the form of suppositories, e.g., for rectal administration of the drug.
  • suppositories e.g., for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • the inhibitor-containing compositions of the present invention may be administered parenterally in a sterile medium.
  • the drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • the amount of inhibitor that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • Dosage unit forms will generally contain between from about 1 mg to about 500 mg of inhibitor.
  • “Pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio or which have otherwise been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • the present invention provides methods for diagnosing rheumatoid arthritis (RA) or pulmonary fibrosis (PF) comprising
  • Subjects at risk of RA are those subjects with any symptoms or risk factors for RA.
  • Symptoms include, but are not limited to inflammation of the joints with the affected joints being swollen, warm, painful and/or stiff, particularly early in the morning on waking or following prolonged inactivity; increased joint stiffness early in the morning that typically lasts for more than an hour; tendon tethering; tendon erosion; joint surface destruction; impaired range of joint movement; deformity involving joint (including hands/fingers, feet/toes, cervical spine, knee, and shoulder); and loss of joint function.
  • PF is the formation or development of excess connective tissue (fibrosis) in the lungs.
  • Symptoms of pulmonary fibrosis include, but are not limited to shortness of breath such as progressive shortness of breath (dyspnea with exertion), chronic dry, hacking coughing, fatigue and weakness, chest discomfort, and/or loss of appetite and rapid weight loss.
  • fine inspiratory crackles can be heard at the lung bases on auscultation.
  • a high resolution CAT scan will generally demonstrate abnormalities.
  • 77 kD GPBP is a pre-inflammatory marker for RA and PF, as the amount of circulating 77 kD increases in the pre-inflammatory stage of RA and PF, and then subsides with the development of pain in a swollen joint(s) characteristic of inflammatory RA, and with the development of dyspnea with exertion characteristic of inflammatory PF.
  • the methods can be used to identify subjects having RA or PF at the pre-inflammatory stage, permitting earlier treatment for subjects suffering from RA or PF.
  • the method can be used to diagnose RA in a subject with symptoms of pre-inflammatory RA, such as one or more swollen joints, one or more stiff joints, one or more warm joints, increased joint stiffness early in the morning that typically lasts for more than an hour, excess synovial fluid, the development of fibrous tissue in the synovium, and/or impaired range of joint movement.
  • the subject is not experiencing pain in all of the one or more swollen joints.
  • the subject does not suffer from destruction of articular cartilage or ankylosis of one or more joints.
  • the method can be used to diagnose PF in a subject with symptoms of pre-inflammatory PF, such as chronic dry, hacking coughing; fatigue and weakness; chest discomfort; loss of appetite and/or rapid weight loss; and/or dyspnea with exertion.
  • the subject is not suffering from dyspnea at rest.
  • the amount of 77 kD GPBP may solely constitute intact 77 kD
  • GPBP may comprise an amount of 77 kD GPBP and 77 kD GPBP fragments in the plasma sample.
  • the subject can be any subject that might benefit from diagnosis, such as a mammal.
  • the subject is a human subject.
  • RA Rheumatoid arthritis
  • synovial a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks (synovial) joints.
  • the process produces an inflammatory response of the capsule around the joints secondary to swelling of synovial cells, excess synovial fluid, and the development of fibrous tissue in the synovium.
  • the pathology of the disease process often leads to the destruction of articular cartilage and ankylosis of the joints.
  • RA can also produce diffuse inflammation in the lungs, pericardium, pleura, sclera, and also nodular lesions, most common in subcutaneous tissue.
  • a “GPBP-binding molecule” is a peptide or nucleic acid molecule that binds selectively to 77 kD GPBP, as opposed to one or more other biological molecules, structures, cells, tissues, etc. Exemplary embodiments of such GPBP-binding molecules include but are not limited to antibodies, aptamers or substrates.
  • a “GPBP substrate” is a target of GPBP biological activity that binds to 77 kD GPBP, or a fragment thereof that retains GPBP-binding activity.
  • Such GPBP substrates include, but are not limited to, 1-20 (SEQ ID NO:18), GPBP-interacting proteins (GIPs) (SEQ ID NOS:19-23), myelin basic protein (MBP) and derivatives thereof (SEQ ID NOS:24-27), prion protein (PrP) (SEQ ID NO:28), type IV collagen ⁇ 3 chain NC1 domain ( ⁇ 3(IV)NC1) (SEQ ID NO:29), and Alzheimer's disease beta peptide (A ⁇ 1-42 ) (SEQ ID NO:30).
  • GIPs GPBP-interacting proteins
  • MBP myelin basic protein
  • PrP prion protein
  • SEQ ID NO:28 type IV collagen ⁇ 3 chain NC1 domain
  • ⁇ 3(IV)NC1 type IV collagen ⁇ 3 chain NC1 domain
  • a ⁇ 1-42 Alzheimer's disease beta peptide
  • a “plasma sample” means blood plasma, the liquid component of blood, and is prepared, for example, by centrifugation of whole blood to remove blood cells.
  • a plasma sample also includes a blood serum sample, in which blood clotting factors have been removed.
  • the plasma sample may be obtained from any suitable subject, preferably from a mammal that is at risk of suffering from RA or PF, including but not limited to a human, dog, cat, horse, or livestock (cow, sheep, etc.). In a most preferred embodiment, the plasma sample is obtained from a human subject. As disclosed herein, the inventors have observed increased circulating 77 kD GPBP levels in animal models of RA and PF.
  • the antibody can be any selective GPBP antibody, whether polyclonal, monoclonal, or humanized monoclonal as described above, although monoclonal antibodies are preferred.
  • Conditions suitable to promote binding of GPBP-binding molecules, such as antibodies, aptamers or substrates, to 77 kD GPBP in the plasma samples can be determined by those of skill in the art based on the teachings herein and the examples provided below.
  • antibody-antigen binding often depends on hydrophobic interactions (the so called hydrophobic bonds); thus, high salt concentrations, such as in the molar range can be used to reduce nonspecific binding and increase specific antigen-antibody binding.
  • further steps may be included to promote selectivity and specificity, including but not limited to one or more wash steps to remove unbound 77 kD GPBP and/or GPBP-binding molecule, or unbound or weakly bound serum proteins; inhibitors of non-specific binding to reduce binding of high concentration serum proteins, control samples known to contain 77 kD GPBP and/or negative controls known not to bind to 77 kD GPBP, and/or inclusion of plasma samples known to not possess 77 kD GPBP (ex: deleted for GPBP).
  • the methods can test for the presence of 77 kD GPBP in the plasma sample by standard techniques including, but not limited to ELISA, immunoflourescence, and chromatography (for example, lateral flow assays where the antibody is immobilized on a surface and plasma proteins are labeled and allowed to flow over the surface under conditions suitable to permit binding of the antibody to GPBP in the plasma).
  • functional beads Becton Dickinson technology coupled to flow cytometry are used; this technique is an emerging method to measure the levels of proteins in biological fluid or cell/tissue extracts.
  • beads made of a fluorescence matrix are coated with one or more 77 kD GPBP antibodies, mixed with the plasma sample and further incubated with a detecting antibody labeled with a phycoerythrins. Finally, beads are analyzed by a flow cytometry program which selects the beads according matrix fluorescence emission and measurement of the level of the analyte through phycoerythrin emission. There are up to thirty different types of beads that can be simultaneously detected and discriminated by the cytometer. This method couples high sensitivity and performance with versatility since a specific bead type coated with GPBP antibody can be mixed with a distinct bead type coated with binding peptides for other analyte (i.e. autoantibodies) and simultaneously measured.
  • analyte i.e. autoantibodies
  • the techniques may determine only the presence or absence of the 77 kD GPBP isoform.
  • the techniques may be quantitative, and provide information about the relative amount of the 77 kD GPBP in the sample. For quantitative purposes, ELISAs are preferred.
  • Detection of immunocomplex formation can be accomplished by standard detection techniques. For example, detection of immunocomplexes can be accomplished by using labeled antibodies or secondary antibodies. Such methods, including the choice of label are known to those ordinarily skilled in the art.
  • the antibodies can be coupled to a detectable substance.
  • the term “coupled” is used to mean that the detectable substance is physically linked to the antibody.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
  • prosthetic-group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • An example of a luminescent material includes luminol.
  • suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • the methods comprise comparison of 77 kD GPBP levels detected in a test plasma sample with a control, such as a control from a plasma sample known to have “normal” levels of 77 kD GPBP or previously determined normal values for 77 kD GPBP in plasma from the subject from whom the plasma is obtained.
  • a control such as a control from a plasma sample known to have “normal” levels of 77 kD GPBP or previously determined normal values for 77 kD GPBP in plasma from the subject from whom the plasma is obtained.
  • the control provides a standard curve using recombinant 77 kD GPBP or a reference value.
  • an increase in 77 kD GPBP in the plasma sample relative to the control indicates the presence of RA or PF.
  • a normal value of 77 kD GPBP as a reference for an standard curve is between ⁇ 1 ng/ml-10 ng/ml in plasma, while pre-inflammatory RA subjects exceed the normal at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold and even higher (i.e. 300-fold) normal values.
  • a subject at risk of RA is diagnosed as having RA if the subject's 77 kD GPBP plasma levels are 100 ng/ml or higher; 200 ng/ml or higher; 300 ng/ml or higher; 400 ng/ml or higher; 500 ng/ml or higher; 600 ng/ml or higher; 700 ng/ml or higher; 800 ng/ml or higher; 900 ng/ml or higher; 1000 ng/ml or higher; 2000 ng/ml or higher; or 3000 ng/ml or higher.
  • a subject at risk of PF is diagnosed as having PF if the subject's 77 kD GPBP plasma levels are 20 ng/ml or higher; 30 ng/ml or higher; 40 ng/ml or higher; 50 ng/ml or higher; 60 ng/ml or higher; 70 ng/ml or higher; or 80 ng/ml or higher.
  • combining 77 kD GPBP determination with analysis of other analytes the methods permit one to perform differential diagnosis or prognosis of RA or PF.
  • other analytes that could be assayed in subjects at risk of RA include rheumatoid factor (RF), anti-CCP, anti-mutated citrullinated vimentin (anti-MCV).
  • RF rheumatoid factor
  • anti-CCP anti-CCP
  • anti-MCV anti-mutated citrullinated vimentin
  • Other analytes that could be assayed in subjects at risk of PF include transforming growth factor beta (TGF- ⁇ ).
  • the method further comprises determining a level of C reactive protein (CRP) in the subject's plasma.
  • CRP is a marker of inflammation.
  • the levels of 77 kD GPBP and CRP in the serum are inversely related in CKD and RA: 77 kD GPBP plasma levels are increased at the pre-inflammatory stage of CKD and RA and then decrease during the inflammatory period.
  • CRP plasma levels increase during the inflammatory phase of RA and CKD while staying at baseline levels during the pre-inflammatory phase of RA and CKD.
  • the methods further comprise measuring the levels of plasma CRP, comparing an amount of plasma CRP to control; and using the CRP comparison to aid in diagnosing the subject as having RA or PF.
  • the invention provides methods for diagnosing CKD or immune complex-mediated GN, comprising
  • the methods of any of these embodiments, or combinations thereof, may further comprising measuring 77 kD GPBP and CRP levels in plasma from the subject at two or more time points (i.e.: 2, 3, 4, 5, or more time points), comparing each to control, and diagnosing the subject as having RA, PF, CKD, or immune complex-mediated GN based on the comparison over two or more measurements.
  • the CRP levels may be measured from the same plasma sample as the 77 kD GPBP levels, or from a different sample. Normal concentrations of CRP in healthy human plasma is usually lower than 3 ⁇ g/mL.
  • CRP levels are at least 4 ⁇ g/ml, such as 10 ⁇ g/ml for mild inflammation and 40 ⁇ g-200 ⁇ g/ml (or greater) in active inflammation.
  • the present invention provides methods for identifying compounds to treat CKD, immune complex-mediated GN, and/or PF, comprising contacting a 77 kD GPBP-77 kD GPBP substrate binding complex with one or more test compounds under binding conditions, wherein those test compounds that displace 77 kD GPBP from the binding complex are candidate compounds for treating CKD, immune complex-mediated GN, and/or PF.
  • the present invention provides methods for identifying compounds to treat CKD, immune complex-mediated GN, and/or PF, comprising contacting a 77 kD GPBP substrate under binding conditions with
  • test compounds that outcompete 77 kD GPBP for binding to the 77 kD GPBP substrate are candidate compounds for treating CKD, immune complex-mediated GN, and/or PF.
  • Suitable 77 kD GPBP substrates in these fifth and sixth embodiments are as disclosed above, including, but are not limited to, I-20 (SEQ ID NO:18), GPBP-interacting proteins (GIPs) (SEQ ID NOS:19-23), myelin basic protein (MBP) and derivatives thereof (SEQ ID NOS:24-27), prion protein (PrP) (SEQ ID NO:28), type IV collagen ⁇ 3 chain NC1 domain ( ⁇ 3(IV)NC1) (SEQ ID NO:29), and Alzheimer's disease beta peptide (A ⁇ 1-42 ) (SEQ ID NO:30).
  • the 77 kD GPBP substrate comprises ⁇ 3(IV)NC1. Exemplary references demonstrating GPBP binding of these substrates can be found in U.S. Pat. Nos. 6,579,969; 7,147,855; and 7,326,768, incorporated by reference herein in their entirety.
  • compounds that displace 77 kD GPBP from binding complexes with 77 kD GPBP substrates, or that compete with 77 kD GPBP for binding to 77 kD GPBP substrates are candidate compounds for treating CKD, immune complex-mediated GN, and/or PF.
  • Suitable conditions to assess binding of the test compounds to ⁇ 3(IV)NC1 or other 77 kD GPBP substrates can be determined by those of skill in the art based on the teachings herein. In one embodiment, conditions such as those used in the examples that follow can be used.
  • microwell plates may be coated with a suitable amount of ⁇ 3(IV)NC1 in a suitable buffer to assess binding interactions. The plates may then be blocked to minimize non-specific binding. In one embodiment, the plates are then incubated with an amount of 77 kD GPBP (such as native 77 kD GPBP) and under suitable conditions to promote binding to the ⁇ 3(IV)NC1 (to form a binding complex), followed by any appropriate wash steps (to remove unbound 77 kd GPBP) and then binding with the one or more test compounds to assess whether any of the test compounds can displace the 77 kD GPBP from the 77 kD GPBP- ⁇ 3(IV)NC1 binding complex in the microwell.
  • an amount of 77 kD GPBP such as native 77 kD GPBP
  • the 77 kD GPBP and the one or more test compounds are contacted under binding conditions at the same time, to identify test compounds that can outcompete 77 kD GPBP for binding to the ⁇ 3(IV)NC1.
  • the 77 kD GPBP may be detectably labelled, and/or the one of more test compounds may be detectably labelled to facilitate identification and analysis of binding events.
  • plates are coated with 1 ⁇ g/mL poly-His- ⁇ 3(IV)NC1 in PBS. Plates are coated for 16 h at 4° C. and blocked with 3% BSA in PBS for 1 h at room temperature.
  • test compounds when the test compounds are antibodies, antibody-antigen binding often depends on hydrophobic interactions (the so called hydrophobic bonds); thus, high salt concentrations, such as in the molar range can be used to reduce nonspecific binding and increase specific antigen-antibody binding.
  • further steps may be included to promote selectivity and specificity, including but not limited to one or more wash steps to remove unbound 77 kD GPBP and/or test compounds; control samples known to contain competitors for GPBP binding to 77 kD GPBP substrates such as ⁇ 3(IV)NC1 and/or negative controls known not to compete for 77 kD GPBP binding to 77 kD GPBP substrates such as ⁇ 3(IV)NC1.
  • the methods can utilize standard techniques including, but not limited to ELISA, immunoflourescence, and chromatography. For quantitative purposes, ELISAs are preferred.
  • Detection of binding events can be accomplished by standard detection techniques. For example, detection of binding complexes can be accomplished by using labeled antibodies or secondary antibodies. Such methods, including the choice of label are known to those ordinarily skilled in the art.
  • the antibodies can be coupled to a detectable substance. The term “coupled” is used to mean that the detectable substance is physically linked to the antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase.
  • suitable prosthetic-group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • An example of a luminescent material includes luminol.
  • suitable radioactive material include 125 I, 131 I, 35 S or 3 H.
  • test compounds comprise polypeptide sequences
  • polypeptides may be chemically synthesized or recombinantly expressed. Recombinant expression can be accomplished using standard methods in the art, as disclosed above.
  • expression vectors can comprise bacterial or viral expression vectors, and such host cells can be prokaryotic or eukaryotic.
  • Synthetic polypeptides prepared using the well-known techniques of solid phase, liquid phase, or peptide condensation techniques, or any combination thereof, can include natural and unnatural amino acids.
  • Amino acids used for peptide synthesis may be standard Boc (N ⁇ -amino protected N ⁇ -t-butyloxycarbonyl) amino acid resin with standard deprotecting, neutralization, coupling and wash protocols, or standard base-labile N ⁇ -amino protected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids. Both Fmoc and Boc N ⁇ -amino protected amino acids can be obtained from Sigma, Cambridge Research Biochemical, or other chemical companies familiar to those skilled in the art.
  • the polypeptides can be synthesized with other N ⁇ -protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, such as by using automated synthesizers.
  • test compounds comprise antibodies
  • such antibodies can be polyclonal or monoclonal.
  • the antibodies can be humanized, fully human, or murine forms of the antibodies.
  • Such antibodies can be made by well-known methods, such as described in Harlow and Lane, Antibodies; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988).
  • nucleic acids may be chemically synthesized or recombinantly expressed as well. Recombinant expression techniques are well known to those in the art (See, for example, Sambrook, et al., 1989, supra).
  • the nucleic acids may be DNA or RNA, and may be single stranded or double.
  • such nucleic acids can be chemically or enzymatically synthesized by manual or automated reactions, using standard techniques in the art. If synthesized chemically or by in vitro enzymatic synthesis, the nucleic acid may be purified prior to introduction into the cell.
  • the nucleic acids can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof.
  • the nucleic acids may be used with no or a minimum of purification to avoid losses due to sample processing.
  • test compounds comprise compounds other than polypeptides, antibodies, or nucleic acids
  • such compounds can be made by any of the variety of methods in the art for conducting organic chemical synthesis.
  • COL4A3BP (GPBP gene) expresses at least three polypeptides including canonical GPBP-1, also called 77-kD GPBP or GPBP (1); GPBP-2, an alternative mRNA exon splicing isoform also called CERT or GPBPA26 (2); and GPBP-3, a variant which results from alternative mRNA translation initiation and also called 91-kD GPBP (3).
  • GPBP-1 is mainly secreted and interacts with type IV collagen (3); GPBP-2 localizes mainly in the cytosol (3), transports ceramide between endoplasmic reticulum and Golgi apparatus (4) and induces protein secretion (5); and GPBP-3 is associated with cellular membranes and promotes GPBP-1 exportation (3).
  • Type IV collagen is composed of six distinct ⁇ chains ( ⁇ 1- ⁇ 6) that form three types of triple-helical molecules [ ⁇ 1. ⁇ 1. ⁇ 2(IV), ⁇ 3. ⁇ 4. ⁇ 5(IV) and ⁇ 5. ⁇ 5. ⁇ b 6 (IV)] (6).
  • Major structural support for the renal glomerulus includes peripheral wrapping membrane-organized ⁇ 3. ⁇ 4. ⁇ 5(IV) network (GBM) and central mesh-organized ⁇ 1. ⁇ 1. ⁇ 2(IV) network (mesangial matrix).
  • the ⁇ 3. ⁇ 4. ⁇ 5(IV) network (epithelial) fuses with a membrane-organized ⁇ 1. ⁇ 1. ⁇ 2(IV) network (endothelial) to yield the backbone of the capillary GBM, a principal component of the glomerular filtration barrier.
  • GPBP-1 is a non-conventional Ser/Thr kinase which targets type IV collagen (1) and regulates its glomerular organization (7).
  • increased expression of GPBP-1 causes dissociation of the ⁇ 3. ⁇ 4. ⁇ 5(IV) and ⁇ 1. ⁇ 1. ⁇ 2(IV) network at the glomerular filtration barrier and induces expansion of the ⁇ 1. ⁇ 1. ⁇ 2(IV) network, thereby mediating capillary collapse (glomerulosclerosis).
  • autoantibody production correlates with glomerular overexpression of GPBP-1 and results in immune complex deposit formation on the disrupted capillary GBM (7).
  • GPBP-1 is secreted to the extracellular compartment (3) and we have demonstrated that GPBP-1 is a constituent of human plasma (cGPBP-1) and that increased levels of cGPBP-1 are associated with immune complex-mediated GN (WO 2010/009856 and U.S. Pat. No. 7,935,492). Here, we show that cGPBP-1 is a therapeutic target for immune complex-mediated GN.
  • cGPBP-1 human plasma was adsorbed onto an affinity column containing immobilized mAb N26, a GPBP-specific monoclonal antibody (mAb), as described in WO 2010/009856 and U.S. Pat. No. 7,935,492. Bound material was eluted and shown to contain a major polypeptide of ca. 77 kDa and minor polypeptides of lower M r reactive with GPBP-specific antibodies confirming that GPBP-1 and derived products are normal constituents of the human plasma.
  • the M r of cGPBP-1 varied between 74- and 80-kDa depending on molecular mass standards used (not shown).
  • NZW mice developed a lupus-prone autoimmune response consisting of IgG and IgA autoantibodies with predominant deposits of IgA immune complexes in the glomerulus (7). In the present study, however, NZW mice developed an IgG-based lupus-prone autoimmune response and no circulating IgA autoantibodies were detected (data not shown).
  • kidneys of aged mice were analyzed by histochemical and immunohistochemical techniques.
  • mesangial proliferation (a), hyaline thrombi (b), subendothelial deposits (c), matrix expansion (d), wire-loop deposits (e), glomerular lobularity (f), endocapillary proliferation (g), glomerular inflammatory infiltrates (h), necrosis, karyorrhexis and pyknosis (i), occasional crescents (j), tubule-interstitial inflammatory infiltrates (k) and tubular atrophy (l).
  • biotinylated mAb N12 or N26 was assessed and found that these mAbs can potentially block cGPBP-1 in vivo ( FIG. 1A ).
  • NZW mice were injected with a single intraperitoneal boost (250 ⁇ g) and their circulating levels monitored ( FIG. 1B ). Both mAbs reached a peak three days after injection (10 ⁇ g/mL). The levels of mAb N12-biotin dropped by half on day 8 and were almost undetectable by day 14, whereas the levels of mAb N26-biotin on day 14 remained elevated (6 ⁇ g/mL), revealing that serum clearance of mAb N26-biotin was lower than mAb N12-biotin.
  • mice 8-10 months of age were injected with PBS (carrier) or with ⁇ 1 ⁇ g/g body weight of murine IgG (Control), mAb N12-biotin or mAb N26-biotin, a dose found not to trigger a rebound effect ( FIG. 2 ).
  • cGPBP-1 levels dropped irrespective of the treatment, suggesting that a certain stress provoked by the manipulation of the mice influenced cGPBP-1 levels at the onset of the study.
  • mice treated with mAb N12-biotin which remained reduced along the treatment, achieving statistical significance between the fourth and sixth week ( FIG. 2A ).
  • the lowering of cGPBP-1 levels in mice treated with mAb N12-biotin was more effective than mice treated with mAb N26-biotin ( FIG. 2B ).
  • Kidneys from control mice displayed abnormalities characteristic of NZW immune complex-mediated GN; however, mice treated with GPBP-specific antibodies did not present relevant glomerular or tubular lesions (HE and Mason). Accordingly, biological treatments reduced glomerular deposits of GPBP-1, did not affect tubular intracellular expression of GPBP, and reduced deposits of immune complexes (IgG, IgM and C3c). The latter was further supported by virtual absence of electron-dense deposits commonly found in the epithelial side of the capillary GBM in control mice. Consistent with all these observations, tubule-interstitial inflammatory infiltrates were sharply reduced in mice treated with GPBP-specific antibodies (HE).
  • HE GPBP-specific antibodies
  • NZW mice develop IgA and IgG lupus-prone autoantibody response with predominant deposits of IgA in the glomerulus (7).
  • NZW mice develop a lupus-prone IgG autoimmune response with predominant glomerular deposit of IgG.
  • NZW mice developed an “inflammatory” lupus nephritis-like (present example) or a “non inflammatory” IgA nephropathy-like (7) depending on whether they were maintained in a pathogen-free or in a less restrictive environment, respectively.
  • blocking antibodies are expected to specifically inhibit the activity of extracellular GPBP-1 which comprises both tissue-bound GPBP-1 and cGPBP-1.
  • therapeutic assays herein represent a unique test to determine the pathogenic role of extracellular GPBP-1 in clinically silent lupus nephritis-like developed by NZW mice.
  • Our results suggest that antibody treatment virtually repairs GBM collagen-based alterations, reduces the glomerular deposits of immune complexes and consequently, attenuates inflammation.
  • Two independent mAbs which display different ability to lower cGPBP-1 levels, but similar activity blocking of GPBP-1 binding to GBM collagen, attenuate GN progression in a similar fashion. The latter indicates that therapeutic effects depend more on blocking than on clearance of cGPBP-1 levels.
  • anti-GPBP therapy apparently reduces glomerular GPBP-1 deposits more efficiently ( FIG. 3 ) than it lowers cGPBP-1 levels ( FIG. 2 ), suggesting that glomerular GPBP-1 is responsible for GBM collagen disorganization.
  • Blocking antibodies are expected to lower glomerular GPBP-1 by reducing the capacity of cGPBP-1 to bind GBM collagen and by enhancing the release of tissue-bound GPBP-1. Although it remains to be determined how cGPBP-1 levels influences tissue-bound GPBP-1 levels, both GPBP-1 sources are likely operative and, in a more general context, account for primary (predominant local production) or secondary (predominant distant production) immune complex-mediated GN.
  • tissue-bound autoantibodies induce GPBP-1 expression and secretion.
  • GPBP-1 either remains bound to tissue in the surrounding extracellular compartment or enters the plasma.
  • tissue-bound GPBP-1 results from local production and entrapment of cGPBP-1.
  • primary GN local production
  • secondary GN distal production and subsequent entrapping
  • GBM collagen undergoes disorganization and promotes additional immune complex deposit formation.
  • Immune complex deposits and lupus-prone cellular stimuli, mediated by elevated cGPBP-1 positively regulate the pathogenesis which perpetuates SLE and GN progression.
  • Anti-GPBP blocking antibodies attenuate GBM collagen disorganization and immune complex deposit formation, and disrupt the pathogenic feedback.
  • Our data identify cGPBP-1, pathogenic factor and therapeutic target in immune complex-mediated GN.
  • cGPBP-1 is a Pre- and Pro-inflammatory Biomarker
  • GPBP-1 is a Pro-inflammatory Factor.
  • macrophages RAW264.7 and LPS and doxorubicin (1) to assess pro-IL-1 ⁇ expression and IL-1 ⁇ release.
  • Many different agents such as asbestos, silicates, uric acid crystals or doxorubicin are inflammasome activators (2), but they are unable to induce expression of pro-IL-1 ⁇ (37 KDa) in macrophages which depends on agonists of the Toll-like receptors (TLR) (i.e. LPS).
  • TLR Toll-like receptors
  • LPS Toll-like receptors
  • RAW264.7 macrophages were primed overnight with 1 ⁇ g/ml of LPS and NLRP3 inflammasome was activated with 10 ⁇ M of doxorubicin.
  • Secreted IL-1 ⁇ and cGPBP-1 in culture media were measured by ELISA at the indicated times, and cytosolic expression of pro-IL-1 ⁇ and GPBP-1 analysed in cell lysates by Western blot ( FIG. 4 ).
  • Silencing of GPBP-1 in RAW264.7 cells caused a reduction in pro-IL-1 ⁇ synthesis and IL-1 ⁇ release ( FIGS. 4A and 4B ).
  • the time pattern of cGPBP-1 release in these cells was similar to that of IL-1 ⁇ ( FIGS. 4B and 4C ) and thus GPBP-1-silenced RAW264.7 cells showed a marked decrease in cGPBP-1 release, indicating that GPBP-1 is the main GPBP isoform being secreted by the cell.
  • cGPBP-1 is a Pre-inflammatory Biomarker in Patients with Proteinuria.
  • CRP C Reactive Protein
  • cGPBP-1 is a Pre-inflammatory Biomarker for RA
  • mice developing a severe CIA [collagen type II-immunized male mice or non-Tg female and Bcl-2-Tg females that are depleted in regulatory T cells after treatment with an anti-CD25 monoclonal antibody (5,6) exhibit very high levels of cGPBP-1 preceding the onset of a clinical CIA (4 weeks after CIA induction; p ⁇ 0.02 in all cases), but these levels are significantly reduced, even below the levels observed in healthy non-immunized controls, once CIA is clinically manifested with high severity (8 weeks after CIA induction; p ⁇ 0.05 in all cases; FIG. 7 ).
  • circulating GPBP-1 is a biomarker for early stage in RA pathogenesis.
  • IL-1 ⁇ is a prototypic multi-functional cytokine, mainly produced by blood monocytes, but also by macrophages, dendritic cells and by a variety of cells of nearly all types (7). IL-1 ⁇ is involved in the pathogenesis of several inflammatory disorders and in human and animal models of GN (7, 8, 9, 10).
  • IL-1 ⁇ and cGPBP-1 release are coordinated upon the same pro-inflammatory stimulus that activate inflammasome.
  • pro-inflammatory conditions such as LPS stimulation
  • pro-IL-1 ⁇ and GPBP-1 are accumulated inside the cell.
  • doxorubicin-mediated inflammasome activation both IL-1 ⁇ and cGPBP-1 are secreted to extracellular media.
  • GPBP-1 Downregulation of GPBP-1 decreases the production of pro-IL-1 ⁇ and delays the release of IL-1 ⁇ in the cultured macrophage-like murine cell line RAW 264.7 revealing that GPBP-1 expression and activation is an earlier step than pro-IL 1 ⁇ expression and activation in the pro-inflammatory cascade
  • cGPBP-1 concentration is under 10 ng/ml in the sera of healthy controls, and is significantly increased in the sera of IgA nephropathy and lupus nephritis patients (see above). This is consistent with our previous findings that GPBP-1 accumulates in the glomerulus of aged NZW kidneys undergoing subclinical immune complex-mediated GN (11).
  • GPBP-2 has been previously proposed as a target for treating-inflammation (12), since its inhibition is expected to limit the availability of ceramides for PGE2 synthesis in response to IL-1 ⁇ , suggesting that both GPBP-1 and GPBP-2 are part of a pro-inflammatory cascade.
  • GPBP-1 Goodpasture Antigen-binding Protein-1
  • Pulmonary fibrosis occurs when the lung tissue becomes damaged and scarred.
  • PF can be developed by unknown causes (idiopathic PF) or be associated or induced by many conditions including chronic inflammatory processes, infections, environmental agents, exposure to ionizing radiation and certain medications (1).
  • idiopathic PF PF
  • bleomycin and doxorubicin have been shown to induce PF in different animal species including humans (2-5).
  • the prognosis of patients with PF is bad and the majority of patients with this disease are dead within 5 years.
  • no medication has been shown to improve the outcome of patients with PF (1) and lung transplantation is the only therapeutic option available at present.
  • GPBP-1 regulates the supramolecular organization of structural proteins both inside and outside of the cell (6, 7). Since the expression of GPBP-1 is regulated by inflammatory stimuli such as TNF ⁇ (8, 9), and inflammation plays a major role in the initiation and or progression of the fibrotic process, we hypothesize here that GPBP-1 may be a key molecule involved in the pathogenesis of diseases characterized by the aberrant accumulation of different types of collagens (ie: collagen type I) such as PF. In our present study, we have characterized two compounds from the Pretswick Chemical Library that have the ability to modulate GPBP-1 activity in vitro.
  • the intratracheal (i.t.) instillation of 75 ⁇ g of doxorubicin into C57BL/6 male mice causes a generalized alteration in the lung architecture 12 days after drug administration that macroscopically is characterized by the intense inflammation of the lungs that appear hemorrhagic ( FIG. 8 ).
  • the histological analysis shows an intense inflammatory cell infiltration that includes the presence of perivascular granulomas and interstitial matrix deposition resulting in the obliteration of alveoli ( FIG. 8 , HE).
  • the collagenous nature of the interstitial matrix deposits is evidenced after Masson's trichrome staining of the lungs ( FIG. 8 ).
  • an increase in the expression of ⁇ 1-collagen I gene is observed in the lungs of doxorubicin-treated mice by RT-qPCR analysis ( FIG. 9 ).
  • mice were instilled i.t. with doxorubicin and treated from the beginning up to the end of the experiment (days 0-12) with different doses of pinacidil.
  • a daily dose of 0.08 mg/kg of pinacidil sharply reduced the severity of doxorubicin-induced pulmonary fibrosis ( FIG. 10 ), and consistently a significant reduced expression of pulmonary ⁇ 1(I)collagen mRNA was observed in pinacidil-treated mice ( FIG. 11A ).
  • Lower doses of pinacidil resulted in a similar reduction of expression of ⁇ 1(I)collagen mRNA ( FIG.
  • FIG. 11A Strikingly, the therapeutic effect of pinacidil was lost when administered higher doses of the drug (0.25 or 1.225 mg/kg/day) ( FIG. 11A ). In contrast when mice were treated with myricetin, efficient reduction in the doxorubicin-induced fibrosis was obtained at all doses tested. ( FIG. 11B ).
  • mAb 14 and mAb N26 Two Monoclonal Antibodies to GPBP-1, Ameliorate Doxorubicin-induced PF In Vivo.
  • mice were i.t. injected with doxorubicin and were treated from the first day after i.t. instillation with a monoclonal antibody against Transforming Growth Factor ⁇ ( ⁇ TGF ⁇ ; 1 mg/week divided in 3 doses), a known anti-fibrotic therapy (10) or with mAb-14 (1 mg/week divided in 3 doses), or with mAb N12 (25 ⁇ g/week in a single injection), or were left untreated (Doxo).
  • ⁇ TGF ⁇ Transforming Growth Factor ⁇
  • mAb-14 1 mg/week divided in 3 doses
  • Doxo mAb N12
  • NZW and C57BL/6 mice were generated and maintained in a pathogen-free environment.
  • Treatments lasted 8 weeks and serum samples were collected immediately before injections.
  • Antibodies or PBS were administered intraperitoneally and serum samples were obtained from mouse tail and used for analytical determinations. We did not find differences between IgG- and PBS-treated mice and both groups were collectively used as controls for statistical analysis.
  • All mice were sacrificed and kidneys were analyzed by standard histological techniques. Sera from 3-month old NZW mice and 8-10 months old C57BL/6 mice were used as controls in analytical studies.
  • mice For induction of PF with doxorubicin (Example 3), eight to ten weeks-old C57BL/6 male mice were purchased from Harlan Ibérica (Barcelona, Spain). For intratracheal (i.t.) instillation of doxorubicin (Sigma), mice were anesthetized by intraperitoneal (i.p.) injection of ketamine (50 ⁇ g/g of body weight), atropine sulphate (0.2 ⁇ g/g) and diazepam (4 ⁇ g/g). The trachea was localized by cutaneous incision in the anterior face of the neck and immobilized with forceps.
  • ketamine 50 ⁇ g/g of body weight
  • atropine sulphate 0.2 ⁇ g/g
  • diazepam 4 ⁇ g/g
  • doxorubicin chlorhydrate (1 mg/ml) were injected i.t. with a 30G needle Skin was then sutured with 3/0 silk.
  • Mice were fed ad libitum with a normal chow diet and bled from the retro-orbital plexus at different time points after doxorubicin i.t. instillation. Pinacidil and Myricetin, were selected from the Pretswick Chemical Library on the basis of their capacity to inhibit the autophosphorylating activity of GPBP in vitro. Mice instilled i.t. with doxorubicin were daily injected i.p.
  • Monoclonal antibodies N12, N26, and N27 were generated against Pichia pastoris human recombinant GPBP-1, which production have been previously reported (1, 2).
  • Monoclonal antibody el 1-2 was generated against GST-e26, a chimeric protein expressed in E. coli containing the protein sequence encoded by exon 11 of GPBP-1, absent in GPBP-2, fused to glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • mAbs N12 and N26 were biotin-labeled with Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Rockford, Ill.) and used for in vivo assays.
  • GPBP-1 was visualized using either mAb N27 labeled with AlexaTM Fluor 546 (Invitrogen, Carlsbad, Calif.), or chicken anti-GPBP-pep 1 polyclonal antibodies previously characterized (3).
  • Goat anti-type IV collagen polyclonal antibodies (Chemicon, Temecula, Calif.) labeled with AlexaTM Fluor 647 (Invitrogen, Carlsbad, Calif.), and biotin-labeled anti- ⁇ 3(IV) mAb3 (Wieslab AB, Lund, Sweden) were used for detection of glomerular ⁇ 1. ⁇ 1. ⁇ 2(IV) and ⁇ 3. ⁇ 4. ⁇ 5(IV) networks, respectively.
  • Both recombinant cGPBP-1 and ⁇ 3(IV)NC1 domain were detected with ANTI-FLAG M2-Peroxidase (Sigma-Aldrich, St Louis, Mo.).
  • Complement component 3c C3c
  • IgA, IgG and IgM deposits were visualized with rabbit polyclonal anti-C3c-FITC (Abcam) and goat anti-mouse IgA-, IgG- or IgM-FITC (Sigma-Aldrich), respectively.
  • Biotinylated antibodies were detected with High Sensitivity NeutrAvidin-HRP (Thermo Fisher Scientific, Rockford, Ill.), Streptavidin-AF488 (Invitrogen) or ExtravidinTM-TRITC (Sigma-Aldrich). Secondary antibodies used were goat polyclonal antibodies to chicken IgY-FITC (Abcam) and donkey anti-mouse IgG-HRP (Jackson ImmunoResearch Europe Ltd, Suffolk, UK).
  • A549 cells were grown with Dulbecco's modified Eagle's F-12, while HEK 293 and RAW 264.7 were cultured with Dulbecco's modified Eagle's medium. All medium were supplemented with 10% fetal calf serum and with 1% penicillin/streptomycin.
  • pSilencerTM 2.1—U6 hygro vector (Ambion) was employed for stable expression of small interfering mRNAs (siRNAs) specific for silencing GPBP-1.
  • the derived construct was named pSi-GPBP-1 and the cDNA target sequence was GCCCTATAGTCGCTCTTCC (SEQ ID NO: 11), as previously described (4).
  • the cDNA of GPBP-1 exon 11 was cloned downstream in-frame with the cDNA of GST in the pGEX-5x-1 vector (GE Healthcare).
  • Wild type RAW 264.7 cells and GPBP-1-silenced RAW 264.7 cells were plated at 2 ⁇ 10 6 cells/well in 6-well culture plates and stimulated with 1 ⁇ g/ml LPS (Sigma-Aldrich, E. Coli serotype 026:B6) overnight (16 h). Then, the LPS-primed macrophages were exposed to 10 ⁇ M doxorubicin (Sigma-Aldrich) during 2, 4 and 6 h. After incubation with doxorubicin, expression of pro-IL-1 ⁇ and cytosolic GPBP-1 was analyzed in cell lysates by Western blot, and mature IL-1 ⁇ and cGPBP-1 in media supernatants were quantified by ELISA.
  • Cell lysates were obtained by scrapping the cells in lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 ⁇ M leupeptin, 1 mM PMSF) and incubating on ice for 10 min. Lysates were clarified by centrifugation at 15,000 ⁇ g for 10 min at 4° C. and analyzed by Western blot.
  • lysis buffer 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 ⁇ M leupeptin, 1 mM PMSF
  • Recombinant poly-His-GPBP-1 used in ELISA was purified from E. coli transformed with GPBP-1 cDNA cloned in frame in the multiple cloning site of the expression plasmid pET-15b (Merck KGaA, Darmstadt, Germany), which adds a tag of six histidines to facilitate its purification with nickel-chelated agarose columns (Clontech Laboratories, Mountain View, Calif.) following standard procedures.
  • cGPBP-1 Recombinant cGPBP-1 (rcGPBP-1) was purified using ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich, St Louis, Mo.) from cell culture media of a HEK 293 cell clone that constitutively secretes FLAG-GPBP-1.
  • GST-e26 expression was induced with 1 mM IPTG in E. coli DH5 ⁇ cells harboring pGEX-5x-1-e26 construct, and purification was performed with GSH-agarose affinity resin (Sigma) following manufacturer's recommendations.
  • poly-His-GPBP-1, rcGPBP-1 or GST-e26 were assessed by SDS-PAGE and Coomassie blue staining and its integrity by Western blot using mAbs to GPBP-1 including mAb N26, mAb N27 and mAb ell-2.
  • mice All mice were sacrificed in a CO 2 chamber. After in vivo experiments with antibodies (Example 1), kidneys were fixed in 10% formalin and embedded in paraffin. Sections of 2 ⁇ m were obtained by an electronic rotary microtome (Microm, Walldorf, Germany) and subjected to conventional Heamatoxylin/Eosin (HE), Masson's staining (Masson).
  • HE Heamatoxylin/Eosin
  • Masson Masson's staining
  • mice were intrabronchially perfused with 4% phosphate buffered formalin, then incubated in this fixative during 24 hours and finally embedded in paraffin.
  • Tissue sections of 5 ⁇ m were stained with HE or with Masson's trichrome following conventional methods.
  • paraffin-embedded lungs were stained with anti-GPBP-pepl antibodies (3), which was detected with anti-chicken-HPR and DAB substrate (Dako Diagnósticos, S. A., Barcelona Spain). Specimens were counterstained with hematoxylin.
  • Kidneys were embedded in OCT (Sakura, Tokyo, Japan) and frozen. Six- ⁇ m sections were obtained with a cryostat (Microm) and stained for conventional and confocal microscopy analysis as previously described (3).
  • Raw data files were background corrected using supplier's methodology and intensity signal standardized across arrays via quantile normalization algorithm.
  • Differential gene expression assessment of all comparisons was carried out using limma moderated t-statistics. Conventional adjustment for multiple testing proposed by Benjamini Hochberg (5) was used to derive adjusted P-values. Also, for each of the comparisons performed in the study, gene set analysis was carried out using logistic regression models, and correcting for multiple testing as before (5). All analyses where carried out using BabelomicsTM (6) web suite at the Bioinformatics Department of Centro de Investigacion Principe Felipe. Array data have been deposited in GEO (accession number GSE32181).
  • RT-qPCR real-time quantitative PCR
  • Primers used were: for ⁇ 1(I) collagen gene, 5′ primer 5′-TCCTGCTGGTGAGAAAGGAT-3′ (SEQ ID NO: 12) and 3′ primer 5′-CTGGAGTCCCATAACGACCT-3′ (SEQ ID NO: 13); for GPBP gene, 5′ primer 5′-GCTGTTGAAGCTGCTCTTGACA-3′ (SEQ ID NO: 14) and 3′ primer 5′-CCTGGGAGCTGAATCTGTGAA-3′(SEQ ID NO: 15); for 18S gene, 5′ primer 5′-GTAACCCGTTGAACCCCATT-3′ (SEQ ID NO: 16) and 3′ primer 5′-GCGATGATGGCTAACCTACC-3′ (SEQ ID NO: 17). Results (in triplicate) were normalized to the ribosomal 18S subunit expression and measured in parallel in each sample. Data were expressed as mean fold change relative to control samples.
  • cGPBP-1 was extracted from human plasma by affinity chromatography. Specifically, the plasma (200 mL) was clarified by centrifugation, filtered (0.45 ⁇ m), and diluted 1:5 (v/v) with 25 mM Tris pH 7.4, 150 mM NaCl (Tris-buffered saline, TBS) supplemented with 0.05% Tween 20 (TBST). The supernatant was loaded onto a column containing 1 mg of mAb N26 immobilized in 1 ml, of cyanogen bromide-activated-Sepharose 4B (Sigma-Aldrich). The bound material was washed with 10 mL of TBST, eluted with ImmunoPureTM Gentle Ag/Ab Elution Buffer (Thermo Fisher Scientific) and extensively dialyzed against TBS.
  • TBS Tris pH 7.4, 150 mM NaCl
  • TBS Tris-buffered saline
  • TBS Tris-buffered s
  • Quantification of cGPBP-1 or of biotinylated mAbs was performed by sandwich ELISA coating plates with mAb N26 or poly-His-GPBP-1 at 2 ⁇ g/mL in PBS, respectively. Plates were blocked and incubated with samples (1:10-1:20 dilutions) and detection performed with mAb N27-HRP or High Sensitivity NeutrAvidinTM-HRP at 1 ⁇ g/mL, respectively.
  • Anti-ss-DNA IgG and CRP titers were measured by indirect sandwich ELISA using a commercial kit from Alpha Diagnostic Intl. (San Antonio, Tex.) and Meditec (Kiel, Germany) respectively, following manufacturer's instructions.
  • Kidneys were excised and fixed by immersion in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium phosphate pH 7.4 (PB) for 24 hours at 4° C. and then washed with PB. Sections of 200 ⁇ m were obtained with a vibratome (Leica VT-1000; Leica Microsystems Heidelberg GmbH, Mannheim, Germany), fixed with 2% osmium tetroxide in PB for 1.5 hours, washed with chilled water (3 ⁇ 5 min), dehydrated through sequential washes with increasing concentrations of chilled ethanol solutions (5 min with 30%, 5 min with 50%, and 10 min with 70%), washed with 2% uranile acetate in 70% ethanol for 2.5 hours at 4° C., and further dehydrated with 2 ⁇ 5 min washes in 70% ethanol, 2 ⁇ 5 min and 10 min washes with 96% ethanol, 2 ⁇ 7 min washes in 100% ethanol, and a single 10 min wash in dried 100% ethanol.
  • PB sodium phosphate pH
  • Prism 4.0 software (GraphPadTM Software, San Diego, Calif.) was used for all calculations. Data were analyzed with two-way ANOVA or Kruskal-Wallis test to assess significant differences between series, and with Spearman's test to determine the statistical significance of the correlation between cGPBP-1 and anti-ssDNA autoantibodies. A P value ⁇ 0.05 was considered significant.

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