WO1989011285A1 - Diagnose et traitement de rhumatismes articulaires - Google Patents

Diagnose et traitement de rhumatismes articulaires Download PDF

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WO1989011285A1
WO1989011285A1 PCT/US1989/002219 US8902219W WO8911285A1 WO 1989011285 A1 WO1989011285 A1 WO 1989011285A1 US 8902219 W US8902219 W US 8902219W WO 8911285 A1 WO8911285 A1 WO 8911285A1
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htlv
sample
rheumatoid arthritis
patient
antibody
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PCT/US1989/002219
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Steffen Gay
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Steffen Gay
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    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A61K31/66—Phosphorus compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • This invention relates to a method for diagnosis of rheumatoid arthritis in an individual. This method is based on the discovery that the presence of a viral peptide or protein is associated with the occurrence of rheumatoid arthritis in an individual.
  • the invention also relates to the treatment of rheumatoid arthritis by administration of an anti-viral agen .
  • RA Rheumatoid arthritis
  • Animals of the autoimmune MRL/1 mouse strain are characterized by proliferation of synovial cells, destruction of articular cartilage and bone, and mononuclear cell infiltration. Most of its serious and debilitating sequelae are derived from erosion and destruction of articular connective tissues. Animals of the autoimmune MRL/1 mouse strain
  • the first stage develops between the ages of 7 and 13 weeks and consists of synovial cell proliferation in the joint recesses.
  • the second stage is characterized by continued proliferation of synovial cells which take on an appearance similar to transformed mesenchymal cells. The earliest destructive changes occur in the second stage and include marginal erosions, followed soon after by progressive destruction of articular and meniscal cartilage.
  • the final stage is characterized by a diminution of synovial cell proliferation, extensive cartilage destruction, formation of scar tissue and fibrocartilage, and a moderate infiltration of the synovial stro a by ononuclear and polymorphonuclear inflammatory cells.
  • MCF mononuclear cell factor
  • the mechanism of cellular attachment is of particular interest, especially since erosions of cartilag and bone occur in the MRL/1 mice in the early inflammatory stage of disease only in areas where the proliferating synovial lining cells appear in close contact to cartilaginous and bony matrices (O'Sullivan, F.X., et al. , 1985, Arthr. Rheum. 28:529-536). The same is true for the primary lesions in human rheumatoid arthritis (Fassbender, H.G., 1983, Collagen Rel. Res. 3:141-155).
  • Electron microscopy of normal synovial lining in human (Wynne-Roberts, C.R. and Anderson, C. , 1978, Arthr. Rheum. 7:279-286; Ghadially, F.N. , 1980, Ultrastruct. Path. 1:249-264), rat (Graabaek, P.M., 1984, Lab. Invest. 50:690-702), and mouse (Linck, G. and Porte, A., 1978, Cell Tissue Res. 187:251-261; Linck, G. and Porte, A., 1978, Cell Tissue Res.
  • Viruses such as Epstein-Barr virus, hepatitis B virus, rubella virus, reovirus, and parvovirus (Brown, K.A., 1984, Nature 309:582) have been implicated in the etiology of rheumatoid arthritis (see Sokoloff, L. , 1984, Intl. Rev. Exp. Pathol. 26:117-119; Smith, C.A. , 1979, J. Rheumatol. 6(2) :113-116; April 7, 1984, The Lancet, pp. ⁇ . ⁇ 772-774; Bacon, P.A. and Tunn, E.J., 1986, Quarterly J. Med.
  • aspirin are used for their anti-inflammatory and analgesic properties.
  • Ibuprofen and similar compounds are salicylate alternatives.
  • Indomethacine is another anti- inflammatory and analgesic drug which is useful for those patients who are not good candidates for treatment with th
  • Gold compounds are used in addition to the prior noted compounds against active joint inflammation, but the compounds are not usually effective where the RA condition is advanced. In addition, toxic side reactions have resulted from the use of gold compounds.
  • 35 D-Penicillamine which is used as an alternative to the gold compounds, is also capable of serious toxic side reactions. Mild to moderate active RA symptoms are treatable with hydroxychloroquine, although irreversible retinal damage can occur with its use. The use of corticosteroids provides short term anti-inflammation effects, but the progression of joint damage is not affected. (Berkow, R. , et al. , 1982,The Merck Manual, 779-786.)
  • RNA tumor viruses constitute the Oncovirinae subfamily of the Retroviridae family.
  • the Retroviridae, o retroviruses are enveloped RNA viruses (for a review, see Hayward, W.S. and Neel, B.G., 1981, Curr. Top. Microbiol. Immunol. 91:217-276) which utilize a DNA intermediate in their replication cycle.
  • the virus particle consists of a ribonucleoprotein core enclosed by an outer membrane envelope derived from the host cell plasma membrane. Vira envelope glycoproteins protrude from the outer envelope.
  • the viral genome consists of a single-stranded RNA molecule.
  • RNA-dependent DNA poly erase or reverse transcriptase.
  • the replication cycle begins with adsorption of the virus to the cell surface, followed by penetration of the cell membrane, uncoating of the virus particle, and the release of viral RNA.
  • the viral RNA the serves as the template for the synthesis of DNA by reverse transcriptase.
  • Some of the linear DNA duplexes are transported to the nucleus where they are converted to covalently closed, circular DNA molecules (proviral DNA) , which are then integrated into the host cell chromosomal DNA.
  • the integrated proviral DNA replicates with the host genome, producing genomic RNA for incorporation into virus particles, and serving as the template for the synthesis of viral messenger RNAs which are processed and translated to produce the viral proteins. Assembly of the virus particle occurs in the cytoplasm at the cell membrane. The immature particles bud through the membrane into the extracellular environment where maturation occurs. The released particles are then ready to begin a new round of infection.
  • the avian RNA tumor viruses can be divided into two groups based on their pathogenic properties.
  • the rapidly t ⁇ transforming viruses are capable of producing neoplasia in vivo within 2-4 weeks and will morphologically transform cells in vitro (Graf, T. and Beug, H. , 1978, Biochem. Biophys. Acta Rev. Cancer 516:269-299) .
  • the slowly transforming retroviruses induce neoplasia after 4-12
  • Avian leukosis virus or ALV
  • ALV is the prototype of the slowly transforming retrovirus. Its genome is compose of the gag, pol, and env genes in a 5' to 3' linear array.
  • the gag gene encodes the structural proteins of the virus
  • 25 core, or group-specific antigens pol encodes the reverse transcriptase, and env encodes the envelope glycoproteins.
  • the oncogenes encompass a variety of cellular locations an functions (for review, see Hunter, T. , 1984, Sci. American 251:70-79) . They can be divided into groups on the basis of sequence and functional similarities. For example, the ras proteins have a GTP-binding property, while both myc
  • the major ras-induced protein in NIH3T3 (mouse fibroblast) cells is the cysteine protease cathepsin L (Joseph, L. , et al. , Nucl. Acids Res. 15(7) :3186) .
  • a positive correlation has been found between the extent of ras expression, the metastatic potential, and the amount of secreted cathepsin L in H-ras-transformed murine fibroblasts (Denhardt, D.T., et al. , 1987, Oncogene 2:55- 59) .
  • the major excreted protein from a Kirsten virus (carrying the ras oncogene) -transformed mouse fibroblast line appears to be a catalytically active precursor form of cathepsin L (Mason, R.W. , et a ⁇ . , 1987, Biochem. J. 248:449-454) .
  • HTLV Human T Cell Leukemia Virus
  • HTLV-I and HTLV-II are associated with certain leukemias and lymphomas.
  • HTLV-I is causatively linked to adult T cell leukemia (Gallo, R.C. and Wong-Staal, F. , 1982, Blood 60:545; Gallo, R.C, 1984, in Cancer Surveys, Vol. 3, Franks, L.M. , et al . . , eds., Oxford Univ. Press, Oxford, pp. 113-159) .
  • HTLV-II was first identified in a patient with a T cell variant of hairy cell leukemia (Kalyanaraman, V.S., et al. , 1982, Science 218:571) . Both viruses have the capacity to transform infected T cells in vitro, in addition to causing other cellular changes (see Arya, S.K., et al., 1984, Science 225:927-930, and references cited therein) .
  • HTLV-I-like retrovirus has been isolated from T cell lines derived from patients with tropical spastic paraparesis, a progressive myelopathy (Jacobson, S., et al. , 1988, Nature 331:540-543) .
  • the causative agent of AIDS is a retrovirus, now termed human immunodeficiency virus type 1 (HIV-1) , and formerly termed HTLV III (Gallo, R.C, et al. , 1984, Science 224:500; Popovic, M. , et al., 1984, Science 224:497), LAV (Barre-Sinoussi, F. , et al. , 1983, Science 220:868; Feorino, P.M., et al., 1984, Science 225:69), and ARV (Levy, J.A., et al. , 1984, Science 225:840) by the three groups which independently isolated viruses which are probably of the same retrovirus subgroup (Levy, J.A. , et al. , Science 225:840).
  • the F (3 7 orf gene) of HIV has been shown to encode a protein, with sequence homologies to oncogene products, that exhibits the GTPase, autophosphorylation, and GTP-binding activities associated with the ras protein (Guy, B. , et al. , 1987, Nature
  • This invention relates to a method for diagnosis of rheumatoid arthritis in an individual. This method is based on the discovery that the presence of a viral peptide or protein is associated with the occurrence of rheumatoid arthritis in an individual. Generally stated, this invention comprises an assay to diagnose RA in a patient suspected of having RA. More specifically, the invention relates to the treatment of rheumatoid arthritis with an anti-viral agent. Such a treatment is directed at the biological particles now known to be associated with the disease's etiology.
  • FIGURE 1 shows fresh frozen section of synovial membrane (233/31) of patient with RA stained with monoclonal antibody to HTLV-I pl9 and secondary FITC- labeled goat - antimouse IgG. Less than 5% of synovial cells show specific intracellular and/or membraneous staining. (x 400)
  • FIGURE 2 shows paraffin section of formalin- fixed RA synovial tissue (R 1892) stained with monoclonal antibody to HTLV-I pl9 labeled by the immunogold silver enhancement technique. Specific staining of synovial cells adjacent to the site of bone erosion is shown. (x 250)
  • FIGURE 3 is an electronmicrograph which shows a typical retroviral structure observed in a sample from a patient afflicted with RA.
  • the serum from this RA patient was negative for antibodies against HTLV-I and HIV-1. (x 285,000)
  • the present invention provides methods for diagnosing rheumatoid arthritis in an individual.
  • the invention also provides methods for treating rheumatoid arthritis by administration of an anti-viral agent.
  • an individual with rheumatoid arthritis contains retroviral protein sequences such as HTLV-related protein sequences which are associated with the proliferating and transformed-appearing synovial cells and immunological response and inflammation of the joint. These protein sequences comprise an epitope that can be recognized by antibody directed against a protein determinant of HTLV.
  • the detection of HTLV-related peptides or proteins associated with rheumatoid arthritis provides means 1) for diagnosis of rheumatoid arthritis in an individual, and 2) for treating rheumatoid arthritis by administration of anti-viral agents.
  • the preferred embodiment of this invention pertains to a method of diagnosing rheumatoid arthritis, by detection in an individual of a peptide or protein comprising an epitope related to that of an retroviral protein such as an HTLV protein.
  • a method of diagnosing rheumatoid arthritis by detection in an individual of a peptide or protein comprising an epitope related to that of an retroviral protein such as an HTLV protein.
  • Such a method comprises: a) obtaining a sample from the individual; b) contacting the sample with an antibody directed against an epitope of a retroviral protein under reaction conditions that permit the antibody to bind the epitope; c) removing unbound antibody molecules from the sample; and d) examining the sample for the presence of bound antibody, wherein the binding of the antibody indicates the presence of rheumatoid arthritis in the individual.
  • a monoclonal antibody directed against an envelope glycoprotein of HTLV is used.
  • the monoclonal antibody can be labeled.
  • the method may further comprise addition of a labeled binding partner of the antibody.
  • the monoclonal antibody for use in the present invention is preferably selected from the group consisting of anti-HTLV I pl9, p24, and anti-HTLV III p24 and gpl20 as well as anti-HTLV reverse transcriptase.
  • any monoclonal antibody having affinity for HTLV may be utilized in carrying out this method.
  • the preferred antibodies are commercially available from Dupont (Billerica, MA) and are identified as anti-HTLV I pl9, anti-HTLV I p24, anti-HTLV III P15/17, anti-HTLV III p24 and anti-HTLV III gpl20 as well as anti-HTLV reverse transcriptase.
  • the peptide or protein comprising an epitope related to that of the HTLV protein can be: 1) uncomplexed; 2) complexed with biological material from said individual; 3) associated with a virus; and 4) a protein of HTLV I, HTLV III, a new HTLV-III-related protein or a new HT " LV-I-related retrovirus. More specifically, the antibody can be directed against an HTLV protein that is selected from the group consisting of HTLV I pl9, HTLV I p24, HTLV III pl5/17, HTLV III p24, and HTLV " III gpl20 as well as anti-HTLV reverse transcriptase.
  • the uncomplexed protein is a protein which is neither covalently or ionically associated with any other biological material.
  • protein is complexed with other biological materials via covalent or ionic bonds.
  • the proteins can be associated with viral replication proteins or proteins produced from viral geno ic material inserted into the host genome. Samples which can be assayed include but are not limited to synovium cells, tissue and fluid, and other joint components such as meniscal and articular cartilage and subchondral bone which are in proximity with the synovium tissue and fluid.
  • the preferred sample for assay is synovial material, wherein the synovial material is selected from the group consisting of the synovial cells and fluid.
  • synovial material is selected from the group consisting of the synovial cells and fluid.
  • other sources of biological samples may be utilized from an affected individual (i.e. , mononuclear peripheral blood cells) .
  • Assaying for the labeled complex is preferably effected by: 1) staining methods such as a) immunofluorescence ( Figure 1) , b) an immuno-peroxidase technique and c) immunogold staining with silver enhancement ( Figure 2) , and 2) an ultrastructural analysis by electron microscopy (as shown in Figure 3).
  • staining methods such as a) immunofluorescence ( Figure 1) , b) an immuno-peroxidase technique and c) immunogold staining with silver enhancement ( Figure 2) , and 2) an ultrastructural analysis by electron microscopy (as shown in Figure 3).
  • i munocytology, immunoblotting, and ELISA (enzyme-linked immunosorbent assay) methods are also used.
  • rheumatoid arthritis can be diagnosed in a patient by an in situ hybridization assay, using as probe a labeled nucleic acid sequence encoding an retroviral protein such as an HTLV protein.
  • nucleic acid probes include but are not limited to DNA, cDNA, or RNA sequences which can encode at least a portion of an envelope glycoprotein, core protein, polymerase, reverse transcriptase or other retrovirus-specific-protein. More specifically, this method is disclosed of diagnosing rheumatoid arthritis in a patient comprising:
  • the retroviral gene is an HTLV gene.
  • the method of diagnosis may comprise isolation of a sample of nucleic acid from the patient followed by hybridization to a nucleic acid probe specific for a retroviral gene.
  • a method may comprise a Northern or Southern hybridization procedure or a cyto dot blot hybridization technique.
  • Another embodiment of this invention is a method for treating rheumatoid arthritis in a patient by administering an anti-viral agent to the patient.
  • the anti-viral agent is preferably selected from the group consisting of acyclovir, phosphonoformate, ribavirin, "anti-sense” oligodeoxy nucleotide, protease inhibitor, 2' ,3'-dideoxynucleoside, methylphosphonate oligonucleotide and interferon.
  • said 2 ' ,3'-dideoxynucleoside is selected from the group consisting of 3'-azido-2 A ,3'-dideoxythymidine and 2' ,3'-dideoxycytidine. 6 .
  • Sections of tissues 4-6 urn thick are prepared from frozen, unfixed biopsy samples by cryostat sectioning. The air-dried sections are incubated with
  • Paraffin-sections are deparaffinized in xylene and alcohol. After trypsin digestion (0.1 mg/ml, Tris- HC1, 60 in) , the sections are treated with 0.3% hydrogen peroxide to inactivate endogenous peroxidase 0 activity and rinsed in 0.05% saponin (2 x 15 min each). Subsequently, sections are pretreated with normal serum diluted 1:5 in 0.2% BSA/PBS, washed and incubated with the primary antibody at 4°C overnight. After washing
  • Paraffin sections are deparaffinized and hydrated as mentioned above. Sections are incubated for 30 min with 5% heat-inactivated blocking serum (goat) and subsequently incubated overnight at 4°C with monoclonal antibody. Thoroughly washed slides are covered with gold-conjugated antibody (AuroProbe LM goat anti-mouse IgG (Janssen, Life Sci. Products, Olen, Belgium), diluted 1:40 with PBS, pH 7.3, containing 0.1% BSA, and are incubated for 2 hours at room temperature.
  • the specimens are cut into slices 0.2 to 0.5 mm thick with 2 razor blades or with a cryostat. They are fixed in 1% glutaraldehyde in 0.16 M cacodylate buffer, pH 7.4, at 4°C for 20 minutes [R. Fleischmajer et al., J. Invest. Der at.
  • Control specimens are treated only with rabbit anti-mouse Ig ferritin-tagged antibodies.
  • the specimens are postfixed in osmium tetroxide, passed through graded alcohols and embedded in Araldite.
  • the blocks are sectioned in a LKB ultratome-4 microtome and the o specimens, about 500-600 A (angstrom) thick, are either left unstained or are stained with a saturated solution of uranyl acetate.
  • the grids are examined with a Hitachi HU-12A or with a Phillips 300 electron microscope.
  • the last wash is performed in PBS with 4% sucrose and 5% glycerol for 1 hour.
  • Cell pellets are then placed in OCT freezing medium with a cork or plastic backing to hold them and quickly frozen by immersing them in a jar of methylbutane (isopentane) placed in a small chamber of liquid nitrogen.
  • the frozen cells are then wrapped in aluminum foil and stored in a closed container at -20°C
  • Frozen sections 8 urn thick are cut and placed in albumin coated slides and air-dried for at least 5 minutes.
  • Slides are then placed in a solution of ice-cold NaBH. (10 mg/100 ml) in PBS for 1 hour with one change. Following this procedure, the slides are washed at 4°C in PBS, 3 changes for 30 minutes each.
  • Protein immunoblot analysis is performed to confirm antibody reactivity seen in immunofluorescent studies.
  • Primary synovial cell cultures are grown-up in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum.
  • Adherent cells from duplicate dishes are treated as follows: (1) One dish is washed with PBS and cells are released by treatment with 0.25% trypsin in PBS. Harvested cells are counted to determine total cell number per dish. (2) A duplicate dish of cells, which has not been treated with trypsin, is washed with PBS and cells are harvested directly into SDS-polyacrylamide gel electrophoresis sample buffer
  • each blot is preincubated in a 1% bovine serum albumin (BSA) solution.
  • BSA bovine serum albumin
  • Monoclonal antibodies are diluted in 1% BSA solution and incubated with blots to allow antibody binding.
  • Bound antibody is detected by incubation with a peroxidase-conjugated goat-anti-mouse antibody (also diluted in 1% BSA solution) and visualized with 4-chloro-l-naphthol.
  • a duplicate blot is stained with an a ido black solution to visualize total transferred proteins as well as molecular weight markers.
  • Sera from patients with rheumatoid arthritis are obtained by bleeding.
  • a single lot of pooled normal human serum is used for negative controls.
  • Patient sera are diluted 1:1, 1:10, 1:100 in PBS and 50 ⁇ l of each diluted serum is added to triplicate wells and incubated at 37°C for 1 hr.
  • the wells are then washed three times with PBS-Tween, and 50 ul of horseradish peroxidase (HRP) conjugated goat anti-human immunoglobulin (New England Nuclear Screening System NE1-602) diluted 1:500 is added to each well and incubated for 1 hr at 37°C.
  • HRP horseradish peroxidase
  • New England Nuclear Screening System NE1-602 New England Nuclear Screening System NE1-602
  • Reverse transcriptase could be detected in about 70% of synovial tissues from RA patients utilizing a monoclonal antibody specific for HIV-1 reverse transcriptase (Cellular Products, Inc., Buffalo, NY). 6.5 TREATMENT OF RA
  • the treatment of rheumatoid arthritis in this invention utilizes an anti-viral agent, such as 3'- azido-2' ,3-dideoxythridine (Zidovudine) .
  • Zidovudine 3'- azido-2' ,3-dideoxythridine
  • This agent has been shown to be active against many human and animal retroviruses in vitro, such as HTLV-I and HTLV-III, has been particularly examined regarding its cytopathic effect on reverse transcriptase activity and on retroviral antigens such as p24 core antigen (gag protein) .
  • the p24 core antigen expression was not detectable when 0.13 ⁇ g/mL of zidovudine was used in connection with a strain of HIV (HTLV-IIIB) .
  • Zidovudine is administered orally.
  • the drug has also been given by intravenous infusion, although no parenteral dosage form of the drug is currently commercially available in the U.S.
  • the initial oral dosage of zidovudine currently recommended is 200 mg every 4 hours 6 times daily; however, this recommendation is based on limited experience, and the optimum dosage and schedule of administration have not been fully established. This dosage corresponds to 2.9 mg/kg every 4 hours in a 70-kg patient.
  • zidovudine usually was administered orally in dosages of 250 mg (2.3-5.9 mg/kg) every 4 hours, however, oral dosages up to 15 mg/kg every 4 hours have been used for short periods (e.g., 4 weeks) in a limited number of patients.
  • Ribavirin is also contemplated for treatment of RA. This agent has been shown to stimulate T cells (T- lymphocytes) and to produce a dose-dependent inhibition of antigen-induced proliferation. In addition, ribavirin exhibits antiviral activity against many RNA viruses, including Retroviridae, and the subfamily, HTLV-III.
  • Ribavirin is administered as a solution by nasal and oral inhalation only, using the Viratek small- particle aerosol generator (SPAG) Model SPAG-2.
  • 4 g of ribavirin as a sterile powder is reconstituted by dissolvin it in 50-100 mL of sterile water
  • This solution is then transferred to a sterile 500-mL flask.
  • the flask acts as the reservoir for the SPAG-2 aerosol generator.
  • the solution is further diluted with sterile water for inhalation to a final volume of 300 mL to provide a solution containing 20 mg/mL.
  • Diluents which contain an additive are not usable for the reconstitution of ribavirin.
  • the ribavirin solution should not contain any particulate matter and discoloration before its administration.
  • nebulization is preferably administered from the SPAG-2 aerosol generator via an oxygen hood, a face mask or oxygen tent.
  • the SPAG-2 aerosol generator delivers a mist containing about 190 ug of ribavirin per L.
  • the recommended administration of the ribavirin dose is delivered to the respiratory tract according to the following formula:
  • mist per minute inhalation therapy with the drug was administered for 16-18 hours. Administration was then continued for 12 hours daily (three 4-hour periods per day) on the second and third days of therapy and for 4 hours on the fourth (final) day of therapy.
  • Acyclovir is another candidate for treatment of RA.
  • This agent in parenteral acyclovir therapy, seemed in immunocompromised individuals to lessen inflamation and pain, prevent new lesions, and promote and healing of lesions.
  • acyclovir's principal therapeutic effect appears to be in preventing the rheumatoid arthritis progression.
  • oral acyclovir has been only recently used, it appears that it may 1) prevent new lesions, 2) possibly decrease the duration of pain, and 3) effect healing of lesions.
  • acyclovir sodium 500 mg of it is added to 10 L of sterile water for injection.
  • 500 mg of acyclovir sodium is added to bacteriostatic water containing no parabens for injection containing benzyl alcohol.
  • These reconstituted solutions should be used within 12 hours.
  • the solution Prior to using a parenteral dose of acyclovir, the solution must be shaken to completely dissolve the drug.
  • the requisite dose of the reconstituted solution is diluted with 50-125 L of a compatible intravenous infusion solution. Slow intravenous infusion using diluted solutions generally takes place over a 1-hour period to avoid the risk of adverse renal effects, which can occur if the infusion takes place in less than 1 hour.
  • An oral dose of 200 mg of acyclovir every 4 hours during waking hours 5 times daily for 10 days is the recommended adult dosage.
  • the recommended adult dosage is 200 mg 3 times daily for up to 26 weeks. In some instances dosages up to 200 mg 5 times daily for up to 26 weeks may be required. Intermittent treatment of recurrent episodes may require a dose of 200 mg every 4 hours while awake 5 times daily for 5 days.
  • Intravenous dosage of acyclovir for adults with normal renal function i.e., creatinine clearance
  • this invention relates to a method for treating rheumatoid arthritis in a patient comprising administering to the patient an agent which inhibits the attachment of the transformed synovial lining cells to cartilage and bone, thereby inhibiting joint erosion and destruction.
  • the agent is a peptide sequence which binds to the cell attachment receptors.

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Abstract

Cette invention concerne un procédé de diagnose de rhumatismes articulaires chez un individu. Ce procédé est basé sur la découverte que la présence d'un peptide ou d'une protéine virale est associée à la manifestation de rhumatismes articulaires chez un individu. L'invention concerne également le traitement de rhumatismes articulaires par l'administration d'un agent antiviral.
PCT/US1989/002219 1988-05-23 1989-05-22 Diagnose et traitement de rhumatismes articulaires WO1989011285A1 (fr)

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US19755488A 1988-05-23 1988-05-23
US197,554 1988-05-23

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WO1989011285A1 true WO1989011285A1 (fr) 1989-11-30

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000047A1 (fr) * 1989-06-30 1991-01-10 The Regents Of The University Of California Detection de retrovirus
EP0797440A1 (fr) * 1994-12-16 1997-10-01 Ray W. Exley Traitement de maladies auto-immunes au moyen de derives de 2-amino purine
WO2001026661A1 (fr) * 1999-10-11 2001-04-19 Yissum Research Development Company Of The Hebrew University Of Jerusalem Compositions comprenant des inhibiteurs de metalloproteinases a base d'oxophosphonate
EP1314429A2 (fr) * 1990-05-21 2003-05-28 The Administrators Of The Tulane Educational Fund Utilisation d'un composé retrovirale dans la fabrication d'un médicament pour le traitement du sydrome de Sjögren, du lupus érythémateux disséminé, de la scléroderme, et de l'arthrite rheumatoide juvenile
US6638909B1 (en) 1996-11-01 2003-10-28 Ethicon, Inc. Wound healing compositions containing alpha-1-antitrypsin

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0185444A2 (fr) * 1984-10-10 1986-06-25 Centocor, Inc. Clonage et expression d'ADN de HTLV-III
US4775636A (en) * 1983-11-25 1988-10-04 Janssen Pharmaceutica N.V. Blot overlay assay using colloidal metal particles
EP0308066A2 (fr) * 1987-08-12 1989-03-22 Hem Pharmaceuticals Corp. Promotion de la défense d'un hôte par traitement systémique avec du RNA double brin

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US4775636A (en) * 1983-11-25 1988-10-04 Janssen Pharmaceutica N.V. Blot overlay assay using colloidal metal particles
EP0185444A2 (fr) * 1984-10-10 1986-06-25 Centocor, Inc. Clonage et expression d'ADN de HTLV-III
EP0308066A2 (fr) * 1987-08-12 1989-03-22 Hem Pharmaceuticals Corp. Promotion de la défense d'un hôte par traitement systémique avec du RNA double brin

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Annals of the Rheumatic Diseases, Vol. 44, No. 11, issued Nov. 19858 E. RODAHL et al, "Antigens Related to the Major Internal Protein, p 27, of a Psoriasis Associated Retrovirus-like Particle are Expressed in Patients with Chronic Arthritis", pages 761-765, see Abstract. *
Annals of the Rheumatic Diseases, Vol. 47, No. 3, issued March 1988, B.K. PELTON et al, "A Search for Retrovirus Infection in Systemic Lupus Erythematosus and Rheumatoid Arthritis", pages 206-2098 see Summary; page 206, col. 1; and page 20., col 1. *
L.E. HOOD et al. Immunology, 2nd Ed. 1984, Benjamin Cummings Publishing Co., Inc., Menlo Park, CA, U.S.A. see pages 66-68. *
Proceedings of the National Academy of Sciences USA, Vol. 82, No. 20 issued October 1985, H. MITSUYA et al, "3-Azido -3'- Deoxythymidine (BW A509U): an Antiviral Agent that Inhibits the Infectivity and Cytopathic Effect of Human T-Lymphotropic Virus Type III/Lymphoadenopathy-Associated Virus in Vitro", pages 7096-7100. see Abstract and Discussion. *
See also references of EP0378621A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991000047A1 (fr) * 1989-06-30 1991-01-10 The Regents Of The University Of California Detection de retrovirus
US5503974A (en) * 1989-06-30 1996-04-02 University Of California Retrovirus detection
EP1314429A2 (fr) * 1990-05-21 2003-05-28 The Administrators Of The Tulane Educational Fund Utilisation d'un composé retrovirale dans la fabrication d'un médicament pour le traitement du sydrome de Sjögren, du lupus érythémateux disséminé, de la scléroderme, et de l'arthrite rheumatoide juvenile
EP1314429A3 (fr) * 1990-05-21 2003-06-04 The Administrators Of The Tulane Educational Fund Utilisation d'un composé retrovirale dans la fabrication d'un médicament pour le traitement du sydrome de Sjögren, du lupus érythémateux disséminé, de la scléroderme, et de l'arthrite rheumatoide juvenile
EP0797440A1 (fr) * 1994-12-16 1997-10-01 Ray W. Exley Traitement de maladies auto-immunes au moyen de derives de 2-amino purine
EP0797440A4 (fr) * 1994-12-16 2000-07-05 Ray W Exley Traitement de maladies auto-immunes au moyen de derives de 2-amino purine
US6638909B1 (en) 1996-11-01 2003-10-28 Ethicon, Inc. Wound healing compositions containing alpha-1-antitrypsin
WO2001026661A1 (fr) * 1999-10-11 2001-04-19 Yissum Research Development Company Of The Hebrew University Of Jerusalem Compositions comprenant des inhibiteurs de metalloproteinases a base d'oxophosphonate
US7468359B2 (en) 1999-10-11 2008-12-23 Yissum Research Develpment Company Of The Hebrew University Of Jerusalem Compositions comprising oxophosphonate-based metalloproteinase inhibitors

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JPH02504429A (ja) 1990-12-13
EP0378621A4 (en) 1991-08-28
EP0378621A1 (fr) 1990-07-25

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