ABSTRACT
Abatacept is the first in a new class of agents that selectively modulates T-cell activation by attenuating CD28-mediated co-stimulation. This study examined the effects of abatacept on disease development in a rat model of collagen-induced arthritis (CIA). The rats were treated with either abatacept (1mg/kg) or control IgG beginning at the time of induction of CIA. By day 16, significant paw swelling was observed in IgG-treated control animals that continued to increase, reaching a plateau on day 21. Prophylactic treatment with abatacept completely abrogated paw swelling throughout the study. Histopathology demonstrated a significant reduction in inflammation, cartilage destruction, bone resorption and pannus formation. Abatacept treatment resulted in 90% inhibition of circulating collagen-specific antibodies and decreased the serum expression of many cytokines and chemokines that were upregulated in diseased animals. Immunohistochemical analysis of the ankle joints demonstrated that interleukin-6 production was reduced in the tissues and the numbers of osteoclasts present in the joints were also decreased. Ankle microcomputer tomography (micro-CT) analyses dramatically demonstrated the protective effects of abatacept on bone destruction in these animals. Data presented here demonstrate that prophylactic administration of abatacept significantly inhibits the onset and progression of disease in a rat CIA model, with reductions in inflammation, inflammatory mediators, and bone and joint destruction.
Subject(s)
Arthritis, Experimental/prevention & control , Bone Resorption/prevention & control , Collagen/immunology , Immunoconjugates/pharmacology , Abatacept , Acid Phosphatase/metabolism , Animals , Arthritis, Experimental/immunology , Autoantibodies/biosynthesis , Bone Resorption/immunology , Bone and Bones/drug effects , Bone and Bones/immunology , Bone and Bones/pathology , Disease Models, Animal , Female , Inflammation/chemically induced , Inflammation/prevention & control , Isoenzymes/metabolism , Osteoclasts/drug effects , Osteoclasts/enzymology , Rats , Tarsal Joints/drug effects , Tarsal Joints/immunology , Tartrate-Resistant Acid PhosphataseABSTRACT
Potent and selective bicyclic heteroaryl hydroxamic acid MMP and TACE inhibitors were synthesized by a novel convergent route. Selectivity and efficacy versus MMPs and TACE could be controlled by appropriate substitution on the scaffolds and by variation of the P1' group. Select compounds were found to be effective in in vivo models of arthritis.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , ADAM Proteins , ADAM17 Protein , Animals , Arthritis/drug therapy , Arthritis/pathology , Biological Availability , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cartilage/drug effects , Cartilage/pathology , Cattle , Disease Models, Animal , Enzyme Inhibitors/chemical synthesis , Hydroxamic Acids/chemical synthesis , Mice , Rats , Structure-Activity RelationshipABSTRACT
The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups was explored. In particular, compound 4t bearing a butynyloxy P1' moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and oral activity in an in vivo model of TNF-alpha production.
Subject(s)
Acetylene/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/pharmacology , ADAM Proteins , ADAM17 Protein , Structure-Activity RelationshipABSTRACT
Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9 and MMP-13.
Subject(s)
Amines/chemistry , Enzyme Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors , ortho-Aminobenzoates/pharmacology , ADAM Proteins , ADAM17 Protein , Animals , Binding Sites , Collagenases/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Hydroxamic Acids/chemistry , Interleukin-1 , Interleukin-1beta , Magnetic Resonance Spectroscopy , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 13 , Matrix Metalloproteinase 9/metabolism , Metalloendopeptidases , Mice , Models, Molecular , Osteoarthritis/drug therapy , Rats , Structure-Activity Relationship , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistryABSTRACT
A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, MMP-13, and TACE was prepared and evaluated. Selective inhibitors of MMP-9, MMP-13, and TACE were identified, including the potent, orally active MMP-13 inhibitor 4p.
Subject(s)
Enzyme Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors , ortho-Aminobenzoates/pharmacology , ADAM Proteins , ADAM17 Protein , Collagenases/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Matrix Metalloproteinase 13 , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinases/metabolism , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Structure-Activity Relationship , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistryABSTRACT
Heteroaryl and cycloalkyl sulfonamide-hydroxamic acid MMP inhibitors were investigated. Of these, the pyridyl analogue 2 is the most potent and selective inhibitor of MMP-9 and MMP-13 in vitro.
Subject(s)
Hydroxamic Acids/pharmacology , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemical synthesis , ortho-Aminobenzoates/pharmacology , Animals , Combinatorial Chemistry Techniques , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Inhibitory Concentration 50 , Matrix Metalloproteinase 13 , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Sulfonamides/pharmacology , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistryABSTRACT
A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, and MMP-13 was prepared and evaluated both in vitro and in vivo. The most potent compound, 6e, has in vivo activity in a rat sponge-wrapped cartilage model.
Subject(s)
Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemical synthesis , ortho-Aminobenzoates/pharmacology , Animals , Combinatorial Chemistry Techniques , Humans , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Inhibitory Concentration 50 , Matrix Metalloproteinase 13 , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Sulfonamides/pharmacology , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistryABSTRACT
It has been suggested that increased collagenase-3 (MMP-13) activity plays a pivotal role in the pathogenesis of osteoarthritis (OA). We have used tetracycline-regulated transcription in conjunction with a cartilage-specific promoter to target a constitutively active human MMP-13 to the hyaline cartilages and joints of transgenic mice. Postnatal expression of this transgene resulted in pathological changes in articular cartilage of the mouse joints similar to those observed in human OA. These included characteristic erosion of the articular cartilage associated with loss of proteoglycan and excessive cleavage of type II collagen by collagenase, as well as synovial hyperplasia. These results demonstrate that excessive MMP-13 activity can result in articular cartilage degradation and joint pathology of the kind observed in OA, suggesting that excessive activity of this proteinase can lead to this disease.
Subject(s)
Cartilage, Articular/enzymology , Collagenases/genetics , Collagenases/metabolism , Osteoarthritis/etiology , Animals , Base Sequence , Cartilage, Articular/pathology , DNA Primers/genetics , Disease Models, Animal , Gene Expression , Humans , Matrix Metalloproteinase 13 , Mice , Mice, Transgenic , Mutation , Osteoarthritis/enzymology , Osteoarthritis/geneticsABSTRACT
The adamalysins are a family of proteins in the metzincin superfamily of metalloproteases, which also includes the matrix metalloproteinases. There are two subfamilies of adamalysins: the snake venom metalloproteases (SVMPs) and the ADAMs (proteins containing a disintegrin and metalloprotease domain). At least 23 ADAMs have been identified to date. The ADAMs are expressed by a wide variety of cell types, and are involved in functions as diverse as sperm-egg binding, myotube formation, neurogenesis, and proteolytic processing of cell surface proteins. An overview of the ADAM family and their functions will be presented. TACE is a unique member of the ADAM family that cleaves membrane-bound TNF-alpha to generate soluble TNF-alpha. Mice lacking proteolytically active TACE have been generated and characterized. The TACE knock-out results in perinatal lethality. Cells from the TACE-deficient mice release 80-90% less soluble TNF-alpha than do wild-type cells. Irradiated mice that are reconstituted with TACE knock-out hematopoeitic stem cells have markedly reduced levels of serum TNF-alpha following LPS challenge, compared to irradiated mice reconstituted with wild-type cells, suggesting that TACE is the major TNF-alpha converting enzyme in vivo. TACE-deficient cells are compromised in the generation of other soluble proteins that are produced as the result of cleavage of a membrane precursor form, suggesting that TACE is involved in multiple shedding events.
Subject(s)
Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , ADAM Proteins , ADAM10 Protein , ADAM17 Protein , Amyloid Precursor Protein Secretases , Animals , Female , Humans , Male , Membrane Proteins/genetics , Metalloendopeptidases/genetics , Mice , Mice, Knockout , Snake Venoms/metabolism , Sperm-Ovum Interactions , Tumor Necrosis Factor-alpha/metabolismABSTRACT
A novel series of matrix metalloproteinase (MMP) inhibitors is described. Incorporation of a terminal alpha-mercaptoketone or alpha-mercaptoalcohol in the zinc binding domain of a series of inhibitors led to compounds exhibiting low nanomolar activity against collagenase-1 (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9).
Subject(s)
Alcohols/chemistry , Ketones/chemistry , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemistry , Sulfhydryl Compounds/chemistry , Alcohols/chemical synthesis , Alcohols/pharmacology , Binding Sites , Drug Design , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Ketones/chemical synthesis , Ketones/pharmacology , Kinetics , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Structure-Activity Relationship , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/pharmacology , Zinc/metabolismABSTRACT
A series of succinyl based mercaptoketones and diastereomeric mercaptoalcohols were prepared and evaluated in vitro as inhibitors of the matrix metalloproteinases collagenase-1 (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9).
Subject(s)
Alcohols/chemical synthesis , Ketones/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Succinates/chemical synthesis , Sulfhydryl Compounds/chemical synthesis , Alcohols/chemistry , Alcohols/pharmacology , Drug Design , Indicators and Reagents , Ketones/chemistry , Ketones/pharmacology , Kinetics , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Molecular Structure , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Structure-Activity Relationship , Succinates/chemistry , Succinates/pharmacology , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/pharmacologyABSTRACT
A novel series of diazepine-based hydroxamic acid inhibitors of MMP-1, MMP-9, and MMP-13 were prepared and evaluated both in vitro and in vivo.
Subject(s)
Azepines/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors , Organic Chemicals , Pyrazines , Animals , Antineoplastic Agents/pharmacology , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 9 , Mice , Protease Inhibitors/pharmacology , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/pharmacologyABSTRACT
Fibroblast collagenase (MMP-1), a 169-residue protein with a molecular mass of 18.7 kDa, is a matrix metalloproteinase which has been associated with pathologies such as arthritis and cancer. The assignments of the 1H, 15N, 13CO and 13C resonances, determination of the secondary structure and analysis of 15N relaxation data of the inhibitor-free catalytic fragment of recombinant human fibroblast collagenase (MMP-1) are presented. It is shown that MMP-1 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel and antiparallel arrangement (residues 13-19, 48-53, 59-65, 82-85 and 94-99) and three alpha-helices (residues 27-43, 112-124 and 150-160). This is nearly identical to the secondary structure determined from the refined X-ray crystal structures of inhibited MMP-1. The major difference observed between the NMR solution structure of inhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is the dynamics of the active site. The 2D 15N-1H HSQC spectra, the lack of information in the 15N-edited NOESY spectra, and the generalized order parameters (S2) determined from 15N T1, T2 and NOE data suggest a slow conformational exchange for residues comprising the active site (helix B, zinc ligated histidines and the nearby loop region) and a high mobility for residues Pro138-Gly144 in the vicinity of the active site for inhibitor-free collagenase. In contrast to the X-ray structures, only the slow conformational exchange is lost in the presence of an inhibitor.
Subject(s)
Collagenases/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Cloning, Organism , Collagenases/metabolism , Crystallography, X-Ray/methods , Escherichia coli , Fibroblasts/enzymology , Humans , Matrix Metalloproteinase 1 , Models, Structural , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
An approach utilizing robotics (automation) for the rapid and reliable determination of protease inhibitor concentration in rat plasma samples is described. The bioassay protocol using an immobilized peptide substrate allows high sample throughput, compatible with parallel synthesis/SAR development strategy.
Subject(s)
Biological Assay/methods , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/pharmacokinetics , Robotics/methods , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid/methods , Dose-Response Relationship, Drug , Extracellular Matrix/enzymology , Fluorescence , Humans , Hydrolysis , Metalloendopeptidases/metabolism , Methanol/chemistry , Molecular Sequence Data , Protease Inhibitors/blood , Rats , Recombinant Proteins/metabolism , Reproducibility of Results , Structure-Activity Relationship , Time FactorsABSTRACT
In the course of screening natural products for antagonists of CD18-mediated cell adhesion, an extract with inhibitory activity was identified from the stem and leaves of Conobea scoparioides. Bioassay-guided fractionation led to a pure compound, identified by spectroscopy as cucurbitacin E [1]. Although many biological activities have been reported for the cucurbitacins, this is the first report of cell adhesion inhibition. Furthermore, closely related cucurbitacin analogues had different potencies, pointing to substructural features that are important for the activity.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cell Adhesion/drug effects , Plant Extracts/pharmacology , Triterpenes/pharmacology , Actins/drug effects , Actins/metabolism , Cell Division/drug effects , Cell Survival/drug effects , Chemotaxis, Leukocyte/drug effects , Cucurbitacins , HeLa Cells , Humans , In Vitro Techniques , Intercellular Adhesion Molecule-1/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Neutrophils/physiology , Thymidine/metabolism , Trees , Triterpenes/isolation & purificationABSTRACT
The effects of the in vivo administration of interleukin 1 (IL 1) on lymphocytes from lymph node and spleen were analyzed. Mice received five daily subcutaneous (s.c.) injections of various doses of human recombinant IL 1 beta. Either 1 or 7 days after IL 1 treatment, spleens, popliteal and inguinal lymph nodes were collected. Lymphadenosis and splenomegaly were observed in the IL 1-treated animals. Lymph nodes from IL 1-treated mice contained a higher percentage of B cells than controls, and B cells from IL 1-treated mice expressed dramatically increased levels of Ia antigen. Lymphadenosis and splenomegaly, as well as the changes in subset distributions and Ia expression were transient. Concomitant treatment of mice with IL 1 and anti-IL 4 monoclonal antibody suppressed IL 1 effects on B cell Ia expression, but not on the B/T cell ratio. In situ hybridization analyses revealed that IL 1 treatment induced the expression of mRNA for IL 4, interferon-gamma, and IL 2 in lymph node and spleen cells. The distribution of cells expressing the various cytokine mRNA was markedly different between the spleens and lymph nodes.
Subject(s)
B-Lymphocytes/immunology , Histocompatibility Antigens Class II/analysis , Interleukin-1/pharmacology , Interleukin-4/physiology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antigens, Differentiation/analysis , CD4-Positive T-Lymphocytes/immunology , Gene Expression Regulation , Immunologic Techniques , Interferon-gamma/genetics , Interleukin-2/genetics , Interleukin-4/genetics , Lymphadenitis/chemically induced , Mice , Mice, Inbred DBA , RNA, Messenger/genetics , Splenomegaly/chemically inducedABSTRACT
1. The subcutaneous administration of recombinant human interleukin-1 beta (rhIL-1 beta) was found to induce an increased incidence and earlier onset of collagen-induced arthritis in mice. 2. The rhIL-1 beta had different effects, depending on when it was administered after collagen-immunization. 3. The effect of rhIL-1 beta may be due, in part, to augmentation of the immune response to type II collagen. 4. Interleukin-1-accelerated, collagen-induced arthritis will provide a useful model for investigating the role of interleukin-1 in the regulation of arthritic diseases, and the development of anti-arthritic therapeutics.
Subject(s)
Arthritis, Rheumatoid/immunology , Interleukin-1/immunology , Animals , Arthritis, Rheumatoid/etiology , Collagen/immunology , Disease Models, Animal , Immunity , Male , Mice , Mice, Inbred DBA , Recombinant Proteins/immunologyABSTRACT
A one-step purification of interleukin-4 is described using an 11B11 monoclonal antibody-Sepharose 4B chromatography column. Beginning with 1,300 ml of supernatant from the murine T-cell clone D10, a homogeneous preparation of IL-4 is obtained (22 micrograms) having a specific activity of 4.75 x 10(6) units/mg (10,650-fold purification) with an overall yield of 45%. The purified protein runs as a single band on silver-stained SDS polyacrylamide gels with a molecular mass of 18,600 +/- 1,000 daltons. Digestion with endoglycosidase F reduces the molecular mass to 15,500 daltons, indicating the presence of N-linked glycosylation. The novelty in this procedure involves the use of native conditions throughout and the absence of a requirement for HPLC resolution. Furthermore, the use of these cells (D10), rather than EL4 cells which have previously been used as a source of IL-4, may facilitate purification since D10 can be stimulated under serum-free conditions.
Subject(s)
Immunosorbent Techniques , Interleukins/isolation & purification , Animals , Chromatography, Affinity , Clone Cells/immunology , Electrophoresis, Polyacrylamide Gel , Interleukin-4 , Mice , Molecular Weight , T-Lymphocytes/immunologyABSTRACT
T cells can be subdivided based on cell surface markers, MHC restriction, function, and production of soluble factors. Analysis of the ability of cloned, Ia-restricted, L3T4+ T cells to induce an in vitro anti-hapten antibody response to hapten-carrier conjugates allowed the definition of three functional subtypes. To examine whether these functional subtypes also differed in the production of soluble mediators, supernatants of the cloned lines were examined for the production of T cell growth factors and factors inducing increased expression of Ia glycoproteins on small resting B cells. All of the cloned lines produced T cell growth factors that could be further differentiated by inhibition with monoclonal antibodies. None of the Ia-restricted, L3T4+ cloned T cell lines that failed to produce IL 4/BSF-1 could provide helper function. Thus, the activation of antigen-specific B cells by helper T cells appears to require IL 4/BSF-1 as a necessary but not sufficient signal for differentiation into antibody-forming cells.