ABSTRACT
OBJECTIVE: To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation. DESIGN: Tumor necrosis factor-alpha (TNFalpha) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFalpha-induced proteoglycan degradation in vivo. RESULTS: (1) IA injection of TNFalpha in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin. CONCLUSION: TNFalpha injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo.
Subject(s)
Aggrecans/metabolism , Arthritis, Experimental/pathology , Cartilage, Articular/pathology , Knee Joint/pathology , Osteoarthritis/pathology , Proteoglycans/metabolism , Tumor Necrosis Factor-alpha/metabolism , Aggrecans/pharmacology , Animals , Arthritis, Experimental/drug therapy , Blotting, Western , Cartilage, Articular/drug effects , Immunohistochemistry , Injections, Intra-Articular , Knee Joint/drug effects , Male , Osteoarthritis/drug therapy , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.
Subject(s)
Heterocyclic Compounds, 1-Ring/chemical synthesis , Hydroxamic Acids/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Tumor Necrosis Factor-alpha/antagonists & inhibitors , ADAM Proteins , ADAM17 Protein , Binding Sites , Heterocyclic Compounds, 1-Ring/chemistry , Heterocyclic Compounds, 1-Ring/pharmacology , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Lipopolysaccharides/pharmacology , Models, Molecular , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Binding , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/metabolismABSTRACT
To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of =0.2 microM in whole blood assay (WBA). Although the P3' area can accommodate a broad array of structurally diversified functional groups including polar residues, hydrophobic residues, and amino and carboxylic acid moieties, in both the cyclophane series and the cyclic carbamate series, a glycine residue at P3' was identified as a critical structural component to achieve both good in vitro potency and good oral activity. With a glycine residue at P3', an N-methylamide at P4' provided the best cyclophane analogue, SL422 (WBA IC(50) = 0.22 microM, LPS-mouse ED(50) = 15 mg/kg, po), whereas a morpholinylamide at P4' afforded the most potent and most orally active cyclic carbamate analogue, SP057 (WBA IC(50) = 0.067 microM, LPS-mouse ED(50) = 2.3 mg/kg, po). Further profiling for SL422 and SP057 showed that these macrocyclic compounds are potent TACE inhibitors, with K(i) values of 12 and 4.2 nM in the porcine TACE assay, and are broad-spectrum MMP inhibitors. Pharmacokinetic studies in beagle dogs revealed that SL422 and SP057 are orally bioavailable, with oral bioavailabilities of 11% and 23%, respectively.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Hydroxamic Acids/chemical synthesis , Lactams/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitors , ADAM Proteins , ADAM17 Protein , Administration, Oral , Animals , Biological Availability , Carbamates/chemical synthesis , Carbamates/chemistry , Carbamates/pharmacokinetics , Carbamates/pharmacology , Dogs , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/pharmacology , In Vitro Techniques , Lactams/chemistry , Lactams/pharmacokinetics , Lactams/pharmacology , Male , Mice , Stereoisomerism , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/analysisABSTRACT
The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Animals , Butadienes/pharmacology , Butadienes/therapeutic use , Edema/prevention & control , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 1 , Male , Mice , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Nitriles/pharmacology , Nitriles/therapeutic use , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.
Subject(s)
Hydroxamic Acids/chemistry , Metalloendopeptidases/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Crystallography, X-Ray , Humans , Inhibitory Concentration 50 , Kinetics , Lipopolysaccharides/metabolism , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/classification , Models, Chemical , Models, MolecularSubject(s)
Biphenyl Compounds/therapeutic use , Heart Transplantation/immunology , Immunosuppressive Agents/therapeutic use , Transplantation, Heterologous/immunology , Animals , Cricetinae , Cyclosporine/therapeutic use , Dose-Response Relationship, Drug , Drug Therapy, Combination , Graft Rejection/prevention & control , Graft Survival/drug effects , Mesocricetus , Mycophenolic Acid/analogs & derivatives , Mycophenolic Acid/therapeutic use , Rats , Rats, Inbred Lew , Rats, Inbred Strains , Structure-Activity Relationship , Transplantation, Homologous/immunologySubject(s)
Hydroxamic Acids/chemical synthesis , Lactams/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Crystallography, X-Ray , Humans , Hydroxamic Acids/blood , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , In Vitro Techniques , Lactams/blood , Lactams/chemistry , Lactams/pharmacology , Lipopolysaccharides/blood , Lipopolysaccharides/pharmacology , Lymphocyte Activation , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 9 , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/blood , Models, Molecular , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacologyABSTRACT
Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.
Subject(s)
Lymphocyte Activation , Protein Kinases/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Animals , CD28 Antigens/immunology , CD3 Complex/immunology , Cell Division/immunology , Clone Cells , Interleukin-2/immunology , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase Kinases , T-Lymphocytes/cytologyABSTRACT
Structure-activity relationships were explored for some analogs of Brequinar having a linking atom between the 2-biphenyl substituent and the quinoline ring. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction were related to the overall shape and lipophilicity of the 2-substituent.
Subject(s)
Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Biphenyl Compounds/chemistry , Carbon/chemistry , Lymphocyte Culture Test, Mixed , Structure-Activity RelationshipABSTRACT
The structure-activity relationships of some tetracyclic heterocycles related to Brequinar were explored. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction are related to ring system, heteroatom placement, and pendant ring substitution.
Subject(s)
Biphenyl Compounds/chemistry , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Oxidoreductases Acting on CH-CH Group Donors , Dihydroorotate Dehydrogenase , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Lymphocyte Culture Test, Mixed , Oxidoreductases/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Prostaglandins have wide-ranging effects in the body and are thought to be important mediators of inflammation. Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis, and occurs in both constitutive (COX-1) and inducible (COX-2) isoforms. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is both inducible and the major isoform of inflammatory cells. Reduction of prostaglandin production by inhibition of cyclooxygenases appears to be the main mechanism of action of most non-steroidal anti-inflammatory drugs (NSAIDS). Here we present an animal model of COX-2 deficiency that was generated by gene targeting. Defects in null mice correlating with reduced viability included renal alterations, characteristic of renal dysplasia (100% penetrance), and cardiac fibrosis (50% penetrance). Female Cox-2-/- mice were infertile. COX-2 deficiency failed to alter inflammatory responses in several standard models, but striking mitigation of endotoxin-induced hepatocellular cytotoxicity was observed.
Subject(s)
Inflammation/enzymology , Kidney/abnormalities , Prostaglandin-Endoperoxide Synthases/drug effects , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Line , Cyclooxygenase Inhibitors , Disease Models, Animal , Female , Fibrosis , Gene Targeting , Heart Diseases/enzymology , Heart Diseases/genetics , Infertility, Female/enzymology , Infertility, Female/genetics , Inflammation/genetics , Kidney/embryology , Kidney/enzymology , Liver/embryology , Liver/enzymology , Mice , Mice, Knockout , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/physiologyABSTRACT
Brequinar sodium (BQR) is a novel immunosuppressive agent that acts by inhibiting the activity of dihydroorotate dehydrogenase, the fourth enzyme in the de novo pyrimidine biosynthetic pathway. The activity of BQR as an immunosuppressive agent is believed to be inhibition of antigen-induced lymphocyte proliferation through inhibition of DNA and RNA synthesis. BQR, therefore, has a different mechanism of action than cyclosporine and may potentiate the immunosuppressive effects of cyclosporine. In this study, we determined the effect of BQR on peripheral blood mononuclear cell (PBMC) activation in a series of in vitro culture systems. In these studies, BQR inhibited PHA-stimulated activation in a dose-dependent fashion beginning at 10(-6) M. The immunosuppressive effect of BQR was similar in magnitude to cyclosporine. Proliferation assays suggested an additive immunosuppression by the combination of BQR and cyclosporine. Similar inhibition of CD2-stimulated or CD3-stimulated activation of PBMC was found. The mechanisms of action of BQR were complex. BQR inhibited interleukin 2 protein production in response to mitogen stimulation. Cell surface interleukin 2 receptor expression was inhibited by BQR. BQR inhibited cell cycle progression, preventing progression from G0/G1 into S and G2 + M phases. BQR had no effect on induction of transcripts for the interleukin 2 receptor, but markedly inhibited the production of transcripts for interleukin 2. Thus, our studies indicate that BQR exerts a potent immunosuppression on mitogen-induced PBMC activation through multiple mechanisms. Consequently, BQR may be an effective agent for immunosuppression in organ transplantation or inflammatory diseases.
Subject(s)
Biphenyl Compounds/pharmacology , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/immunology , DNA/biosynthesis , Depression, Chemical , Humans , Interleukin-2/genetics , Organ Transplantation/physiology , RNA, Messenger/genetics , Receptors, Interleukin-2/physiology , Transcription, Genetic/drug effectsSubject(s)
Biphenyl Compounds/pharmacology , Immunoglobulin A/drug effects , Immunoglobulin G/drug effects , Immunoglobulin M/drug effects , Immunosuppressive Agents/pharmacology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Adult , Biphenyl Compounds/administration & dosage , Cyclosporine/administration & dosage , Cyclosporine/pharmacology , Dose-Response Relationship, Drug , Dose-Response Relationship, Immunologic , Humans , Immunoglobulin A/biosynthesis , Immunoglobulin G/biosynthesis , Immunoglobulin M/biosynthesis , Immunosuppressive Agents/administration & dosage , Lymphocyte Activation/immunology , Pokeweed Mitogens/pharmacology , Tacrolimus/administration & dosage , Tacrolimus/pharmacologySubject(s)
Biphenyl Compounds/pharmacology , Immunosuppressive Agents/pharmacology , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Dermatitis, Contact/immunology , Dinitrofluorobenzene , Epitopes/immunology , Female , Humans , Hypersensitivity, Delayed/chemically induced , Hypersensitivity, Delayed/immunology , Immunity, Cellular/drug effects , Immunity, Cellular/immunology , Lymphocyte Culture Test, Mixed , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Biological , Sheep , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunologySubject(s)
Biphenyl Compounds/pharmacology , Dihydroorotate Oxidase/antagonists & inhibitors , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , Pyrimidines/metabolism , Animals , Cells, Cultured , Concanavalin A/pharmacology , Dihydroorotate Oxidase/metabolism , Humans , Immunity/drug effects , Immunoassay , Lymphocyte Culture Test, Mixed , Lymphocytes/drug effects , Lymphocytes/enzymology , Lymphocytes/immunology , Nucleotides/metabolism , Rats , Rats, Inbred Lew , Swine , Swine, MiniatureSubject(s)
Biphenyl Compounds/pharmacology , Immunosuppressive Agents/pharmacology , Lymphocytes/immunology , Administration, Oral , Animals , Biphenyl Compounds/blood , Biphenyl Compounds/pharmacokinetics , Cells, Cultured , Dose-Response Relationship, Drug , Immunosuppressive Agents/pharmacokinetics , Lymphocyte Activation/drug effects , Lymphocytes/drug effects , Macaca fascicularis , Thymidine/metabolism , Time Factors , TritiumSubject(s)
Biphenyl Compounds/therapeutic use , Graft Rejection/prevention & control , Immunosuppressive Agents/therapeutic use , Liver Transplantation/immunology , Animals , Biphenyl Compounds/administration & dosage , Drug Administration Schedule , Immunosuppressive Agents/administration & dosage , Rats , Time Factors , Transplantation, HomologousABSTRACT
Brequinar sodium (BQR) prevents cell proliferation by virtue of its inhibition of de novo pyrimidine biosynthesis. The immunosuppressive activity of BQR is highly effective in prolonging heart, liver, and kidney allograft survival in the rat. In these experiments, we have tested the ability of BQR to prevent the rejection of concordant cardiac xenografts. LEW inbred rats transplanted with heterotopic hamster hearts were treated orally with brequinar sodium as a single agent. The survival of the cardiac xenografts was significantly prolonged with a variety of treatment regimens. The most effective treatment was the daily oral administration of BQR at 3 mg/kg. At this level, the median graft survival was approximately 25 days. Four animals had hamster heart xenografts that functioned for more than 90 days. The prolonged survival of the xenografts was associated with relatively constant plasma drug levels of approximately 1 to 3 micrograms/ml and a marked suppression of IgM production. At rejection, there was a significant rise in IgM levels compared with those of recipients with stable xenografts. In vitro MLR responses were effectively inhibited by BQR, with an IC50 of 0.08 microgram/ml. The results of these experiments demonstrate that BQR is a new immunosuppressive agent that is highly effective as a single agent in prolonging the survival of hamster-to-rat cardiac xenografts. The prolonged xenograft survival is associated with effective suppression of rat antihamster antibody production, suggesting that brequinar sodium may be an important addition to multidrug immunosuppressive regimes designed to prevent B and T lymphocyte-mediated immune responses.
Subject(s)
Biphenyl Compounds/pharmacology , Heart Transplantation/immunology , Immunosuppressive Agents/pharmacology , Transplantation, Heterologous , Animals , Biphenyl Compounds/blood , Cricetinae , Graft Rejection , Graft Survival/drug effects , Immunoglobulin M/blood , Mesocricetus , Rats , Rats, Inbred LewABSTRACT
The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.