Treatment Of Chronic Inflammatory Diseases Research Articles

Novel substituted pyridinyl imidazoles as potent anticytokine agents with low activity against hepatic cytochrome P450 enzymes.

A series of polysubstituted pyridin-4-yl imidazole inhibitors of p38 MAP (mitogen-activated protein) kinase was prepared as small molecular anticytokine agents and drug candidates for the treatment of chronic inflammatory diseases. The contribution of substituents at the pyridinyl and imidazole moiety to selective inhibition of p38 without concomitant cytochrome P450 interaction was evaluated. Placement of a 1-phenylethyl (7e, p38: IC(50) 0.38 microM) or acetyl substituent at the exocyclic nitrogen of several 2-aminopyridine imidazoles led to the identification of potent p38 inhibitors which exceeded the starting lead ML 3375 (p38: IC(50) 0.63 microM) in potency. A preliminary modeling study related the enhanced bioactivity of 7e to a novel interaction between its 1-phenylethylamino side chain and a hydrophobic pocket close to the linker region of p38. The most active p38 inhibitors in this series maintained their efficacy in functional PBMC (peripheral blood mononuclear cells) and whole blood assays. Moreover, cytochrome P450 interaction, which has been linked to the liver toxicity observed for model p38 inhibitors, was very efficiently reduced through introduction of a tetramethylpiperidine substituent at the 1 position of the imidazole nucleus. Combination of both structural features provided 14c (p38: 0.34 microM, inhibition of CYP1A2 0%, 2C9 2.6%, 2C19 7.6% at 10 microM), which was selected for further development.

Antioxidant Effects of Selenium in Rat Brain and the Stimulating Role of Nitric Oxide

Objective: To evaluate the antioxidant effect of selenium on Na<PRE>+</PRE>, K<PRE>+</PRE>-ATPase in rat brain in the presence of nitric oxide.Methods: Male Wistar rats (70 g) were treated as follows: group 1 received 1 μg of i.p. sodium nitroprusside per kg (SNP), group 2 received 5 μg sodium selenite during 20 days, group 3 received sodium selenite 5 μg+SNP 1 μg and the control group received vehicle 50 μl (0.9% NaCl), same period and route. At the end of treatment, animals were sacrificed and their brain dissected into cortex, hemispheres, cerebellum and brain stem in order to determine lipid peroxidation (TBARS), Na<PRE>+</PRE>, K<PRE>+</PRE> ATPase and total ATPase in each section. Blood hemoglobin concentration (Hb) and prostate weight were also assessed.Results: A significant increase of Hb in blood and of proteins in cortex and hemisphere was detected, but TBARS values fell due to the effect of sodium selenite in all examined regions, except for cerebellum. ATPase activity declined in all groups and regions with and without NTP. We conclude that diet supplementary selenium to inhibit NO generation can be a useful treatment in chronic inflammatory diseases.

Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells.

CD4(+)CD25(+) regulatory T cells have been shown to prevent T cell-mediated immune pathology; however, their ability to ameliorate established inflammation has not been tested. Using the CD4(+)CD45RB(high) T cell transfer model of inflammatory bowel disease, we show that CD4(+)CD25(+) but not CD4(+)CD25(-)CD45RB(low) T cells are able to cure intestinal inflammation. Transfer of CD4(+)CD25(+) T cells into mice with colitis led to resolution of the lamina propria infiltrate in the intestine and reappearance of normal intestinal architecture. CD4(+)CD25(+) T cells were found to proliferate in the mesenteric lymph nodes and inflamed colon. They were located between clusters of CD11c(+) cells and pathogenic T cells and found to be in contact with both cell types. These studies suggest that manipulation of CD4(+)CD25(+) T cells may be beneficial in the treatment of chronic inflammatory diseases.

Impact of topoisomerase II inhibition on cytokine and chemokine production.

In systemic inflammatory diseases such as rheumatoid arthritis, cytokines and chemokines are deeply involved in the development of the disease manifestations. Etoposide is a cytostatic drug, known to deplete the monocyte population in mice and rabbits. We have recently shown that suboptimal doses have a disease-ameliorating effect in collagen II induced arthritis in the absence of monocyte depletion. Anti-arthritic properties parallelled with almost total eradication of production of specific collagen II antibodies. The aim of the present study was to investigate ex vivo and in vitro the function of the mononuclear cells and their production of B cell stimulating cytokines following exposure to etoposide, a topoisomerase II inhibitor. Spleen cells from mice treated during four weeks with etoposide were cultured and the supernatants were analyzed with respect to content of TNF and IL-6. In addition, cells from the murine macrophage cell clone IC-21 were exposed to etoposide and the production of IL-6, using a bioassay, and the production of TNF, MIP-1alpha, RANTES, and IL-1beta, using sandwich ELISAs, was determined. Spleen cells from etoposide-treated mice secreted lower amounts of IL-6 and TNF as compared to the control animals. In addition, in vitro etoposide-exposed macrophages showed reduced capacity to produce TNF, IL-6 and MIP-1alpha. Our results indicate that inhibition of topoisomerase II downregulated the function of monocytes. Owing to its immunoregulatory properties, use of etoposide is suggested in treatment of chronic inflammatory diseases.

PPARsと免疫・炎症システム―関節疾患治療への応用―

Peroxisome proliferator-activated receptors (PPARs), are members of the nuclear hormone receptors superfamily of ligand-activated transcriptional factors that include receptors for steroids, thyroid hormone, vitamin D3 and retinoic acid. PPAR binds to peroxisome proliferator responsive element (PPRE) as a heterodimer with the retinoic receptor (RXR) in the regulation of PPAR target genes. PPARs have been suggested to be important immunomodulatory factors as well as fatty acid regulators. PPARs modulate these activities in different immune cell types such as monocyte/macrophages, lymphocytes, and endothelial cells. PPAR-γ ligands lead to inhibition of the expression of nitric oxide, cytokines, chemokines and adhesion molecules, in part by antagonizing the activities of the transcription factors such as AP-1, and NF-κB. Moreover PPAR-γ ligands including antidiabetic thiazolidinedione and 15-deoxy-Δ12,14-prostaglandin J2 have potent tumor modulatory effects against several cancers. PPAR-γ also expressed in chondrocytes, synovial and bone tissues. Activation of the PPAR-γ induced RA synoviocyte apoptosis and suppression of osteoclast differentiatoin in vitro, and ameliorated adjuvant- induced arthritis with suppression of pannus formation and mononuclear cell infiltration in rats. These findings suggest that PPAR-γ ligands may potentially be useful for the treatment of chronic inflammatory diseases including arthritic joint diseases.

Molecular mechanisms of sulfasalazine-induced T-cell apoptosis.

Impaired apoptosis of T-lymphocytes is involved in the development of chronic inflammatory disorders. Previously we have shown that the anti-inflammatory drug sulfasalazine induces apoptosis in a murine T-lymphocyte cell line. The aims of the present study were to expand these observations to human systems and to analyse the molecular basis for sulfasalazine-induced apoptosis. Sulfasalazine induces apoptosis both in Jurkat cells, a human T-leukaemia cell line (ED50 value approximately 1.0 mM), and in primary human peripheral blood T-lymphocytes (ED50 value approximately 0.5 mM). In contrast SW620 colon carcinoma cells or primary human synoviocytes are not affected at these concentrations suggesting a cell type-specific sensitivity to sulfasalazine. Sulfasalazine triggers the mitochondrial accumulation of Bax and induces a collapse of the mitochondrial transmembrane potential (deltapsi(m)). Sulfasalazine causes cytochrome c release from mitochondria and subsequent activation of caspase-3 and downstream substrates. However, the pan-caspase inhibitor Z-VAD.fmk fails to inhibit sulfasalazine-induced apoptosis. Sulfasalazine stimulates mitochondrio-nuclear translocation of the novel apoptogenic factor apoptosis-inducing factor (AIF) and triggers large-scale DNA fragmentation, a characteristic feature of AIF-mediated apoptosis. Sulfasalazine-induced DeltaPsi(m) loss, AIF redistribution, and cell death are fully prevented by overexpression of Bcl-2. In conclusion, our data suggest that sulfasalazine-induced apoptosis of T-lymphocytes is mediated by mitochondrio-nuclear translocation of AIF and occurs in a caspase-independent fashion. Sulfasalazine-induced apoptosis by AIF and subsequent clearance of T-lymphocytes might thus provide the molecular basis for the beneficial therapeutic effects of sulfasalazine in the treatment of chronic inflammatory diseases.

Effect of taurine chloramine, the product of activated neutrophils, on the development of collagen-induced arthritis in DBA 1/J mice.

Taurine chloramine (TauCl), a product of neutrophil myeloperoxidase - halide system, formed by a reaction of taurine with HOCl, is known as an anti-microbial and anti-inflammatory long-lived oxidant. We previously reported that TauCl inhibits in vitro the production of proinflammatory cytokines (IL-6, IL-8) by RA synoviocytes. Therefore we performed this study to investigate the effect of TauCl treatment on the development of collagen-induced arthritis (CIA) in DBA1/J mice. Early administration of TauCl (after primary immunization) resulted in the delay of the onset of CIA, but had no effect on severity of arthritis. TauCl, given daily for 21 days after booster immunization, did not reduce the symptoms of arthritis in those mice, which already developed CIA, but significantly diminished incidence of the disease (55% vs. 90% of placebo mice). The mechanism of this effect is unknown. This is the first in vivo study suggesting that TauCl may be used for immune intervention in chronic inflammatory diseases.

Protection of marmoset monkeys against EAE by treatment with a murine antibody blocking CD40 (mu5D12).

CD40-CD40 ligand interactions are crucial to cognate interactions between T cells, B cells and antigen-presenting cells (APC), and contribute to non-antigen-specific effector functions of APC in inflammatory disorders. Here we demonstrate that functional blockade of CD40 with an antagonist mouse anti-human CD40 monoclonal antibody (mAb mu5D12) effectively prevents clinical expression of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in outbred marmoset monkeys, a preclinical model of multiple sclerosis. Anti-CD40 mAb interfered with development of clinical symptoms of marmoset EAE during the treatment period, even when treatment was started several weeks after T cell priming. Magnetic resonance imaging demonstrated inflammatory activity in the brain at initiation of antibody treatment, confirming that treatment interfered with the disease process. Access of therapeutic anti-CD40 to potential sites of action, the secondary lymphoid organs and the brain white matter lesions, was visualized in situ. The present data are the first to demonstrate the clinical potential of blocking APC and effector cell functions using murine antagonist anti-CD40 mAb in the treatment of chronic inflammatory diseases.

Small-molecule inhibitors of NF-kappaB for the treatment of inflammatory joint disease.

Recent advances in our understanding of the role of cytokine networks in inflammatory processes have led to the development of novel biological agents for the treatment of chronic inflammatory diseases. At the present time, significant efforts are focused on characterizing the complex signal transduction cascades that are activated by these cytokines and, in turn, regulate their expression. The transcription factor NF-kappaB is a pivotal regulator of the inducible expression of key proinflammatory mediators, and activated NF-kappaB has been observed in several debilitating inflammatory disorders, including rheumatoid arthritis and osteoarthritis. In light of its central role in inflammation, the identification of inhibitors of NF-kappaB should provide novel therapeutics for the treatment of chronic joint disease.

Amelioration of rat adjuvant arthritis by therapeutic treatment with bindarit, an inhibitor of MCP-1 and TNF-alpha production.

This study was designed to evaluate therapeutic effects of bindarit, an indazolic derivative able to inhibit monocyte chemoattractant protein-1 (MCP-1) production, in adjuvant induced arthritis in rats. Arthritis was induced by Freund's complete adjuvant injection. Bindarit was given as a 0.5% medicated diet starting on day 11 after adjuvant injection. The course of arthritis was monitored by sequential paw volume measurement and by radiologic and histologic evaluations. Human osteoblast cell line Saos-2 stimulated with Interleukin-1 (IL-1) was used to assess in vitro bindarit effect on MCP-1 release. In addition, in vivo effects of bindarit on cytokine production were studied in mice injected with lipopolysaccharide (LPS). Immune function studies were performed in mice by evaluating ex vivo antibody response to ovalbumin and splenocytes proliferation to Concanavalin A (Con A). In adjuvant-induced arthritis in rats, bindarit possessed therapeutic activity resulting in a significant inhibition of paw inflammation. Evidence for a disease-modifying activity was also indicated by amelioration of radiologic alterations and by histological evaluation of joints. Additional evidence for beneficial effects in osseous inflammation was provided by an in vitro assay in which bindarit inhibited the release of MCP-1 from IL-1 stimulated osteoblast cells. Moreover, in a murine model of LPS-induced cytokine production bindarit reduced MCP-1 and tumor necrosis factor (TNF)-alpha increase without affecting IL-1 and IL-6 levels. Finally, the drug, given as a 0.5% medicated diet for 14 days, did not affect either anti-ovalbumin serum antibody production or splenocytes proliferative response in mice. Results obtained indicate that bindarit beneficial effects in experimental arthritis are correlated to MCP-1 and TNF-alpha inhibition and suggest that the control of cytokines and chemokines production can have considerable relevance as regards strategies for the treatment of chronic inflammatory diseases.

Endogenous regulation of a therapeutic transgene restores homeostasis in arthritic joints

The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-α, IL-1β, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall–induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.

Endogenous regulation of a therapeutic transgene restores homeostasis in arthritic joints.

The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-alpha, IL-1 beta, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall-induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.

P38 Mitogen-activated protein kinase–induced glucocorticoid receptor phosphorylation reduces its activity: Role in steroid-insensitive asthma

Background: Although glucocorticoids are the most effective treatment for chronic inflammatory diseases, such as asthma, some patients show a poor response. IL-2 combined with IL-4 can alter glucocorticoid receptor (GR) ligand-binding affinity and modulate glucocorticoid function. Objective: We sought to confirm the altered ligand-binding affinity in a distinct group of steroid-dependent asthmatic subjects and examine the mechanism by which IL-2 and IL-4 modify the ligand-binding affinity of the GR. Methods: We examined PBMCs from healthy subjects, subjects with mild asthma, and steroid-dependent subjects with severe asthma using dexamethasone-binding assays and Western blot analysis of GR and phosphorylated activated transcription factor 2 expression. GR phosphorylation was measured after orthophosphate labeling and immunoprecipitation and cytokine production by means of ELISA. Results: GR ligand-binding affinity was reduced in the nucleus but not in the cytoplasm of steroid-dependent asthmatic subjects compared with that seen in healthy subjects (dissociation constant, 39.8 ± 4.6 vs 6.79 ± 0.8 nmol/L). This difference in ligand-binding affinity could be mimicked by IL-2 and IL-4 cotreatment and was blocked by the p38 mitogen-activated kinase (MAPK) inhibitor SB203580. Activation of p38 MAPK by IL-2 and IL-4, as shown by means of phosphorylation of activated transcription factor 2, resulted in GR phosphorylation and reduced dexamethasone repression of LPS-stimulated GM-CSF release. p38 MAPK phosphorylation of CD2+ T cells occurred on serine residues. The ability of dexamethasone to modulate IL-10 release was also inhibited by IL-2 and IL-4 cotreatment. These effects were also inhibited by SB203580. Conclusion: These data show that p38 MAPK inhibitors may have potential in reversing glucocorticoid insensitivity and reestablishing the beneficial effects of glucocorticoids in patients with severe asthma. (J Allergy Clin Immunol 2002;109:649-57.)

The prevention and treatment of murine colitis using gene therapy with adenoviral vectors encoding IL-10.

IL-10-deficient (IL-10(-/-)) mice develop colitis with many similarities to Crohn's disease. Daily IL-10 injections have a short systemic half-life and are unable to induce complete remission in IL-10(-/-) mice with established disease. In this paper, we investigate the duration, potency, and immunogenicity of gene therapy using an adenoviral vector encoding murine IL-10 (AdvmuIL-10). A single systemic injection of AdvmuIL-10 was sufficient not only to prevent the onset of colitis for at least 10 wk but also to induce clinical and histological remission in mice with established disease. In addition, AdvmuIL-10 diminished the systemic manifestations of disease, including elevated acute-phase proteins, as well as the local consequences of inflammation such as raised stool IL-1beta concentrations. Both IL-10 protein and the effects of secreted IL-10 were detectable for 10 wk after AdvmuIL-10 injection. Furthermore, the immunoregulatory effect of a single AdvmuIL-10 injection was manifest both by a reduction in TNF-alpha, IFN-gamma, and RANTES release from stimulated splenocyte cultures, and also by a change in the proportion of CD45RB(high/low) lymphocytes in the spleen compared with control mice. The delivery of AdvmuIL-10 resulted in a significantly diminished host antiadenoviral response compared with control adenoviral vectors. Thus, gene therapy strategies using adenoviral vectors encoding immunoregulatory and antiinflammatory cytokines may prove to be a potent approach for the treatment of chronic inflammatory disease. Antiinflammatory cytokine expression protects against immune responses directed at gene vectors.

Depletion of CCR5-Expressing Cells with Bispecific Antibodies and Chemokine Toxins: A New Strategy in the Treatment of Chronic Inflammatory Diseases and HIV

The chemokine receptor CCR5 is expressed on the majority of T cells and monocytes in the inflammatory infiltrate of diseases such as rheumatoid arthritis, renal diseases, and multiple sclerosis. In contrast, little expression of CCR5 is found on peripheral blood leukocytes. A specific depletion of CCR5(+) cells could therefore be a useful strategy to reduce the cellular infiltrate in chronic inflammations. Moreover, CCR5 is the major coreceptor for M-tropic HIV-1 strains. Depletion of CCR5(+) leukocytes may help to eliminate cells latently infected with HIV-1. We designed two constructs that specifically destroy chemokine receptor-positive cells. The first construct, a bispecific Ab, binds simultaneously to CCR5 and CD3. Thereby it redirects CD3(+) T cells against CCR5(+) target cells. The Ab specifically depletes CCR5(+) T cells and monocytes, but is inactive against cells that do not express CCR5. Furthermore, ex vivo the bispecific Ab eliminated >95% of CCR5(+) monocytes and T cells from the synovial fluid of patients with arthritis. Also, we designed a fusion protein of the chemokine RANTES and a truncated version of Pseudomonas. exotoxin A. The fusion protein binds to CCR5 and down-modulates the receptor from the cell surface. The chemokine toxin completely destroyed CCR5(+) Chinese hamster ovary cells at a concentration of 10 nM, whereas no cytotoxic effect was detectable against CCR5(-) Chinese hamster ovary cells. Both constructs efficiently deplete CCR5-positive cells, appear as useful agents in the treatment of chronic inflammatory diseases, and may help to eradicate HIV-1 by increasing the turnover of latently infected cells.

Cytokine and anti-cytokine therapy in chronic inflammatory bowel diseases

Cytokine and anti-cytokine therapy in chronic inflammatory bowel diseases

The orphan nuclear receptor RORα is a negative regulator of the inflammatory response

Retinoid-related orphan receptor alpha (ROR alpha) (NR1F1) is a member of the nuclear receptor superfamily whose biological functions are largely unknown. Since staggerer mice, which carry a deletion in the ROR alpha gene, suffer from immune abnormalities, we generated an adenovirus encoding ROR alpha1 to investigate its potential role in control of the inflammatory response. We demonstrated that ROR alpha is expressed in human primary smooth-muscle cells and that ectopic expression of ROR alpha1 inhibits TNFalpha-induced IL-6, IL-8 and COX-2 expression in these cells. ROR alpha1 negatively interferes with the NF-kappaB signalling pathway by reducing p65 translocation as demonstrated by western blotting, immunostaining and electrophoretic mobility shift assays. This action of ROR alpha1 on NF-kappaB is associated with the induction of IkappaB alpha, the major inhibitory protein of the NF-kappaB signalling pathway, whose expression was found to be transcriptionally upregulated by ROR alpha1 via a ROR response element in the IkappaB alpha promoter. Taken together, these data identify ROR alpha1 as a potential target in the treatment of chronic inflammatory diseases, including atherosclerosis and rheumatoid arthritis.

Treatment of rheumatoid arthritis with PEGylated recombinant human soluble tumour necrosis factor receptor type I: a clinical update

A recombinant form of the high affinity, natural inhibitor of tumour necrosis factor α (TNFα) is currently under development for the treatment of rheumatoid arthritis (RA).1 This molecule is referred...

Long term azathioprine fails to prevent onset of multiple sclerosis: report of two cases

Azathioprine is an immunosuppressive drug widely used in the treatment of chronic inflammatory diseases, including Multiple Sclerosis (MS). We report two patients who developed the first manifestations of clinically definite multiple sclerosis while on long term (3.5 and 10 years, respectively) treatment with azathioprine for Crohn's disease. Both patients developed the first MS symptoms during a quiescent phase of their inflammatory bowel disease. These cases show that long term azathioprine, while possibly maintaining inflammatory bowel disease under control, could not prevent the onset of MS. Multiple Sclerosis (2000) 6 362 - 363

Identification and Characterization of a Potent, Selective, and Orally Active Antagonist of the CC Chemokine Receptor-1

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.