Seleno-auranofin (Et3PAuSe-tagl): synthesis, spectroscopic (EXAFS, 197Au Mössbauer, 31P, 1H, 13C, and 77Se NMR, ESI-MS) characterization, biological activity, and rapid serum albumin-induced triethylphosphine oxide generation.

Seleno-auranofin (SeAF), an analogue of auranofin (AF), the orally active antiarthritic gold drug in clinical use, was synthesized and has been characterized by an array of physical techniques and biological assays. The Mössbauer and extended X-ray absorption fine structure (EXAFS) parameters of the solid compound demonstrate a linear P-Au-Se coordination environment at a gold(I) center, analogous to the structure of auranofin. The (31)P, (13)C, and (1)H NMR spectra of SeAF in chloroform solution closely resemble those of auranofin. The (77)Se spectrum consists of a singlet at 481 ppm, consistent with a metal-bound selenolate ligand. The absence of (2)J(PSe) coupling in the (31)P and (77)Se spectra may arise from dynamic processes occurring in solution or because the (2)J(PSe) coupling constants are smaller than the observed bandwidths. Electrospray ionization mass spectrometry (ESI-MS) spectra of SeAF in 50:50 methanol-water exhibited strong signals for [(Et(3)P)(2)Au](+), [(Et(3)PAu)(2)-mu-Se-tagl](+), and [Au(Se-tagl)(2)](-), which arise from ligand scrambling reactions. Three assays of the anti-inflammatory activity of SeAF allowed comparison to AF. SeAF exhibited comparable activity in the topically administered murine arachadonic acid-induced and phorbol ester-induced anti-inflammatory assays but was inactive in the orally administered carrageenan-induced assay in rats. However, in vivo serum gold levels were comparable in the rat, suggesting that differences between the in vivo metabolism of the two compounds, leading to differences in transport to the inflamed site, may account for the differential activity in the carrageenan-induced assay. Reactions of serum albumin, the principal transport protein of gold in the serum, demonstrated formation of AlbSAuPEt(3) at cysteine 34 and provided evidence for facile reduction of disulfide bonds at cysteine 34 and very rapid formation of Et(3)P=O, a known metabolite of auranofin.

Long-term activation of TLR3 by poly(I:C) induces inflammation and impairs lung function in mice.

BACKGROUND: The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases. METHODS: TLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C). RESULTS: There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFalpha were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy. CONCLUSION: These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

Dermokine: an extensively differentially spliced gene expressed in epithelial cells.

Studies performed to discover genes overexpressed in inflammatory diseases identified dermokine as being upregulated in such disease conditions. Dermokine is a gene that was first observed as expressed in the differentiated layers of skin. Its two major isoforms, alpha and beta, are transcribed from different promoters of the same locus, with the alpha isoform representing the C terminus of the beta isoform. Recently, additional transcript variants have been identified. Extensive in silico analysis and reverse transcriptase (RT)-PCR cloning has confirmed the existence of these variants in human cells and tissues, identified a new human isoform as well as the gamma isoform in mouse. Recombinant expression and analysis of the C-terminal truncated isoform indicate that the molecule is O-linked glycosylated and forms multimers in solution. In situ hybridization and immunohistochemistry has shown that the gene is differentially expressed in various cells and tissues, other than the skin. These results show that the dermokine gene is expressed in epithelial tissues other than the skin and this expression is transcriptionally and posttranscriptionally complex.

Anti-interleukin-6 monoclonal antibody inhibits autoimmune responses in a murine model of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from dysregulation of the immune system. Interleukin-6 (IL-6) is a multifunctional cytokine produced by macrophages, monocytes and T and B cells. It stimulates B-cell differentiation/maturation, immunoglobulin secretion, and T-cell functions. Elevated levels of IL-6 in serum, urine and renal glomeruli were detected in patients with active SLE and in murine models of SLE. Our study investigated the role of IL-6 in an SLE-like disease in New Zealand Black/White (NZB/W) F1 mice by administration of an anti-murine IL-6 monoclonal antibody (mAb). Intraperitoneal administration of the anti-IL-6 mAb suppressed the production of anti-dsDNA autoantibody. B-cell proliferation induced by anti-IgM and anti-CD40 was lower in the anti-IL-6 mAb-treated mice, ex vivo studies demonstrated that anti-IL-6 mAb treatment inhibited anti-dsDNA production. Anti-CD3-induced T-cell proliferation and mixed lymphocyte reactions were inhibited by anti-IL-6 mAb treatment, indicating a partial down-regulation of T cells. Histological analysis showed that treatment with anti-IL-6 mAb prevented the development of severe kidney disease. These results suggest that treatment with anti-IL-6 mAb has a beneficial effect on autoimmunity in murine SLE and that autoreactive B cells may be the primary target for anti-IL-6 mAb treatment; its effect on autoreactive T cells is also indicated.

Ex vivo activated OVA specific and non-specific CD4+CD25+ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses.

CD4+CD25+ regulatory T cells (Tr) are important in maintaining immune tolerance to self-antigen (Ag) and preventing autoimmunity. Reduced number and inadequate function of Tr are observed in chronic autoimmune diseases. Adoptively transferred Tr effectively suppress ongoing autoimmune disease in multiple animal models. Therefore, strategies to modulate Tr have become an attractive approach to control autoimmunity. Activation of Tr is necessary for their optimal immune regulatory function. However, due to the low ratio of Tr to any given antigen (Ag) and the unknown nature of Ag in many autoimmune diseases, specific activation is not practical for potential therapeutic intervention. It has been shown in animal models that once activated, Tr can exhibit immune suppression in a bystander Ag-non-specific fashion, suggesting the effector phase of Tr is Ag independent. To investigate whether the immune suppression by activated bystander Tr is as potent as that of the Ag specific Tr, Tr cells were isolated from BALB/c or ovalbumin (OVA) specific T cell receptor (TCR) transgenic mice (DO11.10) and their immune suppression of an OVA specific T cell response was compared. We found that once activated ex vivo, Tr from BALB/c and DO11.10 mice exhibited comparable inhibition on OVA specific T cell responses as determined by T cell proliferation and cytokine production. Furthermore, their immune suppression function was compared in a delayed type hypersensitivity (DTH) model induced by OVA specific T cells. Again, OVA specific and non-specific Tr exhibited similar inhibition of the DTH response. Taken together, the results indicate that ex vivo activated Ag-non-specific Tr are as efficient as Ag specific Tr in immune suppression, therefore our study provides additional evidence suggesting the possibility of applying ex vivo activated Tr therapy for the control of autoimmunity.

Modulation of antigen-specific T cell response by a non-mitogenic anti-CD3 antibody.

Suppression of T cell response is the key to enhance graft survival and control autoimmune diseases. A mitogenic anti-CD3 monoclonal antibody (mAb), OKT3, has been used for decades to control acute rejection in organ transplantation. Although effective, the clinical use was limited by its side effects, such as cytokine release mediated by T cell activation. A low mitogenic humanized OKT3 with reduced FcR-binding (hgammaOKT3 Ala-Ala) was generated and tested in several clinical studies. Although hgammaOKT3 Ala-Ala demonstrated maintained efficacy and better safety it still activated T cells. To investigate if a non-mitogenic anti-CD3 mAb can be equally effective in immune suppression, a chimeric non-FcR-binding anti-mouse CD3 mAb (anti-CD3 IgG2a Ala-Ala) was generated. Unlike the hgammaOKT3 Ala-Ala, the mouse IgG2a Ala-Ala anti-CD3 mAb did not induce T cell activation as measured by proliferation, cytokine production and apoptosis. Nevertheless, the IgG2a Ala-Ala anti-CD3 mAb was equally effective in the inhibition of antigen-specific CD4+ T cell activation in vitro to that of the mitogenic anti-CD3 mAb (Anti-CD3 IgG2a). In vivo, the IgG2a Ala-Ala anti-CD3 mAb only induced transient reduction of peripheral and spleen T cells and did not trigger detectable cytokine release. Nonetheless, this non-mitogenic anti-CD3 mAb significantly prolonged islet graft survival as effectively as the mitogenic anti-CD3 mAb in an allogenic islet transplantation model. These results demonstrated that a non-mitogenic anti-CD3 mAb could be used as an effective immune modulator. It may also indicate that a true non-mitogenic version of OKT3 could further improve its safety profile for clinical use.

IL-4 and IL-13 form a negative feedback circuit with surfactant protein-D in the allergic airway response.

The innate immune molecule surfactant protein-D (SP-D) plays an important regulatory role in the allergic airway response. In this study, we demonstrate that mice sensitized and challenged with either Aspergillus fumigatus (Af) or OVA have increased SP-D levels in their lung. SP-D mRNA and protein levels in the lung also increased in response to either rIL-4 or rIL-13 treatment. Type II alveolar epithelial cell expression of IL-4Rs in mice sensitized and challenged with Af, and in vitro induction of SP-D mRNA and protein by IL-4 and IL-13, but not IFN-gamma, suggested a direct role of IL-4R-mediated events. The regulatory function of IL-4 and IL-13 was further supported in STAT-6-deficient mice as well as in IL-4/IL-13 double knockout mice that failed to increase SP-D production upon allergen challenge. Interestingly, addition of rSP-D significantly inhibited Af-driven Th2 cell activation in vitro whereas mice lacking SP-D had increased numbers of CD4(+) cells with elevated IL-13 and thymus- and activation-regulated chemokine levels in the lung and showed exaggerated production of IgE and IgG1 following allergic sensitization. We propose that allergen exposure induces elevation in SP-D protein levels in an IL-4/IL-13-dependent manner, which in turn, prevents further activation of sensitized T cells. This negative feedback regulatory circuit could be essential in protecting the airways from inflammatory damage after allergen inhalation.

Therapeutic dosing with anti-interleukin-13 monoclonal antibody inhibits asthma progression in mice.

In vivo models have demonstrated that interleukin-13 (IL-13) plays an important role in asthma; however, few studies have evaluated the effect of inhibition of IL-13 on established and persistent disease. In the present study, we have investigated the effect of a therapeutic dosing regimen with an anti-IL-13 monoclonal antibody (mAb) in a chronic mouse model of persistent asthma. BALB/c mice were sensitized to allergen [ovalbumin (OVA); on days 1 and 8] and challenged with OVA weekly from day 22. Anti-IL-13 mAb or vehicle dosing was initiated following two OVA challenges when disease was established. At this time, mice exhibited airway hyperresponsiveness (AHR), increased mucus production, inflammation, and initiation of subepithelial fibrosis compared with saline-challenged mice. Mice received four additional OVA challenges. Treatment with anti-IL-13 mAb inhibited AHR and prevented the further development of subepithelial fibrosis and progression of inflammation. Furthermore, mAb treatment reversed the mucus hyperplasia to basal levels. These effects were associated with an inhibition of cytokines, chemokines, and matrix metalloproteinase-9. These data demonstrate that neutralization of IL-13 can inhibit the progression of established disease in the presence of repeated allergen exposures.

Anti-CD3 antibodies OKT3 and hOKT3gamma1(Ala-Ala) induce proliferation of T cells but impair expansion of alloreactive T cells; aspecifc T cell proliferation induced by anti-CD3 antibodies correlates with impaired expansion of alloreactive T cells.

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), has been used for decades to reverse acute transplant rejection. A humanized OKT3 with two alanine replacements on the CH2 domain, hOKT3gamma1(Ala-Ala), was generated to reduce side effects of OKT3. This study reports the effects of OKT3 and hOKT3gamma1(Ala-Ala) on responder T cells in mixed leukocyte cultures (MLC). T cells were purified from peripheral blood mononuclear cells (PBMC) and labeled with CFSE before culture with irradiated allogeneic PBMC in a MLC. Multiparameter flow cytometric analysis of MLC demonstrated logarithmic expansion of T cells with a memory phenotype. Treatment with either OKT3 or OKT3gamma1(Ala-Ala) prevented the logarithmic expansion of these cells. Instead, both OKT3 and hOKT3gamma1(Ala-Ala) induced nonspecific expansion of T cells with both naive and memory phenotype. The proliferating T cells in OKT3 or hOKT3gamma1(Ala-Ala) treated cultures expressed low levels of CD25, and showed a diminished Granzyme B expression compared to that of the untreated MLC, suggesting that these cells may not be fully functional. An array of cytokines in the MLC supernatant was analyzed, and IL-5 and IL-13 were significantly reduced by OKT3 or hOKT3gamma1(Ala-Ala) treatment. These results demonstrated that OKT3 and hOKT3gamma1(Ala-Ala) have similar effects on T cells in MLC. Both antibodies inhibited the logarithmic expansion of allo-reactive T cells, probably through inducing suboptimal proliferation of specific and non-specific T cells. This in vitro study provided one possible mechanism of the therapeutic effects of OKT3 and hOKT3gamma1(Ala-Ala) antibodies.

Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling.

Asthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. Interleukin 13 (IL-13) is a pleiotropic cytokine produced mainly by T cells. A substantial amount of evidence suggests that IL-13 plays a critical role in the pathogenesis of asthma. Therefore, a neutralizing anti-IL-13 monoclonal antibody could provide therapeutic benefits to asthmatic patients. To test the concept we have generated a neutralizing rat anti-mouse IL-13 monoclonal antibody, and evaluated its effects in a chronic mouse model of asthma. Chronic asthma-like response was induced in ovalbumin (OVA) sensitized mice by repeated intranasal OVA challenges. After weeks of challenge, mice developed airway hyperresponsiveness (AHR) to methacholine stimulation, severe airway inflammation, hyper mucus production, and subepithelial fibrosis. When given at the time of each intranasal OVA challenge, anti-IL-13 antibody significantly suppressed AHR, eosinophil infiltration, proinflammatory cytokine/chemokine production, serum IgE, and most interestingly, airway remodeling. Taken together, these results strongly suggest that a neutralizing anti-human IL-13 monoclonal antibody could be an effective therapeutic agent for asthma.

Induction of dendritic cell maturation by IL-18.

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.

Evaluation of potent and selective small-molecule antagonists for the CXCR2 chemokine receptor.

N,N'-Diarylureas were prepared, and the structure-activity relationship relative to the CXCR2 receptor was examined. This led to the identification of a potent and highly selective CXCR2 antagonist, which in addition was shown to be functionally active both in vitro against human neutrophils and in vivo in rabbit models of ear edema and neutropenia.

PGE2 and IL-6 production by fibroblasts in response to titanium wear debris particles is mediated through a Cox-2 dependent pathway.

Aseptic loosening of orthopaedic implants is precipitated by wear debris-induced osteolysis. Central to this process are the pro-inflammatory mediators that are produced in response to wear by the fibroblastic cells, which comprise the majority of periprosthetic membranes. Since this pro-inflammatory cascade is mediated by a plethora of factors with redundant functions, it is imperative to establish a hierarchy. Two well-known fibroblast derived pro-inflammatory factors that stimulate wear debris-induced osteoclastic resorption are prostaglandin E2 (PGE2) and IL-6. However, their relationship to each other in this process is poorly defined. Here we show immunohistochemistry of retrieval membranes indicating that COX-2 is the principal cyclooxygenase responsible for PGE2 production in fibroblasts around failed implants. We also performed in vitro experiments with fibroblasts derived from wild-type (WT), COX-1 (-/-) and COX-2 (-/-) mice, which demonstrated that COX-2 is required for Ti wear debris-induced PGE2 production. Interestingly, COX-2 was also required for IL-6 production in these assays, which could be rescued by the addition of exogenous PGE2 (10(-6) M). Pharmacology studies that utilized the COX-1 selective inhibitor SC 560, the COX-2 selective inhibitor celecoxib, and the nonselective COX inhibitor indomethacin confirmed these results. Taken together, these results indicate that selective inhibition of prostaglandin signaling could favorably impact aseptic loosening beyond its direct effects on PGE2 synthesis, in that it inhibits downstream pro-inflammatory/pro-osteoclastic cytokine production.

What's in the pipeline? Prospects for monoclonal antibodies (mAbs) as therapies for lung diseases.

The striking clinical results from recent studies with Remicade (infliximab, a monoclonal anti-TNFalpha antibody) in rheumatoid arthritis, Crohn's disease and psoriasis demonstrate the disease-altering potential of monoclonal antibodies (mAbs) in chronic inflammation. Chronic obstructive pulmonary disease (COPD) and asthma represent two major chronic pulmonary inflammatory diseases with substantial unmet medical needs. Most of the cells and mediators implicated in the pathophysiology of COPD and asthma are excellent targets for mAb intervention. Indeed, clinical trials with mAbs directed against IL-5, IgE, and CD4 yielded results that are critical in dissecting the pathophysiology of asthma, and reinforce the potential for mAbs as therapeutic agents in treating pulmonary diseases. Furthermore, fundamental advances in the discovery, manufacture and safety of mAbs underscore the enormous therapeutic value of these agents for chronic pulmonary diseases. Indeed, a large number of mAbs are in pre-clinical and clinical development for treating these conditions. In this review, we discuss the scientific rationale for generating mAb therapies directed specifically toward COPD and asthma. We believe that as a therapeutic class, mAbs offer the opportunity to alter symptoms, progression and outcome of chronic pulmonary diseases.

Anti-TNF-alpha antibody allows healing of joint damage in polyarthritic transgenic mice.

Anti-tumor-necrosis-factor-alpha (TNF-alpha) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-alpha (hTNF-alpha) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-alpha antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-alpha treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-alpha prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-alpha and local expression of various proinflammatory mediators were all diminished by anti-TNF-alpha treatment, confirming a critical role of hTNF-alpha in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-alpha treatment was observed in young mice but not in aged mice.

Coming of age: anti-cytokine therapies.

Although cytokines are critical in maintaining normal physiology, excessive production of these proteins can lead to pathological consequences. Inhibitors of cytokine production or action are therefore widely investigated as potential therapeutic agents in a variety of immune and inflammatory diseases. Indeed, the successful application of inhibitors of tumor necrosis factor-alpha in rheumatoid arthritis and Crohn's disease heralds the great therapeutic potential of biopharmaceutical agents to counteract cytokine activities. Emerging opportunities for new therapeutics, as well as the challenges we face in their use and development, are described in this review.

Nonpeptide tachykinin receptor antagonists. III. SB 235375, a low central nervous system-penetrant, potent and selective neurokinin-3 receptor antagonist, inhibits citric acid-induced cough and airways hyper-reactivity in guinea pigs.

In this report the in vitro and in vivo pharmacological and pharmacokinetic profile of (-)-(S)-N-(alpha-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB 235375), a low central nervous system (CNS)-penetrant, human neurokinin-3 (NK-3) receptor (hNK-3R) antagonist, is described. SB 235375 inhibited (125)I-[MePhe(7)]-neurokinin B (NKB) binding to membranes of Chinese hamster ovary (CHO) cells expressing the hNK-3R (CHO-hNK-3R) with a K(i) = 2.2 nM and antagonized competitively NKB-induced Ca(2+) mobilization in human embryonic kidney (HEK) 293 cells expressing the hNK-3R (HEK 293-hNK-3R) with a K(b) = 12 nM. SB 235375 antagonized senktide (NK-3R)-induced contractions in rabbit isolated iris sphincter (pA(2) = 8.1) and guinea pig ileal circular smooth muscles (pA(2) = 8.3). SB 235375 was selective for the hNK-3R compared with hNK-1 (K(i) > 100,000 nM) and hNK-2 receptors (K(i) = 209 nM), and was without effect, at 1 microM, in 68 other receptor, enzyme, and ion channel assays. Intravenous SB 235375 produced a dose-related inhibition of miosis induced by i.v. senktide in the rabbit (ED(50) of 0.56 mg/kg). Intraperitoneal SB 235375 (10-30 mg/kg) inhibited citric acid-induced cough and airways hyper-reactivity in guinea pigs. In mice oral SB 235375 (3-30 mg/kg) was without significant effect on the behavioral responses induced by intracerebral ventricular administration of senktide. Pharmacokinetic evaluation in the mouse and rat revealed that oral SB 235375 was well absorbed systemically but did not effectively cross the blood-brain barrier. The preclinical profile of SB 235375, encompassing high affinity, selectivity, oral activity, and low CNS penetration, suggests that it is an appropriate tool compound to define the pathophysiological roles of the NK-3Rs in the peripheral nervous system.