Immunopharmacology and Inflammation
Development of non-antibiotic macrolide that corrects inflammation-driven immune dysfunction in models of inflammatory bowel diseases and arthritis

https://doi.org/10.1016/j.ejphar.2011.04.036Get rights and content

Abstract

Inflammation-driven immune dysfunction supports the development of several chronic human disorders including inflammatory bowel diseases and rheumatoid arthritis. Macrolides are effective antibiotics endowed with immunomodulatory effects. In this study we report the chemical synthesis and the pharmacological characterization of CSY0073, a non-antibiotic derivative of azithromycin. CSY0073 was tested for efficacy in two experimental models of colitis induced by administering mice with dextran sulfate (DSS) and trinitrobenzene sulphonic acid (TNBS) and in collagen induced arthritis. Like azithromycin, CSY0073 improved clinical, macroscopic and histopathological scores in mice administered DSS (12.5 μmol/kg/day p.o.) and TNBS (45 μmol/kg/day p.o.). When administered to TNBS-treated mice, CSY0073 effectively attenuated influx of neutrophils and macrophages into the colonic mucosa and reduced the intestinal expression pro-inflammatory cytokines TNFα, IL-2 and IFNγ. CSY0073 (0.1 to 10 μM) counter-regulated TNFα, IFNγ, IL-12 and IL-23 release caused by exposure of mouse spleen monocytes and CD11b+ cells isolated from the colonic lamina propria to endotoxin. CSY0073 (25 μmol/kg/day) reduced clinical scores in the collagen induced murine model of rheumatoid arthritis. In myeloid cells, CSY0073 (10 μM) prevented the nuclear translocation of the p65 subunit of NF-κB and its binding to canonical NF-κB responsive elements. In summary, we report a novel class of non-antibiotic 14-member macrocycles with anti-inflammatory and immune-modulatory effects. CSY0073, the prototype of this new class of macrolides exerts counter-regulatory activity on NF-κB signaling. This study suggests the exploitation of non-antibiotic macrolides in the treatment of inflammatory disorders characterized by immune dysfunction.

Graphical abstract

CYS0073 is a macrolide derivative that attenuates intestinal inflammation in rodent model of colitis. CYS0073 attenuates NF-κB activation and release of effector cytokines from intestinal macrophages correcting inflammation-driven immune dysfunction in intestine and systemic macrophages.

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Introduction

The term “macrolide” encompasses a diverse family of unrelated compounds with large macrolactam rings whose activity stems from the presence of a macrolide ring, a large macrocycle lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. The lactone rings are usually 14-, 15-, or 16-membered (Alekshun, 2005). The macrolide antibiotics (macrolides) inhibit RNA-dependent protein synthesis by reversibly binding to the 50S ribosomal subunit of a susceptible microorganism (Alekshun, 2005, Asaka et al., 2003, Wilson, 2009). In addition to erythromycin A, the prototype macrolide, several other macrolides, clarithromycin, roxithromycin, and azithromycin are clinically available (Payne et al., 2007). Besidesits anti-bacterial activity, macrolides exert diverse biological effects including the ability to modulate inflammatory and immune responses without affecting homeostatic immunity (Shinkai et al., 2008). These properties have led to the long-term use of macrolides in treating neutrophil-dominated inflammation including diffuse panbronchiolitis, bronchiectasis, rhinosinusitis, and cystic fibrosis (Rubin and Henke, 2004, Schultz, 2004).

In the setting of chronic inflammation, low-dose macrolides decrease the production of proinflammatory cytokines and chemokines by epithelial and immune cells. The list of these counter-regulatory effects includes the inhibition of secretion of interleukins (IL)-4, IL-5 IL-6, IL-8, GM-CSF and eotaxin, by a variety of lung epithelial cells (Desaki et al., 2004, Ichiyama et al., 2001, Takizawa et al., 1997). In addition, macrolides suppress IL-1β and TNF-α release by endotoxin (LPS)-stimulated monocytes; IL-1β, IL-6, TNF-α and GM-CSF by mast cells, and IL-8, ENA-78 and MIP-1 by macrophages and leukocytes (Bosnar et al., 2009, Yasutomi et al., 2005). Finally, macrolides inhibit the function of Th2-type lymphocytes and suppress IFNγ release in memory T cells, but not in naïve T cells (Aoki and Kao, 1999, Park et al., 2002). These immunomodulatory activities are inhibitory in nature and extend to the modulation of adhesion molecules essential to the process of recruiting neutrophils at the site of inflammation (Culić et al., 2002, Ivetić Tkalcević et al., 2006). Thus, erythromycin inhibits LPS-induced neutrophil recruitment to the rat middle ear by down-regulating l-selectin and Mac-1 expression (Lin et al., 2000). Erythromycin also attenuates upregulation of ICAM-1 and the β2-integrin (CD11b/CD18) induced by the Hemophilus influenzae endotoxin in NHBE cells. Inhibition of the expression of E-selectin and ICAM-1 by azithromycin has also been reported (Lin et al., 2000). These counter-regulatory activities appear to be polymodal, but there is robust evidence linking these effects to interference with the signaling pathways mediated by activator protein-1 (AP-1), extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor (NF)-κB. One important observation is that anti-inflammatory activities exerted by macrolides are time-dependent and become evident in clinical settings only after weeks of treatment (Rubin and Henke, 2004). A concern with this approach however, is that an extended use of low dose macrolides for non-antibiotic indications would increase the emergence of antimicrobial resistance (Ergin et al., 2006, Gogarten and Townsend, 2005, Hawkyard and Koerner, 2007, Vester and Douthwaite, 2001, Warny et al., 2005). Development of non-antimicrobial macrolides is, therefore, of potential clinical interest. Inflammatory bowel disease is a chronic, progressive and destructive disorder of the gastrointestinal tract that may manifest as either Crohn's disease or ulcerative colitis (Xavier and Podolsky, 2007). There is circumstantial evidence to link the pathogenesis of inflammatory bowel diseases to a dysfunctional interaction between bacterial microflora of the gut and the mucosal immune system (Xavier and Podolsky, 2007). The innate immune system has dual roles in host defense, providing a direct and immediate response against microbial invaders as well as playing an instructive role, influencing the nature of adaptive immunity. This instructive role of the innate immune system is served by a subset of intestinal mucosal immune cells, the antigen presenting cells (APCs) that include macrophages and dendritic cells (Niess, 2008, Sturm et al., 2008). A dysregulation in the innate immune function could lead to development of inflammatory bowel diseases, suggesting that attenuation of unbalanced generation of pro-inflammatory mediators by intestinal macrophages might be effective in protecting against inflammatory bowel disease development (Niess, 2008). In this study we report the generation of an azithromycin derivative that lacks anti-bacterial activity while retaining the anti-inflammatory and immunomodulatory activity of conventional macrolides. This agent, CYS0073 (see Fig. 1A for structure), exerts activity in rodent-models of intestinal inflammation and holds potential for treatment of inflammation-driven immune-dysfunction.

Section snippets

Chemistry, design and development of non-antibiotic azithromycin derivative

CSY0073 was prepared by selective oxidation of azithromycin. Briefly, azithromycin was dissolved in HCl to hydrolyse cladinose and was then neutralized, followed by the addition of an acetate protective group via reaction with acetic acid anhydride. The resulting product was added to a mixture of N-chlorosuccinamide and dimethylsulfide at − 40 °C followed by the addition of ethyl diisopropylamine. The dichloromethane soluble portion was dried, hydrolysed in methanol overnight and the residue

CSY0073 is not anti-bacterial and is well tolerated

The chemical structure of the azithromycin derivative CSY0073 is illustrated in panel A. In contrast to azithromycin, CSY0073 did not inhibit growth of B. pumilus at concentrations up to 100 μM (panels B and C). Moreover, CSY0073 was ineffective against E. coli and M. luteus (panel B). The lack of antibacterial activity of CSY0073 was further confirmed by an in vivo study. Administering Balb/c mice with azithromycin 45 μmol/kg/day for 7 days by gavage leads to a significant reduction in fecal

Discussion

Several 14- (including erythromycin, clarithromycin and roxithromycin), 15- (including azithromycin) and 16-membered ring macrolides are broad spectrum antibacterial compounds exhibiting action against Gram-positive and Gram-negative bacteria (Alekshun, 2005, Asaka et al., 2003, Wilson, 2009). Consequently, erythromycin A (and newer macrolides such as clarithromycin, roxithromycin and azithromycin) are alternatives to penicillins and cephalosporins for the treatment of infections, especially

Contributors

AM participated in the design of the study, TNBS colitis, contributed in cell culture experiments, data analysis and drafting the manuscript. MB developed the basic concept using macrolides, designed studies, conducted colitis studies, contributed to interpretation of data, drafting and critical revision of the manuscript. GP contributed to RT-PCR data in colon biopsy. BR contributed to RT-PCR co-IP and EMSA data in cells. ED, J-HG and UH participated in the data interpretation and manuscript

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