Unprecedented Epimerization of an Azithromycin Analogue: Synthesis, Structure and Biological Activity of 2'-Dehydroxy-5″-Epi-Azithromycin.

Certain macrolide antibiotics, azithromycin included, possess anti-inflammatory properties that are considered fundamental for their efficacy in the treatment of chronic inflammatory diseases, such as diffuse pan-bronchiolitis and cystic fibrosis. In this study, we disclose a novel azithromycin analog obtained via Barton-McCombie oxidation during which an unprecedented epimerization on the cladinose sugar occurs. Its structure was thoroughly investigated using NMR spectroscopy and compared to the natural epimer, revealing how the change in configuration of one single stereocenter (out of 16) profoundly diminished the antimicrobial activity through spatial manipulation of ribosome binding epitopes. At the same time, the anti-inflammatory properties of parent macrolide were retained, as demonstrated by inhibition of LPS- and cigarette-smoke-induced pulmonary inflammation. Not surprisingly, the compound has promising developable properties including good oral bioavailability and a half-life that supports once-daily dosing. This novel anti-inflammatory candidate has significant potential to fill the gap in existing anti-inflammatory agents and broaden treatment possibilities.

Preventing lung pathology and mortality in rabbit Staphylococcus aureus pneumonia models with cytotoxin-neutralizing monoclonal IgGs penetrating the epithelial lining fluid.

Staphylococcus aureus pneumonia is associated with high mortality irrespective of antibiotic susceptibility. Both MRSA and MSSA strains produce powerful cytotoxins: alpha-hemolysin(Hla) and up to five leukocidins - LukSF-PV, HlgAB, HlgCB, LukED and LukGH (LukAB) - to evade host innate defense mechanisms. Neutralizing cytotoxins has been shown to provide survival benefit in rabbit S. aureus pneumonia models. We studied the mechanisms of protection of ASN100, a combination of two human monoclonal antibodies (mAbs), ASN-1 and ASN-2, that together neutralize Hla and the five leukocidins, in rabbit MRSA and MSSA pneumonia models. Upon prophylactic passive immunization, ASN100 displayed dose-dependent increase in survival and was fully protective against all S. aureus strains tested at 5 or 20 mg/kg doses. Macroscopic and microscopic lung pathology, edema rate, and bacterial burden were evaluated 12 hours post infection and reduced by ASN100. Pharmacokinetic analysis of ASN100 in bronchoalveolar-lavage fluid from uninfected animals detected efficient penetration to lung epithelial lining fluid reaching peak levels between 24 and 48 hours post dosing that were comparable to the mAb concentration measured in serum. These data confirm that the ASN100 mAbs neutralize the powerful cytotoxins of S. aureus in the lung and prevent damage to the mucosal barrier and innate immune cells.

Precision-cut lung slices from bleomycin treated animals as a model for testing potential therapies for idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a complex lung disease with incompletely understood pathophysiology. Effectiveness of available medicines is limited and the need for new and improved therapies remains. Due to complexity of the disease, it is difficult to develop predictable in vitro models. In this study we have described precision-cut lung slices (PCLS) prepared from bleomycin treated mice as an in vitro model for testing of novel compounds with antifibrotic activity. We have shown that PCLS during in vitro incubation retain characteristics of bleomycin model with increased expression of fibrosis related genes ACTA2 (α-smooth muscle actin), COL1A1 (collagen 1), FN1 (fibronectin 1), MMP12 (matrix metalloproteinase 12) and TIMP1 (tissue inhibitor of metalloproteinases). To further evaluate PCLS as an in vitro model, we have tested ALK5 inhibitor SB525334 which was previously shown to attenuate fibrosis in in vivo bleomycin model and nintedanib which is the FDA approved treatment for IPF. SB525334 and nintedanib inhibited expression of fibrosis related genes in PCLS from bleomycin treated mice. In addition, comparable activity profile of SB525334 was achieved in PCLS and in vivo model. Altogether these results suggest that PCLS may be a suitable in vitro model for compound testing during drug development process.

Immunomodulatory effects of azithromycin on the establishment of lipopolysaccharide tolerance in mice.

Recent reports suggest that azithromycin can shift macrophage polarization towards the alternatively activated M2 phenotype. In order to investigate its immunomodulatory activity in vivo, the influence of azithromycin on survival and cytokine production was assessed in the LPS tolerance model which is characterized by an M2 skewed response. For induction of tolerance, mice received an intraplantar injection of 30 µg LPS, 24 h prior to intravenous challenge with 350 µg LPS. Azithromycin (100 mg/kg) was administered orally, 2 h before LPS application. Influence of treatment on survival and cytokine concentration in serum was monitored. Azithromycin alone, instead of LPS, could not induce an LPS tolerant state. However, when administered before LPS priming it significantly increased survival, which was enhanced by concomitant azithromycin before LPS challenge. Azithromycin had no effect on survival when administered only prior to the LPS challenge. Tolerance induction by LPS priming was associated, upon LPS challenge, with decreased serum concentrations of pro-inflammatory cytokines, TNFα, IL-12p40 and CCL5, and increased serum concentrations of the anti-inflammatory cytokines, IL-10 and IL-1ra. Azithromycin treatment, prior to LPS priming, further reduced serum TNFα and CCL5, yielding the greatest inhibition when the macrolide was also given prior to LPS challenge. Serum concentrations of the anti-inflammatory cytokines, IL-10 and IL-1ra, were unchanged following azithromycin treatment. In summary, we have confirmed the immunomodulatory activity of azithromycin, as reflected in its ability to augment tolerance induction to LPS, promoting increased survival and reduced pro-inflammatory cytokine production, without affecting overt inflammation to LPS or anti-inflammatory cytokine production.

Porphyrins as new endogenous anti-inflammatory agents.

A series of porphyrins, tetrapyrrole natural organic compounds, are evaluated here as endogenous anti-inflammatory agents. They directly inhibit the activity of Fyn, a non-receptor Src-family tyrosine kinase, triggering anti-inflammatory events associated with down-regulation of T-cell receptor signal transduction, leading to inhibition of tumor necrosis factor alpha (TNF-α) production. This is one of the major pro-inflammatory cytokines, associated with diseases such as diabetes, tumorigenesis, rheumatoid arthritis, and inflammatory bowel disease. Porphyrins, as a chemical class, inhibited Fyn kinase activity in a non-competitive, linear-mixed fashion. In cell-based in vitro experiments on polymorphonuclear cells, porphyrins inhibited TNF-α cytokine production, T-cell proliferation, and the generation of free radicals in the oxidative burst, in a concentration-related manner. In vivo, lipopolysaccharide-induced TNF-α production in mice was inhibited by several of the porphyrins. These findings may be very important for the overall understanding of the role(s) of porphyrins in inflammation and their possible application as new anti-inflammatory agents.

N'-substituted-2'-O,3'-N-carbonimidoyl bridged macrolides: novel anti-inflammatory macrolides without antimicrobial activity.

Macrolide antibiotics, like erythromycin, clarithromycin, and azithromycin, possess anti-inflammatory properties. These properties are considered fundamental to the efficacy of these three macrolides in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, long-term treatment with macrolide antibiotics presents a considerable risk for promotion of bacterial resistance. We have examined antibacterial and anti-inflammatory effects of a novel macrolide class: N'-substituted 2'-O,3'-N-carbonimidoyl bridged erythromycin-derived 14- and 15-membered macrolides. A small focused library was prepared, and compounds without antimicrobial activity, which inhibited IL-6 production, were selected. Data analysis led to a statistical model that could be used for the design of novel anti-inflammatory macrolides. The most promising compound from this library retained the anti-inflammatory activity observed with azithromycin in lipopolysaccharide-induced pulmonary neutrophilia in vivo. Importantly, this study strongly suggests that antimicrobial and anti-inflammatory activities of macrolides are independent and can be separated, which raises development plausibility of novel anti-inflammatory therapeutics.

Claudin-3 and Clara cell 10 kDa protein as early signals of cigarette smoke-induced epithelial injury along alveolar ducts.

Smoking-associated chronic obstructive pulmonary disease is characterized by inflammation, changes affecting small airways, and development of emphysema. Various short- and long-term models have been introduced to investigate these processes. The aim of the present study was to identify markers of early epithelial injury/adaptation in a short-term animal model of cigarette smoke exposure. Initially, male BALB/c mice were exposed to smoke from one to five cigarettes and lung changes were assessed 4 and 24 hr after smoking cessation. Subsequently, animals were exposed to smoke from five cigarettes for 2 consecutive days and lungs investigated daily until the seventh postexposure day. Lung homogenates cytokines were determined, bronchioloalveolar fluid cells were counted, and lung tissue was analyzed by immunohistochemistry. Exposure to smoke from a single cigarette induced slight pulmonary neutrophilia. Smoke from two cigarettes additionally induced de novo expression of tight junction protein, claudin-3, by alveolar duct (AD) epithelial cells. Further increases in smoke exposure induced epithelial changes in airway progenitor regions. During the recovery period, the severity/frequency of epithelial reactions slowly decreased, coinciding with the switch from acute to a chronic inflammatory reaction. Claudin-3 and Clara cell 10 kDa protein were identified as possible markers of early tobacco smoke-induced epithelial injury along ADs.

The effect of apigenin on cyclophosphamide and doxorubicin genotoxicity in vitro and in vivo.

The aim of this study was to investigate the mutagenic and antigenotoxic effects of different doses of the flavonoid, apigenin, alone and in combination with the antitumor drugs, cyclophosphamide and doxorubicin, in vitro and in vivo. Using bacterial reverse mutation inhibition in vitro, with and without metabolic activation, the effect of apigenin (10-400 µg/plate) was studied on genotoxicity induced by cyclophosphamide (800 µg/plate) and by doxorubicin (0.2 µg/plate). Subsequent to a dose-finding study in vivo, CD1 mice were treated with either cyclophosphamide (40 mg/kg, i.p.) or doxorubicin (5 mg/kg, i.p.) with or without co-administration of apigenin (1-100 mg/kg, p.o.). Micronuclei were determined microscopically in blood smears and glutathione peroxidase (GPX), superoxide dismutase (SOD) and total antioxidative status (TAS) in whole blood, erythrocytes and plasma, respectively. Apigenin decreased doxorubicin-induced, but not cyclophosphamide-induced mutagenicity in vitro. In vivo, apigenin caused a statistically significant decrease in micronucleus frequency in response to cyclophosphamide, possibly due to active flavonoid metabolite formation or inhibition of cyclophosphamide metabolic activation. In animals treated with apigenin and doxorubicin, a significant decrease in micronucleus frequency was not observed, probably due to interindividual variability. No changes in GPX, SOD or TAS were observed in response to either cytotoxic agents or the flavonoid, possibly due to limited metabolic transformation of the drugs at the doses used. The results of the present study provide further evidence for the chemo-preventative properties of apigenin.

Azithromycin inhibits macrophage interleukin-1ß production through inhibition of activator protein-1 in lipopolysaccharide-induced murine pulmonary neutrophilia.

Macrolide antibiotics, including azithromycin, also possess anti-inflammatory properties. However, the molecular mechanism(s) of activity as well as the target cells for their action have not been unambiguously identified as yet. In this study, the effects of azithromycin on lipopolysaccharide (LPS)-induced pulmonary neutrophilia were investigated in mice. Using immunohistochemistry, mRNA and specific protein assays, we confirmed that azithromycin ameliorates LPS-induced pulmonary neutrophilia by inhibiting interleukin-1ß (IL-1ß) expression and production selectively in alveolar macrophages as well as in LPS-stimulated J774.2 macrophage-derived cells in vitro. Inhibition by azithromycin of neutrophilia and IL-1ß was accompanied by prevention of nuclear expression of activator protein-1 (AP-1) in both alveolar macrophages and J774.2 cells. The macrolide did not alter nuclear factor kappa B (NF-κB) or extracellular signal-regulated kinase 1/2 (ERK1/2) expression, activation or localization in LPS-stimulated lungs or in J774.2 cells. In conclusion, we have shown that inhibition of LPS-induced pulmonary neutrophilia and IL-1ß concentrations in lung tissue following azithromycin treatment is mediated through effects on alveolar macrophages. In addition, we have shown for the first time, in an in vivo model, that azithromycin inhibits AP-1 activation in alveolar macrophages, an action confirmed on J774.2 cells in vitro.

Comparison of pulmonary inflammatory and antioxidant responses to intranasal live and heat-killed Streptococcus pneumoniae in mice.

Inflammatory and antioxidant responses, in male C57Bl6J mice, to single intranasal inoculations with live or heat-killed Streptococcus pneumoniae were studied in order to tease out differences in responses. Heat-killed bacteria elicited weak lung neutrophil infiltration and raised concentrations (peak 6-8 h), in serum or lung tissue, of CXCL1 and 2, tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), and granulocyte-macrophage-colony stimulating factor, with later increases in CCL2 and IL-1ß. Live bacteria induced profound pulmonary neutrophil infiltration and acute chemokine/cytokine elevations. After 72-96 h, live S. pneumoniae induced a delayed rise in chemokines CXCL2 and CCL2, preceded by increases in TNFα, IL-1ß, and IL-6 and mononuclear infiltration of lungs. With both live and heat-killed bacteria, alveolar epithelial type II cells and alveolar macrophages were the main sources of TNFα and IL-1ß. Only live bacteria caused an acute decrease in lung glutathione peroxidase, an increase in superoxide dismutase, and a sustained increase in serum amyloid protein A. Acute innate immune responses to live and heat-killed S. pneumoniae are similar. In response to live bacteria, inflammation is greater, accompanied by changes in antioxidant enzymes and has an additional, later mononuclear component.

Azithromycin and clarithromycin inhibit lipopolysaccharide-induced murine pulmonary neutrophilia mainly through effects on macrophage-derived granulocyte-macrophage colony-stimulating factor and interleukin-1beta.

Macrolide antibiotics possess immunomodulatory/anti-inflammatory properties. These properties are considered fundamental for the efficacy of macrolide antibiotics in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, the molecular mechanisms and cellular targets of anti-inflammatory/immunomodulatory macrolide activity are still not fully understood. To describe anti-inflammatory effects of macrolides in more detail and to identify potential biomarkers of their activity, we have investigated the influence of azithromycin and clarithromycin on the inflammatory cascade leading to neutrophil infiltration into lungs after intranasal lipopolysaccharide challenge in mice. Azithromycin and clarithromycin pretreatment reduced total cell and neutrophil numbers in bronchoalveolar lavage fluid and myeloperoxidase concentration in lung tissue. In addition, concentrations of several inflammatory mediators, including CCL2, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and sE-selectin in lung homogenates were decreased after macrolide treatment. Inhibition of cytokine production observed in vivo was also corroborated in vitro in lipopolysaccharide-stimulated monocytes/macrophages, but not in an epithelial cell line. In summary, results presented in this article confirm that macrolides can suppress neutrophil-dominated pulmonary inflammation and suggest that the effect is mediated through inhibition of GM-CSF and IL-1beta production by alveolar macrophages. Besides GM-CSF and IL-1beta, CCL2 and sE-selectin are also identified as potential biomarkers of macrolide anti-inflammatory activity in the lungs.

Clarithromycin suppresses airway hyperresponsiveness and inflammation in mouse models of asthma.

Macrolide antibiotics, a class of potent antimicrobials, also possess immunomodulatory/anti-inflammatory properties. These properties are considered fundamental for the efficacy of macrolide antibiotics in the treatment of diffuse panbronchiolitis and cystic fibrosis. In patients with asthma, macrolide antibiotics have been reported to reduce airway hyperresponsiveness and improve pulmonary function. However, their beneficial actions in asthmatics possibly could be attributed to antimicrobial activity against atypical pathogens (e.g. Chlamydia pneumoniae), corticosteroid-sparing effect (inhibition of exogenous corticosteroid metabolism), and/or their anti-inflammatory/immunomodulatory effects. In order to investigate whether efficacy of macrolide antibiotics in asthma results from their immunomodulatory/anti-inflammatory activity, the influence of clarithromycin pretreatment (2 h before challenge) was examined on ovalbumin-induced airway hyperresponsiveness and airway inflammation in the mouse. Clarithromycin treatment (200 mg/kg intraperitoneally) decreased IL-4, IL-5, IL-13, CXCL2 and CCL2 concentrations in bronchoalveolar lavage fluid and markedly reduced inflammatory cell accumulation in bronchoalveolar lavage fluid and into the lungs, as revealed by histopathological examination. Furthermore, clarithromycin-induced reduction in inflammation was accompanied by normalization of airway hyperresponsiveness. In summary, in ovalbumin-induced mouse models, clarithromycin efficiently inhibited two important pathological characteristics of asthma, airway hyperresponsiveness and inflammation. These data suggest that the efficacy of clarithromycin, as well as of other macrolide antibiotics, in asthmatic patients could be attributed to their anti-inflammatory/immunomodulatory properties, and not only to their antimicrobial activity or exogenous corticosteroid-sparing effects.

The serum amyloid A response to sterile silver nitrate in mice and its inhibition by dexamethasone and macrolide antibiotics.

Serum amyloid A protein (SAA) is an acute phase protein, known to be a sensitive indicator of inflammation. We have characterized the time course of the SAA response and inflammatory reaction to silver nitrate injection s.c. in mice and studied the effects of dexamethasone and macrolide antibiotics. 2% Sterile silver nitrate solution was injected s.c. into female BALB/c mice and blood collected by capillary action from the tail vein of each mouse at different time points. Hematological variables were determined, albumin by spectrophotometry and SAA and cytokines by ELISA. Animals were treated with either a single i.p. dose of dexamethasone (5-30 mg/kg) 1 h after or daily oral doses of macrolide antibiotics for 3 days. SAA concentrations after silver nitrate injection peaked at 24 h, preceded by increases in serum IL-1 beta and IL-6, associated with decreases in blood leukocytes and local tissue inflammation. Single dexamethasone treatment and daily dosing for 3 days with azithromycin, clarithromycin and roxithromycin (20-80 mg/kg p.o.), but not erythromycin (100-150 mg/kg p.o.), inhibited the increase in SAA but with varying time courses. SAA, measured continuously, is a useful marker of sterile inflammation in mice and is differentially inhibited by macrolide antibiotics.

Enhancement by PL 14736 of granulation and collagen organization in healing wounds and the potential role of egr-1 expression.

Apart from becaplermin (recombinant human platelet-derived growth factor homodimer of B chains, PDGF-BB), for the treatment of lower extremity diabetic ulcers, few agents are available for pharmacological stimulation of wound healing. We have compared the mechanism of action of the potential wound healing agent, PL 14736 (G E P P P G K P A D D A G L V), with that of PDGF-BB on granulation tissue formation following sponge implantation in the normoglycemic rat and in healing full-thickness excisional wounds in db/db genetically diabetic mice. Expression of the immediate response gene, early growth response gene-1 (egr-1) was studied in Caco-2 cells in vitro. While PDGF-BB and PL 14736 had similar selectivity for stimulation of granulation tissue in both sponge granuloma and in healing wounds in db/db mice, PL 14736 was more active in stimulating early collagen organization. It also stimulated expression of egr-1 and its repressor nerve growth factor 1-A binding protein-2 (nab2) in non-differentiated Caco-2 cells more rapidly than PDGF-BB. EGR-1 induces cytokine and growth factor generation and early extracellular matrix (collagen) formation, offering an explanation for the beneficial effects of PL 14736 on wound healing.

Modulation of neutrophil and inflammation markers in chronic obstructive pulmonary disease by short-term azithromycin treatment.

The anti-inflammatory potential of azithromycin in chronic obstructive pulmonary disease (COPD) patients was explored following a standard oral dosing regimen. Patients with moderate and severe COPD were treated with azithromycin (500 mg, n=16) or placebo (n=8) once daily for 3 days in a randomized, double blind design, to compare effects on inflammation markers with those seen in a previous study in healthy volunteers. A battery of tests was made on serum, blood neutrophils and sputum on days 1 (baseline), 3, 4, 11, 18 and 32. In comparison to placebo, azithromycin resulted in an early transient increase in serum nitrites plus nitrates (day 3), associated with a tendency towards an increase in the blood neutrophil oxidative burst to phorbol myristic acetate. Subsequently, prolonged decreases in blood leukocyte and platelet counts, serum acute phase protein (including C reactive protein) and soluble E-selectin and blood neutrophil lactoferrin concentrations and a transient decrease in serum interleukin-8 were observed. Blood neutrophil glutathione peroxidase activity showed a prolonged increase after azithromycin treatment. The biphasic facilitatory-then-inhibitory response to azithromycin seen in healthy volunteers is not so clearly detectable in COPD patients, only potential anti-inflammatory effects. Treatment for longer periods may give therapeutic anti-inflammatory benefit in these patients.

Thrombopoietin and interleukin-6 in children with pneumonia-associated thrombocytosis.

BACKGROUND: The aim of this study was to investigate why some, but not all, children develop thrombocytosis during the course of pneumonia. METHODS: The retrospective study included 40 healthy children and 75 children with pneumonia: 17 patients with platelet count within the reference values, i.e., platelet count 450 x 10(9)/L. Erythrocyte sedimentation rate, leukocyte and platelet counts, and concentrations of hemoglobin, C-reactive protein, interleukin-6 and thrombopoietin were determined in the blood of patients and control groups of children. RESULTS: Patients with thrombocytosis were slightly younger (3.0 +/- 1.8 years and median 2.5 years, respectively) than patients with normal platelet count (3.8 +/- 2.4 years and median 4 years, respectively). Additionally, according to clinical and radiological findings, pneumonia in children with thrombocytosis had a more severe and protracted course. Serum thrombopoietin concentrations were found to be 91.2 +/- 41.7 ng/L (range: 14.3-166.7 ng/L) in patients with normal platelet count (313 +/- 70 x 10(9)/L, range: 206-428 x 10(9)/L). In patients with thrombocytosis (581 +/- 131 x 10(9)/L, range: 450-830 x 10(9)/L) serum thrombopoietin ranged from 63.6 to 1115.9 ng/L (526.6 +/- 268.4 ng/L). In these patients both concentration of hemoglobin (114 +/- 12 g/L) and iron (4.3 +/- 1.3 micromol/L) significantly decreased as compared with the control group. CONCLUSIONS: Study results suggested the possible development of reactive thrombocytosis in children with pneumonia. As platelets are involved in inflammatory reaction, reactive thrombocytosis might be part of the mechanism of defense. Reactive thrombocytosis may develop as a sequel of either anemia or inflammatory reaction (or both).

Azithromycin modulates neutrophil function and circulating inflammatory mediators in healthy human subjects.

Effects on human neutrophils and circulating inflammatory mediators were studied in 12 volunteers who received azithromycin (500 mg/day, p.o.) for 3 days. Blood was taken 1 h before treatment, 2.5, 24 h and 28 days after the last dose. An initial neutrophil degranulating effect of azithromycin was reflected in rapid decreases in azurophilic granule enzyme activities in cells and corresponding increases in serum. The oxidative response to a particulate stimulus was also acutely enhanced. These actions were associated with high plasma and neutrophil drug concentrations. A continuous fall in chemokine and interleukin-6 serum concentrations, within the non-pathological range, accompanied a delayed down-regulation of the oxidative burst and an increase in apoptosis of neutrophils up to 28 days after the last azithromycin dose. Neutrophils isolated from blood at this time point still contained detectable drug concentrations. Acute neutrophil stimulation could facilitate antibacterial effects of azithromycin, while delayed, potentially anti-inflammatory activity may curtail deleterious inflammation.