Biological Activity of Newly Synthesized Benzimidazole and Benzothizole 2,5-Disubstituted Furane Derivatives.

Newly designed and synthesized cyano, amidino and acrylonitrile 2,5-disubstituted furane derivatives with either benzimidazole/benzothiazole nuclei have been evaluated for antitumor and antimicrobial activity. For potential antitumor activity, the compounds were tested in 2D and 3D cell culture methods on three human lung cancer cell lines, A549, HCC827 and NCI-H358, with MTS cytotoxicity and BrdU proliferation assays in vitro. Compounds 5, 6, 8, 9 and 15 have been proven to be compounds with potential antitumor activity with high potential to stop the proliferation of cells. In general, benzothiazole derivatives were more active in comparison to benzimidazole derivatives. Antimicrobial activity was evaluated with Broth microdilution testing (according to CLSI (Clinical Laboratory Standards Institute) guidelines) on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Additionally, Saccharomyces cerevisiae was included in testing as a eukaryotic model organism. Compounds 5, 6, 8, 9 and 15 showed the most promising antibacterial activity. In general, the compounds showed antitumor activity, higher in 2D assays in comparison with 3D assays, on all three cell lines in both assays. In natural conditions, compounds with such an activity profile (less toxic but still effective against tumor growth) could be promising new antitumor drugs. Some of the tested compounds showed antimicrobial activity. In contrast to ctDNA, the presence of nitro group or chlorine in selected furane-benzothiazole structures did not influence the binding mode with AT-DNA. All compounds dominantly bound inside the minor groove of AT-DNA either in form of monomers or dimer and higher-order aggregates.

Macrolide Inspired Macrocycles as Modulators of the IL-17A/IL-17RA Interaction.

Interleukin 17 (IL-17) cytokines promote inflammatory pathophysiology in many autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Such broad involvement of IL-17 in various autoimmune diseases makes it an ideal target for drug discovery. Psoriasis is a chronic inflammatory disease characterized by numerous defective components of the immune system. Significantly higher levels of IL-17A have been noticed in lesions of psoriatic patients, if compared to non-lesion parts. Therefore, this paper is focused on the macrolide inspired macrocycles as potential IL-17A/IL-17RA modulators and covers the molecular design, synthesis, and in vitro profiling. Macrocycles are designed to diversify and enrich chemical space through different ring sizes and a variety of three-dimensional shapes. Inhibitors in the nM range were identified in both target-based and phenotypic assays. In vitro ADME as well as in vivo PK properties are reported.

Preclinical in vitro screening of newly synthesised amidino substituted benzimidazoles and benzothiazoles.

Newly synthesised benzimidazole/benzotiazole derivatives bearing amidino, namely 3,4,5,6-tetrahydropyrimidin-1-ium chloride, substituents have been evaluated for their potential antitumor activity in vitro. Compounds and standard drugs (doxorubicin, staurosporine and vandetanib) were tested on three human lung cancer cell lines A549, HCC827 and NCI-H358. We tested compounds in MTS citotoxicity assay and in BrdU proliferative assay performed on 2 D and 3 D assay format. Because benzmidazole scaffold is similar to natural purines, we tested the most active compounds for ability to induce cell apoptosis of A549 by binding to DNA in comparison with doxorubicin and saturosporine. Additionally, the ADME properties of the most active benzothiazole/benzimidazole and non-active compounds were determined to see if the different ADME properties are the cause of different activity in 2 D and 3 D assays, as well as to see if the tested active compounds have drug like properties and potency for further profilation. ADME characterisation included solubility, lipophilicity, permeability, metabolic stability and binding to plasma proteins. In general, the benzothiazole derivatives were more active in comparison to their benzimidazole analogues. The exception was 2-phenyl substituted benzimidazole 6a being active with very pronounced activity especially towards HCC827 cells. All active compounds have similar mode of action on A549 cell line as standard compound doxorubicin, which binds to nucleic acids with the DNA double helix. Tested active benzothiazole compounds were characterised by moderate to good solubility, good metabolic stability, low permeability and high binding to plasma proteins. One tested active benzimidazole derivative showed ADME properties, but lower lipophilicity resulted in low PPB and higher metabolic instability. In addition, no significant difference was observed in ADME profile between active and non-active compounds.

Antiproliferative activity of amino substituted benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles explored by 2D and 3D cell culture system.

Benzimidazo[1,2-a]quinolines and benzo[b]thieno[2,3-b]pyrido[1,2-a]benzimidazoles with amino chains on the different positions have been evaluated by 2D and 3D assays on the human breast cancer cells. Pentacyclic derivatives were synthesized by microwave assisted amination to study the influence of the thiophene substructure on antitumor activity in comparison to tetracyclic analogues. The results obtained from 2D assay reveals that the antitumor activity is strongly dependent on the nature and position of amino chains. Tetracyclic derivatives displayed selective activity on SK-BR-3 with the 2-amino substituted derivatives as the most active ones while pentacyclic derivatives 6-16 and 21-25 showed more pronounced activity on T-47D. The evaluation of antitumor activity in the 3D assay pointed out that some of the tetracyclic and pentacyclic amino substituted derivatives showed selective activity on the MDA-MB-231 cell line. Influence of physico-chemical properties of the compounds on antiproliferative activity have been investigated by multivariate statistical methods. As a measure of lipophilicity, experimental Chrom LogD values have been determined and number of structural parameters have been calculated for investigated compounds. Main factors contributing to the antiproliferative effect for both 2D and 3D cell cultures are found to be basicity, lipophilicity, molecular weight and number of H-bond donors.

Antitumor activity of amidino-substituted benzimidazole and benzimidazo[1,2-a]quinoline derivatives tested in 2D and 3D cell culture systems.

Due to a poor clinical predictive power of 2D cell cultures, standard tool for in vitro assays in drug discovery process, there is increasing interest in developing 3D in vitro cell cultures, biologically relevant assay feasible for the development of robust preclinical anti-cancer drug screening platforms. Herein, we tested amidino-substituted benzimidazoles and benzimidazo[1,2-a]quinolines as a small platform for comparison of antitumor activity in 2D and 3D cell culture systems and correlation with structure-activity relationship. 3D cell culture method was applied on a human cancer breast (SK-BR-3, MDA-MB-231, T-47D) and pancreatic cancer cells (MIA PaCa-2, PANC-1). Results obtained in 2D and 3D models were highly comparable, but in some cases we have observed significant disagreement indicating that some prominent compounds can be discarded in early phase of researching because of compounds with false positive result. To confirm which of cell culture systems is more accurate, in vivo profiling is needed.

Relative potencies of three glucocorticoids to induce hypoplasia of the physis and concomitant biochemical alterations in the rat.

Although inhaled glucocorticoids are known to have systemic effects on bone metabolism, there is little comparative information on their relative potencies. The effects of three standard glucocorticoids in causing changes in bone metabolism and growth, therefore, were investigated in relation to other systemic effects in the rat. Given to male Sprague-Dawley rats, 4.5-5.5 weeks old, subcutaneously (s.c.), at doses of 0.3-10 mg/kg daily for 7 days, beclomethasone dipropionate, prednisolone and ciclesonide all dose-dependently inhibited thymus body mass index (BMI) (by 57%, 44% and 76% at 3 mg/kg). Ciclesonide, potently and prednisolone, less effectively, also repressed femoral bone growth (by 41% and 18% at 10 mg/kg), significantly reducing body weight gain (both by 100% at 10 mg/kg), and serum concentrations of acid phosphatase (ACP) and tartarate resistant acid phosphatase (TRACP) (by >30% at 10 mg/kg); both increased serum glucose and triglycerides levels. Serum alkaline phosphatase (ALP) was not affected. Beclomethasone dipropionate had little or no effect on these additional variables. In conclusion, ciclesonide showed pronounced bone growth inhibiting activity after s.c. administration to the rat while other two glucocorticoids showed differences in activity on bone metabolism. However, this model is sufficiently sensitive and specific for testing the effect of glucocorticoids on bone metabolism.

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.

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.

Synthesis and biological validation of novel pyrazole derivatives with anticancer activity guided by 3D-QSAR analysis.

Using literature data on anticancer activity of pyrazole derivatives, 3D-QSAR models were developed and 3D-QSAR analysis was performed. The 3D-QSAR analysis enabled identification of molecular properties that have the highest impact on antitumor activity against lung cancer cells. The results of 3D-QSAR analysis were taken into account while new compounds were designed. Obtained 3D-QSAR models were used for prediction of activity of new compounds. In this way, design of new compounds was guided by 3D-QSAR analysis which was performed on literature data. Ten new pyrazole derivatives were synthesised and their antitumor activities against A549 and NCIH23 lung cancer cells were validated. In order to obtain full profile of anticancer activity, cells viability (MTS) assays were combined with cell proliferation (BrdU) assays which measure actively dividing cells in treated sample. Experimental measurements showed good agreement between predicted and measured activities for majority of compounds. Also, anticancer activities of new pyrazole derivatives pointed to the chemical groups that can be useful in designing antitumor molecules. Substitution of hydrazine linker with rigid, 1,2,4-oxadiazole moiety resulted in compound 10, which has low (if any) cytotoxic activity and high potential cytostatic activity. Therefore, compound 10 presents a good starting point for design of new, more potent and safer anticancer therapeutics.

Synthesis of macrolones with central piperazine ring in the linker and its influence on antibacterial activity.

Three macrolides, clarithromycin, azithromycin and 11-O-Me-azithromycin have been selected for the construction of a series of new macrolone derivatives. Quinolone-linker intermediates are prepared by Sonogashira-type C(6)-alkynylation of 6-iodoquinolone precursors. The final macrolones, differing by macrolide moiety and substituents at the position N-1 of the quinolone or by the presence of an ethyl ester or free acid on the quinolone unit attached via a linker. The linker comprises of a central piperazine ring bonded to the 4″-O position of cladinose by 3-carbon ester or ether functionality. Modifications of the linker did not improve antibacterial properties compared to the previously reported macrolone compounds. Linker flexibility seems to play an important role for potency against macrolide resistant respiratory pathogens.

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.

Discovery of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides active against efflux-mediated resistant Streptococcus pneumoniae.

A series of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides, were discovered, structurally characterized and biologically evaluated. They have shown good antibacterial activity against selected Gram-positive and Gram-negative bacterial strains. These include N″ substituted 9a-(N'-carbamoyl-γ-aminopropyl)- (6a,c), 9a-(N'-thiocarbamoyl-γ-aminopropyl)- (7a,e), 9a-[N'-(ß-cyanoethyl)-N'-(carbamoyl-γ-aminopropyl)]- (9a-c, 9g) 9a-[N'-(ß-cyanoethyl)-N'-(thiocarbamoyl-γ-aminopropyl)]-derivatives (10d-f) of 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3). Among the synthesized compounds thiourea 7a and urea 9b have shown substantially improved activity comparable to azithromycin (1) and significantly better activity than the 3-decladinosyl-azithromycin (2) and the parent 3-cladinosyl analogues against efflux-mediated resistant S. pneumoniae.

Intranasal challenge with increasing ovalbumin doses differently affects airway hyperresponsiveness and inflammatory cell accumulation in mouse model of asthma.

OBJECTIVE: To investigate whether challenge with increasing allergen doses could differently affect allergen-induced airway hyperresponsiveness (AHR) and inflammatory cell accumulation in mouse model of asthma, providing an experimental model to investigate their relationship. MATERIAL AND METHODS: AHR and accumulation of inflammatory cells in bronchoalveolar lavage fluid (BALF) and into the lungs were compared in ovalbumin-sensitized mice that were challenged intranasally with 2.5, 10, 25 or 100 microg of ovalbumin/mouse. RESULTS: Both AHR and inflammatory cell accumulation were proportional to the ovalbumin dose used for challenge. However, in group challenged with 10 microg of ovalbumin airway inflammation was present, although allergen-induced AHR was not detected. Additional analysis indicated that neither mucous hyperproduction nor eosinophil degranulation could be correlated to presence of AHR in this model, whereas concentration of interleukin (IL)-13 in BALF was increased only in those groups in which AHR was present. CONCLUSIONS: Altogether, intranasal challenge of mice with increasing allergen doses could serve as a suitable experimental system for investigation of mechanisms by which airway inflammation leads to allergen-induced AHR. Our initial findings are in line with previous reports that dissociate AHR from amount of eosinophil accumulation and imply the role of IL-13 in this process.

Differential evaluation of excisional non-occluded wound healing in db/db mice.

The full-thickness wound in the genetically diabetic (db/db) mouse is a commonly used model of impaired wound healing. We investigated delayed healing of non-occluded, excisional, full-thickness, dermal wounds in db/db mice in comparison to their normal littermate controls and refined methods for monitoring skin wound re-epithelialization, contraction, granulation tissue formation, and inflammation. We have confirmed with a computer-assisted planimetry method the results of previous studies showing that healing of non-occluded full excision wounds in db/db mice does not occur by contraction as much as in healthy mice. In addition, we have developed separate histological methods for the assessment of re-epithelialization, contraction, granulation tissue (mature, immature, fibrosis), and inflammation (lipogranulomas, secondary, nonspecific). Using a new approach to histological assessment, we have shown that wound closure in db/db mice is delayed owing to: (1) delayed granulation tissue maturation; (2) ''laced,'' widely distributed granulation tissue around fat lobules; and (3) obstruction by lipogranulomas, whereas the rate of re-epithelialization seems to be the same as in C57Bl/6 mice. This methodology should permit a more precise differentiation of effects of novel therapeutic agents on the wound healing process in db/db mice.

Influence of Helicobacter pylori infection persistence on bcl-2 expression in gastric mucosa inflammatory cells.

Chronic Helicobacter (H.) pylori infection is an etiological factor related to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The expression of bcl-2 protein significantly decreases as the grade of MALT lymphoma advances. The aim of this study was to evaluate bcl-2 expression in inflammatory cells in lamina propria in gastric biopsy samples collected from two groups of patients with chronic gastritis divided on the basis of the success or failure of H. pylori eradication. Sixty-five patients with chronic gastritis were divided into two groups of 45 and 20 patients according to their therapeutic response. The gastric mucosa samples were analyzed histologically in both groups of patients before and after standard therapy (for eradicated, after one therapeutic cycle; and for non-eradicated, after three therapeutic cycles) for H. pylori density, urease activity and bcl-2 expression. In the eradicated group of patients, H. pylori eradication was accompanied by significantly lower grades of bacterial colonization and lower urease activity in the corpus and antrum. Bcl-2 expression in inflammatory cells showed no statistically significant changes in either patient group at either location. There was no between-group difference in bcl-2 expression either. In conclusion, persistent long-lasting H. pylori infection is associated with higher grades of bacterial colonization and higher urease activity but not with bcl-2 expression in inflammatory cells.

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.

Azithromycin-sulfonamide conjugates as inhibitors of resistant Streptococcus pyogenes strains.

Novel hybrid compounds 6a-6d, conjugates of 15-membered azalides and sulfonamides, i.e. unsubstituted, 4-aryl- and 4-heteroaryl-aminosulfonyl derivatives of 9a-[N'-(phenylcarbamoyl)]-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A were synthesized and characterized by IR, one- and two-dimensional NMR spectroscopies and MALDI-TOF and MS/MS mass spectrometry. The new compounds were evaluated in vitro against a panel of sensitive and resistant Gram-positive and Gram-negative bacterial strains. 9a-{N'-[4-(Aminosulfonyl)phenyl]carbamoyl}--(6a) and 9a-{N'-[4-(phenylaminosulfonyl)phenyl]carbamoyl}--(6b) derivatives showed improvements in activity against inducible resistant Streptococcus pyogenes in comparison with macrolide antibiotic azithromycin and starting material 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2). In addition, the synthesized azithromycin-sulfonamide conjugates 6a-6d showed good antibacterial activity against sensitive S. pyogenes and Streptococcus pneumoniae strains. The kinetics of degradation in the artificial gastric juice showed that the most active compounds, 6a and 6b, exhibited azithromycin like stability. The cleavage of the cladinose sugar was found to be the main decomposition pathway leading to inactive 7a and 7b, prepared also as analytical standards by the alternative synthetic route together with 7c and 7d.

Influence of H. pylori infection and eradication on p53, c-erbB-2 and Ki-67 in gastric mucosa.

BACKGROUND/AIMS: To evaluate the expression of antigens c-erbB-2, p53, and Ki-67 in gastric biopsy, bacteria density and urease activity in two groups of patients with chronic gastritis separated on the basis of the success or failure of H. pylori eradication. METHODOLOGY: Sixty-five patients with chronic gastritis were split in two groups (n=45/20) related to response to the therapy. The gastric mucosa samples (Sydney system) were analyzed histologically in both groups of patients before and after standard therapy (for eradicated, E group after one cycle; for non-eradicated, NE group after three cycles) for H. pylori infection, urease activity, c-erbB-2, p53 and Ki-67 expression. Sera samples taken before and after treatment were also analyzed for specific antibody against H. pylori. RESULTS: The eradication of H. pylori in patients of the E group was accompanied with significant lower colonization grades of bacteria, urease activity, proliferating rate, p53, and Ki-67 expression while c-erbB-2 expression was unchanged. In the NE group, all parameters were the same before and after therapy with exception of p53, which was slightly higher on both locations. Strong expression of c-erbB-2 in corpus of the NE group was determinate. Serum activity of specific antibodies against H. pylori was lower after the therapy in both groups of patients, but in the eradicated group this change was much stronger that in the non-eradicated. CONCLUSIONS: Long persisting infection is related with higher colonization grades of bacteria, urease activity, p53, c-erbB-2 and Ki-67 expression. Changes of those markers are connected with duration of infection and location where these changes were obtained.

Synthesis, characterization and in vitro antimicrobial activity of novel sulfonylureas of 15-membered azalides.

Three series of the novel sulfonylurea derivatives of 15-membered azalides, i.e. 9a-N-[N'-(aryl)sulfonylcarbamoyl] (4a-4f, 5a-5f), 9a-N-{N'-[(aryl)sulfonylcarbamoyl-gamma-aminopropyl]} (10a-10f, 11a, 11c) and 9a-N-{N'-(beta-cyanoethyl)-N'-[(aryl)sulfonylcarabamoyl-gamma-aminopropyl]} (14a-14f, 15a, 15b, 15f) derivatives of 9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin A (2) and 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3) were prepared and their structures elucidated by NMR and IR spectroscopic methods and mass spectrometry. Minimal inhibitory concentration (MIC) of these compounds was determined on a panel of sensitive and resistant Gram-positive and Gram-negative bacterial strains. Several compounds of the series of 9a-N-[N'-(aryl)sulfonylcarbamoyl] derivatives that showed significant improvements in activity against inducible resistant Streptococcus pyogenes strain were suggested for further optimization.

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.