Pharmacological Research 64 (2011) 298–307 Contents lists available at ScienceDirect Pharmacological Research journa l h o me pa ge: www.elsevier.com/locate/yphr s Intensity of macrolide anti-inflammatory activity in J774A.1 cells positively correlates with cellular accumulation and phospholipidosis Vesna Muni ´ c ∗ , Mihailo Banjanac 1 , Sanja Koˇ strun 1 , Krunoslav Nuji ´ c 1 , Martina Bosnar 1 , Nikola Marjanovi ´ c 1 , Jovica Rali ´ c 1 , Mario Matijaˇ si´ c 2 , Mario Hlevnjak 3 , Vesna Erakovi ´ c Haber 1 GlaxoSmithKline Research Center Zagreb Ltd., Prilaz baruna Filipovi´ ca 29, HR-10000 Zagreb, Croatia a r t i c l e i n f o Article history: Received 10 December 2010 Received in revised form 25 March 2011 Accepted 27 March 2011 Keywords: Macrolide J774A.1 Inflammation Cellular accumulation Phospholipidosis Lipid binding Macrophage Uptake a b s t r a c t Some macrolide antibiotics were reported to inhibit interleukin-6 (IL6) and prostaglandin-E2 (PGE 2 ) production by bacterial lipopolysaccharide (LPS) stimulated J774A.1 cells. Macrolides are also known to accumulate in cells and some were proven inducers of phospholipidosis. In the present study, with a set of 18 mainly 14- and 15-membered macrolides, we have investigated whether these macrolide induced phenomena in J774A.1 cells are connected. In LPS-stimulated J774A.1 cells, the extent of inhibition of proinflammatory markers (IL6 and PGE 2 ) by macrolides significantly correlated with their extent of accumulation in cells, as well as with the induction of phospholipidosis, and cytotoxic effects in prolonged culture (with correlation coef- ficients (R) ranging from 0.78 to 0.93). The effects observed were related to macrolide binding to phospholipids (CHI IAM), number of positively charged centres, and were inversely proportional to the number of hydrogen bond donors. Similar interdependence of effects was obtained with chloroquine and amiodarone, whereas for dexamethasone and indomethacin these effects were not linked. The observed macrolide induced phenomena in J774A.1 cells were reversible and elimination of the macrolides from the culture media prevented phospholipidosis and the development of cytotoxicity in long-term cultures. Based on comparison with known clinical data, we conclude that LPS-stimulated J774A.1 cells in presented experimental setup are not a representative cellular model for the evaluation of macrolide anti-inflammatory potential in clinical trials. Nevertheless, our study shows that, at least in in vitro models, binding to biological membranes may be the crucial factor of macrolide mechanism of action. © 2011 Published by Elsevier Ltd. Abbreviations: AGP, alpha-acid glycoprotein; AK, adenylate kinase; AMP, arti- ficial membrane permeability; CHI, chromatographic hydrophobicity index; cpd, compound; DMSO, dimethyl-sulphoxide; HSA, human serum albumin; IAM, immo- bilized artificial membrane; IL6, interleukin-6; LPS, lipopolysaccharide; NBD-PE, nitrobenzoxadiazolyl labelled phosphatidylethanolamine; ns, nonstimulated cells; PCA, principal component analysis; PGE2, prostaglandin E2; PK, pharmacokinetics; SD, standard deviation; TNF, tumour necrosis factor-alpha. ∗ Corresponding author at: Galapagos istraˇ zivaˇ cki centar d.o.o., Prilaz baruna Fil- ipovi ´ ca 29, HR-10000 Zagreb, Croatia. Tel.: +385 1 8886323; fax: +385 1 8886443. E-mail address: vesna.munic@glpg.com (V. Muni ´ c). 1 Present address: Galapagos istraˇ zivaˇ cki centar d.o.o., Prilaz baruna Filipovi ´ ca 29, HR-10000 Zagreb, Croatia. 2 Present address: School of Medicine, Center for Translational and Clinical Research, Department for Intercellular Communication, ˇ Salata 2, HR-10000 Zagreb, Croatia. 3 Present address: Laboratory of Computational Biophysics, Department of Struc- tural and Computational Biology, Max F. Perutz Laboratories, GmbH Campus Vienna Biocenter 5, A-1030 Vienna, Austria. 1. Introduction Macrolide antibiotics with a 14- and 15-membered lactone ring are widely used in the clinic. Isolated in 1952 from the actino- mycete Saccharopolyspora erythraea, erythromycin A was the first member of the class and was succeeded by several semi-synthetic macrolides of which azithromycin has been the most successful commercially [1]. In addition to their therapeutic efficacy as antimicrobials, stan- dard macrolide antibiotics have been reported to exert significant anti-inflammatory/immunomodulatory activity [2,3]. So far, the most pronounced clinical effects have been observed in chronic pulmonary inflammatory diseases with dominant neutrophilic infiltration, in which macrolides inhibit both accumulation of neu- trophils and proinflammatory cytokine release in inflamed tissues. Nowadays, macrolides are used as a standard treatment of dif- fuse panbronchiolitis and cystic fibrosis, and their potential uses in chronic obstructive pulmonary disease, chronic sinusitis, asthma, 1043-6618/$ – see front matter © 2011 Published by Elsevier Ltd. doi:10.1016/j.phrs.2011.03.011