Vesna Munić

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.

Cellular Uptake and Efflux of Azithromycin, Erythromycin, Clarithromycin, Telithromycin, and Cethromycin

ABSTRACT Macrolide antibiotics have an outstanding ability to concentrate within host cells, particularly phagocytes. In the study described in this paper five different macrolide antibiotics were compared regarding the uptake and release kinetics in human peripheral blood polymorphonuclear neutrophils (PMNs) and three different cell lines, two phagocytic cell lines (RAW 264.7 and THP-1) and an epithelial cell line (MDCK). Based on the results obtained, the substances tested could be clustered into different groups. Azithromycin constituted the first group, characterized by rapid and nonsaturable uptake into phagocytic cells and a high degree of retention in the preloaded cells. The second group included erythromycin and clarithromycin. These two substances do not exhibit cell specificity; consequently, they are taken up to a similar extent and are released by all cell types studied. Ketolides constituted the last group. Their uptake was saturable in cells of monocytic lineage as well as in nondifferentiated cells of myeloid lineage, and they were rapidly released from all the cell lines studied. However, in PMNs, ketolide uptake was not saturable; and unlike telithromycin, cethromycin rapidly egressed from the loaded cells.

Cloning and molecular characterization of apical efflux transporters (ABCB1, ABCB11 and ABCC2) in rainbow trout (Oncorhynchus mykiss) hepatocytes

Fish possess similar mechanisms of billiary excretion of xeno(endo)biotics and their metabolites as found in higher vertebrates and various types of ABC efflux proteins expressed in apical membranes of polarized cells appears to be key mediators of this vectorial transport. To test this hypothesis the main goals of this study were identification and cloning of genes coding for different types of ABC transport proteins, determination of the gene transcript (mRNA) levels, and characterization of the related protein transport activities in primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes. We have cloned one partial and two full gene sequences, which show high degree of identity with mammalian Pgp1 (ABCB1), BSEP (ABCB11) and MRP2 (ABCC2) efflux transporters. Using real-time RT-PCR expression levels of the mRNA of these genes were determined. Identical relative expression patterns of identified efflux transporters (BSEP>>MRP2>Pgp1) were observed for both liver and primary hepatocytes, with expression of all three transporter mRNAs approximately 3-4-fold lower in primary hepatocytes in comparison to intact liver. In addition, the presence of Pgp1-, BSEP- and MRP-like transport activities were indicated using putative specific fluorescent substrates (rhodamine 123, calcein-AM, bodipy-verapamil and dihydrofluorescein diacetat), model inhibitors (verapamil, cyclosporine A, MK571, reversine 205, taurocholate and taurochenodeoxycholate) and their combinations. Taken together the results of this study showed that primary trout hepatocytes express critical components of detoxification pathways-phase I and II enzymes, as well as the ABC proteins involved in transport of xenobiotics, affirming this in vitro model as a promising tool in (eco)toxicological research.

Azithromycin distinctively modulates classical activation of human monocytes in vitro

BACKGROUND AND PURPOSE Azithromycin has been reported to modify activation of macrophages towards the M2 phenotype. Here, we have sought to identify the mechanisms underlying this modulatory effect of azithromycin on human monocytes, classically activated in vitro.

Differences in assessment of macrolide interaction with human MDR1 (ABCB1, P-gp) using rhodamine-123 efflux, ATPase activity and cellular accumulation assays

In this study five macrolide antibiotics (azithromycin, erythromycin, clarithromycin, roxithromycin and telithromycin) were compared based on their ability to interact with human MDR1 (ABCB1, P-glycoprotein), studied from two main aspects: by determining the influence of macrolide antibiotics on MDR1 function, as well as the influence of MDR1 on macrolide accumulation in MES-SA/Dx5 cells overexpressing human MDR1. At higher micromolar concentrations five tested macrolides were shown to inhibit MDR1 function in terms of rhodamine-123 efflux and verapamil-activated ATPase function, whereas at lower concentrations they activated MDR1 ATPase. They were confirmed to be substrates of MDR1 and to compete with each other, as well as with verapamil for transport via this transporter. Expression of MDR1 on cells decreased macrolide accumulation in cells from 2- to 80-fold with the most pronounced change observed for azithromycin and erythromycin. Moreover, presence of active MDR1 highly affected the relative ranking of tested macrolides according to their accumulation in cells. In conclusion, out of seven applied methods and assessed parameters, four of them gave similar rough evaluation on the strength of interaction of five macrolides with MDR1, with clarithromycin, roxithromycin and telithromycin showing stronger interaction than azithromycin and erythromycin.

Intensity of macrolide anti-inflammatory activity in J774A.1 cells positively correlates with cellular accumulation and phospholipidosis

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 coefficients (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.

Impairment of lysosomal functions by azithromycin and chloroquine contributes to anti-inflammatory phenotype.

Azithromycin and chloroquine have been shown to exhibit anti-inflammatory activities in a number of cellular systems, but the mechanisms of these activities have still not been clarified unequivocally. Since both drugs are cationic, accumulate in acidic cellular compartments and bind to phospholipids with a consequent increase in lysosomal pH and induce phospholipidosis, we examined the relevance of these common properties to their anti-inflammatory activities. We compared also these effects with effects of concanamycin A, compound which inhibits acidification of lysosomes. All three compounds increased lysosomal pH, accumulation of autophagic vacuoles and ubiquitinated proteins and impaired recycling of TLR4 receptor with consequences in downstream signaling in LPS-stimulated J774A.1 cells. Azithromycin and chloroquine additionally inhibited arachidonic acid release and prostaglandin E2 synthesis. Therefore, impairment of lysosomal functions by azithromycin and chloroquine deregulate TLR4 recycling and signaling and phospholipases activation and lead to anti-inflammatory phenotype in LPS-stimulated J774A.1 cells.

Cloning and mRNA expression analysis of an ABCG2 (BCRP) efflux transporter in rainbow trout (Oncorhynchus mykiss) liver and primary hepatocytes.

As has been recently demonstrated in mammals, apart from the P-glycoprotein (Pgp, ABCB) and the MRP-like proteins (MRPs, ABCC), another efflux transporter - the BCRP (ABCG2) - expressed in polarized epithelial cells of different tissues, is involved in regulation of intestinal absorption and biliary excretion of potentially toxic xenobiotics. However, no study has been directed towards identification of BCRP (ABCG2) in fish species. In the present study we have cloned the full ABCG2 transcript from rainbow trout (Oncorhynchus mykiss) liver, showing a high sequence identity (60%) to human ABCG2 gene. Using Real-Time PCR we measured relative expression of ABCG2 in trout liver and primary hepatocytes and compared these expression levels to the expression of other ABC transporters expressed in apical membrane of hepatocytes (ABCB1, ABCB11 and ABCC2). ABCG2 expression was 6-fold higher than ABCC2 and almost 42 fold higher than ABCB1, indicating that the ABCG2 probably plays a significant role in the overall disposition and excretion of xenobiotics in fish.

A comparison of in vitro ADME properties and pharmacokinetics of azithromycin and selected 15-membered ring macrolides in rodents

The purpose of this study was to evaluate the impact of structural modifications on the 15-membered macrolactone ring and/or substituents on the in vitro ADME properties and in vivo pharmacokinetic (PK) profile for selected derivatives in rodents in comparison to azithromycin. Azithromycin and seven selected 15-membered macrolide derivatives, modified either by removal of the sugar moieties, replacement of the amine with a lactam, or addition of lipophilic substituents, were screened in several in vitro ADME assays and in vivo PK studies in rodents. In vitro ADME profiling included assessment of passive permeability and P-gp substrate, metabolic stability in liver microsomes and hepatocytes, as well as CYP direct inhibition measurements. In vivo PK studies were performed in rats (Sprague–Dawley), mice (Balb/c), and P-gp wild-type and deficient mice (CF-1™). Different structural modifications on the azithromycin scaffold resulted in substantial changes in disposition kinetics and oral bioavailability in both rodent species. However, these differences in vivo cannot be predicted based on in vitro results since most of these molecules are classified in the same category. Therefore, in the case of 15-membered ring macrolides, the in vitro ADME screens presented here seem to have low predictive value for in vivo prediction, making their use as routine in vitro screens prior to PK assessments questionable.

Macrolactonolides: a novel class of anti- inflammatory compounds

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17β-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described.