Development of fiber optic in vitro release testing method for dexamethasone release from the oil solutions.

For many parenteral drugs, there is still no standardized method for in vitro release (IVR) testing available. This article presents the development of a new IVR method for oil solutions using a dialysis membrane and USP II apparatus coupled to a fiber optic UV-Vis spectrometer. Experiments were performed using dexamethasone formulations containing castor oil as a solvent with the addition of cosolvents, 20 % (v/v) of isopropanol or Capryol® 90. Based on solubility testing results, castor oil was chosen as the best solvent amongst other vegetable oils, while a significant increase in solubility was obtained by adding either of the two cosolvents. Partitioning experiments were performed to ensure these formulations could achieve prolonged drug release. IVR testing was performed with model formulations and critical test parameters were varied in order to examine the method's sensitivity. The developed method was sensitive to temperature and stirring rate, while coupling the USP II apparatus with a fiber optic UV-Vis spectrometer enabled complete automation. Moreover, due to the interference of excipients on fiber optic detection of dexamethasone during the release testing, derivative spectroscopy was successfully introduced for the elimination of the interference. The developed IVR method described herein could be useful in preformulation investigations and the early development of novel formulations.

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.

Macrolide antibiotics broadly and distinctively inhibit cytokine and chemokine production by COPD sputum cells in vitro.

Macrolide antibiotics are known to exert anti-inflammatory actions in vivo, including certain effects in COPD patients. In order to investigate the immunomodulatory profile of activity of macrolide antibiotics, we have studied the effects of azithromycin, clarithromycin, erythromycin and roxithromycin on the in vitro production of a panel of inflammatory mediators from cells isolated from human, steroid-naïve, COPD sputum samples. Macrolide effects were compared to three other commonly used anti-inflammatory compounds, the corticosteroid dexamethasone, the PDE4 inhibitor, roflumilast and the p38 kinase inhibitor, SB203580. Three of the four tested macrolides, azithromycin, clarithromycin and roxithromycin, exhibited pronounced, concentration-related reduction of IL-1ß, IL-6, IL-10, TNF-α, CCL3, CCL5, CCL20, CCL22, CXCL1, CXCL5, and G-CSF release. Further slight inhibitory effects on IL-1α, CXCL8, GM-CSF, and PAI-1 production were also observed. Erythromycin was very weakly active. Qualitatively and quantitatively, macrolides exerted distinctive and, compared to other tested classes of compounds, more pronounced immunomodulatory effects, particularly in terms of chemokine (CCL3, CCL5, CCL20, CCL22, and CXCL5), IL-1ß, G-CSF and PAI-1 release. The described modulation of inflammatory mediators could potentially contribute to further definition of biomarkers of macrolide anti-inflammatory activity in COPD.

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.

Anti-inflammatory activity of azithromycin attenuates the effects of lipopolysaccharide administration in mice.

Macrolide antibacterials inhibit the production of various cytokines and the migration of inflammatory cells. These anti-inflammatory actions of macrolides may be beneficial in attenuating inflammatory processes involved in bacterial sepsis. Therefore, we investigated the ability of azithromycin to attenuate the deleterious effects of lipopolysaccharide (LPS), in three different LPS-induced inflammatory models. Our results show that azithromycin (10 and 100 mg/kg) significantly attenuated the intraperitoneal LPS-induced increase in plasma TNF-alpha concentration. It also increased survival rate in a septic shock model in mice challenged with intravenous LPS. Oral treatment with azithromycin (up to 300 mg/kg) was less effective in suppressing neutrophil infiltration into the lungs 24 h after intranasal LPS challenge, possibly because of a slower onset of action or inadequate dosing. In the same model, azithromycin given intraperitoneally significantly improved inflammatory markers (total cell number, neutrophil percentage and MIP-2 concentration) in bronchoalveolar lavage fluid. In conclusion, azithromycin exhibits significant anti-inflammatory properties but the potency of such effects varies depending on the experimental model and route of administration.

Determination of As(III) and As(V) in oilseeds by chronopotentiometric stripping analysis: development of a method.

Chronopotentiometric stripping analysis (CSA) was used for selective determination of As(III) and As(V) in different oilseeds. After the optimization of experimental parameters an appropriate procedure for sample pretreatment was developed. A detection limit of 2 microg/dm3 for As(III) was obtained with an electrolysis time of 600 s. This method was used for arsenic determination in sunflower, pumpkin, and flax seed, as well as for soy flakes and almond.

Atrazine in groundwater of Vojvodina Province.

The objective of this study was to investigate concentrations of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine), deethylatrazine (DEA) (2-amino-4-chloro-6-isopropylamino-1,3,5-triazine), deisopropylatrazine (DIA) (2-amino-4-chloro-6-ethylamino-1,3,5-triazine) and deethyldeisopropylatrazine (DEIA) (6-chloro-2,4-diamino-1,3,5-triazine) in groundwaters of Vojvodina Province. A study was conducted during April 2001. Some 110 samples of groundwater were taken from near surface aquifers. The water samples were first passed through a disk containing solid matrix coated with a chemically bonded C-18 organic phase. The disk was then eluted with supercritical carbon dioxide to remove compounds from the sorbent. Finally the extract was injected into capillary gas chromatograph. Average concentrations were 0.198 microg L(-1) for atrazine, 0.116 microg L(-1) for DEA, 0.043 microg L(-1) for DIA and 0.077 microg L(-1) for DEIA.

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.

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.