Cost-effectiveness analysis of the first-line EGFR-TKIs in patients with advanced EGFR-mutated non-small-cell lung cancer.

AIM: To evaluate the cost-effectiveness of first-line treatments, such as erlotinib, gefitinib, afatinib, dacomitinib, and osimertinib, for patients diagnosed with stage IIIB/IV NSCLC harboring EGFR mutations. MATERIALS & METHODS: A partitioned survival model was developed to estimate quality-adjusted life-year (QALY) and incremental cost-effectiveness ratio (ICER) from the perspective of the Spanish National Health System. Two Bayesian NMAs were performed independently, by using the polynomial fraction method to fit Kaplan-Meier curves for overall survival and progression-free survival. Deterministic and probabilistic sensitivity analyses were performed to evaluate the uncertainty. RESULTS: The ICER was calculated for the four first-line treatments by comparing them with gefitinib, and the ratios obtained were as follows: €166,416/QALY for osimertinib, €183,682/QALY for dacomitinib, €167,554/QALY for afatinib, €36,196/QALY for erlotinib. It was seen that patients who received osimertinib presented higher QALYs (0.49), followed by dacomitinib (0.33), afatinib (0.32), erlotinib (0.31), and gefitinib (0.28). CONCLUSIONS: Gefitinib is the most cost-effective treatment. In terms of QALYs gained, Osimertinib was more effective than all other TKIs. Nevertheless, with a Spanish threshold of €24,000/QALY, the reduction in the acquisition cost of osimertinib will have to be greater than 70%, to obtain a cost-effectiveness alternative.

Phosphodiesterase-4 inhibition improves corticosteroid insensitivity in pulmonary endothelial cells under oxidative stress.

BACKGROUND: Several clinical studies have shown that smoking in asthmatics and chronic obstructive pulmonary disease patients is closely associated with corticosteroid refractoriness. In this work, we have analyzed glucocorticoid insensitivity in human pulmonary artery endothelial cells (HPAECs) under cigarette smoke extract (CSE) exposure as well as the possible additive effects of the combination therapy with a phosphodiesterase (PDE)-4 inhibitor. METHODS: Interleukin (IL)-8 was measured in cell supernatants by ELISA. Histone deacetylase (HDAC), histone acetylase (HAT), and intracellular cAMP levels were measured by colorimetric assays and enzyme immunoassay, respectively. PDE4 isotypes and glucocorticoid receptor (GR)-α and ß expression were measured by real-time RT-PCR. RESULTS: The PDE4 inhibitor rolipram dose dependently inhibited the IL-8 secretion induced by CSE 5%. In contrast, dexamethasone 1 µM did not show inhibitory effect on IL-8 secretion. Combination of subeffective rolipram concentrations at 10 nM increased the inhibitory effect of dexamethasone to ~45% of inhibition. Cigarette smoke extract 5% inhibited HDAC activity and increased HAT activity generating glucocorticoid insensitivity. Rolipram did not modify the HDAC activity, however partially inhibited the increase in HAT activity at 1 µM. PDE4 isotypes were up-regulated by CSE 5% with the consequent cAMP down-regulation. Dexamethasone reduced all PDE4 isotypes expression and showed additive effects with rolipram enhancing cAMP levels. Furthermore, rolipram enhanced GR-α expression and inhibited the increase in GR-ß induced by CSE. CONCLUSIONS: Combination of rolipram and dexamethasone shows additive properties in HPAECs under glucocorticoid insensitive conditions. These results may be of potential value in future anti-inflammatory therapies using combination of PDE4 inhibitors and glucocorticoids.

Sildenafil citrate improves perinatal outcome in fetuses from pre-eclamptic rats.

OBJECTIVE: To evaluate perinatal outcome after sildenafil citrate (SC) administration at the onset of pregnancy in a rat pre-eclampsia model. DESIGN: In vivo animal experimental study. SETTING: Fundación IVI-Instituto Universitario IVI, Valencia, Spain. SAMPLE: Control and pre-eclampsia-induced pregnant Wistar rats exposed to chronic SC administration. METHODS: We evaluated the use of SC, which was tested as a potential therapeutic tool to maintain vasodilatation in complicated pregnancies. We have demonstrated previously that SC shows a hypotensive selective effect in normal rat pregnancies when compared with nonpregnant animals. MAIN OUTCOME MEASURES: Maternal blood pressure, weight and mortality during pre- and postnatal development, maternal blood cellularity and haemodynamic changes with maternal and fetal Doppler quantitative indices. RESULTS: SC restores normal values of blood pressure, cell count and proteinuria for maternal syndrome. In offspring, SC improves weight gain and increases survival rates without fetotoxic effects. According to the haemodynamic results, SC has a significant effect on the resistance index in the uterine artery in pre-eclamptic animals, as it restores normal values to correlate with an increase in fetal perfusion through the ductus venosus. CONCLUSIONS: These results suggest that SC administration during pregnancy may have a potential benefit for the treatment of hypertension during pregnancy by reversing the maternal effects of pre-eclampsia and by improving uteroplacental and fetal perfusion.

Roflumilast N-oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro.

BACKGROUND AND PURPOSE: Mucociliary malfunction occurs in chronic obstructive pulmonary disease (COPD) and compromised functions of ciliated bronchial epithelial cells may contribute to this. Cigarette smoke, a major risk factor for COPD, impairs ciliary beat frequency (CBF). cAMP augments CBF. This in vitro study addressed, in differentiated, primary human bronchial epithelial cells, whether roflumilast N-oxide, a PDE4 inhibitor, (i) augments CBF; (ii) prevents the reduction in CBF induced by cigarette smoke extract (CSE); and (iii) protects against the loss of the ciliated phenotype following long-term CSE exposure. EXPERIMENTAL APPROACH: Air-liquid interface cultured human bronchial epithelial cells were incubated with roflumilast N-oxide and exposed to CSE. CBF was assessed by digital high speed video microscopy (DHSV). Ciliated cells were characterized by ß-tubulin IV staining and analyses of Foxj1 and Dnai2 mRNA and protein (real-time quantitative PCR, Western blotting). KEY RESULTS: Roflumilast N-oxide concentration-dependently triggered a rapid and persistent increase in CBF and reversed the decrease in CBF following CSE. Long-term incubation of bronchial epithelial cells with CSE resulted in a loss in ciliated cells associated with reduced expression of the ciliated cell markers Foxj1 and Dnai2. The PDE4 inhibitor prevented this loss in the ciliated cell phenotype and the compromised Foxj1 and Dnai2 expression. The enhanced release of IL-13 following CSE, a cytokine that diminishes the proportion of ciliated cells and in parallel, reduces Foxj1 and Dnai2, was reversed by roflumilast N-oxide. CONCLUSION AND IMPLICATIONS: Roflumilast N-oxide protected differentiated human bronchial epithelial cells from reduced CBF and loss of ciliated cells following CSE.

Bosentan inhibits cigarette smoke-induced endothelin receptor expression in pulmonary arteries.

The endothelin (ET) system contributes to lung vascular tension and remodelling in smokers and chronic obstructive pulmonary disease (COPD) patients. This study examined the effect of cigarette smoke (CS) on ET receptor A (ET(A)) and B (ET(B)) expression in human pulmonary artery smooth muscle cells (HPASMCs) and human small intrapulmonary arteries, as well as their functional consequences. CS extract (CSE) increased ET(A) and ET(B) expression in HPASMCs and small intrapulmonary arteries, which was attenuated by bosentan, the ET(A) antagonist BQ123 and the ET(B) antagonist BQ788, and by blocking ET-1 with a monoclonal antibody against ET-1, suggesting a feed-forward mechanism mediated by ET-1 release. ET receptor (ETR) antagonism attenuated the CSE-induced HPASMC proliferation. Furthermore, CSE exposure increased the acute ET-1-induced small intrapulmonary artery contraction, which was attenuated by bosentan, BQ123 and BQ788. Pulmonary arteries from smokers and COPD patients showed a higher expression of ET(A) and ET(B) than those of nonsmoker patients. These results show a novel mechanism by which ETR blockade attenuates CS-induced ETR overexpression and, subsequently, small intrapulmonary artery tension. These data may be of potential value to explain therapeutic effects of bosentan in some forms of disproportionate pulmonary hypertension in COPD patients.

Oxidative stress-induced glucocorticoid resistance is prevented by dual PDE3/PDE4 inhibition in human alveolar macrophages.

BACKGROUND: Oxidative stress is present in airway diseases such as severe asthma or Chronic Obstructive Pulmonary Disease and contributes to the low response to glucocorticoids through the down-regulation of histone deacetylase (HDAC) activity. OBJECTIVE: To study the effects of the phosphodiesterase (PDE)-3 and 4 inhibitors and their combination vs. glucocorticoids in a model of lipopolysaccharide (LPS)-induced cytokine release in alveolar macrophages under oxidative stress conditions. METHODS: Differentiated U937 or human alveolar macrophages were stimulated with H(2) O(2) (10-1000 µM) or cigarette smoke extract (CSE, 0-15%) for 4 h before LPS (0.5 µg/mL, 24 h) addition. In other experiments, cells were pre-treated with dexamethasone or budesonide (10(-9) -10(-6) M), with the PDE4 inhibitor rolipram (10(-9) -10(-5) M), PDE3 inhibitor motapizone (10 µM), 3',5'-cyclic monophosphate enhancer PGE(2) (10 nM), or with the combination of rolipram (10(-6) M)+PGE(2) (10 nM)+motapizone (10 µM) 15 min before oxidants. IL-8 and TNF-α were measured by ELISA and HDAC activity by a colorimetric assay. RESULTS: Budesonide and dexamethasone produced a concentration-dependent inhibition of the LPS-induced IL-8 and TNF-α secretion with an E(max) about 90% of inhibition, which was reduced by approximately 30% in the presence of H(2)O(2) or CSE. Pre-treatment with rolipram, motapizone or PGE2 only reached about 20% of inhibition but was not affected by oxidative stress. In contrast, PDE4/PDE3 combination in presence of PGE2 effectively inhibited the LPS-induced cytokine secretion by about 90% and was not affected by oxidative stress. Combined PDE4 and PDE3 inhibition reversed glucocorticoid resistance under oxidative stress conditions. HDAC activity was reduced in the presence of oxidative stress, and in contrast to glucocorticoids, pre-treatment with PDE4/PDE3 combination was able to prevent HDAC inactivity. CONCLUSIONS & CLINICAL RELEVANCE: This study shows that the combination of the PDE3/PDE4 inhibitors prevents alveolar macrophage activation in those situations of glucocorticoid resistance, which may be of potential interest to develop new effective anti-inflammatory drugs in airway diseases.

Haemodynamic effects of long-term administration of sildenafil in normotensive pregnant and non-pregnant rats.

OBJECTIVE: To determine the effects of chronic administration of sildenafil citrate on healthy pregnant rats. DESIGN: In vivo animal experimental study. SETTING: Fundación IVI-Instituto Universitario IVI, Valencia, Spain. SAMPLE: Pregnant and non-pregnant Wistar rats exposed to chronic administration of sildenafil. METHODS: Placental cross-barrier and feto-maternal relationship levels, maternal blood pressure, and haemodymamic effects on uterine arteries were evaluated. The effect of growth on weight and fetal tissues, and on perinatal outcome, was investigated. MAIN OUTCOME MEASURES: Maternal blood pressure, blood viscosity, vascular indices of uterine arteries and fetal ductus venosus, plasmatic levels of sildenafil, embryo/fetal and litter weights, perinatal/postnatal survival rates. RESULTS: Sildenafil citrate crossed the placenta. The maternal and fetal levels of sildenafil, and its metabolite desmethyl-sildenafil, demonstrated a positive linear correlation in treated pregnant animals versus controls; a selective maternal hypotensive effect without changes in uterine vascular resistance was noted on days E8 and E11 (embryonic day). The lower pulsatility index of the ductus venosus on day E18 suggests fetal overflow at the end of the pregnancy. Effects on offspring were placental and liver enlargement, and increased fetal weight gain in the second half of pregnancy (irrespective of liver enlargement) and at birth. Perinatal and postnatal survival rates in the sildenafil group remained unaltered. No haemodynamic effects were evident in non-pregnant animals. CONCLUSIONS: In normotensive rats, sildenafil appears to have a selective effect at the onset of pregnancy, implying increased fetal blood supply, and increased fetal weight, and placental and liver enlargement, but no increased perinatal mortality.

Aclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in human airways.

Mucus hypersecretion and mucin MUC5AC overexpression are pathological features of chronic obstructive pulmonary disease (COPD). This study examines the inhibitory effect of aclidinium, a new long-acting muscarinic antagonist, on MUC5AC expression in human airway epithelial cells. MUC5AC mRNA (RT-PCR) and protein expression (ELISA and immunohistochemistry) were studied in human bronchial tissue and differentiated human airway epithelial cells activated with carbachol (100 µM) or cigarette smoke extract in the absence or presence of aclidinium. Carbachol increased MUC5AC mRNA and protein expression in human bronchus and cultured epithelial cells. Aclidinium inhibited the carbachol-induced MUC5AC mRNA and protein expression with potency (half maximal inhibitory concentration) ~1 nM in human bronchus and cultured airway epithelial cells. AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, inhibited carbachol-induced MUC5AC responses, indicating EGFR transactivation. Aclidinium inhibited carbachol-induced phospho-EGFR and phospho-p44/42 MAPK expression. In cultured airway epithelial cells transfected with small interfering (si)RNA against muscarinic receptor subtypes, siRNA-M3 but not siRNA-M2 blocked carbachol-induced MUC5AC expression. Cigarette smoke-induced MUC5AC upregulation in cultured airway epithelial cells was suppressed by aclidinium. In conclusion, aclidinium decreases carbachol and tobacco smoke-induced MUC5AC overexpression in human airway epithelial cells. This effect may contribute to the clinical efficacy of aclidinium in mucus hypersecretory diseases including COPD.

Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences.

BACKGROUND AND PURPOSE: Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ET(B) ) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction. EXPERIMENTAL APPROACH: ET(B) receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca(2+) , F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A(2) and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phalloidin fluorescence, colorimetric assay, Western blot, elisa and DCFDA fluorescence respectively. KEY RESULTS: Cigarette smoke extract dose-dependently increased ET(B) receptor expression in HPAECs after 24h incubation. CSE-induced ET(B) expression was attenuated by bosentan, the ET(B) receptor antagonist BQ788, the Rho kinase antagonist Y27632 and the antioxidant N-acetylcysteine. A monoclonal antibody to ET-1 prevented CSE-induced ET(B) receptor overexpression. Twenty-four hour exposure to ET-1 dose-dependently increased ET(B) receptor expression, mimicking the effect of CSE. CSE-induced ET(B) receptor overexpression caused greater cell contraction; increased intracellular Ca(2+) ; increased F/G-actin and RhoA activity; increased myosin light chain phosphorylation; augmented TxA(2) and ROS production; and decreased NO after acute ET-1 (10nM). These effects were attenuated by bosentan, BQ788, Y27632 and N-acetylcysteine. CONCLUSIONS AND IMPLICATION: Cigarette smoke extract induced ET(B) receptor overexpression by a feed forward mechanism mediated partly by ET release, promoting HPAEC dysfunction and attenuated by ET(B) receptor blockade, Rho kinase and ROS inhibition. These results provide support for the use of bosentan in CS-related endothelial dysfunction.

Taurine chloramine inhibits functional responses of human eosinophils in vitro.

BACKGROUND: Eosinophils are prominent effectors of allergic inflammation. Taurine-chloramine (TauCl), a derivative of the amino acid taurine, shows antioxidant properties in different cell systems but its effects on eosinophils have not been reported. OBJECTIVE: To study the effects of TauCl and taurine on functional responses of isolated human eosinophils activated by different stimuli. METHODS: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of TauCl and taurine (0.1-1 mM) were investigated on the generation of superoxide anion (ferricytochrome-c reduction microassay), calcium signal (fluorimetry), p47phox-p67phox translocation (Western blot), leukotriene C4 (LTC4) production (enzymeimmunoassay), eosinophil peroxidase (EPO) release (spectrophotometry), eosinophil cationic protein (ECP) release (radioimmunoassay), apoptosis (flow cytometry with annexin V-propidium iodide), and nuclear factor-kappaB (NF-kappaB) activation (Western blot). RESULTS: TauCl inhibited superoxide anion generation triggered by N-formyl-Met-Leu-Phe (fMLP; 30 nM), phorbol myristate acetate (1 nM) and serum opsonized zymosan (0.5 mg/mL) with similar potency (IC50 approximately 200 microM) for the three stimuli, while taurine (0.1-1 mM) was scarcely effective. TauCl but not taurine inhibited p47phox-p67phox translocation. TauCl (200 microM) and taurine (1 mM) did not modify the [Ca2+]i responses to fMLP. TauCl inhibited the release of EPO (IC50 approximately 200 microM) and reduced ECP and LTC4 production from fMLP-activated eosinophils while taurine was without significant effects. TauCl (1 mM) did not change constitutive apoptosis but significantly attenuated the ability of granulocyte-monocyte colony-stimulating factor (GM-CSF) and IL-5 to prevent apoptosis. The activation of eosinophil NF-kappaB induced by GM-CSF and IL-5 was suppressed by TauCl. CONCLUSION: Taurine is without significant in vitro effects on human eosinophil functions but its derivative TauCl inhibits oxidative burst and generation of inflammatory mediators, and reverses the survival effect produced by inflammatory cytokines. Therefore, endogenous TauCl may help to suppress excessive inflammatory response in eosinophils at inflammatory sites.

Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury.

BACKGROUND AND PURPOSE: The effects of a phosphodiesterase 4 (PDE4) inhibitor, roflumilast, on bleomycin-induced lung injury were explored in 'preventive' and 'therapeutic' protocols and compared with glucocorticoids. EXPERIMENTAL APPROACH: Roflumilast (1 and 5 mg.kg(-1).d(-1), p.o.) or dexamethasone (2.5 mg.kg(-1).d(-1), p.o.) was given to C57Bl/6J mice from day 1 to 14 (preventive) or day 7 to 21 (therapeutic) after intratracheal bleomycin (3.75 U.kg(-1)). In Wistar rats, roflumilast (1 mg.kg(-1).d(-1), p.o.) was compared with methylprednisolone (10 mg.kg(-1).d(-1), p.o.) from day 1 to 21 (preventive) or from day 10 to 21 (therapeutic), following intratracheal instillation of bleomycin (7.5 U.kg(-1)). Analyses were performed at the end of the treatment periods. KEY RESULTS: Preventive. Roflumilast reduced bleomycin-induced lung hydroxyproline, lung fibrosis and right ventricular hypertrophy; muscularization of intraacinar pulmonary vessels was also attenuated. The PDE4 inhibitor diminished bleomycin-induced transcripts for tumour necrosis factor (TNFalpha), transforming growth factor (TGFbeta), connective tissue growth factor, alphaI(I)collagen, endothelin-1 and the mucin, Muc5ac, in lung, and reduced bronchoalveolar lavage fluid levels of TNFalpha, interleukin-13, TGFbeta, Muc5ac, lipid hydroperoxides and inflammatory cell counts. Therapeutic. In mice, roflumilast but not dexamethasone reduced bleomycin-induced lung alphaI(I)collagen transcripts, fibrosis and right ventricular hypertrophy. Similar results were found in the rat. CONCLUSIONS AND IMPLICATIONS: Roflumilast prevented the development of bleomycin-induced lung injury, and alleviated the lung fibrotic and vascular remodeling response to bleomycin in a therapeutic protocol, the latter being resistant to glucocorticoids.

[The in vitro effect of the addition of ion exchange resins on the bioavailability of electrolytes in artificial enteral feeding formulas]. / Efecto in vitro de la adición de resinas de intercambio iónico sobre la biodisponibilidad de electrolitos en fórmulas de nutrición enteral artificial.

OBJECTIVE: To determine in vitro free ion concentration in three standard artificial enteral feeding formulas following the addition of ion exchange resins. METHOD: Three standard types of AEF were chosen: Osmolite HN, Nutrison Standard and Isosource Standard. The ion exchange resins used were: Sodium Polystyrene Sulfonate and Calcium Polystyrene Sulfonate. 100 ml of AEF were mixed in a beaker with 1.5 g or 3 g of ion exchange resins for 48 hours at 37 masculineC. Subsequently, the samples were precipitated and the supernatant obtained was used for determining the concentrations of calcium, magnesium, sodium and potassium ions. RESULTS: The addition of Sodium Polystyrene Sulfonate to different types of enteral feeding formulas reduced the concentrations of potassium, calcium and magnesium ions by 70%. 78.2%, and 77.6% in the case of Osmolite HN; by 72.3%, 69.2% and 63.5% in the case of Nutrison Standard, and by 78.3%, 80.5% and 74.5% in the case of Isosource Standard. In contrast, the addition of Calcium Polystyrene Sulfonate reduced the concentration of potassium and magnesium by 50.5% and 55.5% in the case of Osmolite HN; by 49.8% and 43% in the case of Nutrison Standard and by 42.6% and 37.7% in the case of Isosource Standard. CONCLUSIONS: The addition of ion exchange resins to different types of enteral feeding formulas, allows the in vitro free ion content of these to be reduced.

Efecto in vitro de la adición de resinas de intercambio iónico sobre la biodisponibilidad de electrolitos en fórmulas de nutrición enteral artificial / The in vitro effect of the addition of ion exchange resins on the bioavailability of electrolytes in artificial enteral feeding formulas

Objetivo: Conocer la concentración iónica libre in vitro en tres fórmulasde nutrición enteral artificial estándar, tras la adición de resinasde intercambio iónico.Método: Se seleccionaron tres tipos de NEA estándar: OsmoliteHN®, Nutrison Standard® e Issosource Standard®. Las resinas de intercambioiónico empleadas fueron: poliestireno sulfonato sódico ypoliestireno sulfonato cálcico. En un vaso de precipitados se mezclaron100 ml de la NEA con 1,5 g o 3 g de las resinas de intercambioiónico durante 48 h a 37 ºC. Posteriormente se precipitaron lasmuestras y el sobrenadante obtenido se utilizó para determinar lasconcentraciones de los iones calcio, magnesio, sodio y potasio.Resultados: La adición de poliestireno sulfonato sódico a las diferentesnutriciones enterales redujo las concentraciones de los ionespotasio, calcio y magnesio en un 70,9, 78,2, y 77,6% en el caso deOsmolite HN®, en un 72,3, 69,2 y 63,5% en el caso de NutrisonStandard®, y en un 78,3, 80,5 y 74,5% en el caso de IssosourceStandard®. Por el contrario la adición de poliestireno sulfonato cálcicoredujo las concentraciones de potasio y magnesio en un 50,5y un 55,5% en el caso de Osmolite HN®, un 49,8 y un 43% en elcaso de Nutrison Standard® y en un 42,6 y un 37,7% en el caso deIssosource Standard®.Conclusiones: La adición de resinas de intercambio iónico a distintasnutriciones enterales permite reducir el contenido iónico libre invitro de éstas

Objective: To determine in vitro free ion concentration in three standardartificial enteral feeding formulas following the addition of ionexchange resins.Method: Three standard types of AEF were chosen: Osmolite HN®,Nutrison Standard® and Isosource Standard®. The ion exchange resinsused were: Sodium Polystyrene Sulfonate and Calcium PolystyreneSulfonate. 100 ml of AEF were mixed in a beaker with 1.5 g or3 g of ion exchange resins for 48 hours at 37ºC. Subsequently, thesamples were precipitated and the supernatant obtained was usedfor determining the concentrations of calcium, magnesium, sodiumand potassium ions.Results: The addition of Sodium Polystyrene Sulfonate to differenttypes of enteral feeding formulas reduced the concentrations of potassium,calcium and magnesium ions by 70%. 78.2%, and 77.6% inthe case of Osmolite HN®; by 72.3%, 69.2% and 63.5% in the case ofNutrison Standard®, and by 78.3%, 80.5% and 74.5% in the case ofIsosource Standard®. In contrast, the addition of Calcium PolystyreneSulfonate reduced the concentration of potassium and magnesiumby 50.5% and 55.5% in the case of Osmolite HN®; by 49.8% and43% in the case of Nutrison Standard® and by 42.6% and 37.7% inthe case of Isosource Standard®.Conclusions: The addition of ion exchange resins to different types ofenteral feeding formulas, allows the in vitro free ion content of theseto be reduced

Roflumilast inhibits leukocyte-endothelial cell interactions, expression of adhesion molecules and microvascular permeability.

BACKGROUND AND PURPOSE: The present study addressed the effects of the investigational PDE4 inhibitor roflumilast on leukocyte-endothelial cell interactions and endothelial permeability in vivo and in vitro. EXPERIMENTAL APPROACH: In vivo, intravital video-microscopy was used to determine effects of roflumilast p.o. on leukocyte-endothelial cell interactions and microvascular permeability in rat mesenteric venules. In vitro, the effects of roflumilast N-oxide, the active metabolite of roflumilast in humans, and other PDE4 inhibitors on neutrophil adhesion to tumour necrosis factor alpha (TNFalpha)-activated human umbilical vein endothelial cells (HUVEC), E-selectin expression and thrombin-induced endothelial permeability was evaluated. Flow cytometry was used to determine the effect of roflumilast on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced CD11b upregulation on human neutrophils. KEY RESULTS: In vivo, roflumilast, given 1 h before lipopolysaccharide (LPS), dose-dependently reduced leukocyte-endothelial cell interactions in rat mesenteric postcapillary venules. It also diminished histamine-induced microvascular permeability. Immunohistochemical analyses revealed that roflumilast prevented LPS-induced endothelial P- and E-selectin expression. In vitro, roflumilast N-oxide concentration-dependently suppressed neutrophil adhesion to TNFalpha-activated HUVEC and CD11b expression on fMLP-stimulated neutrophils. It also reduced TNFalpha-induced E-selectin expression on HUVEC, when PDE3 activity was blocked. HUVEC permeability elicited by thrombin was concentration-dependently suppressed by roflumilast N-oxide. While roflumilast N-oxide was as potent as roflumilast at inhibiting stimulated endothelial cell and neutrophil functions, both compounds were significantly more potent than the structurally unrelated PDE4 inhibitors, rolipram or cilomilast. CONCLUSIONS AND IMPLICATIONS: These findings further support earlier observations on the inhibition of inflammatory cell influx and protein extravasation by roflumilast in vivo.

Modulatory effects of N-acetyl-L-cysteine on human eosinophil apoptosis.

Eosinophils are oxidant-sensitive cells considered relevant in allergic inflammation. The present study aimed to examine the effects of the antioxidant N-acetyl-L-cysteine (NAC) on constitutive and cytokine-delayed apoptosis in human isolated eosinophils. Human eosinophils were purified from the blood of healthy donors by a magnetic separation system. Apoptosis and cellular glutathione were assessed by cytofluorometric analysis and nuclear factor (NF)-kappaB binding activity assessed by electrophoresis mobility shift assay. The rate of spontaneous apoptosis of human eosinophils after 24 h culture, as assessed by annexin-V-positive staining, was mean+/-sem 48.2+/-1.4%, n = 5. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng.mL(-1)) decreased apoptosis to 19.4+/-1.8%, n = 5. NAC (5 mM) inhibited spontaneous apoptosis (33.6+/-2.7%, n = 5) but augmented apoptosis in the presence of GM-CSF (30.9+/-1.5%, n = 5). NAC (5 mM) also increased the rate of apoptosis in the presence of tumour necrosis factor (TNF)-alpha (10 ng.mL(-1)) and interleukin-5 (5 ng.mL(-1)). NAC (5 mM) increased eosinophil glutathione content. The increase in eosinophil NF-kappaB binding activity induced by GM-CSF and TNF-alpha was suppressed by NAC. In conclusion, N-acetylcysteine modulates eosinophil apoptosis by inhibiting constitutive apoptosis but reversing the survival effect produced by inflammatory cytokines in human eosinophils.

Inhibitory effects of N-acetylcysteine on the functional responses of human eosinophils in vitro.

BACKGROUND: Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. OBJECTIVE: The aim of this work was to study the effects of the clinically used antioxidant N-acetyl-L-cysteine (NAC) on the functional responses of human-isolated eosinophils. METHODS: Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca(2+) signal (fluorimetry), intracellular glutathione (GSH; flow cytometry), p47(phox)-p67(phox) translocation (Western blot) and eosinophil cationic protein (ECP) release (radioimmunoassay). RESULTS: NAC (0.1-1 mm) inhibited the extracellular generation of oxygen species induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and eotaxin (in the presence of IL-5) with -logIC(50) values of 3.61+/-0.03 and 3.36+/-0.09, respectively. Also, the intracellular generation of hydrogen peroxide was virtually abolished by NAC (0.5-1 mm). NAC (1 mm) did not alter the fMLP-induced Ca(2+) signal but augmented the eosinophil content of reduced GSH and inhibited p47(phox)-p67(phox) translocation. NAC inhibited the release of ECP ( approximately 90% inhibition at 1 mm) from fMLP-activated eosinophils. CONCLUSION: Inhibition by NAC of human eosinophil functions in vitro is potentially useful in the treatment of allergic inflammation.

Relaxation of the isolated human internal anal sphincter by sildenafil.

BACKGROUND: Hypertonicity of the internal anal sphincter (IAS) appears to be involved in the pathogenesis of anal fissure. The relaxant effects of sildenafil, a selective phosphodiesterase 5 (PDE5) inhibitor, on isolated human IAS were investigated. METHODS: The efficacy (maximal effect, E(max)) and potency (-log IC(50), where IC(50) is half-maximal inhibitory concentration) of the PDE5 inhibitors, sildenafil and zaprinast, and of nitric oxide donors, sodium nitroprusside and glyceryl trinitrate, as relaxants of histamine (0.1 mmol/l)-induced tone were examined in IAS strips under isometric contraction. The presence of PDE5 isoenzymes and changes in intracellular calcium and cyclic nucleotide levels in IAS muscle were tested by real-time reverse transcriptase-polymerase chain reaction, epifluorescence microscopy and enzyme immunoassay respectively. RESULTS: Sildenafil produced a concentration-related inhibition of the mean(s.e.m.) histamine-induced tone (E(max) 83(2) per cent, - log IC(50) 7.04(0.05); n = 12). Zaprinast produced relaxation to similar degree, but with lower potency. Nitric oxide donors also relaxed IAS. Sildenafil (1 micromol/l) produced a 1.8-fold increase in guanosine 3',5'-cyclic monophosphate content, with no change in adenosine 3',5'-cyclic monophosphate levels. Sildenafil markedly depressed the peak intracellular calcium increase evoked by histamine. PDE5A1, PDE5A2 and PDE5A3 transcripts were expressed in IAS muscle. CONCLUSION: Sildenafil relaxes the augmented tone of human IAS in vitro. These results support the potential use of this PDE5 inhibitor in the treatment of chronic anal fissure.

In vitro tracheal hyperresponsiveness to muscarinic receptor stimulation by carbachol in a rat model of bleomycin-induced pulmonary fibrosis.

1 Bleomycin-induced lung injury is widely used as an experimental model to investigate the pathophysiology of pulmonary fibrosis but the alterations in the pharmacological responsiveness of airways isolated from bleomycin-exposed animals has been scarcely investigated. The aim of this study was to examine the in vitro tracheal responses to muscarinic receptor stimulation with carbachol in a rat bleomycin model. 2 Concentration-response curves to carbachol (10 nm to 0.1 mm) were obtained in tracheal rings isolated from Sprague-Dawley rats 14 days after endotracheal bleomycin or saline. The intracellular calcium signal in response to carbachol (10 microm) was measured by epifluorescence microscopy using fura-2 in primary cultures of tracheal smooth muscle cells from bleomycin- and saline-exposed rats. Circulating plasma tumour necrosis factor (TNF)-alpha/interleukin (IL)-1beta levels were measured by enzyme-linked immunosorbent assay. 3 Maximal contraction in response to carbachol was significantly greater in tracheal rings from bleomycin-exposed rats compared with controls (15.8 +/- 1.3 mN vs. 11.8 +/- 1.4 mN; n = 19, P < 0.05). 4 Carbachol (10 microm) elicited a transient increase of intracellular calcium with greater increment in tracheal smooth muscle cells from bleomycin-exposed rats compared with controls (372 +/- 42 nmvs. 176 +/- 20 nm; n = 7, P < 0.01). 5 Circulating plasma levels of TNF-alpha/IL-1beta were augmented in bleomycin-exposed rats compared with controls. Tissue incubation with TNF-alpha (100 ng ml(-1))/IL-1beta (10 ng ml(-1)) increased in vitro tracheal responsiveness to carbachol. 6 In conclusion, tracheal contraction in response to muscarinic receptor stimulation with carbachol was increased in bleomycin-exposed rats. This in vitro cholinergic hyperresponsiveness may be related to the augmented levels of inflammatory cytokines in bleomycin-exposed rats.

Directly observed treatment for tuberculosis in pharmacies compared with self-administered therapy in Spain.

OBJECTIVES: To compare directly observed treatment (DOT) of tuberculosis through pharmacy offices with self-administered treatment (SAT) in patients at risk for non-adherence. METHODS: Prospective study for DOT (1999-2002) and retrospective study for SAT (1996-1998) in patients at risk for non-adherence (human immunodeficiency virus [HIV] infection, alcoholism, illicit drug use, immigrant or homeless status and/or previous failure to complete). Patients in the DOT programme received medication as out-patients twice a week in pharmacies that supervised adherence and provided socio-sanitary support to patients. RESULTS: There were 101 and 112 patients in the DOT and SAT groups, respectively. Demographic and clinical characteristics were similar in both groups. Differences were observed in risk factors for non-adherence (more immigrants and fewer intravenous drug users in the DOT vs. the SAT groups; P < 0.05). In the DOT group, 76 patients (75.2%) completed treatment and were cured compared to only 30 patients (26.7%) in the SAT group (P < 0.001). Implementation of DOT increased the cost of treatment by 400 Euro per patient compared to SAT. CONCLUSION: In patients at risk for non-adherence, DOT implemented through pharmacy offices was better than SAT; however, completion rates were still low.