[Nebulized drugs: the evolution?]. / Nébulisations médicamenteuses : évolution ?

The revival of nebulization as a drug delivery route is real. The current delivery systems respond to the new European norms, the new mesh-vibrating nebulizers allow delivering drugs more quickly, other nebulizers, more performant because of less drug losses and of a better lung deposition of the drug, are in progress. Only 12 drugs are commercialized for nebulization. All are available in dispensaries, some requiring a first prescription by a physician working in a hospital (cystic fibrosis drugs), others requiring a prescription from only some specialists as paediatricians or pulmonologists (bronchodilators). Works are in progress concerning the diameter and shape of the drug particles (nanotechnology) and also concerning the use of nebulized drugs for a systemic effect (vaccines, insulin, cyclosporine, anticancerous agents, etc.).

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox.

NADPH oxidase is an O2*- -generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes.

Reactive oxygen species activate focal adhesion kinase, paxillin and p130cas tyrosine phosphorylation in endothelial cells.

Reactive oxygen species (ROS), particularly hydroxyl radical (HO*), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 microM) and herbimycin A (0.9 microM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120-130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO*. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 microM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO*, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.

Anethole dithiolethione regulates oxidant-induced tyrosine kinase activation in endothelial cells.

Interaction between neutrophils and endothelial cells is one of the first steps in the functional response of polymorphonuclear neutrophils (PMN), and is necessary for their migration toward damaged tissues. PMN activation, leading to their adhesion to and migration between endothelial cells, is part of a complex phenomenon that can be altered in pathological situations such as the ischemia-reperfusion syndrome, in which large numbers of PMN are recruited to the tissue and release reactive oxygen species (ROS) near the vessel wall. ROS have been implicated in the pathogenesis of various inflammatory diseases. The increased adhesion of PMN to ROS-stimulated endothelial cells involves an increase in tyrosine phosphorylation of a tyrosine kinase focal adhesion kinase (p125FAK) and several cytoskeleton proteins, including paxillin and p130 cas. We examined the role of glutathione (GSH) in the regulation of this adhesion phenomenon and in the increased tyrosine phosphorylation induced by ROS. For this purpose we used anethole dithiolthione (ADT), which increases the glutathione synthesis by activating gamma-glutamyl-cysteine synthetase. We found that ADT reduced both PMN adhesion to ROS-stimulated human umbilical vein endothelial cells (HUVEC) and tyrosine phosphorylation of p125FAK and paxillin. ADT increased redox status by increasing intracellular GSH content in oxidized cells. These results show that GSH can reverse the effect of oxidation on tyrosine kinase activation and phosphorylation, and thus plays an important role in cell signaling. They also confirm the antioxidant activity of ADT.

[Capsular retraction of shoulder: prospective study of imaging benefits in 20 patients]. / Capsulites rétractiles de l'épaule: étude prospective de l'apport de l'imagerie chez 20 patients.

PURPOSE: To evaluate the contribution of principal imaging techniques in diagnosis and treatment in adhesive capsulitis of the shoulder. MATERIALS AND METHODS: In 20 patients presenting adhesive capsulitis of shoulder since mean of 6,7 months, the following examinations were performed: radiographies, angioscintigraphy, MRI as well as an opaque arthrography and a bursography associated with corticosteroid injection. Patients were followed during one year. RESULTS: The opaque arthrography was to affirm the adhesive capsulitis for the inclusion of the patients. Radiographies (patchy demineralization) and scintigraphy (hyperfixation) were often pathological. In MRI, T1 fat-saturated sequences after contrast injection almost always showed enhancement of the articular capsula, the synovia, the miscellaneous bone or the sub-acromial bursa. The latter was often modified and retracted at bursography. In 19 of 20 cases, a functional improvement was observed after the opacifications. CONCLUSION: Therapeutic effect of both arthrography and bursography is almost proved. Post contrast MRI confirms presence of vascular troubles in all the shoulder structures even at this advanced stage.

Characterization of different vitamin E carriers intended for pulmonary drug delivery.

The targeted release of drugs intended for pulmonary delivery is a research field which has been so far rather unexploited but is currently becoming increasingly attractive. Liquid dispersions encapsulating vitamin E (liposomes, micelles, nano-emulsion, and solid lipid particles) were prepared using various methods based on membrane contactor. The dispersions were nebulized and aerodynamic characteristics of the generated aerosols were assessed using two different methods: laser light scattering and cascade impaction. When the laser diffraction technique was used, results showed that fine particle fractions (<5 µm) were 19, 29, 38 and 71% for solid lipid particles, micelles, nano-emulsion and liposomes, respectively. When the impaction method was applied, using a next generation pharmaceutical impactor operated at 30 l/min, results showed that fine particle fractions were 39, 78, 82 and 87% for solid lipid particles, micelles, nano-emulsion and liposomes, respectively. The differences observed between the results obtained from both methods confirm that the laser diffraction method is not always suitable for aerodynamic characterization of aerosols and should be validated against an impaction method. Nebulization of the drug-carrier systems led to an increase of their size most likely due to aggregation phenomena. The size was increased by a factor of 2-26 depending on the encapsulation system. The most important aggregation was obtained with nano-emulsion; the less one with solid lipid particles. The mass median aerodynamic diameter (MMAD) of the generated aerosols ranged from 1.76 to 6.10 µm. The application of a mathematical model, the Multiple-Path Particle Dosimetry (MPPD), for the prediction of the pulmonary deposit gave encouraging results. The rate of vitamin E able to reach the lung ranged from 37.6 (for the liposomes) to 51.6% (for the micelles). The obtained results showed that the different systems developed for vitamin E encapsulation were suitable to target the lung after pulmonary administration by nebulization.

[Inhalation therapy: inhaled generics, inhaled antidotes, the future of anti-infectives and the indications of inhaled pentamidine. GAT aerosolstorming, Paris 2012]. / Aérosolthérapie : les génériques par voie inhalée, les antidotes en aérosol, le futur des anti-infectieux et les indications de la pentamidine en aérosol. Aérosolstorming du GAT, Paris 2012.

The working group on aerosol therapy (GAT) of the Société de pneumologie de langue française (SPLF) organized its third "Aerosolstorming" in 2012. During the course of one day, different aspects of inhaled therapy were discussed, and these will be treated separately in two articles, this one being the first. Inhaled products represent a large volume of prescriptions both in the community and in hospital settings and they involve various specialties particularly ENT and respiratory care. Technical aspects of the development of these products, their mode of administration and compliance with their indications are key elements for the effective therapeutic use of inhaled treatments. In this first article, we will review issues concerning generic inhaled products, the existence of inhaled antidotes, new anti-infective agents and indications for inhaled pentamidine.

Focal adhesion kinase regulation by oxidative stress in different cell types.

Focal adhesion kinase (FAK) is a tyrosine kinase ubiquitously expressed in cells. It was initially shown to be the initiator of focal adhesion formation in adherent cells, after its binding to integrins which induce its autophosphorylation. However, it can be also activated by a great variety of other stimuli able to act on different intracellular signaling. Reactive oxygen species (ROS), which have been shown to act as external or internal cell stimuli, induce tyrosine phosphorylation of FAK. Its autophosphorylation is followed by a submembranous localization which is crucial for many of the biological roles of FAK, including cell spreading, cell migration, cell proliferation, and prevention of apoptosis. It plays an important role in development of tumor cells, its regulation could be thus a way of impairing cell proliferation in cancer. We describe in this review the structure, activity, and functions of FAK in different cells and how ROS are able, like other stimuli, to induce its phosphorylation and modification of cell morphology and structure. The link between ROS and FAK activation could explain the role of ROS in mediating cell proliferation, cell migration, or apoptosis.

Intracellular pool of vascular endothelial growth factor in human neutrophils.

Vascular endothelial growth factor (VEGF ), an endothelial cell mitogen, is a potent angiogenic factor produced by several cell types. Whether human neutrophils are potential producers of VEGF has not yet been described. The present work shows that phorbol-12-myristate 13-acetate (PMA), fMet-Leu-Phe, and tumor necrosis factor-alpha (TNF-alpha) triggered a time-dependent secretion of VEGF by human neutrophils. Cells incubated with 50 ng/mL of PMA released significant amounts of VEGF after 15 minutes. Because the extracellular content of VEGF in human neutrophils supernatants remained constant over a period of 2 to 24 hours and because PMA is a potent inducer of human neutrophil degranulation, the PMA-induced secretion of VEGF may be due to a pre-existing intracellular pool of this molecule. This hypothesis was reinforced by the absence of cycloheximide effect on the PMA-induced secretion of VEGF. The existence of an intracellular pool of VEGF was confirmed by measuring the intracellular content of VEGF in resting neutrophils. A dosedependent inhibition of PMA-induced VEGF secretion was observed when the cells were incubated in the presence of pentoxifylline, a methylxanthine known to inhibit neutrophil degranulation. To confirm the implication of neutrophil degranulation in VEGF release, the effects of two inducers of physiologic degranulation, fMet-Leu-Phe and TNF-alpha, were determined. Both agonists induced a release of VEGF in the absence of cytochalasin B, confirming the involvement of neutrophil degranulation and suggesting the intracellular localization of VEGF in the specific granule fraction. In addition, the kinetics of fMet-Leu-Phe- and TNF-alpha-induced secretion of lactoferrin were similar to those of VEGF release induced by these two both agonists. The subcellular fractionation of human neutrophils showed a granule-specific distribution of the intracellular pool of VEGF in resting neutrophils. The finding that human neutrophils contain an intracellular pool of VEGF, secreted in the extracellular space under PMA-, fMet-Leu-Phe-, and TNF-alpha-induced degranulation, suggests a role for human neutrophils as cellular effectors of physiologic as well as pathologic angiogenesis.

Leukemic pleural effusion in B-cell prolymphocytic leukemia.

The occurrence of leukemic pleural effusion is a rare complication in chronic lymphocytic leukemia and has not been reported in B-cell prolymphocytic leukemia (B-PLL). We report a case of pleural effusion revealing a B-PLL. The diagnosis was made on the cytological and immunological characteristics of cells in the blood and pleural effusion. This patient was treated with fludarabine and was in complete remission after three courses. This observation may have clinical implications for the use of new adenoside nucleotide analogues in symptomatic B-PLL.

Pharmacokinetic evaluation of indomethacin nanocapsules.

Indomethacin-loaded polyisobutylcyanoacrylate nanocapsules were prepared by interfacial polymerization of the alkylcyanoacrylate monomer. Mean particle size of the nanocapsules ranged from 200 to 300 nm. Comparison of the results following intravenous infusion of indomethacin solution and nanocapsules to rats revealed that nanoencapsulation accelerated the extravascular distribution of indomethacin due partly to enhanced uptake of the colloidal carrier by the liver reticuloendothelial system. Following intragastric administration, the oral bioavailability of indomethacin in solution was 90%--indicating complete absorption of this drug from the gastrointestinal tract of the rat. Absorption of indomethacin nanocapsules by this route was more rapid. This was attributed either to an increase in the intensity and/or the duration of contact of the encapsulated drug with the gut wall, or to a more efficient absorption process involving paracellular pathways.

Sulfasalazine induced lupus in rheumatoid arthritis.

We describe 2 cases of a lupus syndrome induced by sulfasalazine in rheumatoid arthritis. All symptoms resolved and antihistone antibodies disappeared when sulfasalazine was discontinued. In one patient, perinuclear antineutrophil cytoplasmic antibodies with specificity for myeloperoxidase were found critically increased just before the occurrence of vasculitis.

Adhesive capsulitis of the shoulder: therapeutic contribution of subacromial bursography.

OBJECTIVE: To demonstrate the therapeutic value of subacromial bursography (with a steroid injection) in adhesive capsulitis of the shoulder inadequately improved by arthrographic glenohumeral distension with steroid injection. METHOD: Twenty cases of adhesive capsulitis documented by glenohumeral arthrography were studied prospectively. A steroid was injected during distension arthrography, which was followed by physical therapy. Subacromial bursography without steroid injection was done routinely for diagnostic purposes. Constant's simplified score and range of motion were determined in each patient at baseline and after one, three, six and 12 months. Patients who were inadequately improved after one to three months underwent repeat subacromial bursography with steroid injection, followed by physical therapy. RESULTS: Of the 20 patients, 13 were noticeably improved 1.7 months on average after the distension arthrography. Of the remaining seven patients, six were improved 0.7 months on average after the bursography with steroid injection. CONCLUSION: Glenohumeral distension arthrography with steroid injection followed by physical therapy is effective in expediting the spontaneously favorable outcome of adhesive capsulitis and also allows to confirm the diagnosis. However, the subacromial bursa is almost consistently involved. Subacromial bursography with steroid injection can be useful in cases that fail to respond to conventional therapy.

A case of primary hypertrophic osteoarthropathy without skin involvement (Currarino's disease).

Hypertrophic osteoarthropathy is characterized by digital clubbing, arthropathy and periostosis of long tubular bones. Currarino's disease is an extremely rare variant of primary hypertrophic osteoarthropathy in which there is delayed closure of the fontanelles and an absence of skin involvement. Most reported cases have been in blacks. We report a case in a Caucasian adolescent.