Long-term treatment with sildenafil in chronic thromboembolic pulmonary hypertension.

For chronic thromboembolic pulmonary hypertension not amenable to pulmonary endarterectomy, effective medical therapy is desired. In an open-label uncontrolled clinical trial, 104 patients (mean +/- sem age 62 +/- 11 yrs) with inoperable chronic thromboembolic pulmonary hypertension were treated with 50 mg sildenafil t.i.d. At baseline, patients had severe pulmonary hypertension (pulmonary vascular resistance 863 +/- 38 dyn.s.cm(-5)) and a 6-min walking distance of 310 +/- 11 m. Eight patients were in World Health Organization functional class II, 76 in class III and 20 in class IV. After 3 months' treatment, there was significant haemodynamic improvement, with reduction of pulmonary vascular resistance to 759 +/- 62 dyn.s.cm(-5). The 6-min walking distance increased significantly to 361 +/- 15 m after 3 months' treatment, and to 366 +/- 18 m after 12 months' treatment. A subset of 67 patients received a single dose of 50 mg sildenafil during initial right heart catheterisation. The acute haemodynamic effect of this was not predictive of long-term outcome. In this large series of patients with inoperable chronic thromboembolic pulmonary hypertension, open-label treatment with sildenafil led to significant long-term functional improvement. The acute effect of sildenafil may not predict the long-term outcome of therapy.

Investigation of the proinflammatory potential of biodegradable nanoparticle drug delivery systems in the lung.

Particulate nanocarriers have been praised for their advantageous drug delivery properties in the lung, such as avoidance of macrophage clearance mechanisms and long residence times. However, instilled non-biodegradable polystyrene nanospheres with small diameters and thus large surface areas have been shown to induce pulmonary inflammation. This study examines the potential of biodegradable polymeric nanoparticles composed of poly(lactic-co-glycolic acid) (PLGA) and the novel PLGA derivative, diethylaminopropylamine polyvinyl alcohol-grafted-poly(lactic-co-glycolic acid) (DEAPA-PVAL-g-PLGA), to provoke inflammatory responses in the murine lung after intratracheal instillation. Lactate dehydrogenase (LDH) release, protein concentration, MIP-2 mRNA induction, and polymorphonucleocyte (PMN) recruitment in the bronchial alveolar lavage fluid (BALF) were used to evaluate an inflammatory response in Balb-C mice. Two sizes of polystyrene (PS) nanospheres (diameters: 75 nm and 220 nm) were included in the study for comparison. All nanoparticle suspensions were instilled at concentrations of 1 microg/microl and 2.5 microg/microl, representative of an estimated "therapeutic dose" and a concentrated "dose" of particles. In all experiments, the 75 nm PS particles exhibited elevated responses for the inflammatory markers investigated. In contrast, biodegradable particles of comparable hydrodynamic diameter showed a significantly lower inflammatory response. The most marked differences were observed in the extent of PMN recruitment. While the 75 nm and 220 nm PS nanospheres exhibited 41 and 74% PMN within the total BALF cell population after 24 h, respectively, PMN recruiting in lungs instilled with both types of biodegradable particles did not exceed values of the negative isotonic glucose control. In conclusion, evidence suggests that biodegradable polymeric nanoparticles designed for pulmonary drug delivery may not induce the same inflammatory response as non-biodegradable polystyrene particles of comparable size.

Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI.

Gene therapy aimed at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and lung cancer. Polyethylenimine (PEI) has been utilized for gene delivery to the airways. In this study, we describe a new modification of PEI, in which an oligopeptide related to the protein transduction domain of HIV-1 TAT was covalently coupled to 25 kDa PEI (PEI) through a heterobifunctional polyethylenglycol (PEG) spacer resulting in a TAT-PEG-PEI conjugate. Improved DNA reporter gene complexation and protection was observed for small (approximately 90 nm) polyplexes as well as significantly improved stability against polyanions, Alveofact, bronchial alveolar lining fluid and DNase. To determine polyplex toxicity in vitro, MTT assays were performed and, for in vivo testing, the mice bronchial alveolar lavage was investigated for total cell counts, quantity of neutrophils, total protein and TNF-alpha concentration. All parameters suggest significantly lower toxicity for TAT-PEG-PEI. Transfection efficiencies of both PEI and TAT-PEG-PEI polyplexes with DNA were studied under in vitro conditions (A549) and in mice after intratracheal instillation. While luciferase expression in A549 cells was much lower for TAT-PEG-PEI (0.2 ng/mg protein) than for PEI (2 ng/mg), significantly higher transfection efficiencies for TAT-PEG-PEI were detected in mice. Reporter gene expression was distributed through bronchial and alveolar tissue. Thus, TAT-PEG-PEI represents a new approach to non-viral gene carriers for lung therapy, comprising protection for plasmid DNA, low toxicity and significantly enhanced transfection efficiency under in vivo conditions.

Modified polyethylenimines as non viral gene delivery systems for aerosol therapy: effects of nebulization on cellular uptake and transfection efficiency.

This study examined the effect of nebulization on the cellular uptake and transfection efficiency of polyplexes from four polyethylenimine (PEI) modifications: branched 25 kDa PEI (bPEI), linear 22 kDa PEI (linPEI), pegylated PEI (pegPEI) and biodegradable PEI (bioPEI). Polyplexes were aerosolized with air-jet and ultrasonic nebulizers. The aerosol was collected and used to determine complex size and zeta potential. Fluorescence-assisted cell sorting (FACS) was used to quantify the cellular association of polyplexes in primary alveolar cells (AEC), A549 cells and primary bronchial cells (BEC). Confocal laser scanning microscopic images provided information about the internalization of polyplexes. Transfection efficiencies of polyplexes were quantified via measurement of luciferase expression. All polymers were stable during nebulization, although size increases were observed after air-jet nebulization. FACS studies showed a two- to three-fold increase in polyplex association with BEC compared to A549 cells, while polyplex association with AEC was negligible. BPEI, linPEI and bioPEI polyplexes were internalized, while pegPEI polyplexes remained predominately attached to the cellular membrane. Luciferase expression was detected only in BEC and A549 cells with transfection efficiencies approximately one order of magnitude higher in BEC. All PEI modifications investigated were suitable for aerosol therapy, although cell type and polymer structure significantly influenced the uptake and transfection efficiency of the polyplexes.

Modified polyethylenimines as non-viral gene delivery systems for aerosol gene therapy: investigations of the complex structure and stability during air-jet and ultrasonic nebulization.

Polyelectrolyte complexes between DNA and polyethylenimine (PEI) are promising non-viral delivery systems for pulmonary inhalation gene therapy and thus require sufficient stability during nebulization. The structure and stability of four different PEI-DNA polyplexes, namely branched (bPEI), linear (linPEI), poly(ethylene glycol)-grafted PEI (PEGPEI), biodegradable (bioPEI) PEI with DNA, were investigated. Using atomic force microscopy, the morphology of DNA and polyplexes before and after both air-jet and ultrasonic nebulization was characterized. The influence of nebulization on physico-chemical properties, particle size and zeta potential, was studied. Efficient DNA condensation to spherical particles was achieved with bPEI (90 nm) and PEGPEI (110 nm). By contrast, incomplete DNA condensations, seen as flower structures, were observed with linPEI (110 nm) and bioPEI (105 nm). Air-jet nebulization altered the polyplex structure to a greater extent than ultrasonic nebulization and resulted mainly in smaller and non-spherical particles (30-200 nm). Ultrasonic nebulization did not change the spherical structure or particle size of the polyplexes. In particular, the shape and size of the PEGPEI polyplexes did not change. We conclude that ultrasonic nebulization is a milder aerosolization method for gene delivery systems based on PEI. Additionally, PEGPEI-DNA polyplexes seem to be more stable than their counterparts, which may be advantageous in pulmonary inhalation gene therapy.

Surfactant-free, biodegradable nanoparticles for aerosol therapy based on the branched polyesters, DEAPA-PVAL-g-PLGA.

PURPOSE: This study describes the development of surfactant-free, biodegradable nanoparticle systems with varying physicochemical properties and their suitability for pulmonary application via nebulization. METHODS: Nanoparticle suspensions were formulated from the branched polyester, diethylaminopropyl amine-poly(vinyl alcohol)-grafted-poly(lactide-co-glycolide) (DEAPA-PVAL-g-PLGA) alone, as well as with increasing amounts of carboxymethyl cellulose (CMC). Particle size, zeta potential, turbidity, and morphology (atomic force microscopy) were characterized. Three formulations were chosen for further study: Cationic nanoparticles without CMC, cationic nanoparticles with CMC, and anionic nanoparticles with an excess of CMC. Nanoparticle degradation was characterized, as well as stability during nebulization. Nanoparticle-cell interactions were investigated and quantified using confocal laser scanning microscopy and fluorescence spectrometry. RESULTS: Nanoparticles ranged in size from 70-250 nm and displayed zeta potentials of +58.9 to -46.6 mV. Anionic nanoparticles showed the highest stability during nebulization. The degradation rate of each nanoparticle formulation decreased with increasing amounts of CMC. Cell association was highest among cationic nanoparticles (57% and 30%, respectively), although these were not internalized. Despite a lower rate of cell association (3%), anionic nanoparticles were internalized by A549 cells. CONCLUSIONS: Surfactant-free nanoparticles from DEAPA-PVAL-g-PLGA are versatile drug delivery systems; however, only the anionic formulations investigated were proven suitable for aerosol therapy.

Bronchoscopic administration of bovine natural surfactant in ARDS and septic shock: impact on biophysical and biochemical surfactant properties.

The purpose of the present study was to investigate the impact of bronchoscopic surfactant administration, on the biochemical and biophysical surfactant properties, in patients with severe and early acute respiratory distress syndrome (ARDS) and septic shock. A total number of 27 ARDS patients received 300-500 mg x kg x body x weight(-1) of a natural bovine surfactant extract (Alveofact) via a flexible bronchoscope. Bronchoalveolar lavages were performed 3 h prior to, and 15-18 h and 72 h after surfactant administration. A comparison to healthy volunteers, undergoing an identical lavage procedure, was made (control, n=12). Severe biophysical and biochemical surfactant abnormalities were encountered throughout in the ARDS patients. These included a massive alveolar protein load, a reduced percentage of large surfactant aggregates (LA), a loss of palmitoylated phosphatidylcholine species and a significant reduction of surfactant apoprotein (SP)-A, SP-B and SP-C in the LA fraction. Both minimum (gammamin) and adsorption (gammaads) surface tension values (pulsating bubble surfactometer) were dramatically increased. Surfactant treatment resulted in a marked increase in the lavagable phospholipid (PL) pool, but predominance of the alveolar surfactant-inhibitory protein load was still encountered. Far-reaching or even complete normalization of the PL profile, the LA fraction and its SP-B and SP-C (but not SP-A) content as well as the fatty acid composition of the phosphatidylcholine class was noted. Surface tension lowering properties (gammamin and gammaads) significantly improved, but were still not fully normalized. Bronchoscopic administration of large quantities of natural bovine surfactant in severe acute respiratory distress syndrome causes far-reaching restoration of biochemical surfactant properties and significant improvement, however not full normalization, of biophysical surfactant function.

Bronchoscopic administration of bovine natural surfactant in ARDS and septic shock: impact on gas exchange and haemodynamics.

The aim of the present study was to investigate the feasibility and efficacy of bronchoscopic surfactant administration in a noncontrolled multicentre study in five university centres. A total number of 27 patients, suffering from severe acute respiratory distress syndrome (mean+/-SEM lung injury score: 3.15+/-0.06) and septic shock (Acute Physiology and Chronic Health Evaluation (APACHE) II score at study entry 33.2+/-1.3, lactate 4.3+/-0.6 mmol x L(-1)) were studied. The patients were ventilated with a mean tidal volume of 11.0+/-0.5 mL x kg(-1) body weight (bw), either volume or pressure controlled, with 16.3+/-2.8 cmH2O positive end-expiratory pressure, for an average of 3.5+/-0.3 days at study entry. A natural bovine surfactant extract (300 mg x kg(-1) bw Alveofact; mean total volume 378 mL) was delivered in divided doses to each segment of the lungs via flexible bronchoscope within approximately 45 min. No untoward effects on gas exchange, lung mechanics and haemodynamics were noted during the procedure of surfactant administration. Within 12 h the oxygen tension in arterial blood/inspiratory oxygen fraction increased from a mean of 109+/-8 mmHg to 210+/-20 mmHg (p<0.001). In seven patients, in whom gas exchange again deteriorated with further progression of the disease, a second surfactant dose of 200 mg x kg(-1) was administered 18-24 h after the first application, again improving arterial oxygenation. A total of 15 patients survived the 28-day study period (mortality rate 44.4%, compared to a calculated risk of death for the given APACHE II scores of 74.0+/-3.5%), with all causes of death being nonrespiratory. The bronchoscopic application of a high dose of natural surfactant in patients with severe acute respiratory distress syndrome and septic shock is both feasible and safe, resulting in a pronounced improvement in gas exchange.

Alveolar epithelial barrier functions in ventilated perfused rabbit lungs.

We employed ultrasonic nebulization for homogeneous alveolar tracer deposition into ventilated perfused rabbit lungs. (22)Na and (125)I-albumin transit kinetics were monitored on-line with gamma detectors placed around the lung and the perfusate reservoir. [(3)H]mannitol was measured by repetitive counting of perfusion fluid samples. Volume of the alveolar epithelial lining fluid was estimated with bronchoalveolar lavage with sodium-free isosmolar mannitol solutions. Sodium clearance rate was -2.2 +/- 0.3%/min. This rate was significantly reduced by preadministration of ouabain/amiloride and enhanced by pretreatment with aerosolized terbutaline. The (125)I-albumin clearance rate was -0.40 +/- 0.05%/min. The appearance of [(3)H]mannitol in the perfusate was not influenced by ouabain/amiloride or terbutaline but was markedly enhanced by pretreatment with aerosolized protamine. An epithelial lining fluid volume of 1.22 +/- 0.21 ml was calculated in control lungs. Fluid absorption rate was 1.23 microl x g lung weight(-1) x min(-1), which was blunted after pretreatment with ouabain/amiloride. We conclude that alveolar tracer loading by aerosolization is a feasible technique to assess alveolar epithelial barrier properties in aerated lungs. Data on active and passive sodium flux, paracellular solute transit, and net fluid absorption correspond well to those in previous studies in fluid-filled lungs; however, albumin clearance rates were markedly higher in the currently investigated aerated lungs.

Ultrasonic versus jet nebulization of iloprost in severe pulmonary hypertension.

Inhalation of iloprost, a stable prostacyclin analogue, is a promising perspective in the treatment of pulmonary hypertension. In initial clinical studies, a conventional jet nebulizer system was successfully used to decrease pulmonary vascular resistance and pressure, requiring however, up to twelve inhalations of 12-15 min per day. The aim of this study was to investigate if the application of an equal dose of iloprost at a drastically reduced duration of inhalation with the use of a more efficient ultrasonic nebulizer, leads to comparable haemodynamic effects, without escalation of side effects. The physical features of the jet nebulizer system (Ilo-Neb) and the ultrasonic nebulizer (Multisonic Compact) were characterized by laser diffractometry and a Tc99m-tracer technique. Mass median aerodynamic diameters were 3.2 microm for the jet and 3.9 microm for the ultrasonic nebulizer. Total output (mean+/-SD) was 60+/-7 microL.min(-1) (jet) and 163+/-15 microL.min(-1)(ultrasonic), and efficiency of the devices was 39+/-3% (jet) and 86+/-5% (ultrasonic). Based on these data, a total inhalative dose of 2.8 microg iloprost was delivered by jet nebulization within 12 min and by ultrasonic nebulization within 4 min, in 18 patients with severe primary and secondary pulmonary hypertension (New York Heart Association class III and IV), in a randomized crossover design. Haemodynamics were assessed by right heart catheterization. Inhalation with the ultrasonic device and jet nebulizer, reduced mean+/-SEM pulmonary artery pressure from 54.3+/-2.1 to 47.1+/-2.0 and from 53.5+/-2.2 to 47.0+/-2.2 mmHg, respectively, and mean+/-SEM pulmonary vascular resistance from 1,073+/-109 to 804+/-87 and from 1,069+/-125 to 810+/-83 dyn.s.cm(-5), respectively. Both modes of aerosolization were well tolerated. In conclusion, due to the markedly higher efficiency and output of the ultrasonic device, wastage of drug is largely avoided and the duration of inhalation can be shortened to one-third, with comparable haemodynamic effects and without enforcing side effects.

Inhaled iloprost to treat severe pulmonary hypertension. An uncontrolled trial. German PPH Study Group.

BACKGROUND: Inhaled aerosolized iloprost, a stable prostacyclin analogue, has been considered a selective pulmonary vasodilator in the management of pulmonary hypertension. OBJECTIVE: To assess the efficacy of inhaled iloprost in the treatment of life-threatening pulmonary hypertension. DESIGN: Open, uncontrolled, multicenter study. SETTING: Intensive care units and pulmonary hypertension clinics at six university hospitals in Germany. PATIENTS: 19 patients who had progressive right-heart failure despite receiving maximum conventional therapy (12 with primary pulmonary hypertension, 3 with pulmonary hypertension related to collagen vascular disease without lung fibrosis, and 4 with secondary pulmonary hypertension). INTERVENTION: Inhaled iloprost, 6 to 12 times daily (50 to 200 microg/d). MEASUREMENTS: Right-heart catheterization and distance walked in 6 minutes at baseline and after 3 months of therapy. RESULTS: During the first 3 months of therapy, New York Heart Association functional class improved in 8 patients and was unchanged in 7 patients. Four patients died, 3 of right-heart failure and 1 of sepsis. The acute hemodynamic response to inhaled iloprost was predominant pulmonary vasodilatation with little systemic effect at baseline and at 3 months (data available for 12 patients). Hemodynamic variables were improved at 3 months, and the distance walked in 6 minutes improved by 148 m (95% CI, 4.5 to 282 m; P = 0.048). Of the 15 patients who continued to use inhaled iloprost, 8 stopped: Four had lung transplantation, 1 switched to intravenous prostacyclin therapy, and 3 died. Seven patients are still receiving inhaled iloprost (mean +/-SD) duration of therapy, 536 +/- 309 days; mean dosage, 164 +/- 38 microg/d). CONCLUSIONS: Inhaled iloprost may offer a new therapeutic option for improvement of hemodynamics and physical function in patients with life-threatening pulmonary hypertension and progressive right-heart failure that is refractory to conventional therapy.

Ultrasonic nebulization for efficient delivery of surfactant in a model of acute lung injury. Impact on gas exchange.

We investigated the effect of ultrasonic nebulization versus instillation of exogenous surfactant on gas exchange abnormalities provoked by detergent inhalation in perfused rabbit lungs. Ventilation-perfusion (VA/Q) distribution was assessed by the multiple inert gas elimination technique. For nebulization of natural bovine surfactant (Alveofact), an ultrasonic device was placed in line with the inspiratory gas flow tubing, manufacturing particles with a mass median aerodynamic diameter of approximately 4.5 microM and high aerosol concentration. In vitro studies demonstrated biochemical and biophysical integrity of postnebulization surfactant. Lung aerosol deposition was monitored by a laser-photometric technique. In lungs with sham inhalation of saline, tracheal instillation of surfactant (approximately 11 mg/kg body weight, infused over 50 min) provoked substantial VA/Q mismatch and limited shunt flow, whereas lung surfactant deposition by ultrasonic nebulization (approximately 7 to 9 mg/kg body weight; nebulization time, 50 min) did not interfere with physiologic gas exchange. Tween 20 inhalation provoked severe VA/Q mismatch with predominant shunt-flow (approximately 21%). This was not reversed by "rescue" application of instilled surfactant, but largely reversed by nebulized surfactant (shunt reduced to 5.5%; p < 0.01). Analysis of postaerosol lavage fluid demonstrated partial reconstitution of surface activity by nebulized surfactant. We conclude that ultrasonic nebulization may be employed for efficient delivery of functionally intact natural surfactant to the distal bronchoalveolar space. This approach effects rapid improvement of gas exchange in a model of acute homogeneous lung injury.

On-line laser-photometric monitoring of aerosol deposition in ventilated rabbit lungs.

A photometric technique was developed for on-line measurement of aerosol deposition in isolated, ventilated, and perfused rabbit lungs. A jet nebulizer was used for aerosolization of saline (hygroscopic particles) and di(2-ethylhexyl) sebacate (nonhygroscopic particles). Aerosol concentration (laser photometer, constructed for measurements in rabbit lungs) and flow rate (commercial pneumotachograph) were continuously monitored at the inlet of the tracheal cannula. Computer-assisted data processing allowed the breath-by-breath calculation of inhaled and exhaled aerosol mass, thus providing the deposition fraction. With the use of hygroscopic particles, however, this approach was hampered by the humidity-induced particle growth in the airways, leading to an overestimation of the aerosol concentration in exhaled air. This effect was corrected by an algorithm using a "particle growth factor" derived breath by breath from the photometer signal. To test the reliability of this approach, saline particles carrying technetium-99m label were aerosolized into rabbit lungs with the use of various ventilator settings, and the aerosol deposition was assessed in parallel by photometry and by radioactivity detection over the lung and over a trap in the exhaled-air circuit. Superimposable curves of cumulative aerosol deposition, with changes in kinetics dependent on the ventilator mode, were obtained. For a given ventilator setting, absolute values of the deposition fraction were 0.32 +/- 0.04 (radiotracer quantification) and 0.36 +/- 0.04 (photometry; means +/- SD; n = 4). We conclude that the presented laser-photometric technique allows reliable on-line monitoring of the deposition of both nonhygroscopic and hygroscopic aerosol particles in ventilated lungs.

Hydrogen peroxide-induced increase in lung endothelial and epithelial permeability--effect of adenylate cyclase stimulation and phosphodiesterase inhibition.

Neutrophil-derived hydrogen peroxide (H2O2) is believed to play an important role in inflammatory lung injury. We investigated the influence of pharmacological agents that increase intracellular c-AMP levels on endothelial and epithelial leakage in response to intravascular H2O2 challenge in buffer-perfused rabbit lungs. Endothelial permeability was assessed by determination of the capillary filtration coefficient (Kfc) and lung weight gain. Measurement of the clearance rate of inhaled aerosolized technetium-99m-labeled diethylenetriamine pentaacetic acid ([99mTc]DTPA) from the lungs into the perfusion fluid was used as an index of alveolar epithelial permeability. Experiments were performed in the presence of acetylsalicylic acid to suppress H2O2-induced lung prostanoid generation and concomitant vasoconstriction. Under these conditions, H2O2 admixture to the perfusate (250 microM) caused a greater than eight-fold increase in Kfc values, resulting in > 30 g lung weight gain within 30 min in the absence of any significant vasopressor response. Pretreatment with the adenylate cyclase activators prostaglandin E1 (0.1 microM) and forskolin (0.1 microM), the dual phosphodiesterase type III/IV inhibitor zardaverine (10 microM) as well as combinations of these drugs all caused a nearly complete suppression of this early Kfc increase; and severe edema formation (> 30 g) was retarded to approximately 50-55 min. In addition to the microvascular leakage response, H2O2 caused a four- to five-fold increase in the [99mTc]DTPA clearance rate, starting within 15 min and culminating after approximately 35 min. Adenylate cyclase activation reduced this epithelial leakage response by approximately 30%, whereas zardaverine exerted no significant effect. We conclude that both microvascular endothelial and alveolar epithelial barrier function are severely compromised by intravascular H2O2 challenge in intact lungs. Pharmacological approaches to increase c-AMP levels, including both adenylate cyclase activation and phosphodiesterase inhibition, partially block the endothelial response and, to a lesser extent, the epithelial response.