Characterization and in vivo testing of a heterogeneous cationic lipid-DNA formulation.

PURPOSE: To identify characteristics of lipid-DNA complexes that correlate with in vivo expression data. METHODS: DOTIM:cholesterol liposomes (1:1 mole ratio) and DNA expressing chloramphenicol acetyl transferase (CAT) were complexed at a 4.2:1 mass ratio (cationic lipid:DNA). Complexes were fractionated by density gradient centrifugation. analyzed for particle size and zeta potential and quantitated using HPLC methods. The unfractionated complexes, "purified" fractions of the complexes, and purified complexes supplemented with liposomes were administered to mice by intravenous injection (i.v.) and intratracheal instillation (i.t.) and their ability to express gene product was assessed. RESULTS: Centrifugation separated two distinct populations within complexes one consisting of free liposomes and the other of lipid complexed with DNA. The vesicle diameter and zeta potential among separated fractions varied from 113 to 354 nm. and + 24 to + 34 mV respectively. Re-centrifugation of the 'purified' fractions containing the lipid-DNA population produced a single band. CAT expression in lung tissue 24 hr post-i.v. was observed with the unfractionated complex, but not the purified form. Some activity was 'restored' with the liposome-supplemented complexes. In contrast, the same series of complexes administered by i.t. resulted in no significant difference in lung expression (p=0.16 ANOVA). CONCLUSIONS: An uncomplexed liposome population exists within DOTIM:cholesterol-DNA complexes that influences the expression of complexes administered i.v. but not i.t..

The influence of sodium glycocholate and other additives on the in vivo transfection of plasmid DNA in the lungs.

PURPOSE: A plasmid containing the luciferase 'marker' cDNA was constructed to test non viral gene delivery formulations in vivo. METHODS: A scale up procedure was devised to produce up to gram quantities of plasmid. Sufficient quantities were generated to process and test the DNA with various additives and to generate a spray-dried powder formulation of the plasmid. Male Sprague-Dawley rats (250 g) were intratracheally instilled with 200-250 microl of solution containing 200 microg plasmid +/- lipid [DC Chol:DOPE 1:1 molar (2mg/kg)] growth factors [KGF (10 mg/kg), EGF (5 mg/kg)], permeation enhancers [sodium glycocholate (0.01 to 10% w/v)), sodium deoxycholate (1% w/v), beta-cyclodextrin (1% w/v)], surfactant [Tween 80 (1% w/v)], a mucolytic [N-acetylcysteine (10% w/v)] and positively charged synthetic polymers [PVAVAM 6 and 14%]. Animals were sacrificed 24 hr post-dose and the lungs were assayed for luciferase using a chemiluminescent assay. RESULTS: The relative ability of the materials to promote luciferase production in the lungs was permeation enhancer >> DNA alone > or = lipid, mucolytic, surfactant, growth factor > polymer. Protein production in the lungs ranged from 10 times below the DNA control (approximately 16 pg) using the polymers (approximately 1.5 pg) to approximately 125 times greater than the control using the permeation enhancer (approximately 2050 pg). The transfection capabilities of the majority of additives was low. The enhancing effects of sodium glycocholate were dose-dependent and perhaps associated with the critical micelle concentration. Although the bile salt was the most successful of the tested compounds, it resulted in significant mortality when used at concentrations greater than 1% w/v. CONCLUSIONS: The results suggest that transfection can be significantly enhanced by additives such as NaGC but some toxicity may be unavoidable.

Systemic absorption and activity of recombinant consensus interferons after intratracheal instillation and aerosol administration.

PURPOSE: The pulmonary pharmacokinetics and bioactivity of E. coli derived recombinant consensus interferon (CIFN) and a modified lactose-conjugated consensus interferon (LacCIFN) were evaluated in animals. METHODS: Estimated doses of 20 and 100 micrograms/kg of the interferons were administered to anesthetized rats by aerosol via ultrasonic nebulizer as well as intravenous injection. Rats also received nominal doses of 50 and 200 micrograms/kg via intratracheal instillation (IT). Hamsters were treated with interferon via various routes including IT. The effectiveness of treatment was assessed by the resistance to development of hind leg paralysis following infection with encephalomyocarditis virus. RESULTS: Significant amounts of CIFN and LacCIFN were found in rat plasma after aerosol administration. Peak plasma levels occurred approximately 25-30 minutes with estimated bioavailabilities approaching 70%. Absorption of CIFN was rate limiting and plasma levels were detectable 12 hr post-dose. The CIFN at IT doses as low as 5 micrograms/kg was effective at reducing paralysis in hamsters but protection was variable and doses of up to 100 micrograms/kg were not 100% effective. CONCLUSIONS: Despite the incomplete protection, the results demonstrate the feasibility of treating systemic viral infections with interferon administered directly to the lungs.

A method for preparing chelate-cytokine conjugates with retention of protein structure, biological activity, and pharmacokinetic properties.

The chelating agent diethylenetriaminepentaacetic acid (DTPA) has been conjugated site-specifically to the N-terminus of recombinant human interleukin-2 (rhIL-2) by reaction with DTPA dianhydride at an initial pH of 6.0, thus demonstrating broader application of the conjugation method previously described for the structurally related cytokine rhG-CSF (Ralph et al., 1995). Purity of the DTPA-rhIL-2 conjugate, isolated by cation-exchange FPLC, and chelation of 111In were revealed by cation-exchange HPLC. Purity of the conjugate as well as chelation of radiometal were also demonstrated by SDS-PAGE and TLC, respectively. The stoichiometric molar ratio of DTPA to protein for the conjugate was approximately 1:1 as determined by TLC and mass spectrometry. Localization of the DTPA moiety was resolved by a peptide mapping procedure. The protein retained > 95% secondary structure (alpha helicity) following the conjugation. Addition of metal induced an approximate 22% loss of secondary structure for the conjugate. The in vitro biological activity of the protein was unaffected by the conjugated DTPA, even with chelated metal. Pharmacokinetic analysis of DTPA-conjugated cytokines, following chelated 111In, showed clearance and pharmacokinetic parameter values comparable to those of the corresponding unmodified cytokine. DTPA-conjugated cytokines may prove useful in cytokine research, and furthermore may represent a novel class of molecules for imaging, diagnosing, and/or treatment of malignancies where the cytokine receptor is overexpressed.

Stability of recombinant consensus interferon to air-jet and ultrasonic nebulization.

The stability of recombinant consensus alpha-interferon (rConIFN) to air-jet and ultrasonic nebulization was evaluated. Volumes of 10 mL of 0.5 mg/mL rConIFN in phosphate-buffered saline (PBS) at pH 6.3 were nebulized with a Collison three-jet nebulizer at 40 psig (10 L/min) for up to 25 min. The effects of pH (3.0, 6.3, and 9.0), additive (0.1% w/v Tween 80, 0.1% w/v Tween 20, and 1% w/v PEG 8000), and ionic strength (0, 0.25, and 1.0) were examined. The effects of ultrasonic nebulization were studied using three devices (DeVilbiss "Aerosonic"; Mountain Medical "Microstat", and Medix "Easimist"). Stability of rConIFN was assessed by size exclusion chromatography and native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Structural effects were examined by circular dichroism spectroscopy and bioactivity was assessed by an in vitro mitogenic inhibition bioassay. rConIFN is destabilized by air-jet nebulization. Insoluble noncovalent aggregates are produced rapidly, and only approximately 25% of the initial monomeric protein remains after 25 min of nebulization. This correlates with a decrease in in vitro bioactivity. Aggregation during nebulization is influenced by pH (9.0 < 6.3 < 3.0) but even at the highest pH, > 25% aggregation is observed. Ionic strength does not appear to influence aggregation. rConIFN is also seen to adhere to glass after nebulization. Samples from a rinse of the emptied reservoir with 0.1% w/v SDS, after thorough rinsing with water (three times), show a strong rConIFN band on SDS-PAGE gels. The use of PEG 8000 and Tween mitigate aggregate formation and adhesion (< 20%). The cumulative output collected as a wet or dry aerosol is not aggregated to the same extent as the residual protein remaining in the nebulizer. Ultrasonic nebulization also results in aggregation, but the extent of denaturation is dependent upon the nebulizer used and is related to the heating of nebulizer solutions. Cooling of the nebulizer solution during operation (< 30 degrees C) minimizes aggregation (< 5%), and bioactivity is retained.

The pulmonary absorption of aerosolized and intratracheally instilled rhG-CSF and monoPEGylated rhG-CSF.

PURPOSE: The objective of this study was to highlight differences in the pulmonary absorption of a monoPEGylated rhG-CSF and rhG-CSF after intratracheal instillation and aerosol delivery. METHODS: Male Sprague Dawley rats (250 g) were anesthetized and intratracheally instilled (IT) with protein solution or were endotracheally intubated and administered aerosol for 20 min via a Harvard small animal ventilator. A DeVilbiss "Aerosonic" nebulizer containing 5 ml of protein solution at approximately 3 mg/ml was used to generate aerosol. The volume of protein solution deposited in the lung lobes was estimated to be approximately 13 microliters after delivery of Tc-99m HSA solutions. The PEGylated proteins consisted of a 6 kDa (P6) or 12 kDa PEG (P12) linked to the N-terminus of rhG-CSF. rhG-CSF also was administered IT in buffers at pH 4 and pH 7 and in dosing volumes ranging from 100 to 400 microliters. Blood samples were removed at intervals after dosing and the total white blood cell counts (WBC) were determined. Plasma was assayed for proteins by an enzyme immuno assay. RESULTS: The plasma protein concentration v. time profiles were strikingly different for aerosol v. IT delivery. The Cmax values for rhG-CSF and P12 after aerosol delivery were greater than found after IT (Aerosol: 598 +/- 135 (ng/ml) rhG-CSF; 182 +/- 14 P12 v. IT: 105 +/- 12 rhG-CSF; 65.9 +/- 5 P12). Similarly, Tmax was reached much earlier after aerosol administration (Aerosol: 21.7 +/- 4.8 (min) rhG-CSF; 168 +/- 31 P12 v. IT: 100 +/- 17 rhG-CSF; 310 +/- 121 P12). Estimated bioavailabilities (F(lung)%) were significantly greater via aerosol delivery than those obtained after IT (Aerosol: 66 +/- 14 rhG-CSF; 12.3 +/- 1.9 P12 v. IT: 11.9 +/- 1.5 rhG-CSF; 1.6 +/- 0.1 P12). An increase in circulating WBC counts was induced by all proteins delivered to the lungs. The rate and extent of absorption of rhG-CSF was not influenced by the pH employed nor the instilled volume. CONCLUSIONS: Estimates of bioavailability are dependent upon the technique employed to administer drug to the lungs. Aerosol administration provides a better estimate of the systemic absorption of macromolecules.

Some factors associated with the ultrasonic nebulization of proteins.

Ultrasonic nebulization of lactate dehydrogenase (LDH) was investigated using a DeVilbiss "Aerosonic" nebulizer. The enzyme (8ml, 0.025 mg/ml Na2HPO4, pH 7.0) was completely inactivated after 20 minutes of operation. However, the inactivation profile observed during ultrasonic nebulization was different from that previously observed using air-jet nebulization. At least two mechanisms are involved, one associated with heating and the other with aerosol production. By preventing heating of the nebulizer fluid during operation, the denaturation profile was dramatically altered. By additionally including 0.01% w/v Tween 80 or 1% w/v PEG 8000, almost all activity was retained. Similar results were obtained by preventing aerosol production and heating. However, 100% of activity was lost when heating was allowed to occur without aerosol formation. The results demonstrate that cooling in conjunction with a surfactant is one approach that could be used to stabilize proteins to ultrasonic nebulization. However, cooling also significantly reduced solute output from the nebulizer. When operated at 10 degrees C output was negligible. At 50 degrees C the output was 5x greater than that found at room temperature. The median droplet size (micron(s)) was not significantly influenced by the operating temperature of the nebulizer fluid (3.6 +/- 0.4, 21 degrees C; 3.9 +/- 0.2, 50 degrees C, p = NS (n = 6)) although the size distribution was noted to increase at the higher temperature.

Delivery of biotherapeutics by inhalation aerosol.

The role of inhalation therapy is adapting to changes brought on by advances in several related disciplines. These range from device technology to the molecular and cell biology of the lungs. Acceptable bioavailability and efficacy have been achieved via the oral route for most traditional pharmaceuticals. Unfortunately, injection is the normal mode of delivery with biotherapeutic agents and alternative delivery approaches are needed. Many preclinical and clinical studies with inhaled proteins, peptides, and DNA have been completed and demonstrate that efficacy can be achieved within the lungs and systemically. Despite the promising results, the development of inhaled biotherapeutics is beset with unique problems that require an integrated and rational approach to development. Aqueous protein formulations are often not stable to aerosolization, while stability of powder formulations can be difficult to evaluate in the solid state. Inhaler efficiency and reproducibility are unacceptable with existing devices and, although improvements in technology have brightened the outlook, new devices are not yet available and remain untried with most biotherapeutics. Once delivered to the lungs, these molecules are also subjected to a variety of efficient clearance mechanisms that can significantly reduce the probability of them being effective. Despite these problems, the number of potential drugs being tested via inhalation continues to increase, suggesting some promise of future success. This review discusses the above issues and highlights a variety of biotherapeutics that have been administered as inhalation aerosols.

Pulmonary delivery of powders and solutions containing recombinant human granulocyte colony-stimulating factor (rhG-CSF) to the rabbit.

Two powder formulations (MMAD < 4 microns) containing rhG-CSF were insufflated (IF) via an endotracheal tube at doses of 5, 75 or 500 micrograms/kg to New Zealand white rabbits. Doses of 5 and 500 micrograms/kg of solutions were administered by intratracheal instillation (IT), subcutaneous (SC) injection in the thigh and intravenous injection (i.v.) via the marginal ear vein. Blood samples were removed at regular intervals from an indwelling jugular catheter. Blood was analyzed directly for total white blood cell counts (WBC). Plasma was assayed for rhG-CSF by a specific ELISA. The distribution of radioactive dose in lung tissue was found after administering Tc99m HSA in solution or when incorporated into powders. The pharmacokinetics and pharmacodynamics were determined for all routes of administration. High dose IV concentration vs. time profiles declined biexponentially (t1/2 alpha = 0.6 +/- 0.2 hrs, t 1/2 beta = 4.6 +/- 0.2 hrs, n = 8). Clearance was does dependent (11.6 +/- 2.6 [500 micrograms/kg, n = 8] vs; 21.8 +/- 3.3 ml/hr/kg [5 micrograms/kg, n = 5]). A normal systemic response was obtained after IF, indicating that rhG-CSF retains activity in the solid state. Dissolution and absorption of rhG-CSF from the powders were not rate limiting. The plasma concentration vs. time profiles peaked at similar times to those after IT (Tmax 1-2 hrs) but were earlier than obtained after SC (Tmax 6-10 hrs). Powders were less efficiently dosed to the lung lobes after insufflation compared with instillates (14.7 +/- 10.5 vs. 60.1 +/- 10.6%), resulting in bioavailabilities ranging from 5 to 33%.(ABSTRACT TRUNCATED AT 250 WORDS)

Prediction and experimental determination of solute output from a Collison nebulizer.

The total output from a nebulizer is made up of aqueous droplets containing solute and a significant component of water vapor. The solvent loss is reflected in an increase in the nebulizer solution concentration over time and this has been described mathematically. This theory, originally described by Mercer et al., was modified to describe the solute output from a three-jet Collison nebulizer. The influence of concentration, air flow (air pressure), volume, and temperature on the output parameters were then studied. Inlet air pressures were 10 (4.1), 20 (6.4), and 40 (10.0) psig (L/min), starting concentrations were 0.1, 2, and 5% (w/w) and initial solution volumes were 10 and 20 mL. To study temperature effects, solutions were nebulized at fixed temperatures ranging from 4 to 50 degrees C. This was achieved by water-jacketing the nebulizer flask. Test solutions consisted of mannitol and a fixed concentration of 11.1 micrograms/mL carboxyfluorescein. Nebulization was carried out for up to 30 min using dry, filtered air at ambient temperature. Total output was determined gravimetrically while solute output was determined by fluorimetry (495-nm excitation, 515-nm emission). Solution concentration changes were also monitored over time by fluorimetry. The results show that the solution and solvent output parameters are independent of concentration, volume, and air flow within the ranges studied but that solvent output, in particular, is highly dependent upon temperature.

Syringe-actuated metered dose inhalers: a quantitative laboratory evaluation of albuterol delivery through nozzle extensions.

The metered dose inhaler (MDI) is a device that can be used to supply aerosolized bronchodilators to the tracheobronchial tree of patients requiring endotracheal intubation. Because direct actuation of the MDI into the breathing circuit is inefficient, a technique of extending the MDI nozzle with small-bore, long, intravenous catheters has been devised. To facilitate connecting the MDI to the extension and actuating the release of aerosol, insertion of the MDI cannister into the barrel of a large syringe attached to the Luer hub of the extension has been proposed but not quantitatively evaluated. The purpose of this study was to test, in a laboratory model, the performance of syringe-actuated MDIs attached to nozzle extensions. The deposition of the bronchodilator albuterol in the delivery device and distal to the extension was quantified spectrophotometrically. Maximum distal delivery of drug from nozzle extensions fashioned from small-bore (19-gauge) catheters was 80% of the actuated release. Loss of drug to the inner surface of the syringe plus extension varied inversely with the number of successive actuations. Syringes from two manufacturers in two sizes were compared and found to perform comparably. Syringes could be attached to the Luer connector of the secondary sampling channel of special pediatric-sized endotracheal tubes (ETTs) and the channels were evaluated as nozzle extensions, with distal delivery > 80% as efficient as the 19-gauge intravenous catheter. We conclude that syringe actuation of MDIs through nozzle extensions is an efficient method for supplying the aerosol distal to the tip of the ETT.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of aerosolized drugs.

The expanding use of inhalation therapy has placed demands on current aerosol generation systems that are difficult to meet with current inhalers. The desire to deliver novel drug entities such as proteins and peptides, as well as complex formulations including liposomes and microspheres, requires delivery systems of improved efficiency that will target the lung in a reproducible manner. These efforts have also been spurred by the phase out of chlorofluorocarbons (CFCs) and this has included a directed search for alternative propellants. Consequently, a variety of new aerosol devices and methods of generating aerosols are being studied. This includes the use of freon replacement propellants, dry powder generation systems, aqueous unit spray systems and microprocessor controlled technologies. Each approach has advantages and disadvantages depending upon each principle of action and set of design variables. In addition, specific drugs may be better suited for one type of inhaler device vs. another. The extent to which aerosol generation systems achieve their goals is discussed together with a summary of selected papers presented at the recent International Congress of Aerosols in Medicine.

Pulmonary absorption of recombinant methionyl human granulocyte colony stimulating factor (r-huG-CSF) after intratracheal instillation to the hamster.

Recombinant methionyl human granulocyte colony stimulating factor (G-CSF), a molecule of 18.8 kDa, has been shown to induce a systemic response after delivery by aerosol. In this work, rate and extent of absorption as well as the response were determined after bolus administration of solutions by intratracheal instillation (IT). The protein was quantified using a specific ELISA and the biological response was assessed by monitoring the increase in numbers of circulating white blood cells (WBC). A dose-response curve was obtained after IT, subcutaneous injection (SC), and intracardiac injection (IC) of 100 microL of a nominal dose ranging from 1 to 1000 micrograms/kg G-CSF (n = 5). WBC numbers were determined 24 hr postadministration. Absorption and clearance kinetics were determined after IT and IC of 500 micrograms/kg protein over a 24-hr time period (n = 5). The response of the lung to G-CSF was monitored by WBC counts and differentials in lung lavage fluid. 73.6 +/- 10.5% (n = 7) of the IT dose reached the lung lobes. The response to single doses of G-CSF by IT or SC was similar, with WBC numbers increasing over 4x baseline at the higher doses. Absorption from the lung was rapid and did not follow first-order kinetics. Clearance after the IC dose was described by a biexponential equation (alpha = 1.41, beta = 0.24 hr-1). Peak serum levels were obtained approximately 1-2 hr after IT. The bioavailability was 45.9% of the administered dose and 62.0% of the dose reaching the lung lobes. These results indicate that G-CSF is rapidly absorbed from the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Small bore nozzle extensions to improve the delivery efficiency of drugs from metered dose inhalers: laboratory evaluation.

Metered dose inhalers (MDIs) are frequently used to supply aerosolized drugs, particularly bronchodilators, to the tracheobronchial tree of patients with endotracheal tubes in the intensive care unit or in the operating room. The efficiency of delivery to the lungs of agents such as the beta 2-adrenergic agonists is known to be low. In an in vitro model, we evaluated a means of improving the delivery of drug released from an MDI beyond the distal tip of the endotracheal tube. Extensions of the MDI nozzle were fashioned from modified intravenous catheters or sections of small bore polyethylene tubing. A model trachea/carina was constructed and suspended above a collecting device. An albuterol MDI was actuated through the nozzle extension and into the model airway. We measured the quantity of albuterol deposited in the nozzle extension, in the trachea/carina and in the distal collecting device. Particle size distribution was determined with a cascade impactor. The results indicate an inverse relationship between the quantity of drug delivered distally and the inner diameter of the nozzle extension, with a marked increase in delivery for an inner diameter < 1 mm. Ninety percent of the actuated dose from the MDI exited a 0.76-mm inner diameter nozzle extension. From 20 to 30% of the nominal MDI dose was recovered from the distal collector, 70% of which deposited in the particle size range of 1 to 5 microns. Deposition in the trachea/carina was high, but this was reduced by introducing a flare in the tip of the nozzle extension, which did not affect the dose reaching the distal collector.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary effects of chronic exposure to liposome aerosols in mice.

Administering liposome-encapsulated drugs by aerosols could be a feasible way of targeting drugs to the lung, specifically to pulmonary alveolar macrophages (AM). In the mouse model, we characterized uptake of carboxyfluorescein- (CF-) labeled liposomes by AM in vivo after acute inhalation of liposome aerosols, and the effects of chronic exposure to liposome aerosols on lung histology and AM function. Mice were placed in a nose-only exposure module and exposed to liposome or saline aerosols for 1 h per day, 5 days per week, for 4 weeks. Five mice of both the experimental and control groups were removed weekly and their lungs examined. Liposomes were made from hydrogenated soy phosphatidylcholine (HSPC) at 50 mg/mL. In vivo uptake of liposomes by AM was documented by fluorescence microscopy and flow cytometry of bronchoalveolar lavage (BAL). A consistent amount of 1-3 micrograms of lipid inhaled per dosing per mouse was estimated from fluorescence measurements. Addition of Triton X-100 to BAL caused a significant increase in fluorescence intensity, indicating that liposomes remained intact in the lung for a period of time. The chronic inhalation study showed no histologic changes of the lung or untoward effects on the general health or survival of animals. AM phagocytic function, intracellular killing, and fatty acid composition were not affected. Transmission electron microscopy and morphometry (computerized image analysis) of AM likewise showed no alterations as a result of the treatment. It was concluded that AM uptake of liposomes delivered by aerosol was operant in vivo. This finding validates the concept of alveolar macrophage-directed delivery of liposome-encapsulated agents to the lung via inhalation. It was also concluded that chronic liposome aerosol inhalation in mice produced no untoward effects on survival, histopathology, and macrophage function. These data confirm and extend prior findings regarding the functional and morphologic interactions of liposomes with AM in vitro (Gonzalez-Rothi et al., Exp. Lung Res. 17:687-705, 1991).

Acetazolamide and furosemide attenuate asthma induced by hyperventilation of cold, dry air.

We investigated the assumption that the efficacy of inhaled diuretics in asthma is dependent upon inhibition of the Na+/K+/2Cl- cotransporter. We compared the protective effect of acetazolamide, a diuretic without significant effect on the loop cotransporter, with the protection provided by inhaled furosemide in a cold, dry air hyperventilation model of asthma. Seven asthmatic subjects underwent a baseline bronchial challenge and then received a nebulized dose of 80 mg of furosemide or 500 mg of acetazolamide or saline placebo in a randomized, double-blind, placebo-controlled crossover design. Repeat challenges were performed immediately and at 2 and 4 h postnebulization. Acetazolamide caused a 47.2% increase in the amount of cold, dry air required to reduce the FEV1, by 20% (expressed in terms of respiratory heat loss as PD20RHL), from 0.79 multiplied or divided by (x/divided by) 1.13 kcal/min (geometric mean x/divided by geometric SEM) at baseline to 1.17 x/divided by 1.09 kcal/min postnebulization (p < 0.025). Furosemide increased the geometric mean PD20RHL by 53.9%, from 0.86 x/divided by 1.12 kcal/min to 1.33 x/divided by 1.12 kcal/min (p < 0.001). There was no significant change after placebo inhalation (0.81 x/divided by 1.15 kcal/min versus 0.87 x/divided by 1.10 kcal/min, NS). Airway responsiveness had returned to baseline by 2 h postnebulization on all 3 days. Furosemide also caused bronchodilatation, producing a 14.1% rise in the mean FEV1 (p < 0.005 versus prenebulization), whereas neither acetazolamide nor placebo altered airway tone significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Nebulization of liposomes. III. The effects of operating conditions and local environment.

Multilamellar liposomes (MLV) of saturated phosphatidylcholine and dipalmitoyl phosphatidylglycerol (DPPG) (9:1 mole ratio) containing 5,6-carboxyfluorescein (CF) were prepared and extruded through 1.0-micron polycarbonate membranes. Diluted aqueous dispersions were aerosolized for a total of 80 min using a Collison nebulizer under a variety of conditions. The effects of air pressure, temperature, buffer osmotic strength, and pH on nebulized liposome dispersions were studied. Changes in air pressure produced large changes in the percentage release of CF and ranged from 1.3% (4 psig) to 88.2% (50 psig) after 80 min of nebulization. The temperature of the nebulizer dispersions dropped during experiments. The extent of the temperature drop varied according to the air pressure used and ranged from 5 degrees C (4 psig) to 11 degrees C (greater than or equal to 30 psig). The temperature of dispersions caused no increase in CF release until the gel-to-liquid crystalline transition temperature was exceeded (54.6 degrees C), whereupon a 20% increase in leakage was observed after 80 min of nebulization. Aerosol mass output was relatively unaffected by the starting temperature of experiments when conducted within the ambient temperature range. Leakage from the liposomes was increased in hypotonic solution but decreased in hypertonic solutions. At a buffer pH of 2.85 the percentage leakage of CF was increased approximately 18% compared to that at pH 7.2 and pH 10.75. Results show that the stability of liposomes composed of saturated phosphatidylcholine and DPPG (9:1 mole ratio) is affected by the operating and environmental conditions under which aerosolization takes place, with air pressure having the greatest effect.

Nebulization of liposomes. II. The effects of size and modeling of solute release profiles.

A series of carboxyfluorescein (CF)-containing multilamellar vesicle (MLV) dispersions was prepared and extruded through polycarbonate membranes ranging in size from 0.2 to 5 microns. Vesicle dispersions were nebulized for 80 min using a Collison nebulizer, and the release of CF was monitored during nebulization. Solute retention was dependent upon the size of the vesicles and leakage ranged from 7.9 +/- 0.4% (N = 3) for vesicles extruded through 0.2-microns filters to 76.8 +/- 5.9% (N = 3) for liposomes that were not filtered. Solute release profiles obtained over greater than or equal to 420-min nebulization periods conformed to a two-compartment kinetic model and exhibited a "fast" initial phase (k1 = 0.052 +/- 0.0043) followed by a "slow" terminal phase (k2 = 0.0034 +/- 0.00018). The results show that CF retention can be increased by nebulizing small vesicles and modeling suggests that the rate of CF leakage from the bilayers is faster than from the core of the liposomes.

Nebulization of liposomes. I. Effects of lipid composition.

A series of multilamellar liposome dispersions was prepared from lipids of soy phosphatidylcholine or hydrogenated soy phosphatidylcholine containing from 0 to 30 mol% of either cholesterol, stearylamine, or dipalmitoyl phosphatidylglycerol. The liposome dispersions were aerosolized with a Collison nebulizer for 80 min at an output flow rate of 4.7 liters of air/min. The effects of nebulization on the vesicles were determined by monitoring the release of encapsulated 5,6-carboxylfluorescein (CF) from dispersions containing approximately 200 micrograms of total CF, of which 93.1 +/- 2.4% (N = 18) was initially encapsulated. In all experiments CF was released from the liposomes while being aerosolized, and this ranged from a mean of 12.7 +/- 3.8 to 60.9 +/- 1.9% of the encapsulated CF, depending upon the lipid composition. The lipid concentration in the dispersions did not affect the rate or percentage release of CF over a range of approximately 0.5 to 50 mg per nebulized dispersion. If liposomes are to be used as drug carriers in an inhalation aerosol a lipid composition should be employed which will minimize the release of encapsulated drug caused by nebulization.

Solute absorption from the airways of the isolated rat lung. III. Absorption of several peptidase-resistant, synthetic polypeptides: poly-(2-hydroxyethyl)-aspartamides.

A series of samples of the synthetic polypeptide poly-alpha, beta-[N(2-hydroxyethyl)-DL-aspartamide] (PHEA), containing covalently bound fluorophore, ethylcarbonyl-6-aminofluorescein, and exhibiting different molecular weight distributions with weight average molecular weights ranging from approximately 4 to 43 kD, was prepared and characterized. Aqueous solutions of the polymers were administered to the airways of isolated perfused rat lung preparations, and transfer to the perfusate was measured. Polymers administered directly to the perfusate were not degraded during the experiment. Polymer transfer rates were dependent upon starting molecular weight distribution, larger molecules being absorbed more slowly. In the case of a polymer with a median molecular weight of 7.2 kD, the absorbed species appeared to be smaller molecules than those which were originally administered. This was not the case for a 3.98-kD polymer; absorbed material had a gel permeation chromatography elution volume equivalent to that of the administered material. Absorption for the 3.98-kD polymer was found to be dose dependent. Approximately 70% absorption of a 0.2-mg dose occurred in 100 min. Much larger polymers (up to 11.65 kD) were also absorbed at finite rates. Results are discussed in the context of macromolecular delivery to the systemic circulation via the lung.