Åke Ryrfeldt

Evidence for the activation of the signal-responsive phospholipase A2 by exogenous hydrogen peroxide.

The intracellular events that lead to arachidonic acid release from bovine endothelial cells in culture treated with hydrogen peroxide were characterized. The hydrogen peroxide-stimulated release of arachidonic acid was time- and dose-dependent, with maximal release achieved at 15 minutes after the addition of 100 microM hydrogen peroxide. Hydrogen peroxide-stimulated release of arachidonic acid was blocked with the phospholipase A2 inhibitor quinacrine. Treatment of the cells with hydrogen peroxide did not result in liberation of oleic acid, indicating that hydrogen peroxide exercised its effect on an arachidonate-specific phospholipase. Pretreatment of the cells with antioxidants, transition metal chelators, and hydroxyl radical scavengers did not affect the hydrogen peroxide-stimulated arachidonic acid release, indicating that the response to hydrogen peroxide is not oxygen radical-mediated. The response to hydrogen peroxide does not appear to be calcium-dependent, due to the following two observations: (a) No increase in intracellular calcium was seen upon exposure of the FURA2-loaded cells to hydrogen peroxide at concentrations sufficient to release arachidonic acid, and (b) no change in the release response was detected in cells loaded with the intracellular calcium chelator BAPTA. Significant inhibition of arachidonic acid release was seen when the cells were pretreated with inhibitors of protein kinase C, but not with inhibitors of tyrosine kinase. The results of these studies indicate that hydrogen peroxide-stimulated arachidonic acid release is mediated by a specific signal-responsive phospholipase A2, and that this process is not mediated via the actions of either lipid peroxidation or calcium but, rather, that a stimulation of intracellular kinase activity is necessary for this response.

Kinetics of the epimeric glucocorticoid budesonide

Budesonide is a potent nonhalogenated glucocorticoid consisting of a 1/1 mixture of the two epimers 22R and 22S. The kinetics of these epimers were studied in six healthy male subjects after intravenous administration of 500 µg 3H‐budesonide. Estimation of the epimer concentrations in plasma was made possible by development of an HPLC method for simultaneous separation and quantification. The plasma t½, distribution volume (Vβ), and plasma clearance for epimer 22R were (X̄ ± SD) 2.66 ± 0.57 hr, 425 ± 100 l, and 117 ± 40 l/hr. The corresponding values for epimer 22S were 2.71 ± 0.69 hr, 245 ± 38 l, and 67 ± 19 l/hr. Differences in Vβ and plasma clearance between the two were significant. The larger Vβ noted for epimer 22R may be a result of higher tissue affinity. The high plasma clearance for both epimers should largely reflect their high rate of liver biotransformation.

In vitro biotransformation of glucocorticoids in liver and skin homogenate fraction from man, rat and hairless mouse

The pharmacological effects of glucocorticoids are greatly influenced by their pharmacokinetic properties. In the present report, the in vitro biotransformation of the topical glucocorticoids [3H]-budesonide ([3H]-BUD). [3H]-triamcinolone acetonide ([3H]-TAAc) and [3H]-hydrocortisone ([3H]-HC) was studied in the 9000 g liver and skin supernatant from man, rat and hairless mouse. The rate of disappearance of the compounds was estimated during the initial 30 min of incubation by high performance liquid chromatography. In human liver the half life (t1/2) rank order was [3H]-BUD (7--23 min) less than [3H]-TAAc (13--68 min) less than [3H]-HC (40--67 min), in rat liver [3H]-HC (14--21 min) less than [3H]-BUD (28--38 min) less than [3H]-TAAc (161--196 min) and in hairless mouse liver [3H]-BUD (17--22 min) less than [3H]-TAAc (21--34 min) less than [3H]-HC (82--165 min). Negligible biotransformation of these glucocorticoids occurred in skin. BUD is a one to one mixture of the [22R]- and [22S]-epimers. It was found that the [22R]-epimer was more susceptible to liver biotransformation than the [22S]-epimer of [3H]-BUD. The results are discussed with particular reference to the extent of systemic side effects of these compounds.

Comparison of Aerosol and Intranasal Challenge in a Mouse Model of Allergic Airway Inflammation and Hyperresponsiveness

Background: The aim was to optimize antigen challenge for induction of airway hyperresponsiveness (AHR) and inflammation in BALB/c mice sensitized to ovalbumin (OVA). Comparisons were made between mice challenged with OVA either as an aerosol or intranasally. The protocol that induced maximal AHR in BALB/c mice was thereafter tested in C57BL/6 mice. Method: Methacholine responsiveness was measured using the flexiVent® system to assess AHR. Inflammatory responses were investigated by histology and cell counts in bronchoalveolar lavage (BAL) fluid. Results: 48 h after challenge with 1 or 6% OVA aerosols, there were similar increments in AHR and BAL cells, predominantly eosinophils. When comparing the effect of 1% OVA aerosol on AHR and cell infiltration at 24 and 48 h after challenge, the responses were similar. At 24 h, intranasal OVA administration (20–200 µg) caused a dose-dependent increase in AHR. BAL cells were increased by all intranasal OVA doses and to a greater extent than after 1% OVA aerosol challenge but without any dose dependency. Histological examination confirmed that there was an increase of eosinophils in lung tissue following either challenge. In C57BL/6 mice, baseline tissue elastance was the only functional outcome that was increased after intranasal OVA challenge. Even though the AHR response was negligible in C57BL/6 mice, a similar infiltration of BAL cells was observed in both strains. Conclusion: Intranasal challenge was more effective than aerosol challenge at inducing both AHR and airway inflammation in BALB/c mice. Although intranasal challenge caused airway inflammation in C57BL/6 mice, this strain is not optimal for studying AHR.

Pulmonary disposition of the potent glucocorticoid budesonide, evaluated in an isolated perfused rat lung model

Budesonide is a potent glucocorticoid developed for the local treatment of respiratory disorders such as bronchial asthma and allergic rhinitis. We now report the lung disposition of 3H-budesonide administered either via the air passages or via the pulmonary circulation using isolated perfused and ventilated rat lungs. A rapid initial absorption was found after intratracheal administration of a clinically relevant dose of the drug. However, about half of the given dose was slowly released into the lung perfusate. The lung uptake of budesonide from the pulmonary circulation was relatively high (lung extraction ratio about 0.12). These data points to a high lung affinity for budesonide. The drug was not biotransformed in the lung. The high lung affinity and absence of lung metabolism can be important factors to explain the good therapeutic effects seen with budesonide in the clinic.

Uptake and biotransformation of ibuterol and terbutaline in isolated perfused rat and guinea pig lungs.

Abstract The lung uptake and biotransformation of [ 3 H]terbutaline and [ 3 H]ibuterol (diisobutyrate ester of terbutaline) was studied using isolated perfused and ventilated rat and guinea pig lungs. The lung extraction ratio, as calculated from the concentration of drug in the inflowing and outflowing medium, in single pass studies of ibuterol ranged from 0.32 to 0.41 at inflowing concentrations 1 × 10 −5 and 1 × 10 −7 M and that of terbutaline ranged from 0.013 to 0.021. Ibuterol was hydrolyzed to terbutaline but no further biotransformation of terbutaline was found. The lung clearance of ibuterol (1 × 10 −7 M) was estimated to 0.092 ml/sec. When ibuterol (1 × 10 −7 M) was infused together with eserine, an esterase inhibitor, the clearance of ibuterol decreased to 0.088 ml/sec (eserine 1 × 10 −5 M) and 0.045 ml/sec (eserine 1 × 10 −4 M). The latter value was significantly lower (P

Pharmacokinetic studies of a potent glucocorticoid (budesonide) in dogs by high-performance liquid chromatography

Pharmacokinetic studies of a potent glucocorticoid (budesonide) in dogs by high-performance liquid chromatography.

A novel method to aerosolize powder for short inhalation exposures at high concentrations: isolated rat lungs exposed to respirable diesel soot.

More efficient methods are needed to aerosolize dry powders for short-duration inhalation exposures at high concentrations. There is an increasing need to reach the peripheral lung with dry powder medications as well as with collected ambient aerosol particulates in environmental research projects. In a novel aerosol generator, a fixed volume of compressed air was used to create a short burst of a highly concentrated aerosol in a 300-ml holding chamber. Collected diesel soot was deagglomerated to a fine aerosol with a mass median aerodynamic diameter (MMAD) of 0.55 μm, not much larger than the 0.25 μm MMAD of diesel exhaust particles measured in air. A fine powder such as 3-μm silica particles was completely deagglomerated to an aerosol with a MMAD of 3.5 μm. Immediately after generation, the aerosol was available for exposure at a chosen flow rate by the use of an automated valve system. Tritium-labeled diesel soot was thus used to expose the isolated perfused rat lung at an air concentration of ∼3 mg/L and a flow rate of 370 ml/min in a 1-min-long exposure. The lungs were ventilated at 75 breaths/min and a tidal volume of 1.13 ± 0.11 ml (SD, n = 3). Results showed that 19.8 ± 1.1 μg (SD, n = 3) soot was deposited in the lungs. This amount constitutes 9.5% of the amount inhaled and is close to literature data on deposition of similar sized particles in the rat lung. More than 97% of the deposited soot was located distal to the extrapulmonary bronchi, indicating that the system delivers a highly respirable aerosol. The aerosol system is particularly useful for peripheral lung delivery of collected ambient aerosols or dry powder pharmaceuticals following a minimal effort in formulation of the powder.

Sulfur Dioxide-Induced Bronchoconstriction in the Isolated Perfused and Ventilated Guinea-Pig Lung

SO2 exposure (50-500 ppm) of isolated, perfused and ventilated guinea pig lungs, via the air passages, caused a concentration-related reduction in dynamic compliance and conductance. No changes in pulmonary perfusion flow was noted at any SO2 concentration. Formed sulfite was detected in lung lavage fluid as well as in the perfusate. Pretreatment of the lungs with a low concentration of SO2 (10 ppm) for 30 min protected against bronchoconstriction by a high concentration of SO2 (250 ppm). A similar protective effect was noted by pretreatment with sodium sulfite (3 mM) in the lung perfusate.

Short Inhalation Exposures of the Isolated and Perfused Rat Lung to Respirable Dry Particle Aerosols; the Detailed Pharmacokinetics of Budesonide, Formoterol, and Terbutaline

There is an increasing interest in using the lung as a route of entry for both local and systemic administration of drugs. However, because adequate technologies have been missing in the preclinical setting, few investigators have addressed the detailed disposition of drugs in the lung following short inhalation exposures to highly concentrated dry powder aerosols. New methods are needed to explore the disposition of drugs after short inhalation exposures, thus mimicking a future clinical use. Our aim was to study the pulmonary disposition of budesonide, formoterol, and terbutaline, which are clinically used for the treatment of bronchial asthma. Using the recently developed DustGun aerosol technology, we exposed by inhalation for approximately 1 min the isolated and perfused rat lung (IPL) to respirable dry particle aerosols of the three drugs at high concentrations. The typical aerosol concentration was 1 mug/mL, and the particle size distribution of the tested substances varied with a MMAD ranging from 2.3 to 5.3 mum. The IPL was perfused in single pass mode and repeated samples of the perfusate were taken for up to 80 min postexposure. The concentration of drug in perfusate and in lung extracts was measured using LC-MS/MS. The deposited dose was determined by adding the amounts of drug collected in perfusate to the amount extracted from the tissues at 80 min. Deposited amounts of budesonide, formoterol fumarate, and terbutaline sulphate were 23 +/- 17, 36 +/- 8, and 60 +/- 3.2 mug (mean +/- SD, n = 3), respectively. Retention in lung tissues at the end of the perfusion period expressed as fraction of deposited dose was 0.19 +/- 0.05, 0.19 +/- 0.06, and 0.04 +/- 0.01 (mean +/- SD, n = 3) for budesonide, formoterol, and terbutaline, respectively. Each short inhalation exposure to the highly concentrated aerosols consumed 1-3 mg powder. Hence, this system can be particularly useful for obtaining a detailed pharmacokinetic characterization of inhaled compounds in drug discovery/development.