Dry powder inhalation exposures of the endotracheally intubated rat lung, ex vivo and in vivo: the pulmonary pharmacokinetics of fluticasone furoate.

BACKGROUND: The isolated perfused rat lung (IPL) is a suitable model for studying lung-specific pharmacokinetics (PK) of inhaled drugs. So far, little has been known, however, whether the PK measured in the ex vivo organ corresponds to the PK measured in similarly exposed animals in vivo, in particular the endotracheally intubated rat (EIR). The purpose of the current research was to compare the PK of inhaled corticosteroid fluticasone furoate (FF) in the IPL and the EIR. METHOD: Aerosols of FF with mass median aerodynamic diameters ranging from 2.2 to 3.2 µm were generated with the DustGun aerosol generator. The IPL, perfused in the single-pass mode, was exposed via inhalation to 5.6 and 46 µg of FF. Following inhalation, the perfusate was repeatedly sampled for 100 min, after which the lungs were recovered for quantitation of remaining FF. Two groups of EIR were also exposed via inhalation to 7 µg of FF. One group was immediately euthanized for determination of the initial deposition of FF in the lungs. From the second group, four venous blood samples were drawn up to 4 hr after exposure. The animals were then sacrificed for determination of FF remaining in the lungs. RESULTS: Following inhalation, FF was slowly disappearing from both the IPL and the lungs of the EIR, with a half-life of pulmonary retention of 4.3-4.9 hr for all three exposure series. For the low exposure levels, the concentration curve of FF in the IPL perfusate was similar in shape to that in venous blood of the EIR, with a Cmax of 1.0 and 0.8 nM for the IPL and the EIR, respectively. CONCLUSIONS: The results indicate that the IPL and the EIR, when used jointly in PK studies, can provide a detailed characterization of inhaled drugs or toxicants.

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.

Delivering horseradish peroxidase as a respirable powder to the isolated, perfused, and ventilated lung of the rat: the pulmonary disposition of an inhaled model biopharmaceutical.

BACKGROUND: Our aim was to investigate the potential of the DustGun aerosol technology integrated with the isolated, perfused, and ventilated lung of the rat (IPL) to study the pulmonary disposition of an inhaled model biopharmaceutical, the 40-kDa protein horseradish peroxidase (HRP). METHOD: The DustGun aerosol technology was used to deliver respirable powder aerosols of HRP (the mass median aerodynamic diameter: 1.7 µm) as an 80-sec bolus to the IPL perfused in a single-pass mode. Lung perfusate was repeatedly sampled for 125 min after the HRP exposure. The amount of active HRP clearing with the perfusate or being retained in the lung was measured enzymatically. RESULTS AND CONCLUSIONS: The total amount of HRP deposited in the lungs was 335 ± 100 µg and 568 ± 47 µg for a low- and high-dose exposure, respectively. After inhalation, the initial appearance of HRP in the perfusate was rapid. However, the total amount of HRP that cleared with the perfusate remained below 0.5% of the deposited dose. The effect of opening the tight junctions between the alveolar epithelial cells on HRP absorption was studied by exposing the IPL to nebulized aerosols of either 0.02, 0.2, or 2% poly-L-Arginine (PLA) (MW 42.5 kDa) in phosphate-buffered saline (PBS) for 5 min, at 40 min after the HRP exposure. Subsequent exposure to 0.02% PLA did not affect HRP absorption. However, exposure to 0.2% PLA increased the absorption rate ninefold, and the total amount of HRP clearing with the perfusate increased to approximately 4% of the deposited dose. No further increase was obtained with 2% PLA, indicating a steep dose-response for the enhancer. It was concluded that the pulmonary absorption of HRP is quite slow, and absorption enhancers affecting tight junctions have a distinctive, yet limited efficiency. The presented inhalation technology can be very useful in studying the pulmonary absorption of biopharmaceuticals.

Prostaglandin modulation of airway inflammation and hyperresponsiveness in mice sensitized without adjuvant.

As adjuvant during sensitization may cause unspecific immune reactions, the aim of the present study was to define the role of cyclooxygenase (COX) activity on airway inflammation and airway hyperresponsiveness (AHR) in an adjuvant-free allergic mouse model. Administration of diclofenac and indomethacin (non-selective COX inhibitors), FR122047 (COX-1 inhibitor) and lumiracoxib (selective COX-2 inhibitor) enhanced AHR. Only diclofenac and lumiracoxib reduced the inflammatory cell content of bronchoalveolar lavage (BAL). Moreover, levels of prostaglandins in BAL were reduced by indomethacin and FR122047 but were unaffected by lumiracoxib. However, compared with antigen controls, none of the COX inhibitors displayed major effects on the production of cytokines, smooth muscle mass, number of goblet cells and eosinophils, or collagen deposition in the airways. These data in mice sensitized without adjuvant support the fact that COX products have a general bronchoprotective role in allergic airway inflammation. Furthermore, the data suggest that COX-1 activity predominantly generates prostanoids in BAL, whereas COX-2 activity is associated with the accumulation of inflammatory cells in BAL. This study further supports that AHR on the one hand, and the inflammatory response and generation of prostanoids on the other, are dissociated and, at least in part, uncoupled events.

Vasoconstriction after inhalation of budesonide: a study in the isolated and perfused rat lung.

INTRODUCTION: Clinical studies have shown that inhaled corticosteroids can induce rapid vasoconstriction in the airways, leading to decreased mucosal blood flow. The aim of this study was to investigate whether vasoconstriction of the pulmonary circulation after short inhalation of a corticosteroid can be detected in the isolated and perfused rat lung (IPL) - a model which could serve as a substitute or a complement to clinical models. METHODS: IPLs were briefly exposed to dry powder aerosol of budesonide. The pulmonary perfusate flow rate was assessed during 100min post-exposure. A reduction in perfusion flow rate was interpreted as vasoconstriction. MAIN RESULTS: Vasoconstriction was more pronounced after brief inhalation of 10 and 50microg budesonide than 2microg. The onset of vasoconstriction became statistically significant within 10-40min after inhalation. Co-administration of a selective alpha(1)-adrenoceptor antagonist (prazosin 50nM added to the perfusate) reduced vasoconstriction by approximately 50% during 100min of perfusion (p=0.003). CONCLUSIONS: Inhaled budesonide rapidly induces pulmonary vasoconstriction suggesting a nongenomic mechanism probably related to disposition of noradrenaline at the neuro-muscular junction. This ex vivo model could serve as a substitute or a complement to clinical models for investigating rapid effects of glucocorticoid receptor agonists on the pulmonary/bronchial circulation.

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites.

Leukotriene D(4) (LTD(4))-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D(2)-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD(4) and PGD(2) were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD(4)-induced bronchoconstriction and thromboxane (TX) A(2) release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT(1) receptor activation by LTD(4) was established by measurements of immunoreactive 6-keto PGF(1alpha) (a stable metabolite of PGI(2)) and PGE(2). In contrast, TP receptor-mediated bronchoconstriction by PGD(2) was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE(2) was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD(2) nor U-46619 actively released PGE(2), but continuous production of PGE(2) and PGI(2) was established, and the response to PGD(2) was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD(4) and PGD(2) in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD(4) was amplified predominantly by secondarily released TXA(2), that induced by PGD(2) was attenuated by bronchoprotective PGE(2) and PGI(2), presumably tonically produced in the airways.

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.

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.

Increasing exposure levels cause an abrupt change in the absorption and metabolism of acutely inhaled benzo(a)pyrene in the isolated, ventilated, and perfused lung of the rat.

The carcinogenic polycyclic aromatic hydrocarbons (PAHs) are active primarily at the site of entry to the body. Lung cancer following inhalation of PAH-containing aerosols such as tobacco smoke is one likely example. A suggested mechanism for this site preference is a slow passage of the highly lipophilic PAHs through the thicker epithelia of the conducting airways, accompanied by substantial local metabolism in airway epithelium. However, it is likely that the airway epithelium will become saturated with PAHs at surprisingly low exposure levels. The purpose of this research was to quantify the level of saturation for inhaled benzo(a)pyrene (BaP) in the isolated, perfused lung (IPL) of the rat. BaP was coated onto carrier particles of silica 3.5 microm diameter at three different levels. The DustGun aerosol generator was then used to deliver respectively 2.2, 36, and 8400 ng of BaP to the IPL with the carrier particles in less than 1 min. For 77 min after the exposure, single-pass perfusate was collected from the lungs. Lungs were then removed and, with the perfusate, analyzed for BaP and metabolites. Results show that the absorption and metabolism of inhaled BaP in the lungs was highly dose dependent. At low exposure levels absorption of BaP in the mucosa was proportional to the concentration in the air/blood barrier and proceeded with substantial local metabolism. At higher exposure levels the capacity of the epithelium to dissolve and metabolize BaP became saturated, and the absorption rate remained constant until crystalline BaP had dissolved, and the process proceeded with much smaller fractions of BaP metabolites produced in the mucosa. This phenomenon may explain the well-known difficulties of inducing lung cancer in laboratory animals with inhalants containing carcinogenic PAHs, where similar lifespan exposures are used as humans may experience but with much higher dose rates.

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 microm, not much larger than the 0.25 microm MMAD of diesel exhaust particles measured in air. A fine powder such as 3-microm silica particles was completely deagglomerated to an aerosol with a MMAD of 3.5 microm. 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 approximately 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 microg (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.

Interactions among three classes of mediators explain antigen-induced bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

Intravascular challenge of isolated perfused and ventilated guinea pig lung (IPL) from actively sensitized guinea pigs, with cumulatively increasing (10-10,000 microg) doses of ovalbumin (OVA), resulted in dose-dependent and reproducible reductions in lung conductance. The antihistamines mepyramine (1 microM) and metiamide (1 microM), the leukotriene antagonist zafirlukast (0.1 microM), or the cyclooxygenase enzyme (COX) inhibitor diclofenac (10 microM) each caused a parallel and rightward shift in the dose-response relation for OVA, providing evidence for contributions of histamine, cysteinyl-leukotrienes, and COX products to the OVA-induced bronchoconstriction in the IPL. Moreover, when all three drugs were combined there was a complete abolishment of the response to OVA. When two antagonists or inhibitors were combined, the results, however, were more complex. The 5-lipoxygenase inhibitor BAY x1005 (30 microM) and the thromboxane (TP) receptor antagonist BAY u3405 (1 microM) given as single treatment did not inhibit the response to OVA. However, combinations of different antagonists/inhibitors, including BAY x1005 and BAY u3405, caused pronounced inhibitions of the antigen responses, suggesting synergism in action. On the basis of these data it was concluded that although histamine and cysteinyl-leukotrienes mediate the major part of the bronchoconstriction, one or several prostanoids other than thromboxane contribute to the bronchoconstriction evoked by OVA. Moreover, the effect of diclofenac involved a dual action because it also made the IPL less sensitive to histamine and LTD4. The findings resemble and extend recent observations in clinical studies of patients with asthma and support the usefulness of this particular model in airway pharmacology.