Introduction

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GCS¹ used in asthma therapy are today primarily administered by inhalation, with the aim of achieving high local concentrations in the airway mucosa and limiting the increase of steroid concentrations in the rest of the body. The inhaled GCS can also ameliorate bronchial hyperresponsiveness, which is much less affected by oral GCS therapy (Jenkins and Woolcock, 1988). It seems that, in addition to high receptor affinity and high first-pass liver and gut inactivation, prolonged high local concentrations in the airways are necessary for the high antiasthmatic efficacy and airway selectivity of inhaled GCS (Brattsand, 1989).

GCS receptor affinity and the preferential inactivation of inhaled GCS by liver metabolism have been studied extensively (Barnes and Pedersen, 1993). In contrast, there are few kinetic data on peak tissue levels and retention of topically applied GCS in the airway and lung tissue (Van den Bosch et al., 1993; Esmailpour et al., 1997). We reported earlier (Miller-Larsson et al., 1994) a prolonged retention of BUD in rat airway tissue in vivo, compared with FP and BDP. These findings seemed to contradict the general positive correlation between the lipophilicity of the GCS and their tissue binding ability, because BUD is less lipophilic than FP and BDP.

In the present study, the prolonged dwell-time of BUD in airway tissue has been confirmed and explained in mechanistic terms. Radiolabeled GCS, at clinically relevant doses, were administered in vivo to rat airways by various methods. In addition, the release of GCS from the exposed airway tissue into the surrounding fluid in vitro was investigated. GCS of the inhaled type (BUD, FP, and BDP), as well as systemic GCS lacking airway efficacy (HC) or airway selectivity (DEX), were studied.

We report here that BUD is conjugated extensively with fatty acids within airway tissue and lung in vivo, resulting in the formation of BUD esters at the C21-hydroxyl group. The results are in agreement with the previously reported formation of BUD esters in vitro in human lung and liver microsomes, where oleate, linoleate, palmitate, palmitoleate, and arachidonate esters of BUD were identified (fig. 1) (Tunek et al., 1997). Fatty acid esters were not detected for FP or BDP. Fatty acid esters of BUD are much more lipophilic than FP or BDP, which explains the prolonged retention of BUD in airway tissue, compared with FP and BDP. These new findings describe a novel mechanism for attaining a high topical selectivity and prolonged activity of GCS at airway levels.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structural formulas of BUD and BUD-21-fatty acid esters. The conjugation is CoA and ATP dependent, and free BUD is regenerated by lipase-catalyzed hydrolysis [reproduced from Tunek et al. (1997), with permission].

	Materials and Methods

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Materials. Male Sprague-Dawley rats were supplied by Møllegaard Breeding Center Ltd. (Skensved, Denmark). Unlabeled BUD was supplied by Astra Draco AB (Lund, Sweden). ³H-labeled GCS (radiochemical purity, >97.0%) were supplied in 99.5% ethanol by Astra Draco AB or by the Radiochemical Center (Amersham, UK). The following ³H-GCS were used: [1,2-³H]BUD, [1,2-³H]FP, [1,2-³H]BDP, [1,2,6,7-³H]HC, and [1,2,4-³H]DEX. Specific radioactivity was 80 Ci/mmol for HC and 30-40 Ci/mmol for the other GCS used (1 tritium atom/molecule yields 29.6 Ci/mmol). One batch of BUD with a specific radioactivity of 17 Ci/mmol was also used.

Study Design. Solutions of ³H-labeled GCS at concentrations between 10⁸ and 10⁵ M were administered into the rat airways by 1) perfusion of a tracheal segment in vivo, 2) intratracheal instillation, or 3) inhalation. The total radioactivity was measured in tissue and plasma at different times after administration. GCS were extracted from the exposed airways and lungs with ethanol, and the extracts were analyzed by HPLC. The release of GCS from the tracheal segments during incubation in vitro in buffer was studied, and buffer extracts were analyzed by HPLC.

Tracheal Perfusion. Before administration, ³H-GCS stock solutions were diluted in 0.9% saline solution in glass vials (final concentration of ethanol, 0.1-0.5%). For concentrations of >3 × 10⁷ M, ethanol solutions of labeled and unlabeled compounds were prepared and stored at 20°C. Dilution in 0.9% saline solution to the final concentration (0.5% ethanol) was performed before each application. Rats (300-400 g) were anesthetized ip with a mixture of fluanisone/fentanyl (Hypnorm veterinary preparation; Janssen Pharmaceutica, Beerse, Belgium), midazolam (Dormicum; Roche, Basel, Switzerland), and water (1:1:2), at a dose of 3.3 ml/kg. Anesthesia was maintained by im injections of fluanisone/fentanyl and occasional iv administration of the initial mixture. A section of trachea between the larynx and sternum was exposed in vivo and cannulated at both ends with two polyethylene catheters, to allow perfusion (Miller-Larsson and Brattsand, 1990). An additional catheter was inserted into the section of trachea connected to the lungs, to allow spontaneous respiration. For blood sampling and anesthesia, the jugular vein was catheterized. A thermostat and heated pad maintained the rat temperatures at 37.5 ± 0.5°C. H-GCS solutions were perfused (0.1 ml/min) for 10 min (t = 0-10 min), followed by continuous perfusion with 0.9% saline solution. All draining perfusate was continuously collected, and its radioactivity was measured. Rats were killed 20, 70, or 120 min after the start of perfusion. The following ³H-GCS were tested: BUD at various concentrations from 7.5 × 10⁸ to 3.0 × 10⁶ M, FP at 2.0 × 10⁸ and 1.5 × 10⁷ M, BDP at 7.2 × 10⁸ M, DEX at 2.8 × 10⁶ M, and HC at 3.4 × 10⁶ M.

Binding of GCS to Plastic Material. To calculate the dose reaching the trachea (perfused dose), the binding of GCS to polyethylene inflow catheters was determined in separate experiments in which ³H-GCS solutions were perfused only through the system of inflow catheters. The binding of FP to inflow catheters was 41.2 ± 3.9% (mean ± SD); hence, FP applied at a concentration of 2.0 × 10⁸ M reached the trachea at a concentration of 1.2 × 10⁸ M. The binding of BDP was 18.9 ± 5.8%, whereas the binding of the other steroids used was below 5%.

Intratracheal Instillation. Ethanol was removed from ³H-GCS stock solutions by evaporation under a N₂ stream, 0.9% saline solution was added to yield the desired concentration, and the solution was sonicated in a glass vial for 1-2 min on ice. Rats (290-410 g) were lightly anesthetized with inhaled enfluran (Efrane; Abbot, Scandinavia AB, Kista, Sweden), which was delivered by vaporizer together with N₂O/O₂, and were placed in the supine position on a board tilted at 30°, with the head uppermost. A solution of either [³H]BUD (1.4 × 10⁵ or 4.8 × 10⁷ M) or [³H]FP (1.5 × 10⁷ M) was instilled intratracheally, in 250 µl, through a metal cannula mounted on a plastic syringe. The amount of radioactive label remaining in the solution after instillation was used for calculation of the administered dose (therefore, there was no need to monitor the binding of GCS to the plastic material of the syringe). After instillation, rats were kept on the tilted board until they awoke (approximately 30 sec). The rats were killed 20 min, 2 hr, 6 hr, or 24 hr after intratracheal instillation.

Inhalation. Rats (210-330 g) were anesthetized with inhaled enfluran as for intratracheal instillation and were intubated with an endotracheal tube (PE-200 tubing) connected to an inhalation chamber (Eirefelt et al., 1992). After intubation, rats breathed spontaneously and inhaled ³H-GCS aerosol (table 1), together with anesthetic gases (delivered to the chamber at 2.6 liters/min), for 5 min. Rats were killed 20 min, 2 hr, or 6 hr after the start of aerosol exposure.

Generation of GCS Aerosol. ³H-GCS stock solutions were diluted 10-fold in 99.5% ethanol, to the concentrations given in table 1. GCS aerosol was generated from the ethanol solutions with a MA2S nebulizer (flow rate, 4 liters/min; Viasol, Malmö, Sweden) connected to the inhalation chamber (chamber pressure, 0.4 kPa; total chamber flow, 6.6 liters/min). Ethanol evaporated during inhalation, and amorphous particles were deposited on the airway mucosa. To estimate the concentrations of ³H-GCS during exposure, samples were drawn from the exposure chamber, at a flow rate of 0.2 liter/min, through a filter with a pore size of 1 µm (FALP02500 filter; Millipore, Bedford, MA). ³H-GCS were then extracted from the filter with ethanol (99.5%), and radioactivity was measured. The inhaled doses of the ³H-GCS were calculated according to the formula ID = CC × ET × RMV (Dahlbäck and Eirefelt, 1994), where ID is the inhaled dose (in nanomoles), CC is the chamber concentration (in nanomoles per liter), ET is the exposure time (in minutes), and RMV is the respiratory minute volume (in liters per minute) [RMV = 4.19 × 10³ × (body weight, in grams)^0.66] (McMahon et al., 1977).

Tissue and Blood Sampling. At the end of each experiment, rats were killed by heart puncture after administration of sodium pentobarbital (60 mg/ml Mebumal veterinary preparation; Pherrovet, Malmö, Sweden) iv (0.1 ml) or ip (1 ml). Blood from the right ventricle was collected, and plasma was separated after centrifugation. After intratracheal instillation and inhalation, the lung vascular system was perfused with two 60-ml aliquots of heparinized (50 IU/ml heparin) 0.9% saline solution, to reduce the impact of the blood contents of the lung. Tissue samples were rinsed briefly in 0.9% saline solution, dried on blotting paper, weighed, and frozen at 70°C.

In Vitro Release of GCS. Segments of trachea were perfused for 10 min with either [³H]BUD (7.1 × 10⁸ M), [³H]FP (1.2 × 10⁷ M), [³H]BDP (9.9 × 10⁸ M), or [³H]HC (7.4 × 10⁸ M), followed by perfusion with 0.9% saline solution for the next 10 min. Because FP and BDP markedly bound to inflow catheters, the concentration reaching the trachea was 7.1 × 10⁸ M for FP and 8.0 × 10⁸ M for BDP (see Binding of GCS to Plastic Material). Immediately after perfusion, the tracheal segments were excised and cut lengthwise into two halves, which were weighed. One half was incubated (37°C) in a glass vessel containing 10 ml of oxygenated (95% O₂/5% CO₂) standard Krebs buffer with 0.2% glucose and 10% rat plasma (to mimic extracellular fluid). The total radioactivity released into the buffer was monitored for 8 hr; 200-µl samples were withdrawn at various time points, and the volume was replaced with fresh incubation medium. After 8 hr, buffer extracts were analyzed by HPLC. The incubated tracheal segment was combusted in a sample oxidizer for determination of total radioactivity or was extracted with ethanol and analyzed by HPLC. The radioactivity present in the nonincubated segment of trachea was measured after combustion, to ensure that during the incubation procedure there was no loss of steroid resulting from binding to the glass vessels.

Extraction of GCS and Conjugates from Tissue. The exposed trachea or the lung (with intrapulmonary bronchi) was frozen in liquid nitrogen and pulverized with a mortar. Ethanol (99.5%) was added (1 ml for the tracheal samples and 5 or 10 ml for the lung samples), and the mixtures were shaken for at least 6 hr, followed by centrifugation for 10 min at 8000g. Supernatants were stored at 70°C until HPLC analysis. Combustion of duplicate samples of either trachea or powdered lung (described below) demonstrated conclusively that complete extraction was achieved with this method.

Measurement of Radioactivity. Tissue samples were weighed, dried overnight at room temperature, and combusted in a sample oxidizer (Packard 307; Packard, Groningen, The Netherlands). Recovery of sample label was >96%. Monophase-S (15 ml) and Ultimagold (10 ml) scintillation cocktails (Packard) were used for oxidized tissue samples and liquid samples, respectively. Radioactivity was measured in a liquid scintillation counter (Packard 460 CD or 300 CD).

HPLC. Sample Preparation. Ethanol extracts of trachea and lung and buffer extracts of trachea (incubation buffer from in vitro experiments) were analyzed using two different HPLC systems. Aliquots (1 ml) of the incubation buffer were extracted with 2 × 2 ml of ethyl acetate, with virtually complete extraction of radioactivity. The pooled extracts were evaporated to dryness and redissolved in ethanol.

LC System 1. Equipment consisted of a 9012 pump, 9010 solvent delivery system, 9100 autosampler, and 9050 variable-wavelength UV/visible detector (all from Varian, Walnut Creek, CA). Mobile phase A was water and mobile phase B was methanol; the gradient was as follows: 0-20 min, 40% A/60% B; at 20 min, stepwise change to 100% B; at 35 min, stepwise change back to the initial conditions. Ethanol extracts were made 60% water/40% ethanol before injection. The flow rate was 1.0 ml/min, and the column was a LiChrosphere® 100 (RP-18) column (125 × 4 mm, 5 µm; Merck, Darmstadt, Germany). Fractions were collected (0.5 min/fraction, for 50 min) and counted in a liquid scintillation counter (Packard Tri Carb 2200CA). Approximate retention times in this system were 17 min for the R-epimer of BUD, 18 min for the S-epimer of BUD, and 28-34 min for fatty acid esters of BUD.

LC System 2. The equipment was as for LC system 1, but with radioactivity detected on-line with a FLO-ONE detector (Radiomatic, C-525-TRX, version 3.01), using Ultima FLO AP scintillation cocktail (HPLC flow/scintillation flow ratio, 1:4; Packard). Ethanol solutions were diluted to 20% water before injection. The column (Supelcosil LC-18-DB, 3.3 cm × 4.6 mm, 3 µm; Supelco Inc., Bellefonte, PA) was operated at a flow rate of 1 ml/min. A three-phase gradient was used, as follows: phase A, 5% ethanol and 0.1% acetic acid; phase B, 95% ethanol and 0.1% acetic acid; phase C, 0.1% acetic acid. Phase C (0.4 ml/min) was added to the HPLC flow, through a T-connection between the injector and the column, during the injection phase and the first 1 min of the gradient. The total flow rate of phases A and B was 0.6 ml/min for the first 1 min and 1 ml/min thereafter. The initial conditions (60% A/40% B) were maintained for 5.5 min and were then changed stepwise to 15% A/85% B. Between 5.5 min and 11.5 min, the conditions were changed linearly to 100% B; at 13.5 min, the system was returned to the initial conditions. In this system, approximate retention times were 5 min for BUD (with a separation between the two epimers of 0.5 min), 10.5 min for BUD palmitoleate, and 11.3 min for both BUD palmitate and BUD oleate.

Data Analysis. The total radioactivity measured in the tissue and plasma was converted to the concentration of GCS according to the equation given above and was expressed as GCS equivalents (picomoles per gram for tissue samples and picomoles per milliliter for plasma samples) per administered nanomole. The tissue concentrations of GCS equivalents were compared among steroids, separately for each administration method, and for each airway level (trachea with main bronchi, bronchi of generations 3 and 4, and lung parenchyma). The analysis was based on analysis of variance with two factors [factor 1, treatment (i.e. steroid) or airway level (difference between steroids or airway levels); factor 2, time]. The interaction between steroid/airway level and time was also analyzed. Comparisons among steroids were performed for concentrations at t = 20 min and for percent decreases in concentration over time. The analysis was performed with a multiplicative model, which implies that the mean values of tissue GCS concentrations discussed are geometric means (presented with 95% confidence intervals). Data for in vitro experiments and for extractions with ethanol are given as arithmetic means ± SD.

	Results

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Demonstration of BUD-C21-Fatty Acid Conjugates in Airway and Lung Tissue. HPLC analysis of ethanol extracts of airways and lungs from rats that had been topically treated with BUD revealed not only free BUD but also large fractions with much higher lipophilicity than BUD (fig. 2), which had been previously shown to be fatty acid conjugates (Tunek et al., 1997). The fatty acid conjugation of BUD in airway and lung tissue was rapid. Within 20 min of topical administration, BUD conjugates represented the majority of total tissue radioactivity, whereas 20-30% of radioactivity represented free BUD (fig. 2A). The ratio between free BUD and the ester fraction decreased with the time between BUD administration and tissue excision (fig. 2); 24 hr after BUD administration, only a small percentage of free BUD was discernible in the HPLC radiochromatograms of tracheal extracts (fig. 2C). However, the tracheal concentration of BUD equivalents after 24 hr was only 3.2% of the level found after 20 min, showing that the bulk of the initially deposited BUD had been exported from the tissue within the 24-hr period (see fig. 6).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Adapted radiochromatograms (LC system 1) of ethanol extracts of trachea (A, B, and C) and lung (D). Tissue was excised at 20 min, 2 hr, and 24 hr after intratracheal instillation of BUD. Already at 20 min, approximately 80% of total retained radioactivity was represented by conjugates and 20% by free BUD, in both trachea and lung. The fraction of free BUD decreased with time after BUD administration; at 24 hr, almost only BUD conjugates were discernible in the radiochromotograms (note different scales in A-D).

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GCS Release In Vitro and Regeneration of Intact BUD from C21-Esters. The rate of radioactivity release from tracheal segments into incubation buffer was lower for BUD than for FP, BDP, and HC (fig. 3). After 2 hr of incubation, approximately 80% of radioactivity was released from tracheas perfused with FP and BDP and 100% of that from tracheas perfused with HC, compared with 40% release from tracheas perfused with BUD. During the next 6 hr, no further release of FP occurred, whereas the release of BUD was increased by an additional 25% and that of BDP by approximately 15%. The area under the curve describing the radioactivity released from the trachea as a function of time (0-8 hr) was nearly 2-fold lower (p < 0.001) for tracheas perfused with BUD, compared with other steroids.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Percentage of total tissue radioactivity released from tracheal segments in an 8-hr in vitro incubation in buffer. Tracheal segments were perfused with 3H-GCS (BUD at 7.1 × 108 M, FP at 1.2 × 107 M, BDP at 9.9 × 108 M, or HC at 7.4 × 108 M) for 10 min, followed by perfusion with saline. The trachea was excised 20 min after the start of perfusion. Values are arithmetic means (N = 4) for each GCS. The BUD AUC is nearly 2-fold lower than those for other GCS (p < 0.001, analysis of variance, followed by Student's t test).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Adapted radiochromatograms (LC system 2) of trachea ethanol extracts (A and B) and trachea incubation buffer (C) from a single experiment. Ethanol extracts were made of two halves of the same, longitudinally divided, tracheal segment, collected 20 min after intratracheal instillation of [3H]BUD; one half was extracted in ethanol directly after excision (A), and the second half was extracted in ethanol after an 8-hr incubation in buffer (B). In A, 15% of total radioactivity was represented by free BUD and 81% by BUD conjugates. During incubation in buffer, the fraction of free BUD was decreased 6-fold and that of BUD conjugates 2-fold (B). The total radioactivity remaining in trachea after incubation consisted of 91% BUD conjugates and 6% free BUD (B). Radioactivity released into the incubation buffer consisted of 93% free BUD and 2% BUD conjugates (C).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Tissue radioactivity in the perfused section of trachea (picomoles of GCS equivalents per gram of tissue), related to 1 nmol of GCS perfused, at various times after 10-min perfusion (0-10 min) with 3H-GCS solutions (BUD at 2.9 × 107 M, FP at 2.0 × 108 M, BDP at 7.2 × 108 M, HC at 3.4 × 106 M, or DEX at 2.8 × 106 M) (GCS perfusion was followed by saline perfusion for 10-120 min). Values are geometric means, with 95% confidence intervals (N = 3-5), for each GCS and time point. The values given as percentages express the ratio of tissue radioactivity at 120 min to that at 20 min.

Uptake and Retention of GCS in the Airways and Lung. The local uptake and retention of tritiated GCS in the airways and lung were expressed as the concentration of total tissue radioactivity (picomoles of GCS equivalents per gram of tissue) related to the administered dose (nanomoles). This was possible because the concentration of radioactivity in the airway and lung tissue was always directly proportional to the administered dose of GCS, regardless of the route of administration (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Tissue radioactivity (picomoles of GCS equivalents per gram of tissue), related to 1 nmol administered, at various times after intratracheal instillation of 3H-GCS solutions (250 µl) (BUD at 4.8 × 107 M or FP at 1.5 × 107 M). Values are geometric means, with 95% confidence intervals (N = 3 or 4), for each GCS and time point. The values given as percentages express the ratio of tissue radioactivity at 24 hr to that at 20 min.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Tissue radioactivity (picomoles of GCS equivalents per gram of tissue), related to 1 nmol administered, in trachea and main bronchi (A), bronchi of generations 3 and 4 (B), and lung parenchyma (C) after 5-min inhalation (0-5 min) of nebulized ethanol solutions of 3H-GCS (BUD at 3.4 × 106 or 6.3 × 106 M, FP at 2.4 × 105 M, or BDP at 1.3 × 105 M). Values are geometric means, with 95% confidence intervals (N = 4), for each GCS and time point. The values given as percentages express the ratio of tissue radioactivity at 6 hr to that at 20 min. The values of tissue radioactivity measured at 6 hr in parenchyma are given in parentheses because they probably represent mainly liver-derived metabolites with weak GCS activity.

View larger version (93K): [in this window] [in a new window]   Fig. 8.   Tape section autoradiogram of a cross-section of a tracheal segment perfused with [3H]BUD at 2.8 × 107 M (10 µCi/ml) for 10 min, followed by continuous perfusion with saline for the next 110 min. The dark areas identify the localization of radioactivity. Maximal radioactivity is seen in the mucosal and serosal compartments. Arrow, position of the tracheal smooth muscle.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Reversible BUD Conjugation with Fatty Acids. Biosynthesis of fatty acid esters of estrogens, ecdysteroids, and, to some extent, mineralocorticoids and GCS has been described in a variety of tissues and species (Pahuja and Hochberg, 1989). It has been proposed that esters of endogenous estrogens are body reservoirs of latent estrogens, requiring only lipase-catalyzed hydrolysis for activation (Pahuja and Hochberg, 1989; Hochberg et al., 1991).

Nonspecific (Non-Receptor) Tissue Affinity. The approximately equal initial local concentrations of BUD, FP, and BDP in the airway and lung tissue after inhalation of doses below or at the lower limit of the clinically relevant range (table 1) show that topical tissue uptake of inhaled GCS is not strictly related to the lipophilicity of the intact steroids (table 2) or to steroid receptor affinity. The receptor affinity for BUD is approximately 2-3-fold lower than that for FP, approximately 1.5-2-fold lower than that for 17-BMP, and approximately 20-fold higher than that for BDP (Dahlberg et al., 1984; Hochberg et al., 1991; Högger and Rohdewald, 1994). Although there were only minor differences among the GCS in initial tissue uptake, BUD had a significantly longer dwell-time in large airway tissue than did FP and BDP, in spite of the shorter half-life ( Högger et al., 1993; Högger and Rohdewald, 1994) of the BUD-receptor complex in vitro (5-6 hr), compared with the receptor complex for FP (10 hr) or 17-BMP (8 hr). Therefore, the differences among BUD, FP, and BDP in local tissue binding are not determined at the receptor level but more likely reflect different degrees of nonspecific binding of GCS to cellular and subcellular membranes (directly related to the lipophilicity of the main GCS storage forms). BUD is approximately 6-8 times less lipophilic than FP and approximately 40 times less lipophilic than BDP (table 2). However, BUD-21-conjugates are 2-4 orders of magnitude more lipophilic than intact BUD, which probably explains the longer dwell-time of BUD in airway tissue.

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Clinical Situation. We have shown that topically applied GCS of the inhaled type (BUD, FP, and BDP) are markedly better taken up from the respiratory lumen and are also better retained within airway tissue, compared with GCS of the noninhaled type (HC and DEX), which helps to explain the relatively poor antiasthmatic activity and selectivity of inhaled HC and DEX. The initial (at t = 20 min) concentrations of BUD equivalents and FP equivalents in the airway tissue were of the order of 10 pmol/g per inhaled nanomole and 100 pmol/g per instilled nanomole, reflecting airway deposition of approximately 10% of the inhaled dose (Eirefelt et al., 1992). Lung depositions of 10-15% are also achieved in human patients with the majority of inhalers (Borgström, 1993). Therefore, the instilled doses of 0.1-10 nmol/kg of body weight used in the present study (table 1) were equivalent to inhaled doses of 1-100 nmol/kg of body weight. The initial GCS equivalent tissue concentration of 10 pmol/g per inhaled nanomole in trachea (with approximately 4-fold lower concentrations in lung parenchyma) was obtained in the present study with rats of approximately 300-g body weight. Allowing for the 250-fold greater body weight and 250-fold larger alveolar surface area (Mercer et al., 1994) of 70-80-kg humans, one can calculate that the inhalation of clinically relevant GCS doses of 0.2-2 mg (400-4000 nmol) would result in initial GCS concentrations of approximately 16-160 pmol/g in the airway tissue and 4-40 pmol/g in lung parenchyma. According to our findings, these concentrations would decrease approximately 2-4-fold in the large airways and 3-10-fold in lung parenchyma 2-6 hr after inhalation. The tissue concentrations obtained are in very good agreement with the results of the human study by Van den Bosch et al. (1993), who reported a concentration of BUD in human peripheral lung averaging 5.5 pmol/g of tissue 1.5-4 hr after inhalation of 1.6 mg.

Conclusion. We have shown that intracellular fatty acid esterification of BUD within airway tissue results in a local depot of latent, slowly regenerable, free BUD. This esterification prolongs the exposure of airway tissue to active GCS, compared with the exposure achieved with FP or BDP. The prolonged tissue exposure, and thus protracted receptor saturation, may explain the prolonged anti-inflammatory activity of BUD within rat large airways, compared with FP. It is tempting to speculate that the described esterification of BUD contributes to the airway selectivity and to the efficacy of BUD in asthma treatment when it is inhaled once daily.