Nasal retention of budesonide and fluticasone in man: Formation of airway mucosal budesonide-esters in vivo

Abstract

Aims

The efficacy of topical glucocorticosteroids in rhinitis and asthma is likely to depend on drug retention in the airway mucosa. With fluticasone propionate, retention may be achieved exclusively by lipophilicity, whereas for budesonide an additional possibility may be provided by its ability to form fatty acid esters in the airway mucosa that release the active drug. The aim of the present study was to determine the nasal mucosal retention of budesonide and fluticasone propionate, and the occurrence of budesonide-esters (budesonide-oleate, budesonide-palmitate) in the nasal mucosa.

Methods

In the present study, involving 24 healthy subjects, we have examined nasal mucosal drug retention of single doses of topical budesonide (256 µg) and fluticasone propionate (200 µg). Treatments were given consecutively and the administration sequence was randomised. Subjects were randomised into four parallel groups and two nasal biopsies were taken from each subject, i.e. before and at 2 h, at 2 and 6 h, at 6 and 24 h, or before and at 24 h after drug administration, resulting in 12 biopsies/time point. The measurement of unesterified budesonide, budesonide-oleate, budesonide-palmitate, and fluticasone propionate was based on microwave extraction procedures combined with liquid-chromatography/tandem mass-spectrometry.

Results

Neither of the analytes was detected in samples taken before glucocorticosteroid administration. After administration, unesterified budesonide, budesonide-esters, and fluticasone propionate were detected in the tissue from 23, 20, and 19 subjects, respectively. The mean tissue levels of budesonide at 2 and 6 h were 1051 and 176 pmol g⁻¹; the mean levels of fluticasone propionate at these time points were 237 and 10 pmol g⁻¹. The dose-corrected budesonide/fluticasone propionate tissue concentration ratios were 3.5 (P = 0.07) and 13.7 (P < 0.0002), respectively. At 24 h, budesonide and fluticasone propionate were detected in 8/12 and 3/12 of the biopsies, respectively.

Conclusions

The present study demonstrates the formation of budesonide-esters in the human nasal mucosa in vivo, and that budesonide is retained in the nasal mucosa to a greater extent than fluticasone propionate. It is suggested that the formation of budesonide-esters and their subsequent release of budesonide contributes to an extended retention of budesonide in the airway mucosa.

Introduction

Topical glucocorticosteroids are highly effective in the treatment of allergic rhinitis and asthma, and have an effect duration allowing once daily dosing in seasonal and perennial rhinitis [1]. It is likely that their clinical efficacy and, particularly, their duration of action are dependent on retention of the drugs in the airway mucosa. Budesonide and especially fluticasone propionate are lipophilic compounds (octanol/phosphate buffered saline partition, expressed as log D, is 3.2 and 4.2 for budesonide and fluticasone propionate, respectively). This characteristic may in part explain a prolonged mucosal retention. In addition, it has been demonstrated that budesonide, but not fluticasone propionate, has a capacity to form fatty acid esters in the airway mucosa [2–4]. Thus, in rat tracheobronchial airways topical budesonide forms pharmacologically inactive, intracellular fatty acid esters. By action of lipases budesonide is released from these esters in a rate limited fashion [2]. Hence, this reversible metabolism may contribute to a prolonged airway mucosal retention of budesonide [3, 4].

In this study, involving healthy subjects receiving intranasal drugs, we have examined the presence of budesonide and fluticasone propionate in nasal biopsies obtained after single dose administration. We have also determined the appearance of budesonide-esters (budesonide-oleate, budesonide-palmitate) in the nasal mucosa of these subjects.

Methods

Study design

Healthy subjects received single doses of budesonide (256 µg; 128 µg per nostril) and fluticasone propionate (200 µg; 100 µg per nostril). The subjects were randomised into four parallel groups and two nasal biopsies were taken from each subject, i.e. before and at 2 h, at 2 and 6 h, at 6 and 24 h, and before and at 24 h after drug administration (resulting in 12 biopsies/time point). Nasal biopsy levels of unesterified budesonide, budesonide-oleate, budesonide-palmitate, and fluticasone propionate were determined. The treatments were given in an open and parallel group design. The order in which the drugs were administered was randomised so that half of the subjects started with budesonide.

Subjects

Twenty-four healthy subjects, 18 men and 6 women, aged 22–33 (mean 25) years, participated in the study. The subjects had no history of general, nasal, or allergic disease. Exclusion criteria were nasal septal deviation, pregnancy or lactation, nasal drug administrations within 2 weeks prior to the study, use of oral contraceptives or hormonal implants, smoking, and strenuous physical exercise within 24 h prior to the study. The study was approved by the National Bioethics Committee (Reykjavik, Iceland) and written informed consent was obtained.

Study drug administration

Budesonide and fluticasone propionate administration took place in a room separated from the biopsy room so as to avoid any contamination of nasal biopsy samples by airborne study drug droplets. The study drugs were administered consecutively. Budesonide nasal aqueous spray (Rhinocort® Aqua, AstraZeneca, Sweden, 64 µg/dose) and fluticasone propionate nasal aqueous spray (Flutide® Nasal, Glaxo Wellcome, 50 µg/dose) were administered as two actuations per nasal cavity of each glucocorticosteroid. The subjects were sitting with the head flexed forward 5–10 degrees from the upright position.

Nasal biopsies

Nasal biopsies were taken on two occasions from each subject (at the time points specified above). Topical anaesthesia was achieved by placing three surgical patties (Johnson & Johnson Professional Inc., MA), each soaked in 1.0 ml of a solution of tetracain (20 mg ml⁻¹) and adrenaline (0.1 mg ml⁻¹), in the nasal cavity for 10 min. The surgical patties were placed in the nasal cavity for 10 min. Nasal biopsies were taken from the inferior aspects of the inferior nasal turbinate about 0.5 cm from its anterior margin. A punch forceps with a 2 mm punch was used for this purpose. The first biopsy was taken from the right nasal cavity and the second from the left nasal cavity. Immediately after the biopsies were obtained they were rinsed in isotonic saline and excess fluids were removed using a sterile tissue. The biopsies were transferred to a polypropylene tube that was sealed with a screw cap and immediately frozen at −20 °C to be transferred to −70 °C as soon as possible.

Analysis

The measurement of unesterified budesonide, budesonide-oleate, budesonide-palmitate, and fluticasone propionate was based on microwave extraction procedures combined with liquid-chromatography/tandem mass-spectrometry [11]. Limit of quantification (LOQ) was defined by the lowest quality control sample found relevant from earlier studies of method characteristics. Limit of detection (LOD) was defined as the lowest concentration where the analyte chromatographic peak was significantly greater than the background in any of the blank samples used as controls. The LOD was 50, 65, 71, and 50 pm, for budesonide, budesonide-oleate, budesonide-palmitate, and fluticasone propionate, respectively, in 0.5 ml of extraction medium. The LOQ in 0.5 ml of extraction medium was 80 pm for all analytes. For budesonide, fluticasone propionate, budesonide-oleate, and budesonide-palmitate, the coefficients of variation were 13%, 14%, 5%, and 10%, respectively, at 150 pm. The accuracies during the study for these analytes at 150 pm were 104%, 100%, 102%, and 108%, respectively.

Statistics

Geometric means were calculated for the tissue levels of unesterified budesonide, budesonide-oleate, budesonide-palmitate, and fluticasone propionate. Logarithmic scales were used in the plots of these variables. Values below the LOD were estimated at LOD/2 and values between LOD and LOQ were estimated at the arithmetic mean of the two. Differences between tissue levels of unesterified budesonide and fluticasone propionate were examined using a t-test (two-sided test) and calculations of confidence intervals (CI) (two-sided test). P levels < 0.05 were considered significant. Randomization order for dosing was not a covariate.

Results

The weight of the nasal mucosal samples ranged from 3 to 25 mg. No analytes were detected in biopsies obtained before administration of the glucocorticosteroids.

Unesterified budesonide, budesonide-esters, and fluticasone propionate were detected in the tissue from 23, 20, and 19 subjects, respectively. At 2 h, all analytes could be quantified in all the samples that were obtained. At 6 h, unesterified budesonide could be quantified in all samples, budesonide-oleate in 11 of 12 samples, budesonide-palmitate in 9 of 12 samples, and fluticasone propionate in 11 of 12 samples. At 24 h, budesonide could be quantified in 8 of 12 samples, budesonide-oleate in 3 of 12 samples, budesonide-palmitate in none of the 12 samples, and fluticasone propionate in 3 of 12 samples (Table 1).

Table 1.

Budesonide (unesterified), budesonide esters and fluticasone propionate in nasal mucosal biopsy samples obtained 2, 6 and 24 h after treatment.

	Geometric means in pmol g−1 tissue (CV%) [number of quantifiable samples/total number of samples]
	2 h	6 h	24 h
Budesonide (unesterified)	1051 (204)	176 (192)	9 (273)
	[12/12]	[12/12]	[8/12]
Budesonide oleate	443 (68)	71 (162)	–
	[12/12]	[11/12]	[3/12]
Budesonide palmitate	29 (69)	6 (91)	–
	[12/12]	[9/12]	[0/12]
Fluticasone propionate	237 (655)	10 (299)	–
	[12/12]	[11/12]	[3/12]

Subjects with high concentrations of budesonide esters at 2 and 6 h also presented high concentrations of unesterified budesonide (Figure 1). The variability (CV%) of budesonide-oleate unadjusted or normalized against unesterified budesonide was at all times lower than that of budesonide (Table 1).

Figure 1.

Individual (connencted by thin lines for budesonide and its esters) and mean concentrations of budesonide (unesterified) (□), budesonide-oleate+budesonide-palmitate (), and fluticasone propionate () in human nasal mucosal biopsies obtained 2, 6, and 24 h after topical administration of budesonide (256 µg) and fluticasone propionate (200 µg). (***denotes P < 0.001.)

The descriptive statistics of the individual analytes are presented in Table 1. The amount of budesonide-esters amounted to about 45% of the unesterified budesonide at 2 and 6 h. At 24 h, estimation of a budesonide-ester/unesterified budesonide ratio was not performed because of few data points. The mean tissue levels of budesonide at 2 and 6 h were 1051 and 176 pmol g⁻¹, and the mean levels of fluticasone propionate at these time points were 237 and 10 pmol g⁻¹, respectively. Since unequal doses of unesterified budesonide and fluticasone propionate were given, a dose-corrected test was performed. The dose-corrected unesterified budesonide/fluticasone propionate ratios at 2 and 6 h were 3.5 (CI 0.9–13.7, P = 0.07) and 13.7 (CI 4.7–40.5, P = 0.0002), respectively. The dose-corrected (unesterified budesonide+budesonide esters)/fluticasone propionate ratios at 2 and 6 h were 5.6 (CI 1.6–19.7, P = 0.00001) and 21.0 (CI 7.5–58.4, P = 0.001), respectively. At 24 h, unesterified budesonide and fluticasone propionate were detected in 8/12 and 3/12 of the biopsies, respectively.

Discussion

The present study demonstrated that budesonide had a prolonged mucosal retention compared with fluticasone propionate in healthy volunteers who received single doses of these drugs as topical nasal treatments. The study further documented the formation of budesonide esters in the human nasal mucosa in vivo. These observations are of interest in relation to the clinical use of these glucocorticosteroids as topical airway drugs.

In the present study, two biopsies (one per nasal cavity) were obtained from each study subject. If more than two biopsies had been obtained this would have resulted in multiple biopsies from the same nasal cavity, allowing the possibility that local mucosal effects (including bleeding) induced by the first biopsy could have affected the outcome of the following biopsy. This consideration, together with ethical and practical reasons, resulted in a design where subjects were randomised into four different sampling groups and where two biopsies were taken per subject. Particular care was taken to rinse the biopsies once taken in isotonic saline. Thus, we regard it likely that the obtained results reflect mucosal tissue levels of glucocorticosteroids and that any levels of these substances in nasal mucosal surface liquids or in circulating blood should have contributed only marginally.

Tissue levels of all analytes were variable, as would be expected for intranasally administered drugs. Contributing factors may include individual administration techniques, differences in anatomy and mucociliary clearance between subjects, and differences in sampling sites. However, the sequential administration of two drugs with subsequent dual analyses of single samples will strongly reduce the variability of between drug comparisons, eliminating patient and sampling site variability as well as variability in sample handling. Theoretically, the present administration scheme may lead to displacement at tissue binding sites of one drug with the other. However, arguing against this was the fact that the dosing sequence appeared not to affect the present tissue drug concentrations. Also, nonspecific tissue binding has a high capacity and is unlikely to become saturated following the present doses [5]. However, saturation of specific binding to, e.g. glucocorticosteroid receptors cannot be excluded. Had this occurred in the present study, the drug with the lowest affinity for the receptor, i.e. budesonide, is the most likely of the two to become displaced. Hence, the combined administration of budesonide and fluticasone propionate will likely reduce drug to drug variability and will if anything favour tissue uptake of fluticasone propionate over budesonide.

In the present study, we have for the first time demonstrated that budesonide, after its topical administration, forms fatty acid esters (budesonide-oleate and budesonide-palmitate) in the human nasal airway mucosa. These results extend previous observations in rat tracheobronchial airways [3, 4]. They further agree with preliminary observations in human bronchial airways of patients undergoing lobe resection for bronchial cancer [6]. The nasal mucosal biopsy levels of budesonide-esters peaked already at 2 h after budesonide administration and then appeared to decline gradually. This finding is compatible with a rate-limiting release of unesterified budesonide from budesonide-esters, corroborating previous observations in rat tracheobronchial airways. The rate of decline in mucosal levels of unesterified budesonide after topical nasal administration would then be slowed down by the release of unesterified budesonide from tissue-bound esters (budesonide-oleate and budesonide-palmitate). Hence, we suggest that the formation and degradation of budesonide fatty acid esters may in part contribute to airway mucosal retention of budesonide and, by that, to the clinical efficacy and duration of action of this airway drug.

The ability to form budesonide esters appeared to be relatively stable. Thus, among the six subjects with the highest budesonide concentrations, five also had the highest levels of budesonide esters. The fact that the variability of budesonide-oleate was lower at all instances than that of unesterified budesonide implies that interindividual variability in ester formation and hydrolysis have relatively less influence on the budesonide exposure than other factors such as dosing, deposition, and distribution.

Topical budesonide and fluticasone propionate, at the recommended doses, are effective treatments for allergic rhinitis, nasal polyposis, and asthma. In the present study, at 24 h after topical administration, unesterified budesonide and fluticasone propionate were detected in 8/12 and 3/12 of nasal mucosal biopsies, respectively. The dose-corrected budesonide/fluticasone propionate ratios at 2 and 6 h were 3.5 (P = 0.07) and 13.7 (P = 0.0002), respectively. The greater ratio at 6 h (c.f. 2 h) and the fact that budesonide was detectable in a greater number of subjects than fluticasone propionate at 24 h, suggest that budesonide is retained by the nasal airway mucosa to a greater extent than fluticasone propionate. Furthermore, budesonide was present in its esterified forms at 6 and 24 h, tentatively contributing also to mucosal levels of unesterified budesonide. Whether the mucosal levels of budesonide at 24 h are sufficient to produce clinical anti-inflammatory effects is unknown. However, the results suggest that budesonide would require less frequent administration than fluticasone propionate to maintain clinical efficacy.

Airway mucosal selectivity is a desired feature of inhaled glucocorticosteroids. The airway selectivity of fluticasone propionate seems to be achieved by a very high lipophilicity coupled with a marginal availability of any swallowed portion of the drug [7]. With budesonide, an additional possibility is provided by its ability to reversibly form fatty acid esters in the airway mucosa that may hold and release the active drug locally. This mechanism has been suggested to contribute to the airway mucosal selectivity of budesonide [8] as well as being one pharmacological explanation for the clinical efficacy of budesonide when administered once daily [9]. Budesonide appears to be esterified in most tissues, although at varying degrees. For example esterification is more abundant in the trachea than in striated muscle of the rat [10]. Hence, differences between tissues in the ability to esterify budesonide may further affect the airway selectivity of this drug. Human pharmacokinetic data, assessed by plasma measurements after nasal and pulmonary dosing, have clearly indicated that the bulk portion of systemically available budesonide shows little accumulation and is readily eliminated with an estimated terminal half-life of 2–3 h [11, 12]. This indicates that peripheral esterification is relatively limited. However, further studies involving human airways and peripheral tissues are warranted to corroborate these findings.

The present study was performed on healthy subjects. However, the overall conclusions that budesonide forms airway fatty acid esters and that budesonide is retained better in the airway mucosa than fluticasone propionate may be valid also for patients with on-going allergic nasal inflammation/disease. Thus, airway inflammation per se appears not to affect fatty acid formation [13]. Furthermore, nasal blockage and secretion, associated with allergic rhinitis, may increase the variability in deposition and uptake of topical glucocorticosteroids, but since budesonide is more readily dissolved and absorbed into the airway tissue than fluticasone propionate [14] symptomatic allergic rhinitis may, if anything, reduce fluticasone propionate uptake more than it would budesonide.

In conclusion, the present study, involving healthy subjects, demonstrates the formation of budesonide-esters in the human nasal mucosa in vivo. Furthermore, this study demonstrates that budesonide, to a greater extent than fluticasone propionate, is retained in the nasal mucosa. It is suggested that the formation of budesonide-esters contributes to the extended mucosal retention of budesonide in the airway mucosa.