Gut mucosal uptake and retention characteristics contribute to the high intestinal selectivity of budesonide compared with prednisolone in the rat

Authors


Dr A. Miller-Larsson, AstraZeneca R&D Lund, 221 87 Lund, Sweden. E-mail: anna.miller-larsson@astrazeneca.com

Abstract

Background:

Budesonide and prednisolone are both effective for the treatment of inflammatory bowel disease, but budesonide produces fewer adverse systemic effects. High first-pass hepatic inactivation of budesonide partially explains its favourable ratio between topical and systemic activity, but it is probable that its uptake and retention in intestinal target tissues are also contributory.

Aim:

To compare the uptake and retention of radio-labelled budesonide and prednisolone in rat ileal mucosa in vivo.

Methods:

3H-Budesonide and 3H-prednisolone were applied for 10 min directly to a perfused segment of rat ileum in vivo, followed by saline lavage every 10 min. Steroid uptake into the mucosa and submucosa was assessed at 20 min and 4 h. The uptake of budesonide was also measured in allergen-challenged animals vs. saline-challenged controls to assess whether inflammation of the mucosa with ongoing plasma exudation would impair its uptake.

Results:

Budesonide was better absorbed into ileal tissue (15-fold at 20 min) than prednisolone and better retained (50-fold at 4 h) after topical administration. The uptake of budesonide was not impaired by exudation processes following allergen challenge.

Conclusions:

The higher uptake and retention characteristics of budesonide in gut mucosa should contribute to its greater intestinal selectivity compared with that of prednisolone.

INTRODUCTION

Inflammatory bowel disease can be effectively treated with oral prednisolone, but at the expense of considerable adverse reactions.1 There is significant therapeutic advantage, therefore, in using more topically selective steroids, such as budesonide, to reduce systemic side-effects.2 Budesonide has been shown to be effective both as an enema3–5 and as controlled-release capsules.6, 7 A major advantage of budesonide over prednisolone is that it is 90% inactivated on first pass through the liver, whereas prednisolone is inactivated by only 20%.8 This efficient hepatic inactivation of budesonide contributes to its topical selectivity, but may not be the only contributing factor, as the uptake and retention characteristics of steroids in target tissues may also play a role in determining their local vs. systemic actions.

Budesonide exhibits a high selectivity in its topical anti-inflammatory action in the rat intestine.9, 10 Oral budesonide was about 30 times more potent in reducing ileal inflammation and produced less systemic effects than oral prednisolone.10 In this study, we have examined the local fate of budesonide and prednisolone in gut mucosa using an animal model in which mucosal barrier function is not impeded by the surgical procedure.11 It involves catheterization of a 10-cm segment of ileum in the rat in vivo, and measurement of the uptake of radioactive steroid into the mucosa and submucosa. The mucosal levels of absorbed budesonide were also measured following allergen challenge to assess whether an ongoing inflammatory exudation process would impair its uptake.

The concentrations of budesonide (3–30 μmol/L) and prednisolone (600 μmol/L) in perfusion solutions selected for this study correspond approximately to the commercial formulation of budesonide in Entocort enema (46 μmol/L, 2.3 mg/115 mL, AstraZeneca) and to prednisolone in Pred-Clysma (516 μmol/L, 31.25 mg/125 mL, Leiras). Furthermore, budesonide concentrations in perfused solutions correspond to the intraluminal peak concentrations of budesonide (3.6–25 μmol/L) measured in ileostomy-operated patients after oral administration of 6 mg Entocort capsules (AstraZeneca).12

METHODS

Steroid preparation

Steroid solutions were prepared with unlabelled steroid dissolved in an ethanol solution of tritiated steroid. Unlabelled and tritiated budesonide ([1,2(n)-3H]-budesonide) were obtained from AstraZeneca (Lund, Sweden). Unlabelled prednisolone (prednisolone 21-disodium phosphate) was purchased from Steraloids Inc. (Wilton, NH, USA), and tritiated prednisolone ([4,6,21(n)-3H]-prednisolone 21-disodium phosphate) from Amersham Pharmacia Biotech Ltd. (Little Chalfont, UK).

General experimental procedures

The uptake and retention characteristics of 3H-budesonide and 3H-prednisolone were studied in vivo using male Sprague–Dawley rats (300–400 g). The following procedures were carried out in all animals regardless of the subsequent experimental design. Food, but not water, was withheld from the animals for 24 h prior to experimentation to minimize the amount of visceral contents and, following anaesthetic, the small intestine was exposed by a midline incision. A 10-cm segment of the distal ileum, 10–20 cm proximal to the caecum, was isolated and perfused with 0.9% saline to remove any luminal contents. It was then ligated and catheterized at each end, care being taken not to disturb its blood supply. Finally, the abdomen was closed leaving only the two catheters protruding. During the experiments, rats were kept on heating pads with heater control to maintain body temperature. All lavage fluids (saline and steroids) were pre-warmed to 37 °C.

Specific experimental protocols

Experiment A: mucosal uptake and retention of 3H-budesonide compared with 3H-prednisolone in the exposed rat ileum.

In order to compare the tissue uptake of 3H-budesonide and 3H-prednisolone, the ileal segment was perfused gently in vivo with either 5 mL of 3H-budesonide (3 × 10–5 mol/L) or 5 mL of 3H-prednisolone (6 × 10–4 mol/L), both dissolved in 0.5% ethanol in saline (5–8 μCi/mL). About 0.5 mL of this solution filled the lumen of the ileal segment without causing undue distension, and this volume was allowed to remain in the lumen after lavage. After 10 min, the remaining solution was gently washed out from the lumen with 4 × 5 mL saline. Following this lavage, the ileal segment was washed with 5 mL saline every 10 min during the entire experiment. The rats were sacrificed either 20 min or 4 h after drug administration.

Experiment B: the influence of allergen challenge-induced plasma exudation on the topical uptake of budesonide.

The rats were sensitized to produce immunoglobulin E antibodies by means of a single intraperitoneal injection of 10 μg ovalbumin with 200 mg Al(OH)3 in saline. They were used in the experiments 3–4 weeks later. In order to investigate whether inflammation with ongoing microvascular–epithelial exudation of plasma, induced by allergen challenge, influenced the tissue uptake of locally applied steroid, 3H-budesonide at 3 × 10–6 mol/L was administered 30 min after allergen challenge (lavage with 5 mL of ovalbumin at 10–6 mol/L) or after saline. This challenge procedure was used, as it has previously been demonstrated to produce a transient 90 min plasma exudation response, peaking at 30 min, that does not injure the mucosa.9, 11 Animals were sacrificed 20 min after 3H-budesonide administration.

Termination of experiments and determination of tissue concentrations of 3H-budesonide and 3H-prednisolone

At the end of each experiment (either 20 min or 4 h after steroid), blood was sampled from the right heart ventricle and the plasma was collected. The whole rat was perfused with heparinized saline, via the aorta, to remove blood from the organs. The lungs and liver received an additional perfusion via the pulmonary artery and portal vein, respectively, to clear them of blood. Next, the gut was sectioned and washed in saline, and the mucosa (villi and lamina propria) was gently scraped from the submucosal layer of the perfused section of ileum. Other organs were removed and washed in saline, and all tissue samples were freeze-dried for 48 h.

The radioactivity of tissue samples was measured after combustion and the steroid concentration was determined from the total radioactivity according to the following formula: Q=DPM/(C × SA × W), where Q is the steroid tissue concentration (pmol/g), DPM is the mean number of disintegrations per minute in the measured sample, C=2.22 × 106 d.p.m./μCi, SA is the specific activity of tritiated steroid (μCi/pmol) and W is the weight (g) of the tissue sample. This equation converts the total radioactivity of a tissue sample into the amount of steroid equivalents (eq.), on the assumption that the measured radioactivity represents unchanged steroid. The tissue concentration of steroid equivalents is expressed as pmol/g (Table 2, see later) or pmol/g per administered nanomole (Table 1, see later). The administered dose in nanomoles was calculated as the steroid concentration in the perfused solution multiplied by the perfused volume (i.e. 5 mL).

Table 2.   Mean distribution of budesonide equivalents (calculated from the total tissue radioactivity in various dry tissues and in plasma), expressed as pmol/g (S.E.M.), and measured 20 min after perfusion of the ileal segment with 3H-budesonide at 3 × 10−6 mol/L (administered 30 min after allergen challenge or saline) Thumbnail image of
Table 1.   Mean distribution of budesonide and prednisolone equivalents (calculated from the total tissue radioactivity) in various dry tissues and in plasma, expressed as pmol/g per perfused nanomole (S.E.M.), and measured 20 min and 4 h after perfusion of ileal segment with tritiated steroid. Perfused dose was approximately 150 nmol (3 × 10−5 mol/L × 5 mL) for budesonide and 3000 nmol (6 × 10−4 mol/L × 5 mL) for prednisolone Thumbnail image of

Determination of budesonide esters

For the determination of budesonide esters and intact budesonide, ethanol extracts of mucosal and submucosal samples of perfused ileum, collected 20 min after the start of perfusion with 3H-budesonide at 3 × 10–6 mol/L, were prepared and analysed by radiochromatography as described by Miller-Larsson et al.13 using a FLO-ONE system. The tissue concentrations of budesonide and budesonide esters were calculated and expressed as pmol/g tissue.

Statistical analysis

Data are presented as the arithmetic means of 4–6 experiments and standard errors of the mean (S.E.M.). The comparison between means was performed using the Wilcoxon–Mann–Whitney test, with the computer software Astute 97, version 1.9 (Microsoft Corporation). Differences were considered to be significant at P < 0.05.

RESULTS

Comparison of uptake and retention of 3H-budesonide and 3H-prednisolone

Twenty minutes after the start of steroid exposure, the local tissue uptake of budesonide was 15 times greater than that of prednisolone (P < 0.05) in both the mucosa (villi and lamina propria) and submucosa of the exposed ileal segment (Table 1, Figure 1). At this time, the absolute concentration of budesonide equivalents in the whole exposed ileal tissue was 5 ± 0.5 nmol/g dry tissue (8 nmol/g in mucosa and 2 nmol/g in submucosa) and 0.8 ± 0.1 nmol/g wet tissue (1 nmol/g in mucosa and 0.4 nmol/g in submucosa; Figure 2, top). At 20 min after administration of either steroid, about 80% of the tissue radioactivity was located in the mucosa and 20% in the submucosa (Figure 1, top). At 4 h, 90% of budesonide radioactivity was in the mucosa and 10% in the submucosa, whereas prednisolone radioactivity had a distribution remaining at 80% vs. 20% (Figure 1, bottom). At this time, 13% of budesonide and 5% of prednisolone radioactivity (of the amounts observed at 20 min) were retained in the exposed ileal tissue. Hence, 4 h after steroid administration, the mucosal concentration of budesonide equivalents was almost 50 times greater than that of prednisolone (P < 0.05), and the submucosal concentration of budesonide equivalents remained at least 10-fold greater than that of prednisolone (P < 0.05) (Table 1; Figure 1, bottom).

Figure 1.

 Concentration of budesonide (BUD) and prednisolone (PRED) equivalents (pmol/g) per perfused nanomole in dry mucosa and submucosa of perfused Ileal segment, 20 min (n=4) and 4 h (n=5–6) after perfusion with 3H-budesonide at 3 × 10–5 mol/L and 3H-prednisolone at 6 × 10–4 mol/L.

Figure 2.

 Absolute concentration of budesonide (BUD) equivalents (pmol/g) in wet tissue of perfused ileal segment, and separately in mucosal and submucosal layers, 20 min (n=4) and 4 h (n=6) after perfusion with 3H-budesonide at 3 × 10–5 mol/L.

At 20 min, the radioactivity in tissues that had not been directly exposed to the drugs (the systemic compartment) was generally 100 times lower for both budesonide and prednisolone and, in plasma, about 500 times lower than that in the exposed ileal tissue (Table 1). The radioactivity in the duodenum, liver and kidneys was about one order of magnitude higher than that in the other non-exposed tissues, whereas the radioactivity in striated muscles (soleus muscle) was close to that in plasma. After 4 h, the ratio between the radioactivity of the exposed ileal segment and other non-exposed organs was, for budesonide, still 50–100 for tissue and 800 for plasma, whereas, for prednisolone, it was only 2–14 for tissue and 70 for plasma (Table 1).

Budesonide esterification

At 20 min after the administration of 3H-budesonide at 3 × 10–6 mol/L, the wet tissue concentration of intact budesonide in mucosa was 160.7 ± 26.9 pmol/g and that of budesonide esters was 11.4 ± 3.8 pmol/g. In the submucosa, the respective values were 33.7 ± 7.0 pmol/g for budesonide and 14.7 ± 2.3 pmol/g for budesonide esters. Thus, in mucosa, budesonide esters made up 6.6% of the total budesonide content (budesonide + budesonide esters), whereas in submucosa the proportion of esters was 31.9%.

The influence of allergen-induced plasma exudation on the local uptake of steroid

The local uptake of 3H-budesonide into the ileal mucosa and submucosa was not impaired by ongoing plasma exudation induced by allergen challenge (budesonide administered 30 min after allergen challenge during the peak of plasma exudation; Table 2) or by post-exudation conditions (budesonide administered 90 min after allergen challenge, data not shown). In fact, the tissue concentration of 3H-budesonide in the 30 min experiments was 37% and 20% higher (6–10% higher in the 90 min experiments) in the inflamed mucosa and submucosa, respectively, compared with that in control rats. However, this difference was not statistically significant (although with a statistical power of only 20–25% to detect 20–40% difference at P=0.05). In addition, the concentrations of budesonide equivalents in other tissue compartments did not differ significantly between animals exposed to allergen before steroid treatment vs. those exposed to saline only.

DISCUSSION

The present in vivo rat experiments have demonstrated that both budesonide and prednisolone, in clinically relevant concentrations, are rapidly taken up into ileal tissue. The concentrations found in the mucosa and submucosa were considerably higher (two to three orders of magnitude) than in tissues not directly exposed to the drugs (Tables 1 and 2). Importantly, budesonide was absorbed from the intestinal lumen to a much greater extent than prednisolone and was also better retained in the intestinal tissue (Figure 1 and Table 1). In addition, the uptake of budesonide was not impaired by inflammation with ongoing plasma exudation (just the opposite; there was a tendency for a higher concentration of budesonide in inflamed mucosa and submucosa, although this was not statistically significant; Table 2). This finding is not surprising as the lipophilic budesonide is absorbed via a broad transcellular uptake, whereas the luminal entry of bulk plasma occurs via a valve-like paracellular epithelial mechanism.14

In this study, the wet tissue concentration of budesonide in the ileal segment was about 0.8 nmol/g (800 nmol/L), 20 min after perfusion with 3 × 10−5 mol/L, and about 0.1 nmol/g (100 nmol/L) after 4 h (Figure 2). The receptor affinity (Kd) determined in vitro for budesonide is approximately1 nmol/L,15 demonstrating that 50% receptor saturation is achieved at this concentration. Thus, the tissue concentration of budesonide at the target application site exceeded that required for receptor saturation by two orders of magnitude even several hours after administration. In contrast, the tissue concentrations in the periphery were approximately two orders of magnitude lower than in the perfused ileum. Thus, in peripheral tissues, the Kd value was either just marginally reached or not reached at all (plasma, striated muscle). Moreover, the total radioactivity data analysed here in peripheral tissues probably represented mostly metabolites (rather than intact budesonide), especially several hours after administration. Polar metabolites in bile and urine may also explain the somewhat higher values of total radioactivity in the duodenum (which contains excreted bile), liver and kidney compared to other peripheral tissues.16

A controlled-release formulation of budesonide (Entocort capsules, AstraZeneca) is now available which is rapidly and almost completely absorbed from the gastrointestinal tract, with approximately 70% of the dose being targeted to the ileum and ascending colon.6 It has been shown to be as effective as prednisolone for the treatment of Crohn’s disease,17 particularly after once-daily (morning) administration, and is associated with a considerable reduction in steroid side-effects compared with prednisolone. A favourable topical vs. systemic potency for budesonide has also been demonstrated in experiments involving inflamed rat intestine.9, 10 This information was important to this study for the selection of a test system suitable for the investigation of the pharmacokinetic behaviour of budesonide in contact with the intestinal mucosa. The present study results demonstrate a desirable profile of absorption and mucosal retention of budesonide in the gut, thus supporting the view that budesonide exerts its effective anti-inflammatory action in Crohn’s disease due to a local topical effect rather than a systemic action. Budesonide accumulation in the local intestinal wall, together with its subsequent 90% first-pass inactivation in the liver,15 provide a comprehensive explanation of why budesonide can exhibit a greater intestinal selectivity and topical potency than prednisolone. In the treatment of ileocaecal inflammatory bowel disease, prednisolone is administered as a plain tablet and, to a large extent, will be absorbed before it reaches the ileum; therefore, its local ileal concentration will be even less than in this experimental study. Furthermore, prednisolone undergoes only a 20% first-pass inactivation in the liver;8 therefore, its therapeutic efficacy relies mainly on its systemic efficacy.

The higher uptake and retention of budesonide in the ileal wall, compared to prednisolone, can probably be explained by budesonide’s higher lipophilicity and its ability to form very lipophilic C-21 esters in the tissue. Budesonide esters are formed in the airways and lung,13, 18–20 and also in the intestinal tissue (this study). High lipophilicity per se increases transmembrane transport and retards tissue binding of steroid within cells,21 whereas it is a drawback for the dissolution of the formulation and for the distribution to mucosal surfaces. Here, budesonide has advantageous properties in that the substance, due to its water solubility (30 μmol/L), can easily be transported to the mucosal surface where, after intracellular uptake, it is partially and reversibly transformed to very lipophilic budesonide esters. Another very potent and lipophilic steroid, fluticasone propionate, has a very low water solubility (approximately 100 times lower than that of budesonide), which probably impairs its intraluminal dissolution. This may explain why fluticasone propionate is not very successful in the treatment of inflammatory bowel disease.22, 23 In contrast, budesonide, with its relatively high water solubility and moderate lipophilicity, is rapidly taken up into the tissue at the local application site and is retained there as an inactive depot in the form of very lipophilic esters; these are gradually hydrolysed, releasing active budesonide, and thereby prolonging its anti-inflammatory activity.24

In conclusion, this study has shown that budesonide is much better absorbed from the intestinal lumen and retained in the intestinal tissue than is prednisolone. The tissue concentration of budesonide is not impaired by inflammation with ongoing plasma exudation, and thus the therapeutic efficacy of budesonide should not be compromised by a highly inflamed mucosa. The present data obtained in rats suggest that local mucosal pharmacokinetics in intestines may influence the therapeutic ratios of budesonide and prednisolone.

Acknowledgements

We wish to thank Elisabeth Hjertberg for radiochromatography analysis, Dr Madeline Frame for assistance with manuscript preparation, and Dr Staffan Edsbäcker for his valuable comments on the manuscript.

Footnotes

  1. *Contributed equally to this work.

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