Colonic bacterial metabolism of corticosteroids

International Journal of Pharmaceutics 457 (2013) 268–274

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Colonic bacterial metabolism of corticosteroids Vipul Yadav, Simon Gaisford, Hamid A. Merchant, Abdul W. Basit ∗ UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK

a r t i c l e

i n f o

Article history: Received 21 June 2013 Received in revised form 12 September 2013 Accepted 15 September 2013 Available online 18 September 2013 Chemical compounds studied in this article: Prednisolone (PubChem CID: 5755) Budesonide (PubChem CID: 5281004) Beclometasone dipropionate (PubChem CID: 21700) Beclometasone monopropionate (PubChem CID: 62965) Keywords: Microbiota Human fecal slurry Degradation Colonic stability Corticosteroids Simulated colonic fluid

a b s t r a c t The aim of this study was to investigate the stability of four corticosteroids in the presence of human colonic bacteria to understand better their luminal behaviour when delivered orally in the treatment of inflammatory bowel disease. The stability of prednisolone, budesonide, beclometasone (17, 21) dipropionate (BDP) and its active metabolite, beclometasone-17-monopropionate (17-BMP), were investigated at three different concentrations following incubation in a mixed faecal inoculum (simulated human colonic fluid) under anaerobic conditions. Prednisolone, at all three concentrations, was completely degraded within 3 h. The degradation of budesonide progressed at a slower rate, with complete degradation occurring within 7 h; the degradation of the S epimer of budesonide was faster than the R epimer. BDP degraded completely within 2 h while its active metabolite 17-BMP was comparatively stable. In contrast to the results in the faecal inoculum, all molecules were stable in the simulated colonic fluid in the absence of human faeces (control). This study demonstrates that prednisolone, BDP and budesonide are completely metabolized in simulated human colonic fluid and confirms the role of colonic bacteria in the metabolism of corticosteroids. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The incidence and prevalence of inflammatory bowel disease (IBD) continues to rise across the world with 505 and 322 per 100,000 persons suffering from ulcerative colitis and Crohn’s disease respectively in Europe, and a further 249 and 319 per 100,000 persons respectively in North America (Molodecky et al., 2012). Corticosteroids have been recommended as the first line of treatment for moderate to severe inflammatory bowel diseases by major IBD therapy guidelines (Dignass et al., 2010; Kornbluth and Sachar, 2004; Mowat et al., 2011; Travis et al., 2008). Generally, topically acting orally administered corticosteroids have low bioavailability and are characterized by rapid mucosal uptake, high glucocorticoid receptor affinity and subsequently high anti-inflammatory activity by binding to the glucocorticoid receptor. This activity leads to the subsequent transactivation of anti-inflammatory proteins in the nucleus, and repression of pro-inflammatory protein expression in

∗ Corresponding author. Tel.: +44 20 7753 5865; fax: +44 20 7753 5865. E-mail addresses: [email protected], [email protected] (A.W. Basit). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.09.007

the cytosol (Barnes and Adcock, 1993; Cato and Wade, 1996; Rhen and Cidlowski, 2005). Prednisolone is a synthetic corticosteroid which is administered in the form of delayed release enteric coated tablets in the treatment of IBD. Following release of the drug in the GI tract, prednisolone is rapidly absorbed with high systemic bioavailability ranging from 75 to 98% (Al-Habet and Rogers, 1980; Bergrem et al., 1983; Tauber et al., 1984; Vogt et al., 2007). While prednisolone is very effective in inducing clinical remission in patients with IBD (Meyers and Sachar, 1990), its systemic absorption from the upper gastrointestinal tract causes severe unwanted side effects, which counterbalances its therapeutic efficacy (Campieri et al., 1997). Therefore, newer glucocorticoids including budesonide and beclometasone dipropionate (BDP) have risen in prominence in the treatment of IBD (Fasci Spurio et al., 2012). Budesonide contains an asymmetric 16␣,17␣-acetal group (Fig. 1) which results in a 1:1 mixture of 22R and 22S configuration epimers due to the chirality at C22 (Albertsson et al., 1978; Cortijo et al., 2001). The 22R epimer is found to exhibit two to three times the anti-inflammatory activity of the 22S epimer (Brattsand et al., 1982; Dahlberg et al., 1984). Budesonide is characterized by high local potency at the site of application with low systemic activity

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269

Fig. 1. Chemical structure of the four corticosteroids tested.

following absorption (Johansson et al., 1982). The high therapeutic ratio and superior side-effect profile of budesonide compared to prednisolone is due to its high glucocorticoid receptor affinity, extensive first-pass metabolism by cytochrome P450 3A (CYP3A) enzymes and gastrointestinal efflux mediated by P-glycoprotein, a product of the multidrug resistance 1 gene (MDR1) (Jonsson et al., 1995; Nunes et al., 2013). These processes in the gut leads to very low systemic bioavailability (approximately 10%) of orally administered budesonide (Jonsson et al., 1995). Budesonide for oral delivery has been developed commercially in gastro-resistant delayed release pellet formulations, which allow the drug to be delivered mainly to the distal small intestine and proximal colon (Campieri et al., 1997; Edsbacker and Andersson, 2004; Edsbacker et al., 2003). Beclometasone dipropionate (BDP) is a synthetic chlorinated corticosteroid diester characterized by potent anti-inflammatory activity (Fasci Spurio et al., 2012). Unchanged BDP has negligible oral bioavailability due to its extensive GI and first pass hepatic metabolism into the pharmacologically active metabolite 17-BMP (Daley-Yates et al., 2001). The oral formulation of beclometasone dipropionate consists of a modified release tablet core with an outer gastro-resistant coating, exhibiting a delayed drug release into the distal small bowel and proximal olon (Steed et al., 1994). The pharmacological effects of corticosteroids are directly influenced by their physiochemical and pharmacokinetic properties. The metabolism and bioavailability of corticosteroids has been previously studied extensively in skin, liver, lung and plasma from animals and humans (Andersson et al., 1982; Edsbacker et al., 1983, 1987; Foe et al., 1998, 2000; Hartiala, 1976; Jonsson et al., 1995; Nugent et al., 1959). For a drug delivered to the colon to be effective, it first must be stable in the lumen. The colon contains large numbers of bacteria (1011 –1012 cfu/mL) far in excess of other regions of the gut (Jung and Kim, 2010). These bacteria are involved in the digestion of proteins and carbohydrates, and are also involved in the biotransformation of drugs. For instance, it has been stated that the intestinal microflora has a greater or equal metabolic potential to that of the liver in the metabolism of exogenous and endogenous compounds (Scheline, 1973). Moreover, at least 30 drugs have been shown to be substrates for colonic bacterial enzymes (Sousa et al., 2008). It is feasible these bacteria and their enzymes may be involved in the degradation of corticosteroids. Hence, the aim of

the present work was to determine the colonic bacterial stability of prednisolone, budesonide, BDP and its active metabolite 17-BMP, in simulated human colonic fluid. This medium contains human faecal slurry, albeit differences in microbial distribution of colon and faeces (Marteau et al., 2001), the simulated human colonic fluid mimics the colonic microbial, enzymatic and electrolyte composition and provides a good surrogate for human colonic fluids (Vieira et al., 2013; Basit et al., 2002; Basit and Lacey, 2001). 2. Materials and methods 2.1. Materials Budesonide was obtained from Sigma Aldrich, UK and BDP was a gift from Glaxo Operations Limited (Ware, Hertfordshire, UK). 17-BMP was from Insight Biotechnology (Middlesex, UK) and prednisolone was obtained from Severn Biotech Limited (Worcestershire, UK). Peptone water and yeast extract were obtained from Oxoid Limited (Hampshire, UK). Sodium chloride and dipotassium hydrogen phosphate were obtained from Fisher Scientific. Magnesium sulphate heptahydrate and calcium chloride hexahydrate were obtained from VWR (Leicestershire, UK). Sodium bicarbonate, haemin, l-cysteine HCl, vitamin K and Resazurin were obtained from Sigma Aldrich (Dorset, UK). All other chemicals and solvents were of HPLC reagent grade and were used without further purification. 2.2. Preparation of basal medium and phosphate buffer Basal medium was used to support aerobic and anaerobic bacterial growth in the faecal slurry for accurate prediction of the degradation of the compounds over short periods of incubation (up to 24 h). It was prepared by weighting peptone water and yeast extract (3 g) into a glass flask containing distilled water (1.3 L), the resultant solution was autoclaved at 125 ◦ C for 20 min. In a 200 mL volumetric flask, sodium chloride (0.15 g), dipotassium hydrogen orthophosphate (0.06 g), of magnesium sulphate heptahydrate (0.015 g) and calcium chloride hexahydrate (0.01 g) were weighed and dissolved in distilled water (ca. 150 mL) under stirring. Following that, Tween 80 (3 mL) was added to the solution and was stirred until completely dissolved. l-Cysteine and

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bile salts (0.75 g) were added and stirred until completely dissolved. To this solution, vitamin K (15 ␮L), haemin (0.0075 g) in two drops of sodium hydroxide and 0.025% resazurin (6 mL) in deionised water solution were added under stirring. Sodium bicarbonate (3 g) was added at the end and the final volume was made up to 200 mL with distilled water. The flask was stoppered during stirring to avoid dissolution of oxygen. The resulting solution was filtered (0.22 ␮m, Millex GP syringe-driven filter units, Millipore, Ireland) into the peptone water and yeast extract solution. This step was performed aseptically in a laminar flow cabinet. The bottle was kept tightly closed and at room temperature until used. The basal medium was discarded after 1 week or if any sediment was observed. Phosphate buffer saline was prepared according to British Pharmacopoeia specifications by adding potassium dihydrogen orthophosphate (0.014 M), dipotassium hydrogen orthophosphate trihydrate (0.017 M) and sodium chloride (0.256 M) in distilled water (2 L) and stirring until dissolved. The pH was adjusted to 6.8 using 0.1 N HCl or 1 N NaOH solutions. 2.3. Preparation of human faecal slurry (simulated human colonic fluid) Fresh faecal samples were collocated in pre-weighed sterile plastic containers from three healthy volunteers. The volunteers were on no medication and had not taken antibiotics for at least the previous six months. The plastic receptacle containing freshly voided human faeces was transferred into the anaerobic workstation kept at 37 ◦ C and relative air humidity of 70%. The faecal material was diluted with pH 6.8 phosphate buffer saline (Section 2.2), to obtain 20% (w/w) slurry (60 g of faecal material was diluted with 240 g of PBS 6.80) by homogenization using an Ultra Turrax (IKA T18 Basic) homogenizer at a speed of 18,000 rpm until no large solid agglomerates were observed. The homogenized faecal slurry was sieved through 350 ␮m nylon mesh (Sefar NitexTM , Heiden, Switzerland) to remove any unhomogenised fibrous material. The basal medium prepared was then added to the sieved homogenized faecal slurry to achieve a 1:1 dilution (∼300 g of the sieved homogenized faecal slurry was diluted with 300 g of basal medium). Hence, the final concentration of faecal slurry used for stability studies was 10% (w/w) (Basit and Lacey, 2001; Basit et al., 2002). 2.4. Drug incubation studies Stability of corticosteroids in simulated human colonic fluid or human faecal slurry was carried out in an anaerobic work station (Electrotek 500TG workstation, Electrotek, UK) to facilitate the growth of anaerobic bacteria in the faecal slurry at 37 ◦ C/70% relative humidity and mixed on a horizontal shaker at 100 rpm (VXR basic Vibrax® , Leicestershire, UK). The control studies were carried out in the absence of faecal bacteria under same conditions. For all the studies, corticosteroid stock solution (0.1 mL) prepared in methanol was added to simulated colonic fluid (10 mL) to achieve final corticosteroids concentrations of 0.00274 mM, 0.0137 mM and 0.0274 mM. These concentrations equate to 0.01, 0.05 and 0.1 mg prednisolone dissolved in 10 mL fermenters. These concentrations were selected on the basis of the typical drug doses administered orally and the assumption that the total fluid volume of the colon is approximately 200 mL (Cummings et al., 1990; McConnell et al., 2008). Incubated sample mixture (150 ␮L) was withdrawn at various time points (Prednisolone and BDP, 0, 15, 30, 45, 60, 90, 120, 150 and 180 min; 17-BMP, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210, 240, 270 and 300 min; budesonide, 0, 15, 30, 60, 90, 120, 180, 240, 300, 360 and 420 min). The withdrawn sample was added to ice-cold acetonitrile (450 ␮L) as stop reagent to precipitate and inactivate the degrading enzymes. The samples were centrifuged at 10,000 rpm

for 10 min and the supernatant was analysed by HPLC to quantify intact drug remaining at each time point. 2.5. HPLC analysis The drug samples were analysed with an HPLC system (Agilent Technologies, 1260 Infinity) equipped with pump (model G1311C), autosampler (model G1329B) and a diode-array UV detector (model G1314B). For prednisolone analysis, a symmetry shield RP8 5 ␮m, 4.6 mm × 250 mm column (Waters® , Hertfordshire, UK) was used operating at 40 ◦ C and detected at 254 nm. Prednisolone eluted isocratically using a mobile phase consisting of water:tetrahydrofuran (THF):methanol (68.8%:25%:6.2%) at a flow rate of 1 mL/min for a total run time of 15 min. Retention time for prednisolone was 7.6 min. For the analysis of budesonide, BDP and beclometasone-17-monoprionate, a Kinetex 2.6 ␮ phenylhexyl 100A, 4.6 mm × 50 mm column (Phenomenex® , Cheshire, UK) was used at 40 ◦ C and detected at 254 nm. Budesonide, BDP and 17-BMP eluted using a gradient system of water (A) and acetonitrile (B) flowing at 1 mL/min. For budesonide the following gradient programme was followed: 0–10 min, 20–60% B; 10–14 min, 60–20% B. For BDP and 17-BDP the following gradient programme was followed: 0–8 min, 20–60% B; 8–12 min, 60–20% B. Retention time for BDP was 8.9 min and 17-BMP was 6.3 min. Budesonide, which is a mixture of epimer R and S, elutes with two distinct peaks. Epimer R elutes before epimer S with the former eluting at 7.3 min and latter eluting at 7.6 min. The identification and quantification of epimers was validated using the British Pharmacopoeia specifications (British Pharmacopoeia, 2013). The peak areas of each epimer were used to calculate the percentage loss of the epimers in the presence and absence of faecal microbiota. 2.6. Data analysis All experiments were performed in triplicates (unless otherwise stated) using same batch of faecal slurry to avoid inter-day variability, and results are presented as mean ± SD. Degradation kinetics were determined by fitting the percent drug remaining vs. incubation time curves to a first-order kinetic model by least squares minimization and rate constant (k) and a half-life (t1/2 ) were calculated using Origin 8.1 (OriginLab Corporation, MA, USA). Statistical differences were calculated non-parametrically at 95% confidence interval (p < 0.05). 3. Results and discussion The in vitro stability of prednisolone in simulated human colonic fluid is shown in Fig. 2. Prednisolone, at all three concentrations, was found to be degraded in simulated human colonic fluid. The two lowest concentrations of prednisolone (0.00274 mM and 0.0137 mM) degraded completely within 45 min (t1/2 = 17.2 ± 1.9 min and 13.5 ± 1.0 min respectively) while the degradation of the highest concentration (0.0274 mM) was significantly slower (p < 0.05) and completed after 150 min (t1/2 = 36.2 ± 3.8 min). No degradation of prednisolone was observed in PBS with ∼99% drug recovery after 2 h (data not shown). Prednisone is a prodrug of prednisolone, which requires conversation into prednisolone by 11-hydroxylation by hepatic ␤-hydroxydehydrogenases for glucocorticoid activity (Madsbad et al., 1980; Powell and Axelsen 1972). Al-Sanea et al. (2009) has shown that prednisone also degrades on incubation with human faecal contents into 9 different metabolites over a 96 h period. The study showed that the probability of these metabolites being pharmacologically active was 78–91%. Fig. 3 shows the metabolism of BDP to its active metabolite 17-BMP in simulated human colonic fluid. At all three

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271

110 100 90 % Drug Content

80 70 60 0.00274 mM

50 40

0.0137 mM

30

0.0274 mM

20 10 0 0 Fig. 2. Stability profile of prednisolone in simulated human colonic fluid. Each value represents mean ± SD (n = 3).

120 110 100

% Drug Content

90 80 70 60 50 40 30 20 10 0 0

15

30

45

60

75

90

105

120

135

150

165

180

Time (mins)

Fig. 3. Metabolism of BDP to active metabolite 17-BMP in simulated human colonic fluid at three concentrations, 0.00274 mM, 0.0137 mM and 0.0274 mM. Each value represents mean ± SD (n = 3). 17-BMP (0.00274 mM) ( BMP (0.0137 mM) ( (

), 17-BMP (0.0274 mM) (

), BDP (0.0137 mM) (

), BDP (0.0274 mM) (

), 17-

), BDP (0.00274 mM) ).

concentrations tested, BDP was degraded completely in simulated human colonic fluid. The lowest concentration of BDP, 0.00274 mM, degraded completely within 90 min to 17-BMP (t1/2 = 18.2 ± 1.8 min) while the intermediate concentration of 0.0137 mM degraded completely within 2 h (t1/2 = 19.0 ± 0.4 min). The highest concentration of 0.0274 mM BDP degraded completely within 3 h (t1/2 = 20.7 ± 0.8 min) to 17-BMP with no intact drug being detected. The stability of BDP in the gastrointestinal tract and subsequent metabolism in the liver influences the pharmacological activity. The rapid hydrolysis of BDP to 17-BMP has previously been shown in simulated gastric and intestinal fluids, human lung slices, cytosol from human lungs and human plasma (Andersson et al., 1982; Wurthwein and Rohdewald, 1990). Wurthwein and Rohdewald (1990) showed no hydrolysis or degradation of BDP being observed in simulated gastric fluid while rapid hydrolysis was observed in simulated intestinal fluid induced by hydrolases enzyme into 17-BMP, which was then slowly transformed to beclometasone following pseudo-first order kinetics. However, the present study is the first to show the metabolism of BDP and the stability of 17-BMP in human colonic conditions. Previous studies have shown that the relative receptor affinity to glucocorticoid receptor of 17-BMP is 26 fold higher than BDP, almost 15 fold higher

30

60

90

120

150 180 Time (mins)

210

240

270

300

Fig. 4. Stability profile of 17-BMP in simulated human colonic fluid. Each value represents mean ± SD (n = 3).

than beclometasone and 154 fold higher than beclometasone-21monopropionate (21-BMP) (Wurthwein and Rohdewald, 1990). It has been shown in a previous study (Ponec et al., 1986) that the introduction of OH group at 21 position, as in case of 17-BMP, leads to a significant enhancement in receptor binding affinity of the corticosteroid while the introduction of OH group at 17 position, as in the case of 21-BMP, significantly decreases receptor binding affinity. The higher receptor binding affinity of BDP as compared to 21-BMP might be due to the higher lipophilicity of BDP, which counteracts the negative steric hindrance effect and the lower lipophilicity of 21-BMP since the introduction of OH group at position 21 has shown to decrease the lipophilicity of the molecule and increase the steric hindrance to binding (Ponec et al., 1986). Previous studies have also shown that the esterase enzyme is responsible for degradation of BDP in gastric and intestinal fluids (Daley-Yates et al., 2001; Wurthwein and Rohdewald, 1990). The colon however has been shown to have a higher esterase activity in faecal extracts from Crohn’s disease patients (51.7 ± 19.7 ␮mol h−1 mg−1 ) and ulcerative colitis patients (39.8 ± 3.3 ␮mol h−1 mg−1 ), as compared to normal healthy volunteers (33.9 ± 3.7 ␮mol h−1 mg−1 ) (Corfield et al., 1988). The colonic lumen also contains a mixture of various bacterial secreted enzymes, apart from esterases, which might contribute to the metabolism of corticosteroids. But in the colonic mucosa the esterase activity is lower in acute (∼500 ng min−1 mg−1 ) and chronic (∼450 ng min−1 mg−1 ) IBD patients as compared to normal healthy volunteers (1447 ± 37 ng min−1 mg−1 ) (Kolios et al., 2002). Hence it is pivotal to study BDP biotransformation in both healthy volunteers and IBD patients due to the varying level of enzyme activities between them which can influence the rate of metabolism in both luminal and mucosal contents. The stability study of the pharmacologically most potent metabolite of BDP was important to understand its influence on the anti-inflammatory activity. 17-BMP when tested individually in simulated human colonic fluid was found to be stable after 5 h at all three concentrations (Fig. 4). Both BDP and its active metabolite 17-BMP were found to be completely stable in PBS solution with more than 95% drug recovery after 3 h (data not shown). Due to the rapid hydrolysis of BDP, only the active metabolite 17-BMP is absorbed through the intestinal mucosa as the C-21 esterified compounds are not absorbed or are deacetylated upon absorption. The stability profile of budesonide in simulated human colonic fluid is shown in Fig. 5. Budesonide was found to be degraded in simulated human colonic fluid with complete degradation of the lowest concentration, 0.00274 mM, within 3 h (t1/2 = 79.13 ± 2.91 min). The higher concentrations, 0.0137 mM

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100

a) 100

90

% Drug Content

0.0137 mM

70 0.0274 mM

60 50 40

% Epimer Content

0.00274 mM

80

30 20

90 80 70 60 50 40 30 20 10 0

Epimer R Epimer S

0

10

30 60 90 120 150 180 210 240 270 300 330 360 390 420

Time (mins)

0 0

30

60

90

120 150 180 210 240 270 300 330 360 390 420

b)

100 90

Time (mins)

Epimer R

70

Epimer S

60 50 40 30 20 10 0 0

30 60 90 120 150 180 210 240 270 300 330 360 390 420

Time (mins)

c)

100 90 80

% Epimer Content

and 0.0274 mM, degraded significantly more slowly (p < 0.05), within 5 h and 7 h respectively (t1/2 = 107.8 ± 11.54 min and 125.03 ± 3.92 min respectively). Budesonide occurs as a 1:1 mixture of 22R and 22S configuration epimers, and since the anti-inflammatory activity of 22R epimer has been found to be 2–3 fold higher than the 22S epimer, it was important to study the degradation behaviour of the individual epimers in simulated human colonic fluid. Fig. 6 shows the stability profile of the two epimers in three concentrations of budesonide tested. At the lowest concentration of budesonide (0.00274 mM), epimer S degraded completely after 2 h (t1/2 = 50.9 ± 4.2 min) with no unchanged epimer being detected, which was significantly faster (p < 0.05) than epimer R which degraded completely after 4 h (t1/2 = 115.5 ± 1.0 min). At 0.0137 mM concentration of budesonide, the degradation of both epimer S and R was significantly slower (p < 0.05) as compared to the lower concentration, with complete degradation occurring after 5 h and 6 h respectively (t1/2 = 73.7 ± 7.5 min and 161.2 ± 8.0 min). At this concentration, the degradation of epimer S was found to be significantly faster than epimer R (p < 0.05). At the highest concentration of budesonide (0.0274 mM), epimer S degraded completely after 6 h (t1/2 = 93.6 ± 3.5 min), which was significantly faster (p < 0.05) than epimer R which degraded completely after 7 h (t1/2 = 187.3 ± 3.5 min). Budesonide and its epimer R and S were found to be completely stable in PBS with more than 96% drug recovery after 7 h. The present results not only show that degradation starts to occur in the lumen before reaching the mucosa, but also showed epimer S degrades faster than R, which is in contrast to its behaviour in colonic mucosa, where the rate of degradation of epimer R was significantly greater than epimer S (Cortijo et al., 2001). The difference in rate of degradation of epimers in simulated colonic fluid and colonic mucosal tissue might be attributed to differences in the type of enzymes present in colonic luminal fluid and mucosal brush border membrane-bound tissue, and may also be a result of cytochrome P450 isozymes catalyzed oxidation or sulphotransferases catalyzed sulphation in the gut wall mucosa (Krishna and Klotz, 1994; Walle et al., 1993; Watkins et al., 1987). The faster rate of degradation of epimer S may be due to its more favourable structural conformation to enzymatic attack, which can have a significant effect on the pharmacological activity of budesonide in inflammatory bowel diseases due to the difference in their relative receptor binding affinities and anti-inflammatory activities. Andersson et al. (1982) have previously shown the metabolism of budesonide and BDP in human liver and it was found that

% Epimer Content

80 Fig. 5. Stability profile of budesonide in simulated human colonic fluid. Each value represents mean ± SD (n = 3).

Epimer R

70

Epimer S

60 50 40 30 20 10 0 0

30 60 90 120 150 180 210 240 270 300 330 360 390 420

Time (mins) Fig. 6. In vitro stability of budesonide epimers R and S in simulated human colonic fluid at concentration. (a) 0.00274 mM, (b) 0.0137 mM and (c) 0.0274 mM. Each value represents mean ± SD (n = 3).

the degradation of BDP was one-fourth as fast as the degradation of budesonide, compared with the present study in which the degradation of BDP was found to be faster than budesonide in simulated human colonic fluid. Hence the stability of drugs in luminal contents represents an important aspect for the development of sustained release formulation drug candidates. Table 1 summarizes the degradation rate constants (k, min−1 ), half-lives (t1/2 , min) and % drug remaining after 60 min, for prednisolone, budesonide and BDP at the different concentrations at which each drug was tested. In the case of prednisolone, degradation was rapid and the limited number of data points at the higher two concentrations precluded model fitting. Rate constants were determined for all other samples. Since the degradation of corticosteroids in simulated human colonic fluid is enzyme-catalyzed, second-order kinetics should be expected. However, because the enzyme was present in excess, degradation was observed to be pseudo first-order with respect to drug concentration. Rate constants were seen to increase with decreasing concentration and it was possible to extrapolate the trends to zero concentration.

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273

Table 1 The estimated degradation rate (k, min−1 ), half-life (t1/2 min) and % drug remaining after 60 min for prednisolone, BDP and budesonide (and its Epimers). Concentration (mM)

k (min−1 )

Prednisolone

0.0274

0.0193 ± 0.002

36.2 ± 3.8

27.83 ± 4.88

BDP

0b 0.00274 0.0137 0.0274

0.0396b 0.0383 ± 0.0036 0.0365 ± 0.0008 0.0336 ± 0.0013

17.5b 18.2 ± 1.8 19.0 ± 0.4 20.7 ± 0.8

– 7.53 ± 1.01 11.60 ± 0.31 13.83 ± 1.63

Budesonide

0b 0.00274 0.0137 0.0274

0.00904b 0.00876 ± 0.0003 0.001 ± 0.0006 0.0055 ± 0.0001

76.7b 79.13 ± 2.9 107.8 ± 11.5 125.0 ± 3.9

– 61.21 ± 1.75 57.36 ± 4.32 59.34 ± 2.35

Epimer R

0b 0.00274 0.0137 0.0274

0.0062b 0.0060 ± 0.0001 0.0043 ± 0.0002 0.0037 ± 0.0001

111.8b 115.5 ± 1.0 161.2 ± 8.0 187.3 ± 3.5

– 73.63 ± 1.10 66.75 ± 5.91 66.12 ± 3.32

Epimer S

0b 0.00274 0.0137 0.0274

0.0135b 0.0136 ± 0.001 0.0094 ± 0.0009 0.0074 ± 0.0003

51.3b 50.9 ± 4.2 73.7 ± 7.5 93.6 ± 3.5

– 48.11 ± 2.94 52.06 ± 4.66 56.19 ± 2.44

Drugs a

a b

t1/2 (min)

% Drug remaining after 60 min

The degradation was rapid and the limited number of data points at the higher two concentrations precluded model fitting. Represents predicted k and t1/2 by extrapolating the concentration to zero on a linear fit between k and corticosteroid concentration.

Although theoretical, these values indicate the fastest possible rates of degradation and so allow determination of worst-case half-lives. When considering drug release in vitro, a sustained release formulation would release drug slowly over an extended time period, and hence the local concentration of active would be low. This would have the effect of increasing the rate of degradation. A burst-release formulation, conversely, would result in a higher local concentration and so the rate of degradation would be comparatively slower. Due to the structural similarity of the corticosteroids (prednisolone, budesonide and BDP, Fig. 1), it is possible that there exists a similar mechanism of colonic enzyme mediated degradation in the simulated human colonic fluid. Sousa et al. (2008) has highlighted 30 drugs which act as substrates for gastrointestinal microbiota and the various metabolic reactions involved to confirm the role of microbiota in the biotransformation of these drugs, for example: reduction, hydrolysis, dehydroxylation, acetylation, deacetylation, proteolysis, deconjugation, and deglycosylation (Sousa et al., 2008). In case of corticosteroids, reduction of the carbonyl group at position 20 to isomeric alcohols, hydroxylation reaction at position 2 and 6 and reduction of double bond at position 4 can possibly lead to biotransformation and loss of biological activity of the corticosteroids by the intestinal microbiota (Tomkins, 2006). This microbiota-mediated degradation of corticosteroids in the lumen may have an influence on the overall anti-inflammatory activity of drugs intended for oral delivery in IBD patients, and therefore emphasizes the need for luminal stability studies in simulated colonic conditions. 4. Conclusions The corticosteroids tested in this study were prone to colonicbacterial degradation. BDP and prednisolone degraded significantly faster than budesonide, whereas 17-BMP, which is the pharmacologically active metabolite of BDP, was found to be stable. This study showed that metabolism of BDP and budesonide starts to occur in the luminal fluids before reaching the mucosa, highlighting the importance of assessing luminal stability of potential drug candidates in simulated colonic conditions. This may have clinical implications in patients with IBD where the drug is targeted to the colon to act locally on the inflamed tissues. Simulated human colonic fluid containing human faecal slurry mimics the microbial, enzymatic and electrolyte composition of colonic fluids in vitro and therefore provides an efficient means for pre-clinical testing of

drug candidates and modified release formulations to assess their luminal stability and drug release.

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