Dr I. R. Wilding, Pharmaceutical Profiles Ltd, Mere Way, Ruddington Fields, Ruddington, Nottingham NG11 6JS, UK. E-mail: firstname.lastname@example.org
Background : There is a growing clinical trend to increase the daily dose of mesalazine, which leads to significant compliance issues associated with multiple dosings of current preparations.
Aim : To examine the gastrointestinal performance and systemic exposure of a 1.5 g sachet (micropellets) mesalazine formulation, compared with three enteric-coated tablets (500 mg each, Claversal).
Methods : A randomized, two-way, cross-over pharmacoscintigraphic (scintigraphy plus pharmacokinetics) study and a two-way, cross-over, pharmacokinetic-only study were performed in 24 healthy volunteers (12 subjects per investigation).
Results : The relative bioavailability of mesalazine was 92% comparing micropellets with Claversal tablets, and the cumulative urine excretion was c. 26% for both preparations, suggesting comparable systemic exposure for the two types of preparation. In the majority of subjects, drug release from the micropellet formulation occurred predominantly in the terminal ileum and ascending colon. The Claversal tablets disintegrated in comparable intestinal sites, albeit at slightly later time points than the micropellets, principally due to slower gastric emptying for the single-unit formulation.
Conclusion : The 1.5 g micropellet formulation offers comparable delivery properties to the marketed tablets, but with greater convenience of dosing.
Mesalazine (5-aminosalicylic acid, 5-ASA) is commonly used in different formulations for the treatment of mild to moderate acute exacerbations of Crohn's disease and ulcerative colitis. The analysis of mesalazine in body fluids, its pharmacokinetics, metabolism and mode of action have been studied extensively.1–3 However, the pathogenesis of inflammatory bowel disease, and thus the precise mechanism of action of mesalazine in subjects, is not fully understood.4–15 It has been shown that the intra-individual variability in the therapeutic effectiveness of 5-ASA is directly correlated with the actual measured 5-ASA concentration in the target tissue.16,17 Therefore, the achievement of sufficiently high therapeutic drug doses at the affected regions of inflammation is an important determinant of topical efficacy in inflammatory bowel disease. The systemic availability of 5-ASA, however, is relevant to the assessment of tolerability and safety. In addition, it is an indirect measure of the systemic uptake of galenic formulations by mirroring the amount of mesalazine that has been in contact with the gut mucosa.3
Plasma concentrations of both the parent drug and the major metabolite, N-acetyl-5-ASA (N-Ac-5-ASA), are relatively high if the drug is released in the duodenum and proximal parts of the small intestine. 5-ASA absorption decreases progressively along the gastrointestinal tract.18 After the oral administration of unmodified formulations, mesalazine is rapidly and almost completely absorbed from the small bowel,19,20 thereby preventing high and therapeutically effective lumen concentrations in typically disease-affected regions, such as the distal small intestine and colon. Various different forms of 5-ASA have been developed to combat this problem, allowing a delayed or, even better, a targeted release of the agent for the oral therapy of ulcerative colitis and Crohn's disease.21
To achieve the targeted delivery of orally administered 5-ASA preparations to the terminal ileum and the colon, several enteric acrylic resin coatings have been developed that dissolve at a range of pH values. Previous scintigraphic imaging has confirmed that disintegration generally takes place in the terminal ileum and ascending colon for such products.21–23 For several 5-ASA preparations, a positive linearity between dose and therapeutic efficacy is well known, with a similar tolerability profile compared to that of placebo. As 5-ASA is, in general, well tolerated, and the incidence of observed adverse events does not appear to be dose related,17 even at therapeutic levels of up to 4 g/day,15 further efforts should be undertaken to improve the quality of life for patients.
Most patients with inflammatory bowel disease have to ingest high doses of 5-ASA, as remission maintenance therapy, for several years. Recent treatment guidelines recommend higher daily dosages of mesalazine (up to 4.8 g/day), making it difficult for many patients to comply with the recommended daily drug intake using currently available formulations.24 Therefore, it is essential to offer patients a preparation in the future that combines all the benefits of the well-established oral treatment with the advantages of a less frequent and more comfortable-to-swallow preparation. An additional rationale for a new 5-ASA micropellet formulation is to enhance patient compliance, as high-dose pellet units are easier and more comfortable to take.
A product containing enteric-coated micropellets consisting of 1.5 g 5-ASA (i.e. equivalent to three 500 mg tablets) has been developed. The 1.5 g mesalazine micropellets can be used for those indications approved for Claversal tablets and are in line with current guidelines for the treatment of inflammatory bowel disease. Using the combined approach of pharmacoscintigraphy and pharmacokinetics, the objective of the present study was to determine the gastrointestinal transit profile of the new micropellet 5-ASA formulation (1.5 g mesalazine) with regard to in vivo drug delivery.
Materials and methods
The clinical study protocol was approved by an independent ethics committee (Quorn Research Review Committee, Nottingham, UK) and all applicable regulatory requirements, including the Administration of Radioactive Substances Advisory Committee requirements. The analytical parts of the study were performed in accordance with German and Organization for Economic Co-operation and Development guidelines of Good Laboratory Practice.
The aim of the study was to investigate the gastrointestinal transit and release properties of 1.5 g 5-ASA micropellets (Merckle GmbH, Germany) vs. 3 × 5-ASA tablets (Claversal 500 mg, Merckle GmbH, Germany) using pharmacoscintigraphic and pharmacokinetic analysis. The study was conducted as a randomized, two-way, cross-over, pharmacoscintigraphic (scintigraphic plus pharmacokinetic assessment) investigation and a two-way, cross-over, pharmacokinetic-only investigation in a target population of 24 healthy male and female subjects (12 subjects in each investigation). Subjects were allocated to the pharmacoscintigraphic or pharmacokinetic-only group following the sequence of enrolment.
Each volunteer allocated to the pharmacoscintigraphic group was scheduled to receive, orally, one sachet of radiolabelled 1.5 g 5-ASA micropellets on one occasion and three radiolabelled Claversal 500 mg tablets in the subsequent dosing period. Each volunteer allocated to the pharmacokinetic-only group was scheduled to receive, orally, one sachet of unlabelled 1.5 g mesalazine (5-ASA) micropellets on one occasion and three unlabelled Claversal 500 mg tablets in the subsequent dosing period.
Radiolabelling of dosage forms
Radiolabelled mesalazine micropellets were prepared by the inclusion of samarium oxide as excipient into the dosage form. Prior to administration, the non-radioactive 152Sm was converted by neutron activation into the gamma-emitting radionuclide 153Sm. Identical drug dissolution characteristics for the clinically used formulation and the samarium oxide-containing study preparation were verified by a validated BIO-DIS in vitro dissolution method. The micropellets were irradiated for 9 min in a neutron flux of 1012 cm−2/s, 72 h prior to dosing, and in vitro testing demonstrated that the neutron activation process did not adversely affect the performance of the dosage form or the stability of the drug. At the time of dosing, the sachet of mesalazine micropellets was radiolabelled with 1 MBq of 153Sm.
The Claversal tablets were radiolabelled by the insertion of 5 mg 153Sm oxide into each tablet through a drilled microhole, which was then sealed with cyanoacrylate. Irradiation of 152Sm was carried out 24 h before tablet administration to the volunteers on the dosing day. At the time of dosing, each Claversal tablet contained 0.33 MBq of 153Sm. Dissolution testing showed that the drill and fill procedure did not alter the release properties of the product.
Subjects were required to fast from midnight on the day prior to dosing and to remain fasting until 4 h post-dose, at which time a light lunch was provided. An evening meal was provided at approximately 9 h post-dose. On subsequent days, meals were provided at 24 h (breakfast), 28 h (lunch) and 33 h (dinner) post-dose. Each subject drank 200 mL of water at 2 h post-dose and decaffeinated fluids were permitted ad libitum after lunch.
For the 12 subjects who received the radiolabelled formulations, scintigraphic images were acquired at approximately 10-min intervals up to 12 h post-dose, and then at 20-min intervals until 16 h post-dose. Thereafter, images were obtained at 18, 20, 24, 36 and 48 h post-dose. For anatomical referencing, markers containing 0.1 MBq 99mTc were taped on to the skin, where the mid-clavicular line met the right costal margin, so that they lay in approximately the same transverse plane as the pylorus. Images were recorded using a gamma camera (General Electric Maxicamera) with a 40-cm field of view and fitted with a low-energy parallel-hole collimator. The volunteers remained moderately active during the study period and all images were acquired with the subjects standing upright in front of the gamma camera. On completion of the study, the scintigraphic images were analysed to obtain the parameters detailed in Table 1.
Table 1. Scintigraphic variables for the two radiolabelled formulations
Time post-dose of gastric emptying
Time when more than 50% (or 90%) of the radiolabel has left the stomach (T50% or T90%)
Average between the time points for the two images between the transition
Time post-dose of arrival at the colon
Time when more than 50% (or 90%) of the radiolabel has reached the colon (T50% or T90%)
Average between the time points for the two images between the transition
Duration of small intestinal transit
Difference in time between colon arrival and gastric emptying
Difference in time between colon arrival and gastric emptying
Anatomical location and time post-dose of initial tablet disintegration
Time (post-dose) and anatomical location of radioactive marker release
Anatomical location and time post-dose of complete tablet disintegration
Time (post-dose) and anatomical location when all of the radiolabelled marker has dispersed and no signs of a distinct ‘core’ remain
For all subjects participating in the study, venous blood samples (8 mL) were withdrawn via an in-dwelling cannula or by venepuncture at 0 (pre-dose) 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 36 and 48 h post-dose. The samples were centrifuged at approximately 1600 × g for 10 min at 4 °C. The resulting plasma fraction was frozen in labelled polypropylene tubes at − 20 °C until the end of each study period, and then at − 80 °C until required for assay. All subjects also collected all urine passed throughout the 48-h study period, according to the following sampling periods: 0 (pre-dose), 0–4, 4–8, 8–12, 12–16, 16–24, 24–36 and 36–48 h post-dose. The total volume of urine collected for each time period was measured, and two 10 mL aliquots were taken and frozen in labelled polypropylene tubes at − 20 °C until the end of each study period, and then at − 80 °C until required for assay.
Plasma samples were analysed by high performance liquid chromatography with fluorescence detection. Frozen samples were thawed and plasma proteins were precipitated with perchloric acid. After centrifugation, the supernatant was neutralized with sodium hydroxide and an aliquot was subjected to high performance liquid chromatographic analysis. Both 5-ASA and N-Ac-5-ASA were quantified by external standardization in the established calibration range between 10 and 2500 ng/mL. Urine samples were mixed with four volumes of 50 mm ammonium acetate prior to high performance liquid chromatographic analysis. The method allowed the determination of 5-ASA and N-Ac-5-ASA in the concentration range between 0.5 and 100 µg/mL. Both methods were validated in accordance with internationally accepted recommendations.25
Plasma concentrations were used to determine the pharmacokinetic parametersCmax (ng/mL), Tmax (h), AUC0–t (ng.h/mL), T1/2 (h) and AUC0–∞ (ng.h/mL). Cmax and Tmax values were directly taken from the plasma concentration–time profiles and AUC0–t values were calculated by application of the linear trapezoidal rule. The elimination rate λ (1/h) was derived by log-linear regression analysis of the last 3–5 concentration/time point data pairs. T1/2 (h) was calculated by ln 2/λ and AUC0–∞ by AUC0–t + Clast/λ. Clast corresponds to the last quantifiable plasma concentration. Pharmacokinetic evaluation was performed employing the non-compartment analysis tool of WinNonlin Pro 3.0 (Pharsight Corporation). The cumulative urine excretion of 5-ASA and N-Ac-5-ASA was determined from the measured concentrations and documented urine volumes.
For statistical analysis, the computer program BIOQ V2.10 was used. With the inclusion of n = 24 subjects, the equivalence test had a power of 80%, when equivalence limits were set to 0.71 and 1.40. In order to achieve a better approximation to a normal distribution, AUC0–t and Cmax, as well as the excretion value Ae, were logarithmically transformed before analysis and tested parametrically by an analysis of variance (anova). anova was calculated by splitting the total variance into the components of subject and treatment. The component subject was further split into sequence and subject within a sequence.
From the result of this procedure, the two one-sided hypotheses at the α = 0.05 level of significance were tested; 90% confidence intervals of two one-sided t-tests were calculated by re-transformation of the shortest confidence interval for the difference of the log10 transformed values. anova and the determination of the confidence intervals were applied to AUC0–t, Cmax and Ae.
The mean pharmacokinetic parameters for 5-ASA and N-Ac-5-ASA after the single administration of mesalazine micropellets or Claversal tablets, both at a dose of 1500 mg 5-ASA, are summarized in Table 2. A relative bioavailability of 92% for mesalazine micropellets vs. Claversal tablets was found. The AUC0–t values of the micropellets and tablets were within the set equivalence limits for point estimates and 90% confidence intervals (Table 3).
Table 2. Pharmacokinetic parameters of 5-aminosalicylic acid (5-ASA) and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) for two mesalazine formulations with an administered dose of 1500 mg (mean ± s.d.; n = 24)
* Owing to difficulties in determining the elimination rate constant λ in a number of subjects, the descriptive statistics of the λ -derived parameters, T1/2 and extrapolated AUC0–∞, are not reported.
Percentage of 1500 mg administered dose of mesalazine (9803.92 µmol).
The AUC0–t values for both 5-ASA and N-Ac-5-ASA did not differ significantly between micropellets and tablets. Overall, the concentration of 5-ASA in plasma from both preparations was low, as expected, whereas the plasma level of the metabolite N-Ac-5-ASA was three times higher due to the known, primarily pre-systemic, 5-ASA metabolization in the gut wall and the different systemic absorption and first-pass performance in the liver when compared with the parent drug (Table 2).
The plasma concentrations of 5-ASA and N-Ac-5-ASA after ingestion of the micropellet formulation increased earlier and showed an earlier peak than the curves for the tablet (Figure 1). After administration, the first time point with quantifiable 5-ASA concentrations varied between 1 and 4 h. The absorption profile of the metabolite N-Ac-5-ASA was even faster compared with the parent drug 5-ASA. However, there was considerable inter-individual variability in the absorption of the parent drug and metabolite for both formulations.
The 5-ASA micropellets produced a bi-phasic absorption profile, with a slow initial drug absorption phase followed by a rapid increase in plasma concentration. For the Claversal tablets, the initial slow phase of drug absorption was not observed. After a prolonged lag time, the plasma concentrations increased with inter-individual variability — more gradually in some subjects and more quickly in others. The mean plasma concentration–time curves (± S.E.M.) for 5-ASA and N-Ac-5-ASA are provided in Figure 1.
After the administration of 5-ASA micropellets, the maximum concentrations varied between 416 and 5893 ng/mL for 5-ASA and between 1249 and 6644 ng/mL for N-Ac-5-ASA [for individual values (scintigraphic plus pharmacokinetic assessment group only), see Table 4]. A similar variability was observed for Claversal tablets, with maximum plasma concentration ranges of 386–5522 ng/mL (5-ASA) and 1174–7011 ng/mL (N-Ac-5-ASA).
Table 4. Correlation between pharmacokinetic parameters and transit characteristics for the micropellet formulation
The drug was primarily excreted as N-Ac-5-ASA. Within 48 h, a mean cumulative drug excretion of 26.5% (5-ASA micropellets) and 26.6% (5-ASA tablets) was determined.
For both formulations, high inter-individual variability was observed, but for the parent drug the values were generally low.
Renal excretion of 5-ASA was limited to 16 h after dosing. In all samples collected after the end of the 16-h collection period, the contribution of 5-ASA to urinary drug excretion was negligible. In contrast, individual cumulative excretion–time profiles for N-Ac-5-ASA indicated that urine excretion was not complete after 48 h. The mean urine excretion profiles are shown in Figure 2.
Scintigraphic images were analysed for all 12 subjects in line with the parameters described in Table 1, and the results are provided in Table 5.
Table 5. Gastrointestinal transit profiles (mean and individual values) for the two mesalazine formulations (h)
Gastric emptying for Claversal tablets occurred, on average, at 0.52 ± 0.36 h post-dose, compared with gastric emptying times for the micropellets of, on average, 1.28 ± 1.10 h (T50%) and 2.29 ± 2.52 h (T90%) post-dose. Mean small intestinal transit times of 3.36 ± 0.78 h were recorded for Claversal tablets. This was similar to the mean small intestinal transit (T50% and T90%) times of 2.96 ± 1.54 and 3.72 ±2.80 h reported for the micropellets. The Claversal tablets disintegrated in all subjects. Initial and complete disintegration occurred at 4.75 ± 1.24 h and at 6.11 ± 1.53 h post-dose, respectively. For Claversal tablets, initial disintegration could be seen in the terminal ileum. In other subjects, disintegration was observed at the ileo-caecal junction and in the ascending colon.
High intraluminal drug concentrations at the site of inflammatory lesions have been shown to be clinically important. At the present time, mesalazine products on the market differ considerably with respect to their drug release behaviour12 and their sensitivity to pH levels in the gut.26–28 Gamma scintigraphy has been described as an ‘elegant technique for phase-I investigation of the locality of in vivo release’,29 and has ‘become the method of choice for investigating the fate of pharmaceutical dosage [forms] in the body’.30
The ability to visualize the drug delivery process in a non-invasive manner acts to fill a significant void in current understanding. In this study, gamma scintigraphy was used to evaluate and compare the gastrointestinal transit profile of the mesalazine micropellet formulation with the anatomical site of disintegration of Claversal tablets. In addition, the relationship between in vivo drug delivery and the subsequent pharmacokinetic profile was investigated. In the present study, gamma scintigraphic analysis demonstrated that, for Claversal tablets, gastric emptying occurred well within the 120–140-min migrating myoelectric complex (MMC) cycle following administration in the fasted state.31 Gastric emptying of micropellets was delayed when compared with Claversal tablets. This is due to the gradual emptying of the micropellets from the stomach, which is typical of a multi-particulate formulation. The Claversal tablets disintegrated in the terminal ileum, ileo-caecal junction and ascending colon. In previous studies, steady state concentrations of 5-ASA, observed in biopsy specimen homogenates with Claversal 500 mg tablets, correlated very well with these sites of disintegration.32 Transit times suggest that the two dosage forms behave similarly in terms of the small intestinal residence times. There is always a high degree of intra- and inter-subject variability observed in gastrointestinal data.31 However, for the majority of subjects dosed, the intestinal transit times were in general agreement with the 3 h (± 1 h; range, 1.3–6 h) reported previously for solutions, pellets and tablets.33
The combination of scintigraphic evaluation and conventional pharmacokinetic assessment to determine the intestinal performance of pharmaceutical formulations is a powerful approach to aid in the understanding of the interaction of drug, delivery system and gastrointestinal tract. This is even more important for drugs that demonstrate their efficacy as topical agents, and that are poorly absorbed from the human intestine, for which any measure of plasma concentration is critically influenced by the location of the formulation in the gut.34
This holds true for mesalazine, which is known to be well absorbed if released high up in the intestine, but poorly absorbed from the colon. Thus, for mesalazine, the location and integrity of the pharmaceutical product will significantly influence systemic exposure. The Committee for Proprietary Medicinal Products (CPMP) has indicated that for ‘locally acting products’[pharmacokinetic] bioequivalence is generally not a suitable way to show therapeutic equivalence, as plasma levels are not relevant for local efficacy, although they may play a role with regard to safety.35 Therefore, in the context of the current study, measurements of plasma levels and pharmacokinetics proved useful for the evaluation of safety, but provided little or no information on the in vivo fate of the therapeutic moiety at its target site.
Using the scintigraphic data to assess both initial and complete disintegration of the three individual Claversal tablets provides important information on the in vivo disposition of mesalazine. Disintegration was observed to occur whilst the preparation was in the terminal ileum, ileo-caecal junction or ascending colon. In those individuals for whom the enteric coating was observed to dissolve prior to arrival in the colon, increased absorption of mesalazine was to be expected. In summary, Claversal tablets deliver mesalazine to the distal ileal and colonic lumen, without exposing the subject to excessive drug plasma levels.
The micropellet formulation has different intestinal transit characteristics from the tablet product. For single-unit preparations, gastric emptying is essentially an all-or-nothing event, i.e. all the tablets are either in the stomach or in the small bowel. However, with multi-particulate formulations, there is a gradual gastric emptying of the drug-containing cores over an extended period of time. As a consequence, the pellets gradually arrive in an area in which the enteric coating can dissolve. The plethora of micropellets spread evenly during transit through the gastrointestinal tract and release mesalazine gradually.
A careful review of the individual scintigraphic and pharmacokinetic relationships suggests that, in the majority of individuals, drug release occurs predominantly in the terminal ileum and ascending colon (Table 4) for the micropellet product.
The objective of the present study was to investigate the gastrointestinal transit and release properties of mesalazine micropellets vs. Claversal tablets using the combined approach of pharmacoscintigraphy and pharmacokinetics. The rationale of the micropellet development was to obtain an oral formulation that allows convenient higher doses and increased residence time at the target site than currently possible with the tablet product.
The overall findings suggest comparable delivery of the therapeutic moiety, mesalazine, to the target sites of likely disease for the two products. As a consequence, the study findings create confidence that, in a separate study, therapeutic equivalence for these two preparations will be shown. The availability of a substantially increased unit dose (1.5 g for the micropellets vs. 0.5 g for the tablet) would constitute a real benefit for subjects with inflammatory bowel disease.