Correspondence to: Dr M. Brunner, Department of Clinical Pharmacology, Division of Clinical Pharmacokinetics, University of Vienna Medical School, Allgemeines Krankenhaus, Währinger Gürtel 18–20, A-1090 Vienna, Austria. E-mail: email@example.com
Background : Mesalazine (5-aminosalicylic acid, 5-ASA)-containing formulations represent a cornerstone in the treatment of inflammatory bowel diseases. Recently, a new formulation has been developed to provide selective and more homogeneous release of 5-ASA compared to traditional systems.
Methods : In a first study, gastrointestinal transit was followed by gamma-scintigraphy after single-dose application of tablets containing 1200 mg mesalazine to 12 healthy male volunteers. 5-ASA release was verified by the assessment of plasma pharmacokinetics. In a second, 7-day, multiple-dose study, the steady state plasma pharmacokinetics, urinary excretion and safety profile were characterized after twice-daily tablet administration to 12 healthy volunteers.
Results : Tablet erosion started after 6.9 ± 1.1 h in the ascending or transverse colon. Radioactivity spread homogeneously throughout the colon, indicating the sustained release of active 5-ASA. Plasma kinetics indicated an earlier initial absorption of 5-ASA, i.e. during transit of the small intestine and ileum. Mean Cmax values (350.6 ± 322.6 ng/mL) were observed during location in the ileo-caecal region. The mean relative absorption of 5-ASA was 19.9 ± 18.2% in the small intestine and ileum and 80.1 ± 18.2% in the colon.
Conclusions : The administration of the new mesalazine formulation was well tolerated, and 5-ASA was continuously released along the whole colon, a favourable prerequisite for the therapy of distally located inflammatory bowel disease.
Mesalazine (5-aminosalicylic acid, 5-ASA), the therapeutically active moiety of sulfasalazine, is routinely employed in the treatment of inflammatory bowel disease, i.e. ulcerative colitis and Crohn's disease.1,2 Orally administered 5-ASA acts locally from the luminal side of the inflamed bowel after absorption by the colonic and ileal mucosa, and is primarily acetylated to its major metabolite N-acetyl-5-ASA (Ac-5-ASA) in the gut wall and the liver.3 On oral ingestion of most uncoated or delayed formulations, 5-ASA is almost completely absorbed from the small intestine, and only a small percentage of intact drug reaches the diseased target regions.4–6 To overcome this problem, different approaches have been pursued to achieve selective drug delivery to the distal intestinal regions. One strategy is the coupling of the mesalazine molecule to a carrier molecule (sulfasalazine, benzalamine, balsalazine) or another mesalazine molecule (olsalazine) to generate unabsorbable, inactive pro-drugs that are activated in the colonic lumen by bacterial cleavage of the diazo bond. Another option is the introduction of galenic modifications, such as enteric-coated, delayed-release tablets (Asacol, Claversal, Salofalk) or ethylcellulose-coated microgranules that release 5-ASA continuously during a period of several hours (Pentasa). Recently, a novel extended-release gastro-resistant tablet has been developed and patented (Cosmo S.p.A., Lainate (MI), Italy, under exclusive licence by Giuliani S.p.A., Milan, Italy),7 which is characterized by a special multi-matrix structure that should provide a more homogeneous and progressive release profile of 5-ASA within the colonic region, compared to the existing delivery systems.
In order to evaluate the in vivo performance of such new formulations, the non-invasive technique of gamma-scintigraphy has been employed.8–12 Gamma-scintigraphy allows the gastrointestinal transit of orally ingested preparations to be followed. In addition, the time and region of disintegration and release of the active compound can be identified. Consequently, it is possible to relate the plasma and urine pharmacokinetics of the ingested formulation to the scintigraphic pattern within the gastrointestinal tract, thereby allowing the determination of the rate and extent of absorption in a defined region of interest (‘pharmacoscintigraphy’). Gamma-scintigraphy requires drug labelling with a radioactive tracer. Radiolabelling can be achieved by either direct incorporation of a radiolabelled compound or by neutron activation of a formulation that contains a non-radioactive tracer. For the evaluation of complex formulations, such as enteric-coated tablets or pellets, labelling is best performed by the addition of a non-radioactive tracer, such as samarium-152 oxide (152Sm2O3), which is not absorbed from the gastrointestinal tract, followed by neutron activation of the finished product.10,12–14
This paper summarizes the results of two studies. In the first, a single-dose pharmacoscintigraphic investigation was performed in healthy volunteers to follow the gastric and intestinal transit of the novel mesalazine extended-release formulation, and to identify the intestinal region in which the release and absorption of 5-ASA occurred. Before oral administration, cold 152Sm was added to the formulation and was activated to radioactive 153Sm. In a subsequent, multiple-dose study, the plasma pharmacokinetics, urinary excretion and safety profile were characterized.
Materials and methods
Both studies were approved by the local ethics committees. All volunteers were given a detailed description of the study and written consent was obtained. Studies were performed in accordance with the Declaration of Helsinki and the Good Clinical Practice Guidelines of the European Commission.
Two phase I studies were conducted in different study populations. The first study was a pharmacoscintigraphic investigation designed to follow the intestinal transit and release of a single oral dose of 153Sm-labelled mesalazine extended-release tablets. The second study aimed to describe the plasma pharmacokinetics and to evaluate the safety and tolerability of the study medication following multiple-dose administration.
Twelve healthy male volunteers participated in each study. The demographic details of the first study population were: mean age, 28 ± 5 years; mean weight, 76 ± 7 kg; mean height, 182 ± 6 cm. The demographic details of the second study population were: mean age, 28 ± 5 years; mean weight, 70 ± 7 kg; mean height, 177 ± 6 cm.
Before the start of each study, each volunteer was subjected to a screening examination including medical history, physical examination, 12-lead electrocardiogram, blood pressure, heart rate, complete blood count with differential blood analysis, urinalysis, urine drug screen, clinical blood chemistry, blood coagulation tests, hepatitis B surface antigen and human immunodeficiency virus antibody test. Subjects were excluded if they had taken any prescribed medication or over-the-counter drugs within a period of 2 weeks before the study. In the scintigraphic study, subjects were excluded if they had undergone any diagnostic analysis with radioactive tracers or X-rays during the 6 months preceding the study.
The study medication for both studies was provided by Cosmo S.p.A., Lainate (MI), Italy, and consisted of gastro-resistant, extended-release tablets, each containing 1200 mg mesalazine (5-amino-2-hydroxybenzoic acid, 5-ASA). For the scintigraphic study, 10 mg of 152Sm2O3 was added to the formulation. 152Sm2O3 was subsequently transformed into the radioactive 153Sm isotope by neutron activation. The study medication was characterized by a multi-matrix structure designed for slow and graded 5-ASA release, especially in the colon. The multi-matrix structure comprised an inner lipophilic matrix consisting of inert substances with a melting point below 90 °C, in which the active ingredient was dispersed, and an outer hydrophilic matrix generated by in situ hydration of selected polymer chains. Tablets were film coated with polymethacrylate to provide gastric resistance.
A series of activation tests was performed on 152Sm2O3-containing tablets before the start of the study to define the optimal activation conditions to obtain the desired radioactivity at the time of administration, without promoting unacceptable changes in the dissolution characteristics of the formulation. Tablets were irradiated for different periods of time at different reactor powers (100–250 kW) with a neutron flux of 1013 neutrons/s.cm2. The optimal conditions determined after the tests were an irradiation period of 0.5 min at a reactor power of 250 kW and a flux of 1013 neutrons/s.cm2. Furthermore, in vitro dissolution tests were performed by Cosmo S.p.A. to verify that the release profiles of labelled and unlabelled mesalazine tablets were comparable.
The administered radioactivity was 1.6 ± 0.6 MBq/ dose. This value complied with the EURATOM directives 80/836, 84/467, 84/466, 89/618, 90/641 and 92/3 and with the general guidelines of the World Health Organization.
Experimental design. Volunteers attended the Clinical Trial Centre on the evening before drug administration and remained under observation for 24 h post-dose. During hospitalization, volunteers received standardized meals according to a fixed time-table based on the normal calorific needs of a healthy male adult of normal weight with slight physical activity. After an overnight fast, the trial medication was administered to the subjects at 07.00 h (± 1 h) with 200 mL of water. Thereafter, volunteers underwent scintigraphic scans, blood and urine sampling at pre-determined intervals up to 24 h post-dose. On the following day, a 24-h plasma sample collection, collection of overnight urine and a last scintigraphic scan were performed. To facilitate the interpretation of the scans, each volunteer had four radioactive point sources taped to the skin in the following sites: lower end of the sternum, umbilicus, left and right iliac spine.
The transit of mesalazine tablets along the gastrointestinal tract was scintigraphically recorded with the volunteers lying under a large field of view, double-head gamma-camera (Siemens E.Cam Dual Detector System) equipped with a low-energy, all-purpose, parallel-hole collimator. Scanning was performed at 3 min post-dose and at approximately 30-min intervals up to 16 h. One additional scan was taken 24 h post-dose. Data were electronically stored. In each subject, the following regions of interest were identified on the basis of anatomic subdivisions: stomach, small intestine, terminal ileum–caecum, ascending colon, transverse colon, descending colon, sigmoid colon.
The location of the labelled formulation in the gastrointestinal tract was established by viewing the images on a monitor. Quantification of the distribution was achieved by measuring the count rates recorded from the regions of interest. The geometric means of the corresponding anterior and posterior count rates were calculated and corrected for radioactive decay. Radioactivity was expressed as the absolute counts and the percentage of the dose in each region at each time point. The appearance or disappearance of the labelled formulation to or from the target region was determined by recording the time of the first and last appearance of radioactivity in the region.
Sampling of venous plasma. Venous blood samples (10 mL) were taken from an arm vein using an indwelling catheter with a switch valve at the following times: 0 (pre-dose), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 24 h. Blood samples were immediately centrifuged at about 2500 × g for 5 min, the cells were discarded and plasma was obtained. Each plasma sample was equally divided into two tubes and frozen at − 80 °C until analysis.
Urine collection. Immediately before drug administration, subjects were asked to empty their bladder (pre-dose urine). After the administration of the study drug, urine was collected over three periods: 0–8, 8–16 and 16–24 h. The total volume of each fraction and the total 24-h urine volume were calculated. For each collection period, two 5-mL aliquots were frozen at − 80 °C for analytical investigation.
Experimental design. Volunteers attended the Clinical Trial Centre on the evening before first drug administration and remained hospitalized until the morning of day 10. On days 1–7, the trial medication was administered twice daily to the fasting volunteers at 07.00 h (± 1 h) and 19.00 h (± 1 h). During hospitalization, volunteers received standardized meals 3, 6 and 13 h after morning drug administration. Starting with the morning administration on day 7, blood and urine samples were collected at fixed time points. The time of defecation was reported and volunteers had to check if parts of the medication were visible in the faeces.
Sampling of venous plasma. Venous blood samples (10 mL) were taken from an arm vein using an indwelling catheter with a switch valve at the following times: days 1–6: pre-dose (morning dose); day 7: 0 (pre-dose), 2, 4, 6, 8, 10, 12, 14, 16, 24, 30, 36, 48, 60 and 72 h. Blood specimens were centrifuged within 20 min at 4 °C for 10 min at 1750 × g to obtain plasma. Plasma samples were immediately divided into two 2-mL aliquots and stored frozen at − 20 °C until analysis.
Urine collection. Immediately before the morning administration on day 7, volunteers were asked to empty their bladder (pre-dose urine). After administration of the study drug, urine was collected over the following time intervals: 0–8, 8–16, 16–24, 24–36, 36–48, 48–60 and 60–72 h. Aliquots (2 × 5 mL) for each collection period were frozen at − 20 °C for subsequent investigation.
Safety assessments. The following assessments for safety and tolerability evaluation were made: (i) physical examination, including body weight, performed at screening and final visits; (ii) vital signs performed every day; (iii) electrocardiogram performed at screening and final visits; (iv) routine haematology, blood chemistry and urinalysis performed, in the fasting condition, at screening and at the end of the study; (v) monitoring of adverse events.
Blood and urine samples from both studies were analysed for their 5-ASA and Ac-5-ASA contents at AAI Industries GmbH (Neu-Ulm, Germany) using a validated high-performance liquid chromatography method.15,16
By employing gamma-scintigraphy, the following time variables could be described: (i) gastric emptying time; (ii) time of arrival in the small intestine; (iii) time of arrival in the ileum; (iv) colonic arrival; (v) time of initial tablet disintegration; (vi) colon transit time. Quantification of the distribution of radioactivity was achieved by measuring the count rates recorded from the regions of interest. The geometric means of the corresponding anterior and posterior count rates were calculated and corrected for radioactive decay. The relative percentage of drug absorption in a region of interest was calculated using the following formula:
where AUCROl is the area under the plasma vs. time curve in the respective region of interest, and AUCt is the area under the plasma vs. time curve up to the last detectable concentration. When plasma samples were missing at the start and end of transit in the relevant regions, plasma levels were obtained by linear interpolation of the concentrations available at the times immediately preceding and following the time of interest.
For both studies, the main pharmacokinetic parameters were calculated using Kinetica Software, Version 2.00.200 (Innaphase Corporation, Philadelphia, PA, USA) for both 5-ASA and Ac-5-ASA.
Data collected during the studies and the calculated parameters were analysed by descriptive statistics (mean, standard deviation) using SAS 6.12 (SAS Institute Inc., Cary, NC, USA).
The gastrointestinal transit and distribution of 153Sm radioactivity incorporated into the extended-release mesalazine formulation were followed by means of scintigraphic imaging. The mean gastric emptying time was 0.4 ± 0.4 h; the mean times of arrival in the small intestine and ileum were 0.8 ± 0.5 and 4 ± 1 h post-dose, respectively. On appearance in the colon (5.8 ± 1.2 h post-dose), the tablets started to dissolve. Initial tablet matrix disintegration/erosion started, on average, after 6.9 ± 1.1 h, either in the ascending colon (n = 8) or the transverse colon (n = 4). Complete tablet disintegration/erosion could not be exactly defined as, after initial tablet disintegration, a ‘tail’ of radioactivity appeared behind the moving tablet, which might be interpreted as ‘spreading’ of the medication on the colon wall. The times of arrival in the transverse and descending colon regions were 7.3 ± 1.0 and 9.5 ± 2.1 h, respectively. The study medication entered the sigmoid colon 13.0 ± 5.9 h post-dose and was still detectable after 24 h. Figure 1 (top panel) depicts the mean plasma vs. time profiles for 5-ASA and Ac-5-ASA and the corresponding mean transit intervals through the different regions of interest.
The initial absorption of 5-ASA was observed during the location of the formulation in the small intestine and ileum, i.e. 5-ASA release started before relevant tablet erosion could be identified from scintigraphic images. Mean maximum plasma concentration (Cmax) values (350.6 ± 322.6 ng/mL) were observed during location of the formulation in the ileo-caecal region. The mean time to reach Cmax(tmax) value was 8.3 ± 6.2 h. The mean AUCt value was 1895 ± 1240 ng/mL. The mean relative absorption of 5-ASA in the colon, as expressed by the ratio AUCcolon/AUCt, was 80.1 ± 18.2%, with the remaining 19.9 ± 18.2% being absorbed earlier in the small intestine and ileum. The cumulative amount of 5-ASA and Ac-5-ASA excreted in the urine was 8.8 ± 4.7% of the total administered dose. However, during the last collection interval, 5-ASA and Ac-5-ASA were still detectable in the samples, indicating that urinary excretion was incomplete after 24 h.
After multiple-dose administration of 1200 mg mesalazine twice daily for 7 days, steady state concentrations were attained after 4–5 days (Figure 1, bottom panel). The main pharmacokinetic parameters are summarized in Table 1. Peak concentrations at steady state were attained after 5.5 ± 3.0 and 4.3 ± 3.3 h (tssmax) for 5-ASA and Ac-5-ASA, respectively, with mean maximum plasma concentration at steady state (Cssmax) values of 2042.0 ± 1846.0 and 2600.0 ± 1729.6 ng/mL, respectively, and mean average plasma concentration at steady state (Caverage) values of 1057.0 ± 821.4 and 1748.7 ± 1007.8 ng/mL, respectively. Peak to trough fluctuations were 152.6% and 90.5% for 5-ASA and its metabolite, respectively.
Table 1. Mean pharmacokinetic parameters of 5-aminosalicylic acid (5-ASA) and N -acetyl-5-ASA (Ac-5-ASA) at steady state after the administration of multi-matrix tablets containing 1200 mg of mesalazine twice daily for 7 days
AUCss , area under the plasma vs. time curve at steady state; AUC0−24 , area under the plasma vs. time curve during the 0−24 hours time interval; Caverage , average plasma concentration at steady state; Cssmax , maximum plasma concentration at steady state; Cssmin , minimum plasma concentration at steady state; CI ss , clearance at steady state; F , fraction of drug absorbed; PTF, peak trough fluctuation; tssmax , time to reach Cssmax .
2042.0 ± 1846.0
2600.0 ± 1729.0
531.0 ± 353.0
1130.3 ± 643.1
1057.0 ± 821.4
1748.7 ± 1007.8
5.5 ± 2.9
4.3 ± 3.3
12684 ± 9857
20985 ± 12093
20784 ± 13148
37009 ± 18722
152.6 ± 109.5
90.5 ± 60.6
3389.8 ± 5516.8
1211.5 ± 1095.9
After the administration of the last mesalazine dose, the mean t1/2 values were 7.1 ± 6.3 h for 5-ASA and 11.4 ± 10.1 h for Ac-5-ASA. The urinary elimination rate during the 0–24-h collection interval on day 7 averaged 4.1 ± 5.6 mg/h for 5-ASA and 23.6 ± 15.9 mg/h for Ac-5-ASA. The cumulative daily urinary elimination rates of 5-ASA and Ac-5-ASA at steady state were 3.6 ± 3.8% and 20.0 ± 12.1%, respectively, of the administered dose of 2400 mg.
No serious adverse events occurred in either study. There were no laboratory abnormalities, relevant abnormalities of vital signs or electrocardiogram changes detected. In two subjects receiving mesalazine in a twice-daily schedule for 7 days, adverse events probably related to the study medication were noted, i.e. slight abdominal pain, nausea, vomiting and flatulence.
A pharmacoscintigraphic approach was employed to describe the gastrointestinal transit and drug leakage from a newly developed mesalazine formulation designed for the homogeneous release of active 5-ASA in the colonic region. Gastric transit was fast and the ileum was reached within 4 h post-dose. During this period, the medication was moving as a point source of radioactivity on the scintigraphic images. Initial tablet disintegration was observed after approximately 7 h, a time consistent with drug transit through the ascending colon. At this time, however, the formulation was not moving along the intestine as before, i.e. as a defined point source of radioactivity. Examination of the scintigraphic images showed a ‘tail’ of radioactivity behind the moving tablet, which might be interpreted as the progressive erosion of small portions of the formulation, resulting, along the whole colonic region, in the ‘spreading’ of the medication on the intestinal wall (Figure 2, right panel).
Although the scintigraphic data identified the colon as the main site of 5-ASA absorption, plasma data indicated that the first release of active 5-ASA occurred during transit through the ileum (Figure 1, top panel), a result that corresponds with the release characteristics of other delayed-release mesalazine formulations.17 Nevertheless, on arrival in the colonic tract, the plasma concentrations of 5-ASA and Ac-5-ASA reached a plateau (characterized by sustained plasma concentrations), which slowly declined. This might be an indication of the continuous absorption of 5-ASA from those parts of the colon in which the medication had spread after disintegration/erosion of the formulation. Furthermore, the high relative absorption of 5-ASA in this region (80.1 ± 18.2%) is consistent with scintigraphic data, which showed a long residence time of the medication in the whole colon. It should be noted that the present data were collected in fasting volunteers. Thus, in principle, the intake of food might modify gastric emptying. Furthermore, the data might not reflect the delivery patterns in patients, although no evidence has been found to indicate that inflammatory bowel diseases have a significant impact on the delivery and release of mesalazine-containing formulations.18
In the 24-h observation period, about 9% of the administered dose was excreted by the kidneys, virtually exclusively in the acetylated form. This value, which is lower than that reported for other mesalazine formulations,19,20 is strongly indicative of the low systemic absorption of mesalazine, due to its short residence time in the small intestine and prolonged residence in the colonic lumen. However, it should be noted that the elimination process was not complete after 24 h; this is also underlined by the rather high 24-h plasma concentrations and the fact that, in some subjects, the start of the decay phase could not be observed at this time point.
To assess the pharmacokinetic profiles of 5-ASA and Ac-5-ASA under therapeutic conditions and to evaluate the terminal elimination phase, the study medication was administered twice daily to healthy volunteers for seven consecutive days; plasma concentrations and urinary excretion were measured up to 72 h following the last dose on day 7. Compared with single-dose administration, there was an accumulation by a factor of 6.7 for 5-ASA and 2.7 for Ac-5-ASA —AUCss values. These results might be overestimated, however, as the multiple-dose data were compared with the data from the scintigraphic study, where the AUCt values refer to the 0–24-h observation period during which recovery was incomplete.
The urinary elimination of 5-ASA and Ac-5-ASA amounted to 3.6 ± 3.8 and 19.9 ± 12.1% of the daily 2400 mg dose and proceeded at a constant rate. A comparison with the steady state pharmacokinetics of other delayed-release mesalazine formulations17 showed that the systemic availability of 5-ASA correlated with the urinary elimination in all studies. However, the peak to trough ratios in the present study were lower than those reported for other formulations, which could be an indication of the sustained absorption of 5-ASA from the site of drug action within different portions of the large intestine.
In conclusion, a new extended-release mesalazine formulation was well tolerated after single- and multiple-dose administration. Furthermore, the active ingredient, 5-ASA, was mainly and selectively delivered to the colonic lumen, with a continuous release along the whole colon, and a lower systemic absorption was observed compared with other mesalazine sustained-release formulations. These characteristics are favourable for the therapy of distally located inflammatory bowel diseases.
The authors wish to thank Dr I. D. Marchioretto, Giuliani S.p.A., Italy, for valuable support.