University of Groningen

Targeting the ileo-colonic region in inflammatory bowel diseaseGareb, Bahez

DOI:10.33612/diss.155874434

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2021

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Gareb, B. (2021). Targeting the ileo-colonic region in inflammatory bowel disease. University of Groningen.https://doi.org/10.33612/diss.155874434

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 03-04-2022

2

Development of a zero-order sustained-release tablet containing mesalazine and budesonide intended to treat the

distal gastrointestinal tract in inflammatory bowel disease

B. Gareb, A.C. Eissens, J.G.W. Kosterink, H.W. Frijlink

Published, Eur J Pharm Biopharm. 2016 Jun;103:32-42. doi: 10.1016/j.ejpb.2016.03.018.

36

Chapter 2

Abstract

Ulcerative colitis (UC) and Crohn’s disease (CD) are diseases affecting the gastrointestinal tract. Treatment depends on the severity of the disease, site of inflammation, and patient’s response. The aim of this study was to develop a zero-order sustained-release tablet containing both the anti-inflammatory drugs mesalazine and budesonide as a new treatment option for ileo-colonic CD and UC. Tablets were attained by wet granulation with hydroxypropyl methylcellulose and direct compression. Our newly developed tablet core was coated with different ColoPulse coating thicknesses and the mesalazine and budesonide release profiles were investigated in a 600-min gastrointestinal simulation system (GISS), together with commercially available MMX-mesalazine and MMX-budesonide. Lag-time, release rate (k

0), completeness of release, and zero-order release

correlation coefficient (R2) could be manipulated by varying the ColoPulse coating thickness. Our newly developed combination preparation (C[4.92]) complied with all conducted European Pharmacopoeia tests as well as an accelerated 6-month stability study and had a lag-time of 250 min (targeted simulated ileum), a zero-order release profile (mesalazine R2=0.9002; budesonide R2=0.9481), and drug release of 100% mesalazine and 77% budesonide. Like C[4.92], MMX-mesalazine had a zero-order (R2=0.9883) and complete release profile (96%). However, C[4.92] lag-time was longer (250 vs. 210 min), assuring simulated ileum specificity. Remarkably, MMX-budesonide lag-time was 480 min and released only 7% with a zero-order release profile (R2=0.9906). The in vitro results suggest that MMX-budesonide effectiveness may be improved if budesonide release in the aqueous phase would be increased and that C[4.92] is a potential, new treatment option for ileo-colonic CD and UC.

37

2

Introduction

Inflammatory bowel diseases (IBD) includes, but are not limited to, Crohn’s disease (CD) and ulcerative colitis (UC). CD and UC are lifelong chronic inflammatory disease characterized by their relapsing behavior. Both diseases can affect the ileo-colonic region of the gastrointestinal tract (GIT) where the inflammation can be diffuse [1–3]. There is currently no definite cure for CD and UC and therefore lifelong anti-inflammatory medication is often needed whereas surgery can be an option for some patients. Treatment options of CD and UC depend on the severity of disease, site of inflammation, and patient’s response. In general, the first treatment step of uncomplicated CD or UC consists of the topically active compounds such as the aminosalicylate mesalazine (5-aminosalicylic acid) and the synthetic glucocorticosteroid (GC) budesonide. Subsequently, if treatment fails to induce a clinical response, other anti-inflammatory compounds such as systemic GC, immunomodulators, and/or biologicals are treatment options [4,5]. Although the mechanism of action of mesalazine and budesonide differ, they both possess a broad range of potent anti-inflammatory properties which in general result in the increase and decrease of respectively anti- and pro-inflammatory mediators [6–13].

Both mesalazine and budesonide act locally [14,15], however, both compounds are effectively absorbed by the upper parts of the GIT if administered in unmodified oral dosage forms [16,17]. Since the inflammation in ileo-colonic CD and UC can be located in the distal part of the GIT, targeted delivery to these sites are required to obtain therapeutic drug levels at the site of inflammation. Therefore, several techniques have been applied to produce commercially available oral mesalazine and budesonide formulations that aim to provide site-specific drug delivery [18].

Results from in vitro studies show that the majority of commercially available oral mesalazine (Pentasa, Salofalk, Claversal, Asacol) [19,20] and budesonide (Entocort, Budenofalk) [20] formulations start to release their content well before the simulated ileo-colonic region, suggesting that the bulk of the dose is delivered to the proximal GIT, and thus, absorbed before reaching the ileo-colonic region. These observations dictate improvements of oral mesalazine and budesonide formulations in view of the treatment of ileo-colonic CD and UC. Hence, enhancing oral ileo-colonic targeting of both compounds would be a valuable addition to the available treatment options of ileo-colonic CD and UC. Interestingly, The Multi Matrix (MMX) system is a relatively new platform technology, which consists of an enteric-coated controlled-release tablet core. This technology is utilized for colonic targeting and controlled release of mesalazine [21] as well as budesonide [22].

Since the inflammation in ileo-colonic CD and UC can be diffuse, a sustained-release dosage form ought to be applied in order to target an entire inflamed region instead of just one site. Zero-order release kinetics ensures time-independent and constant drug release over a period of time and warrants steady drug release during ileo-colonic transit

38

Chapter 2

[23]. In view of potency, combining mesalazine and budesonide in such a dosage form would in theory yield a sustained-release, topically active, potent, anti-inflammatory combination preparation for oral treatment of ileo-colonic CD and UC. The release profiles for both drugs should be characterized by a lag-time, preventing drug release in the upper GIT, followed by a prolonged release of the drugs over several hours. However, assuring content uniformity of such a preparation is challenging because of the great difference in oral dose between mesalazine (1200 mg) and budesonide (9 mg) intended to treat the distal parts of the GIT [4,5]. A further challenge is to realize similar drug dissolution profiles since the water solubilities of both compounds differ greatly as well; 1140 mg/L for mesalazine [24] and 24 mg/L for budesonide [25] determined at 37 °C. Furthermore, mesalazine dissolution is highly pH dependent [24], and therefore, release characteristics can be altered in the GIT, which is characterized by pH fluctuations. Ideally, the release profiles of both compounds should be equivalent in order to guarantee that the dose of both drugs is evenly delivered to the inflamed area.

Ileo-colonic targeting of an oral dosage form can be achieved by the ColoPulse technology, which is a pH-sensitive coating consisting of the pH-sensitive polymer Eudragit S100 with an incorporated super-disintegrant (croscarmellose sodium) in a non-percolating manner [26,27]. In vivo data show that ColoPulse-coated capsules and tablets are suitable for ileo-colonic targeting and that ColoPulse-coated tablets perform comparably in healthy subjects and patients suffering from CD [28–30].

Hydroxypropyl methylcellulose (HPMC), a hydrophilic semi-synthetic polymer, can be used as matrix former in oral controlled-release dosage forms. HPMC is commercially available in a variety of viscosity grades, relatively cheap, non-toxic, and easy to process, making it interesting for sustained-release formulations [31]. Upon contact with water, the HPMC polymer starts to hydrate resulting in the formation of a viscous gel around the dosage form. Subsequently, drug dissolution is governed by the diffusion through the gel layer and the gradual erosion of the gel matrix, resulting in drug release in a controlled manner [32,33].

The aim of this in vitro study was to develop a zero-order sustained-release tablet containing 1200 mg mesalazine and 9 mg budesonide intended to treat the distal GIT in ileo-colonic CD and UC. Our newly developed formulation was compared to the release profiles of commercially available Mezavant 1200 mg (mesalazine-MMX) and Cortiment 9 mg (budesonide-MMX).

39

2

Material and methods

ChemicalsBudesonide (Sofotec Almirall, Bad Homburg, Germany), methacrylic acid–methyl methacrylate copolymer 1:2 ([Eudragit S100], Evonik, Essen, Germany), polyvinyl polypyrrolidone (PVPP), HPMC, polysorbate 80 (P80) (Sigma-Aldrich, St. Louis, USA), polyethylene glycol 6000 ([PEG 6000], Fagron, Capelle aan de Ijssel, The Netherlands), sodium stearyl fumarate ([SSF], JRS Pharma, Rosenberg, Germany), acetonitrile, methanol (Biosolve, Dieuze, France), mesalazine (Syntese, Hvidorve, Denmark), acetone, sodium hydroxide, hydrochloric acid 37% (VWR, Fontenay-sous-Bois, France), talc, potassium dihydrogen phosphate, sodium chloride (Spruyt-Hillen, IJsselstein, The Netherlands), croscarmellose sodium ([CS], FMC, Brussels, Belgium), ortho-phosphoric acid 85%, sodium dihydrogenphophate dihydrate, disodium monohydrogen phosphate dihydrate, trichloroacetic acid (TCA) (Merck, Darmstadt, Germany), microcrystalline cellulose ([MC], DMV Fonterra Excipients, Foxhol, The Netherlands), Mezavant 1200 mg (mesalazine-MMX, Shire Plc, Hampshire, United Kingdom, batch FE077), Cortiment 9 mg (budesonide-MMX, Ferring Pharmaceuticals, Hoofddorp, The Netherlands, batch GF009), and Creon Forte 300-mg capsules (Abbot Laboratories GmbH, Neustadt, Germany), were all used as received from their respective suppliers.

Product developmentA summarized target product profile (TPP) of the to be developed product is given in table 1. The three drug release (Q) values were on the one hand based on the dissolution recommendations of the Ph. Eur. for prolonged-release oral dosage forms, assuring no premature dose dumping (Q180) and adequate (Q360) and complete (Q600) drug release [34]. On the other hand, the Q values were partly based on the aim to treat the ileo-colonic region, and therefore drug release should be sufficient during transit before the chyme in the colon is transformed to a (semi-)solid mass during transit, potentially entrapping the formulation with possibly less or no drug release as a result. Q600 in the gastrointestinal simulation system (GISS) corresponds to drug release during 6 hours of transit in the simulated colon, which presumably results in treating the proximal colon [35,36].

The ColoPulse technology [26,27] was applied to achieve the intended ileo-colonic targeting and HPMC and other commercially available excipients were used to realize the desired zero-order sustained-release profile. Intended ileo-colonic targeting was assessed in the GISS, which is a simple in vitro model that simulates the different stages of the GIT using four phases and is described in detail elsewhere [19]. The dissolution requirements for budesonide were set lower than for mesalazine since it was expected that the low content and low solubility of budesonide would result in a slower dissolution. Moreover, no uniformity of content test for mesalazine was conducted as the bulk of

40

Chapter 2

the tablet mass, approximately 91%, consisted of mesalazine. Therefore, an uniformity of mass test was conducted for mesalazine.

BlendingBlending of dry powder mixtures was performed in a Turbula mixer (Turbula, Bachoven, Basel, Switzerland) at 90 rpm. Budesonide, mesalazine, CS, and MC, were mixed for 10 min. Subsequently, the lubricant SSF was added and blended with the mixture for an additional 2 min.

Table 1: Summary of the desired target product profile (TPP) of the to be developed combination preparation. Qx: % of drug released at x min in the GISS.

Parameter Requirement

Dosage form Tablet, oblong shaped

Tablet mass <1400 mg

Mesalazine tablet content 1200 mg

Budesonide tablet content 9 mg

Tablet friability Mass loss≤1%

Drug content 95-105% for both drugs

Consistency of preparation: budesonide Complies with Ph. Eur. uniformity of content and uniformity of dosage units

Consistency of preparation: mesalazine Complies with Ph. Eur. uniformity of dosage units

Mesalazine GISS dissolution Q180: <10%; Q360: 50-70%; Q600: >80%

Budesonide GISS dissolution Q180: <10%; Q360: 40-60%; Q600: >70%

GranulationMesalazine, and for the combination preparation also micronized budesonide, with varying amounts of disintegrants (intragranular incorporation) were granulated with an aqueous 10% (w/w) HPMC solution in a high shear mixer (Paul, Primax M8, Germany). The granules were dried in an oven (Termarks TS 8056, Salm en Kipp BV, Breukelen, The Netherlands) at 55 °C for approximately 2 h. Finally, the granules were reduced in size to 0.8 mm (Erweka AR40, Apparatenbau GmbH, Heusenstamm, Germany).

CompactionOblong-shaped tablets were obtained by compaction with a hydraulic press (ESH compaction apparatus, Hydro Mooi, Appingedam, The Netherlands). The compaction force, speed, and holding time were 20 kN, 2 kN/s, and 0.1 s respectively during all experiments. Tablet mass containing only 1200 mg mesalazine varied since the content of excipients varied during these experiments. Tablet mass of the uncoated combination preparation was 1317 mg. Table 2 gives the constituents of all the produced formulations during this study.

41

2

Budesonide analysisBudesonide analysis was conducted using reversed phase HPLC (Zorbax Extend-C18 4.6x150 mm 3.5 µm, Agilent Technologies, Santa Clara, USA) coupled to UV detection (Ultimate 3000, Dionex, Germering, Germany). The specifications used during the Ph. Eur., dissolution, and GISS tests were: λ=240 nm; injection volume=50.00 µL; flow rate=1.000 mL/min; column temperature=50.0 °C; run time=30 min; mobile phase consisted of 30:70 (v/v) acetonitrile:phosphate buffer pH=3.2 (29 mM) prepared from ortho-phosphoric acid 85% and sodium dihydrogenphosphate dihydrate. The specifications during the accelerated stability study were: λ=244 nm; injection volume=50.00 µL; flow rate=1.000 mL/min; column temperature=ambient; run time=5 min; mobile phase consisted of 80:20 (v/v) methanol:water.

Mesalazine dissolution experiments at pH=6Dissolution experiments at pH=6 were conducted in an USP dissolution apparatus II (Sotax CH-4123, Allschwil/Basel, Switzerland) coupled to an online UV-VIS spectrophotometer (Thermo Fischer Scientific EVO300 PC, Madison, USA) by a roll pump (Ismatec ISM931C, Wertheim, Germany). The UV-VIS spectrophotometer was equipped with 7 1-mm cuvettes. Path length, wave length, medium temperature, and paddle speed were 1.0 mm, λ=331 nm, 37 °C, and 50 rpm respectively. Dissolution medium consisted of 1 L phosphate buffer pH=6 (67 mM) prepared from potassium dihydrogen phosphate and disodium monohydrogen phosphate dihydrate. pH was measured (Consort R735, Turnhout, Belgium) to ensure the right pH.

Table 2: The content of the different produced formulations during this study. Added intragranular excipients were expressed as % of mass added to mesalazine or mesalazine and budesonide before granulation with HPMC. Added extragranular excipients were expressed as % of mass added to the granules after granulation with HPMC, drying, and size reduction. Abbreviations: HPMC: hydroxypropyl methylcellulose; iPVPP: intragranular polyvinyl polypyrrolidone; iCS: intragranular croscarmellose sodium; eCS: extragranular croscarmellose sodium; eMC: extragranular microcrystalline cellulose; eSFF: extragranular sodium stearyl fumarate

Formulation Mesalazine (mg)

Budesonide (mg)

10% HPMC (mL)

iPVPP (%)

iCS (%)

eCS (%) eMC (%) eSSF

(%)

2.3%eCS 1200 - 10 0.64 - 2.3 - 1.6

1.3%eCS_1.0%eMC 1200 - 10 0.64 - 1.3 1.0 1.6

1.0%eCS_1.3%eMC 1200 - 10 0.64 - 1.0 1.3 1.6

1.15%eCS 1200 - 10 0.64 - 1.15 - 1.6

1.3%iCS_1.0%eMC 1200 - 10 0.64 1.3 - 1.0 1.6

1.3%eCS_1.0%eMC-BUD 1200 9 10 0.64 - 1.3 1.0 1.6

1.5%eCS_1.0eMC-BUD 1200 9 10 0.64 - 1.5 1.0 1.6

42

Chapter 2

Budesonide dissolution experiments at pH=6For budesonide containing tablets, the same USP dissolution apparatus II, dissolution medium, volume, rotational paddle speed, and temperature were applied as described above for mesalazine. Samples of 5-mL were subtracted from the dissolution medium, filtered through a 0.45-µm filter, and analyzed using HPLC-UV, applying the same method as described above. Subtracted samples were replaced with fresh dissolution medium to keep a constant volume.

Uniformity of content and uniformity of dosage units (Ph. Eur.) for budesonideThe budesonide content of 10 individual tablets was determined by transferring a single tablet in a 1000.0-mL volumetric flask filled with ~90% mobile phase. This mixture was magnetically stirred overnight and thereafter diluted till 1000.0 mL with mobile phase. A sample from the resulting mixture was filtered through a 0.45-µm filter and analyzed for budesonide content by HPLC-UV, applying the same method as described above.

Uniformity of dosage units (Ph. Eur.) for mesalazineThe mass of 10 tablets was determined and these tablets were pulverized in a mortar with a pestle. Subsequently, an accurately weighed amount of powder was transferred to a 1000.0-mL volumetric flask. The flask was filled till ~90% with 0.14 M HCl medium solution, magnetically stirred overnight, and diluted till 1000.0 mL with medium. 0.5 mL of the solution was filtered through a 0.20-µm filter and diluted till 25.0 mL with medium. Mesalazine content was determined in a 10.0-mm cuvette at λ=303 (Thermo Spectronics, Unicam UV 540, Waltham, USA). From this assay result and the individual tablet masses, mesalazine content of each tablet was calculated according to Equation 1, as stated in the Ph. Eur. [37]:

(Eq. 1) Xi=Wi*(A/W)

Where Xi is the estimated individual content of the tablets, Wi is the individual masses of the tablets, A is the assay value, and W is the average mass of the 10 tablets.

Tablet friability (Ph. Eur.)Ten tablets were dusted with a brush, weighed and transferred to a multiblade drum (Erweka TA3, Apparatenbau GmbH, Heusenstamm, Germany) of a tablet friability apparatus as stated in the Ph. Eur. [38]. The drum was rotated 100 times at 25 rpm. Thereafter, tablets were inspected for cracks and cleaves, dusted, and weighed. Mass loss was calculated using Equation 2:

(Eq. 2):

28

for mesalazine. Samples of 5-mL were subtracted from the dissolution medium, filtered

through a 0.45-µm filter, and analyzed using HPLC-UV, applying the same method as

described above. Subtracted samples were replaced with fresh dissolution medium to keep a

constant volume.

Uniformity of content and uniformity of dosage units (Ph. Eur.) for budesonide

The budesonide content of 10 individual tablets was determined by transferring a single

tablet in a 1000.0-mL volumetric flask filled with ~90% mobile phase. This mixture was

magnetically stirred overnight and thereafter diluted till 1000.0 mL with mobile phase. A sample

from the resulting mixture was filtered through a 0.45-µm filter and analyzed for budesonide

content by HPLC-UV, applying the same method as described above.

Uniformity of dosage units (Ph. Eur.) for mesalazine

The mass of 10 tablets was determined and these tablets were pulverized in a mortar with

a pestle. Subsequently, an accurately weighed amount of powder was transferred to a 1000.0-

mL volumetric flask. The flask was filled till ~90% with 0.14 M HCl medium solution, magnetically

stirred overnight, and diluted till 1000.0 mL with medium. 0.5 mL of the solution was filtered

through a 0.20-µm filter and diluted till 25.0 mL with medium. Mesalazine content was

determined in a 10.0-mm cuvette at λ=303 (Thermo Spectronics, Unicam UV 540, Waltham,

USA). From this assay result and the individual tablet masses, mesalazine content of each tablet

was calculated according to Equation 1, as stated in the Ph. Eur. [37]:

(Eq. 1) Xi=Wi*(A/W)

Where Xi is the estimated individual content of the tablets, Wi is the individual masses of

the tablets, A is the assay value, and W is the average mass of the 10 tablets.

Tablet friability (Ph. Eur.)

Ten tablets were dusted with a brush, weighed and transferred to a multiblade drum (Erweka

TA3, Apparatenbau GmbH, Heusenstamm, Germany) of a tablet friability apparatus as stated

in the Ph. Eur. [38]. The drum was rotated 100 times at 25 rpm. Thereafter, tablets were

inspected for cracks and cleaves, dusted, and weighed. Mass loss was calculated using Equation

2:

(Eq. 2): Massloss(%) = !"#$%!&("&&#%)*+%!%&!(-)/!"#$%!&("&&")!%+!%&!(-)!"#$%!&("&&#%)*+%!%&!(-) ∗ 100%

Tablet coating

Tablets were coated using the ColoPulse technology [26]. The ColoPulse coating mixture

consisted of Eudragit S100:PEG 6000:CS:talc in a ratio of 7:1:3:2 (w/w) in a 97:3 (v/v) solvent

43

2

Tablet coatingTablets were coated using the ColoPulse technology [26]. The ColoPulse coating mixture consisted of Eudragit S100:PEG 6000:CS:talc in a ratio of 7:1:3:2 (w/w) in a 97:3 (v/v) solvent mixture of acetone:water. This ColoPulse coating mixture was sprayed at ambient temperature on the tablets that were placed in a mini-rotating drum using a roll pump (Gilson, Minipuls 3, Villiers le Bel, France) connected to a spray nozzle. Tablets were cured for 1 h at 55 °C after the coating procedure to promote film formation. The amount of applied coating was expressed as mg Eudragit S100 per cm2.

GISSThe GISS is a simple in vitro model and is described in detail elsewhere [19]. Briefly, the GISS simulates the different stages of the GIT using four phases, simulating consecutively the stomach (pH=1.20 for 2 h), jejunum (pH=6.80 for 2 h), ileum (pH=7.50 for 30 min), and colon (6.00 for as long as needed).

The mesalazine and budesonide release profiles in the GISS were determined using the same apparatus and specifications as described above for the dissolution experiments, with the exception of dissolution medium and volume, since volume and content of dissolution medium vary during the GISS.

For the budesonide release profiles without any additions, 5-mL samples from dissolution medium were subtracted, filtered through a 0.45-µm filter – or centrifuged at 4200 rpm (Salm en Kipp BV, Sigma 3-16L, Breukelen, The Netherlands) for the accelerated stability study – and analyzed by HPLC-UV, applying the same method as described above. Subtracted 5-mL samples were replaced with fresh dissolution medium to keep a constant volume.

Budesonide release profiles with additions were assessed by adding the content of 2 Creon Forte 300-mg capsules to the dissolution medium, starting from phase II of the GISS tests for the digestive enzymes and digestive enzymes and surfactant experiments. This dose was used as the Summary of Product Characteristics (SPC) finds this an effective dose for the treatment of exocrine pancreatic insufficiency. The capsule content was transferred to a small mesh cylinder and submerged in dissolution medium so that the content could dissolve (confirmed by visual inspection) without falling to the bottom of the dissolution vessel. The P80 concentration of the dissolution medium during the digestive enzymes and surfactant experiments was kept constant at 0.5% (w/v) starting from phase II, by dissolving the P80 in the switch solutions before addition to the dissolution medium. Samples of 5mL from the dissolution medium were subtracted and 0.5 mL 20% TCA was added to induce protein denaturation. The samples were centrifuged at 4200 rpm for 10 min and the supernatants were analyzed by HPLC-UV, applying the same method as described above.

44

Chapter 2

Accelerated stability studyA total of 150 1.5%eCS_1.0%eMC-BUD (table 2) tablets were produced and coated as described above. Amount of applied coating was 5.40 mg/cm2. Before starting the stability study, dissolution profiles of the tablets in the GISS were investigated as described above. However, pH of phase II (simulated jejunum) of the GISS was adjusted to 6.90-6.99 (instead of 6.80) with 2.0 M sodium hydroxide during the stability study in order to investigate coating performance at the upper pH limit of phase II in the GISS [19]. This increase of simulated jejunal pH resulted in an additional stress factor since the ColoPulse coating dissolves at a pH of 7 but the formulation needed to release the bulk of drug content in the simulated ileo-colonic region after phase II. This was done so to test the robustness of the developed formulation and coating performance as in vivo jejunal pH’s of 7 or greater have been reported [39].

Assay tests were performed in duplicate for mesalazine as well as budesonide by UV-VIS spectrophotometry and HPLC-UV, respectively, as described above. Thereafter, tablets were stored unprotected from the environment in a closed desiccator with an internal environment of 40 °C/75% RH. Tablet drug content and dissolution profiles in the GISS were investigated after 3 and 6 months, as described in the ICH guideline [40].

The dissolution requirements for the accelerated stability study are given in table 3, which differ from that of the TPP in table 1 since the pH of phase II in the GISS during the stability study was higher than during the non-stability GISS experiments. Therefore, it was expected that drug release started earlier during the stability GISS tests. However, to assure simulated distal GIT specificity, the Q180 requirement was set to <5%.

Table 3: Tablet drug content and release profile requirements in the GISS for the accelerated stability study. Qx: % of drug released at x min in the GISS.

Parameter RequirementMesalazine tablet content 1200 mgBudesonide tablet content 9 mg

Measured drug content 95-105% for both drugsMesalazine GISS dissolution Q180: <5%; Q360: 65-85%; Q600: >85%Budesonide GISS dissolution Q180: <5%; Q360: 50-70%; Q600: >75%

CalculationsZero-order release model (ZORM) parameters [23] were determined by fitting the relevant release profiles to Equation 3 by the least squares method:

(Eq. 3) Qt=Q0+k0*t

Where Qt is the percentage drug released at time t, Q0 is the amount of drug released at t0, and k0 is the zero-order release constant. The zero-order release correlation coefficient (R2) was calculated and served as a measure to depict whether drug release profiles

45

2

could be approximated by the ZORM. A R2≥0.9 was required to be classified as a good approximation of zero-order release. Zero-order release parameters for mesalazine and budesonide dissolution experiments at pH=6 were calculated from t0 till t300 min. Zero-order release parameters for mesalazine in the GISS were calculated from starting from the moment of 1% release till t600 min. Zero-order release parameters for budesonide in the GISS were calculated from t180 till t600 min.

For the Uniformity of dosage units experiments, acceptance values (AV) were calculated using Equation 4 [37]:

(Eq. 4) AV=98.5-Xav+ks

Where Xav is the average (n=10) content expressed as percentage of the claim, k is the acceptability constant (when n=10, k=2.4), and s is the standard deviation of the calculated average content.

The f2 similarity factor was used to compare the dissolution profiles. The calculated f2 value gives a value between 0 and 100. An f2 value of 100 designates that the release profiles are identical and when f2≥50, the dissolution profiles are considered to be similar. The f2 was calculated using Equation 5 [41]:

(Eq. 5)

Where Rt is the time point at time t on the cumulative dissolution profile of the reference formulation, Tt is the time point at time t on the cumulative dissolution profile of the to be compared formulation, and n is the number of time points. The time point t0 min was omitted and only one point after >85% drug release was taken for the f2 calculation [41].

Results and discussion

Tablet core developmentDifferent core formulations containing only 1200 mg mesalazine (table 2) were produced to find a formulation with the desired release profile in a dissolution medium of pH=6, the assumed pH in the colon [19,39] where the bulk of the dose should be released.

Figure 1 presents the release profiles at pH=6.0 of the different core formulations, showing the effects of the different excipients. Table 4 contains the mesalazine release at 300 min (Mt300), R2, and k0 of the formulations. Mesalazine release from the formulations could be well approximated by the ZORM as indicated by the high R2 values, which varied between 0.9569-0.9992. The fastest release was observed from 2.3%eCS. When 1.0% of extragranular CS from 2.3%eCS was replaced with 1.0% extragranular MC (1.3%eCS_1.0%eMC), release became slower whereas completeness of release was

31

Where Xav is the average (n=10) content expressed as percentage of the claim, k is the

acceptability constant (when n=10, k=2.4), and s is the standard deviation of the calculated

average content.

The f2 similarity factor was used to compare the dissolution profiles. The calculated f2 value

gives a value between 0 and 100. An f2 value of 100 designates that the release profiles are

identical and when f2≥50, the dissolution profiles are considered to be similar. The f2 was

calculated using Equation 5 [41]:

(Eq. 5) f2=50*log[(1+∑ (#$%&$)^)!"#$

* )-0.5 * 100]

Where Rt is the time point at time t on the cumulative dissolution profile of the reference

formulation, Tt is the time point at time t on the cumulative dissolution profile of the to be

compared formulation, and n is the number of time points. The time point t0 min was omitted

and only one point after >85% drug release was taken for the f2 calculation [41].

Results and discussion

Tablet core development

Different core formulations containing only 1200 mg mesalazine (table 2) were produced to

find a formulation with the desired release profile in a dissolution medium of pH=6, the

assumed pH in the colon [19,39] where the bulk of the dose should be released.

Figure 1 presents the release profiles at pH=6.0 of the different core formulations, showing

the effects of the different excipients. Table 4 contains the mesalazine release at 300 min

(Mt300), R2, and k0 of the formulations. Mesalazine release from the formulations could be

well approximated by the ZORM as indicated by the high R2 values, which varied between

0.9569-0.9992. The fastest release was observed from 2.3%eCS. When 1.0% of extragranular CS

Table 4: Mesalazine release at 300 min (Mt300), zero-order release correlation coefficients (R2) and release constants

(k0) of the different produced formulations containing 1200 mg mesalazine. Mt300 was expressed as % of 1200 mg.

Formulation Mt300 (%) R2 k0 2.3eCS 87 0.9569 0.3041

1.3eCS_1.0eMC 88 0.9966 0.2498 1.0eCS_1.3eMC 82 0.9992 0.2839

1.15eCS 73 0.9966 0.2498 1.3iCS_1.0eMC 43 0.9899 0.1401

46

Chapter 2

unaffected. However, replacing 1.3% of extragranular CS with 1.3% extragranular MC (1.0%eCS_1.3%eMC) did result in both slower and less complete release. The latter was also observed when 2.3% extragranular CS was halved (1.15%eCS). Interestingly, when 1.3% of extragranular CS was transferred to the intragranular phase (1.3%iCS_1.0%eMC), the slowest and least complete release was observed. These results show that the extragranular disintegrant content in this system has a major impact on release rate and completeness of release.

Table 4: Mesalazine release at 300 min (Mt300), zero-order release correlation coefficients (R2) and release constants (k0) of the different produced formulations containing 1200 mg mesalazine. Mt300 was expressed as % of 1200 mg.

Formulation Mt300 (%) R2 k02.3eCS 87 0.9569 0.3041

1.3eCS_1.0eMC 88 0.9966 0.24981.0eCS_1.3eMC 82 0.9992 0.2839

1.15eCS 73 0.9966 0.24981.3iCS_1.0eMC 43 0.9899 0.1401

Figure 1: The mean (n=3) mesalazine release profiles of the different tablets in phosphate buffer pH=6 (67 mM). The content of the different formulations is depicted in table 2. The dissolution profile of 1200 mg unprocessed 5ASA (n=6) is depicted as well. Bars depict standard deviations.

47

2

The 1.3%eCS_1.0%eMC core was chosen for further development because of the completeness of release and linear character of the release profile (Mt300=88% and R2=0.9966). Mesalazine release rate and completeness of release decreased when 9 mg budesonide was added to the 1.3%eCS_1.0%eMC core formulation (1.3%eCS_1.0%eMC-BUD; f2=57 with 1.3%eCS_1.0%eMC as reference). This was likely the result of the hydrophobicity of budesonide that was distributed throughout the tablet core, resulting in the impediment of water intrusion into the tablet core. Therefore, the extragranular CS was increased to 1.5% which resulted in 1.5%eCS_1.0%eMC-BUD. The mesalazine release profile of 1.5%eCS_1.0%eMC-BUD had an f2=82 with 1.3%eCS_1.0%eMC taken as the reference release profile.

Figure 2 shows the release profiles of mesalazine and budesonide from 1.5%eCS_1.0%eMC-BUD at pH=6 and table 5 summarizes the release characteristics. Budesonide release at 300 min (Bt300) and Mt300 were 76 and 84% respectively. Mesalazine and budesonide R2 were 0.9725 and 0.9616 respectively, indicating that both release profiles had a linear character. Finally, the calculated f2 value was 63, which indicates that both release profiles were similar.

Table 5: Drug release at 300 min (Dt300), zero-order release correlation coefficients (R2) and release constants (k0) of mesalazine and budesonide from 1.5%eCS_1.0eMC-BUD. Dt300 was expressed as % of 1200 mg mesalazine or 9 mg budesonide. The f2 value was calculated using the mesalazine release profile as the reference profile.

Drug Dt300 (%) R2 k0 f2Mesalazine 84 0.9725 0.2887 ReferenceBudesonide 76 0.9616 0.2622 63

Figure 2: The mean mesalazine (n=3) and budesonide (n=3) release profiles from 1.5%eCS_1.0eMC-BUD in phosphate buffer pH=6 (67 mM). The content of 1.5%eCS_1.0eMC-BUD is depicted in table 2. Drug release is expressed as % of 1200 mg mesalazine or 9 mg budesonide. Bars depict standard deviations.

48

Chapter 2

ColoPulse-coated tablets and performance in GISS The developed 1.5%eCS_1.0%eMC-BUD tablet core was coated with either 3.63, 4.92, 9.60, or 14.75 mg/cm2 of ColoPulse coating (C[3.63], C[4.92], C[9.60], and C[14.75] respectively). The release profiles of these tablets were investigated in the GISS [21]. Furthermore, the release profiles of commercially available Mezavant 1200 mg and Cortiment 9 mg were also investigated in the GISS and compared to our newly developed combination preparation. The GISS is a simple, easy, and cheap in vitro model to investigate the dissolution profile of oral dosage forms. The GISS is not a bio-predictive model for the in vitro-in vivo correlation since it only consists of simple aqueous buffers and does not simulate the inter- and intra-individual pH fluctuations in the GIT nor mechanical stress. However, the ColoPulse coating was previously developed in the GISS and in vivo studies in healthy subjects as well as CD patients show that this technology can be applied for the drug targeting of oral dosage forms to the distal GIT [28–30,42]. More complex and bio-predictive as well as patient specific in vitro models have been described elswhere [43–46].

Figures 3 and 4 present the release profiles of respectively mesalazine and budesonide from the different tablets in the GISS. Table 6 summarizes the release characteristics and the calculated f2 values of the different tablets. These data show that the lag-time of our newly developed tablet could be manipulated by adjusting the amount of applied ColoPulse coating, yielding tablets that started to release their content either in the simulated jejunum (C[3.63] lag-time=220 min), ileum (C[4.92] lag-time=250 min), or colon (C[9.60] lag-time=280 min and C[14.75] lag-time=330 min). Mesalazine release after 600 min (Mt600) was 105%, 100%, 83%, and 50% whereas budesonide release after 600 min (Bt600) was 76%, 77%, 40%, and 25% for C[3.63], C[4.92], C[9.60], and C[14.75] respectively. These observations show that an increase in lag-time was accompanied by a decrease in Mt600 and Bt600. For instance, in vitro drug targeting in the simulated colon (C[9.60]) instead of simulated ileum (C[4.92]) resulted in a 17% and 37% decrease of respectively mesalazine and budesonide release. Increasing the amount of applied coating even more from 9.60 to 14.75 mg/cm2 resulted in a 50-min increase of lag-time whereas a further decrease of 15% mesalazine and 33% budesonide release was observed. These results highlight the importance of the balance between on the one hand the in vitro site-specific delivery of the drugs and on the other hand obtaining an adequate drug release rate from the tablet.

Budesonide release from ColoPulse-coated tablets in the GISS was always substantially slower (bud.k0 vs. mes.k0) and lower (Mt600 vs. Bt600) than mesalazine release. This is also illustrated by the low values of the f2 similarity factors indicating that the release profiles of mesalazine and budesonide were no longer equivalent. However, both mesalazine and budesonide were released in a sustained manner with a linear profile as indicated by their respective zero-order correlation coefficients (mes.R2 and bud.R2), and release from C[4.92], C[9.60], and C[14.75] could be well approximated by the ZORM while this was not the case for C[3.63].

49

2

During the experiments it was observed that the coating did not dissolve isotropic nor completely. The coating dissolved the fastest at the curved parts of the tablet and at a certain moment, depending on amount of applied coating, the coating was ruptured and either shed completely or stayed partly intact for some time. The time needed for complete shedding of the coating was greater and varied more as the amount of applied coating increased. This could explain the trend of increasing variability of the release profile (figure 3 and 4) when greater amounts of coatings were applied.

The C[4.92] tablet was chosen as the preferred formulation of the combination preparation that met the dissolution requirements from the TPP the best (table 1). Requirements on lag-time (250 min; simulated ileum targeted), completeness of release (Mt600=100%; Bt600=76%), and zero-order release (mes.R2 =0.9002, bud.R2=0.9481) were met. For the C[4.92] formulation, the release of budesonide was slower than that of mesalazine. However, in view of the lag-time and dissolution characteristics of both drugs, this difference was considered acceptable.

Compared to Mezavant, C[4.92] had a longer lag-time (210 vs. 250 min respectively), releasing its content in the simulated ileum instead of simulated jejunum. Although the lag-time of C[4.92] was 40 min longer, the release after 600 min was virtually the same (100 vs. 96%). Mesalazine release from both formulations had a linear character (R2=0.9002 and 0.9883 respectively). The dose released in the simulated ileum (240-270 min) was three times higher for C[4.92] (12% vs. 4%), even though the Mezavant lag-time was 30 min shorter. The in vitro data show that Mezavant starts to release its content in the simulated jejunum and that release is in a zero-order sustained manner up to the simulated colon. These in vitro results are in accordance with the successful outcome of the two randomized, double-blind, placebo-controlled clinical trials with Mezavant, which showed that mesalazine-MMX 2.4 and 4.8 g/day was superior to placebo for the induction of active mild-to-moderate UC [47]. However, as mentioned before, the GISS is a simple in vitro model and no conclusive statements concerning in vivo release can be made. Nonetheless, the observed in vitro dissolution profile of Mezavant seems favourable in respect of ileo-colonic CD and UC treatment.

50

Chapter 2

Figure 3: The mean (n=4-5) mesalazine release profiles of C[3.63], C[4.92], C[9.60], C[14.75], and Mezavant 1200 mg in the GISS. The pH of the dissolution medium is depicted as well. C[x]: Applied amount of ColoPulse coating thickness expressed as mg Eudragit S100 per cm2. Bars depict standard deviations.

Figure 4: The mean (n=3) budesonide release profiles of C[3.63], C[4.92], C[9.60], C[14.75], and Cortiment 9 mg in the GISS. The pH of the dissolution medium is depicted as well. C[x]: Applied amount of ColoPulse coating thickness expressed as mg Eudragit S100 per cm2. Bars depict standard deviations. DE: digestive enzymes. DE+P80 (0.5% [w/v]): digestive enzymes + polysorbate 80 0.5% (w/v).

Ileum

ColonJejunum

Colon

IleumJejunum

Stomach

Stomach

51

2

Table 6: The lag time, mesalazine release at 600 min (Mt600), budesonide release at 600 min (Bt600), mesalazine zero-order release correlation coefficient (mes.R2), budesonide zero-order release correlation coefficient (bud.R2), mesalazine zero-order release constants (mes.k0), budesonide zero-order release constant (bud.k0), and the calculated f2 value of the different tablets investigated in the GISS experiments. Lag time for ColoPulse-coated tablets and Mezavant was defined as time to 5% mesalazine release. Lag time for Cortiment was defined as time to 5% budesonide release.

Formulation Lag time (min)

Mt600 (%) Bt600 (%) mes.R2 bud.R2 mes.k0 bud.k0 f2

C[3.63] A 220 105 76 0.8806 0.8336 0.2828 0.1683 44 B

C[4.92] A 250 100 77 0.9002 0.9481 0.2802 0.1933 35 B

C[9.60] A 280 83 40 0.9476 0.9296 0.2651 0.1036 26 B

C[14.75] A 330 50 25 0.9780 0.9387 0.1603 0.0636 46 B

Mezavant 210 96 - 0.9883 - 0.2544 - -

Cortiment 480 - 7 - 0.9906 - 0.0170 -

Cortiment+DE 600 - 5 - 0.9957 - 0.0130 93 C

Cortiment+DE+P80 420 - 10 - 0.9928 - 0.0249 84 C

A: Number between brackets depicts applied ColoPulse coating expressed as mg Eudragit S100/cm2.B: Mesalazine release profile was the reference and compared to the budesonide release profile.C: Cortiment release profile was the reference.

Cortiment lag-time was 480 min and thus started to release budesonide only after 210 min in the simulated colon. Remarkably, the release was also rather incomplete with a Bt600 of only 7%. Digestive enzymes (Cortiment+DE) and digestive enzymes and surfactant (Cortiment+DE+P80) were added to the dissolution medium in order to investigate whether this could increase budesonide release from the Cortiment formulation. This was not the case as indicated by the Bt600 values of Cortiment+DE and Cortiment+DE+P80, which were 5% and 10% respectively. Moreover, the calculated f2 values showed that digestive enzymes nor digestive enzymes and surfactant had a significant effect on the release of budesonide from Cortiment.

Compared to C[4.92], Cortiment lag-time was 230 min longer. However, more remarkable was the small amount of drug that was released from Cortiment (7%) compared to C[4.92] (77%). The remarkable low dissolution values found in the GISS for budesonide from the MMX matrix may be explained by the physicochemical nature of the drug and the composition of the MMX matrix, as the MMX matrix contains both hydrophilic and lipophilic components [21,22]. We hypothesized that budesonide release from the MMX matrix was impeded due to the lipophilicity of budesonide, staying in the lipophilic parts of the matrix instead of being released in the hydrophilic dissolution medium. This hypothesis may also explain why drug release from the C[4.92] formulation was substantially higher since this formulation contained hardly any lipophilic components and the hydrophilic HPMC matrix and readily water soluble mesalazine may aid in the wetting and hydration of budesonide.

52

Chapter 2

Although budesonide release from Cortiment in the GISS was low, two randomized, double-blind, double-dummy, placebo-controlled clinical trials showed that budesonide-MMX 9 mg was superior to placebo for the induction of active mild-to-moderate UC [48,49] . In these studies, Cortiment 9 mg was compared to Asacol, [50] Entocort, [48] and placebo. The therapeutic advantage was modest and it can even be questioned why a glucocorticoid was compared to a low dose mesalazine (2.4 g Asacol instead of 4.8 g) in moderately active UC or a budesonide formulation with a completely different release profile (Entocort) [20,51–53]. However, the discrepancy between our in vitro results and in vivo clinical results could be due to the simple nature of the GISS being a simple, non-bio-predictive in vitro model. In vivo, the budesonide release from the MMX matrix may well be substantially higher due to multiple factors. For instance, mechanical stress from chyme on the tablet core induced by the peristaltic movements of the GIT may aid in tablet disintegration, and thus, budesonide release from the MMX matrix. Furthermore, budesonide release may be increased in the presence of lipases potentially metabolizing the lipophilic parts of the MMX matrix as well as lipids, fatty substances, and bile, creating an environment in which the lipophilic budesonide is liberated more readily from the tablet core. However, the in vitro results presented here show that neither the addition of digestive enzymes or the addition of digestive enzymes and surfactant did increase the budesonide release from Cortiment.

The in vitro results of the present study show that budesonide from Cortiment 9 mg is not readily released in the aqueous phase of the GISS. This suggests that the effectiveness of Cortiment 9 mg may be improved if budesonide release in the aqueous phase would be increased, since it is expected that only the dissolved and from the tablet liberated fraction is able to treat the inflamed loci. The C[4.92] formulation in this present study contains hardly any lipidic components that may act as a sink, preventing budesonide release, and showed eleven times (77% vs. 7%) more budesonide release in the aqueous phases of the GISS.

Content uniformity, friability, accelerated stability studyConsistency of our newly developed tablet was assessed by conducting the uniformity of mass, uniformity of content for budesonide, content uniformity for budesonide, and mass variation for mesalazine tests as stated by the Ph. Eur [37]. Furthermore, Ph. Eur. tablet friability test was conducted as well [38]. In addition, an accelerated stability study was also conducted [40]. The GISS dissolution requirements for the accelerated stability study are given in table 3.

Table 7 summarizes the results and requirements of the content uniformity and friability tests. From table 7 it can be observed that the tablets complied with all the Ph. Eur. tests. Table 8 gives the results of the accelerated stability study, showing that tablet drug content was in the predefined range of 95-105% drug after 6 months. Furthermore, no unexplainable peaks on the UV-VIS spectra (λ=200-400 nm) or HPLC-UV

53

2

chromatograms were observed during mesalazine or budesonide, respectively, analysis (data not shown).

Figure 5 and 6 illustrates respectively the mesalazine and budesonide release profiles in the GISS and table 8 summarizes the release characteristics. The tablets started to release their content in the simulated jejunum, although applied coating was greater (5.40 mg/cm2) than that of our newly developed formulation (4.92 mg/cm2). This was due to the relatively high pH of (6.90-6.99 instead of 6.80) during the stability study since we wanted to investigate the coating performance at the upper limit of the pH range of phase II in the GISS [19] as an additional stress factor. More so since jejunal pH’s of 7 or higher have been reported [39]. Moreover, coated tablets were unprotected from the environment (40 ºC/75% RH) during the accelerated stability study. Still, drug content and the Q180, Q360, and Q600 of mesalazine as well as budesonide complied with the predefined requirements (table 3) during the start and after 3 and 6 months of the accelerated stability study (table 8), showing the robustness of our newly developed formulation and the ColoPulse technology.

Altogether, the tablets complied with all the conducted tests, showing that content consistency, structural integrity, drug stability, and coating performance of our newly developed combination preparation could be assured.

Table 7: The results of Ph. Eur. content uniformity and friability test.

Uniformity of mass (g) A

Budesonide uniformity of content (mg) B

Budesonide content uniformity

(%) C

Mesalazine mass variation (%) C

Tablet friability mass loss (%) D

1.315(1.312-1.319) E

8.62(8.57-8.73) E AV=4.0 AV=2.4 0.14

A: Requirement: not more than two individual masses mass should deviate 5%, and not more than one should deviate 10% from the average mass.B: Requirement: each individual content should be between 85-115% of the average content.C: Requirement is ≤15.0%.D: Requirement is ≤1.0%.E: Average value followed by lowest and highest value between parentheses.

Table 8: Results of the accelerated stability study. Assay results are given as % of 1200 mg mesalazine or 9 mg budesonide. Assay tests were conducted in duplicate. Release characteristics were investigated in the GISS (figure 5 and 6). Applied tablet coating was 5.40 mg/cm2. Qx: % of drug released at x min in the GISS.

Mesalazine A Budesonide B

Assay (%) Q180 (%) Q360 (%) Q600 (%) Assay (%) Q180 (%) Q360 (%) Q600 (%)

t0 months 101.9 1 67 90 100.4 1 60 81

t3 months 99.3 3 81 96 98.2 1 66 79

t6 months 103.1 0 76 89 96.0 0 60 80

A: Requirements: Q180<5%; Q360=65-85% ; Q600>85% (table 3).

B: Requirements: Q180<5%; Q360=50-70% ; Q600>75% (table 3).

54

Chapter 2

Figure 5: The mean (n=12) mesalazine release profiles in the GISS of the 6-months accelerated stability study. The pH of the dissolution medium is depicted as well. Applied tablet coating was 5.40 mg Eudragit S100/cm2 and tablets were stored unprotected at 40 °C/75% RH. Bars depict standard deviations.

Figure 6: The mean (n=12) budesonide release profiles in the GISS of the 6-month accelerated stability study. The pH of the dissolution medium is depicted as well. Applied tablet coating was 5.40 mg Eudragit S100/cm2 and tablets were stored unprotected at 40 °C/75% RH. Bars depict standard deviations

Stomach

Colon

Jejunum

Ileum

Stomach

Colon

Jejunum

Ileum

55

2

Potential clinical relevanceIn active mild-to-moderate ileo-colonic CD, oral 9 mg budesonide is superior to placebo [60] and the preferred treatment [4] whereas oral mesalazine is inefficacious [54]. An interesting question is whether adequate mesalazine ileo-colonic mucosal concentrations were achieved with the commercially available mesalazine formulations that were investigated in the ileo-colonic CD clinical trials hitherto, since in vitro results suggest that the bulk of the dose of the majority of commercially available oral mesalazine products does not reach the ileo-colonic region [19,20] but sulfasalazine, an oral colonic-targeted mesalazine formulation, is an effective treatment option of mildly active colonic CD in a dose of 3-6 g (equivalent to 1152-2304 mg mesalazine) [4].

In active mild-to-moderate UC, mesalazine or budesonide as mono-therapy are superior to placebo when the compounds are delivered directly to the site of inflammation, as is the case with enemas and rectal foams [5]. In addition, combination therapy with beclomethasone dipropionate (BDP)—a potent topically active, poorly absorbed GC similar to budesonide [55,56]—and mesalazine is well tolerated and more efficacious than mono-therapy with BDP or mesalazine alone [57,58], even when patients are non-responsive to oral mesalazine treatment [59].

Hence, our newly developed combination preparation could be of clinical importance in treating mild-to-moderately active ileo-colonic CD and UC, or mild-to-moderately active ileo-colonic CD and UC that is non-responsive to single treatment with mesalazine or budesonide alone, if this novel formulation shows a similar release profile in vivo. If so, it is expected that treatment with our newly developed product is more patient friendly than switching to other, systemically active compounds, such as traditional GC, immunomodulators, or biologicals, if mono-therapy with either mesalazine or budesonide fails.

Noteworthy, the release profiles in the GISS of both drugs of our novel product were similar, suggesting that both drugs will treat the same region during transit. This will most likely not be achieved by combining two currently available budesonide- and mesalazine-containing formulations as the in vitro release profiles of these products cannot be considered similar [19,20]. Additionally, our novel product could serve as a starting point for the development of new zero-order sustained-release single-drug containing oral dosage forms intended to treat the distal parts of the GIT by omitting one of the drugs from the formulation. These formulations could be used when either mesalazine or budesonide treatment is effective or when a different daily dose of just one drug is desired during combination treatment, which gives the opportunity to treat patients based on their individual treatment needs.

56

Chapter 2

Conclusion

In summary, the C[4.92] formulation is a novel, easy-to-produce, oral dosage form containing 1200 mg mesalazine and 9 mg budesonide. After the lag-time, the release profiles of both drugs were linear and similar, starting in the simulated ileum and continued to release cilincally relevant amounts of drug in the simulated ileo-colonic region. Although the two drug doses varied greatly, content uniformity for both drugs could be ensured as well as drug, formulation, and coating stability. Based on these in vitro results, this product may be an interesting option for the treatment of ileo-colonic CD and UC. A clinical trial is needed to assess the in vivo release profiles of both drugs as well as the efficacy, safety, and clinical applicability. In addition, results from that study should determine the advantages and disadvantages compared to existing treatment options for ileo-colonic CD and UC. Finally, the C[4.92] could be used as a platform technology for the development of new oral zero-order sustained-release dosage forms containing only mesalazine or budesonide that can be applied in the treatment of ileo-colonic CD and UC in which single-drug treatment is appropriate or when a different daily dose of just one drug is desired during combination treatment.

Acknowledgements

None.

Conflict of interest

None.

57

2

References

1. Baumgart, D.C.; Sandborn, W.J. Crohn’s disease. Lancet 2012, 380, 1590–1605.2. Abraham, C.; Cho, J.H. Inflammatory Bowel Disease. N Engl J Med 2009, 361, 2066–78.3. Hanauer, S.B. Inflammatory bowel disease: epidemiology, pathogenesis, and therapeutic

opportunities. Inflamm. Bowel Dis. 2006, 12 Suppl 1, S3–S9.4. Harbord, M.; Eliakim, R.; Bettenworth, D.; Karmiris, K.; Katsanos, K.; Kopylov, U.; Kucharzik, T.;

Molnár, T.; Raine, T.; Sebastian, S.; et al. Third European evidence-based consensus on diagnosis and management of ulcerative colitis. Part 2: Current management. J. Crohn’s Colitis 2017, 11, 769–784.

5. Gomollón, F.; Dignass, A.; Annese, V.; Tilg, H.; Van Assche, G.; Lindsay, J.O.; Peyrin-Biroulet, L.; Cullen, G.J.; Daperno, M.; Kucharzik, T.; et al. 3rd European evidence-based consensus on the diagnosis and management of Crohn’s disease 2016: Part 1: Diagnosis and medical management. J. Crohn’s Colitis 2017, 11, 3–25.

6. Horváth, K.; Varga, C.; Berkó, A.; Pósa, A.; László, F.; Whittle, B.J.R. The involvement of heme oxygenase-1 activity in the therapeutic actions of 5-aminosalicylic acid in rat colitis. Eur. J. Pharmacol. 2008, 581, 315–323.

7. Greenfield, S.M.; Hamblin, A.S.; Shakoor, Z.S.; Teare, J.P.; Punchard, N.A.; Thompson, R.P. Inhibition of leucocyte adhesion molecule upregulation by tumor necrosis factor alpha: a novel mechanism of action of sulphasalazine. Gut 1993, 34, 252–256.

8. Kaiser, G.C.; Yan, F.; Polk, D.B. Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor κB activation in mouse colonocytes. Gastroenterology 1999, 116, 602–609.

9. Egan, L.J.; Mays, D.C.; Huntoon, C.J.; Bell, M.P.; Pike, M.G.; Sandborn, W.J.; Lipsky, J.J.; McKean, D.J. Inhibition of Interleukin-1-stimulated NF-κB RelA/p65 Phosphorylation by Mesalamine Is Accompanied by Decreased Transcriptional Activity. J. Biol. Chem. 1999, 274, 26448–26453.

10. Mahida, Y.R.; Lamming, C.E.; Gallagher, A.; Hawthorne, A.B.; Hawkey, C.J. 5-Aminosalicylic acid is a potent inhibitor of interleukin 1 beta production in organ culture of colonic biopsy specimens from patients with inflammatory bowel disease. Gut 1991, 32, 50–54.

11. Clark, A.R. Anti-inflammatory functions of glucocorticoid-induced genes. Mol. Cell. Endocrinol. 2007, 275, 79–97.

12. Barnes, P.J. Corticosteroid effects on cell signalling. Eur. Respir. J. 2006, 27, 413–426.13. Rhen, T.; Cidlowski, J.A. Antiinflammatory Action of Glucocorticoids — New Mechanisms for Old

Drugs. N. Engl. J. Med. 2005, 353, 1711–1723.14. Edsbäcker, S.; Andersson, T. Pharmacokinetics of budesonide (EntocortTM EC) capsules for Crohn’s

disease. Clin. Pharmacokinet. 2004, 43, 803–821.15. Desreumaux, P.; Ghosh, S. Review article: mode of action and delivery of 5-aminosalicylic acid -

new evidence. Aliment. Pharmacol. Ther. 2006, 24 Suppl 1, 2–9.16. Edsbäcker, S.; Bengtsson, B.; Larsson, P.; Lundin, P.; Nilsson, Å.; Ulmius, J.; Wollmer, P. A

pharmacoscintigraphic evaluation of oral budesonide given as controlled-release (Entocort) capsules. Aliment. Pharmacol. Ther. 2003, 17, 525–536.

17. Myers, B.; Evans, D.N.; Rhodes, J.; Evans, B.K.; Hughes, B.R.; Lee, M.G.; Richens, A.; Richards, D. Metabolism and urinary excretion of 5-amino salicylic acid in healthy volunteers when given intravenously or released for absorption at different sites in the gastrointestinal tract. Gut 1987, 28, 196–200.

18. Klotz, U.; Schwab, M. Topical delivery of therapeutic agents in the treatment of inflammatory bowel disease. Adv. Drug Deliv. Rev. 2005, 57, 267–79.

19. Schellekens, R.C.A.; Stuurman, F.E.; van der Weert, F.H.J.; Kosterink, J.G.W.; Frijlink, H.W. A novel dissolution method relevant to intestinal release behaviour and its application in the evaluation of modified release mesalazine products. Eur. J. Pharm. Sci. 2007, 30, 15–20.

20. Klein, S.; Stein, J.; Dressman, J. Site-specific delivery of anti-inflammatory drugs in the gastrointestinal tract: an in vitro release model. J. Pharm. Pharmacol. 2005, 57, 709–719.

21. Horst, S.N.; Kane, S. Multi-Matrix System (MMX®) mesalamine for the treatment of mild-to-moderate ulcerative colitis. Expert Opin. Pharmacother. 2012, 13, 2225–32.

58

Chapter 2

22. Gionchetti, P.; Praticò, C.; Rizzello, F.; Calafiore, A.; Capozzi, N.; Campieri, M.; Calabrese, C. The role of Budesonide-MMX in active ulcerative colitis. Expert Rev. Gastroenterol. Hepatol. 2014, 8, 215–22.

23. Costa, P.; Sousa Lobo, J.M. Modeling and comparison of dissolution profiles. Eur. J. Pharm. Sci. 2001, 13, 123–33.

24. French, D.L.; Mauger, J.W. Evaluation of the physicochemical properties and dissolution characteristics of mesalamine: relevance to controlled intestinal drug delivery. Pharm. Res. 1993, 10, 1285–90.

25. Mota, F.L.; Carneiro, A.P.; Queimada, A.J.; Pinho, S.P.; Macedo, E.A. Temperature and solvent effects in the solubility of some pharmaceutical compounds: Measurements and modeling. Eur. J. Pharm. Sci. 2009, 37, 499–507.

26. Schellekens, R.C.A.; Stellaard, F.; Mitrovic, D.; Stuurman, F.E.; Kosterink, J.G.W.; Frijlink, H.W. Pulsatile drug delivery to ileo-colonic segments by structured incorporation of disintegrants in pH-responsive polymer coatings. J. Control. Release 2008, 132, 91–98.

27. Schellekens, R.C.A.; Baltink, J.H.; Woesthuis, E.M.; Stellaard, F.; Kosterink, J.G.W.; Woerdenbag, H.J.; Frijlink, H.W. Film coated tablets (ColoPulse technology) for targeted delivery in the lower intestinal tract: Influence of the core composition on release characteristics. Pharm. Dev. Technol. 2012, 17, 40–47.

28. Schellekens, R.C.A.; Stellaard, F.; Olsder, G.G.; Woerdenbag, H.J.; Frijlink, H.W.; Kosterink, J.G.W. Oral ileocolonic drug delivery by the colopulse-system: A bioavailability study in healthy volunteers. J. Control. Release 2010, 146, 334–340.

29. Maurer, J.M.; Schellekens, R.C.A.; van Rieke, H.M.; Stellaard, F.; Wutzke, K.D.; Buurman, D.J.; Dijkstra, G.; Woerdenbag, H.J.; Frijlink, H.W.; Kosterink, J.G.W. ColoPulse tablets perform comparably in healthy volunteers and Crohn’s patients and show no influence of food and time of food intake on bioavailability. J. Control. Release 2013, 172, 618–624.

30. Maurer, J.M.; Schellekens, R.C.A.; Van Rieke, H.M.; Wanke, C.; Iordanov, V.; Stellaard, F.; Wutzke, K.D.; Dijkstra, G.; Van Der Zee, M.; Woerdenbag, H.J.; et al. Gastrointestinal pH and transit time profiling in healthy volunteers using the IntelliCap system confirms ileo-colonic release of ColoPulse tablets. PLoS One 2015, 10, e0129076.

31. Li, C.L.; Martini, L.G.; Ford, J.L.; Roberts, M. The use of hypromellose in oral drug delivery. J. Pharm. Pharmacol. 2005, 57, 533–546.

32. Tahara, K.; Yamamoto, K.; Nishihata, T. Overall mechanism behind matrix sustained release (SR) tablets prepared with hydroxypropyl methylcellulose 2910. J. Control. Release 1995, 35, 59–66.

33. Bettini, R.; Catellani, P.L.; Santi, P.; Massimo, G.; Peppas, N.A.; Colombo, P. Translocation of drug particles in HPMC matrix gel layer: effect of drug solubility and influence on release rate. J. Control. Release 2001, 70, 383–91.

34. Ph. Eur. Recommendations on Dissolution Testing Available online: https://www-pharmacopoeia-com.proxy-ub.rug.nl/bp-2018/appendices/appendix-12/appendix-xii-b--annex--recommendations-on-dissolution-testing.html?date=2018-07-01&text=recommendations+on+dissolution+testing (accessed on Jul 16, 2018).

35. Haase, A.M.; Gregersen, T.; Christensen, L.A.; Agnholt, J.; Dahlerup, J.F.; Schlageter, V.; Krogh, K. Regional gastrointestinal transit times in severe ulcerative colitis. Neurogastroenterol. Motil. 2016, 28, 217–24.

36. Graff, J.; Brinch, K.; Madsen, J.L. Gastrointestinal mean transit times in young and middle-aged healthy subjects. Clin. Physiol. 2001, 21, 253–259.

37. Ph. Eur. Appendix XII C. Consistency of Formulated Preparations Available online: https://www-pharmacopoeia-com.proxy-ub.rug.nl/bp-2019/appendices/appendix-12/appendix-xii-c--consistency-of-formulated-preparations.html?date=2019-04-01&text=uniformity+of+content (accessed on May 29, 2019).

38. Ph. Eur. Appendix XVII G. Friability Available online: https://www-pharmacopoeia-com.proxy-ub.rug.nl/bp-2018/appendices/appendix-17/appendix-xvii-g--friability.html?date=2018-07-01 (accessed on May 29, 2019).

39. Nugent, S.G.; Kumar, D.; Rampton, D.S.; Evans, D.F. Intestinal luminal pH in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs. Gut 2001, 48, 571–577.

40. ICH Stability Testing of New Drug Substances and Products Q1A(R2). Available online: https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q1A_R2/Step4/Q1A_R2__Guideline.pdf (accessed on Jun 16, 2018).

59

2

41. Shah, V.P.; Tsong, Y.; Sathe, P.; Liu, J.P. In vitro dissolution profile comparison--statistics and analysis of the similarity factor, f2. Pharm. Res. 1998, 15, 889–96.

42. Schellekens, R.C.A.; Baltink, J.H.; Woesthuis, E.M.; Stellaard, F.; Kosterink, J.G.W.; Woerdenbag, H.J.; Frijlink, H.W. Film coated tablets (ColoPulse technology) for targeted delivery in the lower intestinal tract: influence of the core composition on release characteristics. Pharm. Dev. Technol. 17, 40–7.

43. Goyanes, A.; Hatton, G.B.; Basit, A.W. A dynamic in vitro model to evaluate the intestinal release behaviour of modified-release corticosteroid products. J. Drug Deliv. Sci. Technol. 2015, 25, 36–42.

44. Blanquet, S.; Zeijdner, E.; Beyssac, E.; Meunier, J.-P.; Denis, S.; Havenaar, R.; Alric, M. A dynamic artificial gastrointestinal system for studying the behavior of orally administered drug dosage forms under various physiological conditions. Pharm. Res. 2004, 21, 585–91.

45. Andreas, C.J.; Chen, Y.-C.; Markopoulos, C.; Reppas, C.; Dressman, J. In vitro biorelevant models for evaluating modified release mesalamine products to forecast the effect of formulation and meal intake on drug release. Eur. J. Pharm. Biopharm. 2015, 97, 39–50.

46. Dressman, J.B.; Reppas, C. In vitro–in vivo correlations for lipophilic, poorly water-soluble drugs. Eur. J. Pharm. Sci. 2000, 11, S73–S80.

47. Sandborn, W.J.; Kamm, M.A.; Lichtenstein, G.R.; Lyne, A.; Butler, T.; Joseph, R.E. MMX Multi Matrix System mesalazine for the induction of remission in patients with mild-to-moderate ulcerative colitis: a combined analysis of two randomized, double-blind, placebo-controlled trials. Aliment. Pharmacol. Ther. 2007, 26, 205–15.

48. Travis, S.P.L.; Danese, S.; Kupcinskas, L.; Alexeeva, O.; D’Haens, G.; Gibson, P.R.; Moro, L.; Jones, R.; Ballard, E.D.; Masure, J.; et al. Once-daily budesonide MMX in active, mild-to-moderate ulcerative colitis: results from the randomised CORE II study. Gut 2014, 63, 433–441.

49. Sandborn, W.J.; Travis, S.; Moro, L.; Jones, R.; Gautille, T.; Bagin, R.; Huang, M.; Yeung, P.; Ballard, E.D. Once-daily budesonide MMX® extended-release tablets induce remission in patients with mild to moderate ulcerative colitis: results from the CORE I study. Gastroenterology 2012, 143, 1218-1226.e2.

50. Balzola, F.; Cullen, G.; Ho, G.T.; Russell, R.K.; Wehkamp, J. Once-daily budesonide MMX(R) extended-release tablets induce remission in patients with mild to moderate ulcerative colitis: results from the CORE I study. Gastroenterology 2012, 143, 1218–1226.

51. Sherlock, M.E.; MacDonald, J.K.; Griffiths, A.M.; Steinhart, A.H.; Seow, C.H. Oral budesonide for induction of remission in ulcerative colitis. Cochrane Database Syst. Rev. 2015, CD007698.

52. Prantera, C.; Scribano, M.L. Budesonide multi-matrix system formulation for treating ulcerative colitis. Expert Opin. Pharmacother. 2014, 15, 741–743.

53. Prantera, C. Letter: Budesonide MMX for ulcerative colitis? Aliment. Pharmacol. Ther. 2014, 39, 1435.

54. Lim, W.-C.; Wang, Y.; MacDonald, J.K.; Hanauer, S. Aminosalicylates for induction of remission or response in Crohn’s disease. Cochrane Database Syst. Rev. 2016.

55. Daley-Yates, P.T.; Price, A.C.; Sisson, J.R.; Pereira, A.; Dallow, N. Beclomethasone dipropionate: absolute bioavailability, pharmacokinetics and metabolism following intravenous, oral, intranasal and inhaled administration in man. Br. J. Clin. Pharmacol. 2001, 51, 400–9.

56. Harris, D.M. Some properties of beclomethasone dipropionate and related steroids in man. Postgrad. Med. J. 1975, 51 Suppl 4, 20–5.

57. Mulder, C.J.J.; Fockens, P.; Meijer, J.W.R.; van der Heide, H.; Wiltink, H.H.; Tytgat, G.N.J. Beclomethasone dipropionate (3mg) versus 5-aminosalicylic acid (2g) versus the combination of both (3mg/2g) as retention enemas in active ulcerative proctitis. Eur. J. Gastroenterol. Hepatol. 1996, 8, 549–554.

58. Rizzello, F.; Gionchetti, P.; D’Arienzo, A.; Manguso, F.; Di Matteo, G.; Annese, V.; Valpiani, D.; Casetti, T.; Adamo, S.; Prada, A.; et al. Oral beclometasone dipropionate in the treatment of active ulcerative colitis: a double-blind placebo-controlled study. Aliment. Pharmacol. Ther. 2002, 16, 1109–1116.

59. D’Arienzo, A.; Manguso, F.; Castiglione, G.N.; Vicinanza, G.; Scaglione, G.; Bennato, R.; Sanges, M.; Mazzacca, G. Beclomethasone dipropionate (3 mg) enemas combined with oral 5-ASA (2.4 g) in the treatment of ulcerative colitis not responsive to oral 5-ASA alone. Ital. J. Gastroenterol. Hepatol. 1998, 30, 254–7.