University of Groningen
Targeting the ileo-colonic region in inflammatory bowel disease
Gareb, Bahez
DOI:
10.33612/diss.155874434
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Publication date: 2021
Link to publication in University of Groningen/UMCG research database
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Gareb, B. (2021). Targeting the ileo-colonic region in inflammatory bowel disease. University of Groningen. https://doi.org/10.33612/diss.155874434
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General introduction
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Inflammatory bowel disease
Inflammatory bowel disease (IBD) includes Crohn’s disease (CD) and ulcerative colitis (UC). IBD are chronic inflammatory diseases characterized by a relapsing behavior. Despite sharing common characteristics, CD and UC are two different diseases. For instance, the inflammation in UC affects primarily the mucosa whereas the inflammation in CD is transmural. Furthermore, the inflammation in UC is primarily restricted to the rectum and colon in a continuous pattern while the entire gastrointestinal tract (GIT), from mouth to anus, may be affected in a discontinuous pattern in CD. However, the initial location of inflammation in CD is often the ileocecal region. The severity combined with the chronic nature of the disease results in a decrease of health-related quality of life, disability, and frequent hospitalizations. Symptoms of IBD include (bloody) diarrhea, rectal bleeding, abdominal pain, weight loss, malaise, anorexia, and fever [1–3].
The incidence of IBD is increasing over time in different regions around the world. The highest reported annual incidences in Europe are 24.3 and 12.7 per 100,000 person-years for UC and CD, respectively. For North America the highest reported annual incidences are 19.2 and 20.2 per 100,000 person-years for UC and CD, respectively [4].
The exact pathogenesis of IBD is not completely elucidated although research shows that a combination of genetics, environmental factors, and the microbiome play a prominent role in the onset of gut epithelial dysfunction. The intestinal mucosa is constantly in direct contact with potentially pathogenic bacteria, commensal bacteria, and food antigens. Hence, a controlled response against the commensal gut microbiome should ensue and result in tolerance. In IBD however, this intestinal homeostasis is disrupted, which results in an aberrant mucosal immune response to the commensal microflora. This abnormal immune response involves both branches of the innate and adaptive immune system, both contributing to tissue injury as a result of excessive production of proinflammatory mediators such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α). A prolonged inflammatory response against the gut epithelium may result in epithelial injury and therefore could lead to increased exposure to the gut microbiome, which amplifies the immune response. This stronger activation of both the innate and adaptive immune system may perpetuate the inflammatory state resulting in chronic inflammation [2,3,5–8].
There is currently no cure available for IBD. The relapsing inflammatory nature of IBD requires lifelong anti-inflammatory medication whereas surgery can be an option for UC patients and CD patients with complications such as strictures or perianal fistula [9,10]. The initial therapeutic objective is to induce clinical remission and the subsequent aim is to remain in clinical remission. The treatment options for IBD depend on the disease activity, severity, and location. For instance, mild-to-moderately active UC of the rectum should initially be treated with an enema in view of treating the inflammation topically. However, in a severely active disease state intravenous administration of drugs may be necessary to control disease activity and symptoms [2,3,9,10].
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Generally speaking, there are two approaches in treating IBD, namely therapy that aims to treat the inflammation topically in the GIT or systemic therapy that aims to suppress the inflammation systemically. Though systemic suppression induces a more prominent anti-inflammatory response, it is also associated with more and severe therapy-related adverse events [9,10]. Ideally, a site-specific as opposed to systemic anti-inflammatory response is favorable in view of maximizing the site-specific anti-inflammatory effects whilst minimizing the therapy-related side effects. Besides the mentioned enema, topical and site-specific treatment may be realized by oral drug targeting. This approach aims to formulate an oral dosage form in such a manner that upon oral administration it targets a specific part of the GIT. Advantages of this approach are daily mucosal exposure to relatively high drug concentrations with fewer systemic side effects and an easy patient friendly administration of the drug with no trained personnel needed. The latter is a prerequisite for intravenous infusions. However, challenges of this approach are reproducible targeting performance, drug stability at the targeted site, drug penetration into the targeted site with now substantial systemic absorption, and patient compliance since the patient is in control of the therapy.
Oral drug targeting
Oral drug targeting refers to the administration of an oral dosage form that is intended to target a specific site in the body. In light of IBD and this thesis, this means that an oral dosage form targets a drug to a specific site in the GIT for the topical treatment of the inflamed regions. This approach aims to maximize the localized drug concentration at the inflamed sites while minimizing the systemic exposure to the drug in view of reducing adverse events.
A variety of technological approaches have been investigated for their potential to realize oral drug targeting. First, prodrugs that are converted to the active moiety at the targeted site by target-specific initiators (such as site specific enzymes with a unique metabolism activity) have been used in IBD. An example of this approach is sulfasalazine that is only converted to the active moiety (mesalazine) in the colon by colonic bacteria [11]. Second, coating of the oral dosage form can be applied. These coatings may be pH-dependent or degrade by site-specific metabolizers such as the enzymes that are present in colonic bacteria. For instance, a tablet with an enteric coating that has a pH threshold of ≥5.5 survives the acidic environment of the stomach but releases the drug in the small intestine after the pH of the GIT rises to approximately 6.0 [12]. Another example with regards to site-specific initiators are polysaccharides coatings such as alginate, pectin, or chitosan. Due to the rise in the site-specific enzymatic activity in the colon, these polysaccharide are metabolized and drug release ensues as a result of coating degradation [13]. Third, time-dependent systems release the drug after a given lag time, which roughly corresponds to the transit time required to reach the targeted site like, say, the terminal
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ileum. Forth, multi-particulate systems on the macro, micro, or nano scale that may or may not utilize surface modifications such as charge, pegylation, or glycosylation can be used to target specific sites in the GIT. Fifth, drug release characteristics from the core can be modified in view controlled drug release. This approach aims to target an entire region in the GIT instead of just one site since the drug is released in a controlled manner during gastrointestinal (GI) transit. Finally, a combination of these technologies can be used to combine the objectives of these different strategies [13–16].
However, these approaches have limitations that are inherent to the physiological fluctuations in the GIT. The different strategies utilize physiological parameters that vary between individuals. GI pH values vary within in a range and may be influenced by factors such fed state, fluid intake, and microbiome metabolites [17–19]. Likewise, transit times vary as well and may be influenced by factors such as diseases and fed state [20]. Furthermore, the intestinal microbiome varies between individuals that are also influenced by factors such as lifestyle and diet, and may be altered by other external factors such as antibiotics [14,15,17,20]. Therefore, inconsistent drug targeting performance of formulations that utilize these physiological parameters are common [9,10,21].
To achieve a reliable and reproducible drug targeting performance in ileo-colonic IBD, a technology should be used that utilizes a parameter that falls within in relatively narrow range, does not differ greatly within and between individuals, and is not influenced substantially by the disease state. Therefore, drug targeting technologies that depend on GI transit times and site-specific initiators are not optimally suited to achieve reliable and reproducible targeting performance since these parameters may differ greatly between and within individuals [12,18,19,22,23].
Studies show that the intraluminal GI pH values of healthy subjects and IBD patients vary the most in the stomach and colon whereas the pH variability is less in the proximal small intestine. However, the smallest pH variability is observed in the distal small intestine and ileum. Figure 1 shows the different measured pH values from 38 studies that investigated the intraluminal pH of the GIT in healthy humans and patients suffering from IBD or irritable bowel syndrome (IBS) [22]. Figure 1 show the great variability in the pH values of the stomach, duodenum, jejunum, and colon. The pH variability of the ileum is relatively small and falls within in narrow range. This range is approximately 7.0-8.0, although most studies report a pH of approximately 7.5.
A coating technology that uses this pH rise to ≥7.0 in the ileum has great potential for ileo-colonic drug targeting. The reported ileocecal transit times may vary, although the transit time is relatively short compared to the transit times of the small intestines and colon [12,18,19,22,23]. A major challenging in targeting the ileo-colonic region is fast coating disintegration at te targeted site. The coating should protect the dosage form from the GI environment of the stomach and entire small intestine during GI transit. However, upon arrival in the ileum, the coating should disintegrate rapidly even though
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the pH threshold of the coating polymer is maintained for a short period of time. Another challenge is maintaining the coating disintegration kinetics after the dosage form reaches the colon, in which the pH drops below (6.0) the pH threshold of the coating polymer (7.0).
Figure 1: The measured pH values (fasted, fed, or both) of the GIT in healthy and diseased
(IBD, IBS) humans (data from 38 studies). The minimum and maximum pH values are indicated by the left and right limits, respectively, of the depicted bars. A vertical line indicates the mean pH of healthy individuals, and the dots the mean or median pH values of the individual studies. The white squares show the pH values of healthy children 8-14 years old of age (fasted). The gray squares show the pH values of subjects 62-83 years old of age (fasted and fed). Reprinted from [22] with permission from Elsevier.
ColoPulse technology
The ColoPulse technology is a patented coating composition and production technology developed by the University of Groningen and the University Medical Center Groningen [24]. It was developed to produce solid oral dosage forms such as capsules or tablets that specifically target the ileo-colonic region in humans. The coating consists of a pH sensitive polymer (Eudragit S100) in which a superdisintegrant (e.g. croscarmellose sodium or sodium starch glycolate) is incorporated in a non-percolating manner in a meanpHvalueswereusediftheywerementionedinthe
indi-vidualstudies.Meanvalueswerepreferredtopreventdatalossof individualswithoutlyingpHvalues.Ifnomeanvaluewasgiven, themedianvaluewasused.Thenumberofsubjectsintheseparate studieswasnotusedinthecalculations.StudiesonlyreportingpH ranges wereexcludedfromtheoverviewandfromour calcula-tions.Wealsoexcludedstudiesthatusedcolonoscopytoaccess thepHvalueofthelowerGItract,becauseoftheuseofBisacodyl ofKlean-Prep1.WedidnotfindmarkeddifferencesinthepH valuesobtainedwithaspiration,tetheredpH-electrodes,or telem-etrycapsules.
ThepHrisesduringtransitfromthestomachtotheileum,after whichitdropsinthececumandrisesagainslightlyinthecolon (Fig.1).ThepreciselocationofthepostileumpHdropof1.5and 1.2unitsinthefastedandfedstate,respectively,wasfoundtobein theproximalcolon.Thedropcanoccureitherinthececum,the ascending colon, orduring thetransitfromthe cecumto the ascending colon [37]. This pH drop can be explained by the bacterial fermentation of polysaccharides to short-chain fatty acids[53].Thefoodstatusofthesubjectsonlyappearedto influ-encethepHofthestomach;thatis,thepHissubstantiallyhigher
afterfoodintake.However,fewerdatawereavailableonthefed statethanonthefastedstate.Furthermore,inthepostprandial statethestomachhasregionsofdifferentpHvalues,namelya proximalacidlayer(pH2.9),abufferedlayer(pH5.0),andadistal acidlayer(pH2.3)[23].
Fallingborgetal.studiedthepHintheGItractoffastedhealthy children,aged8–14years[26].ThemeanpHvalues,indicatedby whitesquaresinFig.1,onlyslightlydifferedfromthevaluesof fastedhealthyadults.Forhealthyoldersubjects,aged62–83years, threestudieswerefoundinwhichthegastricand/orduodenalpH wasstudied.TheseareindicatedbygraysquaresinFig.1[7,24,40]. Contradictoryresultswerefoundontheinfluenceofageonthe gastric and duodenal pH. Comparative studies from the same group indicated that thegastric fasted and peak-fed pH were significantlylowerfor older (65–83years) [24]thanfor young individuals(21-35years)[19].Bycontrast,Mojaverianetal.found that the postprandial pH values in the stomachof the older subjects (65–79 years) were significantly higher than those of youngindividuals(2–34years)[40].Inthefastedstate,no signifi-cantdifferencesingastricpHbetweenthreedifferentagegroups (20–39,40–59,and60–70years)wasfound[10].TheduodenalpH
REVIEWS DrugDiscoveryToday Volume25,Number8 August2020
Stomach Duodenum Jejunum Ileum Cecum Colon Rectum 0 1 2 3 4 5 6 7 8 Luminal pH Adults Children (8-14 years) Elderly (62-83 years) Healthy - fed Healthy - fasted Diseased - fasted or fed
Drug Discovery Today
FIGURE1
pHvaluesofvariouspartsofthegastrointestinal(GI)tractofhealthyanddiseasedhumans.Themaximum,minimum,andmeanpHofthestomach[9–12,17– 19,21–23,28–33,36,40,42,52],duodenum[6–9,12–14,18–20,25,30–35,39,41,45],jejunum[8,18,30,32,34–36,38,43,45],ileum[25,30–39,41,43–45],cecum
[28,33,34,36,37,44],colon[25,28,30–36,38,41–43,45],andrectum[34,36,43,45]aregivenforhealthyindividualsinthefed(blue)andfasted(orange)state.ThepH valuesofdiseasedhumanindividuals(gray)aregroupedforthefastedandfedstate;themeanpHvalueisnotgiven[33,35,36,44].Themaximumandminimum pHvaluesareindicatedbythebars,inwhichaverticallineindicatesthemeanpHofhealthyindividuals,andthedotsthemeanormedianpHvaluesofthe individualstudies.Thewhitesquares,inthefastedstate,indicatethepHvaluesoftheGItractofhealthychildrenaged8–14years[26].Thegraysquares,inthe fastedandfedstate,indicatethepHvaluesoftheGItractofoldersubjects,aged62–83years[7,24,40].Giventhelimitedamountofstudiesinthefastedstatefor thepHintherectum(onestudy),thebarandverticallineoverlap.
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continues phase consisting of a pH sensitive coating polymer. To obtain a system that specifically targets the ileo-colonic region, the selected pH threshold of the polymer is 7.0 (figure 1) [25,26].
Research shows that the average intraluminal pH of the GIT rises from 6.5 to 7.5 for a short period of time (30 min) during transit from the jejunum to ileum. Thereafter, the intraluminal pH drops to 6.0 in the colon [12,27,28]. The ColoPulse coating was developed to utilize this short period of pH rise in the ileum before reaching the colon as a trigger to initiate coating disintegration and the subsequent drug release. When the coating is applied in an appropriate amount, it resists the pH of the stomach, duodenum, and jejunum. However, when the pH rises to ≥7.0 in the ileum, coating disintegration starts. Water uptake by the coating results in the swelling of the incorporated superdisintegrant, which induces fast coating disintegration even though the pH rises for a short period of time (30 min) to ≥7.0 (figure 2). The disintegration of the coating continues, even when the intraluminal pH drops to values of <7.0.
Figure 2: The mechanism of action of the ColoPulse coating technology. Reprinted from [25]
with permission from Elsevier.
What differentiates the ColoPulse coating formulation from simple, straightforward pH-sensitive polymer coatings is the increased sensitivity of the system to even smaller changes in the pH, which makes the system a truly site-specific delivery system for ileo-colonic drug targeting [25]. Simple pH-sensitive polymers are also pH dependent drug delivery systems. However, adequate functioning of the coating disintegration mechanism requires both a significant change in the environmental pH (preferably 1 to 2 pH units) that is also maintained for a longer period of time (more than 60 to 90
including Eudragit S applied from an organic solution. Their results also show that (thick) Eudragit S-coatings lose their pulsatile release properties. Furthermore, they were able to show that some tablets coated with Eudragit S from an organic solvent failed to disintegrate in some subjects. Therefore it may be expected that these conventional coatings result in too slow and incomplete in vivo in drug release at the wrong moment and at the wrong site in the GIT.
These observations were the reason to set out for the development of a more reliable coating compositions that would give a truly site-specific pulsatile delivery to the ileo-colonic regions. The system should be insensitive to variations in the gastro-intestinal transit time, minor variations in the pH of the GIT and minor variations in coating thickness. Since the slow and irreproducible disruption of the conventional coatings is the major reason for their poor performance, it was hypothesized that a better system could be achieved by a coating with augmented pH-responsiveness once the threshold pH of the polymer is passed. The basic idea to formulate such a system was through the incorporation of disintegrant particles in a non-percolat-ing manner in a pH-responsive polymer. How such a system than
could function is schematically depicted in Fig. 4. Since the
pH-responsive polymers form the continuous phase in the coating, no
drug is released as long as the pH threshold is not passed (Fig. 4A).
However, erosion is triggered once the pH threshold is passed and the
fluid will rapidly reach the incorporated disintegrant particles
(Fig. 4B). The disintegrants may now execute their disruptive effect
on the coating by two different mechanisms (Fig. 4C). First, they may
swell upon contact with water by binding water molecules in their polymeric network. This swelling, which depends on the disintegrant
and may go up to 30 times of the original volume[27,28], will rupture
the polymer and form cracks in the coating through which water can penetrate further into the coating. Secondly, the disintegrant particles may themselves form the route for water penetration. In this way the coating will rapidly (within 60 min) be completely disrupted and the drug can be released fast without any hindrance of coating remnants. Since the disruption of the coating is faster with this technology, the time during which the capsule will be at a pH above the threshold value will no longer be as critical as it is for systems with conventional coatings.
In a coating system as described above it is essential that the disintegrant is incorporated in the polymer in a way that water cannot percolate through a series of neighbouring disintegrant particles directly to the core. Such an ordering is called a non-percolating lattice
[22]. Otherwise the protective activity of the polymer in the upper
parts of the GIT would be lost. To assure this critical structural element in the system, the coating suspension was based on an organic solvent in which the Eudragit S is dissolved while the disintegrant is in a dispersed state. This leads to a coating in which particles of the disintegrant are dispersed in a continuous matrix of coalesced
Eudragit S. The SEM pictures (Fig. 1) confirm this structure of the
coating, a structure of which the existence is further supported by the dissolution results. Spraying from an aqueous based suspension is not feasible for this coating since in such a suspension the disintegrant would form the continuous phase.
The dissolution studies show that indeed the incorporation of disintegrants improves the performance of the coatings. Primojel, Ac-di-sol and Kelacid promoted the pulsatile release behaviour, once the pH trigger had occurred these materials caused rapid and complete erosion of the coating which led to a fast and complete release of the drug. When the coating contained Kelacid 38% w/w or Primojel 56% w/ w release occurred already before the pH threshold was passed. When Primojel or Ac-di-sol at a 38% w/w level was incorporated the release clearly showed the desired profile. The fact that the release from the Ac-di-sol coating was still good in spite of the fact that the coating
thickness was even 8.4 mg/cm2, shows that the new coating system is
indeed less sensitive to variations in the production process. In contrast to the other disintegrants tested, microcrystalline cellulose seems to slow down erosion of the coating, leading to a slow release of the drug. These observations may be explained by the difference in the swelling index (volume increase upon full wetting) of the materials. Avicel has a swelling index which is substantially lower (1.4) than that of alginic acid, croscarmellose sodium or sodium starch glycollate with swelling indices of 30, 12 and 31 respectively. Obviously the swelling of microcrystalline cellulose is insufficient to cause disruption of the coating once the pH threshold is passed.
To investigate the relevance of our in vitro data, the in vivo
proof-of-concept study was performed. In literature [25,26]it has been
described that after a certain period of fasting a subsequent meal activates the gastro-intestinal motility and cause a previously given body such as a capsule to pass the ileo-caecal junction. Since the terminal ileum is the site where the pH would normally pass the threshold value of 7.0 of our pH-responsive coating, it was hypothesized that the moment that the subsequent meal was given would also be the moment that the release would be triggered. In this experimental setup it would consequently be possible to study both whether the release was prevented in the upper GIT (the occurrence of a lag-time), whether the release had a pulsatile character (the
steepness of the13CO
2occurrence in the breath) and finally whether
the release was indeed site-specific and not time dependent (the release would start at a variable moment depending on the time of the subsequent meal).
The study was conducted by applying 13C
6-glucose as the test
compound. Notwithstanding its site of release in the intestine,13C
6
-glucose is a tracer which is (partly) metabolised into13CO
2, which may
be detected in breath. Stable isotope breath tests are a well known technology used in studies to monitor carbohydrate digestion and
glucose absorption[29–33]. The process of absorption of13C
6-glucose
and its metabolism is known to start fast. The appearance of13CO
2in
breath is therefore related to the moment of release and therefore consequently to the site of release. The results obtained after administration of uncoated capsules clearly confirm that the processes of disintegration, dissolution and absorption are fast. When a coated
capsule is administered the PDR5%shifts several hours, depending on
the moment at which the subsequent meal is taken. This clearly shows the ability of the coating to resist deteriorating forces in the stomach and duodenum and delay release until deeper parts of the intestines are reached. Those observations are in agreement with those of Priebe
et al.[25,26] for a liquid meal. Furthermore, the capsule is able to
maintain a pulsatile release profile.
Fig. 4. Schematic presentation of the mode of action of incorporated disintegrants in a pH-responsive polymer coating.
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minutes). Furthermore, coating disintegration and drug release from the core depends on the coating thickness; a thinner coating thickness corresponds to earlier drug release compared to a thicker coating thickness after the pH threshold is reached. As GI transit times and pH values may vary significantly between and within an individual, the use of simple pH-dependent coating formulations may result in drug release starting in different parts of the GIT for one specific formulation of a given coating thickness. If the environmental pH values are not maintained above the pH threshold for a sufficient amount of time, the probability of incomplete drug release is present [12,18,27–29].
Our research group has shown in five clinical trials that ColoPulse-coated tablets and capsules target the ileo-colonic region in healthy subjects as well as CD patients [30–33]. These studies show that oral dosage forms coated with the ColoPulse technology targeted the ileo-colonic region in more than 90% of the tested subjects. In these clinical trials, quality control of the ColoPulse coating was performed in the gastrointestinal simulation system (GISS). The GISS is a simple in vitro model which simulates the intraluminal pH values during the GI transit in an United States Pharmacopeia (USP) dissolution apparatus II, simulating consecutively the stomach (pH=1.2 for 2 h), jejunum (pH=6.8 for 2 h), ileum (pH=7.5 for 30 min), and colon (pH= 6 for as long as needed) by using aqueous buffers. Initial medium volume is 500 ml (stomach) and by consecutively adding multiple buffers on set points in time, the jejunum (630 ml), ileum (940 ml), and colon (1000 ml) phases are simulated [34]. During the development of the GISS, these pH values and transit times were chosen based on published work investigating these parameters in vivo as well as practical feasibility in the laboratory setting [12,18,27–29].
Although the GISS is a simple in vitro model, ColoPulse coating performance in the GISS correlates with coating performance in vivo since this model was used for the quality control of the coating performance in the conducted clinical trials [30–33]. The drug-release controlling mechanism of action of the ColoPulse technology depends mainly on the intraluminal pH values and to a minor extent on transit times. Although these parameters are subject to fluctuation between and within individuals, it was demonstrated that the targeting performance of this coating technology is reliable and reproducible. However, during the clinical development of new products based on this coating technology, the targeting performance should be confirmed since the core may influence the coating performance [26]. Furthermore, failure of the targeting principle should be considered as a possible cause of poor therapeutic efficacy of the investigated, targeted drug.
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Mesalazine
Mesalazine (also known as mesalamine or 5-aminosalicylic acid) is the active moiety [11] of the aminosalicylates used in IBD and possesses a broad range of anti-inflammatory properties. Mesalazine reduces the elevated TNF-α level during experimental colitis [35] and inhibits TNF-α-induced processes like the up-regulation of leucocyte adhesion molecules [36], translocation of nuclear factor kappa B (NF-κB) from the cytosol to the nucleus, the degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκB-α), and the activation of intracellular protein kinases [37]. Furthermore, mesalazine inhibits transcriptional activity [38] and the expression [39] of NF-κB. In addition, mesalazine activates the anti-inflammatory peroxisome proliferator-activated receptor-gamma (PPARγ) pathway [40]. Moreover, mesalazine reduces levels of pro-inflammatory mediators like prostaglandin E2 [41,42], IL-1 [43,44], and the leucocyte chemotactic leukotriene B4 [45,46]. It has also been shown that mesalazine inhibits leucocyte motility [47], secretion of antibodies from mononuclear cells [48], and acts as a cytoprotective agent against leucocyte-derived reactive oxygen species [49–51]. Additionally, the modulation of heat shock proteins (Hsp) [52] and an increase in heme oxygenase-1 activity, which can result in the attenuation of experimental colitis, has also been observed [35]. Lastly, mesalazine possesses antineoplastic properties [53,54]. However, epidemiological studies are unable to provide unambiguous evidence of the chemoprotective properties in the prevention of IBD-associated colorectal cancer [55,56].
Mesalazine is generally well tolerated and acts topically. After oral administration, mesalazine is taken up by the intestinal cells where it undergoes extensive pre-systemic acetylation by the gut wall—approximately 30-40% of the dose [57]—resulting in the major metabolite acetyl-mesalazine [58,59]. Mesalazine and acetyl-mesalazine can be excreted back in the intestinal lumen resulting in the fecal elimination of both compounds. However, both compounds can be systemically absorbed leading to further hepatic acetylation of mesalazine and the subsequent renal elimination of both compounds [58]. The total fecal and urinary excretion of mesalazine is formulation dependent and is on average 12-64% and 10-56%, respectively [60].
According to the European Crohn’s and Colitis Organisation (ECCO) and Cochrane meta-analyses, oral mesalazine therapy is efficacious for the induction [61] and maintenance [62] of remission in UC [9]. However, oral mesalazine is not advised for the treatment of CD [10,63,64].The efficacy of mesalazine is related to the mucosal concentration in the intestinal lumen [55]. Furthermore, absorption of mesalazine depends on the site of release; absorption from the colon is poor compared to absorption from the proximal GIT [56]. This means that mesalazine that is released in the upper GIT is absorbed effectively, metabolized, and excreted. Therefore, for the effective treatment of the distal GIT, oral drug targeting is a prerequisite.
However, in vitro studies on the targeting performance of commercially available mesalazine formulation show that none is targeted to the distal GIT, in particular the
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ileo-colonic region [34,65]. The ECCO states that therapy failure may be the result of subtherapeutic mucosal concentrations of mesalazine [9,10]. Furthermore, a systematic review investigating the medical therapies of IBD suggested that the inconsistent efficacy of oral targeted mesalazine may be the result of ineffective targeting performance to the site of inflammation [21]. Interestingly enough, targeted mesalazine formulations are inefficacious in CD, but sulfasalazine is an effective treatment option [10]. This suggests that the commercially available mesalazine formulations do not effectively target the colon in CD. A downfall of sulfasalazine is the formation of the therapeutically inactive metabolite sulfapyridine by the intestinal bacteria that cleave sulfasalazine into mesalazine. Exposure to sulfapyridine is related to side effects that limit the use of higher doses of sulfasalazine and therefore higher colonic mucosal concentrations of mesalazine [10,63,64]. Higher rectal and colonic mucosal concentrations can be achieved by suppositories and enemas, but may not always be able to reach the inflammation, whereas poor patient compliance may limit their use in IBD [66]. Therefore, an oral ileo-colonic targeted mesalazine formulation for the treatment of IBD would be a valuable addition to the treatment options of IBD.
Budesonide
Glucocorticosteroids (GC) are potent anti-inflammatory compounds. They are ligands of the glucocorticoid receptor (GR), which is a nuclear receptor [67]. The inactive GR resides primarily in the cytoplasm of cells coupled to intracellular proteins such as Hsps and immunophilins that prevent receptor degradation. When a GC binds the GR, it undergoes a conformational change that induces migration to the cell nucleus [68–70]. The activated GR exert the effects through a variety of mechanisms that, in general, result in the induction or inhibition of the expression of certain genes. As depicted in figure 3, these mechanisms are the result of dimerization, DNA binding, tethering, cooperation with or sequestration of transcription factors, and competition for DNA binding domains.
Pathways leading to the anti-inflammatory effects of GCs include, but are not limited to, the inhibition of NF-κB and the activation of mitogen-activated protein kinase (MAPK) phosphatases and Annexin A1 [69–71]. These cellular pathways are involved in inflammatory processes. GC treatment generally results in the upregulation of anti-inflammatory mediators and downregulation of proanti-inflammatory mediators. The up-regulated anti-inflammatory mediators include Annexin A1, IL-10, and inhibitor protein of NF-κB (IκB-α) whereas the downregulated proinflammatory mediators include IL-1, IL-6, IL-8, and TNF-α [68,69,71,72].
A downfall of long-term systemic GC therapy is the manifestation of severe side effects, which render the therapy inappropriate for chronic use. These side effects include Cushing’s syndrome, osteoporosis, diabetes mellitus, hypertension, peptic ulcers, pancreatitis, and muscle atrophy [73]. Therefore, in view of less systemic availability and
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thus fewer severe side effects, GCs that act primarily topically are suitable candidates for the chronic treatment of IBD.
Figure 3: The different mechanism by which the glucocorticosteroid receptor (GR) inhibits
or induces gene expression in the nucleus. X and Y depict transcription factors and (n)GRE are (negative) glucocorticosteroid response elements. Reprinted from [69] with permission from Oxford University Press.
Budesonide is a synthetic GC that is efficacious for the induction of remission in IBD, although long-term therapy for the maintenance of remission is not advised by the ECCO [9,74–76]. Budesonide has a pharmacokinetic profile characterized by an extensive hepatic first-pass metabolism by cytochrome P450 3A4 (CYP3A4). Moreover, budesonide is metabolized by CYP3A4 enzymes that are present in the gut mucosa of the jejunum and ileum, whereas the gut mucosal metabolism in the colon is negligible. Consequently, systemic availability after oral administration is approximately 10%. The principle metabolites of budesonide are 16α-hydroxyprednisolone and 6β-hydroxybudesonide. Budesonide has a 195x greater affinity for the GR compared to hydrocortisone, while the GR affinity and anti-inflammatory properties of the main metabolites are less than 1% compared to budesonide. This renders budesonide a potent and primarily topically active GC for the treatment of IBD [77].
To achieve site-specific therapeutic concentrations of orally administered budesonide, these formulations should be targeted to the inflammation sites. However, as is the case with oral mesalazine formulation, in vitro results show that the commercially available budesonide formulations are not targeted to the ileo-colonic region [65]. Clinical studies show that the majority of the dose is released before the ileo-colonic region [78], that the released dose is incomplete during colonic transit [79], or that the dose is released
other transcription factors and binding to “composite” elements (36, 37). For an overview of the fundamental aspects of GR transcriptional regulation, see Figure 2.
The anti-inflammatory effects of GR are believed to generally result from tethering protein-protein interac-tions between GR and other transcription factors, partic-ularly NF- B and AP-1, which results in TR of a wide variety of proinflammatory genes. On the other hand, the debilitating GC-mediated effects are thought to be caused by TA of simple GRE genes (38, 39). Accordingly, so-called selective GR agonists (SEGRAs) that favor TR were developed as therapeutic agents with reduced side effects. Examples are RU24858, compound A, AL-438, LGD5552, and ZK 216348 (40 – 45). However, more recent data show that the TA potential of GR is indispensable for its anti-inflammatory properties, at least in certain disease settings. Here, we provide an overview of the anti-inflam-matory mechanisms of GR, focusing mainly on the induc-tion of anti-inflammatory genes by GR as a homodimeric transcription factor and with emphasis on in vivo studies.
Current Concept of the Anti-inflammatory Mechanism of GC/GR: Emphasis on TR
Until recently, it was generally be-lieved that TR of transcription fac-tors by monomeric GR is the main determinant of the anti-inflamma-tory properties of GR, whereas its side effects reside in its TA potential (36, 38, 39, 46). This concept has been reviewed extensively (31, 41, 47). Briefly, it is known that TA, through direct DNA binding, in-duces the expression of several en-zymes (eg, phosphoenol pyruvate carboxykinase, tyrosine aminotransfer-ase, and glucose 6-phosphate) involved in glucose and lipid metabolism. Hence, uncontrolled up-regulation of these genes could account for the diabetogenic effects of GCs, which result in hyperglycemia and de-creased carbohydrate tolerance (1, 48). On the other hand, it is believed that the anti-inflammatory actions of GC therapy are predominantly re-lated to the TR effects of GR (11, 49) because some inflammatory pro-cesses could still be restricted in a
mouse strain (GRdim/dim) in which GR is largely
dimeriza-tion defective due to replacement of an alanine by a thre-onine (A458T) (11, 50, 51). This mutation is located in the second zinc finger in the DBD of GR (Figure 1, lower panel) and causes reduced binding to DNA and, more specifically, to the GRE (11, 50, 51).
TR of inflammatory target genes most often involves interference of GR with the activity of DNA-bound pro-inflammatory transcription factors, such as NF- B, cAMP response element-binding protein, interferon regulating factor-3, nuclear factor of activated T cells, signal trans-ducers and activators of transcription, Th1-specific T box transcription factor, GATA3, and AP-1 (52–54). Because these transcription factors regulate the expression of in-flammatory genes, GR-mediated tethering of these tran-scription factors eventually leads to repression of a large number of proinflammatory mediators: cytokines (includ-ing TNF, granulocyte macrophage colony stimulat(includ-ing fac-tor, IL-1 , IL-2, IL-3, and IL-6), chemokines (eg, eotaxin, macrophage inflammatory protein [MIP], and regulated and normal T cell expressed and secreted [RANTES]),
en-Figure 2. GR Signaling Activated GR can lead to either activation or repression of gene
transcription. Left green panel: TA is mediated by (i) binding of GR dimers to GRE, (ii) DNA binding of GR in concert with another transcription factor (TF: XY), or (iii) binding of GR to a TF by a tethering mechanism. Right red panel: TR is mainly achieved by (iv) direct binding of GR dimers to nGRE (simple or IR), (v) DNA-binding cross-talk with another TF, (vi) interference of monomeric GR with the TA activity of TFs by a tethering mechanism, (vii) competition for an overlapping binding site (competitive GRE), (viii) sequestration of a DNA-bound TF, or (ix) competition for binding cofactors with other DNA-bound TFs.
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with an immediate-release profile as opposed to a sustained-release profile [80,81]. This latter is important in view of treating an entire inflamed region during GI transit of the formulation. Therefore, an oral ileo-colonic-targeted budesonide formulation with a sustained-release profile for the treatment of IBD would be a valuable addition to the treatment options of IBD.
Infliximab
TNF-α is a pleiotropic proinflammatory cytokine that is involved in multiple cellular processes, which include cell proliferation, survival, and death. The cells secreting TNF-α are predominantly monocytes, macrophages, and natural killer cells [82–86]. TNF-α is first synthesized as a transmembrane protein (tmTNF-α) and can induce immunological responses in effector cells but also transduce reverse signaling by contact-dependent cell interactions. Additionally, enzymatic cleavage of tmTNF-α by TNF-α-converting enzyme (TACE) yields soluble TNF-α (sTNF-α), which can be distributed in the extracellular space or systemic circulation. Subsequently, sTNF-α can induce immunological effects at distant sites. Effector cell activation by TNF-α under physiological conditions leads to a proinflammatory response that defends the host against infections and site-specific tissue injury [82–86]. However, the elevated TNF-α levels in the mucosa and lamina propria of IBD patients results in an aberrant proinflammatory response that results in clinical symptoms and tissue damage [5,6].
Infliximab (IFX) is a chimeric monoclonal antibody against TNF-α and antagonizes the proinflammatory effects induced by TNF-α. IFX is highly efficacious in the treatment of IBD and induces clinical remission as well as mucosal healing. However, IFX is administered systemically since it is a monoclonal antibody and therefore antagonizes TNF-α systemically. Systemic exposure to anti-TNF-α therapy is associated with adverse events, which include infusion reactions [87,88], psoriasis or psoriasiform lesions [89], osteonecrosis of the jaw [90,91], the development of antinuclear antibodies (ANA) [92– 95], and an increased risk of opportunistic infections [96–98] and developing lymphoma [99]. Additionally, infusion reactions are associated with therapy discontinuation [100]. Systemic administration may also induce the formation of anti-drug antibodies (ADA), which in turn is associated with infusion reactions [101–103]. The ADA generally neutralize IFX, and may thus results in the loss of efficacy or necessitates dose escalation [101–103].
Interestingly, research shows that anti-TNF-α therapy induces localized immunological effects that are associated with the observed clinical response. These localized effects include a decrease in histological and endoscopical disease activity [104–106], inhibition of immune cell activation [107–109], down-regulation of the expression of cell adhesion molecules and proinflammatory cytokines [106,110–115], modulation of monocyte and enterocyte apoptosis [116], restoration of gut barrier function [117,118] and levels
24
of antimicrobial peptides [119] and a favorable effect on the gut microbiome [120,121]. Importantly, it has been shown that anti-TNF-α therapy induces a potent local but not a systemic effect [122] and that gut tissue concentrations may correlate better with a clinical and sustained response compared to serum levels alone [123,124]. This may partly explain anti-TNF-α therapy failure despite therapeutic drug concentrations.
Taken together these data suggest that site-specific as opposed to systemic TNF-α inhibition may be an efficacious treatment option for IBD with expected fewer adverse events related to systemic exposure. However, challenges in realizing site-specific TNF-α inhibition with IFX are drug targeting of the protein to the site of inflammation in a suitable dosage form. Moreover, assuming oral targeted IFX therapy, two major challenges of this approach are the stability of IFX at the targeted site in the GIT and the subsequent penetration of IFX into the inflamed tissue without substantial systemic exposure.
Aim of this thesis
The aim of this thesis was to develop three novel ileo-colonic targeted tablets intended for the topical treatment of IBD in view of maximizing the localized anti-inflammatory effects whilst minimizing the adverse events related to systemic drug exposure. Furthermore, the developmental papers of this thesis may aid as guidance for future research aimed to develop novel formulations targeted to the ileo-colonic region since hydrophilic (mesalazine) and lipophilic (budesonide) drugs and a combination thereof (mesalazine-budesonide) as well as a monoclonal antibody (IFX) have been investigated in a single formulation with or without a sustained-release tablet core, respectively. Additionally, this thesis aims to set the first steps towards the oral targeted treatment of ileo-colonic IBD with monoclonal antibodies.
Outline of this thesis
In chapter 2 we describe an in vitro study on the development of an oral formulation that contained 1200 mg mesalazine and 9 mg budesonide intended for the topical treatment of ileo-colonic IBD. The objective was to develop a zero-order sustained-release tablet with similar release profiles for mesalazine and budesonide. This approach was chosen in view of treating an entire ileo-colonic region during the in vivo GI transit of the formulation. In addition, zero-order release kinetics assures time-independent drug release from the formulation. This study compares the novel formulation with two commercially available formulations that contained 1200 mg mesalazine or 9 mg budesonide, which also aim to target the distal GIT in IBD. Finally, a 6-month accelerated stability study of the formulation was performed. This stability study was performed according to ICH guidelines.
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Chapter 3 describes an in vitro study on the development of two oral formulations
that contained 3 mg or 9 mg budesonide intended for the topical treatment of ileo-colonic IBD. These doses were chosen based on the ECCO guidelines and in view of treatment flexibility. The objective was to develop zero-order sustained-release tablets with similar release profiles for the 3 mg as well as the 9 mg formulation. This approach was chosen in view of treating an entire ileo-colonic region during the in vivo GI transit of the formulation. In addition, zero-order release kinetics assures time-independent drug release from the formulation. The release profiles of the two novel formulations were compared with all the available oral budesonide formulations that are currently used in clinical practice to treat IBD. Finally, a 6-month accelerated stability study of the formulations was performed. This stability study was performed according to ICH guidelines.
In chapter 4 a literature study investigates the available medical literature on the feasibility or effectiveness of localized TNF-α inhibition in IBD. Two search strategies were conducted and the search terms are given in this chapter. One search strategy aimed to find relevant studies on site-specific TNF-α inhibition with drugs that are currently used in clinical practice of IBD, such as IFX and adalimumab. Another search strategy aimed to find relevant studies on more experimental approaches that investigated site-specific TNF-α inhibition, such as antisense oligonucleotides or small interfering RNA (siRNA). The objective of this chapter was to discuss the different approaches of localized TNF-α inhibition in animal studies as well as clinical studies. An important question that will be addressed in this review is whether locally administered anti-TNF-α drugs, which generally are macromolecules, are able to penetrate into the inflamed sites in IBD. The penetration of anti-TNF-α drug into the inflamed region is a prerequisite in view of site-specific TNF-α inhibition in the GIT. However, this is challenging since the absorption mechanisms and kinetics of macromolecules in the GIT differ substantially from smaller chemical entities.
The in vitro study in chapter 5.1 describes the development of an oral formulation that contains 5 mg IFX intended for the topical treatment of ileo-colonic IBD (ColoPulse-IFX). This dose was chosen in view of a practical daily oral dosage regimen that was based on the dosage regimen of intravenously administered IFX, i.e. 5-10 mg/kg per 2-8 weeks. The stresses associated with compounding macromolecules such as IFX into tablets may be detrimental to the protein structure, and thus, the activity and stability of ColoPulse-IFX. Therefore, sugar glass stabilization technology was investigated to overcome this problem. Several protein analysis methods were performed to investigate whether this approach was successful and if IFX could be compounded into ColoPulse-coated tablets. These analyses aim to show that no protein fragments are formed, the tertiary structure is preserved, and that the potency of IFX is maintained. Furthermore, in vitro ileo-colonic targeting of the formulation was investigated. A preliminary stability study was performed to investigate whether this formulation strategy is feasible.
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Chapter 5.2 describes the long-term stability study of ColoPulse-IFX, which was
developed in chapter 5.1. In chapter 5.1 a preliminary stability study was performed to investigate the feasibility of the formulation strategy. Chapter 5.2 describes an extensive long-term stability study of 12 months according to ICH guidelines. During this stability study, ColoPulse-IFX was stored at room temperature (25 °C±2 °C/60% RH±5%) as well as at refrigerated conditions (5 °C±3 °C). This stability study aimed to investigate whether IFX remained stable and potent and whether the ColoPulse targeting performance was maintained during the entire storage period at these two different conditions.
The study design and methodology that aims to investigate the effectiveness of the oral treatment of IBD with ileo-colonic-targeted tablets containing IFX (developed and investigated in chapter 5.1 and 5.2) is presented in chapter 6. This clinical trial is in preparation.
The final chapter (chapter 7) summarizes the results of the earlier chapters and discusses these data in light of the clinical relevance, strengths, limitations, and future perspectives.
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