**Drug Targeting in IBD Treatment – Existing and New Approaches**

Katerina Goracinova, Marija Glavas-Dodov, Maja Simonoska-Crcarevska and Nikola Geskovski *Institute of Pharmaceutical technology, Faculty of Pharmacy University Ss. Cyril and Methodius, Skopje Macedonia* 

## **1. Introduction**

300 Inflammatory Bowel Disease

Yu, H.; Pardoll, D. & Jove, R. (2009). STATs in cancer inflammation and immunity: A

175X.

leading role for stat3. *Nature Reviews Cancer,* Vol.9, No.11, pp.798-809, ISSN 1474-

Recent advances in understanding of inflammatory bowel diseases (IBD) pathogenesis, despite the questions remaining still unanswered, have led to improved approaches in ulcerative colitis (UC) and Crohn`s disease (CD) treatment. In depth investigation of immunopathology of IBD and mucosal inflammation enabled the identification of new strategies for drug targeting, new points of therapeutic attack, cytokine based therapies and new therapeutic agents. Further understanding of the genetic background of this disease will enable discovery of potential gene therapy target molecules related to chronic intestinal inflammation, like new therapeutic targets in IBD.

There is a vast body of information and research associated with current medical treatments, their undesirable effects and limited efficacy. Different drug delivery strategies were employed to overcome limited performance of conventional IBD therapy and many more will be designed to enable safe and efficacious delivery of newly developed therapeutic agents. Both diseases, UC and CD involve different parts of the gastrointestinal (GI) system. CD may involve any part of the GI tract, although most commonly the terminal ileum and colon, while UC usually involves only the colon and always extends proximally from the rectum. T helper 1 (Th1) stimulated immune dysregulation is characteristic for CD while T helper 2 (Th2) stimulated immune dysregulation causes inflammatory mediatory imbalance characteristic for UC (Bouma & Strober, 2003; Hedley, 2000; Sands, 2007; Sellin, 2005). Treatment of UC and CD varies depending on subtype and severity, but significant overlap is seen. The most common therapeutic agents for IBD, aminosalycilates and corticosteroids, have been incorporated into different dosage forms and drug delivery systems (DDS) in order to accomplish successful topical delivery of these agents at the site of inflammation (in CD - terminal ileum, or colon, the site of inflammation for both subtypes) (Green et al., 2002; Haddish-Berhane et al., 2007; Sands, 2007). The most critical step in the development of a reliable DDS for IBD treatment is to achieve improved localization and controlled release of the active substance at the site of inflammation, minimizing the premature release and subsequent absorption in the blood stream. However, the main disadvantage of today's therapy and DDS for management of IBD is the inability to target the drug directly to the site of action (inflammation) and/or to maintain high local concentration. In addition to poor localization extensive metabolism at the level of the epithelial cells of the intestinal wall

Drug Targeting in IBD Treatment – Existing and New Approaches 303

profiling of different phenotypes of IBD (Friend, 2005; Haddish-Berhane et al., 2007;

The rationale behind the utilization of a prodrug approach for drug targeting in IBD is multilateral. Nevertheless, the first 5-ASA prodrug was developed to deliver sulfonamide specifically to the colon, later it was realized that sulfasalazine (sulfapyridyne-azo linkage-5ASA) is efficient for UC treatment as it can successfully perform colon specific delivery of 5-ASA after oral administration, at the same time it can reduce the absorption in the upper intestines due to increased hydrophylicity and/or molecular size (Friend, 2005; Sands, 2007). Azo prodrug approach for colon targeting will improve local concentration of the active substance at the site of therapeutic action, resulting with increased efficacy and fewer side effects of the therapy (Bourgeois, 2005; Oz & Ebersole, 2008; Sands, 2000) . These cannot be linearly applied to all azo conjugates as the side effects may originate from the carrier molecule or degradation products also released

The trigger that releases the active substances from their azo-prodrugs is the colon micoflora enzymatic activity. Contrary to the small intestine (104 CFU/ml mainly gram positive facultative bacteria), colonic flora is of much higher order (1011–1012 CFU/ml) and is mainly consisted of an anaerobic bacteria (Mcconnell et al., 2008; Sinha & Kumria, 2001). For the fermentation of undigested substrates in the small intestine like disaccharides, polysaccharides, mucopolysaccharides, and fulfillment of microflora energy demands, these commensal bacteria produce different types of enzymes like azoreductase, β-galactosidase, β-xylosidase, nitroreductase, glycosidase, deaminase etc. (Bourgeois, 2005; Han & Amidon, 2000; Oz & Ebersole, 2008; Yang, 2008). Most of these anaerobic bacteria are capable of reducing azo linkages, thus releasing the active drug from the azo product. Beside azo prodrugs based on azo linkage of the drug with different carriers, other systems for colon drug targeting based on colonic microflora activity are also developed. The rationale behind glucuronide conjugates development as colon targeting systems is based on the βglucuronidase activity in the lower GI tract; cyclodextrin (CyD) conjugates are based on poor digestion of the complex through the GI tract except in the colon by the colonic microflora; the drug is released from dextran-drug conjugates due to dextranase activity in the region of caecum and colon; amino acid conjugates are probably hydrolyzed by the microbial flora activity at the location of the caecum and colon, etc. (Bourgeois, 2005; Chourasia & Jain, 2003). Examples of the prodrug products available on the market and of

The functionality of these delivery systems is directly influenced by i). the stability of the conjugate in the upper GI tract, ii). colon specific complex degradation and iii). toxicity of the carrier or the cleavage products. Their capacity when applied for targeting in IBD treatment is to release and increase localization and concentration of the drug substance at the specific site in GI tract (site specific delivery). Commercially available 5-ASA conjugates are not able to completely prevent prodrug hydrolysis and 5-ASA absorption in the upper GI tract. Although the approaches presented in the literature using different polymer carriers might probably overcome this weakness, apparently creating a balance among the resilience against hydrolysis in the upper GI tract and required drug release rate in colon is

the prodrug systems still under research are presented in Table 1.

Nugent et al., 2001).

**2.1 Prodrug approach** 

during the azo bond cleavage.

not an easy task.

(ex. hydroxylation of budesonide by cytochrome P450 isoenzyme CYP3A4 in hepatocytes and epithelial cells) might further impair local concentration required for improved drug efficacy (Fedorak & Bistritz, 2005; Klotz & Schwab, 2005).

## **2. Conventional design strategies for GI targeting**

The efficacy of the treatment of IBD depends on the functionality of the strategy for the delivery of therapeutic concentrations of the drug substance at the site of inflammation. In addition, minimizing the intestinal absorption using different formulation design approaches will improve the safety and reduce the adverse effects of the treatments. Various chemical modifications and formulation technologies based on the intestinal physiology (motility, intraluminal pH, and intestinal transit times) as well as distribution of IBD in the GI tract have been developed in order to improve the efficacy and precision of drug release to the affected areas.

These conventional approaches are mainly focused to targeting a particular site in the GI tract and delivery of prodrugs, colonic microflora activated systems, pH dependent and time dependent systems in a form of single or multiunit dosage forms (Gazzaniga et al., 2006; Ishibashi et al., 1998). Delivery strategies and release mechanisms employed in these dosage forms rely on the enzymatic activity of the GI microflora, pH difference between different parts of the GI tract, GI transit time and increased luminal pressure in the colon due to strong peristaltic waves (Leopold, 2001). The conditions in the complex and dynamic GI tract environment are the source of variability of drug release, absorption and patient response even if healthy individuals are concerned. Moreover, due to the pathological changes in response to the IBD, factors like GI tract pH, motility, transit time and microflora activity will become the source of increased variability in the response and effectiveness of different formulations for treatment of the GI diseases. Before mentioned factors and variables suggest that these design approaches might suffer from limited efficacy in concentrating the active substance at the site of inflammation, preventing drug absorption and systemic exposure to these agents. Moreover, if single unit dosage forms are used, their residence time in GI tract will be under constant threat of persistent diarrhea in IBD patients (Bourgeois, 2005; Chourasia & Jain, 2003; Roy & Shahiwala, 2009).

Today it is widely accepted that the delivery of the active substance influences clinical efficacy of the drug product. Based upon this view, clinical studies for the efficacy and safety of different conventional marketed DDS describing the site of drug release, drug release rate, subsequent absorption from the GI tract, systemic absorption and local concentration of the anti-inflammatory agents are the baseline for rational approach in prescribing different doses and dosage forms for IBD, once the location and the type of the disease are known. Due to the clinical efficacy studies valuable information for the usefulness of different dosage forms for the treatment of mild or moderate disease, induction or maintaining remission etc. related to the disease pattern and drug disposition are available (Clemett & Markham, 2000; Prakash, 1999). Moreover, there is increasing number of papers trying to investigate and explain variation in luminal pH in UC and CD patients, mucosal flora profiles for UC and CD which will further improve the understanding of the effect of these variables on the drug release pattern and disposition as well as clinical efficacy of different drug products, but will also help the

profiling of different phenotypes of IBD (Friend, 2005; Haddish-Berhane et al., 2007; Nugent et al., 2001).

## **2.1 Prodrug approach**

302 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

(ex. hydroxylation of budesonide by cytochrome P450 isoenzyme CYP3A4 in hepatocytes and epithelial cells) might further impair local concentration required for improved drug

The efficacy of the treatment of IBD depends on the functionality of the strategy for the delivery of therapeutic concentrations of the drug substance at the site of inflammation. In addition, minimizing the intestinal absorption using different formulation design approaches will improve the safety and reduce the adverse effects of the treatments. Various chemical modifications and formulation technologies based on the intestinal physiology (motility, intraluminal pH, and intestinal transit times) as well as distribution of IBD in the GI tract have been developed in order to improve the efficacy and precision of drug release

These conventional approaches are mainly focused to targeting a particular site in the GI tract and delivery of prodrugs, colonic microflora activated systems, pH dependent and time dependent systems in a form of single or multiunit dosage forms (Gazzaniga et al., 2006; Ishibashi et al., 1998). Delivery strategies and release mechanisms employed in these dosage forms rely on the enzymatic activity of the GI microflora, pH difference between different parts of the GI tract, GI transit time and increased luminal pressure in the colon due to strong peristaltic waves (Leopold, 2001). The conditions in the complex and dynamic GI tract environment are the source of variability of drug release, absorption and patient response even if healthy individuals are concerned. Moreover, due to the pathological changes in response to the IBD, factors like GI tract pH, motility, transit time and microflora activity will become the source of increased variability in the response and effectiveness of different formulations for treatment of the GI diseases. Before mentioned factors and variables suggest that these design approaches might suffer from limited efficacy in concentrating the active substance at the site of inflammation, preventing drug absorption and systemic exposure to these agents. Moreover, if single unit dosage forms are used, their residence time in GI tract will be under constant threat of persistent diarrhea in IBD patients (Bourgeois, 2005; Chourasia & Jain, 2003; Roy &

Today it is widely accepted that the delivery of the active substance influences clinical efficacy of the drug product. Based upon this view, clinical studies for the efficacy and safety of different conventional marketed DDS describing the site of drug release, drug release rate, subsequent absorption from the GI tract, systemic absorption and local concentration of the anti-inflammatory agents are the baseline for rational approach in prescribing different doses and dosage forms for IBD, once the location and the type of the disease are known. Due to the clinical efficacy studies valuable information for the usefulness of different dosage forms for the treatment of mild or moderate disease, induction or maintaining remission etc. related to the disease pattern and drug disposition are available (Clemett & Markham, 2000; Prakash, 1999). Moreover, there is increasing number of papers trying to investigate and explain variation in luminal pH in UC and CD patients, mucosal flora profiles for UC and CD which will further improve the understanding of the effect of these variables on the drug release pattern and disposition as well as clinical efficacy of different drug products, but will also help the

efficacy (Fedorak & Bistritz, 2005; Klotz & Schwab, 2005).

**2. Conventional design strategies for GI targeting** 

to the affected areas.

Shahiwala, 2009).

The rationale behind the utilization of a prodrug approach for drug targeting in IBD is multilateral. Nevertheless, the first 5-ASA prodrug was developed to deliver sulfonamide specifically to the colon, later it was realized that sulfasalazine (sulfapyridyne-azo linkage-5ASA) is efficient for UC treatment as it can successfully perform colon specific delivery of 5-ASA after oral administration, at the same time it can reduce the absorption in the upper intestines due to increased hydrophylicity and/or molecular size (Friend, 2005; Sands, 2007). Azo prodrug approach for colon targeting will improve local concentration of the active substance at the site of therapeutic action, resulting with increased efficacy and fewer side effects of the therapy (Bourgeois, 2005; Oz & Ebersole, 2008; Sands, 2000) . These cannot be linearly applied to all azo conjugates as the side effects may originate from the carrier molecule or degradation products also released during the azo bond cleavage.

The trigger that releases the active substances from their azo-prodrugs is the colon micoflora enzymatic activity. Contrary to the small intestine (104 CFU/ml mainly gram positive facultative bacteria), colonic flora is of much higher order (1011–1012 CFU/ml) and is mainly consisted of an anaerobic bacteria (Mcconnell et al., 2008; Sinha & Kumria, 2001). For the fermentation of undigested substrates in the small intestine like disaccharides, polysaccharides, mucopolysaccharides, and fulfillment of microflora energy demands, these commensal bacteria produce different types of enzymes like azoreductase, β-galactosidase, β-xylosidase, nitroreductase, glycosidase, deaminase etc. (Bourgeois, 2005; Han & Amidon, 2000; Oz & Ebersole, 2008; Yang, 2008). Most of these anaerobic bacteria are capable of reducing azo linkages, thus releasing the active drug from the azo product. Beside azo prodrugs based on azo linkage of the drug with different carriers, other systems for colon drug targeting based on colonic microflora activity are also developed. The rationale behind glucuronide conjugates development as colon targeting systems is based on the βglucuronidase activity in the lower GI tract; cyclodextrin (CyD) conjugates are based on poor digestion of the complex through the GI tract except in the colon by the colonic microflora; the drug is released from dextran-drug conjugates due to dextranase activity in the region of caecum and colon; amino acid conjugates are probably hydrolyzed by the microbial flora activity at the location of the caecum and colon, etc. (Bourgeois, 2005; Chourasia & Jain, 2003). Examples of the prodrug products available on the market and of the prodrug systems still under research are presented in Table 1.

The functionality of these delivery systems is directly influenced by i). the stability of the conjugate in the upper GI tract, ii). colon specific complex degradation and iii). toxicity of the carrier or the cleavage products. Their capacity when applied for targeting in IBD treatment is to release and increase localization and concentration of the drug substance at the specific site in GI tract (site specific delivery). Commercially available 5-ASA conjugates are not able to completely prevent prodrug hydrolysis and 5-ASA absorption in the upper GI tract. Although the approaches presented in the literature using different polymer carriers might probably overcome this weakness, apparently creating a balance among the resilience against hydrolysis in the upper GI tract and required drug release rate in colon is not an easy task.

Drug Targeting in IBD Treatment – Existing and New Approaches 305

(dialdehyde dextran) coupled to alpha NH2 groups from 5- ASA to form imine bonds which are further reduced to secondary amine bonds to improve stability in water

5-ASA Oxidised dextran

5-ASA PAMAM (drug is

bound to the polymer *via* spacers containing azo bonds: p-

spacer)

**2.2 pH, time and microbiologically dependent systems for colon targeting** 

aminobenzoic acid spacer and paminohippuric acid

Drug delivery approaches for pH and time dependent systems currently on the market are based on polymer coating or matrix technology for single or multiple unit dosage forms. Eudragit S coated 5-ASA tablets (Asacol®) and Eudragit L coated 5-ASA tablets (Salofalk®, Claversal®, Mesazal®, Calitoflak®) represent purely pH dependent delayed release systems which release the active substance upon the dissolution of the polymer coating, generally at pH 7 or pH 6 for Eudragit S and Eudragit L coating, respectively (Klotz & Schwab, 2005; Leopold, 2001; Wilding et al., 2000). Entocort® is designed as Eudragit L100-55 coated budesonide/ethylcellulose beads in gelatin capsule in order to delay the release of the active substance till pH 5.5 and further sustain the drug release through GI tract due to the presence of ethylcellulose (Fedorak & Bistritz, 2005; Friend, 2005; Klotz & Schwab, 2005). The coating of combined Eudragit enterosoluble and swelling polymers (Eudragit L/S/RL an RS) in Budenofalk microgranules is supposed to delay the release until pH 6.4. It is assumed that multiunit coated beads will provide

Prednisolone α-CyD Enzymatic hydrolysis -

Bacterial enzymatic degradation of CyD rings to small

saccharides followed by ester hydrolysis in the lower parts of GI tract

Enzymatic hydrolysis of the amine complex by deaminases and the dextrane glycoside bonds in the distal ileum and proximal colon by dextranases

Enzymatic cleavage of the azo-bond by colonic azo-reductases

to oligomers which are further split by colonic esterases

(Yano et al., 2001)

(Ahmad et al., 2006)

(Wiwattana patapee et al., 2003)

**Cyclodextrin (CyD) conjugates** 

**Polysaccharide conjugates** 

**Dendrimer conjugates**

Table 1. Conjugates for colon drug targeting

Hydrophylic polyamidoamine dendrimer (PAMAM)

Prednisolone Succinate-CyD Ester Conjugate

Dextran conjugate


(azoreduction) of the azo-bond by azoreductases in the colon

of the amide bond

cleavage of the polar moiety by bacterial glycosidases in the

Deglucuronidation of the drug – glucuronic acid prodrug by the β −glucuronidase secreted by GI bacteria in the

by the GI microorganisms in the caecum and

colon

colon

colon

hydrolysis -

β-D-glucoside Enzymatic hydrolysis -

Marketed product

Marketed product

Marketed product

(Brown et al., 1983)

(Kopecek, 1990)

(Kopecek et al., 1992)

(Jung et al., 1998)

(Friend & Chang, 1985)

(Friend, 1991; Nolen et al., 1995)

**Conjugates Drug Carrier molecule Release mechanism Source** 

Sulfasalazine 5-ASA Sulfapyridine Enzymatic cleavage

alanine

ASA is used as a carrier for the other


methacrylamide copolymer

methacrylamide copolymers with bioadhesive moiety

(fucosylamine)

Budesonide β-D-glucuronide Enzymatic

5-ASA Glycine Enzymatic hydrolysis

5-ASA One molecule of 5-

5-ASA Sulphanilamidoethyl

5-ASA *N*-(-hydroxypropyl)

5-ASA *N*-(2-hydroxypropyl)

Balsalazide 5-ASA 4-aminobenzoyl-β-

**Azo prodrugs** 

Olsalazine (disodium azodisalicylate)

Non-absorbable polymer-drug azo conjugate

Water soluble polymer-drug azo conjugate

Bioadhesive polymer-drug azo conjugate

**Amino-acid conjugates**  Drug-amino acid conjugate

Drug-glycoside conjugate (coupling through β−glycosidic bond)

Drugglycoronide conjugate

**Glycoside and glucuronide conjugates** 

or

Prednisolone

Dexamethasone


Table 1. Conjugates for colon drug targeting

#### **2.2 pH, time and microbiologically dependent systems for colon targeting**

Drug delivery approaches for pH and time dependent systems currently on the market are based on polymer coating or matrix technology for single or multiple unit dosage forms. Eudragit S coated 5-ASA tablets (Asacol®) and Eudragit L coated 5-ASA tablets (Salofalk®, Claversal®, Mesazal®, Calitoflak®) represent purely pH dependent delayed release systems which release the active substance upon the dissolution of the polymer coating, generally at pH 7 or pH 6 for Eudragit S and Eudragit L coating, respectively (Klotz & Schwab, 2005; Leopold, 2001; Wilding et al., 2000). Entocort® is designed as Eudragit L100-55 coated budesonide/ethylcellulose beads in gelatin capsule in order to delay the release of the active substance till pH 5.5 and further sustain the drug release through GI tract due to the presence of ethylcellulose (Fedorak & Bistritz, 2005; Friend, 2005; Klotz & Schwab, 2005). The coating of combined Eudragit enterosoluble and swelling polymers (Eudragit L/S/RL an RS) in Budenofalk microgranules is supposed to delay the release until pH 6.4. It is assumed that multiunit coated beads will provide

Drug Targeting in IBD Treatment – Existing and New Approaches 307

Compared to pH dependent single unit dosage forms that release the drug in a short period after the dissolution of the coating, multiple dose units spread all over the region of interest

pH and time dependent systems were developed in order to combine delayed dissolution and sustained diffusion through swellable or non-swellable coatings or matrices. Apriso® is formulated as enteric coated microgranules with delayed release starting at pH 6.0 and polymer matrix core which will provide extended release of the active substance and deliver the drug continuously from the small bowel through the colon (Brunner et al., 2003;

Time dependent delivery systems like TIME CLOCK™ and Pulsincap™ are developed as colon drug delivery systems based on the observation that the small intestinal transit time doesn't exceed a mean of 3-4 hours (Bourgeois, 2005; Chourasia & Jain, 2003; Gazzaniga et al., 2008). These systems usually show burst drug release after the lag time, mainly due to the superdisintegrants and highly swellable agents which act upon the dissolution and permeability of the protective coating. TIME-CLOCK™ system, proposed by Pozzi et al. (Pozzi et al., 1994) is composed of tablet core containing the drug and bulking agents like lactose, polyvinyl pyrrolidone, corn starch and lubricant magnesium stearate, coated with hydrophobic dispersion of carnauba wax, bees' wax, polyoxyethylene sorbitan monooleate and hydroxypropyl methylcellulose in water. Drug release is not dependent to normal physiological conditions, pH, digestive state and anatomical position. The lag time can be modulated by altering the thickness of the coating. Pulsincap™ system consists of water insoluble capsule containing the formulation closed at the open end with a swellable hydrogel plug. Lag time is controlled by the type, dimensions and position of the plug and rapid drug release at particular site in GI tract is ensured by incorporation of disintegrants or effervescent agents. Pulsatile pH and time dependent multiple unit dosage forms composed of enterosolvent outer layer and a second membrane of water insoluble and enteric polymers are also suitable for colon drug targeting. OROS-CT™ system can be a single osmotic unit or may incorporate as many as 5-6 push-pull units, each 4 mm in diameter, encapsulated within a hard gelatin capsule (Leopold, 2001; Verma et al., 2000, 2002). Because of its drug-impermeable enteric coating, the release from each push-pull unit is prevented and delayed until higher pH values. When the acid resistant coating dissolves, water enters the unit, causing the osmotic push compartment to swell. Drug gel is forced out through the orifice due to the swelling effect and increased osmotic pressure in the push compartment at a rate precisely controlled by the rate of water transport through the semipermeable membrane. For treating ulcerative colitis, each push pull unit is designed with a 3-4 hours post gastric delay to prevent drug delivery in the small intestine. OROS-CT™ units can maintain a constant release rate for up to

24 hours in the colon or can deliver drug over a period as short as four hours.

Except the previously presented examples, approaches based on modification and combination of two or more conventional designs in order to improve the delivery and site specificity in the GI tract are presented through literature. Sinha and Kumria developed conventional enteric/coated time dependent single unit dosage form for colon specific delivery of water insoluble drugs or slightly soluble drugs (Sinha & Kumria, 2002). Time dependent delivery was achieved using xanthan gum, guar gum, chitosan and Eudragit E as binders. The most successful in sustaining the drug release in the upper GI tract was chitosan. Moreover, application of chitosan for site specific targeting of less soluble substances was favorable as the release was retarded only till microbial degradation or polymer solubilization took place in the colon. Another variation of pH and time dependent

and release the drug in a sustained manner at the site of action.

Oliveira, L. & Cohen, 2011; Sandborn et al., 2010).

more uniform transit and distribution through the GI tract and accordingly more uniform drug release as they are less subjected to the differences in the transit time due to the environmental changes. Accordingly, the exposition of the dosage forms to an acidic environment due to the differences in the gastric emptying process for pellets and tablets might be different. Pellets are continuously emptied from the stomach (unless settled at the greater curvature or float because of the high or low density, respectively) during the digestive period, and non-desintegrating tablets, like enteric coated tablets, are emptied during the interdigestive period. The description of the drug release site according to the solubility of the enterosolvent coating is a valuable orientation point, but at the same time very general as it can be only applied in ideal circumstances and is subjected to number of inter-, intra-individual variables as well as different disease factors. However, it might be expected that patients with UC will have the greatest benefit as by design pH dependent dosage forms will deliver most of the dose in the distal ileum and colon. Latest published research data on small bowel pH in patients with ileal CD (3 patients with ileal CD, 8 patients with operated ileal CD and 4 normal controls) point that small bowel pH was similar to the control group and sufficiently high to allow the dissolution of enterosolvent delayed release dosage forms coated with polymer that requires pH less than 7 to be dissolved (ex. Eudragit L) (Nugent et al., 2000). As these systems usually need at least 30 minutes (max. 1 hour) to complete the dissolution in vitro it seems that the drug will be released during the transit through the small bowel towards the colon. Another study that measured the luminal pH and mean transit time in patients with mild to moderate UC clearly state that more than 50% of examined patients (10 males, 4 with extensive and 6 with distal colitis) failed to achieve the sustained pH level needed for dissolution of some delayed release 5-ASA preparations (proximal, distal small bowel pH as well as right and left colon pH were measured). A different study confirmed that colonic pH is lower in patients with mild to moderate UC compared to the control group, but small bowel pH was not significantly changed compared to the controls (Friend, 2005; Oliveira, L. & Cohen, 2011). Opposite findings, from confirmed efficacy to incomplete drug release, are also found through the reports from clinical studies in the literature. As pH dependent systems are widely used in today's practice careful and individual approach in prescribing of the delayed release systems for IBD would benefit the patient.

Time based delivery systems release the drug in a sustained manner as they pass down the GI tract. Pentasa® is based on ethylcellulose coated beads that release 5-ASA slowly and continuously throughout the small bowel and colon in a time-dependent manner (Klotz & Schwab, 2005; Larouche, 1995; Wilding et al., 2000). Scintigraphic evaluation of the disposition, dispersion and movement of the Pentasa® microgranules through the GI tract (app. 1 mm in diameter) point that the particles showed fluid like properties in a fasted stomach, rapid exponential emptying in a progressive manner over 30-60 min period with no signs of delay. Colon arrival was observed within 4-6 hours on average followed by subsequent wide distribution of the beads through the colon. Accumulation of time controlled system in the ileo-caecal junction might present serious problem and should be carefully avoided by stimulating the colonic activity and gastrocolonic responce with a carefully scheduled light meal (Adkin et al., 1993; Price et al., 1993; Wilding et al., 2000). If the release of 5-ASA is successfully postponed or minimized by the presence of a ethylcellulose polymeric membrane till reaching the lower parts of the GI tract, this type of dosage form due to its wide multiunit distribution at the site of inflammation would be beneficial as the release is expected in a sustained manner through the entire colon.

more uniform transit and distribution through the GI tract and accordingly more uniform drug release as they are less subjected to the differences in the transit time due to the environmental changes. Accordingly, the exposition of the dosage forms to an acidic environment due to the differences in the gastric emptying process for pellets and tablets might be different. Pellets are continuously emptied from the stomach (unless settled at the greater curvature or float because of the high or low density, respectively) during the digestive period, and non-desintegrating tablets, like enteric coated tablets, are emptied during the interdigestive period. The description of the drug release site according to the solubility of the enterosolvent coating is a valuable orientation point, but at the same time very general as it can be only applied in ideal circumstances and is subjected to number of inter-, intra-individual variables as well as different disease factors. However, it might be expected that patients with UC will have the greatest benefit as by design pH dependent dosage forms will deliver most of the dose in the distal ileum and colon. Latest published research data on small bowel pH in patients with ileal CD (3 patients with ileal CD, 8 patients with operated ileal CD and 4 normal controls) point that small bowel pH was similar to the control group and sufficiently high to allow the dissolution of enterosolvent delayed release dosage forms coated with polymer that requires pH less than 7 to be dissolved (ex. Eudragit L) (Nugent et al., 2000). As these systems usually need at least 30 minutes (max. 1 hour) to complete the dissolution in vitro it seems that the drug will be released during the transit through the small bowel towards the colon. Another study that measured the luminal pH and mean transit time in patients with mild to moderate UC clearly state that more than 50% of examined patients (10 males, 4 with extensive and 6 with distal colitis) failed to achieve the sustained pH level needed for dissolution of some delayed release 5-ASA preparations (proximal, distal small bowel pH as well as right and left colon pH were measured). A different study confirmed that colonic pH is lower in patients with mild to moderate UC compared to the control group, but small bowel pH was not significantly changed compared to the controls (Friend, 2005; Oliveira, L. & Cohen, 2011). Opposite findings, from confirmed efficacy to incomplete drug release, are also found through the reports from clinical studies in the literature. As pH dependent systems are widely used in today's practice careful and individual approach in

prescribing of the delayed release systems for IBD would benefit the patient.

Time based delivery systems release the drug in a sustained manner as they pass down the GI tract. Pentasa® is based on ethylcellulose coated beads that release 5-ASA slowly and continuously throughout the small bowel and colon in a time-dependent manner (Klotz & Schwab, 2005; Larouche, 1995; Wilding et al., 2000). Scintigraphic evaluation of the disposition, dispersion and movement of the Pentasa® microgranules through the GI tract (app. 1 mm in diameter) point that the particles showed fluid like properties in a fasted stomach, rapid exponential emptying in a progressive manner over 30-60 min period with no signs of delay. Colon arrival was observed within 4-6 hours on average followed by subsequent wide distribution of the beads through the colon. Accumulation of time controlled system in the ileo-caecal junction might present serious problem and should be carefully avoided by stimulating the colonic activity and gastrocolonic responce with a carefully scheduled light meal (Adkin et al., 1993; Price et al., 1993; Wilding et al., 2000). If the release of 5-ASA is successfully postponed or minimized by the presence of a ethylcellulose polymeric membrane till reaching the lower parts of the GI tract, this type of dosage form due to its wide multiunit distribution at the site of inflammation would be beneficial as the release is expected in a sustained manner through the entire colon. Compared to pH dependent single unit dosage forms that release the drug in a short period after the dissolution of the coating, multiple dose units spread all over the region of interest and release the drug in a sustained manner at the site of action.

pH and time dependent systems were developed in order to combine delayed dissolution and sustained diffusion through swellable or non-swellable coatings or matrices. Apriso® is formulated as enteric coated microgranules with delayed release starting at pH 6.0 and polymer matrix core which will provide extended release of the active substance and deliver the drug continuously from the small bowel through the colon (Brunner et al., 2003; Oliveira, L. & Cohen, 2011; Sandborn et al., 2010).

Time dependent delivery systems like TIME CLOCK™ and Pulsincap™ are developed as colon drug delivery systems based on the observation that the small intestinal transit time doesn't exceed a mean of 3-4 hours (Bourgeois, 2005; Chourasia & Jain, 2003; Gazzaniga et al., 2008). These systems usually show burst drug release after the lag time, mainly due to the superdisintegrants and highly swellable agents which act upon the dissolution and permeability of the protective coating. TIME-CLOCK™ system, proposed by Pozzi et al. (Pozzi et al., 1994) is composed of tablet core containing the drug and bulking agents like lactose, polyvinyl pyrrolidone, corn starch and lubricant magnesium stearate, coated with hydrophobic dispersion of carnauba wax, bees' wax, polyoxyethylene sorbitan monooleate and hydroxypropyl methylcellulose in water. Drug release is not dependent to normal physiological conditions, pH, digestive state and anatomical position. The lag time can be modulated by altering the thickness of the coating. Pulsincap™ system consists of water insoluble capsule containing the formulation closed at the open end with a swellable hydrogel plug. Lag time is controlled by the type, dimensions and position of the plug and rapid drug release at particular site in GI tract is ensured by incorporation of disintegrants or effervescent agents. Pulsatile pH and time dependent multiple unit dosage forms composed of enterosolvent outer layer and a second membrane of water insoluble and enteric polymers are also suitable for colon drug targeting. OROS-CT™ system can be a single osmotic unit or may incorporate as many as 5-6 push-pull units, each 4 mm in diameter, encapsulated within a hard gelatin capsule (Leopold, 2001; Verma et al., 2000, 2002). Because of its drug-impermeable enteric coating, the release from each push-pull unit is prevented and delayed until higher pH values. When the acid resistant coating dissolves, water enters the unit, causing the osmotic push compartment to swell. Drug gel is forced out through the orifice due to the swelling effect and increased osmotic pressure in the push compartment at a rate precisely controlled by the rate of water transport through the semipermeable membrane. For treating ulcerative colitis, each push pull unit is designed with a 3-4 hours post gastric delay to prevent drug delivery in the small intestine. OROS-CT™ units can maintain a constant release rate for up to 24 hours in the colon or can deliver drug over a period as short as four hours.

Except the previously presented examples, approaches based on modification and combination of two or more conventional designs in order to improve the delivery and site specificity in the GI tract are presented through literature. Sinha and Kumria developed conventional enteric/coated time dependent single unit dosage form for colon specific delivery of water insoluble drugs or slightly soluble drugs (Sinha & Kumria, 2002). Time dependent delivery was achieved using xanthan gum, guar gum, chitosan and Eudragit E as binders. The most successful in sustaining the drug release in the upper GI tract was chitosan. Moreover, application of chitosan for site specific targeting of less soluble substances was favorable as the release was retarded only till microbial degradation or polymer solubilization took place in the colon. Another variation of pH and time dependent

Drug Targeting in IBD Treatment – Existing and New Approaches 309

On the other hand, advances in the understanding of the pathophysiology of IBD led to a great interest in the evaluation of new therapeutic agents with novel and improved therapeutic actions and new therapeutic targets. Biological therapy came about as a consequence of improved understanding of the mechanism and pathophysiology of the disease and it was the most important addition to the IBD therapy in 50 years. Development of sophisticated drug targeting carriers for per oral delivery of new protein and peptide therapeutic agents for the treatment of IBD is by no means essential not only to provide stability, efficacy and improved targeting at the site of inflammation but to decrease the serious side effect of the biological therapeutics when administered through conventional parenteral dosage forms. The underlying mechanism of the novel disease oriented experimental strategies for drug targeting is based on complete understanding of

In order to cover the basic principles of the disease oriented strategies for GI targeting based on micro- and nano-sized carriers and to emphasize the advantages and disadvantages of this design approach, short summary of the disease ethyology and pathogenesis will be given. Common working hypothesis for explanation and understanding of etiology and pathogenesis of IBD is that IBD results from inappropriate and exaggerated mucosal immune response of the innate and adaptive immune system to normal constituents of the mucosal microflora that is in part determined by the genetic factor. Immunopathogenesis results from secretion of toxic peroxide anions, proteases, and oxygen/nitrogen radicals by activated macrophages and T-cells that kill the invading bacteria. But, these substances, except destroying the antigen, will also cause indiscriminate damage to the surrounding tissue. In healthy GI tract the inflammation ceases once the antigen is eliminated and the immune cells are no longer directly stimulated. But in IBD the immune cells are stimulated from commensal bacteria or GI tract bacterial microflora which is a trigger for continuous inflammation, mounting and accumulating inflammatory mediators and inflammatory substances with increasing potential for inflammation induced damage to the epithelial barrier. Inflammation induced damage will allow increased permeability and infiltration of bacteria into the lamina propria causing further stimulation of the immune cells, magnifying the inflammatory response and creating a vicious circle of continuous tissue damage. Increased permeability of the epithelial barrier, accompanied with increase of M-cell number as well as increased uptake activity of the immunoregulatory cells at the site of inflammation are the main disease related factors resulting with increased interaction with the physical systems like micro- and nano-particles (MPs and NPs) and increased concentration of these polymeric carriers loaded with drug substance at the site of action (Babbs, 1992; Beckman & Ames, 1997; Cuvelier et al., 1994; Grisham & Granger, 1988; Ina et al., 1999; Nikolaus et al., 1998; Oz & Ebersole, 2008; Uguccioni et al., 1999). During inflammation the particles will be concentrated in an increased manner in the lamina propria and in the follicle region not only through the usual gateway like antigen sampling microfold cells (M-cells) overlying the lymphoid follicles of Payer's patches in the small intestines and colonic mucosal lymphoid organs in the colon but through the leaky inflamed epithelium as well (Fujimura et al., 1992; Van Assche & Rutgeerts, 2002; Yeh et al., 1998). Further, the interaction of the physical systems with the aberrantly present enormously active immunorelated cells (macrophages, dendritic cells) at the site of inflammation will increase the concentration of the active substance in the inflammation related elements which actually represent therapeutic targets for the anti-inflammatory

the mechanisms of the disease and drug action.

agents.

single unit system for colonic delivery was published by Ishibashi et al (Ishibashi et al., 1998). Drug release from the capsule in the upper parts of the GI tract was postponed by the acidoresistant layer at the capsule surface. To prevent the contact among the outer anionic layer and inner cationic (Eudragit E) polymer, an intermediate water soluble layer was introduced. After gastric empting both layers dissolve quickly, exposing the cationic layer to the intestinal environment. The cationic layer delayed the release till its complete dissolution due to the presence of the organic acid in the inner capsule body together with the drug. Microgranular system coated with outer layer of enterosolvent Eudragit FS (dissolves at pH higher than 6.8) and inner layer composed of combination of pH independent cationic polymers Eudragit RL and Eudragit RS demonstrated the potential for delayed release till pH 6.5 and sustained release through the colon for approximately 12 hours (Gupta et al., 2000 as cited in Gupta et al., 2001). Different enzymatically cleavable polymers are also reported to be synthesized for application in colonic microflora activated systems. First biodegradable enzymatically cleavable polymers for colon drug targeting are the azo polymers composed of hydrophobic and hydrophilic moiety connected by an azo segment. Their microbial degradation and consequently drug release rate from the coated drug dosage forms depends upon their hydrophilicity. Careful adjustment among the hydrophilic and hydrophobic part is a necessity for maintaining gastric resistance and sustained release in the lower GI tract. However, reduction of the azo compound is usually very slow which might lead to incomplete release of the drug substance. Major drawback of these compounds is coming from the toxicity of the primary aromatic amines resulting from the microbial reduction of the more hydrophilic azo compounds and with more hydrophobic polymers reduction will be stopped at the hydrazo compounds instead of leading to the amines which will influence the drug release rate and mechanism. Azo crosslinked copolymers of styrene and hydroxyethylmethacrylate and methyl methacrylate polymers crosslinked through bifunctional azo aromatic compounds, azo aromatic group containing polyurethanes and pH sensitive terpolymers containing hydroxyethylmethacrylate, methyl methacrylate and methacrylic acid were also investigated as sustained release coatings and water insoluble hydrogels for colon targeting (Bourgeois, 2005; Leopold, 2001). More examples of technologies and combined formulation approaches for pH, time,

**3. Disease oriented strategies for drug targeting in IBD** 

for colon targeting are presented in Table 2.

During the past fifteen years vast body of research has been done on CD and UC complex cascade of immunologically driven interactions by inflammatory substances and cytokines. Detailed knowledge of different stages of these pathways (Rivkin, 2009; Rutgeerts et al., 2004; Van Deventer, 1999; Wong et al., 2008) is very useful for identifying new therapeutic targets for IBD therapy as well as clarification of the mechanisms of action of current therapeutic agents. Improved understanding of the mechanisms of disease and mechanism of action of the active substances at the molecular levels brought new ideas and models for rational drug targeting and drug delivery at the site of action (organ, tissue, and cell) at the same time reducing the concentration at the non-targeted sites. It has been proven that development of rational delivery approaches for old therapeutic agents might improve the efficacy, decrease the side effects of the therapy, and even improve therapeutic potential of the drug substance.

microbiologically and pressure dependent single and multiple unit drug delivery systems

single unit system for colonic delivery was published by Ishibashi et al (Ishibashi et al., 1998). Drug release from the capsule in the upper parts of the GI tract was postponed by the acidoresistant layer at the capsule surface. To prevent the contact among the outer anionic layer and inner cationic (Eudragit E) polymer, an intermediate water soluble layer was introduced. After gastric empting both layers dissolve quickly, exposing the cationic layer to the intestinal environment. The cationic layer delayed the release till its complete dissolution due to the presence of the organic acid in the inner capsule body together with the drug. Microgranular system coated with outer layer of enterosolvent Eudragit FS (dissolves at pH higher than 6.8) and inner layer composed of combination of pH independent cationic polymers Eudragit RL and Eudragit RS demonstrated the potential for delayed release till pH 6.5 and sustained release through the colon for approximately 12 hours (Gupta et al., 2000 as cited in Gupta et al., 2001). Different enzymatically cleavable polymers are also reported to be synthesized for application in colonic microflora activated systems. First biodegradable enzymatically cleavable polymers for colon drug targeting are the azo polymers composed of hydrophobic and hydrophilic moiety connected by an azo segment. Their microbial degradation and consequently drug release rate from the coated drug dosage forms depends upon their hydrophilicity. Careful adjustment among the hydrophilic and hydrophobic part is a necessity for maintaining gastric resistance and sustained release in the lower GI tract. However, reduction of the azo compound is usually very slow which might lead to incomplete release of the drug substance. Major drawback of these compounds is coming from the toxicity of the primary aromatic amines resulting from the microbial reduction of the more hydrophilic azo compounds and with more hydrophobic polymers reduction will be stopped at the hydrazo compounds instead of leading to the amines which will influence the drug release rate and mechanism. Azo crosslinked copolymers of styrene and hydroxyethylmethacrylate and methyl methacrylate polymers crosslinked through bifunctional azo aromatic compounds, azo aromatic group containing polyurethanes and pH sensitive terpolymers containing hydroxyethylmethacrylate, methyl methacrylate and methacrylic acid were also investigated as sustained release coatings and water insoluble hydrogels for colon targeting (Bourgeois, 2005; Leopold, 2001). More examples of technologies and combined formulation approaches for pH, time, microbiologically and pressure dependent single and multiple unit drug delivery systems

for colon targeting are presented in Table 2.

the drug substance.

**3. Disease oriented strategies for drug targeting in IBD** 

During the past fifteen years vast body of research has been done on CD and UC complex cascade of immunologically driven interactions by inflammatory substances and cytokines. Detailed knowledge of different stages of these pathways (Rivkin, 2009; Rutgeerts et al., 2004; Van Deventer, 1999; Wong et al., 2008) is very useful for identifying new therapeutic targets for IBD therapy as well as clarification of the mechanisms of action of current therapeutic agents. Improved understanding of the mechanisms of disease and mechanism of action of the active substances at the molecular levels brought new ideas and models for rational drug targeting and drug delivery at the site of action (organ, tissue, and cell) at the same time reducing the concentration at the non-targeted sites. It has been proven that development of rational delivery approaches for old therapeutic agents might improve the efficacy, decrease the side effects of the therapy, and even improve therapeutic potential of On the other hand, advances in the understanding of the pathophysiology of IBD led to a great interest in the evaluation of new therapeutic agents with novel and improved therapeutic actions and new therapeutic targets. Biological therapy came about as a consequence of improved understanding of the mechanism and pathophysiology of the disease and it was the most important addition to the IBD therapy in 50 years. Development of sophisticated drug targeting carriers for per oral delivery of new protein and peptide therapeutic agents for the treatment of IBD is by no means essential not only to provide stability, efficacy and improved targeting at the site of inflammation but to decrease the serious side effect of the biological therapeutics when administered through conventional parenteral dosage forms. The underlying mechanism of the novel disease oriented experimental strategies for drug targeting is based on complete understanding of the mechanisms of the disease and drug action.

In order to cover the basic principles of the disease oriented strategies for GI targeting based on micro- and nano-sized carriers and to emphasize the advantages and disadvantages of this design approach, short summary of the disease ethyology and pathogenesis will be given. Common working hypothesis for explanation and understanding of etiology and pathogenesis of IBD is that IBD results from inappropriate and exaggerated mucosal immune response of the innate and adaptive immune system to normal constituents of the mucosal microflora that is in part determined by the genetic factor. Immunopathogenesis results from secretion of toxic peroxide anions, proteases, and oxygen/nitrogen radicals by activated macrophages and T-cells that kill the invading bacteria. But, these substances, except destroying the antigen, will also cause indiscriminate damage to the surrounding tissue. In healthy GI tract the inflammation ceases once the antigen is eliminated and the immune cells are no longer directly stimulated. But in IBD the immune cells are stimulated from commensal bacteria or GI tract bacterial microflora which is a trigger for continuous inflammation, mounting and accumulating inflammatory mediators and inflammatory substances with increasing potential for inflammation induced damage to the epithelial barrier. Inflammation induced damage will allow increased permeability and infiltration of bacteria into the lamina propria causing further stimulation of the immune cells, magnifying the inflammatory response and creating a vicious circle of continuous tissue damage. Increased permeability of the epithelial barrier, accompanied with increase of M-cell number as well as increased uptake activity of the immunoregulatory cells at the site of inflammation are the main disease related factors resulting with increased interaction with the physical systems like micro- and nano-particles (MPs and NPs) and increased concentration of these polymeric carriers loaded with drug substance at the site of action (Babbs, 1992; Beckman & Ames, 1997; Cuvelier et al., 1994; Grisham & Granger, 1988; Ina et al., 1999; Nikolaus et al., 1998; Oz & Ebersole, 2008; Uguccioni et al., 1999). During inflammation the particles will be concentrated in an increased manner in the lamina propria and in the follicle region not only through the usual gateway like antigen sampling microfold cells (M-cells) overlying the lymphoid follicles of Payer's patches in the small intestines and colonic mucosal lymphoid organs in the colon but through the leaky inflamed epithelium as well (Fujimura et al., 1992; Van Assche & Rutgeerts, 2002; Yeh et al., 1998). Further, the interaction of the physical systems with the aberrantly present enormously active immunorelated cells (macrophages, dendritic cells) at the site of inflammation will increase the concentration of the active substance in the inflammation related elements which actually represent therapeutic targets for the anti-inflammatory agents.

Drug Targeting in IBD Treatment – Existing and New Approaches 311

This phenomenon is equable to epithelial EPR effect (enhanced permeability and retention due to increased tumor capillary endothelial permeability) employed for drug targeting in solid tumors, as the potential strategy for targeting the inflamed tissue in GI tract is based on quite similar principles of increased permeability and retention by the endothelial tissue (Lamprecht, 2010; Pastorelli et al., 2008). Compared to conventional GI site targeting using pH, time dependent, pressure or microbiologically dependent systems, this approach is a an improvement in the principle of accumulation as it targets directly the site of inflammation. The fact that often the exact location of the site of inflammation is not known is not an issue for this design approach as drug delivery systems accumulate at the site of inflammation due to the increased permeability of the inflamed mucosa as well as particle uptake due to the interaction with aberrantly present macrophages and dendritic cells at the site of inflammation (Nakase et al., 2000; Tabata et al., 1996). The DDS designed by this targeting approach have to be fabricated with specific physicochemical properties and to be able to overcome the barriers including steep pH gradient, premature binding to the mucus layer, premature uptake or absorption and premature clearance, in order to reach the site of

inflammation and accumulate according to the epithelial EPR effect in the GI tract.

Fig. 1. Translocation of the particles through GI tract epithelium – mechanistic approach (the sieving effect and partitioning between mucus/glycocalyx and GI tract epithelium is not presented), 1. Un-inflamed mucosa: endocytotic uptake and/or transcytosis through enterocytes (particles size<500 nm); lymphatic uptake - particles adsorbed by M-cells of the Peyer's patches (particle size <5 µm) and enhanced adhesion of the MPs and NPs to the intestinal epithelium elicited by the adequate muco/bioadhesive coating 2. Inflamed mucosa: increased particle uptake due to cytokine induced disruption and leaky epithelium; presence of large intercellular pores due to the lower expression of tight junction proteins; improved

macrophages, dendritic cells and natural killer cells in lamina propria and in the mucus layer. Sophisticated manipulation of the physicochemical properties during the fabrication of the targeted DDS will provide functionality of the proposed targeting mechanism. Among them in addition to particle size and particle size distribution are the stability in GI tract, zeta potential, hydrophylicity, hydrophobicity, swelling properties, muco/bioadhesivity, surface

Targeting IBD using disease oriented strategy requires particle stability and inertness in the upper GI tract, increased retention time in the lower parts of the GI tract, specific interaction

lymphatic uptake due to the increased M-cell population and large population of

**3.1 Physicochemical properties affecting the efficacy of the DDS** 

active groups, density, porosity, etc.


Table 2. Conventional approaches for colon targeting

CODES™ - colon specific drug delivery technology for single unit (tablets) and multiple unit (pellets) dosage forms - developed as a combination of pH, (Katsuma et al., 2004, Omar et al., 2007)

(Khan et al., 2000)

(Akhgari et al., 2006; Patel, 2010)

(Bott et al., 2004; Ibekwe et al., 2006; Kshirsagar, 2009)

(Alvarez-Fuentes et al., 2004)

(Wei et al., 2008)

(Jeong et al., 2001)

(Oliveira, G.F., 2010)

**Polymer Design approach Sources** 

Enteric coating polymer/s (delayed release):

Inner acid soluble coating (sustained release):

pH dependent polymers (delayed release): Eudragit

Combined pH and time dependent reservoir system pH dependent polymer (delayed release): Eudragit

Time dependent polymers (sustained release):

disintegrants acting upon the increased

pH dependent reservoir systems with or without

permeability or dissolution of the acid resistant

ethylcellulose/hydroxyethylcellulose Enterosolvent

Pressure controlled system: disintegrates due to the colon luminal inner pressure composed of HPMCP enterosolvent coating over ethylcellulose coating

multiparticulated system composed of HPMCP,

Time and microbiologically dependent multi-

Time dependent coating was composed of ethylcellulose combined with microbiologically

pH and microbiologically controlled

time and microbiological approach

Polysacharide containing core

pH dependent reservoir system

Eudragit RL and Eudragit RS

pH and time dependent system Time dependent polymer matrix:

polymer coat: Eudragit S 100

reservoir drug delivery system

degradable pectin

pectin and chitosan

Table 2. Conventional approaches for colon targeting

L100 and Eudragit S100

Composition:

Eudragit E

S, Eudragit L

layer

Eudragit L, HPMCP

**Eudragit E 100**  Cationic copolymer soluble in water

**Eudragit RL 100 Eudragit RS 100** 

**Eudragit FS 30D** 

**Ethylcellulose**  Insoluble in water

**Hydroxypropyl methylcellulose phthalate (HPMCP) HP50; HP 55**  Soluble in: water (pH>5.0; pH >

5.5)

Neutral copolymers insoluble in water

with pH independent swelling

**Anionic copolymer dissolves at pH above 7.0** 

(<pH 5) **Eudragit L 100**  Anionic copolymer dissolves at pH≥6.0 **Eudragit S 100** Anyonic copolymer dissolves at pH≥7.0

This phenomenon is equable to epithelial EPR effect (enhanced permeability and retention due to increased tumor capillary endothelial permeability) employed for drug targeting in solid tumors, as the potential strategy for targeting the inflamed tissue in GI tract is based on quite similar principles of increased permeability and retention by the endothelial tissue (Lamprecht, 2010; Pastorelli et al., 2008). Compared to conventional GI site targeting using pH, time dependent, pressure or microbiologically dependent systems, this approach is a an improvement in the principle of accumulation as it targets directly the site of inflammation. The fact that often the exact location of the site of inflammation is not known is not an issue for this design approach as drug delivery systems accumulate at the site of inflammation due to the increased permeability of the inflamed mucosa as well as particle uptake due to the interaction with aberrantly present macrophages and dendritic cells at the site of inflammation (Nakase et al., 2000; Tabata et al., 1996). The DDS designed by this targeting approach have to be fabricated with specific physicochemical properties and to be able to overcome the barriers including steep pH gradient, premature binding to the mucus layer, premature uptake or absorption and premature clearance, in order to reach the site of inflammation and accumulate according to the epithelial EPR effect in the GI tract.

Fig. 1. Translocation of the particles through GI tract epithelium – mechanistic approach (the sieving effect and partitioning between mucus/glycocalyx and GI tract epithelium is not presented), 1. Un-inflamed mucosa: endocytotic uptake and/or transcytosis through enterocytes (particles size<500 nm); lymphatic uptake - particles adsorbed by M-cells of the Peyer's patches (particle size <5 µm) and enhanced adhesion of the MPs and NPs to the intestinal epithelium elicited by the adequate muco/bioadhesive coating 2. Inflamed mucosa: increased particle uptake due to cytokine induced disruption and leaky epithelium; presence of large intercellular pores due to the lower expression of tight junction proteins; improved lymphatic uptake due to the increased M-cell population and large population of macrophages, dendritic cells and natural killer cells in lamina propria and in the mucus layer.

Sophisticated manipulation of the physicochemical properties during the fabrication of the targeted DDS will provide functionality of the proposed targeting mechanism. Among them in addition to particle size and particle size distribution are the stability in GI tract, zeta potential, hydrophylicity, hydrophobicity, swelling properties, muco/bioadhesivity, surface active groups, density, porosity, etc.

## **3.1 Physicochemical properties affecting the efficacy of the DDS**

Targeting IBD using disease oriented strategy requires particle stability and inertness in the upper GI tract, increased retention time in the lower parts of the GI tract, specific interaction

Drug Targeting in IBD Treatment – Existing and New Approaches 313

GI tract includes the following processes i). endocytotic uptake - particles absorbed by intestinal enterocytes through endocytosis (particles size<500 nm); ii). lymphatic uptake particles adsorbed by M cells of the Peyer's patches (particle size <5 μm) and iii). an enhanced adhesion of the microparticles and nanoparticles to the intestinal epithelium elicited by the adequate muco/bioadhesive coating, resulting, overall in a marked improvement of the absorption into the intestinal cells due to the ability of creating favorable concentration gradient for absorption or escaping from the multi-drug resistance pump proteins. But usually, because of the low endocytic activity of the enterocytes and the presence of tight junctions, translocation is mainly performed across the M-cells. Macrophages in M-cells invaginate the basolateral membrane to an extent that they come very close to the apical membrane, sometimes even protruding into the lumen. Literature data point that further biological fate after internalization depends on the size and chemistry as well. It is reported that internalized particles between 2 – 5 μm will remain longer in the Payer's patches, consequently showing very small systemic distribution compared to smaller nano sized particles. Particles bellow 2 μm migrated from the patches to mesenteric lymph nodes. Altered mucus layer during IBD, leaky epithelium and increased activity of the immunoregulatory cells in the inflamed mucosa are additional variables contributing to the improved localization of MPs and NPs at the site of inflammation but at the same time they assist the translocation and biological fate of the MP/NP-DDS to be even less

Implementing previously stated targeting principles we have designed microparticulated polyelectrolyte muco/bioadhesive DDS for inflammation targeting using the enhanced permeability effect as targeting strategy (Crcarevska et al., 2009; Glavas Dodov et al., 2009; Mladenovska et al., 2007; Mladenovska et al., 2007; Simonoska Crcarevska et al., 2008). We hypothesized that polyelectrolyte particles with a size from 1-5 μm, narrow particle distribution, positive surface charge, pH and crosslinking dependent swelling/bioadhesion and release might be suitable DDS for interaction and increased accumulation in the inflamed tissue. However, the distribution is not only size related property, but a complex interrelationship among size, shape, density, hydrophylicity/ hydrophobicity, swelling properties, surface active groups, surface charge of the drug carrier etc. Consequently, only complex combination of different attributes of the DDS might result with efficacious

*Surface active groups, zeta potential and muco/bioadhesion:* Number of polymers with muco/bioadhesive properties are cited in literature. Anionic polymers (polyacrylates and cellulose derivatives) and cationic polymers (chitosan) interact with mucus layer through non-covalent interactions (hydrophobic interactions, hydrogen binding, van der Waals interactions, electrostatic interactions) modulated by pH and ionic strength of the environment. Alginate (anionic polymer) is also citied among polymers with mucoadhesive properties involving hydrogen bonding of alginate carboxylic groups with mucus layer as a mechanism of mucoadhesive interaction (Chickering, 1995; Deacon et al., 2000; Fiebrig, 1994; 1995; Gombotz & Wee, 1998; Hejazi & Amiji, 2003; Wittaya-Areekul et al., 2006). Thiolated polymers of polyacrylates and cellulose derivatives as well as chitosan thiolated polymers exibit cationic covalent bonding building strong covalent disulfide bonds with the cysteine domains of mucins (Bernkop-Schnurch et al., 1999). Other synthetic polymers used in bioadhesive formulations are: polyvinyl alcohol, polyamides, polycarbonates, polyalkylene glycols, polyvinyl ethers, esters and halides, polymethacrylic acid, polymethyl methacrylic acid, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl

predictable (Lamprecht, 2010; Nixon et al., 1996; Reece et al., 2001).

targeting and performance.

of the particles with the inflamed mucosal tissue and immunoregulatory elements as well as controlled release at the site of action. Improved concentration of the DDS and controlled release of the drug substance at the site of therapeutic action will contribute to improved efficacy as well as decreased systemic exposure and side effects from the therapy. The importance and tailoring of the physicochemical properties of the DDS according to selected targeting mechanism as well as physiological and patophysiological conditions at the therapeutic site of action will be discussed through design, production and physicochemical characterization of budesonide loaded chitosan-Ca-alginate MPs intended for targeting and treatment of IBD.

*Particle size:* Very well known fact about the particle size of the DDS is that accelerated elimination and premature clearance due to the diarrhea, a major symptom of IBD, will be circumvented by size reduction effect and formulation of MPs or NPs for inflammation targeting (Lamprecht et al., 2005; Nakase et al., 2000; Nakase et al., 2001). In order to achieve improved localization and prolonged residence time due to increased epithelial permeability and enormous immunoregulatory cells activity at the site of inflammation, the beads should have an optimal particle size, probably between 4 and 15 µm (Coppi et al., 2001, 2002; Lamprecht et al., 2001, 2001a). Carrier systems in that size range are able to attach more efficiently to the mucus layer and accumulate in the inflamed region even without the need for macrophage uptake. NPs have also shown potential for specific accumulation in the areas with inflamed tissue increasing the selectivity of local drug delivery. When particles of different sizes are compared one simple conclusion can be drawn; that increased retention of particles of all sizes bellow 10 μm is noticed in inflamed tissue and with further size reduction the retention effect is maximized and the clearance minimized at the size of approximately 100 nm (Lamprecht et al., 2001, 2001a).

The mucus gel layer covering the intestinal/colonic mucosa is the first barrier to overcome in order to achieve increased localization in the Payer patches, intestinal lymphoid tissue and lamina propria. It is well known that UC and, to a lesser extent, CD is associated with an alteration and reduction of the protective mucus layer in the large intestine. In active UC there was a trend for the mucus layer to become progressively thinner and significantly more discontinuous as disease severity increases. The number of goblet cells in UC, which synthesize both mucin and intestinal trefoil factor, is reduced in active disease and the gel layer is consequently thinner. Mucin quality is also affected by the depletion or decreased sulfation and by increased quantity of sialic acid residues (Fujimura et al., 1992; Nakase et al., 2001; Yeh et al., 1998). Recently, data developed mainly through the investigation of most common UC induced model, dextran sulphate model, were published, pointing that the defects in the inner mucus layer may allow massive bacterial penetration into the normal sterile crypts and trigger the inflammation. Probably this pathology of the outer and inner compact and protective mucus layer contributes to the effect of increased permeability of the intestinal mucosa and improved localization of the particulate systems during inflammation. CD, unlike UC, is deep seated, therefore cytokines may initially stimulate mucus secretion, and increase thickness, but as the inflammation becomes more extensive it might begin to impair mucus production by the epithelium (Dieleman et al., 1998; Kojouharoff et al., 1997; Ni et al., 1996).

Translocation of the particles across the enterocytes/colonocytes and M cells after diffusion through mucus/glycocalyx layer as a diffusional and enzymatic barrier for healthy intestinal tissue is also affected by size and surface chemistry. Plausible mechanistic explanation for size dependent disposition and translocation of MP and NP-DDS in healthy

of the particles with the inflamed mucosal tissue and immunoregulatory elements as well as controlled release at the site of action. Improved concentration of the DDS and controlled release of the drug substance at the site of therapeutic action will contribute to improved efficacy as well as decreased systemic exposure and side effects from the therapy. The importance and tailoring of the physicochemical properties of the DDS according to selected targeting mechanism as well as physiological and patophysiological conditions at the therapeutic site of action will be discussed through design, production and physicochemical characterization of budesonide loaded chitosan-Ca-alginate MPs intended for targeting and

*Particle size:* Very well known fact about the particle size of the DDS is that accelerated elimination and premature clearance due to the diarrhea, a major symptom of IBD, will be circumvented by size reduction effect and formulation of MPs or NPs for inflammation targeting (Lamprecht et al., 2005; Nakase et al., 2000; Nakase et al., 2001). In order to achieve improved localization and prolonged residence time due to increased epithelial permeability and enormous immunoregulatory cells activity at the site of inflammation, the beads should have an optimal particle size, probably between 4 and 15 µm (Coppi et al., 2001, 2002; Lamprecht et al., 2001, 2001a). Carrier systems in that size range are able to attach more efficiently to the mucus layer and accumulate in the inflamed region even without the need for macrophage uptake. NPs have also shown potential for specific accumulation in the areas with inflamed tissue increasing the selectivity of local drug delivery. When particles of different sizes are compared one simple conclusion can be drawn; that increased retention of particles of all sizes bellow 10 μm is noticed in inflamed tissue and with further size reduction the retention effect is maximized and the clearance

minimized at the size of approximately 100 nm (Lamprecht et al., 2001, 2001a).

The mucus gel layer covering the intestinal/colonic mucosa is the first barrier to overcome in order to achieve increased localization in the Payer patches, intestinal lymphoid tissue and lamina propria. It is well known that UC and, to a lesser extent, CD is associated with an alteration and reduction of the protective mucus layer in the large intestine. In active UC there was a trend for the mucus layer to become progressively thinner and significantly more discontinuous as disease severity increases. The number of goblet cells in UC, which synthesize both mucin and intestinal trefoil factor, is reduced in active disease and the gel layer is consequently thinner. Mucin quality is also affected by the depletion or decreased sulfation and by increased quantity of sialic acid residues (Fujimura et al., 1992; Nakase et al., 2001; Yeh et al., 1998). Recently, data developed mainly through the investigation of most common UC induced model, dextran sulphate model, were published, pointing that the defects in the inner mucus layer may allow massive bacterial penetration into the normal sterile crypts and trigger the inflammation. Probably this pathology of the outer and inner compact and protective mucus layer contributes to the effect of increased permeability of the intestinal mucosa and improved localization of the particulate systems during inflammation. CD, unlike UC, is deep seated, therefore cytokines may initially stimulate mucus secretion, and increase thickness, but as the inflammation becomes more extensive it might begin to impair mucus production by the epithelium (Dieleman et al., 1998;

Translocation of the particles across the enterocytes/colonocytes and M cells after diffusion through mucus/glycocalyx layer as a diffusional and enzymatic barrier for healthy intestinal tissue is also affected by size and surface chemistry. Plausible mechanistic explanation for size dependent disposition and translocation of MP and NP-DDS in healthy

treatment of IBD.

Kojouharoff et al., 1997; Ni et al., 1996).

GI tract includes the following processes i). endocytotic uptake - particles absorbed by intestinal enterocytes through endocytosis (particles size<500 nm); ii). lymphatic uptake particles adsorbed by M cells of the Peyer's patches (particle size <5 μm) and iii). an enhanced adhesion of the microparticles and nanoparticles to the intestinal epithelium elicited by the adequate muco/bioadhesive coating, resulting, overall in a marked improvement of the absorption into the intestinal cells due to the ability of creating favorable concentration gradient for absorption or escaping from the multi-drug resistance pump proteins. But usually, because of the low endocytic activity of the enterocytes and the presence of tight junctions, translocation is mainly performed across the M-cells. Macrophages in M-cells invaginate the basolateral membrane to an extent that they come very close to the apical membrane, sometimes even protruding into the lumen. Literature data point that further biological fate after internalization depends on the size and chemistry as well. It is reported that internalized particles between 2 – 5 μm will remain longer in the Payer's patches, consequently showing very small systemic distribution compared to smaller nano sized particles. Particles bellow 2 μm migrated from the patches to mesenteric lymph nodes. Altered mucus layer during IBD, leaky epithelium and increased activity of the immunoregulatory cells in the inflamed mucosa are additional variables contributing to the improved localization of MPs and NPs at the site of inflammation but at the same time they assist the translocation and biological fate of the MP/NP-DDS to be even less predictable (Lamprecht, 2010; Nixon et al., 1996; Reece et al., 2001).

Implementing previously stated targeting principles we have designed microparticulated polyelectrolyte muco/bioadhesive DDS for inflammation targeting using the enhanced permeability effect as targeting strategy (Crcarevska et al., 2009; Glavas Dodov et al., 2009; Mladenovska et al., 2007; Mladenovska et al., 2007; Simonoska Crcarevska et al., 2008). We hypothesized that polyelectrolyte particles with a size from 1-5 μm, narrow particle distribution, positive surface charge, pH and crosslinking dependent swelling/bioadhesion and release might be suitable DDS for interaction and increased accumulation in the inflamed tissue. However, the distribution is not only size related property, but a complex interrelationship among size, shape, density, hydrophylicity/ hydrophobicity, swelling properties, surface active groups, surface charge of the drug carrier etc. Consequently, only complex combination of different attributes of the DDS might result with efficacious targeting and performance.

*Surface active groups, zeta potential and muco/bioadhesion:* Number of polymers with muco/bioadhesive properties are cited in literature. Anionic polymers (polyacrylates and cellulose derivatives) and cationic polymers (chitosan) interact with mucus layer through non-covalent interactions (hydrophobic interactions, hydrogen binding, van der Waals interactions, electrostatic interactions) modulated by pH and ionic strength of the environment. Alginate (anionic polymer) is also citied among polymers with mucoadhesive properties involving hydrogen bonding of alginate carboxylic groups with mucus layer as a mechanism of mucoadhesive interaction (Chickering, 1995; Deacon et al., 2000; Fiebrig, 1994; 1995; Gombotz & Wee, 1998; Hejazi & Amiji, 2003; Wittaya-Areekul et al., 2006). Thiolated polymers of polyacrylates and cellulose derivatives as well as chitosan thiolated polymers exibit cationic covalent bonding building strong covalent disulfide bonds with the cysteine domains of mucins (Bernkop-Schnurch et al., 1999). Other synthetic polymers used in bioadhesive formulations are: polyvinyl alcohol, polyamides, polycarbonates, polyalkylene glycols, polyvinyl ethers, esters and halides, polymethacrylic acid, polymethyl methacrylic acid, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl

Drug Targeting in IBD Treatment – Existing and New Approaches 315

the site of inflammation, it is obvious that by utilization of one step spray drying procedure

Fig. 2. Different structural and surface properties of chitosan-Ca-alginate MPs prepared by:

As crosslinked polyelectrolyte matrices posses different properties compared to the starting polymers, physicochemical stability in the bio-environment of the upper GI tract, at the same time increased site-specificity and interaction with the bio-environment in the lower parts of GI tract (swelling, muco/bioadhession and controlled drug release) were adjusted through the degree of crosslinking during the production process. As mucoadhesiveness of the polymers and physicochemical stability of the chitosan-Ca-alginate MPs depend on their

A. one step spray-drying procedure; B. two step spray-drying procedure

expected mucoadhesivity of prepared MPs will be obtained.

methylcellulose, sodium carboxymethylcellulose and various biodegradable polymers like poly(lactides), poly(glycolides), poly(lactide-co-glycolides), polycaprolactones, polyalkyl cyanoacrylates, polyorthoesters etc.

Having in mind bio/mucoadhesive properties of natural biopolymers, cationic chitosan and anionic alginate were selected for formulation of the budesonide loaded microparticulated DDS with a potential for IBD targeting. Sodium alginate LF 10/60 which consists of 65–75% of guluronic acid (G) and 25–35% of manuronic acid (M) was used for particle preparation because MG types compared with MM and GG types of sodium alginate have better flexibility (Smidsrød, 1973), and polymer gels formed from alginate with high percentage of guluronic acid (>70%) have highest mechanical strength and stability towards monovalent ions (Martinsen, 1991). Chitosan with low viscosity, highly deacetylated was chosen for polyelectrolyte complexation with sodium alginate. The fact that the deacetylated chains are fully stretched by the electrostatic repulsion among the –NH3+ groups (and the acetylated blocks are micelle-like agglomerates because of the hydrophobic forces), leads to a conclusion that higher degree of deacetylation might contribute to more efficient process of coating. Additional physicochemical stability of the alginate-chitosan polyelectrolyte complex was provided by crosslinking with inorganic calcium chloride. The method of preparation was simple and highly reproducible one step spray-drying procedure (Goracinova, 2005) carried out through concomitant spraying of core (budesonide containing alginate solution) and coating solutions (chitosan/calcium chloride solution) through adopted two fluid-nozzle. Processes of ionotropic gelation/polyelectrolite complexation are simultaneously performed during the short contact of the core and coating solution at the tip of the nozzle followed by drying in a spray drying chamber. Theoretically, during one-step procedure (Fig. 2.A) both chitosan molecules and calcium ions are competing with each other at the same time for the negatively charged groups of the alginate molecules and this competition may result in slightly bound chitosan molecules at the particle surface, hence keeping their flexibility when the particles are suspended in aqueous milieu. As a result, they are able to interact with the mucin chains and show good mucoadhesivity. Zeta potential of the particles is positive and in a magnitude of 30 – 45 mV (buffer solutions from pH 2.0 till pH 6.8) providing good stability against agglomeration. Considering, that chitosan–Ca–alginate MPs showed a positive value of the zeta potential at all pH media an assumption for the presence of chitosan at the surface of the particles can be made (Borges et al., 2005). 14C sodium acetate was used for quantitative determination of amino groups at the MPs surface and 14C glycine ethyl ester was used for carboxyl group quantification. Although amino groups were prevalent at the surface, also carboxyl groups were present at the surface of the carrier. Compared to the one step procedure, when two step spray drying procedure was applied for MPs production (preparation of alginate particles by spray drying with subsequent crosslinking in a solution of calcium chloride and chitosan) particle's zeta potential was negative, pointing that most of the chitosan amino groups are crosslinked with the carboxyl alginate groups or that the chitosan is deeply infiltrated within the pores of the microspheres without forming a continuous coating layer at the surface (Fig. 2.B). Surface properties of prepared beads are essential for efficacy of the DDS, since positive charge originating from chitosan is crucial for the interaction with negatively charged mucus and cell membranes. During the inflammation these surfaces are becoming even more negative due to the increased production of sialic acid and sialic acid residues during inflammation (Martinac et al., 2005). Considering the expecting performance of the DDS under development and the benefit of increase residence time at

methylcellulose, sodium carboxymethylcellulose and various biodegradable polymers like poly(lactides), poly(glycolides), poly(lactide-co-glycolides), polycaprolactones, polyalkyl

Having in mind bio/mucoadhesive properties of natural biopolymers, cationic chitosan and anionic alginate were selected for formulation of the budesonide loaded microparticulated DDS with a potential for IBD targeting. Sodium alginate LF 10/60 which consists of 65–75% of guluronic acid (G) and 25–35% of manuronic acid (M) was used for particle preparation because MG types compared with MM and GG types of sodium alginate have better flexibility (Smidsrød, 1973), and polymer gels formed from alginate with high percentage of guluronic acid (>70%) have highest mechanical strength and stability towards monovalent ions (Martinsen, 1991). Chitosan with low viscosity, highly deacetylated was chosen for polyelectrolyte complexation with sodium alginate. The fact that the deacetylated chains are fully stretched by the electrostatic repulsion among the –NH3+ groups (and the acetylated blocks are micelle-like agglomerates because of the hydrophobic forces), leads to a conclusion that higher degree of deacetylation might contribute to more efficient process of coating. Additional physicochemical stability of the alginate-chitosan polyelectrolyte complex was provided by crosslinking with inorganic calcium chloride. The method of preparation was simple and highly reproducible one step spray-drying procedure (Goracinova, 2005) carried out through concomitant spraying of core (budesonide containing alginate solution) and coating solutions (chitosan/calcium chloride solution) through adopted two fluid-nozzle. Processes of ionotropic gelation/polyelectrolite complexation are simultaneously performed during the short contact of the core and coating solution at the tip of the nozzle followed by drying in a spray drying chamber. Theoretically, during one-step procedure (Fig. 2.A) both chitosan molecules and calcium ions are competing with each other at the same time for the negatively charged groups of the alginate molecules and this competition may result in slightly bound chitosan molecules at the particle surface, hence keeping their flexibility when the particles are suspended in aqueous milieu. As a result, they are able to interact with the mucin chains and show good mucoadhesivity. Zeta potential of the particles is positive and in a magnitude of 30 – 45 mV (buffer solutions from pH 2.0 till pH 6.8) providing good stability against agglomeration. Considering, that chitosan–Ca–alginate MPs showed a positive value of the zeta potential at all pH media an assumption for the presence of chitosan at the surface of the particles can be made (Borges et al., 2005). 14C sodium acetate was used for quantitative determination of amino groups at the MPs surface and 14C glycine ethyl ester was used for carboxyl group quantification. Although amino groups were prevalent at the surface, also carboxyl groups were present at the surface of the carrier. Compared to the one step procedure, when two step spray drying procedure was applied for MPs production (preparation of alginate particles by spray drying with subsequent crosslinking in a solution of calcium chloride and chitosan) particle's zeta potential was negative, pointing that most of the chitosan amino groups are crosslinked with the carboxyl alginate groups or that the chitosan is deeply infiltrated within the pores of the microspheres without forming a continuous coating layer at the surface (Fig. 2.B). Surface properties of prepared beads are essential for efficacy of the DDS, since positive charge originating from chitosan is crucial for the interaction with negatively charged mucus and cell membranes. During the inflammation these surfaces are becoming even more negative due to the increased production of sialic acid and sialic acid residues during inflammation (Martinac et al., 2005). Considering the expecting performance of the DDS under development and the benefit of increase residence time at

cyanoacrylates, polyorthoesters etc.

the site of inflammation, it is obvious that by utilization of one step spray drying procedure expected mucoadhesivity of prepared MPs will be obtained.

Fig. 2. Different structural and surface properties of chitosan-Ca-alginate MPs prepared by: A. one step spray-drying procedure; B. two step spray-drying procedure

As crosslinked polyelectrolyte matrices posses different properties compared to the starting polymers, physicochemical stability in the bio-environment of the upper GI tract, at the same time increased site-specificity and interaction with the bio-environment in the lower parts of GI tract (swelling, muco/bioadhession and controlled drug release) were adjusted through the degree of crosslinking during the production process. As mucoadhesiveness of the polymers and physicochemical stability of the chitosan-Ca-alginate MPs depend on their

Drug Targeting in IBD Treatment – Existing and New Approaches 317

study indicated that complete gastric emptying was reached within 2 hours, while after 6th hour most of the MPs were located in the colon where the radioactive material deposits remained detectable even after 24 hours. When correlated with GI time distribution studies the *in vitro* swelling behavior, mucoadhesion and *in vitro* drug release correlated with the

Fig. 4. **A.** GI tract distribution of 99mTc labeled MPs after peroral administration to Wistar rats with TNBS induced colitis (mean ± SD, *n*=12), swelling and mucoadhesive properties of EMPB (mean ± SD, *n*=3). **B.** *In vitro* release profiles of BDS suspension, MPB and EMPB (2 hours at pH 2.0; additional 4 hours at pH 6.8 and up to 24 hours

B. C.

at pH 7.4) (mean ± SD, *n*=3); Colon/body weight ratio and total score points after the treatment with EMPB, MPB, BDS suspension, blank uncoated and Eudragit coated MPs ( MP and EMP), as well as non treated animals sacrificed 6th day after colitis induction, (mean ± SD; *n*=5), **○** statistically significant difference (*P*<0.05) compared to MPB, • statistically significant difference (*P*<0.05) compared to EMPB. **C.** Photographs and histology of a representative colon specimens of animals with TNBS induced colitis: non treated group–severe inflammation with complete destruction of mucosa structure followed by loss of epithelium; treated with BDS suspension - focal ulcerations, necrosis with demarcation, loss of the necrotic epithelium, and formation of granulation tissue; treated with MPB - lower degree of necrosis with distinct

boundary of necrotic lesions, focal erosive changes, but also, formation of granulation tissue, regeneration and parts with normal proliferating mucosa; treated with EMPB - focal ulcerative lesions, necrosis with focal character and distinct boundary from the normal tissue, lower parts of mucosa with large granulation tissue. Regeneration tendency

expected performance *in vivo* (Fig. 4A, B).

A.

could be observed easily.

solubility, flexibility of the polymer backbone and its polar functional groups, it is obvious that it can be modified during the cross-linking procedure (Huang et al., 2000; Wittaya-Areekul et al., 2006). By controlling the degree of crosslinking, through optimization of the process conditions, concentration of polymer and calcium chloride solutions, the polyelectrolyte bio-matrices were tailored to be inert in the bio-environment of the upper parts of GI tract, showing relatively low degree of swelling and high physicochemical stability. This slightly swollen matrices travel freely through the GI tract until reaching biological fluids with higher pH values and composition which will favor de-crosslinking of the matrix, inducing swelling and controlled drug release of the active substance.

Fig. 3. Schematic presentation of coating procedure of budesonide loaded chitosan–Ca– alginate MPs (EMPB)

Physicochemical changes in the hydrogel environment induce relaxation of the polymer network which initiates mucus layer interaction as a result of pH, ion exchange and microbiologically induced swelling of the polymer (chitosan enzymatic degradation in colon) network. Prolonged residence time of the bioresponsive matrices at the site of action will further improve targeting of the inflammation facilitating and improving the contact with the mucosal tissue, providing better conditions for particle uptake by the inflamed tissue or improved absorption. Increased localization and uptake as well as controlled drug release at the site of action will provide significant improvement of the therapeutic efficacy. In order to avoid any undesirable erroneous performance in the upper GI tract, the chitosan-Ca-alginate MPs loaded with budesonide (MPB) were additionally coated with enterosolvent polymer (EMPB), as a second control barrier to the drug release at pH range from 2.0 to 6.8 (Fig. 3).

In order to test the suitability of prepared particles for efficient treatment of IBD *in vivo,* studies on rat model of TNBS induced colitis were performed. GI tract time distribution

solubility, flexibility of the polymer backbone and its polar functional groups, it is obvious that it can be modified during the cross-linking procedure (Huang et al., 2000; Wittaya-Areekul et al., 2006). By controlling the degree of crosslinking, through optimization of the process conditions, concentration of polymer and calcium chloride solutions, the polyelectrolyte bio-matrices were tailored to be inert in the bio-environment of the upper parts of GI tract, showing relatively low degree of swelling and high physicochemical stability. This slightly swollen matrices travel freely through the GI tract until reaching biological fluids with higher pH values and composition which will favor de-crosslinking of

the matrix, inducing swelling and controlled drug release of the active substance.

Fig. 3. Schematic presentation of coating procedure of budesonide loaded chitosan–Ca–

Physicochemical changes in the hydrogel environment induce relaxation of the polymer network which initiates mucus layer interaction as a result of pH, ion exchange and microbiologically induced swelling of the polymer (chitosan enzymatic degradation in colon) network. Prolonged residence time of the bioresponsive matrices at the site of action will further improve targeting of the inflammation facilitating and improving the contact with the mucosal tissue, providing better conditions for particle uptake by the inflamed tissue or improved absorption. Increased localization and uptake as well as controlled drug release at the site of action will provide significant improvement of the therapeutic efficacy. In order to avoid any undesirable erroneous performance in the upper GI tract, the chitosan-Ca-alginate MPs loaded with budesonide (MPB) were additionally coated with enterosolvent polymer (EMPB), as a second control barrier to the drug release at pH range

In order to test the suitability of prepared particles for efficient treatment of IBD *in vivo,* studies on rat model of TNBS induced colitis were performed. GI tract time distribution

alginate MPs (EMPB)

from 2.0 to 6.8 (Fig. 3).

study indicated that complete gastric emptying was reached within 2 hours, while after 6th hour most of the MPs were located in the colon where the radioactive material deposits remained detectable even after 24 hours. When correlated with GI time distribution studies the *in vitro* swelling behavior, mucoadhesion and *in vitro* drug release correlated with the expected performance *in vivo* (Fig. 4A, B).

Fig. 4. **A.** GI tract distribution of 99mTc labeled MPs after peroral administration to Wistar rats with TNBS induced colitis (mean ± SD, *n*=12), swelling and mucoadhesive properties of EMPB (mean ± SD, *n*=3). **B.** *In vitro* release profiles of BDS suspension, MPB and EMPB (2 hours at pH 2.0; additional 4 hours at pH 6.8 and up to 24 hours at pH 7.4) (mean ± SD, *n*=3); Colon/body weight ratio and total score points after the treatment with EMPB, MPB, BDS suspension, blank uncoated and Eudragit coated MPs ( MP and EMP), as well as non treated animals sacrificed 6th day after colitis induction, (mean ± SD; *n*=5), **○** statistically significant difference (*P*<0.05) compared to MPB, • statistically significant difference (*P*<0.05) compared to EMPB. **C.** Photographs and histology of a representative colon specimens of animals with TNBS induced colitis: non treated group–severe inflammation with complete destruction of mucosa structure followed by loss of epithelium; treated with BDS suspension - focal ulcerations, necrosis with demarcation, loss of the necrotic epithelium, and formation of granulation tissue; treated with MPB - lower degree of necrosis with distinct boundary of necrotic lesions, focal erosive changes, but also, formation of granulation tissue, regeneration and parts with normal proliferating mucosa; treated with EMPB - focal ulcerative lesions, necrosis with focal character and distinct boundary from the normal tissue, lower parts of mucosa with large granulation tissue. Regeneration tendency could be observed easily.

Drug Targeting in IBD Treatment – Existing and New Approaches 319

Vinogradov et al., 1999). The most exploited ligands for GI targeting are different types of lectins due to their specificity for the membrane associated carbohydrate rich material mainly composed of olligosacharides conjugated with membrane lipids, proteins or peptide glycans. When conjugated to DDS and macromolecular drugs, depending on the lectin structure and sugar specificity, lectins may adhere and bind to the cellular surface or induce cellular uptake and internalization routing of the DDS. However, there are two distinct layers present in the intestinal mucosa, mucus layer and the glycocalix that are reach with oligosaccharides. Carbohydrate domains of glycolipids and glycoproteins protrude outwards the cell membrane to create, together with the acidic mucopolysaccharides, a thick meshwork or glycocalyx. Adhesion of the lectinised DDS at the mucus layer will produce effect similar to non-specific mucoadhesives, prolonging the residence time at the site of absorption, and dependent on the physicochemical properties of the carrier as well as the type and intensity of mucoadhesive interaction increased concentration gradient between the lumen and enterocytes and facilitated absorption might be also provided (Gao et al., 2007; Gupta, 2009; Irache et al., 2008; Smart, 2004). Anyway, this highly viscous mucus layer is also a barrier to the diffusion of the DDS towards transmembrane mucin associated oligosaccharides into the glycocalyx contributing to low accessibility and low predictability of cytoadhesion and/or cytoinvasion with lectin mediated DDS. If the physicochemical properties of the carrier (particle size, polymer properties, surface charge, surface active groups as well as the nature of attached ligand) promote mucus diffusion, partitioning of the formulation to the cell surface is possible due to the reversibility of the lectin-mucin interaction, even if the similar oligosaccharides in the mucus layer and glycocalyx are the targeted one. Interaction with the specific carbohydrates of the glycocalyx is also possible and it will induce cytoadhesion increasing the concentration gradient and improving absorption. Apart from this interaction, lectins might interact with carbohydrate domains of glycolipids and glycoproteins protruding outwards the cell membrane into the glycocalyx (glycosylated cell receptor interaction). Lectins interacting wih the glycocalix of certain region or certain cell types in GI tract are so called "bioadhesives of second generation" (Lehr, 2000; Tao et al., 2003). Direct adhesion to the cell wall will certainly overcome the limitation of mucoadhesion contact time improvement limited to few hours, extending the residence time and the interaction time to several days. However, glycocalyx sieve function is again important for the receptor accessibility and the interaction with glycosylated receptors at the cell membrane which for some lectines as wheat germ agglutinin (WGA) for epidermal growth factor (EGF)-receptor might induce receptor mediated endocytosys and internalization of nano-scaled carrier systems into acidic endosomal compartments, releasing the drug into the cytoplasm or part of the nano-carriers can also follow transcytotic

The features of different sites and cell types as well as characteristics of the overlying mucus layer and glycocalyx (thickness and glycosylation pattern) are well documented through literature. Folicle associated epithelium covering the Payer patches with its M-cells specialized in transcytosis; weak mucus production, unique ultrastructure of the glycocalyx and glycosylation pattern, number of infiltrated B cells, T cells, macrophages and dendritic cells, lack of subepithelial myofibroblast sheat and its basal lamina much more porous compared to regular epithelia are characteristics that support different translocation pattern and membrane receptor accessibility (Gabor et al., 2004; Gupta, 2009). Also, although Mcells highly express diverse terminaly glycosylated glycoconjugates which may be exploited as receptors, the uptake of particles by M-cells is not entirely dependent on specific ligand

patway (Lochner et al., 2003).

Efficacy of prepared MPs (selected uncoated and Eudragit coated formulations, CaCl2:alginate=1:0.625) was evaluated on male Wistar rats using experimentally TNBS induced colitis (Crcarevska et al., 2009). For comparison, adequate blank MPs, as well as budesonide (BDS) suspension were used. After 5 days of daily administration by oral gavage of prepared formulations, the rats were sacrificed and colon/body weight ratio, gross morphological and histological evaluation, and clinical activity score as inflammatory indices were determined. Individual clinical and histological evaluation showed that colitis severity was suppressed in following order BDS suspension < MPB < EMPB (Fig. 4C). Clinical activity score decreased in the same order (Fig. 4B). Statistical analysis of total score points indicated that the incorporation of budesonide into MPs showed significant differences in favor of efficacy of the DDS with expected accumulation at the site of inflammation, mucoadhesive properties and controlled release at the site of action (one-way ANOVA, P<0.05) (Crcarevska et al., 2009). Fig. 4B comparatively presents the drug dissolution profiles of the tested formulations to the efficacy of designed systems in the treatment of TNBS induced colitis.

It is obvious that the system showing postponed release until colon and controlled release during colon residence time or at the site of action demonstrated highest *in vivo* efficacy. In fact this is most likely due to the unique combination of bio/mucoadhesive properties of designed system along with physicochemical and biopharmaceutical properties, hence, by its design providing improved localization in the inflamed tissue and/or prolonged residence time in colon. Namely, carrier system with such properties possess ability to attach more efficiently to the mucus layer in the lower GI tract and accumulate in the inflamed region even without need for macrophage uptake, although the particles were designed to be taken up by macrophages easily as well. The system is inert in the upper GI tract, showing minimal adhesion and swelling but, anyway, second line defense was added with Eudragit coating in order to prevent any nonspecific adherence in the upper GI tract and provide increased drug control release through GI tract. Thus, budesonide is effectively delivered in a controlled manner to the colon due to the increased accumulation effect in the inflamed tissue of the MPs itself and controlled release at the site of action. Controlled drug release allows pharmacological effects to be extended due to the prolonged residence time of the carrier system at the targeted inflamed area. In fact, enteric coated MPs are specific complex system different from uncoated MPs, by their *in vitro* and hence *in vivo* performance. Although both systems show improved *in vivo* efficacy, the Eudragit coated one outperformed non coated MPs during the *in vivo* studies.

## **4. Specific colon targeting**

Theoretically, the selectivity of inflammation targeting in GI tract can be improved by attachment of various ligands at the surface of the carriers. Specific interactions with the receptors uniformly present at large areas or only in a specialized areas in the GI tract will improve bioadhesion and absorption, capacity for endocytosis and cell localization. Number of ligands and ligand-receptor pairs are discovered and examined for targeting the healthy and diseased tissue. Among them are receptor-recognizable ligands, such as lectins, toxins, viral haemagglutinins, invasins, transferrin, and vitamins (Vitamin B12, folate, riboflavin and biotin), which may improve the specificity of the delivery systems for the target cells (Brayden et al., 2005; Clark et al., 1998; De Boer, 2007; Foster & Hirst, 2005; Leamon & Low, 2001; Lee et al., 2005; Ota et al., 2002; Roth-Walter et al., 2004, 2005; Russell-Jones et al., 1999;

Efficacy of prepared MPs (selected uncoated and Eudragit coated formulations, CaCl2:alginate=1:0.625) was evaluated on male Wistar rats using experimentally TNBS induced colitis (Crcarevska et al., 2009). For comparison, adequate blank MPs, as well as budesonide (BDS) suspension were used. After 5 days of daily administration by oral gavage of prepared formulations, the rats were sacrificed and colon/body weight ratio, gross morphological and histological evaluation, and clinical activity score as inflammatory indices were determined. Individual clinical and histological evaluation showed that colitis severity was suppressed in following order BDS suspension < MPB < EMPB (Fig. 4C). Clinical activity score decreased in the same order (Fig. 4B). Statistical analysis of total score points indicated that the incorporation of budesonide into MPs showed significant differences in favor of efficacy of the DDS with expected accumulation at the site of inflammation, mucoadhesive properties and controlled release at the site of action (one-way ANOVA, P<0.05) (Crcarevska et al., 2009). Fig. 4B comparatively presents the drug dissolution profiles of the tested formulations to the efficacy of designed systems in the

It is obvious that the system showing postponed release until colon and controlled release during colon residence time or at the site of action demonstrated highest *in vivo* efficacy. In fact this is most likely due to the unique combination of bio/mucoadhesive properties of designed system along with physicochemical and biopharmaceutical properties, hence, by its design providing improved localization in the inflamed tissue and/or prolonged residence time in colon. Namely, carrier system with such properties possess ability to attach more efficiently to the mucus layer in the lower GI tract and accumulate in the inflamed region even without need for macrophage uptake, although the particles were designed to be taken up by macrophages easily as well. The system is inert in the upper GI tract, showing minimal adhesion and swelling but, anyway, second line defense was added with Eudragit coating in order to prevent any nonspecific adherence in the upper GI tract and provide increased drug control release through GI tract. Thus, budesonide is effectively delivered in a controlled manner to the colon due to the increased accumulation effect in the inflamed tissue of the MPs itself and controlled release at the site of action. Controlled drug release allows pharmacological effects to be extended due to the prolonged residence time of the carrier system at the targeted inflamed area. In fact, enteric coated MPs are specific complex system different from uncoated MPs, by their *in vitro* and hence *in vivo* performance. Although both systems show improved *in vivo* efficacy, the Eudragit coated

Theoretically, the selectivity of inflammation targeting in GI tract can be improved by attachment of various ligands at the surface of the carriers. Specific interactions with the receptors uniformly present at large areas or only in a specialized areas in the GI tract will improve bioadhesion and absorption, capacity for endocytosis and cell localization. Number of ligands and ligand-receptor pairs are discovered and examined for targeting the healthy and diseased tissue. Among them are receptor-recognizable ligands, such as lectins, toxins, viral haemagglutinins, invasins, transferrin, and vitamins (Vitamin B12, folate, riboflavin and biotin), which may improve the specificity of the delivery systems for the target cells (Brayden et al., 2005; Clark et al., 1998; De Boer, 2007; Foster & Hirst, 2005; Leamon & Low, 2001; Lee et al., 2005; Ota et al., 2002; Roth-Walter et al., 2004, 2005; Russell-Jones et al., 1999;

treatment of TNBS induced colitis.

**4. Specific colon targeting** 

one outperformed non coated MPs during the *in vivo* studies.

Vinogradov et al., 1999). The most exploited ligands for GI targeting are different types of lectins due to their specificity for the membrane associated carbohydrate rich material mainly composed of olligosacharides conjugated with membrane lipids, proteins or peptide glycans. When conjugated to DDS and macromolecular drugs, depending on the lectin structure and sugar specificity, lectins may adhere and bind to the cellular surface or induce cellular uptake and internalization routing of the DDS. However, there are two distinct layers present in the intestinal mucosa, mucus layer and the glycocalix that are reach with oligosaccharides. Carbohydrate domains of glycolipids and glycoproteins protrude outwards the cell membrane to create, together with the acidic mucopolysaccharides, a thick meshwork or glycocalyx. Adhesion of the lectinised DDS at the mucus layer will produce effect similar to non-specific mucoadhesives, prolonging the residence time at the site of absorption, and dependent on the physicochemical properties of the carrier as well as the type and intensity of mucoadhesive interaction increased concentration gradient between the lumen and enterocytes and facilitated absorption might be also provided (Gao et al., 2007; Gupta, 2009; Irache et al., 2008; Smart, 2004). Anyway, this highly viscous mucus layer is also a barrier to the diffusion of the DDS towards transmembrane mucin associated oligosaccharides into the glycocalyx contributing to low accessibility and low predictability of cytoadhesion and/or cytoinvasion with lectin mediated DDS. If the physicochemical properties of the carrier (particle size, polymer properties, surface charge, surface active groups as well as the nature of attached ligand) promote mucus diffusion, partitioning of the formulation to the cell surface is possible due to the reversibility of the lectin-mucin interaction, even if the similar oligosaccharides in the mucus layer and glycocalyx are the targeted one. Interaction with the specific carbohydrates of the glycocalyx is also possible and it will induce cytoadhesion increasing the concentration gradient and improving absorption. Apart from this interaction, lectins might interact with carbohydrate domains of glycolipids and glycoproteins protruding outwards the cell membrane into the glycocalyx (glycosylated cell receptor interaction). Lectins interacting wih the glycocalix of certain region or certain cell types in GI tract are so called "bioadhesives of second generation" (Lehr, 2000; Tao et al., 2003). Direct adhesion to the cell wall will certainly overcome the limitation of mucoadhesion contact time improvement limited to few hours, extending the residence time and the interaction time to several days. However, glycocalyx sieve function is again important for the receptor accessibility and the interaction with glycosylated receptors at the cell membrane which for some lectines as wheat germ agglutinin (WGA) for epidermal growth factor (EGF)-receptor might induce receptor mediated endocytosys and internalization of nano-scaled carrier systems into acidic endosomal compartments, releasing the drug into the cytoplasm or part of the nano-carriers can also follow transcytotic patway (Lochner et al., 2003).

The features of different sites and cell types as well as characteristics of the overlying mucus layer and glycocalyx (thickness and glycosylation pattern) are well documented through literature. Folicle associated epithelium covering the Payer patches with its M-cells specialized in transcytosis; weak mucus production, unique ultrastructure of the glycocalyx and glycosylation pattern, number of infiltrated B cells, T cells, macrophages and dendritic cells, lack of subepithelial myofibroblast sheat and its basal lamina much more porous compared to regular epithelia are characteristics that support different translocation pattern and membrane receptor accessibility (Gabor et al., 2004; Gupta, 2009). Also, although Mcells highly express diverse terminaly glycosylated glycoconjugates which may be exploited as receptors, the uptake of particles by M-cells is not entirely dependent on specific ligand

Drug Targeting in IBD Treatment – Existing and New Approaches 321

positivity in a small proportion of UC and CD rectal biopsies, but not in normal rectal mucosa, may be due to reduced sialylation or increased fucosylation. Other lectins used in this study like wheat germ agglutinin (WGA), soy bean (SBA), grifonia seed (GSI) showed

In the study of Melo-Junior et al. (Melo-Junior et al., 2004), it was found that WGA presented recognition pattern for diseased tissue. The authors claim that Nacetylglucosamine was absent or not accessible for lectin recognition in normal tissues, as well as mannosides and galactose. L-fucose was found in the intestinal crypts of normal glands and UC intestinal biopsies showed intense WGA binding in the gland cells of intestinal crypts, indicating high expression of N-acetylglucosamine in these cells in UC. Also, fucose binding Lotus tetragonolobus agglutinin was highly bound to UC gland

Although investigations of IBD pathogenesis did not clear up all misunderstandings of this disease and causes of IBD are still unknown, in depth studies of immunopathology of IBD and mechanism driving the uncontrolled inflammation enabled the development of design strategies for improvement of the efficacy of the conventional therapeutic agents as well as identification of new therapeutic targets and novel therapeutic active agents. Genetic factors and defects in innate and adaptive immune pathways have been identified, and biological therapies that target specific pathophysiological mechanisms of IBD selectively blocking the

The fundamentals of biological treatment strategies involve neutralization of proinflammatory cytokines that plays central role in pathogenesis of CD and UC, use of antiinflammatory cytokines and inhibition of neutrophil adhesion or T-cell signaling. Since the discovery of the central role of the proinflammatory cytokine TNF α in the inflammatory cascade of UC and CD, based on large randomized clinical trials, anti-TNF-α agents have substantially extended the therapeutic armamentarium in IBD. A variety of biological agents have been used to inhibit TNF-α in patients with IBD, including the mouse/human chimeric monoclonal antibody (infliximab), the humanized monoclonal antibody CDP571, the human soluble TNF-α p55 receptor (onercept), the human monoclonal antibody D2E7 (adalimumab), the p75 soluble TNF receptor fusion protein (etanercept), and the polyethylene glycol (PEG)ylated anti-TNF-α antibody fragment CDP-870. Among these, infliximab (formerly cA2) and CDP571 have shown the most promise, particularly in CD. However, up to date with few isolated approaches for local administration (Worledge et al., 2000; AVX-470 in preclinical studies) most of these agents are administered through conventional parenteral dosage forms resulting with lower concentration at the site of inflammation as well as direct intrusion in the human immune system, number of contraindications and serious adverse effects. In addition to these agents that directly antagonize and block the activity of TNF-α, alternative pathways for improved therapeutic approach are investigated. First of all, gene delivery that will provide sustained production of anti-inflammatory proteins has significant promise for local treatment of IBD. Also, transcription factors that regulate the synthesis of TNF-α and other proinflammatory cytokines are identified like new therapeutic targets. Among them the key transcription factor of lymphocytes and macrophages, NF-κB that plays a major role in regulating more than hundred proinflammatory cytokines, including TNF-α, is becoming an attractive

**5. Biological and gene therapies for inflammatory bowel diseases** 

similar affinity to normal, UC and CD biopsies.

epithelium pointing to increased L-fucose levels.

inflammatory mechanisms have been designed.

binding, since adherence to M-cells by any mechanism leads to endocytosis, phagocytosis, pinocytosis, and macropinocytosis or any other mechanism used for the ingestion of the extracellular material. Colonic mucosa doesn't contain Payer's patches but it contains large lymphoid follicles of a dome-type configuration, extended as far as the lamina propria of the mucosa and associated with massive lymphoid aggregations spreading beyond the muscularis mucosa from the submucosa. The epithelium covering these follicles, is associated with a few goblet cells, contains M-cells and many migrating lymphocytes crossing through discontinuities of the basal lamina in the vicinity of the M-cells, and is specialized, differing from the surrounding mucosa (Fujimura et al., 1992).

In order to integrate this concepts of glycotargeting into the inflammation targeting the influence of mucus production impairment and reduction of the protective mucus layer in the intestines during inflammation, increased epithelial permeability, characteristic increased immunoregullatory cells activity in the inflamed tissue (section 3), presence of occasional erosions for ex. at the apical surface of the colonic lymphoid follicles in a size range of 2–6 μm in CD, revealing the naked surface of the dome beneath the epithelium and alteration of the glycolation pattern during the inflammation, have to be considered as additional factors influencing the design of the DDS. Additional decoration of the microand nano-carriers for inflammation targeting designed for increased accumulation due to the epithelial EPR effect might further improve the concentration of the active substance in the targeted cells due to the effect of cytoadhesion and cytoinvasion. E. coli K99 fimbriae adhesin was used to target 6-methyl prednisolone to the inflamed tissue in GI tract of the Chron's patients. Peptide, protein and DNA therapeutics delivery to the sites of therapeutic action will be also possible through the design of these specialized decorated cytoadhesive and cytoinvasive nanocarriers (section 5). Targeting the lectine molecules expressed at the mammalian cell surface, like galectins which are β-galactoside binding proteins, or direct lectine targeting, is also used for normal and diseased colon targeting.

Alteration of glycolylation pattern is seen during inflammation and neoplastic colonic disease. Abnormality in epithelial cell glycoconjugates is commonly present in both UC and CD and it may reflect abnormality in mucus glycoprotein synthesis in IBD. As a result altered lectin binding by colonic epithelial glycoconjugates in UC and CD can be seen. Up to date only limited data are available on the "sugar code" of the GI tract inflammation (Gabius, 2000). It is well known that the enterocytes, follicle-associated epithelial cells, Mcells, immunoregulatory elements and colonocites differ by their glycosylation pattern, but the data on the abnormality, differences and characteristics of epithelial glycoconjugates during UC and CD are very scarce (Yeh et al., 1998). Even less data can be found about the type of interaction mediated by certain oligosaccharide sequence and possible homing of the carrier payload into the cell or cell routing triggered by receptor ligand linking.

Histochemical studies are useful for understanding the altered lectin binding and changes in the glycosylation map during cancer and inflammation. In the study of Rhodes et al. high proportion of binding of the lectins of peanut agglutinin (PNA), Ulex europeus I (UEAI) and Griffonia simplicifolia II (GSII) to UC and CD mucosal samples was shown (Kiss et al., 1997; Rhodes et al., 1986, 1988, 2008). It was shown that PNA exhibited specificity for inflamed biopsies without binding to the mucosa or free mucus of the normal biopsies. PNA positivity, when present, was most marked in the surface epithelium, particularly in the supranuclear region of the epithelial cells. PNA identifies Gal(β1,3)GalNAc which is normally obscured by the terminal sialic acid that is added to mucus sialoglycoprotein in the Golgi apparatus as the final step in mucin synthesis. The finding of UEAI (fucose binding)

binding, since adherence to M-cells by any mechanism leads to endocytosis, phagocytosis, pinocytosis, and macropinocytosis or any other mechanism used for the ingestion of the extracellular material. Colonic mucosa doesn't contain Payer's patches but it contains large lymphoid follicles of a dome-type configuration, extended as far as the lamina propria of the mucosa and associated with massive lymphoid aggregations spreading beyond the muscularis mucosa from the submucosa. The epithelium covering these follicles, is associated with a few goblet cells, contains M-cells and many migrating lymphocytes crossing through discontinuities of the basal lamina in the vicinity of the M-cells, and is

In order to integrate this concepts of glycotargeting into the inflammation targeting the influence of mucus production impairment and reduction of the protective mucus layer in the intestines during inflammation, increased epithelial permeability, characteristic increased immunoregullatory cells activity in the inflamed tissue (section 3), presence of occasional erosions for ex. at the apical surface of the colonic lymphoid follicles in a size range of 2–6 μm in CD, revealing the naked surface of the dome beneath the epithelium and alteration of the glycolation pattern during the inflammation, have to be considered as additional factors influencing the design of the DDS. Additional decoration of the microand nano-carriers for inflammation targeting designed for increased accumulation due to the epithelial EPR effect might further improve the concentration of the active substance in the targeted cells due to the effect of cytoadhesion and cytoinvasion. E. coli K99 fimbriae adhesin was used to target 6-methyl prednisolone to the inflamed tissue in GI tract of the Chron's patients. Peptide, protein and DNA therapeutics delivery to the sites of therapeutic action will be also possible through the design of these specialized decorated cytoadhesive and cytoinvasive nanocarriers (section 5). Targeting the lectine molecules expressed at the mammalian cell surface, like galectins which are β-galactoside binding proteins, or direct

Alteration of glycolylation pattern is seen during inflammation and neoplastic colonic disease. Abnormality in epithelial cell glycoconjugates is commonly present in both UC and CD and it may reflect abnormality in mucus glycoprotein synthesis in IBD. As a result altered lectin binding by colonic epithelial glycoconjugates in UC and CD can be seen. Up to date only limited data are available on the "sugar code" of the GI tract inflammation (Gabius, 2000). It is well known that the enterocytes, follicle-associated epithelial cells, Mcells, immunoregulatory elements and colonocites differ by their glycosylation pattern, but the data on the abnormality, differences and characteristics of epithelial glycoconjugates during UC and CD are very scarce (Yeh et al., 1998). Even less data can be found about the type of interaction mediated by certain oligosaccharide sequence and possible homing of the

Histochemical studies are useful for understanding the altered lectin binding and changes in the glycosylation map during cancer and inflammation. In the study of Rhodes et al. high proportion of binding of the lectins of peanut agglutinin (PNA), Ulex europeus I (UEAI) and Griffonia simplicifolia II (GSII) to UC and CD mucosal samples was shown (Kiss et al., 1997; Rhodes et al., 1986, 1988, 2008). It was shown that PNA exhibited specificity for inflamed biopsies without binding to the mucosa or free mucus of the normal biopsies. PNA positivity, when present, was most marked in the surface epithelium, particularly in the supranuclear region of the epithelial cells. PNA identifies Gal(β1,3)GalNAc which is normally obscured by the terminal sialic acid that is added to mucus sialoglycoprotein in the Golgi apparatus as the final step in mucin synthesis. The finding of UEAI (fucose binding)

specialized, differing from the surrounding mucosa (Fujimura et al., 1992).

lectine targeting, is also used for normal and diseased colon targeting.

carrier payload into the cell or cell routing triggered by receptor ligand linking.

positivity in a small proportion of UC and CD rectal biopsies, but not in normal rectal mucosa, may be due to reduced sialylation or increased fucosylation. Other lectins used in this study like wheat germ agglutinin (WGA), soy bean (SBA), grifonia seed (GSI) showed similar affinity to normal, UC and CD biopsies.

In the study of Melo-Junior et al. (Melo-Junior et al., 2004), it was found that WGA presented recognition pattern for diseased tissue. The authors claim that Nacetylglucosamine was absent or not accessible for lectin recognition in normal tissues, as well as mannosides and galactose. L-fucose was found in the intestinal crypts of normal glands and UC intestinal biopsies showed intense WGA binding in the gland cells of intestinal crypts, indicating high expression of N-acetylglucosamine in these cells in UC. Also, fucose binding Lotus tetragonolobus agglutinin was highly bound to UC gland epithelium pointing to increased L-fucose levels.

## **5. Biological and gene therapies for inflammatory bowel diseases**

Although investigations of IBD pathogenesis did not clear up all misunderstandings of this disease and causes of IBD are still unknown, in depth studies of immunopathology of IBD and mechanism driving the uncontrolled inflammation enabled the development of design strategies for improvement of the efficacy of the conventional therapeutic agents as well as identification of new therapeutic targets and novel therapeutic active agents. Genetic factors and defects in innate and adaptive immune pathways have been identified, and biological therapies that target specific pathophysiological mechanisms of IBD selectively blocking the inflammatory mechanisms have been designed.

The fundamentals of biological treatment strategies involve neutralization of proinflammatory cytokines that plays central role in pathogenesis of CD and UC, use of antiinflammatory cytokines and inhibition of neutrophil adhesion or T-cell signaling. Since the discovery of the central role of the proinflammatory cytokine TNF α in the inflammatory cascade of UC and CD, based on large randomized clinical trials, anti-TNF-α agents have substantially extended the therapeutic armamentarium in IBD. A variety of biological agents have been used to inhibit TNF-α in patients with IBD, including the mouse/human chimeric monoclonal antibody (infliximab), the humanized monoclonal antibody CDP571, the human soluble TNF-α p55 receptor (onercept), the human monoclonal antibody D2E7 (adalimumab), the p75 soluble TNF receptor fusion protein (etanercept), and the polyethylene glycol (PEG)ylated anti-TNF-α antibody fragment CDP-870. Among these, infliximab (formerly cA2) and CDP571 have shown the most promise, particularly in CD. However, up to date with few isolated approaches for local administration (Worledge et al., 2000; AVX-470 in preclinical studies) most of these agents are administered through conventional parenteral dosage forms resulting with lower concentration at the site of inflammation as well as direct intrusion in the human immune system, number of contraindications and serious adverse effects. In addition to these agents that directly antagonize and block the activity of TNF-α, alternative pathways for improved therapeutic approach are investigated. First of all, gene delivery that will provide sustained production of anti-inflammatory proteins has significant promise for local treatment of IBD. Also, transcription factors that regulate the synthesis of TNF-α and other proinflammatory cytokines are identified like new therapeutic targets. Among them the key transcription factor of lymphocytes and macrophages, NF-κB that plays a major role in regulating more than hundred proinflammatory cytokines, including TNF-α, is becoming an attractive

Drug Targeting in IBD Treatment – Existing and New Approaches 323

offer tailor made control of immune responses and inflammatory reactions for an individual patient, contributing to the overall success of the anti-inflammatory therapy during IBD.

For the experimental work presented the authors would like to acknowledge the support of

Adkin, D.A., Davis, S.S., Sparrow, R.A. & Wilding, I.R. (1993). Colonic transit of different sized

Ahmad, S., Tester, R.F., Corbett, A. & Karkalas, J. (2006). Dextran and 5-aminosalicylic acid (5-

*Research,* Vol. 341, No. 16, (November 2006), pp. 2694-2701, ISSN 0008-6215 Akhgari, A., Sadeghi, F. & Garekani, H.A. (2006). Combination of time-dependent and ph-

Alvarez-Fuentes, J., Fernandez-Arevalo, M., Gonzalez-Rodriguez, M.L., Cirri, M. & Mura, P.

Babbs, C.F. (1992). Oxygen radicals in ulcerative colitis. *Free Radical Biology and Medicine,* Vol.

Beckman, K.B. & Ames, B.N. (1997). Oxidative decay of DNA. *Journal of Biological Chemistry,*

Bernkop-Schnurch, A., Schwarz, V. & Steininger, S. (1999). Polymers with thiol groups: A new

Bhavsar, M. D. & Amiji, M. M. (2007). Gastrointestinal distribution and in vivo gene

*of Pharmaceutics,* Vol. 299, No. 1-2, (August 2005), pp. 155-166, ISSN 0378-5173 Bott, C., Rudolph, M.W., Schneider, A.R., Schirrmacher, S., Skalsky, B., Petereit, H.U.,

Bourgeois, S. (2005). Polymer colon drug delivery systems and their application to peptides,

*Therapeutics,* Vol. 20, No. 3, (August 2004), pp. 347-353, ISSN 0269-2813 Bouma, G. & Strober, W. (2003). The immunological and genetic basis of inflammatory bowel

*of controlled release,*Vol. 119, No. 3, (June 2007), pp. 339-348, ISSN 0168-3659 Borges, O., Borchard, G., Verhoef, J.C., De Sousa, A. & Junginger, H.E. (2005). Preparation of

Vol. 272, No. 32, (August 1997), pp. 19633-19636, ISSN 0021-9258

13, No. 2, (August 1992), pp. 169-181, ISSN 0891-5849

tablets in healthy subjects. *Journal of Controlled Release,* Vol. 23, No. 2, (February 2003),

asa) conjugates: Synthesis, characterisation and enzymic hydrolysis. *Carbohydrate* 

dependent polymethacrylates as a single coating formulation for colonic delivery of indomethacin pellets. *International Journal of Pharmaceutics,* Vol. 320, No. 1-2, (August

(2004). Development of enteric-coated timed-release matrix tablets for colon targeting. *Journal of Drug Targeting,* Vol. 12, No. 9-10, (December 2004), pp. 607-612,

generation of mucoadhesive polymers? *Pharmaceutical Research,* Vol. 16, No. 6, (June

transfection studies with nanoparticles-in-microsphere oral system (NiMOS). *Journal* 

coated nanoparticles for a new mucosal vaccine delivery system. *International Journal* 

Langguth, P., Dressman, J.B. & Stein, J. (2004). In vivo evaluation of a novel ph- and time-based multiunit colonic drug delivery system. *Alimentary Pharmacology &* 

disease. *Nature Reviews Immunology,* Vol. 3, No. 7, (October 2003), pp. 521-533, ISSN

proteins, and nucleic acids. *American journal of drug delivery,* Vol. 3, No. 3, (n.d.), pp.

**6. Acknowledgments** 

**7. References** 

NATO Science for Peace program: grant No. 978023

pp. 147-156, ISSN 0168-3659

2006), pp. 137-142, ISSN 0378-5173

1999), pp. 876-881, ISSN 0724-8741

ISSN 1061-186X

14741733

171-204, ISSN 1175-9038

target for therapeutic intervention in IBD. A NF-κB decoy therapeutic system using a synthetic double stranded oligonucleotide to competitively inhibit binding and interaction of NF-κB to their target genoms and prevent the gene induction, transcription and production of the proinflammatory cytokines, is already presented in the literature as promising therapy for IBD and other inflammatory diseases. Successful intracellular and intranuclear delivery of the stable NF-κB decoy to the site of inflammation and action in GI tract is a field yet to be explored (Tahara et al., 2011). Finaly, RNA interference therapy utilizing short interfering (siRNA), usually composed of 20-25 nucleotides targeted to cytosol will trigger gene silencing mechanism through RNA interference where siRNA can block the expression of a specific gene (TNF-α or different proinflammatory gene expression in IBD) and proinflammatory protein synthesis, thus providing for successful therapeutic approach in IBD (Kriegel & Amiji, 2011).

Gene therapy can be delivered to local sites in GI tract, produce and concentrate a therapeutic protein in intestinal tissue, and release negligible amounts into the circulation (Kriegel & Amiji, 2011). Examples presented through literature for design approaches for gene, peptide and protein targeting in IBD relay on the previous experience with nano- and micro-carriers for inflammation and vaccine non specific or specific targeting. Higher concentration of the carrier in the inflamed tissue due to enhanced permeability of GI tract epithelium as well as increased activity of immune regulatory cells during UC and CD, increased residence time and improved carrier/cell non-specific or specific interaction are processes assisting the uptake and endosomal release in cytosol or different trafficking pathways after triggering internalization. Non-viral nano-sized vectors based on natural and/or synthetic polymers for tissue and cell specific delivery with encapsulated DNA, siRNA or oligonucleotide payload have shown promising stability, intracellular uptake, further trafficking (endosomal/lysosomal escape) and successful transfection efficacy. NiMOS (nanoparticles in microspheres system) is based on 200 nm non-condensing type B gelatin NPs encapsulated into pH and enzyme attack protective 1-5 μm poly(epsiloncaprolactone) (PCL) microspheres (Xu et al., 2011). As PCL is degraded by lipases in the small and large intestine it is expected that plasmid DNA loaded NPs might be internalized by the enterocytes or other cells in GI tract for transfection of the encoded protein. These particles loaded with anti-inflammatory murine IL-10 expressing plasmid DNA were evaluated for efficacy of transfection, through measurement of the mRNA and antiinflammatory protein levels in TNBS induced colitis in Balb/c mice's. Concomitant effect of reduction of pro-inflammatory cytokines and chemokines together with increased messenger RNA (mRNA) and antiinflamatory IL-10 levels were reported by the authors (Bhavsar & Amiji, 2007). It is well known that successful delivery of siRNAs in the cytoplasm, will initiate a process that cleaves the complementary mRNA to prevent its processing and translation, blocking the expression of a specific gene eg. those expressed in a disease (Plevy & Targan, 2011). NiMOS was also used for oral TNF-α specific siRNA delivery (Kriegel & Amiji, 2011) and the system was evaluated for the efficacy of oral TNF-α gene silencing using Balb/c mice's TNBS induced colitis model. It was pointed that the system is promising and that lower expression of TNF-α due to silencing preceded the downregulation of other inflammatory cytokines and within time showed similar effect on the chemokine production. The concept of gene therapy for oral delivery and treatment of IBD has received significant attention, while the GI tract offers an ideal target due to large surface area and access to the luminal site of inflammation after oral administration. As the research in this field is growing day by day successful local gene delivery will probably offer tailor made control of immune responses and inflammatory reactions for an individual patient, contributing to the overall success of the anti-inflammatory therapy during IBD.

## **6. Acknowledgments**

For the experimental work presented the authors would like to acknowledge the support of NATO Science for Peace program: grant No. 978023

## **7. References**

322 Inflammatory Bowel Disease – Advances in Pathogenesis and Management

target for therapeutic intervention in IBD. A NF-κB decoy therapeutic system using a synthetic double stranded oligonucleotide to competitively inhibit binding and interaction of NF-κB to their target genoms and prevent the gene induction, transcription and production of the proinflammatory cytokines, is already presented in the literature as promising therapy for IBD and other inflammatory diseases. Successful intracellular and intranuclear delivery of the stable NF-κB decoy to the site of inflammation and action in GI tract is a field yet to be explored (Tahara et al., 2011). Finaly, RNA interference therapy utilizing short interfering (siRNA), usually composed of 20-25 nucleotides targeted to cytosol will trigger gene silencing mechanism through RNA interference where siRNA can block the expression of a specific gene (TNF-α or different proinflammatory gene expression in IBD) and proinflammatory protein synthesis, thus providing for successful

Gene therapy can be delivered to local sites in GI tract, produce and concentrate a therapeutic protein in intestinal tissue, and release negligible amounts into the circulation (Kriegel & Amiji, 2011). Examples presented through literature for design approaches for gene, peptide and protein targeting in IBD relay on the previous experience with nano- and micro-carriers for inflammation and vaccine non specific or specific targeting. Higher concentration of the carrier in the inflamed tissue due to enhanced permeability of GI tract epithelium as well as increased activity of immune regulatory cells during UC and CD, increased residence time and improved carrier/cell non-specific or specific interaction are processes assisting the uptake and endosomal release in cytosol or different trafficking pathways after triggering internalization. Non-viral nano-sized vectors based on natural and/or synthetic polymers for tissue and cell specific delivery with encapsulated DNA, siRNA or oligonucleotide payload have shown promising stability, intracellular uptake, further trafficking (endosomal/lysosomal escape) and successful transfection efficacy. NiMOS (nanoparticles in microspheres system) is based on 200 nm non-condensing type B gelatin NPs encapsulated into pH and enzyme attack protective 1-5 μm poly(epsiloncaprolactone) (PCL) microspheres (Xu et al., 2011). As PCL is degraded by lipases in the small and large intestine it is expected that plasmid DNA loaded NPs might be internalized by the enterocytes or other cells in GI tract for transfection of the encoded protein. These particles loaded with anti-inflammatory murine IL-10 expressing plasmid DNA were evaluated for efficacy of transfection, through measurement of the mRNA and antiinflammatory protein levels in TNBS induced colitis in Balb/c mice's. Concomitant effect of reduction of pro-inflammatory cytokines and chemokines together with increased messenger RNA (mRNA) and antiinflamatory IL-10 levels were reported by the authors (Bhavsar & Amiji, 2007). It is well known that successful delivery of siRNAs in the cytoplasm, will initiate a process that cleaves the complementary mRNA to prevent its processing and translation, blocking the expression of a specific gene eg. those expressed in a disease (Plevy & Targan, 2011). NiMOS was also used for oral TNF-α specific siRNA delivery (Kriegel & Amiji, 2011) and the system was evaluated for the efficacy of oral TNF-α gene silencing using Balb/c mice's TNBS induced colitis model. It was pointed that the system is promising and that lower expression of TNF-α due to silencing preceded the downregulation of other inflammatory cytokines and within time showed similar effect on the chemokine production. The concept of gene therapy for oral delivery and treatment of IBD has received significant attention, while the GI tract offers an ideal target due to large surface area and access to the luminal site of inflammation after oral administration. As the research in this field is growing day by day successful local gene delivery will probably

therapeutic approach in IBD (Kriegel & Amiji, 2011).


Drug Targeting in IBD Treatment – Existing and New Approaches 325

Fedorak, R.N. & Bistritz, L. (2005). Targeted delivery, safety, and efficacy of oral enteric-coated

Fiebrig, I. (1994). Sedimentation analysis of potential interactions between mucins and a

Fiebrig, I. (1995). Transmission electron microscopy studies on pig gastric mucin and its

Foster, N. & Hirst, B.H. (2005). Exploiting receptor biology for oral vaccination with

Friend, D.R. (1991). Colon-specific drug delivery. *Advanced drug delivery reviews,* Vol. 7, No. 1,

Friend, D.R. (2005). New oral delivery systems for treatment of inflammatory bowel disease.

Friend, D.R. (2005). New oral delivery systems for treatment of inflammatory bowel disease.

Friend, D.R. & Chang, G.W. (1985). Drug glycosides: Potential prodrugs for colon-specific

Fujimura, Y., Hosobe, M. & Kihara, T. (1992). Ultrastructural study of m cells from colonic

Gabius, H.J. (2000). Biological information transfer beyond the genetic code: The sugar code. *Naturwissenschaften,* Vol. 87, No. 3, (March 2000), pp. 108-121, ISSN 0028-1042 Gabor, F., Bogner, E., Weissenboeck, A. & Wirth, M. (2004). The lectin-cell interaction and its

Gao, X., Chen, J., Tao, W., Zhu, J., Zhang, Q., Chen, H. & Jiang, X. (2007). Uea i-bearing

Gazzaniga, A., Maroni, A., Sangalli, M.E. & Zema, L. (2006). Time-controlled oral delivery

Gazzaniga, A., Palugan, L., Foppoli, A. & Sangalli, M.E. (2008). Oral pulsatile delivery systems

*Biopharmaceutics,* Vol. 68, No. 1, (January 2008), pp. 11-18, ISSN 0939-6411 Glavas Dodov, M., Calis, S., Crcarevska, M.S., Geskovski, N., Petrovska, V. & Goracinova, K.

2005), pp. 303-316, ISSN 0169-409X

2005), pp. 431-450, ISSN 0169-409X

2006), pp. 583-597, ISSN 1742-5247

(July-August 1991), pp. 149-199, ISSN 0169-409X

Vol. 37, No. 7, (July 1992), pp. 1089-1098, ISSN 0163-2116

*Review,* Vol. 56, No. 4, (March 2004), pp. 459-480, ISSN 0169-409X

pp. 66-73, ISSN 0340-255X

239-244, ISSN 0144-8617

409X

3476

0169409X

ISSN 0022-2623

formulations of budesonide. *Advanced Drug Delivery Review,* Vol. 57, No. 2, (January

putative bioadhesive polymer. *Progress in colloid & polymer science,* Vol. 94, No. (n.d.),

interactions with chitosan. *Carbohydrate polymers,* Vol. 28, No. 3, (December 1995), pp.

biodegradable particulates. *Advanced Drug Delivery Review,* Vol. 57, No. 3, (January

*Advanced Drug Delivery Review,* Vol. 57, No. 2, (January 2005), pp. 247-265, ISSN 0169-

*Advanced drug delivery reviews,* Vol. 57, No. 2, (January 2005), pp. 247-265, ISSN

drug delivery. *Journal of Medicinal Chemistry,* Vol. 28, No. 1, (January 1985), pp. 51-57,

lymphoid nodules obtained by colonoscopic biopsy. *Digestive diseases and sciences,*

implications to intestinal lectin-mediated drug delivery. *Advanced Drug Delivery* 

nanoparticles for brain delivery following intranasal administration. *International Journakl of Pharmaceutics,* Vol. 340, No. 1-2, (August 2007), pp. 207-215, ISSN 0378-5173

systems for colon targeting. *Expert Opinion on Drug Delivery,* Vol. 3, No. 5, (September

based on swellable hydrophilic polymers. *European Journal of Pharmaceutics and* 

(2009). Wheat germ agglutinin-conjugated chitosan-ca-alginate microparticles for local colon delivery of 5-fu: Development and in vitro characterization. *International Journal of Pharmaceutics,* Vol. 381, No. 2, (November 2009), pp. 166-175, ISSN 1873-


Brayden, D.J., Jepson, M.A. & Baird, A.W. (2005). Keynote review: Intestinal peyer's patch m

Brown, J.P., Mcgarraugh, G.V., Parkinson, T.M., Wingard, R.E., Jr. & Onderdonk, A.B. (1983).

Chickering, D.E. (1995). Bioadhesive microspheres: I. A novel electrobalance-based method to

Clark, M.A., Hirst, B.H. & Jepson, M.A. (1998). M-cell surface beta1 integrin expression and

Clemett, D. & Markham, A. (2000). Prolonged-release mesalazine: A review of its therapeutic

Coppi, G., Iannuccelli, V., Bernabei, M. & Cameroni, R. (2002). Alginate microparticles for

Coppi, G., Iannuccelli, V., Leo, E., Bernabei, M.T. & Cameroni, R. (2001). Chitosan-alginate

Crcarevska, M.S., Dodov, M.G., Petrusevska, G., Gjorgoski, I. & Goracinova, K. (2009).

Cuvelier, C. A., Quatacker, J., Mielants, H., De Vos, M., Veys, E. & Roels, H. J. (1994). M-cells

Deacon, M.P., Mcgurk, S., Roberts, C.J., Williams, P.M., Tendler, S.J., Davies, M.C., Davis, S.S.

Dieleman, L.A., Palmen, M.J., Akol, H., Bloemena, E., Pena, A.S., Meuwissen, S.G. & Van Rees,

*Histopathology,*Vol. 24, No. 5, (May 1994), pp. 417-426, ISSN 0309-0167 De Boer, A.G. (2007). Drug targeting to the brain. *Annual review of pharmacology and toxicology,*

Vol. 47, No. 1, (February 2007), pp. 323-355, ISSN 0362-1642

No. 3, (December 1998), pp. 385-391, ISSN 0009-9104

*Chemistry,* Vol. 26, No. 9, (September 1983), pp. 1300-1307, ISSN 0022-2623 Brunner, M. , Greinwald, R. , Kletter, K., Kvaternik, H., Corrado, M. E., Eichler, H. G. &

2005), pp. 1145-1157, ISSN 1359-6446

(January-April 2003), pp. 33-66, ISSN 1482-1826

(August 2002), pp. 263-266, ISSN 0378-5173

No. 5, (May 2001), pp. 393-400, ISSN 0363-9045

9567

pp. 929-956, ISSN 0012-6667

pp. 788-802, ISSN 1029-2330

ISSN 0264-6021

cells and oral vaccine targeting. *Drug Discovery Today,* Vol. 10, No. 17, (September

A polymeric drug for treatment of inflammatory bowel disease. *Jornal of Medicinal* 

Müller, M. (2003). Gastrointestinal transit and release of 5-aminosalicylic acid from 153Sm-labelled mesalazine pellets vs. Tablets in male healthy volunteers. *Alimentary pharmacology & therapeutics,* Vol. 17, No. 9, (May 2003), pp. 1163-1169, ISSN 0269-2813

study adhesive interactions between individual microspheres and intestinal mucosa. *Journal of controlled release,* Vol. 34, No. 3, (June 1995), pp. 251-261, ISSN 0168-3659 Chourasia, M.K. & Jain, S.K. (2003). Pharmaceutical approaches to colon targeted drug

delivery systems. *Journal of. Pharmacy & Pharmaceutical Sciences,* Vol. 6, No. 1,

invasin-mediated targeting of yersinia pseudotuberculosis to mouse peyer's patch m cells. *Infection and Immunity,* Vol. 66, No. 3, (March 1998), pp. 1237-1243, ISSN 0019-

potential in ulcerative colitis and crohn's disease. *Drugs,* Vol. 59, No. 4, (April 2000),

enzyme peroral administration. *International Journal of Pharmaceutics,* Vol. 242, No. 1-2,

microparticles as a protein carrier. *Drug Development and Industrial Pharmacy,* Vol. 27,

Bioefficacy of budesonide loaded crosslinked polyelectrolyte microparticles in rat model of induced colitis. *Journal of Drug Targeting,* Vol. 17, No. 10, (December 2009),

are damaged and increased in number in inflamed human ileal mucosa.

& Harding, S.E. (2000). Atomic force microscopy of gastric mucin and chitosan mucoadhesive systems. *Biochemical Journal,* Vol. 348, No.3 (June 2000), pp. 557-563,

E.P. (1998). Chronic experimental colitis induced by dextran sulphate sodium (dss) is characterized by th1 and th2 cytokines. *Clinical & Experimental Immunology,* Vol. 114,


Drug Targeting in IBD Treatment – Existing and New Approaches 327

Jeong, Y.I., Ohno, T., Hu, Z., Yoshikawa, Y., Shibata, N., Nagata, S. & Takada, K. (2001).

Jung, Y.J., Lee, J.S., Kim, H.H., Kim, Y.M. & Han, S.K. (1998). Synthesis and evaluation of 5-

Katsuma, M., Watanabe, S., Takemura, S., Sako, K., Sawada, T., Masuda, Y., Nakamura, K.,

Khan, M.Z., Stedul, H.P. & Kurjakovic, N. (2000). A ph-dependent colon-targeted oral drug

Kiss, R., Camby, I., Duckworth, C., De Decker, R., Salmon, I., Pasteels, J.L., Danguy, A. &

Klotz, U. & Schwab, M. (2005). Topical delivery of therapeutic agents in the treatment of

Kojouharoff, G., Hans, W., Obermeier, F., Mannel, D.N., Andus, T., Scholmerich, J., Gross, V. &

Kopecek, J., Kopecková, P., Brøndsted, H., Rathi, R., Ríhová, B., Yeh, P.Y. & Ikesue, K. (1992).

Kriegel, C. & Amiji, M. (2011). Oral tnf-α gene silencing using a polymeric microsphere-based

Kshirsagar, S.J. (2009). In vitro in vivo comparison of two ph sensitive eudragit polymers for

Lamprecht, A. (2010). Ibd: Selective nanoparticle adhesion can enhance colitis therapy. *Nature* 

*release,*Vol. 150, No. 1, (February 2011), pp. 77-86, ISSN 01683659

*Science,* Vol. 93, No. 5, (May 2004), pp. 1287-1299, ISSN 0022-3549

*Pharmacy,* Vol. 26, No. 5, (May 2000), pp. 549-554, ISSN 0363-9045

5173

ISSN 0168-3659

ISSN 0017-5749

2005), pp. 267-279, ISSN 0169-409X

3, (March 1992), pp. 121-130, ISSN 0168-3659

4, (December 2009), pp. 61-70, ISSN 0975-1459

279-290, ISSN 0168-3659

1759-5053

*International Journal of Pharmaceutics,* Vol. 168, No. 1, (June 1998), pp. 31-40, ISSN 0378-

Evaluation of an intestinal pressure-controlled colon delivery capsules prepared by a dipping method. *Jornal of Controlled Release,* Vol. 71, No. 2, (April 2001), pp. 175-182,

aminosalicyl-glycine as a potential colon-specific prodrug of 5-aminosalicylic acid. *Archives of pharmacal research,* Vol. 21, No. 2, (April 1998), pp. 174-178, ISSN 0253-6269

Fukui, M., Connor, A.L. & Wilding, I.R. (2004). Scintigraphic evaluation of a novel colon-targeted delivery system (codes) in healthy volunteers. *Journal of Pharmaceutical* 

delivery system using methacrylic acid copolymers. Ii. Manipulation of drug release using eudragit l100 and eudragit s100 combinations. *Drug Development and Industrial* 

Yeaton, P. (1997). In vitro influence of phaseolus vulgaris, griffonia simplicifolia, concanavalin a, wheat germ, and peanut agglutinins on hct-15, lovo, and sw837 human colorectal cancer cell growth. *Gut,* Vol. 40, No. 2, (February 1997), pp. 253-261,

inflammatory bowel disease. *Advanced Drug Delivery Review,* Vol. 57, No. 2, (January

Falk, W. (1997). Neutralization of tumour necrosis factor (tnf) but not of il-1 reduces inflammation in chronic dextran sulphate sodium-induced colitis in mice. *Clinical & Experimental Immunology,* Vol. 107, No. 2, (February 1997), pp. 353-358, ISSN 0009-9104 Kopecek, J. (1990). The potential of water-soluble polymeric carriers in targeted and site-

specific drug delivery. *Journal of Controlled Release,* Vol. 11, No. 1-3, (January 1990), pp.

Polymers for colon-specific drug delivery. *Journal of Controlled Release,* Vol. 19, No. 1-

delivery system for the treatment of inflammatory bowel disease. *Journal of controlled* 

colon specific drug delivery. *Journal of pharmaceutical sciences and research,* Vol. 1, No.

*Reviews Gastroenterology and Hepatology,* Vol. 7, No. 6, (June 2010), pp. 311-312, ISSN


Gombotz, W.R. & Wee, S. (1998). Protein release from alginate matrices. *Advanced drug delivery* 

Goracinova, K. Formulation and preparation of spray-dried alginate–Ca microparticles. Patent

Green, J.R., Mansfield, J.C., Gibson, J.A., Kerr, G.D. & Thornton, P.C. (2002). A double-blind

Grisham, M.B. & Granger, D.N. (1988). Neutrophil-mediated mucosal injury. Role of reactive

Gupta, V. K., Beckert, T. E. & Price, J. C. (2001). A novel ph- and time-based multi-unit

*Pharmaceutics,* Vol. 213, No. 1-2, (February 2001), pp. 83-91, ISSN 0378-5173 Gupta, A. (2009). Targeting cells for drug and gene delivery: Emerging applications of

*Delhi, India: 1963),* Vol. 68, No. 6, (June 2009), pp. 465-483, ISSN 0022-4456 Haddish-Berhane, N., Farhadi, A., Nyquist, C., Haghighi, K. & Keshavarzian, A. (2007).

Han, H.K. & Amidon, G.L. (2000). Targeted prodrug design to optimize drug delivery. *AAPS* 

Hedley, M.L. (2000). Gene therapy of chronic inflammatory disease. *Advanced Drug Delivery Review,* Vol. 44, No. 2-3, (November 2000), pp. 195-207, ISSN 0169-409X Hejazi, R. & Amiji, M. (2003). Chitosan-based gastrointestinal delivery systems. *Journal of Controlled Release,* Vol. 89, No. 2, (April 2003), pp. 151-165, ISSN 0168-3659 Huang, Y., Leobandung, W., Foss, A. & Peppas, N.A. (2000). Molecular aspects of muco- and

Ibekwe, V.C., Liu, F., Fadda, H.M., Khela, M.K., Evans, D.F., Parsons, G.E. & Basit, A.W. (2006).

Ina, K., Kusugami, K., Hosokawa, T., Imada, A., Shimizu, T., Yamaguchi, T., Ohsuga, M.,

Irache, J.M., Salman, H.H., Gamazo, C. & Espuelas, S. (2008). Mannose-targeted systems for

Ishibashi, T., Hatano, H., Kobayashi, M., Mizobe, M. & Yoshino, H. (1998). Design and

*Hepatology,* Vol. 14, No. 1, (January 1999), pp. 46-53, ISSN 0815-9319

*PharmSci,* Vol. 2, No. 1, (March 2000), pp. E6, ISSN 1522-1059

*Release,* Vol. 65, No. 1-2, (March 2000), pp. 63-71, ISSN 0168-3659

*Therapeutics,* Vol. 16, No. 1, (January 2002), pp. 61-68, ISSN 0269-2813

comparison of balsalazide, 6.75 g daily, and sulfasalazine, 3 g daily, in patients with newly diagnosed or relapsed active ulcerative colitis. *Alimentary Pharmacology and* 

oxygen metabolites. *Digestive diseases and sciences,* Vol. 33, No. 3 Suppl, (March 1988),

potential colonic drug delivery system. I. Development. *International Journal of* 

mannans and mannan binding lectins. *Journal of scientific & industrial research (New* 

Biological variability and targeted delivery of therapeutics for inflammatory bowel diseases: An in silico approach. *Inflammation & Allergy*-*Drug Targets,* Vol. 6, No. 1,

bioadhesion: Tethered structures and site-specific surfaces. *Journal of Controlled* 

An investigation into the in vivo performance variability of ph responsive polymers for ileo-colonic drug delivery using gamma scintigraphy in humans. *Jornal of Pharmaceutical Science,* Vol. 95, No. 12, (December 2006), pp. 2760-2766, ISSN 0022-

Kyokane, K., Sakai, T., Nishio, Y., Yokoyama, Y. & Ando, T. (1999). Increased mucosal production of granulocyte colony-stimulating factor is related to a delay in neutrophil apoptosis in inflammatory bowel disease. *Journal of Gastroenterology and* 

the delivery of therapeutics. *Expert Opinion on Drug Delivery,* Vol. 5, No. 6, (June

evaluation of a new capsule-type dosage form for colon-targeted delivery of drugs.

*reviews,* Vol. 31, No. 3, (May 1998), pp. 267-285, ISSN 0169-409X

No. MP/MK/05/01/FF/BE/01/IP, 2005

(March 2007), pp. 47-55, ISSN 1871-5281

2008), pp. 703-724, ISSN 1742-5247

pp. 6S-15S, ISSN 0163-2116

3549

*International Journal of Pharmaceutics,* Vol. 168, No. 1, (June 1998), pp. 31-40, ISSN 0378- 5173


Drug Targeting in IBD Treatment – Existing and New Approaches 329

Mladenovska, K., Raicki, R.S., Janevik, E.I., Ristoski, T., Pavlova, M.J., Kavrakovski, Z., Dodov,

Nakase, H., Okazaki, K., Tabata, Y., Uose, S., Ohana, M., Uchida, K., Matsushima, Y.,

*Therapeutics,* Vol. 292, No. 1, (January 2000), pp. 15-21, ISSN 0022-3565 Nakase, H., Okazaki, K., Tabata, Y., Uose, S., Ohana, M., Uchida, K., Nishi, T., Debreceni, A.,

*Therapeutics,* Vol. 297, No. 3, (June 2001), pp. 1122-1128, ISSN 0022-3565 Ni, J., Chen, S.F. & Hollander, D. (1996). Effects of dextran sulphate sodium on intestinal

Nikolaus, S., Bauditz, J., Gionchetti, P., Witt, C., Lochs, H. & Schreiber, S. (1998). Increased

Nixon, D.F., Hioe, C., Chen, P.D., Bian, Z., Kuebler, P., Li, M.L., Qiu, H., Li, X.M., Singh, M.,

Nugent, S.G., Kumar, D., Rampton, D.S. & Evans, D.F. (2001). Intestinal luminal ph in

Nugent, S.G., Kumar, D., Yazaki, E.T., Evans, D.F. & Rampton, D.S. (2000). Small intestinal

Oliveira, G.F. (2010). Chitosan–pectin multiparticulate systems associated with enteric

Oliveira, L. & Cohen, R.D. (2011). Maintaining remission in ulcerative colitis--role of once daily

Omar, S., Aldosari, B., Refai, H. & Gohary, O.A. (2007). Colon-specific drug delivery for

*Vaccine,* Vol. 14, No. 16, (November 1996), pp. 1523-1530, ISSN 0264-410X Nolen, H., 3rd, Fedorak, R.N. & Friend, D.R. (1995). Budesonide-beta-d-glucuronide: A

1-2, (September 2007), pp. 124-136, ISSN 0378-5173

42, No. 4, (April 1998), pp. 470-476, ISSN 0017-5749

Vol. 84, No. 6, (June 1995), pp. 677-681, ISSN 0022-3549

241, ISSN 0017-5749

0017-5749

A780-A780, ISSN 0016-5085

111-116, ISSN 1177-8881

2010), pp. 1004-1009, ISSN 0144-8617

2007), pp. 691-700, ISSN 1061-186X

M.G. & Goracinova, K. (2007). Colon-specific delivery of 5-aminosalicylic acid from chitosan-ca-alginate microparticles. *International Journal of Pharmaceutics,* Vol. 342, No.

Kawanami, C., Oshima, C., Ikada, Y. & Chiba, T. (2000). Development of an oral drug delivery system targeting immune-regulating cells in experimental inflammatory bowel disease: A new therapeutic strategy. *Journal of Pharmacology and Experimental* 

Itoh, T., Kawanami, C., Iwano, M., Ikada, Y. & Chiba, T. (2001). An oral drug delivery system targeting immune-regulating cells ameliorates mucosal injury in trinitrobenzene sulfonic acid-induced colitis. *Journal of Pharmacology and Experimental* 

epithelial cells and intestinal lymphocytes. *Gut,* Vol. 39, No. 2, (August 1996), pp. 234-

secretion of pro-inflammatory cytokines by circulating polymorphonuclear neutrophils and regulation by interleukin 10 during intestinal inflammation. *Gut,* Vol.

Richardson, J., Mcgee, P., Zamb, T., Koff, W., Wang, C.Y. & O'hagan, D. (1996). Synthetic peptides entrapped in microparticles can elicit cytotoxic t cell activity.

potential prodrug for treatment of ulcerative colitis. *Journal of Pharmaceutical Science,*

inflammatory bowel disease: Possible determinants and implications for therapy with aminosalicylates and other drugs. *Gut,* Vol. 48, No. 4, (April 2001), pp. 571-577, ISSN

luminal ph in crohn's disease: Implications for release of antiinflammatory drugs from ph-dependent capsules. *Gastroenterology,* Vol. 118, No. 4, Part 1, (April 2000), pp.

polymers for colonic drug delivery. *Carbohydrate polymers,* Vol. 82, No. 3, (October

extended-release mesalamine. *Drug Design, Development and Therapy,* Vol. 5, (n.d.), pp.

mebeverine hydrochloride. *Journal of Drug Targeting,* Vol. 15, No. 10, (December


Lamprecht, A., Schafer, U. & Lehr, C.M. (2001). Size-dependent bioadhesion of micro- and

Lamprecht, A., Ubrich, N., Yamamoto, H., Schafer, U., Takeuchi, H., Maincent, P., Kawashima,

*Therapeutics,* Vol. 299, No. 2, (November 2001), pp. 775-781, ISSN 0022-3565 Lamprecht, A., Yamamoto, H., Takeuchi, H. & Kawashima, Y. (2005). Nanoparticles enhance

Larouche, J. (1995). Release of 5-asa from pentasa in patients with crohn's disease of the small

Leamon, C.P. & Low, P.S. (2001). Folate-mediated targeting: From diagnostics to drug and

Lee, E.S., Na, K. & Bae, Y.H. (2005). Super ph-sensitive multifunctional polymeric micelle. *Nano Letters,* Vol. 5, No. 2, (February 2005), pp. 325-329, ISSN 1530-6984 Lehr, C.M. (2000). Lectin-mediated drug delivery: The second generation of bioadhesives. *J Control Release,* Vol. 65, No. 1-2, (March 2000), pp. 19-29, ISSN 0168-3659 Leopold, C.S. (2001). A practical approach in the design of colon-specific drug delivery

Lochner, N., Pittner, F., Wirth, M. & Gabor, F. (2003). Wheat germ agglutinin binds to the

Martinac, A., Filipovic-Grcic, J., Voinovich, D., Perissutti, B. & Franceschinis, E. (2005).

Martinsen, A. (1991). Comparison of different methods for determination of molecular weight

Mcconnell, E.L., Short, M.D. & Basit, A.W. (2008). An in vivo comparison of intestinal ph and

Mladenovska, K., Cruaud, O., Richomme, P., Belamie, E., Raicki, R.S., Venier-Julienne, M.C.,

*Laboratorial,* (April 2004), Vol. 40, No. 2, pp. 123-125, ISSN 1676-2444

18, No. 6, (June 2001), pp. 788-793, ISSN 0724-8741

2005), pp. 196-202, ISSN 0022-3565

2003), pp. 833-839, ISSN 0724-8741

(n.d.), pp. 171-193, ISSN 0144-8617

pp. 69-77, ISSN 0378-5173

320, ISSN 0269-2813

1878-5832

9783527600069

nanoparticulate carriers to the inflamed colonic mucosa. *Pharmaceutical Research,* Vol.

Y. & Lehr, C.M. (2001). Biodegradable nanoparticles for targeted drug delivery in treatment of inflammatory bowel disease. *Journal of Pharmacology and Experimental* 

therapeutic efficiency by selectively increased local drug dose in experimental colitis in rats. *Journal of Pharmacology and Experimental Therapeutics,* Vol. 315, No. 1, (October

intestine. *Alimentary pharmacology & therapeutics,* Vol. 9, No. 3, (June 1995), pp. 315-

gene delivery. *Drug Discovery Today,* Vol. 6, No. 1, (January 2001), pp. 44-51, ISSN

systems, In:Book *Drug targeting* pp. (157-170), Wiley-VCH Verlag GmbH, ISBN

epidermal growth factor receptor of artificial caco-2 membranes as detected by silver nanoparticle enhanced fluorescence. Pharmaceutical Research,Vol. 20, No. 5, (May

Development and bioadhesive properties of chitosan-ethylcellulose microspheres for nasal delivery. *International Journal of Pharmaceutics,* Vol. 291, No. 1-2, (March 2005),

and molecular weight distribution of alginates. *Carbohydrate polymers,* Vol. 15, No. 2,

bacteria as physiological trigger mechanisms for colonic targeting in man. *Jornal of Controlled Release,* Vol. 130, No. 2, (September 2008), pp. 154-160, ISSN 1873-4995 Melo-Junior, M. R.; Lelles, A. M. S. & Albuquerque, F E. B. (2004). Altered lectin-binding sites

in normal colon and ulcerative colitis. *Jornal Brasileiro de Patologia e Medicina* 

Popovski, E., Benoit, J.P. & Goracinova, K. (2007). 5-asa loaded chitosan-ca-alginate microparticles: Preparation and physicochemical characterization. *International Journal of Pharmaceutics,* Vol. 345, No. 1-2, (December 2007), pp. 59-69, ISSN 0378-5173


Drug Targeting in IBD Treatment – Existing and New Approaches 331

Roy, P. & Shahiwala, A. (2009). Multiparticulate formulation approach to pulsatile drug

Russell-Jones, G.J., Arthur, L. & Walker, H. (1999). Vitamin b12-mediated transport of

Rutgeerts, P., Van Assche, G. & Vermeire, S. (2004). Optimizing anti-tnf treatment in

Sandborn, W.J., Korzenik, J., Lashner, B., Leighton, J.A., Mahadevan, U., Marion, J.F., Safdi, M.,

Sands, B.E. (2007). Inflammatory bowel disease: Past, present, and future. *Journal of Gastroenterology,* Vol. 42, No. 1, (January 2007), pp. 16-25, ISSN 0944-1174 Sellin, J. (2005). Treatment targets in inflammatory bowel disease. *Advanced Drug Delivery* 

Simonoska Crcarevska, M., Glavas Dodov, M. & Goracinova, K. (2008). Chitosan coated ca-

Sinha, V.R. & Kumria, R. (2001). Colonic drug delivery: Prodrug approach. *Pharmaceutical* 

Sinha, V.R. & Kumria, R. (2002). Binders for colon specific drug delivery: An in vitro

Smart, J.D. (2004). Lectin-mediated drug delivery in the oral cavity. *Advanced Drug Delivery* 

Smidsrød, O. (1973). The relative extension of alginates having different chemical composition. *Carbohydrate research,* Vol. 27, No. 1, (March 1973), pp. 107-118, ISSN 0008-6215 Tabata, Y., Inoue, Y. & Ikada, Y. (1996). Size effect on systemic and mucosal immune responses

Tahara, K., Samura, S., Tsuji, K., Yamamoto, H., Tsukada, Y., Bando, Y., Tsujimoto, H.,

Tao, S.L., Lubeley, M.W. & Desai, T.A. (2003). Bioadhesive poly(methyl methacrylate)

Uguccioni, M., Gionchetti, P., Robbiani, D.F., Rizzello, F., Peruzzo, S., Campieri, M. &

alginate microparticles loaded with budesonide for delivery to the inflamed colonic mucosa. *European Journal of Pharmaceutics and Biopharmaceutics,* Vol. 68, No. 3, (March

evaluation. *International Journal of Pharmaceutics,* Vol. 249, No. 1-2, (December 2002),

induced by oral administration of biodegradable microspheres. *Vaccine,* Vol. 14, No.

Morishita, R. & Kawashima, Y. (2011). Oral nuclear factor-[kappa]b decoy oligonucleotides delivery system with chitosan modified poly(d,l-lactide-coglycolide) nanospheres for inflammatory bowel disease. *Biomaterials,*Vol. 32, No. 3,

microdevices for controlled drug delivery. *Journal of Controlled Release,* Vol. 88, No. 2,

Baggiolini, M. (1999). Increased expression of ip-10, il-8, mcp-1, and mcp-3 in ulcerative colitis. The *American Journal of Pathology,* Vol. 155, No. 2, (August 1999), pp.

*Review,* Vol. 57, No. 2, (January 2005), pp. 217-218, ISSN 0169-409X

*Research,* Vol. 18, No. 5, (May 2001), pp. 557-564, ISSN 0724-8741

*Review,* Vol. 56, No. 4, (March 2004), pp. 481-489, ISSN 0169-409X

17-18, (December 1996), pp. 1677-1685, ISSN 0264-410X

(January 2011), pp. 870-878, ISSN 0142-9612

(March 2003), pp. 215-228, ISSN 0168-3659

331-336, ISSN 0002-9440

2009), pp. 74-80, ISSN 1873-4995

2008), pp. 565-578, ISSN 0939-6411

pp. 23-31, ISSN 0378-5173

1610, ISSN 0016-5085

(March 1999), pp. 247-255, ISSN 0378-5173

Suppl 1, (February 2000), pp. S68-82, ISSN 0016-5085

delivery: Current perspectives. *Journal of Controlled Release,* Vol. 134, No. 2, (March

nanoparticles across caco-2 cells. *International Journal of Pharmaceutics,* Vol. 179, No. 2,

inflammatory bowel disease. *Gastroenterology,* Vol. 126, No. 6, (May 2004), pp. 1593-

Sninsky, C.A., Patel, R.M., Friedenberg, K.A., Dunnmon, P., Ramsey, D. & Kane, S. (2010). Once-daily dosing of delayed-release oral mesalamine (400-mg tablet) is as effective as twice-daily dosing for maintenance of remission of ulcerative colitis. *Gastroenterology,* Vol. 138, No. 4, (April 2010), pp. 1286-1296.e1283, ISSN 0016-5085 Sands, B.E. (2000). Therapy of inflammatory bowel disease. *Gastroenterology,* Vol. 118, No. 2


Ota, T., Maeda, M. & Tatsuka, M. (2002). Cationic liposomes with plasmid DNA influence

Oz, H.S. & Ebersole, J.L. (2008). Application of prodrugs to inflammatory diseases of the gut. *Molecules,* Vol. 13, No. 2, (February 2008), pp. 452-474, ISSN 1420-3049 \ Pastorelli, L., Saibeni, S., Spina, L., Signorelli, C., Celasco, G., De Franchis, R. & Vecchi, M.

Patel, J.M. (2010). Colon targeted oral delivery of ornidazole using combination of ph and time

Plevy, S. E. & Targan, S. R. (2011). Future therapeutic approaches for inflammatory bowel diseases. *Gastroenterology,*Vol. 140, No. 6, (May 2011), pp. 1838-1846, ISSN 0016-5085 Pozzi, F., Furlani, P., Gazzaniga, A., Davis, S.S. & Wilding, I.R. (1994). The time clock system: A

Prakash, A. (1999). Oral delayed-release mesalazine a review of its use in ulcerative colitis and

Price, J.M., Davis, S.S., Sparrow, R.A. & Wilding, I.R. (1993). The effect of meal composition on

Reece, J.C., Vardaxis, N.J., Marshall, J.A., Crowe, S.M. & Cameron, P.U. (2001). Uptake of hiv

Rhodes, J. M., Black, R. R. & Savage, A. (1986). Glycoprotein abnormalities in colonic

Rhodes, J. M., Black, R. R. & Savage, A. (1988). Altered lectin binding by colonic epithelial

Rivkin, A. (2009). Certolizumab pegol for the management of crohn's disease in adults. *Clinical Therapeutics,* Vol. 31, No. 6, (June 2009), pp. 1158-1176, ISSN 1879-114X Roth-Walter, F., Bohle, B., Scholl, I., Untersmayr, E., Scheiner, O., Boltz-Nitulescu, G., Gabor,

Roth-Walter, F., Scholl, I., Untersmayr, E., Fuchs, R., Boltz-Nitulescu, G., Weissenbock, A.,

*Sciences,*Vol. 33, No. 11, (November 1988) pp. 1359-1363, ISSN 0163-2116 Rhodes, J. M., Campbell, B. J. & Yu, L. G. (2008). Lectin-epithelial interactions in the human

Vol. 100, No. 2, (September 2005), pp. 182-188, ISSN 0165-2478

*Research,* Vol. 10, No. 5, (May 1993), pp. 722-726, ISSN 0724-8741

*Cell Biology,* Vol. 79, No. 3, (June 2001), pp. 255-263, ISSN 0818-9641

December 2002), pp. 4049-4052, ISSN 0250-7005

2, No. 1, (January-March 2010), pp. 28-35, ISSN 1674-0440

ISSN 1365-2036

ISSN 0012-6667

1334, ISSN 0021-9746

1486, ISSN 1470-8752

cancer metastatic capability. *Anticancer Research,* Vol. 22, No. 6C, (November-

(2008). Oral, colonic-release low-molecular-weight heparin: An initial open study of parnaparin-mmx for the treatment of mild-to-moderate left-sided ulcerative colitis. *Alimentary Pharmacology & Therapeutics,* Vol. 28, No. 5, (September 2008), pp. 581-588,

dependent drug delivery system. *International Journal of Pharmaceutical Research,* Vol.

new oral dosage form for fast and complete release of drug after a predetermined lag time. *Journal of Controlled Release,* Vol. 31, No. 1, (April 1994), pp. 99-108, ISSN 0168-3659

Crohn`s disease. *Drugs (New York, N.Y.),* Vol. 57, No. 3, (March 1999), pp. 383-408,

the gastrocolonic response: Implications for drug delivery to the colon. *Pharmaceutical* 

and latex particles by fresh and cultured dendritic cells and monocytes. *Immunology &* 

carcinomata, adenomata, and hyperplastic polyps shown by lectin peroxidase histochemistry. *Journal of Clinical Pathology,*Vol. 39, No. 12, (December 1986), pp. 1331-

glycoconjugates in ulcerative colitis and crohn's disease. *Digestive Diseases and* 

colon. *Biochemical Society Transactions,*Vol. 36, No. Pt 6, (December 2008), pp. 1482-

F., Brayden, D.J. & Jensen-Jarolim, E. (2005). Targeting antigens to murine and human m-cells with aleuria aurantia lectin-functionalized microparticles. *Immunology Letters,*

Scheiner, O., Gabor, F. & Jensen-Jarolim, E. (2004). M cell targeting with aleuria aurantia lectin as a novel approach for oral allergen immunotherapy. *Journal of Allergy and Clinical Immunology,* Vol. 114, No. 6, (December 2004), pp. 1362-1368, ISSN 0091-6749


Van Assche, G. & Rutgeerts, P. (2002). Antiadhesion molecule therapy in inflammatory bowel

Van Deventer, S.J. (1999). Anti-tnf antibody treatment of crohn's disease. *Annals of the Rheumatic Diseases,* Vol. 58, No. Suppl 1 (November 1999), pp. I114-120, ISSN 0003-4967 Verma, R.K., Krishna, D.M. & Garg, S. (2002). Formulation aspects in the development of

Verma, R.K., Mishra, B. & Garg, S. (2000). Osmotically controlled oral drug delivery. *Drug* 

Vinogradov, S., Batrakova, E., Li, S. & Kabanov, A. (1999). Polyion complex micelles with

Wei, H., Qing, D., De-Ying, C., Bai, X. & Li-Fang, F. (2008). In-vitro and in-vivo studies of

Wittaya-Areekul, S., Kruenate, J. & Prahsarn, C. (2006). Preparation and in vitro evaluation of

Wiwattanapatapee, R., Lomlim, L. & Saramunee, K. (2003). Dendrimers conjugates for colonic

Wong, M., Ziring, D., Korin, Y., Desai, S., Kim, S., Lin, J., Gjertson, D., Braun, J., Reed, E. & Singh,

*Digestive Diseases and Sciences,*Vol. 45, No. 12, pp. 2298-2305, ISSN 0163-2116 Xu, J., Ganesh, S. & Amiji, M. Non-condensing polymeric nanoparticles for targeted gene and

Yang, L. (2008). Biorelevant dissolution testing of colon-specific delivery systems activated by

Yano, H., Hirayama, F., Arima, H. & Uekama, K. (2001). Prednisolone-appended alpha-

Vol. 34, No. 2-3, (December 1998), pp. 123-133, ISSN 1872-8294

No. 1-3, (Feb 2002), pp. 7-27, ISSN 0168-3659

0998

0363-9045

1043-1802

ISSN 0269-2813

113-118, ISSN 0378-5173

2003), pp. 1-9, ISSN 0168-3659

2011.05.036, ISSN 0378-5173

86, ISSN 1873-4995

disease. *Inflammatory Bowel Disease,* Vol. 8, No. 4, (July 2002), pp. 291-300, ISSN 1078-

osmotically controlled oral drug delivery systems. *Jornal of Controlled Release,* Vol. 79,

*Development and Industrial Pharmacy,* Vol. 26, No. 7, (July 2000), pp. 695-708, ISSN

protein-modified corona for receptor-mediated delivery of oligonucleotides into cells. *Bioconjugate Chemistry,* Vol. 10, No. 5, (September-October 1999), pp. 851-860, ISSN

pectin/ethylcellulosefilm-coated pellets of 5-fluorouracil for colonic targeting. *Journal Pharmacy and Pharmacology,* Vol. 60, No. 1, (January 2008), pp. 35-44, ISSN 0022-3573 Wilding, I.R., Kenyon, C.J. & Hooper, G. (2000). Gastrointestinal spread of oral prolonged-

release mesalazine microgranules (pentasa) dosed as either tablets or sachet. *Alimentary Pharmacology & Therapeutics,* Vol. 14, No. 2, (February 2000), pp. 163-169,

mucoadhesive properties of alginate/chitosan microparticles containing prednisolone. *International Journal of Pharmaceutics,* Vol. 312, No. 1-2, (April 2006), pp.

delivery of 5-aminosalicylic acid. *Journal of Controlled Release,* Vol. 88, No. 1, (February

R.R. (2008). Tnfalpha blockade in human diseases: Mechanisms and future directions. *Clinical Immunology,* Vol. 126, No. 2, (February 2008), pp. 121-136, ISSN 1521-6616 Worledge, K. L., Godiska, R., Barrett, T. A. & Kink, J. A. (2000). Oral administration of avian

tumor necrosis factor antibodies effectively treats experimental colitis in rats.

sirna delivery. *International Journal of Pharmaceutics,* DOI: 10.1016/j.ijpharm.

colonic microflora. *Journal of Controlled Release,* Vol. 125, No. 2, (January 2008), pp. 77-

cyclodextrin: Alleviation of systemic adverse effect of prednisolone after intracolonic administration in 2,4,6-trinitrobenzenesulfonic acid-induced colitis rats. *Journal of Pharmaceutical Science,* Vol. 90, No. 12, (December 2001), pp. 2103-2112, ISSN 0022-3549 Yeh, P., Ellens, H. & Smith, P.L. (1998). Physiological considerations in the design of

particulate dosage forms for oral vaccine delivery. *Advanced Drug Delivery Review,*

## *Edited by Sami Karoui*

This book is dedicated to inflammatory bowel disease, and the authors discuss the advances in the pathogenesis of inflammatory bowel disease, as well as several new parameters involved in the etiopathogeny of Crohn's disease and ulcerative colitis, such as intestinal barrier dysfunction and the roles of TH 17 cells and IL 17 in the immune response in inflammatory bowel disease. The book also focuses on several relevant clinical points, such as pregnancy during inflammatory bowel disease and the health-related quality of life as an end point of the different treatments of the diseases. Finally, advances in management of patients with inflammatory bowel disease are discussed, especially in a complete review of the recent literature.

Photo by jacquesdurocher / iStock

Inflammatory Bowel Disease - Advances in Pathogenesis and Management

Inflammatory Bowel Disease

Advances in Pathogenesis and Management

*Edited by Sami Karoui*