Modied Chitosans for Oral Drug Delivery

RCH2 MARTIN WERLE,1 HIROFUMI TAKEUCHI,1 ANDREAS BERNKOP-SCHNU Department of Drug Delivery Technology and Science, Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi, Gifu 502-8585, Japan
2 1

Department of Pharmaceutical Technology, Leopold-Franzens University Innsbruck, Innrain 52, 6020 Innsbruck, Austria

Received 6 May 2008; accepted 3 July 2008 Published online 9 September 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21550

ABSTRACT: The cationic polysaccharide chitosan has been extensively studied for oral drug delivery. In recent years, chemically modied chitosans developed in order to improve the properties of chitosan for oral drug delivery have gained increasing attention. Representatives of these novel polymers are trimethyl-chitosans, thiolated chitosans, carboxymethyl chitosan and derivatives, hydrophobic chitosans, chitosan succinate and phthalate, PEGylated chitosans and chitosan-enzyme inhibitor conjugates. Besides their use for oral delivery of therapeutic peptides and proteins, they have recently been evaluated regarding their potential for the delivery of other substance classes, including genes and efux pump substrates. Within the current review, various modied chitosan derivatives, their properties and synthesis are discussed. 2008 WileyLiss, Inc. and the American Pharmacists Association J Pharm Sci 98:16431656, 2009

Keywords: chitosan; oral drug delivery; polymeric drug delivery systems; biomaterials; polymer synthesis

INTRODUCTION
Oral drug administration is generally the most convenient and preferred application route. Besides painless administration, high patient compliance is another main advantage. In addition, pharmaceutical companies have a strong interest in the development of oral drug delivery systems for drugs which are currently on the market only as injections as well as for new chemical entities. After oral administration, a drug has to overcome several barriers before reaching systemic circulation. Physico-chemical properties of the drug such

Correspondence to: Andreas Bernkop-Schnu rch (Telephone: 43-512-507-5383; Fax: 43-512-507-2933; E-mail: andreas.bernkop@uibk.ac.at)
Journal of Pharmaceutical Sciences, Vol. 98, 16431656 (2009) 2008 Wiley-Liss, Inc. and the American Pharmacists Association

as low water solubility or high molecular mass and aggregation can lead to negligible drug plasma levels after oral administration.1 Especially macromolecular hydrophilic drugs such as therapeutic peptides, proteins and genes are often degraded by the harsh environment during gastro-intestinal (GI) passage.2 In this context, enzymatic degradation caused by a broad variety of luminally secreted and membrane bound digestive enzymes that occur in the GI tract, play a pivotal role.3 Moreover, the mucus layer itself comprises a physical barrier for drugs.4 It is well known that certain drugs cannot permeate well through the intestinal membrane. This can be either due to their hydrophilicity/lipohilicity ratio or their size, but also to active transporter systems. In particular transporters of the ABC gene family such as P-glycoprotein or the multidrug-resistant transporter proteins (MRP) have
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been identied to constitute a barrier for orally administered substrates of these transmembrane located transporter proteins.5 Several strategies have been developed in order to overcome these barriers. The polymer chitosan has been extensively studied in oral peptide/ protein delivery and recently in gene delivery. Chitosan is a cationic polysaccharide composed of randomly distributed b-(14)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) (Fig. 1). It is produced via deacetylation of the naturally occurring chitin which is derived from crustacean. The degree of deacetylation has a direct impact on the solubility of the polymer. The amine group of the polymer has a pKa in the range of 5.56.5. Therefore, chitosan is commonly insoluble at pH values above 6.5. It has been reported that chitosan can accelerate gastric ulcer healing6 and that pretreatment with chitosan did prevent ulcerogenic effects in rats.7 Moreover, it has been shown that chitosan also displays antimicrobial activity.8,9 Generally, it must be considered that chitosan refers to polymers of a broad range of molecular mass, different salts and various sources. Chitosan offers many advantages for oral drug delivery. First, it is regarded as safe and biocompatible. Moreover, due to its cohesive properties it can be used in matrix tablets in order to provide a controlled release.10 Chitosan is capable of opening tight junctions and can therefore improve the oral uptake of hydrophilic drugs such as peptides.11 Furthermore, it is widely used because of its mucoadhesive properties.12 The mucoadhesive properties can mainly be regarded

to the ionic interactions, in particular between the cationic amino groups of chitosan and negative functional groups such as sialic acid of the mucus. Concerning gene delivery, chitosan offers the advantage that nanoparticles can be prepared easily by mixing negatively charged DNA/RNA with the cationic chitosan, leading to stable nanoparticles.13 According to these aforementioned features, chitosan is of great interest for researchers in the oral drug delivery eld. Nevertheless, several chemical modications of chitosan have been synthesized and evaluated regarding their potential for oral drug delivery. Usually, chitosan is modied in order to improve the features of chitosan or to add additional functionality to chitosan. As way of example, certain derivatives such as trimethylated chitosan (TMC) have been developed in order to make feasible the solubility of chitosan at neutral and slightly basic pH. Chitosan conjugates with additional benecial properties for oral drug delivery are for example chitosan-inhibitor conjugates, thiolated polymers, PEGylated chitosans or hydrophobically modied chitosans.

CHEMISTRY AND FEATURES OF MODIFIED CHITOSANS

Most chemical modications of chitosan are performed at the free amino groups of the glucosamine units. For example, the formation of amide bonds between these amino groups and activated carboxylic groups can be initiated easily. Besides, there are also reports about modications of chitosan hydroxyl groups.14 Purication of modied chitosans can often be done by simple dialysis, using molecular weight cut off tubings which allow the diffusion of low molecular weight compounds used for the synthesis such as coupling reagents and modiers, but which keep the polymers concentrated inside of the tubings. In large-scale production, purication is mainly based on ultraltration and precipitation techniques. The most important chemically modied chitosan derivatives for oral drug delivery will be introduced briey, and their synthesis will be discussed as well.

Figure 1. Subunit of chitosan displaying b-(14)linked D-glucosamine (deacetylated unit) and Nacetyl-D-glucosamine (acetylated unit).
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Trimethyl-Chitosan N-trimethyl chitosan chloride (TMC) is a quaternized chitosan derivative which has been develDOI 10.1002/jps

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Figure 2. Modied glucosamine-units of trimethylated chitosan (TMC) and the thiomer chitosan-thiobutylamidine (Chito-TBA).

oped in order to extend the water solubility of chitosan to a broader pH range (Fig. 2). In brief, TMC is synthesized via a modication of chitosan amino groups with methyl-iodide. After precipitation and centrifugation, the product is puried. Finally, 1 M HCl is added in order to substitute iodide with chloride. The research group of Junginger investigated the inuence of conditions such as the number and duration of reaction steps and the amount of methyl iodide on TMC which was characterized via NMR.15 Although several water soluble chitosan-derivatives have been synthesized previously (e.g., cyclodextrin chitosan,16 carboxymethyl-chitosan17), TMC gained so far most attention for a use in oral drug delivery. The above-mentioned insolubility of chitosan at neutral and alkaline pH of course interferes with its potential use for oral drug delivery. Therefore, it was anticipated that modied chitosan derivatives which are soluble at higher pH, would be more benecial for oral drug delivery. It was demonstrated, that TMC is soluble up to pH 9.18 In the same study the effect of various concentrations of TMC on the transepithelial resistance (TEER) of Caco-2 cells was investigated. A reversible decrease of the TEER was observed, which was attributed to the opening of tight junctions. This theory was supported by the observation, that the permeation of the hydrophilic compounds FD4, buserelin and [14C]-mannitol was strongly increased in the presence of TMC. The apical to basolateral transport of these compounds increased when increasing TMC concentration. Also a correlation between drug-size and permeation enhancing
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effect was observed. The smaller the drug, the more pronounced the permeation enhancing effect. Cells were stained with trypan-blue after transport studies, and no toxicity was observed.18 In addition, for the use of TMC as a nasal delivery system, the toxicity was determined in vivo by investigating the acute microscopic toxic potential on the morphology of rat nasal epithelium with transmission electron microscopy (TEM) and in vitro by measurement of the ciliary beat frequency (CBF) of human ciliated nasal epithelium. According to the results, TMC did not show any toxic effects using the evaluated concentrations.19 The inuence of the positive charge of chitosan on the tight junction opening process has been thoroughly investigated.20 A specic interaction of the quaternized amino moieties with the tight junctions can be anticipated. In contrast, the mucoadhesive properties of TMC are lower than that of unmodied chitosan and a correlation between decrease in mucoadhesiveness and increase in the degree of quaternization of the TMC polymers was found.21

Thiolated Chitosans Thiolated chitosans constitute an integral part of designated thiomers, which are thiolated polymers that are widely used in noninvasive drug delivery research. In brief, small molecular mass thiol group bearing compounds are covalently bound to polymeric backbones. Therefore, thiomers are polymers which display thiol groups on their surface. Exemplarily, the substructure of chitosan-thiobutylamidine (Chito-TBA) is depicted in Figure 2. The synthesis of thiomers is mainly based on the formation of an amide bond between the free amino groups of chitosan and the activated carboxylic groups of a low molecular mass thiol group bearing compound. A typical procedure can be summarized as follows: EDAC is added to a solution of the low molecular mass thiol group bearing compound in order to activate the carboxylic groups. After a certain incubation period, the activated thiol group bearing compound is added to a chitosan solution. The reaction mixture is stirred and incubated at room temperature for several hours. Finally, the reaction mixture is dialyzed to remove unbound low molecular mass thiol group bearing compounds and EDAC and lyophilized. There are many reports describing the synthesis of thiomers, discussing also the impact of various conditions during the synthesis
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on the nal product as well as optimized conditions for the synthesis.22 The initial reason for the introduction of the thiol groups was based on the hypothesis, that free thiol groups which are bound to a polymer will form disulde bonds with thiol groups of the mucus, consequently leading to high mucoadhesive properties. In fact, the GI mucus comprises around 80% glycoproteins, in which cysteine-rich subdomains occur. It could be demonstrated, that these novel polymers display highly improved mucoadhesive features and that this phenomenon can be attributed to the proposed formation of disulde bonds between thiomers and the mucus covering GI epithelia.23,24 Thorough investigations revealed that thiomers offer various further properties which are benecial for an intended use in oral drug delivery. Due to the inter- and intramolecular formation of disulde bonds, a tight 3-dimensional network is formed which leads to high cohesiveness and allows a controlled drug release.25 Moreover, it could be demonstrated that a reversible opening of tight junctions occurs in the presence of thiomers, leading to a more pronounced permeation enhancing effect of thiomers in comparison to the unmodied polymer.26 Recently, it has been demonstrated that thiomers are capable of inhibiting efux pumps such as P-gp, which renders them likely as promising tools for oral delivery of efux pump substrates.27 Various different polymeric backbones and introduced thiol group bearing compounds have been used so far. The following chitosan based thiomers have been synthesized and characterized: chitosan-thioglycolicacid (ChitoTGA),28 chitosan-thiobutylamidine (Chito-TBA),29 chitosan-thioethylamidine (Chito-TEA),22,30 chitosancysteine (Chito-Cys)31 and chitosan-glutathione (Chito-GSH).25 The toxicity prole of Chito-TBA has been assessed using different tests including red blood cell lysis test and MTT as well as BrdU assays. Results revealed that the toxicity prole was comparable to that of unmodied chitosan.32

Figure 3. Modied glucosamine-units of mono-Ncarboxymethyl chitosan (MCC) and N-sulfonato-N,Ocarboxymethylchitosan (SNOCC).

MCC and SNOCC Mono-N-Carboxymethyl Chitosan (MCC) is a polyampholytic chitosan derivative, which is soluble at neutral and alkaline pH33 (Fig. 3). It has been synthesized by adding glyoxolic acid to a chitosan solution. After imine formation, sodium borhydride was added in order to reduce the imine. Finally, ethanol was added to precipitate
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the polymer and the product was puried and underwent a second cycle of carboxymethylation as described above. As determined by NMR, high degrees of modication (around 90%) can be achieved by this method.33 Besides its improved solubility, one reason for the development of this derivative was to make an administration of polyanionic drugs feasible. It has been reported that chitosan but also TMC forms aggregates with polyanionic drugs such as low molecular weight heparin (LMWH), leading to precipitation. Contrary, MCC has been evaluated to be a suitable delivery system for such compounds.33 Moreover, in the same study it was demonstrated that MCC can improve LMWH transport through Caco-2 cells. This effect can most likely be explained by an opening of tight junctions. A reversible decrease in TEER was observed, which supports the theory of opened tight junctions. As it was shown that cationic unmodied chitosan as well as polyampholytic derivatives such as MCC act as potent absorption enhancers, it was of interest to investigate whether anionic chitosan derivatives might display similar features. Therefore, N-sulfonato-N,O-carboxymethylchitosan (SNOCC) was developed34 (substructure is provided in Fig. 3). For the synthesis of SNOCC, N,O-carboxymethylchitosan (NOCC) was used as an educt. This polymer retains around 50% of its nitrogen centers on the glucose subunits as free amino groups.35 The synthesis was performed by dissolving NOCC in water and repeatedly adding sulfur trioxide-pyridine in alkaline medium at room temperature. After ltration, the ltered solution was precipitated in isopropanol, dried, dissolved in water and puried via dialysis. The product was dried via lyophilization.34 Similar to
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TMC and MCC, SNOCC was capable of improving the transport of hydrophilic drugs across Caco-2 cells. The effect was attributed to an opening of tight junctions.

the free amino-groups of the glucosamine unit with fatty acids (C6C16). Changes in the mechanical features of tablets based on these novel chitosan derivatives were observed.38

Hydrophobically Modied Chitosans The hydrophobic character of chitosan can be increased by the covalent attachment of hydrophobic excipients. Dodecylated chitosan (substructure depicted in Fig. 4) was synthesized by a slightly modied method described previously.36 Chitosan was dissolved in an acetic acid solution and ethanol was added. Dodecyl aldehyde, dissolved in ethanol, was added as well as an excess amount of sodium cyanoborohydride, which acts as a catalyst. After an incubation period of 24 h at room temperature, the reaction solution was ltrated, the product was precipitated with ethanol, separated via centrifugation and dried.37 Dodecylated chitosan was investigated regarding its potential as a coating material for liposomes.37 It was found to be more suitable for the coating of neutral liposomes than unmodied chitosan. This was attributed to interactions between the liposomal bilayer and the dodecyl-chains. Moreover, liposomes coated with this novel polymer displayed mucoadhesive properties. Another research group synthesized various chitosan derivatives with increased hydrophobicity by N-acylation of

Chitosan-Succinate and Chitosan-Phthalate Chitosan-succinate and chitosan-phthalate are two chitosan derivatives, in which primary amino groups of chitosan form amide bonds with substructures that carry a free carboxylic group. A detailed description of chitosan-succinate and chitosan-phthalate has been published,39 exemplarily, the structure of chitosan-succinate is provided in Figure 4. The introduction of the carboxylic groups to the polymer leads to changes in the solubility behavior. Both, chitosan-succinate and chitosan-phthalate are soluble in an alkaline environment. This behavior can be explained by the fact that under acidic conditions the carboxylic groups exist in nonionized form and are therefore poorly hydrophilic. Contrary, in alkaline conditions, the polymer is ionized and is considerably more hydrophilic.40 Chitosan-succinate and chitosan-phthalate have been proposed as useful tools for colonic drug delivery.39 It has been demonstrated that insulin encapsulated in chitosan-phthalate microspheres was quickly released in phosphate buffer pH 7.4, whereas only a small amount of insulin was released at pH 2.0.

PEGylated Chitosans Chitosan-PEG (shown in Fig. 4) for oral peptide delivery was synthesized by Prego et al., according to the following procedure. In a rst step, MeOPEG-CH2-CO2H was synthesized from MeO-PEGOH as described previously.41 In the following step, chitosan hydrochloride was dissolved in water and MeO-PEG-CH2-CO2H as well as Nhydroxysuccinimide was added to the solution. Finally, a carbodiimide was added and the solution was stirred for 22 h at room temperature. After ultraltration, the solution was lyophilized to obtain the PEGylated chitosan.42 Using this synthesis, chitosan with two different degrees of modication were prepared (0.5% and 1%). PEGylation of chitosan for oral peptide/protein delivery has two main reasons. The rst reason is, that generally, PEGylation is believed to improve the biocompatibility of chitosan.43 The second reasons is, that nanocapsules based on PEGylated
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Figure 4. Modied glucosamine-units of dodecylated chitosan, chitosan-succinate and PEGylated chitosan.

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chitosan show an improved stability in GI uid.42 Apart from an intended use as promising excipient for oral peptide/protein delivery, PEGylated chitosan might be interesting for the delivery of other substance classes. It has been demonstrated, for example, that PEGylated chitosan can improve the solubility of hydrophobic substances.44

Chitosan-Enzyme Inhibitor Conjugates It has been demonstrated several years ago, that coadministration of enzyme inhibitors can improve the oral uptake of drugs susceptible to enzymatic digestion in the GI tract.45 This relates in rst instance to peptide and protein drugs, but also to, for example, pDNA. However, due to feedback mechanisms, prolonged administration of enzyme inhibitors can lead to severe sideeffects, including hyperplasia of the pancreas and carcinoma.46 Therefore, strategies to minimize toxic side effects of orally administered enzyme inhibitors have been developed. One approach is the covalent attachment of enzyme inhibitors to polymers such as poly(acrylic acid) or chitosan. Various advantages of such a chitosan-enzyme inhibitor conjugate have been discussed. First, the covalent attachment of the enzyme-inhibitor to chitosan would prevent the oral absorption of the inhibitor. Second, due the mucoadhesive properties of chitosan, the drug would be released at the same intestinal segments where the enzyme inhibition takes place. Therefore, lower amounts of enzyme-inhibitor can be coadministered, which would consequently lead to a reduction of toxic side-effects. The combination of the mucoadhesive properties of chitosan and a controlled release of the drug out of the polymeric matrix minimizes

the contact-time of the drug with luminally secreted proteins. Third, the coabsorption of potentially harmful substances of the GI tract would be minimized, because of the targeted inhibition and the steric hindrance of the chitosan matrix. Last but not least, the permeation enhancing effect of chitosan might further contribute to an improved drug uptake. Various inhibitors, in rst instance proteaseinhibitors including Bowman-Birk-Inhibitor (BBI), elastatinal or aprotinin, but also complexing agents with the ability to inhibit a broader range of enzymes such as EDTA have been covalently bound to chitosan. So far synthesized and evaluated chitosan-inhibitor conjugates are summarized in Table 1. Similar to several other discussed modications, a covalent bond is formed between carboxylic groups of the inhibitor and the free amino groups of chitosan. Analogue to the synthesis of most thiolated polymers, the carboxylic groups of the inhibitors are activated with, for example, EDAC in order to gain sufcient yields. Unbound amounts of coupling agent and inhibitor can be removed via dialysis utilizing membranes with a suitable molecular mass cut off. It was demonstrated in vitro, that chitosanaprotinin conjugate shows strong inhibitory activity towards trypsin and chymotrypsin, whereas chitosan which was pretreated in the same way as chitosan-aprotinin but omitting EDAC in the activation step shows no inhibitory activity.47 This demonstrates the covalent attachment of aprotinin as well as that the inhibitory activity remains after such conjugation.47 To our knowledge, the rst synthesized chitosan-inhibitor conjugate was chitosan-EDTA, which initially was developed for oral peptide delivery.48 Recently, chitosan-EDTA has been investigated as a novel and promising tool for the oral adminis-

Table 1. Various Chitosan-Enzyme Inhibitor Conjugates, Their Corresponding Inhibited Enzymes and the Evaluated Protected Drug Inhibitor Conjugate Chitosan-ATA Chitosan-aprotinin Chitosan-BBI Chitosan-EDTA Inhibitor of Lysozyme, DNase, enzymes of collected porcine intestinal uid Trypsin, chymotrypsin Trypsin, chymotrypsin DNAseI, enzymes of collected porcine gastric and intestinal uid, enzymes of small intestinal homogenate Elastase Pepsin Protected Drug and Reference pDNA51 Insulin47 Insulin,62,77 calctionin63 pDNA,49 insulin72

Chitosan-elastatinal Chitosan-pepstatin

Insulin,62,77 calcitonin63 Calcitonin64

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tration of pDNA.49,50 Another evaluated chitosanenzyme inhibitor conjugate for the oral delivery of pDNA is chitosan-aurintricarboxylic acid (ATA).51

MODIFIED CHITOSAN DERIVATIVES FOR ORAL DRUG DELIVERY

Substance Classes Modied chitosans have, in rst instance, been used for the oral delivery of therapeutic peptides and proteins. As unmodied chitosan itself already displays benecial properties for oral peptide and protein delivery (e.g., capability to open tight junctions, mucoadhesive properties), the use of modied derivatives is a logical consequence. Recently, the potential of certain modied chitosans including TMC,52 thiolated chitosan53,54 and chitosan-enzyme inhibitor conjugates,4951 for noninvasive gene delivery gains increasing interest. In addition, thiolated chitosan has been shown to be capable of inhibiting efux pumps, in particular P-glycoprotein (P-gp). Therefore, thiolated chitosan has been used for the oral delivery of P-gp substrates.5557 The potential of chitosan, TMC and MCC for oral vaccine delivery has been reviewed previously.58

Figure 5. Time-dependent buserelin serum levels after intraduodenal administration of buserelin acetate with * 1% chitosan, & 1% TMC60 and * 1% TMC40; all experiments were performed at pH 7.2; each point represents the mean SD of six experiments; gure modied from Ref. 59.

Formulations Liquid Formulations The effect of 1% solutions of two different TMC on the oral absorption of the peptide drug buserelin after intraduodenal administration in rats was investigated.59 Both evaluated formulations clearly increased buserelin plasma levels in comparison to a dispersion of unmodied chitosan (pH of all administered formulations was 7.2). Moreover, buserelin plasma levels after administering TMC60 (60% trimethylation) were signicantly more increased in comparison to plasma levels after TMC40 (40% trimethylation) administration. The enhanced absorption in the presence of TMC60 was attributed to the more pronounced ability of TMC60 to open tight junctions. Results of the in vivo study are provided in Figure 5. In another study, the effect TMC solutions on octreotide in vitro permeation and in vivo absorption in rats was investigated.60 An intrajejunally administered 1% (w/v) TMC solution signicantly increased the absorption of
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octreotide vefold compared to a solution of octreotide only. Contrary, coadministration of 1% (w/v) chitosan hydrochloride did not enhance octreotide bioavailability. Moreover, the effect of various liquid formulations on the oral bioavailability of octreotide was studied in pigs.61 In this study, the efcacy of a 1.5% gel of chitosan HCl (pH 5.5.) as well as two solutions of TMC (5% and 10% at pH 7.4, respectively) were compared with a control solution of octreotide in buffer (pH 7.4). Formulations were administered intrajejunally. In presence of all evaluated polymers, absorption was enhanced in comparison to the control solution. Rank order was TMC 10% > TMC 5% > Chitosan 1.5%.61 Similar studies were performed with MCC and SNOCC focusing on the oral delivery of LMWH.33,34 In both studies, chitosan derivatives in a concentration of 3% were administered intraduodenally to rats. Both formulations were highly effective in improving the oral bioavailability of LMWH in comparison to the control formulation. Matrix Tablets In vivo studies using tablets based on thiolated chitosan have been performed using peptide drugs as well as efux pump substrates. Enteric coated mini matrix tablets based on chitosan-TBA containing the drug salmon calcitonin for the oral administration to rats were developed. In addition to chitosan-TBA, the tablets also contained two
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different chitosan-enzyme inhibitor conjugates, (chitosan-BBI conjugate and chitosan-elastatinal).62 At predetermined time-points after oral administration, the calcium plasma level was measured. In the control group which received an oral solution containing salmon calcitonin, no decrease of the calcium level could be observed. Moreover, the oral administration of tablets containing calcitonin and chitosan only did not decrease blood calcium levels. However, after oral administration of chitosan-TBA/chitosan-enzyme inhibitor conjugate based tablets containing salmon calcitonin, the plasma calcium level decreased for several hours for more than 5% of the initial value.63 In another study, a stomach targeted delivery system for salmon calcitonin was investigated. The tablets contained chitosan-TBA as well as chitosan-pepstatin. A pharmacological efcacy of 1.35% versus i.v.-injection was calculated.64 The results of this study are depicted in Figure 6. Furthermore, the efcacy of chitosan-TBA/GSH for oral peptide delivery has been studied using the peptide drug antide. The drug was administered i.v., s.c. and orally in solution as well as in chitosan-TBA/GSH tablets. Antide was not absorbed after oral administration of the solution, whereas the absolute and relative bioavailability of antide after oral administration of the chitosanTBA/GSH tablets was determined to be 1.1% and 3.2%, respectively.65 Besides peptides and proteins, also the oral bioavailability of efux pump substrates can be improved using matrix tablets based on thiolated chitosan. The oral bioavailability of the P-gp

substrate Rhodamine 123 (Rho-123) was investigated after administration of Rho-123 in buffer, Rho-123 tablets based on chitosan as well as Rho-123 tablets based on chitosan-TBA and GSH. An approximately twofold increase of the AUC was observed after oral administration of chitosan tablets, whereas a threefold increase of Rho-123 AUC was observed after administration of chitosan-TBA tablets.55 In another study, optimized tablets comprising of chitosan-TBA with lower molecular mass (75150 kDa instead of 400 kDa) even led to a 5.5-fold increase in Rho-123 AUC in comparison to the Rho-123 buffer solution. In vivo studies with chitosan-inhibitor conjugates only are rather sparse. As described above, Guggi et al. investigated the effect of various calcitonin containing minitablets on the blood calcium level of rats after oral administration. In comparison to tablets containing calcitonin and chitosan only, which showed no pharmacological effect, a minor reduction of the calcium level was observed after administration of tablets additionally containing chitosan-pepstatin conjugate64 (Fig. 6). In another study focusing on oral insulin delivery, a reduced blood glucose level for at least 8 h was observed after oral administration of tablets containing insulin and chitosan-aprotinin conjugate.66

Micro- and Nanoparticulate Oral Drug Delivery Systems Based on Modied Chitosans TMC based insulin loaded nanoparticles have been prepared by a two step procedure. In the rst step, an ion complex between the cationic TMC and the anionic insulin was formed. In a second step, crosslinking mediated by tripolyphosphate (TPP) was performed.67 Depending on the preparation conditions, nanoparticles displayed a size between around 150 and 250 nm, low polydispersity index (PI 0.10.4) and positive zeta potential. The properties of these nanoparticles were compared to the properties of insulin-TMC polyelectrolyte complexes (PEC) formed between the oppositely charged insulin and TMC. PECs displayed a size between around 100 and 200 nm, low PI and positive zeta potential. It turned out, that insulin-TMC polyelectrolyte complexes display not only higher colloidal stability than TMC nanoparticles in simulated intestinal uid, but were also more efcient in protecting insulin from trypsinic degradation.67 Another group studied the potential of TMC nanoparticles for oral
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Figure 6. Time-dependent calcemia after peroral administration of calcitonin containing tablets based on & chitosan/chitosan-pepstatin and & chitosanTBA/chitosan-pepstatin; each point represents the mean SD of at least three experiments; gure modied from Ref. 64.
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vaccine delivery. Intragastrical (i.g.) administration of TMC-nanoparticles containing the model vaccine urease led to higher IgG and IgA levels than the i.g. administration of urease solution with and without TMC.68 Recently, the efciency of TMC as vector in gene delivery was investigated using in vitro and in vivo techniques.69 Three different TMC based nanoparticles were used to encapsulate pDNA encoding green uorescent protein (GFP) using the complex coacervation technique. GFP expression in the mucosa of the stomach, duodenum, jejunum, ileum, and large intestine were found and the expression was most pronounced in the stomach and the upper intestinal part. Nanoparticles based on chitosan-TGA and pDNA for an intended use as oral delivery systems have been prepared. Particles in the size-range of 100200 nm and positive zeta potential were obtained.53 Moreover, acrylic nanoparticles have been successfully coated with chitosan-TBA. Also in this case, particle size was around 200 nm and the zeta potential of the particles was positive. The presence of free thiol groups on the particle surface could be determined by using the Ellmans reagent.70 To our knowledge no in vivo studies using thiolated chitosan nanoparticles for oral delivery have been published yet, however, data regarding oral insulin delivery using thiolated poly(acrylic acid) nanoparticles71 as well as data of studies focusing on intranasal gene delivery using chitosan-TGA nanoparticles are available.54 Intragastric administration of calcitonin containing liposomes coated with dodecylated chitosan to rats was more efcient in decreasing the blood calcium level than unmodied chitosan.37 Moreover, liposomes have been coated with two other modied chitosan derivatives, TMC and chitosan-EDTA. In vivo studies focusing on the oral absorption of insulin revealed that chitosanEDTA coated liposomes showed the most pronounced effect regarding a decrease in blood glucose.72 Microspheres based on chitosan-succinate have been investigated regarding their potential for the oral delivery of insulin.40 Average diameter of the microspheres was around 50 mm, and insulin loading capacity was around 60%. Only a small drug amount was released in articial gastric uid, whereas insulin was quickly released in articial intestinal uid of pH 7.4. Moreover, the delivery system was evaluated in vivo in diabetic rats. By using chitosan-succinate microspheres, the relative pharmacological efcacy was about
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fourfold improved in comparison to a chitosansuccinate solution.40 Comparable results were achieved using chitosan-phthalate microspheres.73 PEGylated chitosan was investigated regarding its potential for the oral delivery of salmon calcitonin. In detail, a nanoemulsion which was prepared by solvent displacement technique74 was used. This delivery system then was coated with two different PEGylated chitosans (0.5% and 1%). Whereas the control nanoemulsion (appr. size was 200 nm) displayed a negative zeta potential, the carrier systems coated with PEGylated chitosan (appr. size was 250 nm) displayed a positive zeta potential. In vivo studies in rats showed, that the oral uptake of salmon calcitonin when administered in carriers coated with PEGylated chitosan was higher than when administering the control nanoemulsion.42 The results of this study are shown in Figure 7. Various modied chitosans used in oral drug delivery, their properties as well as drugs used in vivo studies are summarized in Table 2.

DISCUSSION
Despite the benecial properties of chitosan for oral drug delivery, various derivatives of chitosan have been synthesized and found to exhibit improved features in comparison to unmodied chitosan. Besides the chitosan derivatives dis-

Figure 7. Time-dependent calcemia after intragastric administration of a calcitonin containing nanoemulsion & and * chitosan-PEG nanocapsules with a pegylation degree of 0.5%; each point represents the mean SD of at least six experiments; gure modied from Ref. 42.
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Table 2. Various Chitosan Derivatives Used in Oral Drug Delivery, Their Main Features and Therapeutic Drugs Used in Oral Absorption Studies Chitosan Derivative TMC Main Features and Comments Soluble at neutral and alkaline pH, reversible permeation enhancing effect,18 decreased mucoadhesion21 Improved mucoadhesion,29,30 cohesion,29 controlled release,29 permeation enhancing effect,25 efux pump inhibitory properties27,5557 Soluble at neutral and alkaline pH, permeation enhancing effect33 Soluble at neutral and alkaline pH, permeation enhancing effect34 Due to the hydrophobic chains, dodecylated chitosan interacts with lipophilic surfaces such as lipid bilayers37 Soluble at neutral and alkaline pH40,73 Reduced cytotoxicity42 Capable of inhibiting various enzymes, depending on the attached enzyme inhibitor (see Table 1)
18

Delivered Drug Buserelin,78 insulin,67,72 octreotide60,61 Antide,65 calcitonin,63,64 a insulin,77 a saquinavir56

Thiolated chitosan

MCC SNOCC Dodecylated chitosan

LMWH33 LMWH34 Calcitonin37

Chitosan succinate, chitosan phthalate PEGylated chitosan Chitosan-enzyme inhibitors

Insulin40,73 Calcitonin42 Insulin,47,77 a calcitonin63 a,64 a

Used in combination with other excipients.

cussed within the current manuscript, a number of other chitosan derivatives have been synthesized and might be interesting for oral drug delivery. Representatives are for example chitosan-cyclodextrin conjugates.75,76 Due to several factors, there is no one ts all solution and the chitosan derivative as well as the formulation itself must be chosen deliberately. Such a decision is in rst instance based on the drug itself. For example, if degradation by GI enzymes can be anticipated, specic chitosan-enzyme inhibitor conjugates might be useful whereas they might not be useful for the delivery of efux pump substrates. But even if the barriers for a certain drug are known, the choice of the appropriate derivative remains challenging. In cases were the physico-chemical features of the modied chitosan does not markedly differ from unmodied chitosan, a direct comparison of the features of chitosan and its derivatives is possible. This refers especially to modications, which do not inuence the solubility behavior and which can therefore be compared with unmodied chitosan under the same conditions. Such derivatives usually have a low degree of modication, which is highly effective. Representatives are thiomers
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and chitosan-inhibitor conjugates. Contrary, a direct comparison of derivatives with a different solubility prole than unmodied chitosan, such as, for example, TMC, MCC, SNOCC or chitosansuccinate/phthalate seems more challenging. For example, it was demonstrated that MCC precipitates at pH values below 6.0, whereas unmodied chitosan often precipitates above this pH range.33 Besides the changes in the solubility prole, also changes in the rheological properties have been reported. The viscosity of unmodied chitosan at pH 5.5 was signicantly higher than that of MCC33 measured at pH 7.4. Contrary, a correlation between an increase in the amount of thiol groups in thiomer chitosan-TBA and an increase in viscosity was reported.29 Factors such as viscosity can inuence the drug release behavior which might consequently lead to an altered pharmacological prole of an orally administered drug. This is only one example that should illustrate the complexity of a direct comparison between different modied chitosans. Another important factor when comparing different chitosan derivatives is of course the used drug formulation. As the discussed derivatives differ in their properties, one derivative might be superior
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to others when used in a certain formulation. When comparing in vivo data, the administration route peroral, intragastrically or intraduodenally as well as the administered dose and volume should be critically examined; especially in regard to a possible use in human. Another critical point is the design of control experiments. Also economic factors such as cost-benet calculations are important in later stages of drug development. In conclusion, various modied chitosans have been developed and proven to be effective for the oral delivery of peptides, proteins, vaccines and efux-pump substrates. Moreover, modied chitosans recently gained increasing attention regarding their use for oral gene delivery. Although a plethora of data concerning the properties of modied chitosans is available, the choice for a certain application remains challenging. The progress in the development of modied chitosans might lead to tailor-made delivery systems, making feasible the successful oral delivery of a broad variety of drugs.

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