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In this study four quaternized derivatives of chitosan: trimethyl chitosan (TMC), dimethylethyl chitosan (DMEC), diethylmethyl chitosan (DEMC) and triethyl chitosan (TEC) with degree of substitution of approximately 50+/-5% were synthesized and their effect on the permeability of insulin across intestinal Caco-2 monolayers was studied and compared with chitosan both in free-soluble form and in nanoparticulate systems. Transepithelial electrical resistance (TEER) studies revealed that all four chitosan derivatives in free-soluble forms were able to decrease the TEER value in the following order TMC>DMEC>DEMC=TEC>chitosan, indicating their abilities to open the tight junctions. Recovery studies on the TEER showed that the effect of the polymers on Caco-2 cell monolayer is reversible and proves the viability of cells after incubation with all polymers. A similar rank order was also observed when measuring the zeta-potentials of the various polymers in solution form. Transport studies of insulin together with the soluble polymers across Caco-2 cell layers showed the following ranking: TMC>DMEC>DEMC>TEC>chitosan which is in agreement with the strength of the cationic charge of the polymer. In comparison to the free-soluble polymers, the nanoparticles prepared by ionic gelation of the chitosan and its quaternized derivatives had much lower effect on decreasing the TEER by opening of the tight junctions. This can be explained by the reduced available amount of positive charge at the surface of the nanoparticles. In accordance with these results, the insulin loaded nanoparticles showed much less permeation across the Caco-2 cell monolayer in comparison to the free-soluble polymers. Mass balance transport studies revealed that a substantial amount of the nanoparticles has been entrapped into the Caco-2 monolayer or attached to the cell surface. It can thus be stated that while free-soluble polymers can reversibly open the tight junctions and increase the permeation of insulin, the nanoparticles had basically only a low effect on the opening of the tight junction and the paracellular transport of insulin across the Caco-2 cell monolayer. These data convincingly show that nanoparticles consisting of chitosan and its quaternary ammonium derivatives loaded with insulin are less effective in facilitating paracellular transport across Caco-2 cell monolayers than the corresponding free polymers.

TMC and DEMC, quaternized derivatives of chitosan, have been shown to have penetration enhancement properties and able to open the tight junctions of the intestinal epithelia at neutral and alkaline pH environments. The use of the nanoparticulate systems has the advantage of protecting the peptidic drugs from the harsh environment of the gastrointestinal tract. Hence, the aim of this study was to synthesize and characterize TMC and DEMC, both with quaternization degrees of 50+/-5%, which were then used to prepare insulin nanoparticles with two different methods: ionotropic gelation and the polyelectrolyte complexation (PEC) techniques. The obtained nanoparticles were then characterized for size, zeta potential, insulin loading and release as well as antibacterial activities. The results showed that nanoparticles prepared by the PEC method had higher insulin loading efficiency and zeta potential than those made by the ionotropic gelation method and may subsequently be used for further in vitro, ex vivo and in vivo studies. Moreover, the antibacterial studies suggest that the polymers in free form have higher antibacterial activity against Gram-positive bacteria than in the nanoparticulate form.

The absorption enhancing effects of chitosan and its derivatives have been intensively studied in recent years. It has been shown that these compounds are potent absorption enhancers. Chitosan is only soluble in acidic environments and is therefore incapable of enhancing absorption in the small intestine, the main absorption area in the gastrointestinal tract. Special emphasis has been placed on the absorption enhancing properties of N-trimethyl chitosan chloride (TMC), a partially quaternised derivative of chitosan, due to its solubility in neutral and basic environments. TMC is prepared by the reductive methylation of chitosan. The degree of quaternisation can be altered by increasing the number of reaction steps or by increasing the reaction time. Although the molecular weight of the polymer increases with addition of the methyl groups, a net decrease in the molecular weight is observed due to a decrease in the chain length of the polymer. TMC, like chitosan, possesses mucoadhesive properties. In vitro studies performed on Caco-2 cell monolayers showed a pronounced reduction in the transepithelial electrical resistance (TEER). TMC is also able to increase the permeation of hydrophilic compounds such as [14C]-mannitol and [14C] polyethylene glycol 4000 ([14C] PEG 4000, MW4000) across the cell monolayers. It was also shown that the degree of quaternisation of the polymer plays an important role on its absorption enhancing properties, especially in neutral environments where chitosan is ineffective as an absorption enhancer. The reduction in TEER is an indication of the opening of the tight junctions located between epithelial cells. Opening of the tight junctions will result in enhancement of absorption via the paracellular route. Confocal laser scanning microscopy confirmed transport of large hydrophilic compounds via the paracellular route as well as the mechanism of action of the polymer in which redistribution of the cytoskeletal F-actin is provoked, which leads to the opening of the tight junctions. Various in vivo studies in different animal models confirmed the ability of TMC to increase the absorption of the peptide drugs buserelin and octreotide after intraduodenal or -jejunal administration. However, TMC has always been administered as a solution in these studies. The impracticality of administering a solution, as well as the fact that most peptides are unstable in the presence of water, have led to the need for a solid oral dosage form with which TMC can be administered together with peptide drugs. Recent studies have focused on the development and in vivo evaluation of solid oral dosage forms.

N-Trimethyl chitosan chloride (TMC) is a soluble chitosan derivative that shows effective enhancing properties for peptide and protein drug transport across mucosal membranes. TMC was synthesized by reductive methylation of chitosan in an alkaline environment at elevated temperature. The number of methylation process steps and the base used in the process was demonstrated to affect the degree of quaternization of the primary amino group and methylation of 3- and 6-hydroxyl groups. 1H-Nuclear magnetic resonance spectra showed that the degree of quaternization of TMC was higher when using sodium hydroxide as the base compared to using dimethyl amino pyridine. The degrees of quaternization as well as O-methylation of TMC increased with the number of reaction steps. O-Methylation resulted in decreased solubility of TMC. The high degree of quaternization of TMC with a low degree of O-methylation was prepared by employing one reaction step with two subsequent addition steps and a controlled alkaline environment of the mixture reaction.

In this study, minitablet and granule formulations were developed as solid oral dosage forms for the delivery of peptide drugs with the absorption enhancer N-trimethyl chitosan chloride (TMC). Minitablets were deemed suitable as a dosage form due to their ability, as components of multiple unit dosage forms (MUDFs), to disperse from each other, before disintegration, effectively increasing the area in which the polymer can assert its absorption-enhancing effect. The polymer should be released from the dosage forms prior to the release of the peptide, which was, together with achieving maximum release of both ingredients, the main focus of this study. Desmopressin (1-(3-mercaptopropionic acid)-8-D-arginine vasopressin monoacetate (DDAVP) was used as model peptide drug. The optimized minitablet formulation consisted of two types of granules, namely DDAVP and TMC granules. DDAVP granules, containing tetraglycerol pentastearate (TGPS), were specifically aimed at delaying the release of the peptide from the dosage form. Burst release of TMC was attempted with TMC granules. Both these granule types were included in the granule formulation. Release profiles for both the optimized minitablet formulation as well as the granule formulation showed that the release of DDAVP was effectively delayed from the formulation compared to the formulation where no attempt at delaying the release was made. In comparison, more TMC was released, and at a faster rate, from the granule formulation than the optimized minitablet formulations. Both the optimized minitablet formulation and the granule formulation show suitable release profiles for the delivery of peptide drugs with TMC as absorption enhancer in solid oral dosage forms.

Purpose: To investigate the enhancing effect of trimethyl chitosan chloride (TMC) on the enteral absorption of octreotide and to delineate the required doses of both TMC and peptide in vivo in juvenile pigs., Methods: Six female pigs (body weight, 25 kg) were operated to induce a stoma at the beginning of their jejunum and to insert an in-dwelling fistula for intrajejunal (IJ) administration of the formulations. A silicone cannula was inserted at the jugular vein for blood sampling. One week after surgery the pigs received IJ octreotide solution administrations with or without TMC at pH 7.4 or chitosan HCl at pH 5.5. For determining bioavailability (F) values, the pigs also received an octreotide solution intravenously (IV). Blood samples were taken from the cannulated jugular vein and subsequently analyzed by radioimmunoassay., Results: Intrajejunal administration of 10 mg octreotide without any polymer (control solution) resulted in F values of 1.7 +/- 1.1% (mean +/- SE). Chitosan HCl 1.5% (w/v) at pH 5.5 led to a 3-fold increase in F compared to the control (non-polymer containing) formulations. Co-administration of octreotide with 5 and 10% (w/v) TMC at pH 7.4 resulted in 7.7- and 14.5-fold increase of octreotide absorption, respectively (F of 13.9 +/- 1.3% and 24.8 +/- 1.8%). IJ administration of 5 mg octreotide solutions resulted in low F values of 0.5 +/- 0.6%, whereas co-administration with 5% (w/v) TMC increased the intestinal octreotide bioavailability to 8.2 +/- 1.5%., Conclusions: Cationic polymers of the chitosan type are able to enhance the intestinal absorption of the peptide drug octreotide in pigs. In this respect, TMC at neutral pH values of 7.4 appears to be more potent than chitosan HCl at a weak acidic pH of 5.5.

The synthesis and evaluation of mono-N-carboxymethyl chitosan (MCC) as an intestinal permeation enhancer for macromolecular therapeutics is presented. MCCs were synthesized from two different viscosity grade chitosans to yield both high and low viscosity grade products. These MCCs were tested on Caco-2 cells for their efficiency to decrease the transepithelial electrical resistance (TEER) and to increase the paracellular permeability of the anionic macromolecular anticoagulant low molecular weight heparin (LMWH). For in vivo studies, LMWH was administered intraduodenally with or without MCC to rats. Both types of experiments were performed at pH 7.4. Results show that both viscosity grade MCCs managed to significantly decrease the TEER of Caco-2 cell monolayers when they were applied apically at concentrations of 3-5% (w/v). Transport studies with Caco-2 cells revealed substantial increases of LMWH permeation in the presence of both viscosity grade MCCs compared with controls. In rats, 3% (w/v) low viscosity MCC significantly increased the intestinal absorption of LMWH, reaching the therapeutic anticoagulant blood levels of LMWH. Both in vitro and in vivo results indicate that the polyampholytic chitosan modification MCC is a suitable and functional polymer for the delivery and intestinal absorption of anionic macromolecular therapeutics like LMWH., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90: 38-46, 2001)

Octreotide acetate is a somatostatin analogue used for the control of endocrine tumors of the gastrointestinal (GI) tract and the treatment of acromegaly. The oral absorption of octreotide is limited because of the limited permeation across the intestinal epithelium. Both chitosan hydrochloride and N-trimethyl chitosan chloride (TMC), a quaternized chitosan derivative, are nonabsorbable and nontoxic polymers that have been proven to effectively increase the permeation of hydrophilic macromolecules across mucosal epithelia by opening the tight junctions. This study investigates the intestinal absorption of octreotide when it is coadministered with the polycationic absorption enhancer TMC. Caco-2 cell monolayers were used as an in vitro intestinal epithelium model, and male Wistar rats were used for in vivo studies. Octreotide with or without polymers (TMC; chitosan hydrochloride) was administered intrajejunally in rats, and serum peptide levels were measured by radioimmunoassay. All applications and administrations were performed at neutral pH values (i.e., pH = 7.4). In vitro transport studies with Caco-2 cells revealed an increased permeation of octreotide in the presence of TMC. Enhancement ratios ranged from 34 to 121 with increasing concentrations of the polymer (0.25-1.5%, w/v). In rats, 1.0% (w/v) TMC solution significantly increased the absorption of the peptide analogue, resulting in a 5-fold increase of octreotide bioavailability compared with the controls (octreotide alone). Coadministration of 1.0% (w/v) chitosan hydrochloride did not enhance octreotide bioavailability. These results in combination with the nontoxic character of TMC suggest that this polymer is a promising excipient in the development of solid dosage forms for the peroral delivery and intestinal absorption of octreotide.

N-Trimethyl chitosan chloride (TMC) is a permanently quaternized chitosan derivative with improved aqueous solubility compared to native chitosan. TMC is able to open the tight junctions of intestinal epithelia at physiological pH values, where chitosan is insoluble and therefore ineffective. TMCs with degrees of substitution of 40 and 60% were synthesized according to a novel synthesis procedure and their effect on the permeability of the tight junctions of the intestinal Caco-2 monolayers was studied, measuring the transepithelial electrical resistance and the transport of a mainly paracellularly transported compound, [14C]-mannitol. Toxicity studies using nucleic stains were done to establish the transport as a cause of opening of the tight junctions and not of possible cytotoxicity. TMC60 showed higher transport enhancement ratios than TMC40 in all concentrations tested (0.05-1. 0%, w/v). Both derivatives did not affect the viability of the Caco-2 cell monolayers. These results suggest that high charge density is necessary for TMC to substantially improve the paracellular permeability of intestinal epithelia. It is expected that TMC40 and TMC60 will enhance the intestinal permeation of hydrophilic macromolecular drugs such as peptides and proteins.

Purpose: To evaluate N-trimethyl chitosan chloride (TMC) of high degrees of substitution as intestinal permeation enhancers for the peptide drug buserelin in vitro using Caco-2 cell monolayers, and to investigate TMCs as enhancers of the intestinal absorption of buserelin in vivo, in rats., Methods: TMCs were tested on Caco-2 cells for their efficiency to increase the paracellular permeability of the peptide buserelin. For the in vivo studies male Wistar rats were used and buserelin was administered with or without the polymers intraduodenally. Both types of experiments were performed at pH 7.2., Results: Transport studies with Caco-2 cell monolayers confirmed that the increase in buserelin permeation is dependent on the degree of trimethylation of TMC. In agreement with the in vitro results, in vivo data revealed highly increased bioavailability of buserelin following intraduodenal co-administration with 1.0% (w/v) TMCs. Intraduodenally applied buserelin resulted in 0.8% absolute bioavailability, whereas co-administrations with TMCs resulted in mean bioavailability values between 6 and 13 %. Chitosan HCl (1.0%; pH = 7.2) did not significantly increase the intestinal absorption of buserelin., Conclusions: Both the in vitro and in vivo results indicate that TMCs are potent mucosal permeation enhancers of the peptide drug buserelin at neutral pH values.

N-trimethyl chitosan (TMC) polymers are quaternized chitosans in different degrees of trimethylation. These polymers enhance the absorption of macromolecules through mucosal epithelia by triggering the reversible opening of tight junctions and only allow for paracellular transport. To investigate the safety of these novel absorption enhancers cytotoxicity and ciliotoxicity studies have been performed. Intestinal Caco-2 cell monolayers were chosen to study possible membrane damaging effects of these polymers, using confocal laser scanning microscopy visualization of nuclear staining by a membrane impermeable fluorescent probe during transport of the paracellular marker Texas red dextran (MW 10 000). Ciliated chicken embryo trachea tissue was used to study the effect of the polymers on the ciliary beat frequency (CBF) in vitro. In both studies the TMC polymers of different degrees of substitution (20, 40 and 60%) were tested at a concentration of 1.0% (w/v). No substantial cell membrane damage could be detected on the Caco-2 cells treated with TMCs, while the effect on the CBF in vitro was found to be marginal. TMC60 and TMC40 enhance paracellular transport of Texas red dextran in Caco-2 cell monolayers, whereas TMC20 is ineffective. In conclusion, TMCs of high degrees of substitution may be effective and safe absorption enhancers for peptide and protein drug delivery.

N-trimethyl chitosan chloride (TMC), a partially quaternized derivative of chitosan with superior water solubility, was synthesized with different degrees of quaternization [12.6% quaternized (TMC-L) and 19.9% quaternized (TMC-H)] and the effects of these novel polymers on the permeability of intestinal epithelial cells were investigated in Caco-2 cell monolayers. Transepithelial electrical resistance (TEER) measurements showed that both polymers in 1.5-2.5% w/v concentrations caused a pronounced, concentration dependent lowering in TEER values, but that TMC-H was more effective than TMC-L at similar concentrations (36 +/- 3% reduction with TMC-L and 53 +/- 6% reduction with TMC-H at 2.0% concentrations). Similar results were obtained in transport studies with the hydrophilic radioactive markers [14C]mannitol (MW 182.2) and [14C]polyethylene glycol 4000 ([14C]PEG 4000, MW 4000). The transport of [14C]mannitol was increased 51-fold (TMC-L) and 97-fold (TMC-H) at 2.5% concentrations. No deleterious effects to the cells could be demonstrated with trypan blue exclusion studies. The results show that TMC is able to open the tight junctions of intestinal epithelial cells to allow for paracellular transport of hydrophilic molecules. It is concluded that the charge density of TMC, as determined by the degree of quaternization, is an important factor determining its potential use as an absorption enhancer across intestinal epithelia.

Previous studies have established that a partially quaternized derivative of chitosan, N-trimethyl chitosan chloride (TMC), can be used as an absorption enhancer for large hydrophilic compounds across mucosal surfaces. This study evaluates and compares the effects of the degree of quaternization of TMC, in a neutral environment, on the permeability of intestinal epithelial cells in vitro, where normal chitosan salts are ineffective as absorption enhancers. The effects of TMC-H [61.2% quaternized, (0.05-1.5% w/v)], TMC-L [12.3% quaternized, (0.5-1.5% w/v)], and chitosan hydrochloride [0.5-1.5% w/v] on the transepithelial electrical resistance (TEER) and permeability, for the hydrophilic model compound [14C]mannitol, of intestinal epithelial Caco-2 cell monolayers, were investigated at pH values of 6.20 and 7.40. The viability of the monolayers was checked with the trypan blue exclusion technique. At a pH of 6.20, all the polymers caused a pronounced reduction (37-67% at 0.5% w/v concentrations) in the TEER of Caco-2 cells. On the contrary, at a pH of 7.40, only TMC-H was able to decrease the TEER values, even in a concentration as low as 0.05% w/v (35% reduction). Comparable results were obtained with the permeation of [14C]mannitol. Large increases in the transport rate (18-23-fold at 0.5% w/v concentrations) were found at pH 6.20, whereas only TMC-H was able to increase the permeation of [14C]mannitol at pH 7.40 (31-48-fold at 0.05-1.5% w/v concentrations of TMC-H). For all the polymers studied, no deleterious effects to the cells could be demonstrated with the trypan blue exclusion technique. It is concluded that highly quaternized TMC is a potent absorption enhancer and the potential use of this polymer, especially in neutral and basic environments where normal chitosan salts are not effective, is expected to be an important contribution to the development of effective delivery systems for hydrophilic compounds such as peptide drugs.

Chitosan, a biodegradable and biocompatible polymer, has attracted great attention in the pharmaceutical and biomedical fields especially due to its properties to reversibly open the tight junctions of the epithelial tissues and to allow for paracellular transport of hydrophilic macromolecules. However, chitosan exhibits low solubility at pH values above 6 that may prevent its enhancing effects at the sites of intestinal absorption. Hence, a number of alkylated chitosan salts have been synthesized and characterized. These derivatives have been shown to have good solubility at neutral pH and act as effective permeation enhancers. In this study, two new derivatives of chitosan, C2-C6 trimethyl 6-amino-6-deoxy chitosan and C2-C6 triethyl 6-amino-6-deoxy chitosan were synthesized and characterized using 1H-NMR and FTIR spectra. Moreover, the zeta potential and the antibacterial properties of these polymers were compared to chitosan, trimethyl chitosan (TMC) and triethyl chitosan (TEC). Our results suggest that both C2-C6 trimethyl and triethyl 6-amino- 6-deoxy chitosan, as highly water soluble polymers, have higher positive surface charge than chitosan, TMC, and TEC. Moreover, the new synthesized polymers show higher antibacterial activity against Gram-positive Staphylococcus aureus bacteria. Consequently, these polymers with substitution degrees of 50-60% may be good candidates for the enhancement of peptides in mucosal drug delivery. [ABSTRACT FROM AUTHOR]

N,N-dimethyl N-ethyl chitosan (DMEC), a quanternized derivative of chitosan was synthesized based on a modified two-stop method via a 2² factorial design to optimize the preparative conditions. The degree of deacetylation of the starting chitosan was determined by FTIR and NMR methods and was 95%. In the first step of the synthesis, mono-ethyl chitosan was prepared by introducing an ethyl group onto the amine group of chitosan via a Schiff base and in the next step methyl iodide was added to produce DMEC which was water soluble in a pH range of 4–8. The DMEC polymers with different degrees of quaternization were obtained and fully characterized using FTIR and ¹H-NMR spectroscopic methods. Based on ¹H-NMR calculations, the degree of quaternization was 52% by optimizing the two-step process. [ABSTRACT FROM AUTHOR]

Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo , could be tested in vitro . The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14 C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres ® ) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres ® (size 0.2 μm) and chitosan microparticles (size 1.7 μm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro , the targeting of these cells can be studied more efficiently and can now be done in cells from human origin. [ABSTRACT FROM AUTHOR]