J.C. Verhoef

Oral drug absorption enhancement by chitosan and its derivatives.

Chitosan is a non-toxic, biocompatible polymer that has found a number of applications in drug delivery including that of absorption enhancer of hydrophilic macromolecular drugs. Chitosan, when protonated (pH<6.5), is able to increase the paracellular permeability of peptide drugs across mucosal epithelia. Chitosan derivatives have been evaluated to overcome chitosans limited solubility and effectiveness as absorption enhancer at neutral pH values such as those found in the intestinal tract. Trimethyl chitosan chloride (TMC) has been synthesized at different degrees of quaternization. This quaternized polymer forms complexes with anionic macromolecules and gels or solutions with cationic or neutral compounds in aqueous environments and neutral pH values. TMC has been shown to considerably increase the permeation and/or absorption of neutral and cationic peptide analogs across intestinal epithelia. The mechanism by which TMC enhances intestinal permeability is similar to that of protonated chitosan. It reversibly interacts with components of the tight junctions, leading to widening of the paracellular routes. Mono-carboxymethylated chitosan (MCC) is a polyampholytic polymer, able to form visco-elastic gels in aqueous environments or with anionic macromolecules at neutral pH values. MCC appears to be less potent compared to the quaternized derivative. Nevertheless, MCC was found to increase the permeation and absorption of low molecular weight heparin (LMWH; an anionic polysaccharide) across intestinal epithelia. Neither chitosan derivative provokes damage of the cell membrane, and therefore they do not alter the viability of intestinal epithelial cells.

Preparation and NMR characterization of highly substitutedN-trimethyl chitosan chloride

N,N,N-Trimethyl chitosan chloride (TMC) is a chemically modified chitosan with improved aqueous solubility, compared with the native chitosan. It is essential to follow a synthesis procedure in which the degree of substitution of the final product can be controlled by means of the number of reaction steps, the duration of each reaction step and the amount of methyl iodide as reagent. A two-step reaction yields products with high degrees of substitution (40–80%). Comparison of the NMR spectra of the product TMC, after a two-step reaction, indicates that there is a peak assigned to the substituted amino group that shifts from 2.5 to 3.1 ppm upon acidification. This peak must be assigned to N(CH3)2 and not to N(CH3)+3. A three-step reaction procedure yields products with a degree of substitution > 80%, but with substantially decreased water-solubility.

Chitosan for mucosal vaccination

The striking advantage of mucosal vaccination is the production of local antibodies at the sites where pathogens enter the body. Because vaccines alone are not sufficiently taken up after mucosal administration, they need to be co-administered with penetration enhancers, adjuvants or encapsulated in particles. Chitosan easily forms microparticles and nanoparticles which encapsulate large amounts of antigens such as ovalbumin, diphtheria toxoid or tetanus toxoid. It has been shown that ovalbumin loaded chitosan microparticles are taken up by the Peyers patches of the gut associated lymphoid tissue (GALT). This unique uptake demonstrates that chitosan particulate drug carrier systems are promising candidates for oral vaccination. Additionally, after co-administering chitosan with antigens in nasal vaccination studies, a strong enhancement of both mucosal and systemic immune responses is observed. This makes chitosan very suitable for nasal vaccine delivery. In conclusion, chitosan particles, powders and solutions are promising candidates for mucosal vaccine delivery. Mucosal vaccination not only reduces costs and increases patient compliance, but also complicates the invasion of pathogens through mucosal sites.

Chitosan and its derivatives as intestinal absorption enhancers

Chitosan is a non-toxic, biocompatible polymer that has found a number of applications in drug delivery including that of absorption enhancer of hydrophilic macromolecular drugs. Chitosan, when protonated (pH<6.5), is able to increase the paracellular permeability of peptide drugs across mucosal epithelia. Chitosan derivatives have been evaluated to overcome chitosans limited solubility and effectiveness as absorption enhancer at neutral pH values such as those found in the intestinal tract. Trimethyl chitosan chloride (TMC) has been synthesized at different degrees of quaternization. This quaternized polymer forms complexes with anionic macromolecules and gels or solutions with cationic or neutral compounds in aqueous environments and neutral pH values. TMC has been shown to considerably increase the permeation of neutral and cationic peptide analogs across Caco-2 intestinal epithelia. The mechanism by which TMC is enhancing the intestinal permeability is similar to that of protonated chitosan. It reversibly interacts with components of the tight junctions, leading to widening of the paracellular routes. This chitosan derivative does not provoke damage of the cell membrane, and does not alter the viability of intestinal epithelial cells. Co-administrations of TMC with peptide drugs were found to substantially increase the bioavailability of the peptide in both rats and juvenile pigs compared with administrations without the polymer.

Chitosan microparticles for oral vaccination : preparation, characterization and preliminary in vivo uptake studies in murine Peyer's patches

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine in the gut and low uptake in the lymphoid tissue of the gastrointestinal tract still complicate the development of oral vaccines. In this study chitosan microparticles were prepared and characterized with respect to size, zeta potential, morphology and ovalbumin-loading and -release. Furthermore, the in vivo uptake of chitosan microparticles by murine Peyers patches was studied using confocal laser scanning microscopy (CLSM). Chitosan microparticles were made according to a precipitation/coacervation method, which was found to be reproducible for different batches of chitosan. The chitosan microparticles were 4.3+/-0.7 microm in size and positively charged (20+/-1 mV). Since only microparticles smaller than 10 microm can be taken up by M-cells of Peyers patches, these microparticles are suitable to serve as vaccination systems. CLSM visualization studies showed that the model antigen ovalbumin was entrapped within the chitosan microparticles and not only associated to their outer surface. These results were verified using field emission scanning electron microscopy, which demonstrated the porous structure of the chitosan microparticles, thus facilitating the entrapment of ovalbumin in the microparticles. Loading studies of the chitosan microparticles with the model compound ovalbumin resulted in loading capacities of about 40%. Subsequent release studies showed only a very low release of ovalbumin within 4 h and most of the ovalbumin (about 90%) remained entrapped in the microparticles. Because the prepared chitosan microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyers patches. Initial in vivo studies demonstrated that fluorescently labeled chitosan microparticles can be taken up by the epithelium of the murine Peyers patches. Since uptake by Peyers patches is an essential step in oral vaccination, these results show that the presently developed porous chitosan microparticles are a very promising vaccine delivery system.

Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal caco-2 cell monolayers.

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.

Mucoadhesive polymers in peroral peptide drug delivery. IV. Polycarbophil and chitosan are potent enhancers of peptide transport across intestinal mucosae in vitro

The purpose of the study was to evaluate the inhibitory effect of the mucoadhesive polymers polycarbophil, chitosan and chitosan glutamate on trypsin and carboxypeptidase B (CPB) activity as well as their potential to improve the intestinal transport of the peptide drug 9-desglycinamide, 8-l-arginine vasopressin (DGAVP) in vitro. The degradation of the model substrates N-α-benzoyl-l-arginine ethylester by trypsin and hippuryl-l-arginine by CPB in the presence of the polymers was studied. Furthermore, the effect of the polymers on intestinal DGAVP transport was investigated using Caco-2 cell monolayers and the rat vertically perfused intestinal loop model. Uniquely, polycarbophil in a concentration of 1% (w/v) was able to inhibit both trypsin and CPB activities. Chitosan glutamate in concentrations of 0.4 and 1% (w/v) strongly increased the transport of DGAVP across Caco-2 cell monolayers, whereas 1% (w/v) polycarbophil showed only low transport enhancement. All polymers in concentrations of 1% (w/v), however, showed a pronounced and comparable improvement of DGAVP transport across intestinal mucosae in the vertically perfused loop model. It is concluded that the chitosans enhance the transport of DGAVP solely by increasing the paracellular permeability due to opening of intercellular junctions. The observed comparable transport effect of polycarbophil in the intestinal loop model is mainly ascribed to protection of DGAVP against proteolytic degradation in the intestinal lumen, which allows for sufficient concentration and thus transport of the peptide drug when polycarbophil induced paracellular transport is less enhanced.

Mono‐N‐carboxymethyl chitosan (MCC), a polyampholytic chitosan derivative, enhances the intestinal absorption of low molecular weight heparin across intestinal epithelia in vitro and in vivo

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.

Bioadhesive polymers for the peroral delivery of peptide drugs

Abstract Two different classes of bioadhesive excipients which have been approved by the FDA, the anionic charged poly (acrylic acid) derivatives and the cationic charged chitosans, have been investigated with respect to their ability to improve intestinal peptide drug absorption. It was found that both polycarbophil and the chitosan derivatives Daichitosan ® VH and chitosan-glutamate (SeaCure ® + 210) enhance the absorption of the peptide drug 9-desglycinamide, 8-arginine vasopressin (DGAVP) in the vertically perfused intestinal loop model of the rat. Recent studies demonstrated that the two poly (acrylates) polycarbophil and Carbopol ® 934P are able to inhibit the activity of the proteolytic enzyme trypsin at pH 6.7, which may lead to an increased stability of the peptide drug in the intestine. The depletion of Ca 2+ out of the incubation medium due to the Ca 2+ binding properties of the poly (acrylates) is discussed as a possible mechanism of action. Because of the observation that depletion of Ca 2+ can additionally cause an opening of tight junctions, the influence of polycarbophil on the paracellular integrity of Caco-2 monolayers was also investigated by measurements of transepithelial electrical resistance (TEER) as well as by visualization studies using confocal laser scanning microscopy. At pH 4.0, apically applied polycarbophil tended to decrease TEER values stronger than the control solution, whereas at pH 7.0 no pronounced changes of TEER could be observed. At pH 7.4, polycarbophil was only able to increase the paracellular permeability of the hydrophilic model compound fluorescein-isothiocyanate-dextran ( M w 4000) when applied to the basolateral side of the Caco2 cell monolayer. In conclusion, bioadhesive polymers are promising absorption promoting agents for peroral delivery of peptide drugs, and their mechanism of action is probably a combination of inhibiting protease activities and modulating the intestinal epithelial permeability.

N-Trimethylated Chitosan Chloride (TMC) Improves the Intestinal Permeation of the Peptide Drug Buserelin In Vitro (Caco-2 Cells) and In Vivo (Rats)

AbstractPurpose. To evaluate N-trimethyl chitosan chloride (TMC) of highdegrees of substitution as intestinal permeation enhancers for thepeptide drug buserelin in vitro using Caco-2 cell monolayers, and toinvestigate TMCs as enhancers of the intestinal absorption of buserelinin vivo, in rats. Methods. TMCs were tested on Caco-2 cells for their efficiency toincrease the paracellular permeability of the peptide buserelin. For thein vivo studies male Wistar rats were used and buserelin wasadministered with or without the polymers intraduodenally. Both types ofexperiments were performed at pH 7.2. Results. Transport studies with Caco-2 cell monolayers confirmed thatthe increase in buserelin permeation is dependent on the degree oftrimethylation of TMC. In agreement with the in vitro results, in vivodata revealed highly increased bioavailability of buserelin followingintraduodenal 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 bioavailabilityvalues between 6 and 13 %. Chitosan HCl (1.0% pH = 7.2) did notsignificantly increase the intestinal absorption of buserelin. Conclusions. Both the in vitro and in vivo results indicate that TMCsare potent mucosal permeation enhancers of the peptide drug buserelinat neutral pH values.