A.F Kotzé

Comparison of the effect of different chitosan salts and N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2)

A partially quaternized chitosan derivative, N-trimethyl chitosan chloride (TMC) (degree of quaternization 12.28%), was synthesized and the effects of this novel polymer on the permeability of intestinal epithelial cells, using Caco-2 cell monolayers, were investigated and compared with those of chitosan hydrochloride and chitosan glutamate. Transepithelial electrical resistance (TEER) measurements at pH 6.20 revealed that all these polymers (0.25-1.5% w/v) caused an immediate and pronounced lowering in TEER values in the order chitosan hydrochloride (84% reduction after 2 h incubation) > chitosan glutamate (60% reduction) > TMC (24% reduction) at 0.25% w/v concentrations. At higher concentrations (up to 2.5% w/v), TMC was able to decrease the TEER further. Similar results were obtained in transport studies, using the hydrophilic radioactive markers, [14C]-mannitol (MW 182.2) and [14C]-polyethylene glycol 4000 (PEG-4000, MW 4000). Large increases in the permeation of these markers were found. The transport of [14C]-mannitol was increased 34-fold (chitosan hydrochloride), 25-fold (chitosan glutamate) and 11-fold (TMC) at 0.25% w/v concentrations. Further increases in the permeation of both markers were found at higher concentrations of TMC. Due to its quaternary structure, TMC is better soluble than the other chitosan salts, and its higher solubility may compensate for its lesser effectivity at similar concentrations. It is also soluble at pH 7.40, where the chitosan salts are insoluble and therefore ineffective. No deleterious effects to the cells could be demonstrated with trypan blue exclusion studies and confocal laser scanning microscopy (CLSM). CLSM confirmed that these polymers increase the transport of large hydrophilic compounds (using the fluorescent markers FD-4, MW 4400 and FD-20, MW 19,600) through opening of tight junctions to allow for paracellular transport. It is concluded from this study that the charge, charge density and the structural features of chitosans and chitosan derivatives are important factors determining their potential use as absorption enhancers.

Chitosans for enhanced delivery of therapeutic peptides across intestinal epithelia: in vitro evaluation in Caco-2 cell monolayers

The aim of the study was to evaluate the transport enhancing effects of two chitosan salts, chitosan hydrochloride and chitosan glutamate (1.5% w/v), and the partially quaternized chitosan derivative, N-trimethyl chitosan chloride (TMC) (1.5 and 2.5% w/v), in vitro in Caco-2 cell monolayers. The transport of the peptide drugs buserelin, 9-desglycinamide, 8-arginine vasopressin (DGAVP) and insulin was followed for 4 h at pH values between 4.40 and 6.20. All the chitosans (1.5%) were able to increase the transport of the peptide drugs significantly in the following order: chitosan hydrochloride>chitosan glutamate>TMC. Due to its quaternary structure, TMC is better soluble than the chitosan salts and further increases in peptide transport were found at higher concentrations (2.5%) of this polymer. The better solubility of TMC may compensate for its lower efficacy at similar concentrations. The increases in peptide drug transport are in agreement with a lowering of the transepithelial electrical resistance (TEER) measured in the cell monolayers. No deleterious effect to the cell monolayers could be detected with the trypan blue exclusion technique. The enzyme inhibitory effect of chitosan hydrochloride (1.5%) was compared with carbomer (1.5%) [Carbopol® 934P] in transport studies with buserelin in the presence of the endoprotease, α-chymotrypsin. In the presence of α-chymotrypsin the transport of buserelin was decreased markedly (from 4.3 to 1.3% of the total dose applied) with chitosan hydrochloride (1.5%), in contrast with carbomer (1.5%) where the transport remained constant (1.4% of the total dose applied). Also the chitosan derivative TMC was not able to inhibit α-chymotrypsin. It is concluded from this study that chitosans are potent absorption enhancers, and that the charge, charge density and the structural futures of chitosan salts and N-trimethyl chitosan chloride are important factors determining their potential use as absorption enhancers for peptide drugs, but that they are unable to prevent degradation from proteolytic enzymes. Structural modification of the chitosan molecule may compensate for this shortcoming.

Effect of the degree of quaternisation of N-trimethyl chitosan chloride on absorption enhancement: in vivo evaluation in rat nasal epithelia

Five TMC polymers with different degrees of quaternisation (12-59%) were synthesised and administered together with [14C]-mannitol in the nasal route of rats at a pH of 6.20 and 7.40, respectively. All the TMC polymers increased the nasal absorption of [14C]-mannitol significantly at pH 6.20, but only TMC polymers with higher degrees of quaternisation (>36%) were able to increase the absorption of this hydrophilic model compound at pH 7.40. The absorption of [14C]-mannitol at pH 7.40 increased with an increase in the degree of quaternisation of TMC until a maximum absorption value was reached with TMC with a degree of quaternisation of 48%. The absorption of [14C]-mannitol did not increase further, even when TMC with a higher degree of quaternisation (59%) was used. This can probably be explained by steric effects caused by the attached methyl groups and changes in the flexibility of the TMC molecules with an increase in the degree of quaternisation above an optimum value for absorption enhancement in a neutral environment. It was concluded that the degree of quaternisation of TMC plays an important role in the absorption enhancement properties of this polymer across nasal epithelia in a neutral environment.

The relationship between the absolute molecular weight and the degree of quaternisation of N-trimethyl chitosan chloride

N-Trimethyl chitosan chloride (TMC) is a partially quaternised derivative of chitosan with enhanced water solubility and superior drug absorption enhancing properties, especially in neutral and basic environments where chitosan is insoluble. The degree of quaternisation of TMC plays an important role on the solubility, the absorption enhancing ability and mucoadhesive properties of this polymer. A range of TMC polymers with different degrees of quaternisation were synthesised by varying the number and duration of reaction steps. Size exclusion chromatography (SEC) and multi-angle laser light scattering (MALLS) were used to determine the absolute molecular weight, radius and polydispersity of these TMC polymers. The viscosities of TMC polymer solutions were measured at different concentrations and the calculated intrinsic viscosity values were used as a further indication of the molecular weight of each polymer. The decrease in absolute molecular weight correlated well with the intrinsic viscosities of the TMC polymers and was related to the increase in their degrees of quaternisation. This polymer degradation was explained by increased exposure time of the polymer molecules to reaction conditions, such as the strong alkaline environment and elevated temperatures, that is necessary to produce higher degrees of quaternisation during the synthesis of TMC polymers.

Evaluation of the mucoadhesive properties of N-trimethyl chitosan chloride.

Abstract Previous studies have established that N-trimethyl chitosan chloride (TMC) is a potent absorption enhancer for peptides and large hydrophilic compounds across mucosal surfaces, especially in neutral and basic environments where chitosan is ineffective as an absorption enhancer. The degree of quaternization of TMC plays an important role on its absorption-enhancing properties. Several TMC polymers with different degrees of quaternization were synthesized and the molecular mass of the polymers was determined by SEC/MALLS. The mucoadhesive properties of the TMC polymers were measured with a modified tensiometer based on the Willhelmy plate method. The effect of the TMC polymers on the surface tension of a mixture of polymer and mucus was measured with a Du Noüy tensiometer. The degrees of quaternization of the synthesized TMC polymers were between 22.1% and 48.8% and the molecular mass was above 100,000 g/mole for all the polymers. A decrease in mucoadhesivity with an increase in the degree of quaternization of the TMC polymers was found. Surface-tension analysis of a mixture of polymer and mucus showed the effect of excessive polymer hydration on mucoadhesion. The results show that the degree of quaternization of TMC had a pronounced effect on the mucoadhesive properties of this polymer. Although the mucoadhesive profiles for the TMC polymers were lower than the original chitosan, they still retained sufficient mucoadhesive properties for successful inclusion into mucoadhesive dosage forms.

N-trimethyl chitosan chloride: optimum degree of quaternization for drug absorption enhancement across epithelial cells.

Abstract N-trimethyl chitosan chloride (TMC) is a polycation that enhances drug transport across epithelia by opening tight junctions. The degree of quaternization of TMC determines the number of positive charges available on the molecule for interactions with the negatively charged sites on the epithelial membrane and thereby influences its drug absorption-enhancing properties. The effects of six different TMC polymers (degree of quarternization between 12% and 59%) on the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers and on the transport of hydrophilic and macromolecular model compounds across Caco-2 cells were determined. All the TMC polymers were able to decrease the TEER markedly in a slightly acidic environment (pH 6.2). However, only TMC polymers with higher degrees of quaternization (>22%) were able to reduce the TEER in a neutral environment (pH 7.4). The maximum reduction in TEER (47.3 ± 6.0% at a concentration of 0.5% w/v and pH 7.4) was reached with TMC with a degree of quaternization of 48%, and this effect did not increase further with higher degrees of quaternization of TMC. In agreement with the TEER results, the transport of model compounds across Caco-2 cell monolayers increased with an increase in the degree of quaternization of TMC. However, the transport reached a maximum for TMC with a degree of quaternization of 48% (25.3% of the initial dose for [14C]mannitol and 15.2% of the initial dose for [14C]PEG 4000), and this effect did not increase further with higher degrees of quaternization of TMC. Therefore, the increase in the effects of TMC on intestinal epithelia did not directly correlate up to the maximum quaternization degree of this polymer, but reached an optimum value already at an intermediate degree of quaternization (ca. 48%).

Effect of the Type of Base and Number of Reaction Steps on the Degree of Quaternization and Molecular Weight of N-Trimethyl Chitosan Chloride

N-Trimethyl chitosan chloride (TMC), a chemically modified derivative of chitosan, is the first chitosan derivative shown to be an effective absorption enhancer for peptide and protein drugs across mucosal epithelia. TMC is synthesized by reductive methylation with methyl iodide in the presence of a strong base such as sodium hydroxide. In this reaction, the primary amino group on the C-2 position of chitosan is changed to a quaternary amino group. The charge density, as determined by the degree of quaternization, and probably also the molecular weight of TMC are important factors that influence the absorption enhancement effect and toxicity of this polymer. The molecular weight of the starting polymer decreases during the synthesis procedure due to factors such as the strong alkaline environment and elevated experimental temperatures. This study investigated the effects of two different bases, sodium hydroxide and dimethyl amino pyridine, together with a varying number and duration of reaction steps, on the degradation and the degree of quaternization of TMC polymers. 1H-NMR (nuclear magnetic resonance) spectra showed a major increase in the degree of quaternization (21%–59%) of TMC with an increase in the number of reaction steps when sodium hydroxide was used as the base. Intrinsic viscosity values indicated that the use of dimethyl amino pyridine did not cause polymer degradation to the same extent as sodium hydroxide, but that the degree of quaternization of TMC stayed low (7.3%–9.6%) even when the number of reaction steps was increased. A combination of the two bases did not reduce polymer degradation, while the degree of quaternization was limited to relatively low values (12.5%–34.4%).

Intestinal paracellular permeation enhancement with quaternised chitosan: in situ and in vitro evaluation

Previous studies have shown that N-trimethyl chitosan chloride (TMC) is a potent absorption enhancer for hydrophilic and macromolecular compounds across mucosal surfaces. TMC proved to be effective in neutral and basic pH environments where the absorption enhancing ability of chitosan is severely hampered by its insolubility in these environments. The absorption enhancing characteristics of TMC polymers with different degrees of quaternisation were investigated in vitro and in situ to identify the most effective polymer in a neutral pH environment. Different degrees of quaternisation were obtained by varying the number and duration of the reaction steps in the synthesis process of TMC. The TMC polymers were characterised with 1H-NMR spectroscopy and the degrees of quaternisation were between 22.1 and 48.8%. Everted intestinal sacs (rats) were used to determine the effect of the polymers (0.0625-0.5% w/v) on the permeation of the hydrophilic model compound, [14C]mannitol, at a pH value of 7.4. A single pass intestinal perfusion method was also used to evaluate the permeation enhancing properties of the TMC polymers under the same conditions. The results obtained from both methods clearly showed a pronounced enhancement of [14C]mannitol permeation when administered with the different TMC polymers. It was shown that the permeation enhancing effects depend on the degree of quaternisation of TMC. In both models the best permeation enhancing results were obtained with the highest degree of quaternisation of TMC (48.8%) at a concentration of 0.5% w/v.

Applications of Lipid based Formulation Technologies in the Delivery of Biotechnology-based Therapeutics

In the last decades several new biotechnologically-based therapeutics have been developed due to progress in genetic engineering. A growing challenge facing pharmaceutical scientists is formulating these compounds into oral dosage forms with adequate bioavailability. An increasingly popular approach to formulate biotechnology-based therapeutics is the use of lipid based formulation technologies. This review highlights the importance of lipid based drug delivery systems in the formulation of oral biotechnology based therapeutics including peptides, proteins, DNA, siRNA and vaccines. The different production procedures used to achieve high encapsulation efficiencies of the bioactives are discussed, as well as the factors influencing the choice of excipient. Lipid based colloidal drug delivery systems including liposomes and solid lipid nanoparticles are reviewed with a focus on recent advances and updates. We further describe microemulsions and self-emulsifying drug delivery systems and recent findings on bioactive delivery. We conclude the review with a few examples on novel lipid based formulation technologies.

Direct Compression of Chitosan: Process and Formulation Factors to Improve Powder Flow and Tablet Performance

Chitosan is a polymer derived from chitin that is widely available at relatively low cost, but due to compression challenges it has limited application for the production of direct compression tablets. The aim of this study was to use certain process and formulation variables to improve manufacturing of tablets containing chitosan as bulking agent. Chitosan particle size and flow properties were determined, which included bulk density, tapped density, compressibility and moisture uptake. The effect of process variables (i.e. compression force, punch depth, percentage compaction in a novel double fill compression process) and formulation variables (i.e. type of glidant, citric acid, pectin, coating with Eudragit S®) on chitosan tablet performance (i.e. mass variation, tensile strength, dissolution) was investigated. Moisture content of the chitosan powder, particle size and the inclusion of glidants had a pronounced effect on its flow ability. Varying the percentage compaction during the first cycle of a double fill compression process produced chitosan tablets with more acceptable tensile strength and dissolution rate properties. The inclusion of citric acid and pectin into the formulation significantly decreased the dissolution rate of isoniazid from the tablets due to gel formation. Direct compression of chitosan powder into tablets can be significantly improved by the investigated process and formulation variables as well as applying a double fill compression process.