J. Coos Verhoef

Chitosan and its derivatives in mucosal drug and vaccine delivery.

Numerous studies have demonstrated that chitosan and their derivatives (N-trimethyl chitosan, mono-N-carboxymethyl chitosan) are effective and safe absorption enhancers to improve mucosal (nasal, peroral) delivery of hydrophylic macromolecules such as peptide and protein drugs and heparins. This absorption enhancing effect of chitosans is caused by opening of the intercellular tight junctions, thereby favouring the paracellular transport of macromolecular drugs. Chitosan nano- and microparticles are also suitable for controlled drug release. Association of vaccines to some of these particulate systems has shown to enhance the antigen uptake by mucosal lymphoid tissues, thereby inducing strong systemtic and mucosal immune responses against the antigens. The aspecific adjuvant activity of chitosans seems to be dependent on the degree of deacetylation and the type of formulation. From the studies reviewed it is concluded that chitosan and chitosan derivatives are promising polymeric excipients for mucosal drug and vaccine delivery.

The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro

Two mucoadhesive polymers, chitosan-glutamate and carbomer, were studied in an in vitro model (Caco-2 cell monolayers) with respect to their ability to enhance intestinal peptide drug delivery. Preparations of the polymers at concentrations of 0.5, 1.0, and 1.5% w/v (chitosan), and of 0.5 and 1.0% w/v (carbomer) were applied to the apical side of Caco-2 cell monolayers. The effects on transepithelial electrical resistance (TEER), paracellular transport of a FITC-dextran of a molecular weight of 4400 (FD-4) and [14C]mannitol were measured. Paracellular transport of FD-4 was visualized by means of confocal laser scanning microscopy (CLSM). Furthermore, the impact of lowering the pH of the polymer solutions to pH 4 on the integrity of the cell layer was determined. The results show that both polymers were able to decrease TEER of Caco-2 cell layers significantly. In the case of carbomer, CLSM revealed a partial opening of epithelial tight junctions. Lowering of the pH in the control and polymer solutions to pH 4 resulted in every case in the irreversible damage of a large percentage of the cells, as shown by CLSM. Transport studies with [14C]mannitol and FD-4 showed only during co-application of carbomer significantly increased fluxes, whereas no difference from the control solution could be detected for chitosan-glutamate. A threshold value of about 50% of TEER reduction has been identified, which allows for transport of hydrophilic compounds across the cell monolayers of the Caco-2 cell model.

Mucoadhesive Polymers in Peroral Peptide Drug Delivery. VI. Carbomer and Chitosan Improve the Intestinal Absorption of the Peptide Drug Buserelin In Vivo

AbstractPurpose. To evaluate the effect of the crosslinked poly(acrylate) carbomer 934P (C934P) and its freeze-dried neutralized sodium salt (FNaC934P) as well as chitosan hydrochloride on the intestinal absorption of the peptide drug buserelin. Methods. Buserelin was applied intraduodenally in control buffer, 0.5% (w/v) C934P, 0.5% (w/v) FNaC934P, 1.5% (w/v) chitosan hydrochloride or FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture in rats. Results. All polymer preparation showed a statistically significant improvement of buserelin absorption compared to the control solution. The absolute bioavailabilities for the different polymer preparations were: control, 0.1%; 0.5% FNaC934P, 0.6%; 0.5% C934P, 2.0%; chitosan hydrochloride, 5.1% and FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture, 1.0%. The higher bioavailability with chitosan hydrochloride compared to C934P and FNaC934P indicates that for buserelin the intestinal transmucosal transport enhancing effect of the polymer plays a more dominant role than the protection against proteases such as α-chymotrypsin. Conclusions. The mucoadhesive polymers carbomer 934P and chitosan hydrochloride are able to enhance the intestinal absorption of buserelin in vivo in rats, and may therefore be promising excipients in peroral delivery systems for peptide drugs.

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.

Nasal mucociliary clearance as a factor in nasal drug delivery

The nasal mucociliary clearance system transports the mucus layer that covers the nasal epithelium towards the nasopharynx by ciliary beating. Its function is to protect the respiratory system from damage by inhaled substances. Impairment of nasal mucociliary clearance can result in diseases of the upper airways. Therefore, it is important to study the effects of drugs and drug excipients on nasal mucociliary clearance. A large number of methods are used to assess mucociliary clearance. These methods study the effects of drug and excipients on the mucociliary system in vitro or in vivo in animals and humans. In some cases, the results of different in vitro and in vivo measurements do not correlate well. In vitro methods, especially ciliary beat frequency measurements, have been demonstrated to be valuable tools for toxicity screening. However, in vivo studies are essential to confirm the safety of nasal drug formulations. Nasal mucociliary clearance also has implications for nasal drug absorption. Drugs are cleared rapidly from the nasal cavity after intranasal administration, resulting in fast systemic drug absorption. Several approaches are discussed to increase the residence time of drug formulations in the nasal cavity, resulting in improved nasal drug absorption. However, more experimental evidence is needed to support the conclusion that this improved absorption is caused by a longer residence time of the nasal drug formulation.

N-Trimethyl Chitosan Chloride as a Potential Absorption Enhancer Across Mucosal Surfaces: In Vitro Evaluation in Intestinal Epithelial Cells (Caco-2)

AbstractPurpose. Previous studies have established that chitosan hydrochloride and glutamate are potent absorption enhancers for large hydrophilic compounds across mucosal surfaces. However, these compounds lack solubility at neutral pH values. A partially quaternized and well-soluble derivative of chitosan, N-trimethyl chitosan chloride, was synthesized and the effects of this polymer on the transepithelial electrical resistance and permeability of intestinal epithelial cells were investigated in vitro.Methods. N-trimethyl chitosan chloride was synthesized by reductive methylation and characterized with NMR. The effect of this polymer (1.0−2.5% w/v) on the transepithelial electrical resistance of intestinal epithelial cells, using Caco-2 cell monolayers, was investigated. Permeation of the hydrophilic model compounds [l4C]-mannitol (MW 182.2), FITC-Dextran (MW 4400) and the peptide drug buserelin (MW 1299.5), in the presence of N-trimethyl chitosan chloride (1.5−2.5% w/v), was followed for 3 hours. The transport process of the fluorescent marker, FITC-Dextran 4400, across the cell monolayers was visualised with confocal laser scanning microscopy. Viability of the cells was checked with the trypan blue exclusion technique. Results. N-trimethyl chitosan chloride was found to be a perfectly water-soluble, partially quaternized (about 12%) derivative of chitosan. This polymer (1.5−2.5% w/v) caused a pronounced and immediate reduction (25−85%) in the transepithelial electrical resistance of Caco-2 cells. Large increases in the transport rate of [!4C]-mannitol (32−60 fold), FITC-Dextran 4400 (167−373 fold) and buserelin (28−73 fold) were demonstrated. Confocal laser scanning microscopy confirmed that N-trimethyl chitosan chloride opens the tight junctions of intestinal epithelial cells to allow increased transport of hydrophilic compounds through the paracellular transport pathway. No deleterious effects to the cells could be demonstrated with trypan blue. Conclusions. The potential use of N-trimethyl chitosan chloride as an absorption enhancer across mucosal surfaces could be an important contribution towards the development of effective delivery systems for hydrophilic drugs.

Chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice.

In this study, the ability of chitosan microparticles to enhance both the systemic and local immune responses against diphtheria toxoid (DT) after oral and nasal administration in mice was investigated.Firstly, DT was associated to chitosan microparticles to determine antigen loading and release. Then DT loaded chitosan microparticles, DT in phosphate buffered saline (PBS) and empty chitosan microparticles (as controls) were fed intragastrically and administered nasally to mice. Mice were also subcutaneously immunised with DT associated with alum. All mice were vaccinated in week 1 and boosted in week 3. Sera were analysed for anti-DT IgG and nasal washings and faeces for anti-DT IgA titres using an enzyme linked immunosorbent assay. Loading capacities of about 25% and loading efficacies of about 100% were obtained after loading the chitosan microparticles with DT. No DT was released at 37 degrees C in PBS. Compared to intragastrical feeding with DT in PBS, a strong enhancement of the systemic and local immune responses against DT were found in mice fed with DT loaded chitosan microparticles. In addition, a dose-dependent immune reaction was observed for mice vaccinated with different doses of DT associated to chitosan microparticles. Significant systemic humoral immune responses were also found after nasal vaccination with DT associated to chitosan microparticles.DT associated to chitosan microparticles results in protective systemic and local immune response against DT after oral vaccination, and in significant enhancement of IgG production after nasal administration. Hence, these in vivo experiments demonstrate that chitosan microparticles are very promising mucosal vaccine delivery systems.

Mucoadhesive Polymers in Peroral Peptide Drug Delivery. II. Carbomer and Polycarbophil Are Potent Inhibitors of the Intestinal Proteolytic Enzyme Trypsin

AbstractPurpose. The evaluation of the inhibitory action of two mucoadhesive poly(acrylates), polycarbophil and carbomer, registered by the Food and Drug Administration (FDA), on the intestinal proteolytic enzyme trypsin. Methods. The effect of the polymers on trypsin activity by measuring the degradation of a trypsin specific substrate. Binding of Ca2+ ions and proteins (125I-BSA) to the poly(acrylates). The influence of the polymers on the secondary trypsin structure by circular dichroism. Results. Trypsin inhibition was found to be time-dependent upon addition of Ca2+ in the degradation experiment. Only when Ca2+ was added within 10 min after trypsin incubation, recovery of the enzyme could be observed. Both polymers showed a strong Ca2+ binding ability. Carbomer, which had a higher inhibitory effect on trypsin activity, also revealed a higher Ca2+ binding affinity than polycarbophil. The amount of Ca2+ depleted out of the trypsin structure and the reduction of enzyme activity were comparable. Immobilization of trypsin by binding to the polymers could not be observed at pH 6.7. Circular dichroism studies suggested that, under depletion of Ca2+ from trypsin, the secondary structure changed its conformation, followed by an increased autodegradation of the enzyme. Conclusions. The poly(acrylates) investigated may have potential to protect peptides from tryptic degradation and may be used to master the peroral delivery of peptide drugs.

The Nasal Mucociliary Clearance: Relevance to Nasal Drug Delivery

Mucociliary clearance is an important physiological defense mechanism of the respiratory tract to protect the body against noxious inhaled materials. This process is responsible for the rapid clearance of nasally administered drugs from the nasal cavity to the nasopharynx, thereby interfering with the absorption of drugs following intranasal application. This review describes the mucociliary system and the methods used for its characterization. Examples are given of the effects of drugs and additives on its functioning. Further, possible approaches are presented for increasing the residence time of drugs in the nasal cavity, thereby improving intranasal drug delivery.

ABSORPTION ENHANCING EFFECT OF CYCLODEXTRINS ON INTRANASALLY ADMINISTERED INSULIN IN RATS

The absorption enhancing effect of α-, β-, and γ-cyclodextrin (CD), dimethyl-β-cyclodextrin (DMβCD), and hydroxypropyl-β-cyclodextrin (HPβCD) on intranasally administered insulin was investigated in rats. Coadministration of 5% (w/v) DMβCD to the insulin solution resulted in a high bioavailability, 108.9 ± 36.4% (mean ± SD, n = 6), compared to i.v. administration, and a strong decrease in blood glucose levels, to 25% of their initial values. Coadministration of 5% α-CD gave rise to an insulin bioavailability of 27.7 ± 11.5% (mean ± SD, n = 6) and a decrease in blood glucose to 50% of its initial value. The rate of insulin absorption and the concomitant hypoglycemic response were delayed for the α-CD-containing solution as compared to the DMβCD preparation. The other CDs, HPβCD (5%), β-CD (1.8%), and γ-CD (5%), did not have significant effects on nasal insulin absorption. DMβCD at a concentration of 5% (w/v) induces ciliostasis as measured on chicken embryo tracheal tissue in vitro, but this effect is reversible. In conclusion, DMβCD is a potent enhancer of nasal insulin absorption in rats.