NTrimethyl Chitosan as Absorption Enhancer of Peptide Drugs

TMC was first investigated for permeation enhancing properties and toxicity by Kotze and coworkers (1997,1999), using the Caco-2 cells as a model for intestinal epithelium. Initially a trimethylated chitosan having a degree of trimethylation of 12% (dimethylation 80%) was tested. This polymer (1.5-2.5%, w/v; pH 6.7) caused large increases in the transport rate of [14C]mannitol (32- to 60-fold), fluorescently labeled dextran 4,400 (167- to 373-fold), and the peptide drug buserelin (28- to 73-fold). CLSM confirmed that TMC opens the tight junctions of intestinal epithelial cells to allow increased transport of hydrophilic compounds along the paracellular transport pathway. No intracellular transport of the fluorescent marker could be observed (Kotze et al., 1999).

Chitosan HCl and TMCs of different degrees of trimethylation were tested by Kotze and collaborators (1999) for enhancing the permeability of [14C]mannitol in Caco-2 intestinal epithelia at a pH value of 7.2. Chitosan HCl failed to increase the permeability of these monolayers and so did TMC with a degree of methyla-tion of 12.8%. However, TMC with a degree of trimethylation of 60% increased significantly the [14C]mannitol permeability across Caco-2 intestinal monolayers, indicating that a threshold value at the charge density of the polymer is necessary to trigger the opening of the tight junctions at neutral values.

TMC polymers were further investigated by Thanou et al. (1999, 2000a) to see if they provoke cell membrane damage on Caco-2 cell monolayers during enhancement of the transport of hydrophilic macromolecules. Using cell membrane impermeable fluorescent probes and CLSM, it was visualized that TMC polymers widen the paracellular pathways without cell damage. From such visualization studies it also appears that the mechanism of opening the tight junctions is similar to that of protonated chitosan (Thanou et al., 2001c). Because of the absence of significant toxicity, TMC polymers (particularly with a high degree of trimethylation) are expected to be safe absorption enhancers for improved transmucosal delivery of peptide drugs (Thanou et al., 1999).

The effects of TMC60 (degree of trimethylation 60%) polymers were subsequently studied in vivo in rats, using the peptide drug buserelin (pH = 6.8) and octreotide (pH = 8.2) (Thanou et al., 2000b, 2000c). Buserelin formulations with or without TMC60 (pH 7.2) were compared with chitosan dispersions at neutral pH values after intraduodenal administration in rats. A remarkable increase in buserelin serum concentrations was observed after coadministration of the peptide with TMC60, whereas buserelin alone was poorly absorbed. In the presence of TMC60 buserelin was rapidly absorbed from the intestine having tmax at 40min, whereas chitosan dispersions (at pH 7.2) showed a slight increase in buserelin absorption compared to the control. Chitosan did not manage to increase the buserelin concentrations to the levels achieved with TMC60. The absolute bioavailability of buserelin after coadministration with 1.0% TMC60 was 13.0%. Similar to the buserelin studies (Thanou et al., 2000b), octreotide absorption after intrajejunal administration was substantially increased, resulting in peptide absolute bioavailability of 16%.

Octreotide was also administered to juvenile pigs with or without TMC60 at a pH of 7.4. The solutions were administered intrajejunally through an in-dwelling fistula that was inserted one week prior to the octreotide. Intrajejunal administration of 10 mg of octreotide, coadministered with 5 and 10% (w/v) TMC60, resulted in a 7.7- and 14.5-fold increase in octreotide absorption with absolute bioavailabilities of 13.9 ± 1.3% and 24.8 ± 1.8%, respectively (Fig. 6.4) (Thanou et al., 2001a).

It is stated by the authors that a gel was obtained with the 10% (w/v) concentration of the polymer. This high concentration of the TMC60 polymer was chosen to counteract the dilution of the 20 ml administration volume by the luminal fluids and mucus of the intestinal tract and to ensure that substantial amounts of both peptide and enhancer could reach the absorptive site of the intestinal mucosa (Thanou et al., 2001a). Although the results show very high bioavailabilities (also taking into account the small absorptive area which is created by only widening of the tight junctions), the impracticality of administering such high concentrations in a solid dosage form cannot be overlooked as concentrations of 1-2 g of the polymer have to be administered in an attempt to obtain the same results (Van der Merwe et al., 2004a).

In order to overcome these problems a completely new and different approach has been chosen by Dorkoosh and coworkers (2002). The platform of their delivery systems consists of superporous hydrogels (SPH) and superporous hydrogels composite (SPHC). These hydrogels can swell very rapidly and have the capacity to take up between 100 and 200 times of intestinal liquid of their original volume. Arriving in the intestine those SPHs swell quickly and bring the delivery systems (small tablet in which the drug is incorporated) which is attached to the outside of the SPH platform in direct contact to the absorbing surface. TMC at the outside of the small tablet will interfere at the interface between swollen SPH and intestinal wall as a polymeric penetration enhancer widening locally the tight junctions

Figure 6.4. Plasma octreotide concentration (mean ± SE) versus time curves after intra-jejunal administration in pigs (10 mg/20 ml/pig) with the polymers chitosan HCl [CS1.5, 1.5% (w/v), pH 5.5; n = 6] and TMC [TMC 10, 10% (w/v); pH 7.4; n = 6, and TMC, 5% (w/v); pH 7.4; n = 3] or without any polymer [OA 10, octreotide in 0.9% NaCl; pH 7.4; n = 5]. With permission from Thanou et al. (2001a)

to allow for paracellular absorption of the peptide drug. In an in vivo study with pigs the achieved absolute bioavailabilities of octreotide were between 8.7 ± 2.4% and 16.1 ± 3.3% depending on the type of delivery system used. The value of 16.1 ±3.3% was achieved with TMC60 as absorption enhancer. After the peptide's release from the dosage form the SPH platforms get overhydrated and are easily broken down by the peristaltic forces of the gut. Scintigraphic studies in human have shown the good performance of these oral peptide drug delivery systems with prolonged residence times in the gut. Incorporating the SPH© delivery platforms in enteric coated gelatin capsules of size 000 lead to various stomach transit times (2-6h in pigs) and 1.5-3 h in human volunteers (Dorkoosh et al., 2004). Capsules of smaller size (00) may reduce the variability in gastric transit times. Monocarboxymethyl Chitosan

An usual approach to increase chitosan's solubility at neutral pH values is the substitution of the primary amine. Whereas N-substitution with alkyl groups (i.e. —CH3 groups) can increase the aqueous solubility without affecting its cationic character, substitution with moieties bearing carboxyl groups can yield polymers with polyampholytic properties (Muzzarelli et al., 1982). Monocar-boxymethylated chitosan (MCC) was synthesized and further evaluated as potential absorption enhancer (Thanou et al., 2001d). This chitosan derivative (degree of substitution 87-90%) has polyampholytic (zwitterionic) character, which allows the formation of clear gels or solutions (dependent on the

Figure 6.4. Plasma octreotide concentration (mean ± SE) versus time curves after intra-jejunal administration in pigs (10 mg/20 ml/pig) with the polymers chitosan HCl [CS1.5, 1.5% (w/v), pH 5.5; n = 6] and TMC [TMC 10, 10% (w/v); pH 7.4; n = 6, and TMC, 5% (w/v); pH 7.4; n = 3] or without any polymer [OA 10, octreotide in 0.9% NaCl; pH 7.4; n = 5]. With permission from Thanou et al. (2001a)

concentration of the polymer) even in the presence of polyanionic compounds like heparins at neutral and alkaline pH values, whereas it aggregates at acidic pH. Chitosan and the quaternized derivative TMC form complexes with polyanions that precipitate out of the solution. in contrast, MCC appeared to be compatible with polyanions.

Two viscosity grades MCC (high and low) were initially investigated to see if they are able to increase the permeation of LMWH (4,500 Da) across Caco-2 intestinal cell monolayers. However, the MCC concentrations necessary to open the tight junctions were several times higher than that of TMC60 at neutral pH value. Low viscosity MCC induced higher transport of LMW when compared with the high viscosity derivative. Cell viability tests at the end of the experiments showed that this type of polymer had no damaging effect on cell membranes, whereas recovery of the transepithelial electrical resistance values to initial levels indicated the functional integrity of the monolayer. The mechanism by which polyampholytic chitosans interacts with the tight junctions is not clear yet.

For in vivo studies, LMWH was administered intraduodenally with or without MCC to rats. Three percent (w/v) low viscosity MCC significantly increased the intestinal absorption of LMWH, reaching the therapeutic anticoagulant blood levels of LMWH for at least 5 h determined by measuring anti-Xa levels (Thanou et al., 2001d). Thiolated Polymers

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