H.L. Lueßen

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.

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.

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.

Chitosan for enhanced intestinal permeability: prospects for derivatives soluble in neutral and basic environments.

In this study the effects of two chitosan salts, namely chitosan hydrochloride and chitosan glutamate (0.5 and 1.5% w/v), on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cell monolayers, using the radioactive marker [14C]-mannitol, were investigated in a slightly acidic (pH 6.2) and neutral (pH 7.4) environment. Both salts are soluble in acidic conditions up to a concentration of 1.5% w/v and solutions of this strength, at a pH of 6.2, caused a pronounced lowering in the TEER of Caco-2 cell monolayers in the order of 70+/-1% (chitosan glutamate) and 77+/-3% (chitosan hydrochloride), 20 min after incubation started. In agreement with the TEER results the transport of the radioactive marker, [14C]-mannitol, was increased 25-fold (chitosan glutamate) and 36-fold (chitosan hydrochloride), respectively, at this pH. However, at a pH of 7.4 both salts are insoluble and prove to be ineffective since no reduction in the TEER values or increase in the transport of [14C]-mannitol were found. The results show that these chitosan salts are potent absorption enhancers in acidic environments. We conclude that there is a need for chitosan derivatives with increased solubility, especially at neutral and basic pH values, for use as absorption enhancers aimed at the delivery of therapeutic compounds in the more basic environment of the large intestine and colon.

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.

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 quaternization of N-trimethyl chitosan chloride on the permeability of intestinal epithelial cells (Caco-2).

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.

Mucoadhesive polymers in peroral peptide drug delivery. I. Influence of mucoadhesive excipients on the proteolytic activity of intestinal enzymes

Abstract In the present study the potency of mucoadhesive excipients to inhibit intestinal proteases has been evaluated. Among the different mucoadhesive polymers investigated, uniquely the poly(acrylates) polycarbophil and carbomer 934P were able to inhibit the activities of trypsin, α-chymotrypsin, carboxypeptidase A and cytosolic leucine aminopeptidase. However, they failed to inhibit microsomal leucine aminopeptidase and pyroglutamyl aminopeptidase. Carbomer was found to be more efficient to reduce proteolytic activity than polycarbophil. The pronounced binding properties of polycarbophil and carbomer for bivalent cations such as zinc and calcium was demonstrated to be a major reason for the observed inhibitory effect. These polymers were able to deprive Ca2+ and Zn2+, respectively, from the enzyme structures, thereby inactivating their activities. Carboxypeptidase A and α-chymotrypsin activities were observed to be reversible upon addition of Zn2+ and Ca2+ ions, respectively. It is concluded that poly(acrylates) may be promising excipients to protect peptide drugs from intestinal degradation. In combination with their low toxicity risk they are expected to be suitable excipients for improved peroral delivery of peptide drugs.