Davide Guggi

Review articleThiolated chitosans

The derivatization of the primary amino groups of chitosan with coupling reagents bearing thiol functions leads to the formation of thiolated chitosans. So far, three types of thiolated chitosans have been generated: chitosan-cysteine conjugates, chitosan-thioglycolic acid conjugates and chitosan-4-thio-butyl-amidine conjugates. Various properties of chitosan are improved by this immobilization of thiol groups. Due to the formation of disulfide bonds with mucus glycoproteins, the mucoadhesiveness is 6-–100-fold augmented (I). The permeation of paracellular markers through intestinal mucosa can be enhanced 1.6-–3-fold utilizing thiolated instead of unmodified chitosan (II). Moreover, thiolated chitosans display in situ-gelling features, due to the pH-dependent formation of inter- as well as intra-molecular disulfide bonds (III). This latter process provides a strong cohesion and stability of carrier matrices being based on thiolated chitosans (IV). Consequently, thiolated chitosans can guarantee a prolonged controlled release of embedded therapeutic ingredients (V). The potential of thiolated chitosans has meanwhile also been demonstrated in vivo. A significant pharmacological efficacy of 1.3% of orally given salmon calcitonin, for instance, could be achieved utilizing thiolated chitosan as polymeric drug carrier matrix, while no effect was reached using unmodified chitosan. According to these results thiolated chitosans represent a promising new category of polymeric excipients in particular for the non-invasive administration of hydrophilic macromolecules. Further applications such as their use as scaffold materials in tissue engineering or as coating material for stents seem feasible.

Systemic peptide delivery via the stomach: in vivo evaluation of an oral dosage form for salmon calcitonin

It was the aim of this study to investigate the potential of stomach targeted delivery systems for systemic peptide administration using salmon calcitonin as a model drug. Chitosan was modified by the immobilization of thiol groups utilizing 2-iminothiolane in order to obtain a chitosan-4-thiobutylamidine conjugate (chitosan-TBA). Furthermore, a chitosan-pepstatin A conjugate was synthesized by a carbodiimide mediated linkage of the pepsin inhibitor to the polymer. The protective effect of this novel conjugate for calcitonin towards pepsin was evaluated in vitro. Minitablets (5 mg) were generated by direct compression of calcitonin, chitosan, chitosan-TBA, chitosan-pepstatin A conjugate and glutathione (GSH), respectively (A, 1:0:69:20:10; B, 1:79:0:20:0; C, 1:99:0:0:0). The drug release was investigated in an artificial gastric fluid. Biofeedback studies were performed in rats by determining the decrease in plasma calcium level after oral administration. The novel chitosan-pepstatin A conjugate displayed 291+/-58 nmol inhibitor per gram polymer (mean+/-S.D., n = 5). The chitosan-inhibitor conjugate showed a very strong protective effect for salmon calcitonin towards pepsinic degradation. A controlled drug release was provided by all tested dosage forms-A, B and C. Dosage form B led only to a slight reduction of the plasma calcium level, displaying a pharmacological efficacy versus i.v. injection of 0.41%, while dosage form C did not lead to any significant effect. In contrast, dosage form A led to a decrease in the plasma calcium level of 10% for at least 12 h. The pharmacological efficacy of this formulation was determined to be 1.35%. The study suggests that stomach targeted oral delivery might be a promising novel approach for noninvasive systemic peptide administration.

In Vivo Evaluation of an Oral Salmon Calcitonin-Delivery System Based on a Thiolated Chitosan Carrier Matrix

AbstractPurpose. To develop and evaluate an oral delivery system for salmon calcitonin. Methods. 2-Iminothiolane was covalently bound to chitosan in order to improve the mucoadhesive and cohesive properties of the polymer. The resulting chitosan-TBA conjugate (chitosan-4-thiobutylamidine conjugate) was homogenized with salmon calcitonin, mannitol, and a chitosan-Bowman-Birk inhibitor conjugate and a chitosan-elastatinal conjugate (6.75 + 0.25 + 1 + 1 + 1). Optionally 0.5% (m/m) reduced glutathione, used as permeation mediator, was added. Each mixture was compressed to 2 mg microtablets and enteric coated with a polymethacrylate. Biofeedback studies were performed in rats by oral administration of the delivery system and determination of the decrease in plasma calcium level as a function of time. Results. Test formulations led to a significant (p < 0.005) decrease in the plasma calcium level of the dosed animals in comparison to control tablets being based on unmodified chitosan. The addition of glutathione in the tablets led to a further improvement in the oral bioavailability of salmon calcitonin with an earlier onset of action and a decrease in the calcium level of about 10% for at least 10 h. Conclusions. The combination of mucoadhesive thiolated chitosan, chitosan-enzyme-inhibitor conjugates and the permeation mediator glutathione seems to represent a promising strategy for the oral delivery of salmon calcitonin.

Development and in Vivo Evaluation of an Oral Delivery System for Low Molecular Weight Heparin Based on Thiolated Polycarbophil

AbstractPurpose. It was the purpose of this study to develop a new oral drug delivery system for low molecular weight heparin (LMWH) providing an improved bioavailability and a prolonged therapeutic effect. Methods. The permeation enhancing polycarbophil-cysteine conjugate (PCP-Cys) used in this study displayed 111.4 ± 6.4 μM thiol groups per gram polymer. Permeation studies on freshly excised intestinal mucosa were performed in Ussing chambers demonstrating a 2-fold improved uptake of heparin as a result of the addition of 0.5% (w/v) PCP-Cys and the permeation mediator glutathione (GSH). Results. Tablets containing PCP-Cys, GSH, and 279 IU of LMWH showed a sustained drug release over 4 h. To guarantee the swelling of the polymeric carrier matrix in the small intestine tablets were enteric coated. They were orally given to rats. For tablets being based on the thiomer/GSH system an absolute bioavailability of 19.9 ± 9.3% (means ± SD; n = 5) vs. intravenous injection could be achieved, whereas tablets comprising unmodified PCP did not lead to a significant (p < 0.01) heparin concentration in plasma. The permeation enhancing effect and subsequently a therapeutic heparin level was maintained for 24 h after a single dose. Conclusions. Because of the strong and prolonged lasting permeation enhancing effect of the thiomer/GSH system, the oral bioavailability of LMWH could be significantly improved. This new delivery system represents therefore a promising tool for the oral administration of heparin.

In vitro evaluation of polymeric excipients protecting calcitonin against degradation by intestinal serine proteases

The oral bioavailability of salmon calcitonin is strongly reduced due to the enzymatic degradation by luminally secreted serine proteases. Apart from being degraded by trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1), it was shown in this study that calcitonin is also digested by elastase (EC 3.4.21.36). It was therefore the aim of this study to generate polymeric excipients protecting perorally administered salmon calcitonin from degradation by these enzymes. Mediated by a carbodiimide trypsin and chymotrypsin inhibitor Bowman-Birk inhibitor (BBI) and elastase inhibitor elastatinal were each covalently attached to the mucoadhesive polymer chitosan. The share of the Bowman-Birk inhibitor in the resulting conjugate was 3.5+/-0.1% (w/w, mean+/-S.D., n=4) and that of elastatinal 0.5+/-0.03% (w/w, mean+/-S.D., n=4). Enzyme assays with synthetic substrates demonstrated a strong inhibitory effect of the chitosan-BBI conjugate towards trypsin and chymotrypsin as well as of the chitosan-elastatinal conjugate towards elastase. In an artificial intestinal fluid containing physiological concentrations of trypsin, alpha-chymotrypsin and elastase, calcitonin being incorporated in unmodified chitosan (0.5%, w/v) was degraded by 99.7+/-0.1% (mean+/-S.D., n=3) within 2h at 37 degrees C. On the contrary, incorporating the drug in chitosan-BBI conjugate and chitosan-elastatinal conjugate (1+1, 0.5%, w/v) led to a degradation of only 36.4+/-0.9% (mean+/-S.D., n=3). Hence, the chitosan-inhibitor conjugates described in this study seem to be promising tools for the oral delivery of salmon calcitonin.