Thiolated chitosan microparticles: a vehicle for nasal peptide drug delivery.

The goal of this study was to develop a microparticulate delivery system based on a thiolated chitosan conjugate for the nasal application of peptides. Insulin was used as model peptide. For thiolation of chitosan 2-iminothiolane was covalently linked to chitosan. The resulting chitosan-TBA (chitosan-4-thiobutylamidine) conjugate featured 304.89+/-63.45 micromol thiol groups per gram polymer. 6.5% of these thiol groups were oxidised. A mixture of the chitosan-TBA conjugate, insulin and the permeation mediator reduced glutathione were formulated to microparticles. Control microparticles comprised unmodified chitosan and insulin. As second control served mannitol-insulin microparticles. All microparticulate systems were prepared via the emulsification solvent evaporation technique. In 100 mM phosphate buffer pH 6.8 chitosan-TBA-insulin microparticles swelled 4.39+/-0.52-fold in size, whereas chitosan based microparticles did not swell at all. Chitosan-TBA microparticles showed a controlled release of fluorescein isothiocyanate (FITC)-labelled insulin over 6 h. Nasal administered chitosan-TBA-insulin microparticles led to an absolute bioavailability of 7.24+/-0.76% (means+/-S.D.; n=3) in conscious rats. In contrast, chitosan-insulin microparticles and mannitol-insulin microparticles exhibited an absolute bioavailability of 2.04+/-1.33% and 1.04+/-0.27%, respectively (means+/-S.D.; n=4). Because of these results microparticles comprising chitosan-TBA and reduced glutathione seem to represent a useful formulation for the nasal administration of peptides.

Comparison of the mucoadhesive properties of various polymers.

In this study the mucoadhesive potential of nineteen different, most often referred mucoadhesive polymers was evaluated and characterized by adhesion time and total work of adhesion (TWA) of the polymer to porcine small intestinal mucosa. In addition, the influence of pH of the polymer and of method of drying on adhesion was evaluated. Aqueous polymer solutions were therefore adjusted to pH 3.0 and 7.0. Solutions were either dried by lyophilization (lyo.) or precipitated (pr.) in organic solvent and air-dried. Results of this study led to the following rank order of adhesion time: chitosan-4-thiobuthylamidine pH 3 lyo. >chitosan-4-thiobuthylamidine pH 6.5 pr.>polycarbophil-cysteine pH 3 lyo.>chitosan-4-thiobuthylamidine pH 6.5 lyo.>PAA450-cysteine pH 3 lyo.>pH 7 pr.>Carbopol 980 pH 7 pr.>Carbopol 974P pH 7 pr.>polycarbophil pH 7 pr.>980 pH 3 lyo. The rank order obtained for adhesion time was in agreement with the rank order obtained for total work of adhesion. The highest mucoadhesion was shown by thiolated polymers at pH 3.0, dried by lyophilization. In contrary, polyacrylates were most mucoadhesive in form of precipitated neutral sodium salts. Other tested polymers like natural polysaccharides, cellulose derivatives, polyvinylpirrolidone and polyethylenglycole, although previously reported as good mucoadhesives, showed low to almost no mucoadhesion. The pH of polymer and drying method were found to be important factors influencing the mucoadhesive potential of polymers.

The use of thiolated polymers as carrier matrix in oral peptide delivery--proof of concept.

It was the aim of this study to develop an oral delivery system for the peptide drug antide. The stability of the therapeutic peptide towards gastrointestinal peptidases was evaluated. The therapeutic agent and the permeation mediator glutathione were embedded in the thiolated polymer chitosan-4-thio-butylamidine conjugate (chitosan-TBA conjugate) and compressed to tablets. Drug release studies were performed in the dissolution test apparatus according to the Pharmacopoeia Europea using the paddle method and demineralized water as release medium. In order to avoid mucoadhesion of these delivery systems already in the oral cavity and oesophagus tablets were coated with a triglyceride. These tablets were orally given to pigs (weight: 50+/-2 kg; Edelschwein Pietrain). Moreover, antide was administered intravenously, subcutaneously and orally in solution. Results showed stability of antide towards pepsin, trypsin and chymotrypsin. In contrast, antide was rapidly degraded by elastase. Consequently a stomach-targeted delivery system was designed. Drug release studies demonstrated an almost zero-order controlled release of antide over 8 h. In vivo studies demonstrated a relative bioavailability of 34.4% for the subcutaneous administration. Oral administration of antide in solution led to no detectable concentrations of the drug in plasma at all. In contrast, administering antide being incorporated in the thiolated polymer resulted in a significant uptake of the peptide. The absolute and relative bioavailability was determined to be 1.1% and 3.2%, respectively.

Improved paracellular uptake by the combination of different types of permeation enhancers.

This study had the purpose to improve the paracellular uptake of drugs by combining the thiomer/reduced glutathione (GSH) permeation-enhancing system with a proteolytic enzyme. Due to the covalent binding of 2-iminothiolane to chitosan the thiomer chitosan-TBA (chitosan-4-thiobutylamidine) was obtained. Permeation studies were performed with freshly excised intestinal mucosa of guinea pigs mounted in Ussing-type chambers using on the one hand the low-molecular size marker flurescein (Na-Flu) and on the other hand the high-molecular size marker FITC-dextran. Apparent permeability coefficient (P(app)) as well as enhancement ratios (=P(app) permeation-enhancing system/P(app) control) were calculated. Trypsin, papain and bromelain displayed a permeation-enhancing effect for Na-Flu on the small intestinal mucosa. Enhancement ratios of 1.84, 1.63 and 1.78 were identified for 2% trypsin, 0.5% papain and 2% bromelain solutions, respectively. However, only bromelain could guarantee a significant permeation enhancement of FITC-dextran with a P(app) of 4.45+/-0.44 x 10(-6) cm/s representing an enhancement ratio of 1.57. A similar enhancement of FITC-dextran permeation was reached by the use of the chitosan-TBA (0.5%)/GSH (5%) system. Moreover, an additive permeation-enhancing effect of the chitosan-TBA/GSH system in combination with bromelain (2%) was observed, leading to a maximum P(app) of 5.91+/-0.51 x 10(-6) cm/s, which corresponds to an enhancement ratio of 2.1. According to these results, the combination of the thiomer/GSH system with bromelain might represent a new promising strategy in order to raise the in vivo efficacy of non-invasive administered hydrophilic macromolecular drugs.

Nasal delivery of human growth hormone: in vitro and in vivo evaluation of a thiomer/glutathione microparticulate delivery system.

It was the aim of this study to develop and evaluate a nasal microparticulate delivery system for human growth hormone (hGH) based on the thiomer polycarbophil-cysteine (PCP-Cys) in combination with the permeation mediator glutathione (GSH). Microparticles were prepared by dissolving PCP-Cys/GSH/hGH (7.5:1:1.5), PCP/hGH (8.5:1.5), and mannitol/hGH (8.5:1.5) in demineralized water, followed by lyophilization and micronization. Particles were evaluated with regard to size distribution and swelling behavior using a laser diffraction particle size analyzer. The release of fluorescence-labelled hGH from microparticles was determined in Franz diffusion chambers. In vivo studies in rats were performed comparing the nasal bioavailability achieved by PCP-Cys/GSH/hGH microparticles with that of unmodified PCP/hGH microparticles and mannitol/hGH powder. PCP-Cys/GSH/hGH and PCP/hGH microparticles showed a comparable size distribution (80% in the range of 4.8-23 microm) and swelled to almost fourfold size in phosphate-buffered saline (PBS). Both formulations exhibited almost identical sustained drug release profiles. The intranasal administration of the PCP-Cys/GSH/hGH microparticulate formulation resulted in a relative bioavailability of 8.11+/-2.15%, which represents a 3-fold and 3.3-fold improvement compared to that of PCP/hGH microparticles and mannitol/hGH powder, respectively. The study suggests that the PCP-Cys/GSH/hGH nasal microparticulate formulation might represent a promising novel tool for the systemic delivery of hGH.

Thiomers in noninvasive polypeptide delivery: in vitro and in vivo characterization of a polycarbophil-cysteine/glutathione gel formulation for human growth hormone.

This study was aimed at investigating the potential of a new polycarbophil-cysteine (PCP-Cys)/glutathione (GSH) gel formulation to enhance the permeation of the model drug human growth hormone (hGH) across nasal mucosa in vitro and in vivo. The aqueous nasal gel contained PCP-Cys, GSH, and hGH in a final concentration of 0.3%, 0.5%, and 0.6% (m/v), respectively. In vitro permeation studies were performed in Ussing chambers on freshly excised bovine nasal mucosa using fluorescence-labeled dextran (molecular mass: 4.3 kDa; FD-4) and hGH (FITC-hGH). The release profile of FITC-hGH from the gel formulation and an unmodified PCP control formulation was determined. Furthermore, in vivo studies in rats were performed comparing the PCP-Cys/GSH/hGH gel with PCP/hGH control gel and physiological saline. The permeation of FD-4 and FITC-hGH across the nasal mucosa was improved two-fold and three-fold, respectively, in the presence of PCP-Cys/GSH. The PCP-Cys/GSH/hGH gel and the PCP/hGH control gel showed the same biphasic and matrix-controlled drug release. The nasal administration of the PCP-Cys/GSH/hGH gel formulation to rats resulted in a significantly increased and prolonged hGH plasma concentration-time profile versus unmodified PCP gel and physiological saline. According to these results, PCP-Cys gels might represent a promising new strategy for systemic nasal polypeptide delivery.

Comparative evaluation of cytotoxicity of a glucosamine-TBA conjugate and a chitosan-TBA conjugate.

D-glucosamine and chitosan were modified by the immobilization of thiol groups utilizing 2-iminothiolane. The toxicity profile of the resulting D-glucosamine-TBA (4-thiobutylamidine) conjugate, of chitosan-TBA conjugate and of the corresponding unmodified controls was evaluated in vitro. On the one hand, the cell membrane damaging effect of 0.025% solutions of the test compounds was investigated via red blood cell lysis test. On the other hand, the cytotoxity of 0.025, 0.25 and 0.5% solutions of the test compounds was evaluated on L-929 mouse fibroblast cells utilizing two different bioassays: the MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide), which assess the mitochondrial metabolic activity of the cells, and the BrdU-based enzyme-linked immunosorbent assay, which measures the incorporation in the DNA of 5-bromo-2'-deoxyuridine and consequently the cell proliferation. Results of the red blood cell lysis test showed that both thiolated compounds displayed a lower membrane damaging effect causing a significantly lower haemoglobine release than the unmodified compounds. Data obtained by the MTT assay and the BrdU assay revealed a concentration dependent relative cytotoxicity for all tested compounds. The covalent linkage of the TBA-substructure to D-glucosamine did not cause a significant increase in cytotoxicity, whereas at higher concentrations a slightly enhanced cytotoxic effect was caused by the derivatisation of chitosan. In conclusion, the -TBA derivatives show a comparable toxicity profile to the corresponding unmodified compounds, which should not compromise their future use as save pharmaceutical excipients.

Matrix tablets based on thiolated poly(acrylic acid): pH-dependent variation in disintegration and mucoadhesion.

This study examined the influence of the pH on the mucoadhesive and cohesive properties of polyarcylic acid (PAA) and thiolated PAA. The pH of PAA (molecular mass: 450 kDa) and of a corresponding PAA-cysteine conjugate was adjusted to 3, 4, 5, 6, 7 and 8. The amount of immobilised thiol groups and disulfide bonds was determined via Ellman's reagent. Tablets were compressed out of each pH-batch of both thiolated and unmodified PAA and the swelling behaviour, the disintegration time and the mucoadhesiveness were evaluated. The amount of thiol/disulfide groups per gram thiolated PAA of pH 3 and pH 8 was determined to be 332 +/- 94 micromol and 162 +/- 46 micromol, respectively. The thiolated PAA tablets displayed a minimum four-fold higher water uptake compared to unmodified PAA tablets. A faster and higher water uptake of both polymer types was observed above pH 5. Thiolated polymer tablets showed a 3-20-fold more prolonged disintegration time than unmodified PAA tablets. The cohesiveness of PAA-cysteine conjugate increased at higher pH, whereas the unmodified PAA behaved inversely. A 3-7-fold stronger mucoadhesiveness was observed for the PAA-cysteine conjugate tablets compared to unmodified PAA tablets. For both thiolated and unmodified polymer the mucoadhesiveness was 2-4-fold enhanced below pH 5. The difference in mucoadhesion between the two polymer types was most pronounced at these lower pH values. In this study substantial information regarding the pH-dependence of mucoadhesion and cohesion of unmodified polyacrylates and of thiolated polyacrylates is provided, representing helpful basic information for an ameliorated deployment of these polymers.

Oral insulin delivery: the potential of thiolated chitosan-insulin tablets on non-diabetic rats.

It was the aim of this study to develop a delivery system providing an improved efficacy of orally administered insulin utilizing a thiolated polymer. 2-Iminothiolane was covalently linked to chitosan. The resulting chitosan-TBA (chitosan-4-thiobutylamidine) conjugate exhibited 453.5+/-64.1 micromol thiol groups per gram polymer. 3.1% of these thiol groups were oxidised. Additionally, the enzyme inhibitors BBI (Bowman-Birk-Inhibitor) and elastatinal were covalently linked to chitosan representing 3.5+/-0.1% and 0.5+/-0.03% of the total weight of the resulting polymer conjugate, respectively. Chitosan-TBA conjugate (5 mg), insulin (2.75 mg), the permeation mediator reduced glutathione (0.75 mg) and the two inhibitor conjugates (in each case 0.75 mg) were compressed to so-called chitosan-TBA-insulin tablets. Control tablets consisted of unmodified chitosan (7.25 mg) and insulin (2.75 mg). Chitosan-TBA-insulin tablets showed a controlled release of insulin over 8 h. In vitro mucoadhesion studies showed that the mucoadhesive/cohesive properties of chitosan were at least 60-fold improved by the immobilisation of thiol groups on the polymer. After oral administration of chitosan-TBA-insulin tablets to non-diabetic conscious rats, the blood glucose level decreased significantly for 24 h corresponding to a pharmacological efficacy of 1.69+/-0.42% (means+/-S.D.; n=6) versus s.c. injection. In contrast, neither control tablets nor insulin given in solution showed a comparable effect. According to these results the combination of chitosan-TBA, chitosan-enzyme-inhibitor conjugates and reduced glutathione seems to represent a promising strategy for the oral application of insulin.

Thiolated 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.

Thiolated chitosans: development and in vitro evaluation of a mucoadhesive, permeation enhancing oral drug delivery system.

It was the aim of this study to develop a mucoadhesive, permeation enhancing delivery system for orally administered poorly absorbed drugs. Chitosan was modified by the immobilisation of thiol groups utilising 2-iminothiolane (Traut's reagent). The permeation enhancing effect of the resulting chitosan-4-thio-butylamidine conjugate (chitosan-TBA conjugate) in combination with the permeation mediator glutathione (GSH) was evaluated in Ussing chambers on freshly excised small intestinal mucosa from guinea pigs using rhodamine 123 as marker for passive drug uptake. The mucoadhesive properties of the chitosan-TBA conjugate adjusted to pH 3, 5 and 7 were evaluated via the rotating cylinder method and via tensile studies. Release studies were performed with tablets comprising 10% cefadroxil used as model drug, 10% GSH and 80% chitosan-TBA conjugate pH 3 in 100 mM phosphate buffer pH 6.8 at 37 degrees C. Results showed a 3-fold higher permeation enhancing effect of the chitosan-TBA conjugate/GSH system in comparison to unmodified chitosan. Mucoadhesion studies revealed that the lower the pH of the thiolated chitosan is, the higher are its mucoadhesive properties. Release studies showed a sustained release of both cefadroxil and GSH over several hours. This delivery system might represent a promising novel tool in order to improve the therapeutic efficacy of various drugs which are poorly absorbed from the gastrointestinal tract.

Spiro-fused (C2)-azirino-(C4)-pyrazolones, a new heterocyclic system. Synthesis, spectroscopic studies and X-ray structure analysis.

Reaction of 1-substituted 4-acyl-5-hydroxy-3-methyl-1H-pyrazoles (2) with hydroxylamine gives the corresponding "oximes" 3, which are mainly present as (Z)-2,4-dihydro-4-[(hydroxyamino)methylene]-3H-pyrazol-3-ones. Treatment of compounds 3 with trichloroacetyl isocyanate/potassium carbonate in anhydrous diethyl ether affords 7-methyl-1,5,6-triazaspiro[2.4]hepta-1,6-dien-4-ones (4). The structure of compounds 4 was elucidated by means of single-crystal X-ray analysis (4f, 4h) and confirmed by NMR spectroscopic investigations ((1)H, (13)C).

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.

Development and in vivo evaluation of an oral delivery system for low molecular weight heparin based on thiolated polycarbophil.

PURPOSE: 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 microM 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.

In vivo evaluation of an oral salmon calcitonin-delivery system based on a thiolated chitosan carrier matrix.

PURPOSE: 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.