Comparative in vitro study of different chitosan-complexing agent conjugates.

In this study we analysed the bioadhesive properties and the enzyme inhibitory effects of different chitosan-complexing agent conjugates. Etylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), respectively, were covalently attached to chitosan by the formation of amide bonds between the primary amino group of the polymer and the carboxylic acid groups of the complexing agents. Whereas almost each primary amino groups of chitosan could be modified by EDTA, DTPA was bound to only 63.8 +/- 5.8% (n = 3; +/- SD) of the amino groups of chitosan. The remaining primary amino groups of the chitosan-DTPA conjugate lead to strongly reduced adhesive properties, with a maximum detachment force of 3.0 +/- 1.3 mN in contrast to the chitosan-EDTA conjugate with 81.7 +/- 9.9 mN in the tensil studies described here (n = 4; +/- SD). However, both polymer conjugates displayed an inhibitory effect towards the zinc-dependent proteases carboxypeptidase A (EC 3.4.17.1) and aminopeptidase N (EC 3.4.11.2). The results of this comparative study should provide substantial knowledge for the development of bioadhesive polymers as auxiliary agents for the peroral administration of peptide and protein drugs.     

In vitro evaluation of chitosan-EDTA conjugate polyplexes as a nanoparticulate gene delivery system

It was the purpose of this study to evaluate the potential of different molecular-weight chitosan-EDTA conjugates as a carrier matrix for nanoparticulate gene delivery systems. Covalent binding of EDTA to more than one chitosan chain provides a cross-linked polymer that is anticipated to produce stabilized particles. pDNA/chitosan-EDTA particles, generated via coazervation, were characterized in size and zeta potential by electrophoretic light scattering and electron microscopy. Stability was investigated at different pH values by enzymatic degradation and subsequent gel retardation assay. Lactate dehydrogenase assay was performed to determine toxicity. Furthermore, transfection efficiency into Caco-2 cells was assessed using a beta-galactosidase reporter gene. Chitosan-EDTA produced from low-viscous chitosan with 68% amino groups being modified by the covalent attachment of EDTA showed the highest complexing efficacy resulting in nanoparticles of 43 nm mean size and exhibiting a zeta potential of +6.3 mV. These particles were more stable at pH 8 than chitosan control particles. The cytotoxicity of chitosan-EDTA particles was below 1% over a time period of 4 hours. These new nanoplexes showed 35% improved in vitro transfection efficiency compared with unmodified chitosan nanoparticles. According to these results, the chitosan-EDTA conjugate may be a promising polymer for gene transfer.     

Synthesis and In Vitro Evaluation of Chitosan-EDTA-Protease-Inhibitor Conjugates Which Might Be Useful in Oral Delivery of Peptides and Proteins

Purpose. To develop a novel mucoadhesive polymer that protects peptide drugs from degradation by secreted as well as membrane-bound proteases in the intestine, and to evaluate this polymer in vitro. Methods. The serine protease inhibitors antipain, chymostatin and elastatinal were covalently linked to chitosan (poly-[l 4]--D-glucosamine). Thereafter, the complexing agent ethylenediaminete-traacetic acid (EDTA) was bound to the remaining primary amino groups of the polymer. The inhibitory effect of the resulting polymer-conjugate towards trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), elastase (3.4.21.36), carboxypeptidase A (EC 3.4.17.1), carboxypeptidase B (EC 3.4.17.2) and aminopeptidase N (EC 3.4.11.2) as well as its mucoadhesive properties were evaluated in vitro. Results. Whereas the novel polymer-conjugate exhibited excellent swelling properties, its adhesive force was under our assay conditions 42% lower than that of unmodified chitosan. However, the polymer-conjugate showed a strong inhibitory activity towards all tested serine proteases. Due to its additional high binding affinity towards bivalent metal ions, it also inhibited the Zn²⁺-dependent exopeptidases carboxypeptidase A, B and aminopeptidase N. Conclusions. The novel mucoadhesive polymer-conjugate described in this study seems to be a useful tool in overcoming the enzymatic barrier to perorally administered therapeutic peptides and proteins.     

Development and in Vitro Evaluation of Systems to Protect Peptide Drugs from Aminopeptidase N

Purpose. Develop and evaluate systems to prevent aminopeptidase N caused enzymatic degradation of perorally administrated peptide drugs. Methods. Bacitracin was covalently bound to the unabsorbable carrier matrix poly(acrylic acid) (paa) in order to avoid any dilution effects of the inhibitor in the intestine as well as systemic toxic side effects. The inhibitory effect of this conjugate, of neutralized paa and N-acetylcysteine was evaluated using a brush border membrane model. Results. Whereas within 6 h of incubation 65.3 ± 3.7 mol/1 of the substrate (L-leucine p-nitroanilide) was hydrolyzed under our assay conditions, this metabolism was reduced to 44.5 ± 6.3 mol/1 and 49.0 ± 8.8 mol/1 (n = 3–5; ± S.D.) using 1.5% bacitracin-polymer conjugate and 0.5% N-acetylcysteine, respectively. The same amount of bacitracin as immobilized to the polymer exhibited a comparably weaker inhibitory effect. Neutralized paa did not inhibit membrane bound aminopeptidase N. Covering the membrane with a thin mucus layer led to a significantly lowered inhibitory effect of all tested agents. Conclusions. The immobilization of enzyme inhibitors to a carrier matrix and the use of N-acetylcysteine as a novel inhibitor are promising strategies in order to overcome the enzymatic barrier caused by membrane bound peptidases. However the use of effective mucolytic agents seems to be a prerequisite.     

Oral gene delivery: Strategies to improve stability of pDNA towards intestinal digestion

Purpose: Gastrointestinal (GI) nucleases are responsible for a rapid presystemic degradation of orally administered transgenes. Within the current study, the activity of these degrading enzymes as well as the effect of various nuclease inhibitors on the degradation process were evaluated in order to assess their potential as auxiliary agents in oral gene delivery.

Methods: Digestion assays of pDNA with DNaseI and in GI juices were performed in absence and presence of inhibitors. Consequently, a chitosan conjugate with covalently bound ethylendiaminetetraacetic acid disodium salt dihydrat (EDTA) was synthesized and its nuclease inhibitory properties were evaluated.

Results: Small intestinal juice was shown to possess a nuclease activity per millilitre corresponding to 0.02 Kunitz units of DNaseI. Inhibition studies revealed that inhibitory activity followed the ranking: EDTA > sodium dodecyl sulfate (SDS) > aurintricarboxylic acid (ATA) > poly (acrylic acid) > cysteine. The chitosan–EDTA conjugate offered good nuclease inhibiting properties.

Conclusion: This study determined the nuclease activity of native porcine small intestinal juice as well as enterocytes homogenate. Moreover, several promising strategies to overcome this enzymatic barrier were identified.     

Oral gene delivery: Strategies to improve stability of pDNA towards intestinal digestion

PURPOSE: Gastrointestinal (GI) nucleases are responsible for a rapid presystemic degradation of orally administered transgenes. Within the current study, the activity of these degrading enzymes as well as the effect of various nuclease inhibitors on the degradation process were evaluated in order to assess their potential as auxiliary agents in oral gene delivery. METHODS: Digestion assays of pDNA with DNaseI and in GI juices were performed in absence and presence of inhibitors. Consequently, a chitosan conjugate with covalently bound ethylendiaminetetraacetic acid disodium salt dihydrat (EDTA) was synthesized and its nuclease inhibitory properties were evaluated. RESULTS: Small intestinal juice was shown to possess a nuclease activity per millilitre corresponding to 0.02 Kunitz units of DNaseI. Inhibition studies revealed that inhibitory activity followed the ranking: EDTA > sodium dodecyl sulfate (SDS) > aurintricarboxylic acid (ATA) > poly (acrylic acid) > cysteine. The chitosan-EDTA conjugate offered good nuclease inhibiting properties. CONCLUSION: This study determined the nuclease activity of native porcine small intestinal juice as well as enterocytes homogenate. Moreover, several promising strategies to overcome this enzymatic barrier were identified.     

Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N.

The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides.     

Insulin Aggregation in Aqueous Media and Its Effect on Alpha-Chymotrypsin-Mediated Proteolytic Degradation

Self-association of zinc–insulin monomers into dimers and hexamers may lead to enhanced protection of the peptide from proteolytic degradation. The present study has been undertaken to investigate the relationship, if any, between the rate of enzymatic degradation of insulin by a protease, alpha-chymotrypsin, and the extent of insulin aggregation in aqueous solutions. Insulin solutions (0.6 mg/ml) containing varying proportions of dimer and hexamer were obtained by adding ethylene diamine tetraacetic acid (EDTA) within a concentration range of 0.005 to 0.040 mM. As the EDTA concentration was increased above 0.040 mM, a complete dissociation of hexamers to dimers occurred and the rate of enzymatic degradation reached its maximum. The overall first-order rate constants appeared to be linearly related to the square of EDTA concentrations. The apparent first-order rate constants for dimer and hexamer degradation obtained from a linear plot of rate constant versus EDTA squared concentration were found to be 0.02800 ± 0.00065 and 0.00798 ± 0.00075 min^–1, respectively. Two major insulin degradation products were also detected and the kinetics of product appearance agreed well with the disappearance kinetics of insulin. The results indicated that the degradation of insulin dimers by alpha-chymotrypsin is about 3.5 times faster than the degradation of the hexamer. The second-order dependency of degradation rate on EDTA concentration might be due to the fact that insulin hexamers contain two zinc ions which are sequestered by two EDTA molecules. Chelation of zinc ions by EDTA lead to hexamer deaggregation to dimers as was evidenced from a circular dichroism study. Formation of three dimer species from one hexamer aggregate should theoretically enhance the rate of degradation threefold, a value consistent with the experimentally determined ratio of 3.5.     

Vaginal Permeability and Enzymatic Activity Studies in Normal and Ovariectomized Rabbits

Purpose. This study was initiated to develop an animal model, using ovariectomized rabbits, for the post-menopausal human, based on in vitro vaginal tissue permeability and aminopeptidase activity. Methods. An enkephalin derivative [D-ala²,N-methyl-phe⁴-glycol⁵][tyrosyl-3,5-³H] enkephalin {[³H] RX 783006), which has relative enzymatic stability to aminopeptidases and dipeptidyl peptidase, was used as a model peptide drug for permeability experiments. Aminopeptidase activity in vaginal homogenates, as well as in tissue pieces, was determined using 4-methoxy-2-naphthylamides of leucine, alanine, arginine, and glutamic acid as specific substrates. In addition, histological examination of normal and ovariectomized vaginal tissues was performed. Results. Vaginal permeability of the drug was significantly increased in the ovariectomized compared to the intact animal. The full vaginal tissue became thinner and mucosal epithelial thickness was reduced about twofold after ovariectomization and vaginal cells from the castrated rabbit were typically immature. Aminopeptidase activity, leucine aminopeptidase, aminopeptidase B and A, was the same in vaginal tissue homogenates and whole-tissue specimens in both normal and ovariectomized rabbits whereas the activity of aminopeptidase N was significantly decreased in ovariectomized as compared to normal rabbits. Conclusions. Based on the present data, the ovariectomized rabbit may be useful as an animal model for postmenopausal vaginal studies.     

Polymers with Thiol Groups: A New Generation of Mucoadhesive Polymers?

Purpose. To improve the mucoadhesive properties of polycarbophil by the introduction of sulfhydryl groups. Methods. Mediated by a carbodiimide, cysteine was covalently bound to polycarbophil (PCP) forming amide bonds between the primary amino group of the amino acid and the carboxylic acid moieties of the polymer. The amount of covalently attached cysteine and the formation of disulfide bonds within the modified polymer were determined by quantifying the share of thiol groups on the polymer conjugates with Ellman's reagent. The adhesive properties of polycarbophil-cysteine conjugates were evaluated in vitro on excised porcine intestinal mucosa by determining the total work of adhesion (TWA). Results. Depending on the weight-ratio of polycarbophil to cysteine at the coupling reaction, e.g., 16:1 and 2:1, 0.6 ± 0.7 mole and 5.3 ± 2.4 mole cysteine, respectively, were covalently bound per g polymer. The modified polymer displayed improved internal cohesive properties due to the formation of interchain disulfide bonds within the polymer in aqueous solutions at pH-values above 5. Adhesion studies revealed strongly improved adhesive properties. Whereas the TWA was determined to be 104 ± 21 J for the unmodified polymer, it was 191 ± 47 J for the polymer-cysteine conjugate 16:1 and 280 ± 67 J for the polymer-cysteine conjugate 2:1. Conclusions. Polymers with thiol groups might represent a new generation of mucoadhesive polymers displaying comparatively stronger adhesive properties.     

Synthesis and <Emphasis Type="BoldItalic">in Vitro</Emphasis> Evaluation of a Novel Chitosan–Glutathione Conjugate

Purpose It was the aim of this study to synthesize and characterize a novel chitosan–glutathione (GSH) conjugate providing improved mucoadhesive and permeation-enhancing properties.Methods Mediated by carbodiimide and N-hydroxysuccinimide, glutathione was covalently attached to chitosan via the formation of an amide bond. The adhesive properties of chitosan–GSH conjugate were evaluated in vitro on freshly excised porcine mucosa via tensile studies and the rotating cylinder method. The cohesive properties and stability of the resulting conjugate were evaluated by disintegration test and by oxidation experiments, respectively. The permeation-enhancing effect of the chitosan–GSH/GSH system was evaluated in Ussing chambers by using rhodamine 123 as model compound.Results The obtained conjugate displayed 265.5 μmol immobilized free thiol groups and 397.9 μmol disulfide bonds per gram polymer. Because of the formation of disulfide bonds within the polymer, the stability of matrix tablets could be strongly improved. In tensile studies, the total work of adhesion of the conjugate was determined to be 9.9-fold increased in comparison to unmodified chitosan. Results from the rotating cylinder method showed more than 55-fold increase in the adhesion time of thiolated chitosan vs. unmodified chitosan. In addition, the conjugate in combination with GSH displayed a 4.9-fold higher permeation-enhancing effect compared with unmodified chitosan.Conclusions Because of the improved mucoadhesive and cohesive properties, and the strong permeation-enhancing effect of the chitosan–GSH conjugate/GSH system, the novel thiolated chitosan seems to represent a promising multifunctional excipient for various drug delivery systems.     

Design and evaluation of a chitosan–aprotinin conjugate for the peroral delivery of therapeutic peptides and proteins susceptible to enzymatic degradation

One main barrier for the peroral administration of therapeutic peptides and proteins is the enzymatic barrier, that is mediated by luminally secreted and membrane bound proteolytic enzymes. It was the aim of the study to synthesise, characterise and evaluate a novel polymer–inhibitor conjugate in order to improve the bioavailability of orally-administered peptides and proteins. The trypsin/chymotrypsin inhibitor aprotinin was covalently bound to chitosan. The percentage of the inhibitor in the polymer–inhibitor conjugate (m/m) was determined to be between 1.11 ± 0.36 and 1.92 ± 0.05%. In vitro enzyme assays clearly demonstrated the potential of the novel conjugate to inhibit trypsin and chymotrypsin. Moreover, studies in rats were performed to evaluate the efficacy of the conjugate in vivo. Eight hours after oral administration of tablets containing insulin and the novel chitosan–aprotinin conjugate, the mean blood glucose level decreased to 84 ± 6%. In contrast, the mean blood glucose level in the control group increased to 121 ± 8% of the initial measured blood glucose level. In conclusion it was demonstrated that chitosan–aprotinin conjugate represents a novel and promising tool for the oral administration of therapeutic peptides and proteins susceptible to enzymatic degradation caused by trypsin and chymotrypsin.     

In Vitro-In Vivo Evaluation of a Controlled Release Buccal Bioadhesive Device for Oral Drug Delivery

Purpose. To investigate the use of buccal bioadhesive device in targeting controlled drug delivery to the gastrointestinal tract. Methods. A three-leg crossover study was designed to evaluate the application of buccal bioadhesive device for providing controlled drug delivery to the gastrointestinal tract of a model drug cyanocobalamin in four healthy adult male beagle dogs. Results. In vitro dissolution studies using deionized water as the medium indicated that 100% of the drug was released within 15 min from a immediate release oral capsule formulation, whereas 90% of the drug was released within a period of 18 hrs from a buccal bioadhesive device formulation. Drug release from the buccal bioadhesive devices appeared to follow Higuchi's square root of time dependent model. The terminal half-life of the drug following I.V. administration in four dogs was found to be 16.4 ± 2.4 hrs. Following immediate release oral capsule administration of the drug C_max, t_max and bioavailability were 2333 ± 1469 ng/L, 2.5± 1.0 hrs and 14.1 ± 7.9%, respectively. Following buccal bioadhesive device administration of the drug C_max, t_max and bioavailability were 4154 ± 1096 ng/L, 11 ± 1.2 hrs and 35.8 ± 4.1%, respectively. Significantly higher bioavailability of the drug was observed with the buccal bioadhesive device administration when compared to the immediate release oral capsule. Conclusions. The buccal bioadhesive device appears to improve the oral bioavailability of cyanocobalamin by providing controlled delivery of the drug to the gastrointestinal tract.     

Development and in vitro evaluation of a drug delivery system protecting from trypsinic degradation

We have been developing a delivery system based on a novel polymer conjugate protecting perorally administered (poly)peptide drugs from trypsinic degradation. The trypsin inhibitor antipain was, therefore, covalently attached to the mucoadhesive polymer chitosan. The protective effect of resulting chitosan–antipain conjugates was quantified by an enzyme assay. In contrast to the unmodified polymer, chitosan–antipain conjugates exhibited a significant inhibitory effect towards enzymatic activity of trypsin (EC 3.4.21.4). Moreover, the mucoadhesive force of chitosan was not influenced by the slight modification. Based on a chitosan–antipain conjugate, a drug delivery system was generated using insulin as model drug. Tablets containing 5% polymer conjugate demonstrated after incubation with trypsin (180 spectrophotometric BAEE units/ml) for 1.5 h in lateral parts of the swelled dosage form a 13.3±2.3% (mean±S.D., n=3) minor proteolysis of matrix embedded insulin compared to tablets lacking the polymer conjugate. In the inner part of the swelled dosage form containing the conjugate proteolysis was completely inhibited, whereas in control tablets 11.3±9.5% (mean±S.D., n=3) insulin was degraded. Furthermore, a controlled drug release over a period of 6 h was guaranteed by the delivery system. According to these results, the novel chitosan–antipain conjugates shielding from luminal enzymatic attack may be a useful tool for the peroral administration of mainly trypsinic degraded peptide and protein drugs.     

In Vitro Evaluation of Microparticles and Polymer Gels for Use as Nasal Platforms for Protein Delivery

Purpose. Nasal delivery of protein therapeutics can be compromised by the brief residence time at this mucosal surface. Some bioadhesive polymers have been suggested to extend residence time and improve protein uptake across the nasal mucosa. We examined several potential polymer platforms for their in vitro protein release, relative bioadhesive properties and induction of cytokine release from respiratory epithelium. Methods. Starch, alginate, chitosan or Carbopol^® microparticles, containing the test protein bovine serum albumin (BSA), were prepared by spray-drying and characterized by laser diffraction and scanning electron microscopy. An open-membrane system was used to determine protein release profiles and confluent, polarized Calu-3 cell sheets were used to evaluate relative bioadhesion, enhancement of protein transport and induction of cytokine release in vitro. Results. All spray-dried microparticles averaged 2–4 m in diameter. Loaded BSA was not covalently aggregated or degraded. Starch and alginate microparticles released protein more rapidly but were less adhesive to polarized Calu-3 cells than chitosan and Carbopol^® microparticles. Protein transport across polarized Calu-3 cells was enhanced from Carbopol^® gels and chitosan microparticles. A mixture of chitosan microparticles with lysophosphatidylcholine increased protein transport further. Microparticles prepared from either chitosan or starch microparticles, applied apically, induced the basolateral release of IL-6 and IL-8 from polarized Calu-3 cells. Release of other cytokines, such as IL-l, TNF-, GM-CSF and TGF-, were not affected by an apical exposure to polymer formulations. Conclusions. We have described two systems for the in vitro assessment of potential nasal platforms for protein delivery. Based upon these assessments, Carbopol^® gels and chitosan microparticles provided the most desirable characteristics for protein therapeutic and protein antigen delivery, respectively, of the formulations examined.     

Design and In Vivo Evaluation of An Oral Delivery System for Insulin

Purpose. To develop an oral controlled release system for insulin. Methods. The polymer-inhibitor conjugates carboxymethylcellulose (CMC)-Bowman-Birk inhibitor and CMC-elastatinal were homo- genized with polycarbophil-cysteine conjugate, insulin, and mannitol, compressed to 2 mg microtablets and enteric coated with a polymethacrylate. The protective effect of this delivery system for insulin towards enzymatic degradation, as well as the release profile, was evaluated in vitro. In addition, the effect of the dosage form on glucose levels of diabetic mice was determined. Results. Tablets containing the CMC-inhibitor conjugates showed a strong protective effect for insulin. Whereas 91.6 ± 7.4% (mean ± SD, n = 3) of insulin in the dosage form without the inhibitor conjugates has been degraded within 3 h of incubation in an artificial intestinal fluid containing physiological concentrations of trypsin, chymotrypsin, and elastase, 49.7 ± 5.5% (mean ± SD, n = 3) of insulin remained stable in the delivery system containing the polymer-inhibitor conjugates. Additionally, polycarbophil-cysteine (PCP-Cys) provides high cohesiveness of the dosage form, due to the formation of inter- as well as intramolecular disulfide bonds within the polymer matrix. According to this, a controlled release of insulin could be achieved over a time period of 10 h. Furthermore, in vivo studies in diabetic mice showed a decrease in basal glucose levels of 20% to 40% during a time period of 80 h. Conclusions. Mucoadhesive polymer-inhibitor conjugates might represent a promising excipient in delivery systems for oral (poly)peptide delivery.     

Development of buccal drug delivery systems based on a thiolated polymer

The purpose of the present study was to investigate the benefit of thiolated polymers (thiomers) for the development of buccal drug delivery systems. L-Cysteine was thereby covalently attached to polycarbophil (PCP) mediated by a carbodiimide. The resulting conjugate displayed 140.5+/-8.4 microM thiol groups per gram polymer. Disintegration studies were carried out with tablets based on unmodified polymer and conjugated polymer, respectively. Due to the formation of disulfide bonds within the thiolated polymer, the stability of matrix-tablets based on this polymer was strongly improved. Additionally tensile studies were carried out, which were in good correlation with further results obtained by mucoadhesion studies, using the rotating cylinder method. These results showed that tablets based on thiolated PCP remained attached on freshly excised porcine mucosa 1.8 times longer than the corresponding control. Moreover, the enzyme inhibitory properties of polymers were evaluated as well. Thiolated PCP increased the stability of the synthetic substrate for aminopeptidase N-leu-p-nitroanilide (N-leu-pNA) and the model drug leucin-enkephalin (leu-enkephalin) against enzymatic degradation on buccal mucosa. Due to the use of thiolated polymers also a controlled drug release for leu-enkephalin was guaranteed over a time period for more than 24 h. Results of the present studies suggest that thiolated polymers represent a very useful tool for buccal delivery of peptide drugs.     

Stabilization of methionine enkephalin in various rabbit mucosal extracts by enzyme inhibitors

The inhibition of the enzymatic degradation of methionine enkephalin (Met-Enk) was investigated kinetically in nasal, rectal, and vaginal extracts of rabbits with and without inhibitors, such as puromycin (PM), amastatin (AM), thiorphan (TP), Na₂EDTA, and thimerosal (TM), alone or in combination, by analyzing the parent peptide and its hydrolytic fragments by HPLC. The effects of variation of pH in the nasal extracts, and the addition of 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CyD) on the stabilization of Met-Enk were also studied. The degradation of Met-Enk was found to be fastest at around pH 7, indicating that the activity of enkephalin-degrading enzymes is optimal at this pH. Addition of 2-HP-β-CyD (10%) to the nasal, rectal, and vaginal extracts was noted to reduce the first-order degradation rate constants for Met-Enk by 2.5-2.8-fold, compared to the control. AM alone inhibited the enzymatic degradation of Met-Enk with IC₅₀ values of 3.5 and 0.22 μM for the rectal and vaginal extracts, respectively, whereas PM was found to be approx. 14.2- and 26.8-fold less potent than AM, respectively. The effects of both aminopeptidase inhibitors in the nasal extracts were smaller. Even at 50 μM, TP (a potent enkephalinase A inhibitor) alone revealed only a small increase of Met-Enk stability in the various mucosal extracts, however, EDTA (5 μM) was observed to inhibit enzymatic hydrolysis considerably by blocking both enkephalinase A and B and, to some extent, aminopeptidase. On the other hand, TM (0.05%) was found to be a new and potent inhibitor for enkephalinase B and aminopeptidases, which was more potent than AM (50 μM) in inhibiting the degradation of Met-Enk in various mucosal extracts. Furthermore, the addition of TM (0.01%) to a combination of AM (50 μM) and EDTA (5 μM) was observed to protect Met-Enk from enzymatic degradation in nasal, rectal, and vaginal extracts by more than 90%, after 24 h of incubation, by inhibiting almost completely all the enkephalin-degrading enzymes present in the incubation mixtures.     

Neutral endopeptidase and alcohol consumption, experiments in neutral endopeptidase-deficient mice

Alcohol consumption was investigated in mice which were rendered deficient in the peptide-degrading enzyme neutral endopeptidase (EC 3.4.24.11) (NEP-/-) by gene targeting and compared to alcohol consumption in corresponding wild type mice (NEP+/+). Mice were offered a free choice to drink tap water or 10% alcohol. The NEP-/- mice consumed significantly more alcohol ( approximately 42%) than the NEP+/+ mice, whereas no significant differences were observed in the total fluid consumption. The daily food consumption of alcohol naive NEP-/- animals was elevated ( approximately 29%). Furthermore, the activities of peptidases closely related to neutral endopeptidase were analysed ex vivo in several brain regions from NEP-/- and NEP+/+ mice not treated with alcohol. There was no obvious compensation for the total loss of neutral endopeptidase by the functionally related peptidases angiotensin-converting enzyme and aminopeptidase N. In vitro, the degradation of exogenously applied [Leu(5)]enkephalin was not reduced in membrane preparations of those brain regions assayed in NEP-/- mice. A small reduction in [Leu(5)]enkephalin degradation was detected in striatal membrane preparations of NEP-/- mice, if aminopeptidase N was additionally blocked by bestatin or amastatin.     

Niosomes and Polymeric Chitosan Based Vesicles Bearing Transferrin and Glucose Ligands for Drug Targeting

Purpose. To prepare polymeric vesicles and niosomes bearing glucose or transferrin ligands for drug targeting. Methods. A glucose-palmitoyl glycol chitosan (PGC) conjugate was synthesised and glucose-PGC polymeric vesicles prepared by sonication of glucose-PGC/ cholesterol. N-palmitoylglucosamine (NPG) was synthesised and NPG niosomes also prepared by sonication of NPG/ sorbitan monostearate/ cholesterol/ cholesteryl poly-24-oxyethylene ether. These 2 glucose vesicles were incubated with colloidal concanavalin A gold (Con-A gold), washed and visualised by transmission electron microscopy (TEM). Transferrin was also conjugated to the surface of PGC vesicles and the uptake of these vesicles investigated in the A431 cell line (over expressing the transferrin receptor) by fluorescent activated cell sorter analysis. Results. TEM imaging confirmed the presence of glucose units on the surface of PGC polymeric vesicles and NPG niosomes. Transferrin was coupled to PGC vesicles at a level of 0.60 ± 0.18 g of transferrin per g polymer. The proportion of FITC-dextran positive A431 cells was 42% (FITC-dextran solution), 74% (plain vesicles) and 90% (transferrin vesicles). Conclusions. Glucose and transferrin bearing chitosan based vesicles and glucose niosomes have been prepared. Glucose bearing vesicles bind Con-A to their surface. Chitosan based vesicles are taken up by A431 cells and transferrin enhances this uptake.