Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties.

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

Gelatin and Glycerine-Based Bioadhesive Vaginal Hydrogel.

AIM: The work's aim was the preparation and characterization of a hydrogel based on gelatin and glycerine, useful for site-specific release of benzydamine, an anti-inflammatory drug, able to attenuate the inflammatory process typical of the vaginal infection. OBJECTIVE: The obtained hydrogel has been characterized by Electronic Scanning Microscopy (SEM) and Differential Scanning Calorimetry (DSC). In addition, due to the precursor properties, the hydrogel exhibits a relevant mucoadhesive activity. METHODS: The swelling degree was evaluated at two different pHs and at defined time intervals. In particular, phosphate buffers were used at pH 6.6, in order to mimic the typical conditions of infectious diseases at the vaginal level, particularly for HIV-seropositive pregnant women, and pH 4.6, to simulate the physiological environment. RESULTS: The obtained results revealed that the hydrogel swells up well at both pHs. CONCLUSION: Release studies conducted at both pathological and physiological pHs have shown that benzydamine is released at the level of the vaginal mucosa in a slow and gradual manner. These data support the hypothesis of the hydrogel use for the site-specific release of benzydamine in the vaginal mucosa.

3D printing of multilayered orodispersible films with in-process drying.

The aim of this study was to prepare benzydamine hydrochloride loaded orodispersible films using modified semisolid extrusion 3D printing method. An innovative approach was developed where thin layer of drug loaded dispersion is printed and dried before printing of subsequent layers. Layer-by-layer drying as the in process step improves mechanical properties of films, uniformity of drug content and allows faster preparation of films in compounding settings due to shortening of drying time. Orodispersible films consisted of film forming maltodextrin, sorbitol as a plasticizer and hydroxyethylcellulose as a thickening agent. The height of the digital model showed excellent correlation with the disintegration time, weight, thickness and mechanical properties of prepared films. Drug content, predefined by volume of digital model and concentration of drug in print dispersion, showed excellent uniformity. The modified printing method shows great promise in a compounding production of personalized film dosage forms, and brings in possibilities such as one step preparation of films with compartmented drugs and incorporation of taste masking or release control layers.

Three-dimensional finite element analysis of silk protein rod implantation after core decompression for osteonecrosis of the femoral head.

BACKGROUND: Several methods are available for the treatment of early-stage osteonecrosis of the femoral head. Core decompression with implantation is a widely-used treatment. However, no single implant is recognized as the most effective way to prevent disease progression. Silk has high strength and resiliency. This study explored the possibility of a strong and resilient silk protein biomaterial as a new alternative implant. METHODS: We investigated the biomechanical properties of the silk protein material by regular compression, torsion, and three-point bending tests. We established three-dimensional finite element models of different degrees of femoral head osteonecrosis following simple core decompression, fibula implantation, porous tantalum rod implantation, and silk protein rod implantation. Finally, we compared the differences in displacement and surface stress under load at the femoral head weight-bearing areas between these models. RESULTS: The elastic modulus and shear modulus of the silk protein material was 0.49GPa and 0.66GPa, respectively. Three-dimensional finite element analyses demonstrated less displacement and surface stress at the femoral head weight-bearing areas following silk protein rod implantation compared to simple core decompression (p < 0.05), regardless of the extent of osteonecrosis. No differences were noted in the surface deformation or surface stress of the femoral head weight-bearing areas following silk protein rod, fibula or tantalum rod implantation (p > 0.05). CONCLUSIONS: When compared with simple core decompression, silk protein rod implantation demonstrated less displacement and surface stress at the femoral head weight-bearing area, but more than fibula or tantalum rod implantation. Similar effects on the surface stress of the femoral head between the silk rod, fibula and tantalum rod implantations, combined with additional modifiable properties support the use of silk protein as a suitable biomaterial in osteonecrosis surgery.

Development of Orodispersible Films Containing Benzydamine Hydrochloride Using a Modified Solvent Casting Method.

The aim of this study was to develop benzydamine hydrochloride-loaded orodispersible films using the modification of a solvent casting method. An innovative approach was developed when the drying process of a small-scale production was used based on a heated inert base for casting the film. During this process, two types of film-forming maltodextrins for rapid drug delivery were used. They were plasticized with two different polyols (xylitol and sorbitol). Superdisintegrant Kollidon® CL-F was tested as an excipient that can induce faster disintegration of the prepared films. The influence of the formulation parameters (dextrose equivalent of film-forming maltodextrins, a type of plasticizer, and the presence of superdisintegrant) on the disintegration time, mechanical properties, and moisture content of films was statistically evaluated using a multivariate data analysis. Orodispersible films containing maltodextrin with lower dextrose equivalent value showed better mechanical properties (tensile strength ranged from 886.6 ± 30.2 to 1484.2 ± 226.9 N cm-2), lower moisture content (0.5 ± 0.0 to 1.2 ± 0.2%), and shorter disintegration time (17.6 ± 2.9 to 27.8 ± 2.8 s). Films plasticized with xylitol showed shorter disintegration time (17.6 ± 2.9 to 29.2 ± 3.8 s) than films containing sorbitol (23.8 ± 2.9 to 31.7 ± 3.9 s). With the addition of superdisintegrant Kollidon® CL-F, a significant influence on disintegration time was not observed. The modified solvent casting method shows great promise in a small-scale laboratory production of orodispersible films, e.g., in a pharmacy lab.

Design, Development and In vitro/Ex vivo Evaluation of Mucoadhesive Buccal Film of Benzydamine Hydrochloride for the Effective Treatment of Aphthous Stomatitis.

BACKGROUND: During the recent two decades, the development of mucoadhesive drug delivery system has been gained tremendous importance to cure many recurrent diseases of the oral cavity. The drug delivery through the buccal route is quite challenging due to limited absorption area, movements of the target region and regular flow of saliva lead to the sub-therapeutic drug level in the buccal region. OBJECTIVE: The aim is to develop unidirectional release mucoadhesive buccal film for the mucosal delivery of Benzydamine Hydrochloride (WO2016126217Al, EP0812193B1) and evaluate the effects of a different grade of HPMC polymer (US5980942A) with the presumption to prolong the residence time and therapeutic effectiveness at the target site. METHODS: Mucoadhesive buccal films were prepared by solvent casting evaporation method by employing different grades of HPMC as mucoadhesive and rate controlling polymer. Total twenty-four formulations were developed using ethyl cellulose as a backing layer. The prepared films were subjected to various physicochemical parameters. RESULTS: The physicochemical parameters were found to be varied according to the type and concentration of polymer used. On the basis of in vitro drug release, desired ex-vivo mucoadhesive time, mucoadhesive force the formulations F4, F9, F14 and F19 were subjected to ex vivo drug permeation study. The F14 film containing 1% w/v of HPMC K50M was considered optimized final formulation due to higher ex vivo drug permeation. Drug-excipient compatibility was confirmed by FTIR and DSC. XRD of final formulation revealed the amorphous nature of drug. SEM indicated the perfect binding between backing and adhesive layer. CONCLUSION: The developed mucoadhesive buccal film having adequate physicochemical properties was capable to provide prolonged residence time and sustained delivery as compared to existing conventional therapies.

Vaginal Delivery of Benzydamine Hydrochloride through Liposomes Dispersed in Mucoadhesive Gels.

Liposomal vaginal drug delivery systems are important strategy in the treatment of both topical and systemic diseases. The aim of this study was to develop a vaginal delivery system for benzydamine hydrochloride (BNZ) loaded liposomes dispersed into mucoadhesive gels. The delivery system was also designed for a once a day dosage and to obtain controlled release of the BNZ. For this purpose BNZ containing gel formulations using hydroxypropyl methylcellulose (HPMC) K100M and Carbopol® 974P, which are composed of polymers that show promising potential as mucoadhesive vaginal delivery systems, were developed. In addition, a BNZ containing liposome formulation was developed for vaginal administration. To improve the vaginal retention time, liposome was incorporated in HPMC K100M and Carbopol® 974P gel formulations. This system is called lipogel. The developed BNZ liposomes have a slightly negative zeta potential (-1.50±0.16 mV), a 2.25±0.009 µm particle size and a 34% entrapment efficiency. These gels and lipogels have appropriate pH, viscosity, textural properties and mucoadhesive value for vaginal administration. Lipogels were found to be the best formulations for in vitro diffusion and ex vivo mucoadhesion. The work of mucoadhesion obtained from liposomes was in the range of 0.027±0.045 and 0.030±0.017 mJ/cm2, while the value obtained from lipogels was between 0.176±0.037 and 0.243±0.53 mJ/cm2. N1 and N2 lipogel formulations diffused 57 and 67% of BNZ respectively at the end of 24 h. Moreover, a higher mucoadhesion, which increases drug residence time in comparison to liposomes, could improve BNZ efficacy. In conclusion, BNZ mucoadhesive vaginal lipogel formulations can be promising alternatives to traditional dosage forms for vaginal topical therapy.

Identification of human flavin-containing monooxygenase 3 substrates by a colorimetric screening assay.

Human hepatic flavin-containing monooxygenase 3 is a phase I drug-metabolizing enzyme that is responsible for the oxidation of a variety of drugs and xenobiotics. This work reports on a high throughput rapid colorimetric assay for the screening of substrates or inhibitors of this enzyme. The method is based on the competition of two substrates for access to the active site of hFMO3 whereby the enzymatic product of the first drug converts nitro-5-thiobenzoate (TNB, yellow) to 5,5'-dithiobis (2-nitrobenzoate) (DTNB, colourless). Upon addition of a competing substrate, the amount of detected DNTB is decreased. The assay is validated testing three known substrates of hFMO3, namely benzydamine, tozasertib and tamoxifen. The latter drugs resulted in 41%-55% inhibition. In addition, two other drugs also classified as doping drugs, selegiline and clomiphene, were selected based on their chemical structure similarity to known substrates of hFMO3. These drugs showed 21% and 60% inhibition in the colorimetric assay and therefore were proven to be hFMO3 substrates. LC-MS was used to confirm their N-oxide products. Further characterisation of these newly identified hFMO3 substrates was performed determining their Km and kcat values that resulted to be 314 µM and 1.4 min-1 for selegiline and, 18 µM and 0.1 min-1 for clomiphene. This method paves the way for a rapid automated high throughput screening of nitrogen-containing compounds as substrates/inhibitors of hFMO3.

Potential for drug interactions mediated by polymorphic flavin-containing monooxygenase 3 in human livers.

Human flavin-containing monooxygenase 3 (FMO3) in the liver catalyzes a variety of oxygenations of nitrogen- and sulfur-containing medicines and xenobiotic substances. Because of growing interest in drug interactions mediated by polymorphic FMO3, benzydamine N-oxygenation by human FMO3 was investigated as a model reaction. Among the 41 compounds tested, trimethylamine, methimazole, itopride, and tozasertib (50 µM) suppressed benzydamine N-oxygenation at a substrate concentration of 50 µM by approximately 50% after co-incubation. Suppression of N-oxygenation of benzydamine, trimethylamine, itopride, and tozasertib and S-oxygenation of methimazole and sulindac sulfide after co-incubation with the other five of these six substrates was compared using FMO3 proteins recombinantly expressed in bacterial membranes. Apparent competitive inhibition by methimazole (0-50 µM) of sulindac sulfide S-oxygenation was observed with FMO3 proteins. Sulindac sulfide S-oxygenation activity of Arg205Cys variant FMO3 protein was likely to be suppressed more by methimazole than wild-type or Val257Met variant FMO3 protein was. These results suggest that genetic polymorphism in the human FMO3 gene may lead to changes of drug interactions for N- or S-oxygenations of xenobiotics and endogenous substances and that a probe battery system of benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities is recommended to clarify the drug interactions mediated by FMO3.

Human flavin-containing monooxygenase 3 on graphene oxide for drug metabolism screening.

Human flavin-containing monooxygenase 3 (hFMO3), a membrane-bound hepatic protein, belonging to the second most important class of phase-1 drug-metabolizing enzymes, was immobilized in its active form on graphene oxide (GO) for enhanced electrochemical response. To improve protein stabilization and to ensure the electrocatalytic activity of the immobilized enzyme, didodecyldimethylammonium bromide (DDAB) was used to mimic lipid layers of biological membranes and acted as an interface between GO nanomaterial and the hFMO3 biocomponent. Grazing angle attenuated total reflectance Fourier transform infrared (GATR-FT-IR) experiments confirmed the preservation of the protein secondary structure and fold. Electrochemical characterization of the immobilized enzyme with GO and DDAB on glassy carbon electrodes was carried out by cyclic voltammetry, where several parameters including redox potential, electron transfer rate, and surface coverage were determined. This system's biotechnological application in drug screening was successfully demonstrated by the N-oxidation of two therapeutic drugs, benzydamine (nonsteroidal anti-inflammatory) and tamoxifen (antiestrogenic widely used in breast cancer therapy and chemoprevention), by the immobilized enzyme.

Discoloration of provisional restorations after oral rinses.

PURPOSE: Oral rinses are widely used to promote periodontal health with provisional restorations during the interim period. The aim of this study was to compare the discoloration of provisional restoration materials with different oral rinses. MATERIAL AND METHODS: A total of 140 disc-shaped specimens (shade A2) (10 mm x 2 mm) were prepared from one PMMA-based (TemDent Classic(®)) and three different bis-acrylic-based (Protemp II(®), Luxatemp(®) and Fill-In(®)) provisional restoration materials (n=7). The color values (L*, a*, and b*) of each specimen were measured before and after exposure with a colorimeter, and the color changes (∆E) were calculated according to the CIE L*a*b* system. The specimens were immersed in each of the 4 oral rinses (alcohol-containing mouthwash, chlorhexidine, benzydamine HCl, benzydamine HCl and chlorhexidine) twice a day for 2 minutes. After 2 minutes of immersion in the oral rinses, the specimens were immersed in artificial saliva. The specimens were exposed to the oral rinses and the artificial saliva for 3 weeks. Two-way ANOVA, the Bonferroni test and the paired sample t-test were used for statistical analyses (p<0.05). RESULTS: Comparison of the discoloration from the oral rinses after immersion for three weeks revealed no significant differences (p>0.05). The lowest color change was observed in PMMA-based Temdent in all oral rinses (p<0.05). There were no significant differences between the bis-acryl composites after immersion in saliva or the mixture of benzydamine HCl and chlorhexidine and the alcohol-containing mouthwash for 3 weeks (p>0.05). After immersion in chlorhexidine, the color change values of Protemp II and Fill-in showed significant differences (p=0.018). Protemp II also showed less discoloration than the other bis-acryl composites, and this color change was statistically significant (p <0.05). For all oral rinses, the L* value decreased while b* values increased, and this color change was found to be statistically significant (p <0.05). A* values were found to be significantly higher with oral rinses (p<0.05), except Protemp II immersed in benzydamine HCl or alcohol-containing mouthwash. CONCLUSIONS: The type of the oral rinse did not affect the discoloration process. For long-term esthetic results, choosing MMA-based materials for provisional restorations appears to be more effective.

In vitro drug metabolism by C-terminally truncated human flavin-containing monooxygenase 3.

Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs and xenobiotics which contain a soft-nucleophiles, usually sulfur or nitrogen. As the release from the microsomal membranes can facilitate the in vitro experimental determination of drug metabolism by hFMO3, in this work we identified and eliminated the membrane anchoring sequence without affecting the activity of the enzyme and producing a soluble active enzyme. The truncated hFMO3 carrying a C-terminal deletion of 17 amino acids (tr-hFMO3) was expressed and purified from the cytosolic fraction. The tr-hFMO3 proves to be detached from the membrane, properly folded and fully active towards well-known marker substrates such as benzydamine and sulindac sulfide with measured apparent K(m) values of 45 ± 8 µM and 25 ± 4 µM, respectively. Its activity was further tested with newly discovered Aurora kinase inhibitors, Tozasertib and Danusertib, and compared to those of the wild type enzyme. The use of this soluble form of the hFMO3 enzyme as opposed to the usual microsomal preparations is advantageous for in vitro drug metabolism studies that are a requirement in the early phases of drug development by pharmaceutical industry.

Fluorescence quenching of biologically active carboxamide by aniline and carbon tetrachloride in different solvents using Stern-Volmer plots.

Fluorescence quenching of biologically active carboxamide namely (E)-2-(4-chlorobenzylideneamino)-N-(2-chlorophenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide [ECNCTTC] by aniline and carbon tetrachloride (CCl4) quenchers in different solvents using steady state method and time resolved method using only one solvent has been carried out at room temperature to understand the role of quenching mechanisms. The Stern-Volmer plot has been found to be linear for all the solvents studied. The probability of quenching per encounter p (p') was determined in all the solvents and was found to be less than unity. Further, from the studies of rate parameters and life time measurements in n-heptane and cyclohexane with aniline and carbon tetrachloride as quenchers have been shown that, the phenomenon of quenching is generally governed by the well-known Stern-Volmer (S-V) plot. The activation energy Ea (or E'a) of quenching was determined using the literature values of activation energy of diffusion Ed and the experimentally determined values of p (or p'). It has been found that, the activation energy Ea (E'a) is greater than the activation energy for diffusion Ed in all solvents. Hence, from the magnitudes of Ea (or E'a) as well as p (or p') infer that, the quenching mechanism is not solely due to the material diffusion, but there is also contribution from the activation energy.

New solid mucoadhesive systems for benzydamine vaginal administration.

The aim of this work was the realization of new formulations for vaginal application to improve the pharmacological effect of benzydamine, displaying both anti-inflammatory and antiseptic activities. For this reasons, this drug was formulated in solid dispersions, by using the mucoadhesive polymers HPMC and/or Carbopol(®), then compressed. Tablets were characterized by studies of friability, hardness, hydration, DSC, mucoadhesion and in vitro release. Kinetics, responsible for drug delivery, was investigated as well. Tablets prepared by using only HPMC showed the best results in terms of swelling and mucoadhesion (time and force) together with prolonged and complete drug release, by diffusive mechanism, through gelled layer. Despite the good mucoadhesive properties, Carbopol(®) does not represent a good excipient because, after the contact with water, it generates a spongy gel layer, not homogeneous, stiff, brittle and with breaking tendency when highly swelled. This kind of gel does not guarantee a linear drug release and could provoke discomfort because of fragment release. HPMC mucoadhesive tablets could be a proper delivery system for benzydamine administration representing a good alternative to traditional dosage forms for vaginal topical therapy.

Direct electrochemistry of drug metabolizing human flavin-containing monooxygenase: electrochemical turnover of benzydamine and tamoxifen.

This communication reports on the first electrochemical study of the human flavin-containing monooxygenase 3 (hFMO3) either absorbed or covalently linked to different electrode surfaces. Glassy carbon and gold electrodes gave reversible electrochemical signals of an active hFMO3. The midpoint potential measured for the immobilized enzyme on a glassy carbon electrode was -445 +/- 8 mV (versus Ag/AgCl). A monolayer coverage was obtained on gold functionalized with dithio-bismaleimidoethane that covalently linked surface accessible cysteines of hFMO3. A structural model of the enzyme was generated to rationalize electrochemistry results. The turnover of the active enzyme was measured with two specific drugs: tamoxifen and benzydamine. For tamoxifen, 1.7 and 8.0 microM of its N-oxide product were formed by the enzyme immobilized on glassy carbon and gold electrodes, respectively. In the case of benzydamine, a K(M) of 44 +/- 5 microM was measured upon application of a -600 mV bias to the enzyme immobilized on the glassy carbon electrode that is in good agreement with the values published for microsomal hFMO3 where NADPH is the electron donor.

Thermally sensitive gels based on chitosan derivatives for the treatment of oral mucositis.

The aim of the present work was the development of a thermally sensitive mucoadhesive gel based on chitosan derivatives for the treatment of oral mucositis. Trimethyl chitosan (TMC) and methylpyrrolidinone chitosan (MPC) were considered. They were mixed with glycerophosphate (GP) according to different polymer/GP molar ratios and characterized for gelation properties by means of rheological analysis in comparison with chitosan. The influence of molecular weight and substitution degree (SD) of TMC on gelation temperature and time was investigated. The mucoadhesive properties of the mixtures were also assessed using porcine buccal mucosa. The best properties were shown by TMC with high MW and low SD mixed with GP according to 1:2molar ratio. Such mixture was loaded with benzydamine hydrochloride, an anti-inflammatory drug with antimicrobial properties and subjected to in vitro drug release and wash away test. The formulation based on TMC/GP mixture was able to prolong drug release and to withstand the removal physiological mechanisms. The antimicrobial properties of both vehicle and formulation were investigated. Also in absence of drug, TMC/GP mixture was characterized by antimicrobial properties.

Benzydamine hydrochloride buccal bioadhesive gels designed for oral ulcers: preparation, rheological, textural, mucoadhesive and release properties.

This study developed and examined the characterization of Benzidamine hydrochloride (BNZ) bioadhesive gels as platforms for oral ulcer treatments. Bioadhesive gels were prepared with four different hydroxypropylmethylcellulose (HPMC) types (E5, E15, E50 and K100M) with different ratios. Each formulation was characterized in terms of drug release, rheological, mechanical properties and adhesion to a buccal bovine mucosa. Drug release was significantly decreased as the concentration and individual viscosity of each polymeric component increased due to improved viscosity of the gel formulations. The amount of drug released for the formulations ranged from 0.76 +/- 0.07 and 1.14 +/- 0.01 (mg/cm2 +/- SD). Formulations exhibited pseudoplastic flow and all formulations, increasing the concentration of HPMC content significantly raised storage modulus (G'), loss modulus (G''), dynamic viscosity (eta') at 37 degrees C. Increasing concentration of each polymeric component also significantly improved the hardness, compressibility, adhesiveness, cohesiveness and mucoadhesion but decreased the elasticity of the gel formulations. All formulations showed non-Fickian diffusion due to the relaxation and swelling of the polymers with water. In conclusion, the formulations studied showed a wide range of mechanical and drug diffusion characteristics. On the basis of the obtained data, the bioadhesive gel formulation which was prepared with 2.5% HPMC K 100M was determined as the most appropriate formulation for buccal application in means of possessing suitable mechanical properties, exhibiting high cohesion and bioadhesion.

Formulation studies of benzydamine mucoadhesive formulations for vaginal administration.

BACKGROUND: Vaginal cavity represents a good site for drug administration and delivery. AIM: The aim of this work was the design of new mucoadhesive semisolid dosage forms for vaginal delivery of benzydamine. METHOD: Simple gels, obtained by using sodium carboxymethylcellulose (NaCMC) and hydroxyethylcellulose (HEC), were employed as water phase of an oil-in-water emulsion (O/W cream) to obtain emulgels, more stable and manageable than gels. Successively, in order to modify the emulgel consistency, the ingredient cetostearylic alcohol was replaced by the same amount of gel or vaseline. All the preparations were submitted to mucoadhesion and rheological, extrusion, and release studies and compared to market vaginal cream Tantum Rosa. RESULTS: HEC formulations showed good drug release profiles and good rheological behavior but low mucoadhesion strength, whereas NaCMC (4% gel) formulations had better drug release and very high mucoadhesive strength. However, the presence of NaCMC 4% conferred too much viscosity to the preparation. Taking into consideration all performances, the most suitable formulations for vaginal applications resulted in those containing NaCMC (3% gel) and with gel replacing cetostearylic alcohol as they showed good ex vivo performances in terms of manageability and high bioadhesion to vaginal mucosa.

Spectrophotometric determination of benzydamine HCl, levamisole HCl and mebeverine HCl through ion-pair complex formation with methyl orange.

A simple, rapid and sensitive spectrophotometric method has been proposed for the assay of benzydamine HCl (BENZ), levamisole HCl (LEV) and mebeverine HCl (MBV) in bulk and pharmaceutical formulations. The method based on the reaction of the selected drugs with methyl orange (MO) in buffered aqueous solution at pH 3.6. The formed yellow ion-pair complexes were extracted with dichloromethane and measured quantitatively with maximum absorption at 422 nm. The analytical parameters and their effects on the reported systems are investigated. The extracts are intensely colored and very stable at room temperature. The calibration graphs were linear over the concentration range of 2-10 microg ml(-1) for BENZ, 6-24 microg ml(-1) for LEV and 4-14 microg ml(-1) for MBV. The stoichiometry of the reaction was found to be 1:1 in all cases and the conditional stability constant (K(f)) of the complexes have been calculated. The proposed method was successfully extended to pharmaceutical preparations-tablets. Excipients used as additive in commercial formulations did not interfere in the analysis. The proposed method can be recommended for quality control and routine analysis where time, cost effectiveness and high specificity of analytical technique are of great importance.

Novel hydrogels via click chemistry: synthesis and potential biomedical applications.

A novel procedure for the in situ rapid chemical gelation of aqueous solutions of hyaluronan has been employed. In brief, water-soluble polysaccharide derivatives bearing side chains endowed with either azide or alkyne terminal functionality have been prepared. When the latter two types of derivatives are mixed together in aqueous solution they give rise to a 1,3-dipolar cycloaddition reaction resulting in fast gelation (in the presence of catalytic amounts of Cu(I)) at room temperature. Gel formation has been characterized rheologically and could also be followed qualitatively by means of IR spectroscopy. The resulting gels have been studied in terms of swelling properties and, in particular, NMR spectral features. Carrying out the gelation process in aqueous solutions of benzidamine and doxorubicin, respectively, the polysaccharide networks acted as drug reservoirs. The doxorubicin release resulted in well controllable acting upon the gels degree of cross-linking. Finally, formation of the click-gels using aqueous suspensions of Saccharomices cerevisiae yeast cells allowed the obtainment of scaffolds inside which cells were homogeneously distributed and smoothly adhered to the inner pores surfaces, according to SEM analysis. After 24 h about 60% of the entrapped cells exhibited proliferating activity. Click-gels prepared as detailed herein do have a number of positive features that make them, in perspective, materials of choice for drug release and tissue engineering manipulations.