Ylenia Zambito

Gel-forming erodible inserts for ocular controlled delivery of ofloxacin

A new application of high molecular weight (400 kDa) linear poly(ethylene oxide) (PEO) in gel-forming erodible inserts for ocular controlled delivery of ofloxacin (OFX) has been tested in vitro and in vivo. Inserts of 6 mm diameter, 20 mg weight, medicated with 0.3 mg OFX, were prepared by powder compression. The in vitro drug release from inserts was mainly controlled by insert erosion. The erosion time scale was varied by compounding PEO with Eudragit L100 (EUD) 17% neutralized (EUDNa17) or 71% neutralized (EUDNa71). The insert erosion rate depended on the strength of interpolymer interactions in the compounds, and on the hydrophilic-hydrophobic balance of compounds. Immediately after application in the lower conjunctival sac of the rabbit eyes, the inserts based on plain PEO, PEO-EUDNa17 or PEO-EUDNa71 formed mucoadhesive gels, well tolerated by the animals; then the gels spread over the corneal surface and eroded. The gel residence time in the precorneal area was in the order PEO-EUDNa71 < PEO < PEO-EUDNa17. Compared to commercial OFX eyedrops, drug absorption into the aqueous humor was retarded by the PEO-EUDNa71 inserts, and both retarded and prolonged by the PEO-EUDNa17 inserts, while C(max) (maximal concentration in the aqueous) and AUC(eff) (AUC in the aqueous for concentrations > MIC) were barely altered by either insert type. On the other hand, C(max), AUC(eff) and t(eff) (permanence time in the aqueous at concentrations > MIC) were strikingly increased by plain PEO inserts with respect to commercial eyedrops (5.25 +/- 0.56 vs. 1.39 +/- 0.05 microg ml(-1); 693.6 vs. 62.7 microg ml(-1) min; and 290 vs. 148 min, respectively). Bioavailability increase has been ascribed to PEO mucoadhesion and/or increased tear fluid viscosity.

Effect of chitosan on in vitro release and ocular delivery of ofloxacin from erodible inserts based on poly(ethylene oxide)

The effects of chitosan hydrochloride (CH-HCl) on in vitro release of ofloxacin (OFX) from mucoadhesive erodible ocular inserts and on the relevant ocular pharmacokinetics have been studied both to contribute evidence of the ability of CH-HCl to enhance transcorneal penetration of drugs and to increase the therapeutic efficacy of topically applied OFX. Circular inserts of 6 mm in diameter, 0.8-0.9 mm in thickness and 20 mg in weight, medicated with 0.3 mg drug, were prepared by powder compression. The addition of 10, 20 or 30% medicated CH-HCl microparticles, obtained by spray-drying, to formulations based on poly(ethylene oxide) of MW 900 kDa (PEO 900) or 2000 kDa (PEO 2000) produced changes in the insert microstructure which accelerated both insert erosion and OFX release from inserts. The effect was stronger with higher CH-HCl fractions. Of the CH-HCl-containing formulations based on either PEO 900 or PEO 2000, PEO 900-CH-HCl (9:1 w/w) was more suitable for a prolonged OFX release. Following insertion in the lower conjunctival sac of the rabbits eye, such an insert produced no substantial increase of AUC(eff) (AUC in the aqueous humour for concentrations >MIC(90%)) with respect to inserts based on plain PEO; however, it produced a concentration peak in the aqueous significantly higher than that produced by any of the CH-HCl-free PEO inserts, and well higher than the MIC(90%) for the more resistant ocular pathogens (7 microg/ml vs. 4 microg/ml). It has been argued that the increase was due to the ability of CH-HCl to enhance the transcorneal permeability of the drug.

Is dialysis a reliable method for studying drug release from nanoparticulate systems?-A case study.

The kinetics of in vitro drug release from nanoparticulate systems is extensive, though uncritically, being studied by dialysis. Evaluating the actual relevance of dialysis data to drug release was the purpose of this study. Diclofenac- or ofloxacin-loaded chitosan nanoparticles crosslinked with tripolyphosphate were prepared and characterized. With each drug, dynamic dialysis was applied to nanoparticle dispersion, solution containing dissolved chitosan·HCl, and solution of plain drug. Drug kinetics in receiving phase (KRP), nanoparticle matrix (KNM) and nanoparticle dispersion medium (KDM) were determined. Release of each drug from nanoparticles was also assessed by ultracentrifugation. Although KRP data may be interpreted in terms of sustained release from nanoparticles, KNM and KDM data show that, with both drugs, the process was in fact controlled by permeation across dialysis membrane. Analysis of KRP data reveals a reversible interaction of diclofenac with dispersed nanoparticle surface, similar to the interaction of this drug with dissolved chitosan·HCl. No such interactions are noticed with ofloxacin. The results from the ultracentrifugation method agree with the above interpretation of dialysis data. This case study shows that dialysis data from a nanoparticle dispersion is not necessarily descriptive of sustained-release from nanoparticles, hence, if interpreted uncritically, it may be misleading.

Improved synthesis of quaternary ammonium-chitosan conjugates (N+-Ch) for enhanced intestinal drug permeation

The pH-induced structural modifications of the reaction product between chitosan and 2-diethylaminoethyl chloride are studied with the purpose of testing and comparing the resulting chitosan derivatives on the basis of their intestinal drug permeability-enhancing properties. The reaction reproducibly yielded conjugates comprising short pendant chains of n adjacent diethyl-dimethylene-ammonium groups substituted onto the primary amino group of the chitosan repeating unit. The more significant derivatives, N(+)-Ch-7 (degree of substitution, DS=41%; n=3) and N(+)-Ch-8 (DS=59%; n=1.7) were prepared at pH 7 and 8, respectively. The apparent permeability (P(app)) of excised rat intestine was determined by means of Ussing type chambers. The hydrophilic fluorescein sodium (NaFlu) and fluorescein isothiocyanate dextran (MW 4400 Da) (FD-4), and the lipophilic rhodamine 123 (Rh-123), were applied in Ringer buffer to the apical side. Apical to basolateral transport was measured in the absence or presence of 0.5% (w/v) of the polymer under test. N(+)-Ch-7 and N(+)-Ch-8 were more effective P(app) enhancers than N-trimethyl-chitosan. Both N(+)-Ch-7 and N(+)-Ch-8 enhanced the P(app) of NaFlu (enhancement ratio, ER=1.84 and 1.33, respectively), while N(+)-Ch-8 was the more effective enhancer for FD-4 (ER=2.14). The P(app) of Rh-123 was significantly enhanced only by N(+)-Ch-7 (ER=1.37). Permeant-polymer binding counteracted the enhancement effect of polymer on transmembrane permeant flux. Contact with the chitosan conjugates did not impair the mucosal epithelium integrity.

In vitro evaluation of a system for pH-controlled peroral delivery of metformin

Oral absorption of the antihyperglycaemic agent metformin (MF x HCl) is confined to the upper part of the intestine, therefore controlled-release oral formulations of this drug should ensure a complete release during transit from stomach to jejunum. Compressed matrix tablets based on pH-sensitive poly(ethylene oxide) (PEO)-Eudragit L100 (EUD L) compounds have shown in vitro a compliance with the above requirement. The polymer compounds were prepared by a coevaporation process. The release pattern of MF x HCl from matrices depended on the PEO-EUD L ratio in the coevaporate. The 1:1 (w/w) ratio was unable to control MF x HCl release in simulated gastric fluid (SGF, pH 1.2), because the matrix material was excessively hydrophilic. Nevertheless, the release rate in SGF could be modulated by increasing the EUD L fraction in the coevaporate. With a PEO (M(w), 400 kDa)-EUD L (1:2, w/w) ratio the percent dose released in 2 h to SGF, where the coevaporate was insoluble, was around 23 or 50% with 10 or 20% loading dose. The release was then completed within the successive 2 h of elution with simulated jejunal fluid (SJF, pH 6.8) where EUD L and the coevaporate gradually dissolved. Release in SGF was controlled by matrix swelling and/or drug diffusion in matrix, whereas matrix dissolution controlled release in SJF. The unique release-controlling properties of the polymer compounds were due to PEO-EUD L macromolecular interactions. Matrices show promise of a gradual and complete release of MF x HCl from stomach to jejunum, unaffected by gastric pH fluctuations. This mode of administration might allow the use of lower therapeutic doses compared to existing immediate- or sustained-release products, thus minimising side effects.

A study of release mechanisms of different ophthalmic drugs from erodible ocular inserts based on poly(ethylene oxide).

When topical controlled delivery of ophthalmic drugs is realised via erodible inserts, drug bioavailability is maximised, if release is controlled exclusively by insert erosion, since parallel mechanisms which increase the release rate, also increases the dose fraction cleared from the precorneal area by tear fluid draining. The respective contributions of diffusion and erosion to the release mechanism of different drugs, namely, prednisolone (PDS), oxytetracycline hydrochloride (OTH) and gentamicin sulfate (GTS), from erodible ocular inserts based on poly(ethylene oxide) (PEO) of molecular weight 400 or 900kDa was determined by an in vitro technique adequate to predict the release mechanism in vivo. PDS and OTH were released with erosion-controlled kinetics. With therapeutic doses of these drugs in the inserts (0.3mg, 1.5%), the possibility of a purely erosive mechanism was shown to rely upon drug-PEO molecular interactions, which limit drug diffusion in the swollen matrix. This was the case with OTH, for which strong interactions with PEO were measured, whereas some contribution from the parallel diffusive mechanism was evidenced for PDS, which showed weaker interactions with polymer. Such a contribution disappeared when the PDS concentration in the insert was increased to 6%, which suggested that the erosive mechanism is favoured by a drug concentration in the hydrated insert substantially higher than solubility. On the other hand, the release of about 50% GTS dose was controlled by diffusion, due to the high water solubility of this drug, accompanied by weak drug-PEO interactions. In this case the residence time of drug in the precorneal area is expected to be significantly shorter than that of the PEO carrier.

Red grape skin and seeds polyphenols: Evidence of their protective effects on endothelial progenitor cells and improvement of their intestinal absorption.

SCOPE To evaluate the ability of grape skin and seeds to protect endothelial progenitor cells (EPC) from oxidative stress induced by hyperglycemia (HG) compared to red wine (RW) and prepare innovative pharmaceutical systems for the oral administration of red grape extract allowing the overcoming of its poor intestinal absorption. METHODS AND RESULTS Human EPC were characterized by expression of cell surface markers. Cells were incubated with different concentrations of total polyphenols from grape components or RW in the presence or absence of HG. Cell viability, migration, adhesion, and reactive oxygen species (ROS) production were assayed. Intestinal permeation of polyphenols was studied in the absence or presence of a quaternary ammonium-chitosan conjugate (N⁺(60)-Ch). Grape components and RW increased EPC viability, adhesion and migration, and prevented the HG effect (P < 0.01). ROS production induced by HG was significantly reduced only by grape seed extract and RW (P < 0.01). N⁺(60)-Ch acted as an effective enhancer of polyphenol permeability across the excised rat intestine. CONCLUSIONS Red grape components are a source of antioxidant compounds that ameliorate EPC viability and function, while preventing endothelial dysfunction. The use of polycationic chitosan derivatives can promote the absorption of polyphenols across intestinal epithelium, thus increasing their bioavailability and potential therapeutic value in atherosclerosis.

Relevance of polymer molecular weight to the in vitro/in vivo performances of ocular inserts based on poly(ethylene oxide).

A previous study of the present authors on gel-forming erodible inserts, based on high molecular weight (MW, 400 kDa) poly(ethylene oxide) (PEO), for ocular controlled delivery of ofloxacin (OFX) has been extended to investigate the effects of PEO MW, in the 200-2000 kDa range, on insert properties relevant to therapeutic efficacy. Mucoadhesion has shown a dependence on MW, with a maximum for PEO 400. The in vitro drug release from inserts based on PEO 200, PEO 400 and PEO 900 was mainly controlled by insert erosion, whereas with PEO 2000 it was mainly diffusion-controlled in a first phase, followed by an erosion-controlled phase. The erosion time scale depended directly on MW. Immediately after application in the lower conjunctival sac of the rabbit eye, the inserts based on PEO of whichever MW formed mucoadhesive gels, well tolerated by the animals; then the gels spread over the corneal surface and eroded. PEO 2000 was unsuitable as an insert material, since the resulting gel spilled from the eye, due to excessive swelling. The gel residence time in the precorneal area, the drug permanence time in the aqueous humor at concentrations > MIC and the time to reach the maximal drug concentration in the aqueous humor (C(max)) depended directly on MW, indicating that transcorneal absorption was governed by gel erosion. All inserts increased Cmax and AUCeff (AUC for concentrations > MIC) with respect to the commercial eyedrops. The increases caused by PEO 400 and PEO 900 were similar (3.78- and 3.16-fold, respectively, for Cmax; 11.06- and 12.37-fold, respectively, for AUCeff), whereas smaller increases were produced by PEO 200. The PEO 400 and PEO 900 inserts have shown a potential for a topical treatment of endophthalmitis.

Synergistic interaction between TS-polysaccharide and hyaluronic acid: implications in the formulation of eye drops.

An interaction between tamarind seed polysaccharide (TSP) and hyaluronic acid (HA) in aqueous solution has been ascertained. Various TSP/HA mixtures have been studied as the basis for the development of a potential excipient for eye drops synergistically improved over those of the separate polymers. Information about the nature of interpolymer interactions, and their dependence on TSP/HA ratios were obtained by NMR spectroscopy in solution. Superior mucin affinity of TSP/HA mixtures with respect to the single polysaccharides was assessed by NMR proton selective relaxation rate measurements. The mucoadhesivity of the TSP/HA (3/2) mixture, evaluated in vitro by NMR or viscometry, and in vivo by its mean and maximum residence time in rabbit precorneal area, is stronger than that of the component polysaccharides or the TSP/HA mixtures of different composition. TSP/HA (3/2) is little viscous and well tolerated by rabbit eyes. It stabilizes the tear film, thereby prolonging the residence of ketotifen fumarate and diclofenac sodium in tear fluid, but is unable to permeabilize the cornea. In conclusion, mucoadhesivity is responsible for the TSP/HA (3/2) synergistic enhancement of either extra- or intra-ocular drug bioavailability.

New chitosan derivatives for the preparation of rokitamycin loaded microspheres designed for ocular or nasal administration.

Acanthamoeba spp. are the causative agents of granulomatous amoebic encephalitis (GAE) and amoebic keratitis. Recent studies performed by Rassu et al. showed that, compared with the free drug, the loading of rokitamycin in chitosan microspheres improves and prolongs the in vitro antiamoebic activity of rokitamycin. This could be useful in transporting the drug for either ocular application to treat amoebic keratitis or nasal administration as an alternative route for the administration of the drug to the brain in GAE therapy. Starting from the previous study, our goal was to optimize the technological parameters in order to obtain chitosan microparticles loaded with rokitamycin and to evaluate the use of new quaternary ammonium chitosan derivatives in the preparation of spray dried microspheres containing the macrolide; these derivatives showed better characteristics (solubility, penetration enhancement) compared with chitosan itself. Toxicity studies on new polymers were performed. Spray dried loaded microspheres based on chitosan or chitosan derivatives were obtained by using appropriate preparative parameters. Microparticles containing chitosan derivatives showed similar or often better properties than formulations made of chitosan with respect to size, in vitro release behaviour and mucoadhesiveness thus making them more suitable for ocular or nasal administration. New polymers did not demonstrate cytotoxicity.