Acidic mammalian chitinase in dry eye conditions.

PURPOSE: An acidic mammalian chitinase (AMCase) seems to be implicated in allergic asthma and allergic ocular pathologies. The aim of this work was to investigate the role of AMCase during Sjögren's Syndrome (SS) and Meibomian Gland Dysfunction (MGD) dry eye diseases. METHODS: Six patients with MGD dry eye (20-58 years, median 40) and six patients with dry eye associated to SS (32-60 years, median 47) were enrolled in this study. AMCase activity was measured in tears and AMCase mRNA expression was evaluated by real-time polymerase chain reaction from RNA extracted from epithelial cells of the conjunctiva. Six healthy adult subjects of the same age (34-44 years, median 39) were also studied as the control group. RESULTS: AMCase activity was significantly increased in patients affected by MGD dry eye (18.54 +/- 1.5 nmol/ml/h) and SS dry eye (8.94 +/- 1.0 nmol/ml/h) respectively, compared to healthy controls (1.6 +/- 0.2 nmol/ml/h). AMCase activity was higher in the tears of subjects with MGD dry eye (P < 0.001). AMCase mRNA was detected in conjunctival epithelial cells and the expression was significantly higher in MGD dry eye than SS dry eye. A significant correlation between AMCase activity in the tears and mRNA in conjunctival epithelial cells was found. CONCLUSION: AMCase may be an important marker in the pathogenesis of dry eye, suggesting the potential role of AMCase as a therapeutic target in these frequent pathologies.

New coumarin-based anti-inflammatory drug: putative antagonist of the integrins alphaLbeta2 and alphaMbeta2.

This study was conducted to investigate putative antagonism of integrin receptors alphaMbeta2 and alphaLbeta2 by a novel coumarin derivative (BOL-303225-A), its efficacy in-vivo after retinal ischaemia-reperfusion injury, and its bioavailability in rat plasma. A cellular adhesion assay in Jurkat and U937 cells, and a flow cytometry assay with an antibody against the beta2 subunit were conducted. BOL-303225-A bioavailability in rat plasma and the retinal levels of myeloperoxidase (MPO) after ischaemia-reperfusion injury were evaluated after oral administration (10 mg kg(-1)). In-vitro cell viability assays revealed no cytotoxicity for BOL-303225-A over a wide dose range, and IC50 values of 32.3 +/- 1.5 muM and 84.95 +/- 2.3 muM were found for Jurkat and U937 cells, respectively. The drug showed specific binding to the alphaMbeta2 and alphaLbeta2 integrin receptors expressed by U937 and Jurkat cells, respectively, producing a fluorescence shift towards lower values in a concentration-dependent manner. The pharmacokinetic profile of BOL-303225-A exhibited rapid absorption following oral administration in the rat. A significant reduction of retinal MPO levels was observed in drug-treated rats. This study demonstrated that BOL-303225-A acts as an antagonist of the integrin alphaLbeta2 and alphaMbeta2 receptors, suggesting that this drug could be used for ocular diseases such as diabetic retinopathy.

Retinal and systemic pharmacokinetics of the anti-inflammatory drug cloricromene following oral administration in the rat and rabbit.

PURPOSE: The aim of this study was to evaluate the retina and plasma distribution of cloricromene, a coumarin derivative, and its active metabolite (MET) after an oral administration in rabbits and rats. METHODS: A single dose of cloricromene was orally administered to rabbits (10 or 100 mg/kg) and to rats (100 mg/kg). Retina and plasma samples were collected at 15, 30, 60, and 90 min following administration. Drug concentrations in the retina and plasma were measured by high-performance liquid chromatography. RESULTS: As anticipated, only the active metabolite was found in all samples. The retina and plasma showed the same T(max); peak levels of the drug were achieved at 15 min in rats and at 30 min in rabbits. In rabbits, MET exposure was approximately dose-proportional in both retina and plasma between the 10- and 100-mg/kg dose. Substantial retinal exposure was observed in both the rat and rabbit, at exposures approximately nine- to sixteenfold lower in the retina than in plasma. CONCLUSIONS: The results showed that the active metabolite of cloricromene reached the retina after a single oral dose with exposures proportional to those in plasma. These data, along with the previously published potency data for cloricromene, suggest that cloricromene could be potentially useful in ischemic-retinal diseases where amelioration of blood flow and inflammation is desirable.

Hydroxyl radical scavenging activity of a new ophthalmic viscosurgical device.

PURPOSE: To quantify the hydroxyl radical scavenging activity of a new ophthalmic viscosurgical device (OVD) based on sodium hyaluronate and hydroxypropylmethylcellulose (named VISC28) in comparison with Viscoat, Healon, and Amvisc Plus. METHODS: The hydroxyl radicals that represent the principal free-radical species generated during phacoemulsification were produced by the Fenton reaction, and the scavenging activity of the tested viscoelastic substances was evaluated in vitro by the 2-deoxy-D-ribose (2-DR) oxidation method that produces the thiobarbituric acid-malondialdehyde (TBA-MDA), complex. An aliquot of viscosurgical formulation was added to phosphate buffer and mixed with 2-DR, Fe2 +/ethylenediaminetetraacetic acid (EDTA), and H2O2. The sample mix was incubated and thiobarbituric acid-trichloroacetic acid solution was added. The sample was then incubated for 30 min, and a chromatographic analysis was performed to quantify the TBA-MDA complex. The data were expressed as micromoles of MDA per milliliter of sample. RESULTS: All tested OVDs showed a marked hydroxyl radical scavenging activity. The MDA level was significantly lower in VISC28 (0.045 +/- 0.007 micromol/ml) compared with Viscoat (0.070 +/- 0.012 micromol/ ml, p < 0.05), Amvisc Plus (0.111 +/- 0.008 micromol/ml, p < 0.001), and Healon (0.175 +/- 0.016 micromol/ml, p < 0.001). A reduced scavenging activity was shown by VISC28 phosphate-buffered solution (PBS) (no TRIS and no sorbitol) compared with VISC28 (p < 0.001). CONCLUSIONS: The new OVD, VISC28, showed significantly higher hydroxyl radical inhibition compared with the other viscosurgical formulations. The following rank order for the scavenging activity was established: VISC28 > Viscoat > Amvisc Plus > Healon.

Development and validation of an RP-HPLC-UV method for the determination of BOL-303225-A, a new coumarin-based anti-inflammatory drug, in rat plasma.

A simple and rapid high-performance liquid chromatographic method was developed and validated for the analysis in rat plasma of BOL-303225-A, a new coumarin-based anti-inflammatory drug. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and water containing 1% triethylamine pH 3.5, adjusted with orthophosphoric acid (35.5:64.5 v/v) as mobile phase. The UV detector was set at 324 nm. The method proved to be linear (r(2) > 0.99) and precise (RSD < 7%) over the concentration range 29-940 ng/mL, and was suitable for the support of pharmacokinetic studies in rats.

Novel polysaccharides-based viscoelastic formulations for ophthalmic surgery: rheological characterization.

Different formulations based on bioadhesive and biocompatible polymers, hydroxypropylmethylcellulose (HPMC), sodium hyaluronate (SH) and chitosan glutamate (CG), were prepared to be potentially used as ophthalmic viscosurgical device (OVD) during cataract surgery. Their rheological properties were analyzed in terms of flow and oscillation properties and compared to a commercially available OVD, widely employed in cataract surgery, named Viscoat. All the formulations tested presented a pseudoplastic behavior during flow. Primary systems containing HPMC or CG and HPMC/CG binary systems behaved as viscous solution (G''>G') over the range of oscillatory frequencies observed, while the primary systems containing SH and HPMC/SH binary formulations and showed an entangled network behavior when subjected to a sinusoidal stress. By increasing the SH concentration in the binary systems, the viscoelastic parameters, G'and G'', and zero frequency viscosity (derived from the Cross model) increased. Viscoat presents viscoelastic parameters values lower than the corresponding values of all the binary formulations of HPMC/SH and higher than all the formulations made up of CG and HPMC. As regard to HPMC/SH binary system, the cross-over frequency decreased by increasing SH concentration in the systems and it was the highest for Viscoat and thus the opposite occurred for the relaxation time. The rheological synergy in the binary formulations was assessed by calculating the interaction parameters which increased as a function of SH and CG concentration in the binary systems. The values of the interaction parameters of the formulations based on CG, are lower than 10 Pa indicating that they did not interact synergically while the formulations based on SH show high values of the interactions parameters (in the range from 55 to 130 Pa). This indicates that secondary bonds formation occurs between SH and HPMC. From the rheological analysis it can be concluded that the binary formulations based on CG do not possess appropriate features to be used as OVD while both the viscoelastic and the flow properties of the binary formulations made up of SH and HPMC are suitable for their application as OVD being able to maintain the ocular spaces and to be easily administrated. Moreover, thank to the adhesive properties of both components, the binary formulation should be able to interact with corneal endothelium so offering a durable protection to ocular tissue. On the basis of the rheological characterization presented in this work, we concluded that the binary system named VISC26 (HPMC at 0.8% and SH at 2.3%) represents the formulation that better fulfill the OVD requirements.

Preparation and characterization of eudragit retard nanosuspensions for the ocular delivery of cloricromene.

The purpose of this study was to improve the stability of cloricromene (AD6) in ophthalmic formulations and its drug availability at the ocular level. To this end, AD6-loaded polymeric nanoparticle suspensions were made using inert polymer resins (Eudragit RS100 and RL100). We modified the quasi-emulsion solvent diffusion technique by varying some formulation parameters (the drug-to-polymer ratio, the total drug and polymer amount, and the stirring speed). The chemical stability of AD6 in the nanosuspensions was assessed by preparing some formulations using (unbuffered) isotonic saline or a pH 7 phosphate buffer solution as the dispersing medium. The formulations were stored at 4 degrees C, and the rate of degradation of AD6 was followed by high performance liquid chromatography (HPLC). The obtained nanosuspensions showed mean sizes and a positive surface charge (zeta-potential) that make them suitable for an ophthalmic application; these properties were maintained upon storage at 4 degrees C for several months. In vitro dissolution tests confirmed a modified release of the drug from the polymer matrixes. Nanosuspensions prepared with saline solution and no or lower amounts of surfactant (Tween 80) showed an enhanced stability of the ester drug for several months, with respect to an AD6 aqueous solution. Based on the technological results, AD6-loaded Eudragit Retard nanoparticle suspensions appear to offer promise as a means to improving the shelf life and bioavailability of this drug after ophthalmic application.

Preparation and characterization of Eudragit Retard nanosuspensions for the ocular delivery of cloricromene.

The purpose of this study was to improve the stability of cloricromene (AD6) in ophthalmic formulations and its drug availability at the ocular level. To this end, AD6-loaded polymeric nanoparticle suspensions were made using inert polymer resins (Eudragit RS100 and RL100). We modified the quasi-emulsion solvent diffusion technique by varying some formulation parameters (the drug-to-polymer ratio, the total drug and polymer amount, and the stirring speed). The chemical stability of AD6 in the nanosuspensions was assessed by preparing some formulations using (unbuffered) isotonic saline or a pH 7 phosphate buffer solution as the dispersing medium. The formulations were stored at 4°C, and the rate of degradation of AD6 was followed by high performance liquid chromatography (HPLC). The obtained nanosuspensions showed mean sizes and a positive surface charge (ζ-potential) that make them suitable for an ophthalmic application; these properties were maintained upon storage at 4°C for several months. In vitro dissolution tests confirmed a modified release of the drug from the polymer matrixes. Nanosuspensions prepared with saline solution and no or lower amounts of surfactant (Tween 80) showed an enhanced stability of the ester drug for several months, with respect to an AD6 aqueous solution. Based on the tecnological results, AD6-loaded Eudragit Retard nanoparticle suspensions appear to, offer promise as a means to improving the shelf life and bioavailability of this drug after ophthalmic application.

Simple determination of riluzole in rat brain by high-performance liquid chromatography and spectrophotometric detection.

A simple method was developed for separation and quantification of riluzole in rat brain. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Hypersil ODS) with UV detection at 264 nm. The mobile phase consisted of methanol-water containing 1% triethylamine adjusted with orthophosphoric acid to pH 3.2. The retention time was 8.6 min. A simple liquid-liquid extraction with ethyl acetate was used to obtain riluzole from brain samples. The limit of quantification was 10 ng/g. The recovery was about 80%. The relationship between peak areas and concentrations was linear over the range between 0.01 and 0.8 microg/g, with r2 value over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of riluzole.

Eudragit RL100 nanoparticle system for the ophthalmic delivery of cloricromene.

A Eudragit RL100 polymer nanoparticle system loaded with cloricromene was prepared and characterized on the basis of physicochemical properties, stability and drug release features. To investigate the ocular bioavailability of cloricromene after inclusion in the polymer matrix, the new nanoparticle system was topically administered in the rabbit eye and compared with an aqueous solution of the same drug. The nanoparticle system showed interesting size distribution and surface charge values, suitable for ophthalmic application. The results indicated that the dispersion of cloricromene within Eudragit RL100 polymer nanoparticles increased its ocular bioavailability and enhanced the biopharmaceutical profile. The new cloricromene-loaded nanoparticle system described here may be useful in clinical practice.

Rapid determination of nimesulide in rabbit aqueous humor by liquid chromatography.

A rapid method was developed for quantification of nimesulide (methanesulfonamide, N-[4-nitro-2-phenoxyphenil]) in rabbit aqueous humor. The analyses were performed by high-performance liquid chromatography using a C(18) reversed-phase column (Ultracarb ODS) with UV detection at 300 nm. The mobile phase consisted of acetonitrile-water containing 1% triethylamine (TEA) adjusted to pH 3.2 with orthophosphoric acid. The retention time was 4.5 min. A simple pre-treatment with acetonitrile was used to deproteinize aqueous humor samples. The limit of quantitation was 50 ng/ml. The recovery was over 90%. The relationship between peak areas and concentration was linear over the range between 0.05 and 2.5 microg/ml, with r(2) values over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of nimesulide.