Dopamine-3 receptor modulates intraocular pressure: implications for glaucoma.

The aim of the present study was to investigate the role of D3 receptor on intraocular pressure regulation using WT and KO D3R⁻/⁻ mice. Both mice were used with normal eye pressure or steroid-induced ocular hypertension. As measured by tonometry, the topical application of 7-OH-DPAT, a dopamine D3-preferring receptor agonist, significantly decreased, in a dose-dependent manner, the intraocular pressure in WT mice both in an ocular normotensive group and an ocular hypertensive group. Pretreatment with U-99194A, a D3 receptor antagonist, reverted 7-OH-DPAT induced ocular hypotension in WT mice. No change of intraocular pressure was observed after topical application of 7-OH-DPAT in KO D3R⁻/⁻ mice. PCR analysis demonstrated the presence of all dopamine receptor genes in eye tissues obtained from WT mice, and the lack of D3R mRNAs in KO mice. The present study identified the D3R subtype as the most important receptor of the dopaminergic system to modulate intraocular pressure with relevant implications for glaucoma that represents one of the most crippling optic neuropathies.

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.

Effect of chitinase inhibitors on endotoxin-induced uveitis (EIU) in rabbits.

The acidic mammalian chitinase (AMCase) is significantly increased in tears of human allergic conjunctivitis. The aim of the study was to investigate the effects of chitinase inhibitors, allosamidin and caffeine versus dexamethasone, in rabbit endotoxin-induced uveitis (EIU). EIU was induced in rabbits by a single intravitreal injection of 100ng/10microl lipopolysaccharide (LPS). Drugs at four different concentrations (0.1, 0.01, 0.001 and 0.0001mM) were topically applied to the rabbit eye five times in 24h. Tears were collected at 0, 6 and 24h after LPS to measure the AMCase activity. The effect of treatment was also evaluated at the same time by slit lamp examination. Tear AMCase activity increased 6 and 24h after LPS injection. The AMCase activity was significantly inhibited in all treated groups with all doses of allosamidin and caffeine except with the lowest concentration. A higher AMCase inhibition at 24h was found with allosamidin and caffeine compared to dexamethasone. Moreover, topical administration of allosamidin, caffeine and dexamethasone produced a remarkable reduction of inflammatory signs, in the order: dexamethasone>caffeine>allosamidin. AMCase inhibitors showed in this rabbit model of uveitis a notable control of inflammatory response with a significant reduction of AMCase activity in tears with caffeine and allosamidin. These results support the key role of AMCase in the pathogenesis of human ocular inflammatory diseases and the therapeutic effect of AMCase inhibitors on experimental uveitis.

Chitinase levels in the tears of subjects with ocular allergies.

PURPOSE: Chitin is abundant in the structural coatings of fungi, insects, and parasitic nematodes. The host defense against chitin-containing pathogens includes production of chitinases. An acidic mammalian chitinase (AMCase) is produced in human epithelial cells of lower airways through a TH2-specific, interleukin-13-dependent pathway and appears to be associated with allergic asthma. The role of AMCase in allergic ocular pathologies has never been studied previously. METHODS: Six patients with vernal keratoconjunctivitis (VKC), 7 patients with season allergic conjunctivitis (SAC), and 8 healthy controls (4 children and 4 adults) were enrolled in this study. AMCase activity was measured in tears, RNA was extracted from epithelial cells of the conjunctiva, and AMCase mRNA expression was evaluated by real-time polymerase chain reaction. RESULTS: AMCase activity was increased in patients affected by VKC (33.7 +/- 10.8 nmol/mL/h) and SAC (7.3 +/- 4.1 nmol/mL/h) compared with healthy controls (1.6 +/- 0.2 nmol/mL/h), and AMCase activity was higher in subjects with VKC (P = 0.0001). Receiver operating characteristic analysis showed that the sensitivity and specificity were 100%, addressing the use of AMCase assay in the biochemical diagnosis of VKC and SAC. AMCase mRNA was detected in epithelial cells of the conjunctiva, and the expression was significantly higher in VKC and SAC. CONCLUSIONS: AMCase may be an important mediator in the pathogenesis of TH2 inflammation eye diseases, suggesting a potential diagnostic and therapeutic target in these pathologies.

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.

Pharmacokinetic profile of topical flunarizine in rabbit eye and plasma.

The purpose of this study was to determine the aqueous humor, cornea, iris-ciliary body, retina and plasma levels and pharmacokinetics of a new topical formulation containing 0.05% flunarizine upon single drop application. Albino rabbits were used and tissue samples were collected at 15, 30, 60, 120 and 240 min after instillation. Drug concentrations in ocular tissues and plasma were measured by gas chromatography assay. After single dose application peak levels of drug were achieved at 15 min in cornea and at 30 min in aqueous humor, iris-ciliary body and retina. Unilateral topical flunarizine caused a significant reduction of intraocular pressure in rabbits. The pharmacokinetic profile showed a good ocular bioavailability of the drug providing that the new topical formulation containing 0.05% flunarizine reach the target tissues at effective concentrations and therefore may be use in the treatment of glaucoma.

Influence of preparation conditions on acyclovir-loaded poly-d,l-lactic acid nanospheres and effect of PEG coating on ocular drug bioavailability.

PURPOSE: The evaluation of nanosphere colloidal suspensions containing acyclovir as potential ophthalmic drug delivery systems was carried out. The influence of polymer molecular weight and type and concentration of various surfactants on nanosphere properties was studied. The ocular pharmacokinetics of acyclovir-loaded nanoparticles was evaluated in vivo and compared with an aqueous suspension of the free drug. METHODS: Nanospheres were made up of poly-d,l-lactic acid (PLA). The colloidal suspension was obtained by a nanoprecipitation process. The surface properties of PLA nanospheres were changed by the incorporation of pegylated 1,2-distearoyl-3-phosphatidylethanolamine. The mean size and zeta potential of the nanospheres were determined by light scattering analysis. The acyclovir loading capacity and release were also determined. In vivo experiments were carried out on male New Zealand rabbits. The ocular tolerability of PLA nanospheres was evaluated by a modified Draize test. The aqueous humor acyclovir levels were monitored for 6 h to determine the drug's ocular bioavailability for the various formulations. RESULTS: A reduction of the mean size and a decrease of the absolute zeta potential of PLA nanospheres resulted from increasing the surfactant concentration. The higher the polymer molecular weight, the smaller the nanosphere mean size. PEG-coated and uncoated PLA nanospheres showed a sustained acyclovir release and were highly tolerated by the eye. Both types of PLA nanospheres were able to increase the aqueous levels of acyclovir and to improve the pharmacokinetics profile, but the efficacy of the PEG-coated nanospheres was significantly higher than that of the simple PLA ones. CONCLUSIONS: PEG-coated PLA nanospheres can be proposed as a potential ophthalmic delivery system for the treatment of ocular viral infections.

Enhanced ocular anti-inflammatory activity of ibuprofen carried by an Eudragit RS100 nanoparticle suspension.

PURPOSE: To investigate the ocular pharmacodynamic profile of a polymer nanoparticle system loaded with sodium ibuprofen (IBU-RS) in comparison to an aqueous solution of ibuprofen lysinate (IBL) in the rabbit eye both being applied topically. METHODS: Ocular inflammation was elicited by topical application of sodium arachidonate. Inflammation was quantified according to a modified Draize test. The protein level and the number of polymorphonuclear leukocytes in the aqueous humor were assessed after 2 h from arachidonate instillation. The ibuprofen concentration in the aqueous humor was evaluated by HPLC assay. The physico-chemical properties of nanoparticles were also evaluated. RESULTS: The IBU-RS nanosuspension formulation significantly reduced the primary signs of ocular inflammation as well as significantly reducing the protein level and the number of polymorphonuclear leukocytes in the aqueous humor compared with the IBL formulation. Furthermore, the aqueous humor drug concentration from the group treated with IBU-RS was significantly higher compared to the IBL-treated group. The IBU-RS nanosuspensions showed very interesting size and surface charge values, adequate for ophthalmic administration. CONCLUSIONS: The pharmacological profile of the topical IBU-RS nanosuspension formulation described in this study indicates that the dispersion of the drug within RS polymer nanoparticles increased its ocular bioavailability and ultimately its pharmacological activity.

Eudragit RS100 nanosuspensions for the ophthalmic controlled delivery of ibuprofen.

Topical application of non-steroidal anti-inflammatory drugs on the eye is a common treatment used to contrast the miosis induced by surgical traumas, such as cataract extraction. With the aim of improving the availability of sodium ibuprofen (IBU) at the intraocular level, IBU-loaded polymeric nanoparticle suspensions were made from inert polymer resins (Eudragit RS100). The nanosuspensions were prepared by a modification of the quasi-emulsion solvent diffusion technique using variable formulation parameters (drug-to-polymer ratio, total drug and polymer amount, stirring speed). Nanosuspensions had mean sizes around 100 nm and a positive charge (zeta-potential of +40/+60 mV), this makes them suitable for ophthalmic applications. Stability tests (up to 24 months storage at 4 degrees C or at room temperature) or freeze-drying were carried out to optimize a suitable pharmaceutical preparation. In vitro dissolution tests indicated a controlled release profile of IBU from nanoparticles. In vivo efficacy was assessed on the rabbit eye after induction of an ocular trauma (paracentesis). An inhibition of the miotic response to the surgical trauma was achieved, comparable to a control aqueous eye-drop formulation, even though a lower concentration of free drug in the conjunctival sac was reached from the nanoparticle system. Drug levels in the aqueous humour were also higher after application of the nanosuspensions; moreover, IBU-loaded nanosuspensions did not show toxicity on ocular tissues.

Rapid high-performance liquid chromatographic assay of dorzolamide in rabbit aqueous humor.

A rapid high-performance liquid chromatographic method using a C(18) reversed-phase column (Hypersil ODS) with UV detection at 254 nm and a simple pre-treatment of samples is presented for the analysis of dorzolamide, a carbonic anhydrase inhibitor, in rabbit aqueous humor. A water solution containing 2% ZnSO(4) small middle dot7H(2)O was used to deproteinize aqueous humor samples. The mobile phase consisted of 7% CH(3)CN and 93% of a solution containing 1% TEA adjusted to pH = 3.5 with H(3)PO(4). Paracetamol was found to be a suitable internal standard. The standard curves were linear in the detection range. The precision and the accuracy were <5% for both intra- and inter-day assays.

Pharmacological profile of oxaprozin eye drops.

The effects of eye drops containing a propionic acid derivative (oxaprozin) at 0.1% concentration on ocular inflammation produced by sodium arachidonate in the rabbit's eye were evaluated. Furthermore, the aqueous bioavailability of the drug formulation in the uninflamed and inflamed eyes was evaluated. Oxaprozin eye drops significantly reduced the signs of ocular inflammation elicited by sodium arachidonate on conjunctiva and iris. Oxaprozin treatment significantly reduced the levels of polymorphonuclear leukocytes and protein concentration in aqueous samples obtained from the eyes treated with arachidonate. Present data suggest, for the first time, that oxaprozin may be employed topically to prevent ocular reactions where the arachidonic acid cascade is activated.

Simultaneous determination of cloricromene and its active metabolite in rabbit aqueous humor by high-performance liquid chromatography.

A rapid and simple method was developed for the simultaneous separation and quantification of cloricromene, a coumarine derivative, and its active metabolite, cloricromene acid, in rabbit aqueous humor. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Hypersil ODS) with UV detection at 318 nm. The mobile phase consisted of acetonitrile-water containing 1% triethylamine pH 3.5, adjusted with orthophosphoric acid. An acetonitrile gradient was necessary to achieve good separation within 13 min. Timolol was found to be a suitable internal standard. The retention times ranged from 5.72 to 11.25 min. A simple pre-treatment with acetonitrile containing 0.6% HCIO4 was used to deproteinize aqueous humor samples. The limit of quantitation ranged between 10 and 20 ng/ml. The recovery was >90%. The relationship between peak areas and concentration was linear over the range between 0.01 and 3.8 microg/ml, with r2 > 0.99. The assay provided good reproducibility and accuracy for both analytes and proved to be suitable for pharmacokinetic studies of cloricromene.