Effect of textured eye drop bottles on the photostability of pranoprofen 0.1% ophthalmic solution.

CONTEXT: Ophthalmic solutions are usually filled in a plastic bottle due to its durability and disposability. In Japan, photostability is one of the concerns for the quality control because an eye drop bottle must be a transparent container. OBJECTIVE: The present work studied the effect of textured eye drop bottles on its light blocking to improve the photostability of ophthalmic solutions. MATERIALS AND METHODS: We investigated the photostability of Pranoprofen ophthalmic solution filled in a variety of textured eye drop bottles. Pranoprofen content was analyzed by high-performance liquid chromatography and surface structure of textured eye drop bottles was evaluated by transmittance, calculated average roughness (Ra) and haze intensity. RESULTS: We observed that eye drop bottle which had greater than Ra value of 1.0 µm and haze intensity 62% clearly showed photostability improvement. CONCLUSIONS: This report is the first one which shows that photostability of ophthalmic solution is improved by using textured eye drop bottle. Moreover, this approach is a simple and effective method to improve the photostability. This method is available for not only various ophthalmic applications but also other liquid pharmaceuticals or food products.

Evaluation of the Safety and Tolerability of Conjunctival Ring for Posterior Segment of the Eye.

PURPOSE: To evaluate the safety and tolerability of conjunctival rings (CRs), a novel device for drug delivery to the posterior segment of the eye. METHODS: In animal studies, CRs containing 5% dexamethasone sodium phosphate (DSP) or vehicle solution were placed on the right and left eyes of C57BL/6J mice, respectively. Contact lenses (CLs) containing vehicle solution were used as a control. Twenty-four hours after placement of the CRs, corneal fluorescein staining was graded based on the McDonald-Shadduck scoring system, ranging from 0 to 4. In humans, CRs containing vehicle solution were placed on the right eye of healthy volunteers for 9 hours. The corneal curvature, corneal thickness, intraocular pressure, visual acuity, tear production (Schirmer I test), tear film break-up time and fluorescein staining scores of the cornea (scores ranging from 0 to 3) and conjunctiva (scores ranging from 0 to 6) were assessed before and after wearing the CRs. The release characteristics of DSP from CRs were also evaluated. RESULTS: In animal experiments, corneal fluorescein staining scores were 1 or less in all the groups, and there was no significant difference between the CR group and the CL group. In the preclinical safety evaluation of CR for humans, ophthalmic examination revealed that CR caused no significant changes in all the parameters investigated including corneal curvature (p = 0.77), corneal thickness (p = 0.96), intraocular pressure (p = 0.59), visual acuity (p = 0.14), Schirmer I test results (p = 0.76), tear film break-up time (p = 0.68), corneal fluorescein staining scores (p = 0.64), and conjunctival fluorescein staining scores (p = 0.52). The DSP release from CRs occurs within a few hours, which is similar to the drug-release property of medicated CL, as reported previously. CONCLUSIONS: The current data showed the safety and tolerability of CR as a drug delivery device for the treatment of posterior segment diseases.

Stability of latanoprost in an ophthalmic lipid emulsion using polyvinyl alcohol.

Latanoprost in water is not stable against heat stress due to hydrolysis of the isopropyl ester in the latanoprost molecule. Therefore, the storage condition of latanoprost ophthalmic solution, Xalatan brand, was in a low temperature (2-8 degrees C). We formulated a favorable ophthalmic lipid emulsion of latanoprost using polyvinyl alcohol as emulsifier which showed a good heat stability. The assays of the latanoprost ophthalmic lipid emulsions adjusted to pH 5.0, 6.0 and 7.0 were 100.4%, 100.7% and 99.2% after storage for 4 weeks at 60 degrees C, respectively. The possibility of room temperature storage for the latanoprost ophthalmic lipid emulsion was demonstrated.

Formulation of an ophthalmic lipid emulsion containing an anti-inflammatory steroidal drug, difluprednate.

Preparation of oil-in-water (o/w) type lipid emulsion is one of the approaches to formulate drugs that are poorly water-soluble but can be dissolved in the oil phase of the emulsions. A synthetic glucocorticoid medicine, difluprednate (DFBA), is a water-insoluble compound. We formulated DFBA (0.05%, w/v) ophthalmic lipid emulsion containing 5.0% (w/v) caster oil and 4.0% (w/v) polysorbate 80. The appearance of the emulsion was blue and translucent lipid emulsion, and the median particle size of the lipid emulsion was 104.4 nm. Neither separation nor change in particle size was observed after 6 months at 40 degrees C. Furthermore, when compared with DFBA (0.05%, w/v) ophthalmic suspension, the lipid emulsion showed 5.7-fold higher concentration of DFB that was an active metabolite of DFBA in aqueous humor at 1h after instillation. Ophthalmic lipid emulsion enhances the intraocular penetration of drugs, and it is useful as a delivery system for the ophthalmic preparations of lipophilic drugs.

Decreased mucin concentrations in tear fluids of contact lens wearers.

Characteristics of tear-film may be influenced by contact lens wear, because contact lenses present the habitual, direct rubbing action of the lids upon the covered ocular surface and may cause changes of tear-film. In the present paper, influence of contact lens on proteins in tear samples was studied using carbohydrates attached to the protein as a marker. We found that N-acetylneuraminic acid (Neu5Ac) was significantly decreased in tear samples of volunteers wearing contact lens (wearing, 86.1 +/- 57.7 nmol/ml; normal, 190.2 +/- 121.9 nmol/ml). Analysis by polyacrylamide gel electrophoresis demonstrated that the amounts of major proteins in tear fluids, such as lactoferrin and secretory immunoglobulin A were not changed upon wearing contact lenses. In contrast, cellulose acetate membrane electrophoresis revealed that mucin band in tear samples from contact lens wearers showed significant decrease as examined by lectin staining.

Hyaluronic acid: separation and biological implications.

Hyaluronic acid (hyaluronan) is a ubiquitous extracellular matrix component, and present at high concentrations in skin, joints and cornea. In the skin, it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes. Hyaluronic acid usually exists as a high molecular mass (600,000-1,000,000) and non-sulfated glycosaminoglycan composed of a disaccharide unit of [bond]3GlcNAc beta 1[bond]4GlcA beta 1[bond]. Hyaluronic acid has been widely used not only for osteoarthritis and ophthalmology but also for cosmetics for skin care. To examine the biological activities of hyaluronic acid, we have to accurately determine the quantity and molecular masses in biological samples. We review recent development in the analysis of hyaluronic acid having various molecular sizes using electrophoretic and chromatographic techniques. Recently, interactions between hyaluronic acid oligomers and hyaluronic acid-binding proteins have attracted the interest for understanding the biological functions. We show some interesting reports on biological interactions of hyaluronic acid and its oligomers with some proteins.

Evaluation of ophthalmic suspensions using surface tension.

Uniformity and precision of single dose are required for ophthalmic suspensions including water-insoluble ingredients. Solid sediments formed after standing still must be immediately re-dispersible and distributed homogeneously before use. However, selection of an appropriate water-soluble polymer as suspending agent is a challenging problem. In this report, the relationship between the surface tension and the re-dispersibility of suspensions was investigated. The surface tension of 0.1 w/v% fluorometholone suspensions began to decline from 74 mN/m at 0.0001 w/v% of hydroxypropylmethylcellulose (HPMC) and became almost constant at 52 mN/m at 0.01 w/v% of HPMC. Re-dispersion time was less than 4 s when HPMC was present at concentrations between 0.0001 w/v% and 0.01 w/v%. At these concentrations, aggregation of suspended particles was not observed. When indomethacin suspensions at 1.0 w/v% concentration were used, the surface tension began to decline from 73 mN/m at 0.0005 w/v% HPMC and became constant at 50 mN/m at 0.005 w/v% HPMC. The suspension also showed good re-dispersibility, and a uniform suspension was obtained between 0.0005 w/v% and 0.005 w/v% of HPMC. The time required for re-dispersion was less than 17 s. The change of surface tension showed a good correlation with the concentration of HPMC in ophthalmic suspensions having good re-dispersibility. Measurement of the surface tension of suspensions provided the optimal concentration of the water-soluble polymers for the suspensions of well re-dispersible characteristics. Evaluation of ophthalmic suspension using surface tension is a good strategy for formulation of suspending pharmaceutical products in the ophthalmic area.

An HPLC method to evaluate purity of a steroidal drug, loteprednol etabonate.

Validation of an analytical method for impurities and degradation products in an active pharmaceutical ingredient is important to assessment of quality and safety in a new pharmaceutical product. In the present study, a high-performance liquid chromatographic method was validated to evaluate purity of loteprednol etabonate (LE). LE and its four related substances, major process impurities and degradation products (PJ-90, PJ-91, LE-11-keto and LE-methyl ester) were well resolved using a phenyl-stationary phase under isocratic conditions. Two photo-degradation products were identified as chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-5alpha-methyl-2-oxo-19-norandrosta-1(10),3-diene-17beta-carboxylate and chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-1-methyl-3-oxo-6(5-->10alpha)-abeo-19-norandrosta-1,4-diene-17beta-carboxylate. A photo-degradation product, chloromethyl 1beta,11beta-epoxy-17alpha-ethoxycarbonyloxy-2-oxo-10alpha-androsta-4-ene-17beta-carboxylate, was not abundant by ultraviolet detector. The risk depending on only ultraviolet detection should be noted. Calibration curves for PJ-90, PJ-91, LE-11-keto and LE-methyl ester showed linearity over the range of 0.05-2.0% levels in LE with correlation coefficient of 0.999. Accuracy (n = 3) at the concentration of 0.5% level in LE for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 2.0, 2.0, 2.3 and 2.0%, respectively. Intra-day repeatability (n = 6) at the concentration of 0.5% level in LE for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 1.4, 1.4, 1.8 and 1.4%, respectively. The lower limits of detection for PJ-90, PJ-91, LE-11-keto and LE-methyl ester were 0.002, 0.001, 0.004 and 0.003% levels in LE, respectively.

Analysis of an anti-inflammatory steroidal drug, difluprednate, in aqueous humor by combination of semi-micro HPLC and column switching method.

A specific and sensitive method for the determination for difluprednate (DFBA) and its metabolite (deacetylated DFBA, DFB) in aqueous humor was developed. DFBA and DFB were initially absorbed on a Pinkerton-type column, then analyzed by high-performance liquid chromatography using a semi-micro column after column switching. Under the optimized conditions, calibration curves for DFBA and DFB showed good linearity over the range of 1.0-50 and 0.5-50 ng/ml, respectively. We applied the method to the analysis of DFBA and DFB in rabbit aqueous humor, and found that DFBA in rabbit aqueous humor 1 h after instillation of 0.002% DFBA ophthalmic emulsion was not detected, but DFB was present at the concentration of 4.3+/-3.1 ng/ml.

Comparative studies of N-glycans and glycosaminoglycans present in SIRC (Statens Seruminstitut rabbit cornea) cells and corneal epithelial cells from rabbit eyes.

PURPOSE: We compared cultured Statens Seruminstitut rabbit cornea (SIRC) cells and corneal epithelial cells from rabbit eyes by analyzing their N-glycans and glycosaminoglycans (GAGs). This work is a fundamental study on the efficacy of using cultured cells instead of animals for drug development. MATERIALS AND METHODS: N-Glycans and GAGs from SIRC cell monolayers and corneal epithelial cells of rabbit eyes were analyzed by capillary electrophoresis (CE) and a combination of high-performance liquid chromatography (HPLC) and mass spectrometry. RESULTS: High mannose-type glycans and a hybrid-type glycan were the common N-glycans in SIRC cells and corneal epithelial cells of rabbit eyes. Mono-fucosylated biantennary glycans with or without one N-acetylneuraminic acid residue were observed only in SIRC cells. Hyaluronic acid was the only measurable GAG in the corneal epithelial cells of rabbit eyes. In contrast, hyaluronic acid and chondroitin sulfates were abundantly present in SIRC cells. CONCLUSIONS: Profiles of both N-glycans and GAGs were conspicuously different between SIRC cells and corneal epithelial cells of rabbit eyes. This report will be useful for the evaluation of pharmaceutical candidates when animals or cultured cells are employed in drug development studies.

Comparative studies of HPLC-fluorometry and LC/MS method for the determination of N-acetylneuraminic acid as a marker of deteriorated ophthalmic solutions.

Methods for determining the deterioration of ophthalmic solutions using both high-performance liquid chromatography (HPLC) with fluorescence detection and liquid chromatography coupled with selected ion monitoring mass spectrometry (LC/MS) are described. The methods are based on the determination of N-acetylneuraminic acid (NeuAc) released by the hydrolysis of foreign bodies that contaminate eye drops during use. The released NeuAc was either labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB) for fluorometric detection or detected without derivatization by mass spectrometry. The calibration curves for NeuAc showed good linearity between 1.2 ng/mL and 39 ng/mL for fluorometric HPLC and 5.0 ng/mL and 100 ng/mL for LC/MS, respectively. Detection limits for fluorometric HPLC and LC/MS were 0.20 ng/mL and 0.88 ng/mL, respectively. The NeuAc content of some model glycoproteins determined by LC/MS method were 62-78% of those determined by fluorometry. The differences are attributed to matrix effects but the LC/MS method afforded sufficiently high sensitivity that NeuAc in the foreign bodies could be determined in eight of nine test samples.

Corneal critical barrier against the penetration of dexamethasone and lomefloxacin hydrochloride: evaluation by the activation energy for drug partition and diffusion in cornea.

The cornea is a solid barrier against drug permeation. We searched the critical barrier of corneal drug permeation using a hydrophobic drug, dexamethasone (DM), and a hydrophilic drug, lomefloxacin hydrochloride (LFLX). The activation energies for permeability of DM and LFLX across the intact cornea were 88.0 and 42.1 kJ/mol, respectively. Their activation energies for permeability across the cornea without epithelium decreased to 33.1 and 16.6 kJ/mol, respectively. The results show that epithelium is the critical barrier on the cornea against the permeation of a hydrophobic drug of DM as well as a hydrophilic drug of LFLX. The activation energy of partition for DM (66.8 kJ/mol) was approximately 3-fold larger than that of diffusion (21.2 kJ/mol). The results indicate that the partition for the hydrophobic drug of DM to the corneal epithelium is the primary barrier. Thermodynamic evaluation of activation energy for the drug permeation parameters is a good approach to investigate the mechanism of drug permeability.

Capturing of acidic macromolecules from biological samples using a temperature-responsive polymer modified with poly-l-lysine.

We synthesized a temperature-responsive polymer, N-(isopropylacrylamide)-methacrylic acid copolymer, to which poly-l-lysine was introduced. The synthesized polymer as well as the parent polymer showed reversible soluble-insoluble changes in response to temperature changes across the lower critical solution temperature at 32 degree C in an aqueous solution. We found that the polymer efficiently captured acidic bio-macromolecules such as RNA, glycosaminoglycans and mucin-type glycoproteins in biological samples, and the captured molecules were recovered using aqueous NaCl solutions at high concentration. The target acidic molecules thus obtained will be employed for further studies such as structural analysis after brief desalting procedure. The proposed method does not require any chromatographic separations, but only needs a small volume of an aqueous salt solution for releasing captured molecules. Overall procedures are quite easy and simple, and are completed at least within 1 h. We show a few examples for capturing RNA and glycosaminoglycans from cultured cells using the polymer.