N-Acetylcysteine amide (NACA) and diNACA inhibit H2O2-induced cataract formation ex vivo in pig and rat lenses.

Oxidative stress plays a central role in cataract formation suggesting that antioxidants might slow cataract progression. The anticataract activity of N-acetylcysteine amide (NACA) and (2 R, 2 R')-3,3'-disulfanediyl bis(2-acetamidopropanamide) (diNACA) and/or N-acetylcysteine (NAC), were evaluated in porcine and rat lens models. Cataractogenesis via oxidation was induced with H2O2 and/or glucose oxidase (GO). Porcine lenses were incubated in 0.1 mM, 1 mM, or 10 mM NAC, NACA or diNACA for 24 h. Lenses were then transferred to media containing 0.75 mM H2O2 and 4.63U of GO in order to maintain a constant H2O2 level for an additional 8 h. At the end of incubation, lenses were imaged under darkfield microscopy. Separately, rat lenses were extracted from 3-week-old Wistar rats and incubated with either 10 mM NACA or 10 mM diNACA for 24 h prior to treatment with 0.2U GO to generate a steady source of ∼0.6 mM H2O2. Rat lenses were analyzed by LC-MS/MS to quantify changes in cysteine, cystine, glutathione (GSH) or oxidised glutathione (GSSG) levels in the lens epithelium, cortex or core. Pre-treatment with NACA or diNACA followed by oxidation with H2O2 and/or GO to stimulate cataract formation afforded rapid assessment in ex vivo porcine (32 h) and rat (48 h) lens models. Pre-treatment of isolated porcine lenses with 0.1 mM, 1 mM or 10 mM of either NAC, NACA or diNACA followed by H2O2/GO treatment resulted in reduced lens opacity relative to the lenses exposed to H2O2/GO, with NACA and diNACA reducing opacities to a greater extent than NAC. Rat lenses incubated with 10 mM NACA or 10 mM diNACA without exposure to H2O2 showed no signs of opacities. Pre-treatment of rat lenses with 10 mM NACA or 10 mM diNACA, followed by GO cataract induction resulted in reduced opacities compared to control (GO alone). LC-MS/MS analyses revealed that NACA, but not diNACA, increased cysteine, cystine and GSH levels in rat lens epithelium and cortex regions. Taken together, both NACA and diNACA inhibited cataract formation to a greater extent than NAC (all at 1-10 mM) in an ex vivo porcine lens model. Both NACA and diNACA (both at 10 mM) reduced cataract formation in rat lenses. Based on LC-MS/MS analyses, NACA-induced reduction in opacity observed in rat lenses was attributed to enhanced cysteine and GSH levels while the diNACA-induced reduction in opacity induced did not consistently increase cysteine, cystine and GSH levels and, therefore, appears to involve a different antioxidant mechanism. These screening studies warrant further testing of NACA and diNACA as anticataract agents.

Stability storage and testing of ophthalmic products for global registration.

Regulatory stability testing requirements for various types of ophthalmic products, including eyedrops, eye ointments, ophthalmic inserts, injections, irrigating solutions, lens washes, and lenses, are not always straightforward. The International Conference on Harmonization (I CH) guidelines do not completely address all of the stability conditions required for these types of pharmaceutical products and medical devices; regulatory agencies around the world still differ on the exact data requirements, storage conditions (especially temperature and humidity conditions), and certain testing methods. These uncertainties make the development of a global ophthalmic product very difficult. This article reviews many of the appropariate internationally recognized, regulatory guidelines and many of the important points to consider in stability studies for new ophthalmic products. Discussions of regulatory experiences and Expert Reports provide insight to common questions frequently asked by regulatory agencies on these types of products.

Determination of benzalkonium chloride in ophthalmic solutions containing tyloxapol by solid-phase extraction and reversed-phase high-performance liquid chromatography.

A procedure using solid-phase extraction (Supelcoclean CN) followed by HPLC [Beckman Ultrasphere CN, acetonitrile:phosphate solution (60:40, v/v)] was developed and validated to quantitate the quaternary ammonium preservative benzalkonium chloride in an experimental ophthalmic formulation containing the polymeric material tyloxapol. This procedure makes routine determinations of benzalkonium chloride at concentrations of 0.0035 to 0.01% simpler than the traditional ion-pairing colorimetric methods. This method is quick, specific, and especially useful for drug product stability studies. In addition, because the method distinguishes each homologue, it can be extended to routinely determine the homologue ratio for quality control purposes.

Deposition of ciprofloxacin, prednisolone phosphate, and prednisolone acetate in SeeQuence disposable contact lenses.

We noted opaque deposits in SeeQuence disposable contact lenses in three patients with persistent epithelial defects who were being treated with topical ciprofloxacin and prednisolone acetate. In each patient, the contact lenses with deposits were removed and replaced. High performance liquid chromatography analysis revealed the deposits to be precipitates of ciprofloxacin and prednisolone acetate. We incubated new SeeQuence disposable contact lenses in ciprofloxacin, prednisolone phosphate, and prednisolone acetate alone and in combination. Precipitates did form when ciprofloxacin was combined with either prednisolone acetate or prednisolone phosphate. We recommend removal and replacement of contact lenses should these deposits develop to prevent the possibility of corneal toxicity.

Metabolism of 3-(p-chlorophenyl)pyrrolidine. Structural effects in conversion of a prototype gamma-aminobutyric acid prodrug to lactam and gamma-aminobutyric acid type metabolites.

By use of rat liver or brain homogenate supernatants containing microsomes and/or mitochondria, it was found that the prototype GABAergic prodrug [3-(p-chlorophenyl)pyrrolidine (1)] underwent a series of alpha-oxidation transformations to a pair of amino acid metabolites and a pair of lactam metabolites [4-amino-3-(p-chlorophenyl)butanoic acid, baclofen (5); 4-amino-2-(p-chlorophenyl)butanoic acid (10); 4-(chlorophenyl)pyrrolidin-2-one and 3-(p-chlorophenyl)pyrrolidine-2-one (11)]. With the liver homogenates, the formation of the lactam metabolites was approximately 2 orders of magnitude greater than that of the amino acid metabolites, while with the brain homogenates, the amino acid and lactam pathways were of similar magnitude. For either tissue, for both the lactam and the amino acid series, attack at the less sterically hindered 5-position of the pyrrolidine ring was greater than the attack at the 2-position (5 greater than 10 and 6 greater than 11) with the exception of the liver homogenate mitochondrial fraction (6 less than 11). The parenteral administration of the prodrug 1 was found to give detectable brain levels of 5 as well as activity in an isoniazid-induced (GABA-inhibited) convulsion model.

Determination of baclofen and alpha-baclofen in rat liver homogenate and human urine using solid-phase extraction, o-phthalaldehyde-tert.-butyl thiol derivatization and high-performance liquid chromatography with amperometric detection.

Methods were developed for the determination of the zwitterionic compounds baclofen and alpha-baclofen in complex biological samples (rat liver homogenates and human urine) in concentration ranges that would be suitable for pharmacokinetic studies of these compounds. In the procedure, the biological samples along with an internal standard were selectively concentrated using C18 solid-phase extraction cartridges, evaporated, derivatized with o-phthalaldehyde and tert.-butyl thiol at room temperature for 2 min, then subjected to high-performance liquid chromatographic analysis. The reversed-phase (C18) chromatographic analysis with amperometric detection (glassy carbon electrode in oxidative mode, +0.6 V vs. Ag/AgCl) was found to be useful for the measurement of both baclofen and alpha-baclofen from 10 ng/ml to 10 micrograms/ml in these complex biological samples. The primary advantage of the method was that the derivatives formed using tert.-butyl thiol were markedly more stable than the previously reported derivatives prepared using mercaptoethanol.