Box-Behnken design was applied for the development of a novel, precise, simple, and accurate high-performance thin-layer chromatography method for simultaneous estimation of mentioned drugs in the formulation. Optimization was performed using Quality by Design. Principles of risk assessment followed by application of design for optimization of chromatographic conditions to determine critical parameters: volume of hexane, chamber saturation time, solvent front and their effects on critical method attributes: R f values of both drugs. Optimized conditions were determined through Derringer's desirability approach of the multi-criteria decision-making technique. Good resolution using optimized mobile phase, hexane:ethyl acetate:ammonium acetate in methanol (5.1:3:2) with R f values 0.34 and 0.67 for Ivabradine Hydrochloride and Carvedilol, respectively, was obtained. The method was validated as per International Council on Harmonization having a linear range of 1000-5000 ng/band of Ivabradine and 1250-6250 ng/band for Carvedilol, respectively. %Recovery was in range of 99.23-101.3 and 99.8-100.5, respectively. Precision and robustness were found to be in an acceptable range. Thus, Quality by Design based method development assisted in making design space with knowledge of all method performance characteristics with a better understanding of high-performance thin-layer chromatography. The Analytical GREEnness profile was assessed using AGREE tool with an overall reliable score of 0.68.

Background: Glaucoma is widely treated using eye drops, but around 95% of the drug is lost by the ocular barrier resulting in low bioavailability. The incorporation of polymeric nanoparticles into mucoadhesive polymer containing in situ gel is generally helpful in the retention of nanoparticles on the eye and improves the efficacy of the formulation. Objective: The objective of the present investigation has to develop polymeric brinzolamide (BRZ) nanoparticles laden with timolol maleate (TM) in situ gel formulation. Method: The optimized BRZ nanoparticles were prepared using PLGA by nanoprecipitation technique utilizing 32 full factorial designs (FFD). Healthy New Zealand White Rabbit (250-300 g) was used for the pharmacokinetic and pharmacodynamic study. Design of the experiment was applied to optimize formulation and validate the model. Some evaluation parameters related to BRZ nanoparticles as well as in-situ gel, have been done. Result: The results of FFD reveal that the optimized condition for drugs to polymer ratio (1:7) containing 0.98 %w/v for poloxamer 188 results in higher entrapment efficiency and drug release with 156.7 nm particle size. The in-situ gel formulation has been prepared using Gelrite (0.5%w/v), and HPMC K4M (0.5%w/v) shows acceptable results with sustained drug release up to 6±0.1 h. The rabbit model's in-vivo pharmacokinetics and pharmacodynamic data showed sustained release of drugs longer than the marketed formulation. Conclusion: The proposed formulation could successfully deliver therapeutic concentrations in the eye with prolonged resident time and serve as a potential alternative for the treatment of glaucoma.