Development and Evaluation of Lyophilized Methotrexate Nanosuspension using Quality by Design Approach

Abstract

With the application of the quality by design (QbD) approach, a high-pressure homogenizer (HPH) methodology was employed to develop methotrexate nanosuspension (MTX-NS) to boost bioavailability. The Ishikawa diagram was used to analyze potential risk factors in formulation development. To screen and study the impact of various formulation and process factors on the critical quality attributes (CQA), the Placket–Burman design and central composite design were utilized. The number of HPH cycles, poloxamer 188 concentration, and tween 80 concentration were shown to be significant parameters (P<0.05), that were further optimized using Central Composite Design. The zeta potential of optimized lyophilized MTX-NS was determined to be –11.6 ± 7.52 mV and the average particle size was 260 ± 0.25 nm. In vitro cytotoxicity experiments revealed a greater than 80% inhibition, with apoptotic cells shrinking, fragmentation, and cell death. Furthermore, the C_max and AUC_0–t were increased by 2.53 and 8.83 folds, respectively. The relative bioavailability of MTX-NS was found to be 8.83 times higher than that of MTX-aqueous dispersion. As a result, the QbD method resulted in the development of a lyophilized MTX-NS with process understanding and control based on quality risk management.