Lipase-based in vitro release assays for pharmaceutical lipid formulations

Sammanfattning: The purpose of this project was to develop and test lipase-based in vitro release (IVR) assays for model and active pharmaceutical ingredients (API) entrapped in lipid formulations based on mixtures of phosphatidylcholine (PC) and glycerol dioleate (GDO). Two dyes, Fluorescein disodium salt and Patent Blue V sodium salt, functioning as model drug compounds, were investigated separately in the PC/GDO formulation. As the lipid formulation containing one of the two dyes was injected in vitro into a buffered saline solution, the lipids self-assembled into liquid crystal (LC) phases which trap the dye inside a nearly spherical LC depot. The dye was then released slowly over time. In the presence of lipases, the dye release was accelerated by biodegradation of the lipids. The IVR rate was followed by spectrophotometric measurements of the dye concentration in the aqueous release medium. This thesis describes the investigation of many parameters such as the suitability of the two dyes as model APIs, the effect of two different triacyl glycerol lipases (TGL) and a phospholipase, different concentrations of lipases as well as mixtures of different lipases, different lipid formulation types, the difference between a single vial experimental setup and a 96 well plate setup, as well as an overall evaluation of the project. It was shown that the addition of lipases dramatically increases the IVR rate, even at low concentrations. The effect on IVR was much higher for the phospholipase than for the TGLs. This research is of interest to the research- and development based pharmaceutical company Camurus AB in Lund, Sweden, currently developing pharmaceutical products based on the lipid liquid crystal formulation technology. The results will be used in the assessment and development of in vitro in vivo correlation (IVIVC) assays which are useful for formulation development and for regulatory purposes in conjunction with post registration changes related to Chemistry, Manufacturing and Controls (CMC).