Validation et conditionnement d'un test PAMPA amélioré pour l'évaluation de la perméabilité membranaire de médicaments
PAMPA tests and Caco-2 tests are in vitro assays used in the evaluation of the intestinal permeability of drugs. They are performed during the discovery phase of a drug. PAMPA tests are not biologically representative of the intestinal membrane, but they can be done quickly and are low cost. On the other hand, Caco-2 tests require a cell culture time of 21 days and specific installations. They are composed of a confluent enterocyte monolayer and, therefore, more biologically representative. There is a need to develop a hightroughput biologically representative assay of the human intestinal membrane at low cost. The first goal of this project is to develop an analytical method for the simultaneous quantification of eight control drugs used in the assay validation. The second goal of this project is to optimize an improved PAMPA membrane, the neoPAMPA. This new membrane is constituted of three components: (1) a porous filter that acts as a support, (2) a negatively charged polydopamine cushion that serves as an anchor and ensures the bilayer fluidity (3) a lipid bilayer formed by the fusion of vesicles. An HPLC-MS/MS analytical method was validated according to FDA and EMA specifications. This method allowed to simultaneously quantify eight standard drugs used in the neoPAMPA test. A PAMPA test was also performed as a control assay. The permeability coefficients measured through the PAMPA membrane for the eight drugs compared favorably to literature results. The components of the neoPAMPA membrane were optimised. The optimal conditions were polycarbonate hydrophilic filters having pores of 15 nm, Costar 12 wells plates used as the device for the permeability assays, a lipid bilayer composed of 70 % DOPC and of 30 % cationic cholesterol and a deposition of liposomes in presence of 150 mM NaCl followed by an equilibration time of 1 h in presence of a saturated DOPC solution. The stability of the eight drug cassette and the liposomes was insufficient for the conditioning of commercial neoPAMPA membranes. Different optimisation parameters improved the neoPAMPA membrane, although without making it functional. The neoPAMPA membrane was not sufficiently able to discriminate molecules as a function of their expected permeability.
PAMPA Polydopamine Permeability Lipid bilayer hplc-ms/ms