CHROMATOGRAPHIC METHODS FOR PHYSICO-CHEMICAL EVALUATION AND PERMEABILITY PREDICTION IN EARLY DRUG DISCOVERY

In the preclinical phase, developing new analytical methods and new assays to improve the throughput, success rates, and the efficiency of the discovery projects, to minimize waste of resources and the attrition, which is the loss of the compound during the late stages of development, is very important and crucial. The physico-chemical properties determine the performance of the New Chemical Entities (NCEs) during absorption, distribution, metabolism and excretion processes. Experimental methods remain the most widely used and reliable approach to measure the physico-chemical properties; however, nowadays many software tools are available to calculate and predict physico-chemical properties of the compounds. Despite these advances, mathematical models and software cannot fully replace the experimental measurements; as well as being more reliable, experimental data are necessary for refining predictive algorithms through iterative processes. This project, carried out in collaboration with Chiesi Farmaceutici S.p.A., is the result of studies and analysis conducted within the Analytics and Early Formulations (A&EF) department, under the supervision of Dr. Valentina Mileo, Dr. Elisa Moretti, Prof. Marco Mor from University of Parma. This department is located in the preclinical area, on the Global Research and Preclinical Development site based in Parma. This work stems from my Master’s thesis, and it is aimed at the setup and implementation in Chiesi’s screening cascade of chromatographic methods for physico-chemical evaluation in early drug discovery stage. Specifically, 19 pharmaceutical standards and hundreds of Chiesi’s compounds, belonging to Rule of Five (Ro5) and beyond Rule of Five (bRo5) classes, including peptides and macrocycles, were fully evaluated to assess the capability of these experimental methods to describe passive permeability better than, or as well as, the gold standard assays. The main physico-chemical properties usually determined to adequately characterize NCEs and to guide chemical synthesis, which include acid dissociation constant (pKa), lipophilicity, polarity, and propensity to form Intramolecular Hydrogen Bonds (IMHBs), will be described in detail below. Each physico-chemical property investigated will be presented in a dedicated chapter, outlining the aims, materials and methods used, and the results obtained. These results include comparison with established golden standard. Furthermore, lipophilicity and polarity will be analyzed to evaluate their capability to predict the formation of IMHBs using the NMR technique as “positive control assay”. A specific section of this thesis will also provide a brief description of the experimental method used to determine permeability, performed in collaboration with the Department of Pharmacokinetics, Biochemistry and Metabolism (PKBM) at Chiesi Farmaceutici S.p.A and will be followed by an assessment of polarity and lipophilicity as predictors of passive permeability. The results will be presented, including comparison with the gold standard data, and conclusions will be drawn based on these findings.