Browsing by Subject "PAMPA"

This thesis work was carried out in Biology Section of Experimental Therapeutics Programme in Spanish National Cancer Research Center in Madrid. The aim of this work was to carry out characterization of ADME profile of novel protein kinase inhibitors synthesized by the Medicinal Chemistry Section of Experimental Therapeutics Programme. ADME refers to absorption, distribution, metabolism and excretion. ADME screening provides crucial information when choosing new chemical entities (NCEs) and lead compounds with desirable properties for further development and for clinical studies. ADME screening is carried out in the early discovery phase in order to avoid costly failures in the later stages. The protein kinase inhibitors used in this work were designed against three different targets. However, the targets cannot be disclosed in this work due to confidentiality reasons and thus they will be referred to as X, Y, and Z. The ADME characterization was performed in a high-throughput format to screen fast compounds with desired properties. To carry out ADME characterization for the novel compounds, several in vitro assays and in vivo pharmacokinetic studies were carried out. The work was started by setting up an LC-MS/MS detection method for each compound. All in all, LC-MS/MS detection method was set up for 63 new compounds. The detection method was used to analyze the results of different in vitro and in vivo studies. In vitro assays included kinetic solubility assay, parallel artificial membrane permeability assay (PAMPA), microsomal metabolic stability assay, plasma protein binding assay, and solubility in biological fluids. The solubility in biological fluids assay was performed only for the two compounds that were selected for the pharmacokinetic (PK) study. Pharmacokinetic properties of the compounds selected for the pharmacokinetic study using the Balb/C mice were further analyzed by their pharmacokinetic parameters. These parameters were calculated by applying non-compartmental model in the WinNonlin software. One compound, ETP-871, showed promising results in the pharmacokinetic study. Another compound ETP-827 was cleared too fast from the body. Too fast excretion is undesirable since low plasma concentration of the drug is insufficient to reach the therapeutic effect. For the compound ETP-827 a new PK study with higher dose was carried out. Due to the confidentiality reasons, these further studies are not presented in this work.

Prodrugs are pharmacologically inactive molecules which undergo metabolic bioactivation in vivo to form pharmaceutically active agents. Prodrugs have been designed to improve so called drug-like properties of active parent compounds (APC) i.e. to increase solubility or absorption and to reduce first-pass metabolism etc. In this master's thesis the goal was to establish non-cell-based in vitro methods to study prodrug bioactivation. Four commercially available prodrugs (bambuterol, olmesartan medoxomil (OM), candesartan cilexetil (CC) and famciclovir) were used as test compounds. The prodrugs were incubated in liver and intestinal S9 fractions and blood plasma to study in vitro bioactivation of these prodrugs. Other metabolism of the prodrug and APC (nonproductive metabolism) was studied by comparing incubation with and without cofactors of metabolizing enzymes. Species differences was studied using human, rat and dog matrices. Prodrug concentrations were quantified from the incubation samples using liquid chromatography- tandem mass spectrometry (LC-MSMS) methods developed for this study. Additionally the effect of promoiety on passive permeability was studied with parallel artificial membrane permeability assay (PAMPA). All of the studied prodrugs produced at least low concentrations of APC in one or more incubations. Terbutaline (APC of bambuterol) formation was observed in human plasma and was concentration dependent which is consisted with the literature. Olmesartan and candesartan were formed in S9 fraction in high rate, but not in buffer: indicating enzyme mediated hydrolysis. However, based on literature CC hydrolysis was not expected to occur in intestinal S9 fractions. Penciclovir (APC of famciclovir) was formed only in presence of human or rat liver S9 fraction which was in line with the pre-existing literature. With the method used the nonproductive metabolism could not be estimated. In PAMPA bambuterol, famciclovir and OM had higher permeability than corresponding APCs whereas CC was only more permeable than candesartan in pH 7.4. The in vitro incubation used in this study can be used for screening prodrugs. However both low and high activation rates were observed thus the clinically relevant in vivo APC formation can be achieved with both high and low bioactivation in vitro. Studying the rate of prodrug formation alone estimations about clinically relevant bioactivation rates cannot be concluded. No clear signs of nonproductive could be seen with the prodrugs studied with current method. For the estimation of nonproductive metabolism, metabolite screening studies would need to be developed and conducted parallel to studies prescribed in this master's thesis.