Seite - (000118) - in Biomedical Chemistry: Current Trends and Developments

Advances in techniques for chemical synthesis allow the synthesis of hundreds to thousands compounds every month. As described in Chapter 2.1.2, several high-throughput in vitro assays have been extensively used in early discovery to select the NCEs most likely to have favorable pharmacokinetic parameters. The early ADME screening of a larger number of samples is therefore required, which in turn calls for high-throughput bioanalytical approaches. Although most publications on in vitro studies hardly pay attention to the bioanalytical aspects involved in obtaining data, it becomes clear considering the examples depicted in Table 2.1.6 that bioanalytical techniques used to support these investigations commonly employ HPLC-UV, LC-MS or LC-MS/MS. LC-UV techniques are mainly used to collect data from in vitro absorption methods, which aim at investigating the in vitro transport of drugs through intestinal or other cell layers, predicting the in vivo transport of a drug through the intestinal membrane and the involvement of certain transporter proteins, particularly the efflux transporter P-glycoprotein and the oligopeptide transporter (PepT1). The most common in vitro systems are the colorectal carcinoma cell line (Caco-2) and other cell lines, like the MDCK (Volpe, 2011). The Ussing chamber technique has also been employed, incorporating a healthy intestinal membrane. In both techniques, after a specific incubation time, samples are taken from both sides of the monolayers for bioanalytical determination of the drug concentration. Most of these in vitro techniques are calibrated within the laboratory using a set of commercial drugs with known human absorption fraction, which are plotted against the experimentally obtained apparent permeability (Fortuna, 2012). In our laboratory, we have developed an Ussing chamber technique employing mouse jejunum segments in order to predict the human intestinal absorption fraction and the involvement of P-glycoprotein in drug absorption. The technique was initially validated with reference compounds and then applied to compare the absorption of nine derivatives of carbamazepine, a classical antiepileptic drug (Fortuna, 2012). An HPLC-UV technique was successfully employed in order to quantify each compound in our samples. However, a major drawback is one frequently ascribed to the use of HPLC-UV in these analyses, which is that for poorly permeable drugs, the LLOQ achieved