Browsing by Subject "drug sequestration"

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  • Huusko, Karoliina (Helsingfors universitet, 2013)
    Intracellular drug sequestration is useful to understand when designing new drugs with intracellular targets. The knowledge of the intracellular distribution can also help to understand the side effects and pharmacokinetics of a drug, as well as the lack of response in e.g. some multidrug resistant cancer cells. Intracellular concentrations are also important to know when predicting the role of active transport in the overall transport process when binding site of the transporter is intracellular. The literature review describes the mechanisms causing intracellular drug sequestration along with the consequences of intracellular drug sequestration and methods that are used to study it. Alterations of intracellular distribution of anticancer drugs in multidrug resistant cancer cells are also described as an example of the many factors affecting the distribution pattern of the drugs inside cells. Understanding these mechanisms is valuable when designing strategies to overcome the multidrug resistance. The most commonly applied methods for studying intracellular concentrations of drugs are based on fluorescence microscopy. In experimental work, subcellular fractionation protocol is introduced and applied to determine the concentration of CDCF, clotrimazole and celiprolol in vitro in the plasma membrane and cytoplasm of MDCKII cells. CDFC and celiprolol are substrates of the MDR1 transporter and clotrimazole is an inhibitor. Concentrations in the fractions were measured in wild type cells and in MDR1-transfected cells with and without MDR1 inhibitor verapamil to see if the transporter had an effect on the concentrations. Also the effect of lipophilicity of the drug on partition between plasma membrane and cytoplasm was reviewed. Celiprolol showed a typical behaviour of the MDR1 substrate whereas CDCF and clotrimazole did not. Clotrimazole as a lipophilic compound was accumulated more to the plasma membrane than less lipophilic CDCF and celiprolol. Lipophilicity affected also to the ratio of Km (or Ki)(determined from the concentration in extracellular fluid) and Km (or Ki)(membrane) (determined from the plasma membrane concentration) values, with clotrimazole Ki(membrane) value being larger than respective Ki value, and CDCF and celiprolol Km(membrane) values being smaller than their respective Km values.