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The importance of genomic variation in the biotransformation of drugs is well known. Only in the last two decades, the big importance of drug membrane transporters and of genomic variation in their genes became apparent. For this reason and because of the importance in drug-drug interactions, the identification of the relevant membrane transporters has meanwhile become an essential part of pre-clinical and early clinical drug development. Particularly for hydrophobic drugs the ATP consuming efflux transporters like the multidrug resistance transporter 1 (MDR1) are highly important. In addition to the efflux transporters, there are influx transport proteins which accelerate the uptake of hydrophilic cationic molecules into the cell. One group of these influx transporters are the organic cation transporters (OCTs). In order to measure substrate concentrations in transport experiments within the cell, the analytical method of the high pressure liquid chromatography (HPLC) is the most commonly used procedure alongside to the still widespread use of radioactive labels. However, not every previously published method can be easily transfered to the existing conditions. The specific challenges of this present work resulted from the small volumes and low concentrations to be analyzed in the transport experiments. The thesis had the following objectives: Firstly, the HPLC methods for quantitative analysis of drugs such as proguanil, cycloguanil, amisulpride and debrisoquine for membrane transport experiments should be optimized and validated. The cellular uptake of proguanil and cycloguanil should be measured in HEK293 cells which overexpress the organic cation transporters OCT1, -2, and -3 and OCTN1 and -N2. The data should be further validated by inhibition of the transport using typical inhibitors of OCTs and drugs relevant as comedication. In addition, the impact on the relevant inherited variants of organic cation transporters should be analyzed. Finally, the transport of proguanil into non-infected erythrocytes should be characterized. This should include inhibition experients with prototypical inhibitors of organic cation transporters and with substances relevant as co-medication.
Based on validation and optimization of HPLC methods, the concentration of the drugs proguanil, cycloguanil, amisulpride and debrisoquine could be determined quantitatively. The drugs were measured and detected in the low nanogram/ml concentration range. In-vitro experiments showed that the antimalarial drug proguanil and its active metabolite cycloguanil can be transported with different maximum transport velocities and affinities by the electrogenic organic cation transporters OCT1, OCT2 and OCT3. In contrast to them, the organic cation transporter OCTN1 and OCTN2 overexpressed in HEK cells didn´t show any increase in the cellular uptake of proguanil and cycloguanil. Selected inherited variants of OCT1 existing in Caucasian populations with a relatively high frequency and differing in their transport functions with many other drug also showed altered uptake of the drug proguanil and its active metabolite cycloguanil here. Coincubation with other drugs or model substrates in different concentrations showed significant reductions in the transport of proguanil and cycloguanil by OCT1. In particular, transport activity was reduced by quinine, mefloquine and omeprazole. A carrier-mediated transport of the antimalarial drug proguanil through the erythrocyte membrane could also be shown. This process could be inhibited and followed a Michaelis-Menten kinetics. Interestingly, the erythrocyte uptake could be inhibited by the OCTN substrate carnitine, a finding which requires further studies considering that overexpression of OCTN1 and OCTN2 in HEK cells did not increase proguanil or cycloguanil uptake. In conclusion, the results showed that the organic cation transporters OCT1, OCT2 and OCT3 may play a role in the absorption and secretion of proguanil and cycloguanil within the human organism.