Überprüfung des Drug Delivery Konzepts von „Lipid-Nucleosid- und Polysaccharid-Konjugaten von 5-Fluorouracil“

In 2012 malignant diseases were ranked at second position in German mortality statistics (Becker & Holzmeier 2012). Chemotherapy is one of the most important components in the therapy of malignant tumors. However, despite great advances in recent years only a marginal improvement of the survival of the patients could be achieved (Bourret et al. 1979, Krook et al. 1991). 5-Fluorouracil (5-FU) - an uracil analogue - is an antimetabilte, which is widely used in the therapy of malignant illnesses (Bodner-Adler et al. 2007, Carrato 2008, Khosravi Shahi et al. 2007, Kish et al. 1994, Zhao et al. 2013). One of the major handicaps of many chemotherapeutics- like 5-FU- is the insufficient penetration through cell membranes due to high hydrophobicity (Malecki et al. 2013a). Therefore, Prof. Dr. Rosemeyer und Dr. Malecki developed in a cooperation project (Prof. Rosemeyer, Osnabrück/Prof. Kinscherf, Marburg) several derivatives of 5-FUrd (2a) carrying lipophilic moieties at the N(3)- and/or in the 2‘3‘-O-position (3a-7a, 3c, 2c, 3b-7b) with the aim to improve the penetration and to achieve less side-effects due to higher lipophilicity (Malecki 2013, Köstler et al. 2013, Malecki et al. 2013b, Malecki et al. 2014, Malecki & Rosemeyer 2010, Rosemeyer, H., Malecki, E., Rosemeyer, H., Malecki, E., Werz, E., Korneev, S., Gall, K., Werz et al. 2013). Additionally, the 2‘3‘-O-Nonadecylidene derivative of uridine, 3c, was compounded for comparative reasons (Köstler et al. 2013). This dissertation describes the in vitro testing of the drug delivery concept of the „lipid-nucleoside- and polysaccharide-conjugate of 5-FU (2a, 3a-7a, 3c, 2c, 3b-7b) towards three carcinoma cell lines HT-29 (human colon carcinoma), HepG2 (human hepatoblastoma), RENCA (murine renal adenocarcinoma) and on differentiated human THP-1 macrophages. We incubated HT-29 cells for 24 h with ATTO 245®-conjugated substances (2c, 3b-7b) in order to find out, if the substances are able to pass the cell membrane and if so, in which cell compartment they were located after penetration. All derivatives passed the cell membrane and accumulated in the cytoplasm, all avoiding the nuclei, except derivative 5-FUrd (2c), which showed a granulated distribution in the cytoplasm (Malecki et al. 2013a). The impact of the absorption of the derivatives (3a-7a, 3c) on proliferation and apoptosis of the cells (in comparison with 5-FU (1) and 5-FUrd (2a)) set up priorities in this dissertation. After 24 h, 48 h or 72 h of incubation of HT-29 human colon carcinoma cells with the derivatives 1 (5-FU) (14-23%, 35-46% and 30-58%) or 2a (5-FUrd) (33-45%, 60-67% and 58-67%) the survival of the cells was significantly decreased, in comparison to the negative control. Additionally, after 24 h treatment of the HT-29 cells at concentrations of 40 µm and 80 µm with the derivatives 3a (77% and 95%), 5a (30% and 86%) and 3c (89% and 96%) significantly reduced the viability of the HT-29 cells in comparison to the negative control. These three derivatives showed after 24 h of incubation of HT-29 cells at concentrations of 40 µm and 80 µm a significant reduction of the viability in comparison to the positive control (1) (61-81%, 73% and 80-82%), too. Incubation (48 h) of the HT-29 cells with the derivatives showed that four derivatives at concentrations of 40 µm and 80 µm significantly reduced the viability in comparison to 5-FU (~positive control): at the derivatives 3a (9-54%), 5a (5-48%), 7a (2-30%) and 3c (53-63%). Derivatives 4a and 6a did not affect the viability of the HT-29 cells. The derivatives 3a, 5a, 7a and 3c also showed a significant decrease of viability in other cancer cell lines (RENCA and HepG2): After 48 h of incubation of the RENCA cells, the derivatives 3a (51-57%) and 5a (56%) showed a significant decrease of viability compared to 5-FU. The derivative 3c (38-49%) and 7a (25-44%) revealed similar cytotoxic effects on the viability of RENCA cells like substance 1 (29-48%) and 2a (41-46%). After 48 h incubation of HepG2 cell lines, the derivatives 3a (72-75%), 5a (53%), 7a (43%) and 3c (22-74%) showed a significant decrease of viability in comparison to 5-FU. Investigations of the derivatives using differentiated macrophages (THP1) were of major importance for the discrimination between cytotoxic- and side-effects. The derivative 5a showed a significant 6-27% increase of the viability in comparison to 5-FU (~positive control) after 48 h of incubation. The ATTO 425®-conjugated derivatives 3b, 4b, 5b, 6b and 7b were tested regarding their anticancer activity in human HT-29 cells after 24 h and 48 h of treatment. All of the derivatives lost their anticancer activity after conjugation with ATTO 425® at all concentrations under test (10-80 µm) in comparison to the control. Using concentrations of 10-80 µM only the ATTO 425®-conjugated derivative 5-FUrd (2c) showed a significant 31-52% or 41-57% reductions of the viability in comparison to the control . Furthemore, we tested the impact of the derivatives on induction of apoptosis or proliferation in HT-29 cells after 1 h, 2 h, 4 h or 6 h of incubation in comparison to 5-FU (1) and 5-FUrd (2a). The derivative 5a showed the highest increase of apoptosis at a concentration of 80 µM - after 2 h (3.1-fold), 4 h (3-fold) and 6 h (4-fold). After 6 h of treatment at concentration 40 µM a significant 3.3-fold increased apoptosis rate was observed. Moreover, the effects of the treatment of HT-29 cells with the substances on gene signatures were part of the investigation. In real time PCR (qRT-PCR) the derivative 5a (40 µM) showed a significant 2-fold increase of the tumor suppressor gene p53 expression compared to the negative and positive control (5-FU), and a 1.5-fold increased expression of the proapoptotic Caspase-3 gene. This dissertation shows that the cytotoxicity of several derivatives of 5-FUrd (2a) was more effective than 5-FU, respectively, 5-FUrd using several cancer cell lines. Especially the derivative 5a is of major interest, because it has no cytotoxic effects on differentiated human macrophages (~ immune cell), despite of its high cytotoxic activity on several cancer cell lines. Thus, the derivative 5a could be a novel potential cytotoxic multipotent chemotherapeutic drug. For the final assessment of the pharmacological/therapeutic potential, additional investigations, such as microtumors/-spheres, animal studies, and prospective clinical trials are needed.