In rapidly growing tumors such as glioma, the nucleoside analogue 5-lodo-2-deoxyuridine is used as efficient diagnostic agent and provides a means to determine the proliferative activity of the tumor. On the other hand, (radiolabeled) 5-ethyl-uridine analogues (targeting the viral thymidine kinase 1: HSV1 TK) can be used to assess the viral replication and the tumor response to therapy in "suicide gene therapy" and/or to inhibit gene therapy. Thus there is a prominent interest for inexpensive 99m Tcbased nucleoside analogues for non-invasive diagnosis.
In this study we present the first quantitative in vitro and the first in vivo evaluation rhenium/ technetium-tricarbonyl complexes of thymidine. Furthermore, in silico molecular dynamic and molecular docking experiments are described based on the structure of the structure of viral TK with selected compounds.
Thymidine was functionalized at position C5โฒ. For tridentate coordination of [M(H 2 O) 3 (CO) 3 ] + (M = Re, 99m Tc), 1, an iminodiacetic acid chelating system was chosen and spacer entities of different length have been introduced between the pharmacophor and the metal core. Enzyme kinetic studies revealed inhibition exclusively of the human cytosolic thymidine kinase (hTK1). The compounds revealed different K i -values (7-334 ยตM). On the other hand, complex 2 with a spacer of โผ30 ร
(see figure) evinced also inhibition of the HSV1 TK (K i = 2 ยตM for HSV1 TK and 30 ยตM for hTK1). Molecular docking and dynamics (MD) studies corroborate these observations. Stable simulations of HSV1 TK-Re-thymidine complex were only found in case of 2. In vitro studies with compound 2 (M = 99m Tc) using two different osteosarcoma cell lines expressing (TK+) or lacking (TK-) HSV1 TK revealed statistical differences in internalization (0.5 % vs. 2.5 % of total activity 4 h post incubation). In vivo studies in athymic nu/nu mice bearing TK+/-osteoblastoma cells, however, revealed no differences in tumor uptake (0.2ยฑ0.1 % ID/g in both cases). On the other hand in MG63 osteoblastoma cells expressing hTK1 displayed a significant higher uptake in tumor tissue (0.8ยฑ0.4 % ID/g, 3 h p.i.). Route of clearance was predominantly via the hepatobiliary pathway due to the pronounced lipophilicity of complex 2 (liver: 4.8ยฑ1.3 %, intestines: 2.6ยฑ0.9 % ID/g, 3 h p.i.). Radioactivity in other organs and tissues was < 0.4 % ID/g except for the stomach (0.9ยฑ0.6 % ID/g). In vivo experiments with more hydrophilic complexes and different cell lines expressing hTK1 are currently in progress.
At the present time our data preclude the use of 99m Tc-thymidine derivatives functionalized at position C5โฒ for use in suicide gene therapy because of low tumor uptake or lack of selective interaction with HSV1 TK. However, the compounds can potentially be employed to monitor tumor proliferation.
In vivo studies with different other tumor cell lines are currently in progress. MD experiments based on the recently published structure of human TK will be presented. These calculations should aid to develop more potent and more selective 99m Tc-based nucleoside inhibitors and substrates respectively.