¹⁸F-labelling, cellular uptake and biodistribution of novel acyclic purine nucleosides for monitoring HSV1-tk based gene therapy

Aim
Suicide gene therapy utilizing herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) requires careful monitoring of the successful transfection. ¹⁸F-labelled nucleoside analoga, especially acyclic nucleosides such as 9-[(3-[¹⁸F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([¹⁸F]FHPG) and 9-[(4-[¹⁸F]fluoro)-3-hydroxymethylbutyl]guanine ([¹⁸F]FHBG) are currently used as PET tracers for monitoring and quantification of HSV1-tk expression. To improve the tracer properties, two new precursors have been synthesized by introduction of bromine in order to get the new substrates 8-bromo-9-[(3-[¹⁸F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([¹⁸F]FHBrPG) and 8-bromo-9-[(4-[¹⁸F]fluoro)-3-hydroxymethylbutyl]guanine ([¹⁸F]FHBrBG) for monitoring gene expression of HSV1-tk.

Materials and methods
The precursors as well as the nonradioactive reference compounds were prepared by introduction a p-toluenesulphonyl leaving group and trityl protecting groups into the brominated derivatives of ganciclovir and penciclovir. In a further step the precursors reacted with a K[¹⁸F]F kryptofix complex to trityl-protected intermediates. Then, the protecting groups were removed by heating in methanolic acetic acid solution. Both tracers were purified by RP HPLC separation. The identity of the tracers with the nonradioactive reference compounds was proved by HPLC. The in vitro uptake of all compounds was evaluated in a glioblastoma cell line stably transfected with the HSV1-tk gene and in the respective non-transfected cell line. The biodistribution and biokinetics of the radiotracers have been studied in mice and rats by small animal PET.

Results
The radiochemical yield of the ¹⁸F-tracers amounts to 10-15% (decay corrected) after a synthesis time of 85 95 min, the radiochemical purity was > 98%. The average specific activity was 19 GBq/µmol at the end of synthesis. Cell-uptake studies were carried out with both the brominated and the non-brominated derivatives of ganciclovir and penciclovir as standards. The relative cellular uptake (ratio HSV-tk-positive/controls) was 12.4±4.5 after 1 h of incubation with [¹⁸F]FHBrBG. In contrast, the uptake ratio of the other tracers [¹⁸F]FHBrPG (1.7±0.4), [¹⁸F]FHBG (4.0±2.3) and [¹⁸F]FHPG (1.6±0.3) was significantly lower than that of [¹⁸F]FHBrBG. Renal elimination was the main route of tracer clearance from the body, however with increasing lipophilicity of the tracers, increasing amounts were also eliminated through liver and bile into the intestine.

Conclusions
The novel tracer [¹⁸F]FHBrBG showed a superior uptake in HSV1-tk transfected cells compared to the clinically tested PET-tracer [¹⁸F]FHBG. Therefore [¹⁸F]FHBrBG appears to be a novel tracer for an improved monitoring of HSV1-tk based gene therapy approaches with PET.