N¹-Methyl-9-[(4-[¹⁸F]fluoro-3-hydroxymethylbutyl]guanine [¹⁸F]MFHBG as new substrate of HSV-1-thymidine kinase to monitor gene expression?

Aim
Gene therapy with the transfer of Herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) has shown significant potential in treating several cancers. Many nucleoside analogues are known to localize selectively HSV1-tk transfected cells and show a high specificity for the viral enzyme, especially acyclic nucleosides such as ganciclovir or penciclovir. The penciclovir derivative [¹⁸F]FHBG is one of the proposed tracer for monitoring the gene expression by positron emission tomography. In order to improve the tracer properties, we modified the lipophilicity of [¹⁸F]FHBG by introduction of a methyl group into the N¹-position to get N¹-methyl-9-[(4-[18F]fluoro-3-hydroxymethylbutyl]guanine 3.

Materials and methods
The precursor 1 and the nonradioactive reference substance (MFHBG) were prepared by methylation of N¹-Methyl-N²-(p-anisyldiphenylmethyl)-9-[(4-(p-toluene-sulphonyloxy))-3-(p-anisyldiphenylmethoxy)-methyl-butyl]guanine with methyl iodide in the N¹ position. The precursor 1 reacts with a [¹⁸F]KF kryptofix complex to the intermediate 2 in a 65-75 % radiochemical yield. In a second step the protecting groups are removed from 2 by heating in methanolic HCl solution. The tracer is purified by RP HPLC separation. The identity of the tracer [¹⁸F]MFHBG with the nonradioactive reference compound was also proved by HPLC. The in vitro uptake of [¹⁸F]FHBG and [¹⁸F]MFHBG was evaluated into transduced and non-transduced human MC 38 cells.

Results
The labelling yield amounts to 70%, and the radiochemical purity of the tracer is greater than 98 %. Biodis-tribution data of [¹⁸F]MFHBG show a fast blood clearance without remarkable erythrocyte binding. An ini-tial high uptake of the tracer was found in the spleen, kindney and liver, which decreased rapidly in time. No metabolites were found. The rapid blood and organ clearance (faster than for [¹⁸F]FHBG) and the high in vivo stability of [¹⁸F]MFHBG are considered advantageous for in vivo monitoring of the gene transfer in tumours. The uptake of [¹⁸F]MFHBG is however lower than the uptake of [¹⁸F]FHBG in HSV1-tk trans-fected cell lines.

Conclusion
The novel tracer 9-[(4-[¹⁸F]fluoro-3-hydroxymethyl-butyl]-guanine ([¹⁸F]MFHBG) seems to be inferior to [¹⁸F]FHBG because of lower uptake in the target cells. It is assumed that the molecule position chosen for methylation is unsuitable for the anticipated improvement of the tracer.