Rapid and reproducible radiosynthesis of [¹⁸F] FHBG

Abstract

9-(4-[¹⁸F] Fluoro-3-hydroxymethylbutyl) guanine ([¹⁸F] FHBG), an imaging agent for gene therapy using PET, was prepared in a one-pot, two-step synthesis. Microwave (MW) mediated nucleophilic fluorination of N², monomethoxytrityl-9-[4-(tosyl)-3-monomethoxytrityl-methylbutyl] guanine using no-carrier-added [¹⁸F] fluoride, followed by deprotection with hydrochloric acid and HPLC purification, gave [¹⁸F] FHBG. The radiochemical yield (decay corrected) was 12±5% (n = 35), the synthesis time was 55-60 min, and the radiochemical purity was >99%. The compound was used for lung imaging and was injected into Sprague-Dawley rats previously infected with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene. MicroPET imaging showed accumulation confined to the lungs.

Introduction

9-(4-[¹⁸F] Fluoro-3-hydroxymethylbutyl) guanine ([¹⁸F] FHBG) [(3) Scheme 1] has been used as a reporter probe to image the expression of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene in living organisms [1], [2]. HSV1-tk phosphorylates [¹⁸F] FHBG to its monophosphate form, leading to intracellular accumulation [3]. Cellular retention of radioactivity is, therefore, an indicator of HSV1-tk gene expression. When HSV1-tk is linked to another (e.g. therapeutic) transgene, the accumulation of [¹⁸F] FHBG in tissues can be used to measure the magnitude, location and timing of expression of the target transgene. Positron emission tomography (PET) has been used successfully in conjunction with [¹⁸F] FHBG to assess gene expression in vivo. Eventually, use of these methods may be useful in the planning of gene therapy in humans.

Various research groups have presented methods for the radiosynthesis of [¹⁸F] FHBG [4], [5], [6]. As the use of [¹⁸F] FHBG increases, there is a need to develop more efficient approaches for its radiosynthesis. In order to shorten the synthesis time and thereby increase the yield, we have developed a method for the radiosynthesis of [¹⁸F] FHBG using a custom-designed microwave cavity (MWC) [7]. There are advantages of microwave heating other than time gain, such as a cleaner reaction mixture due to decreased sample decomposition and improved chemical flexibility due to the ability of microwaves to accelerate typically sluggish reactions of less activated substrates. Since our group first reported the use of a commercial microwave to shorten reaction times for short-lived radiolabeled pharmaceuticals [8], the application of microwave heating has grown in popularity [9], [10], [11]. In the current study, we have applied heating with a microwave cavity [7] to shorten the synthesis time and improve the radiochemical yield.

At our institution, demand for [¹⁸F] FHBG (2-4 times per week) is currently focused on studies of pulmonary gene expression in rodents using an enhanced mutant of HSV1-tk as the PET reporter gene. Typically, these studies show a low level of radiotracer uptake by the lungs, and thus require a radioactive tracer with very high radiochemical purity and with very low levels of free fluoride. We report here the details of the radiosynthesis of [¹⁸F] FHBG based on this MWC method and our improved solid-phase purification (SP) approach for the final product, by exchanging the commonly used C18 Sep-Pak for the more efficient Oasis® HLB (Hydrophilic-Lipophilic Balance) media.