Semiautomated radiosynthesis and biological evaluation of [18F]FEAU: a novel PET imaging agent for HSV1-tk/sr39tk reporter gene expression

Abstract

2'-deoxy-2'-[(18)F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil ([(18)F]FEAU) is a promising radiolabeled nucleoside designed to monitor Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene expression with positron emission tomography (PET). However, the challenging radiosynthesis creates problems for being able to provide [(18)F]FEAU routinely. We have developed a routine method using a commercial GE TRACERlab FX-FN radiosynthesis module with customized equipment to provide [(18)F]FEAU. All radiochemical yields are decay corrected to end-of-bombardment and reported as means +/- SD. Radiofluorination (33 +/- 8%; n = 4), bromination (85 +/- 8%; n = 4), coupling reaction (83 +/- 6%; n = 4), base hydrolysis steps, and subsequent high-performance liquid chromatography purification afforded purified [(18)F]FEAU beta-anomer in 5 +/- 1% overall yield (n = 3 runs) after approximately 5.5 h and a beta/alpha-anomer ratio of 7.4. Radiochemical/chemical purities and specific activity exceeded 99% and 1.3 Ci/micromol (48 GBq/micromol), respectively. In cell culture, [(18)F]FEAU showed significantly (P < 0.05) higher accumulation in C6 cells expressing HSV1-tk/sr39tk as compared to wild-type C6 cells. Furthermore, [(18)F]FEAU showed slightly higher accumulation than 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butylguanine ([(18)F]FHBG) in cells expressing HSV1-tk (P < 0.05), whereas [(18)F]FHBG showed significantly higher (P < 0.05) accumulation than [(18)F]FEAU in HSV1-sr39tk-expressing cells. micro-PET imaging of mice carrying tumor xenografts of C6 cells stably expressing HSV1-tk or HSV1-sr39tk are consistent with the cell uptake results. The [(18)F]FEAU mouse images also showed very low gastrointestinal signal with predominant renal clearance as compared to [(18)F]FHBG. The routine radiosynthesis of [(18)F]FEAU was successfully semiautomated using a commercial module along with customized equipment to provide the beta-anomer in modest yields. Although further studies are needed, early results also suggest [(18)F]FEAU is a promising PET radiotracer for monitoring HSV1-tk reporter gene expression.

Fig. 1

Chemical structures of 2′-deoxy-2′-[¹⁸F]fluoro-5-iodo-1-β-d-arabinofuranosyluracil ([¹⁸F]FIAU), 2′-deoxy-2′-[¹⁸F]fluoro-5-methyl-1-β-d-arabinofuranosyluracil ([¹⁸F] FMAU), 2′-deoxy-2′-[¹⁸F]fluoro-5-ethyl-1-β-d-arabinofurano-syluracil ([¹⁸F]FEAU; 8), and 9-[4-[¹⁸F]fluoro-3-(hydroxy-methyl)butylguanine ([¹⁸F]FHBG).

Fig. 2

UV absorbance (λ=254 nm) and radioactivity HPLC chromatograms for [¹⁸F]FEAU and carrier: a preparative purification and b QC analysis.

Fig. 3

Accumulation of [¹⁸F]FEAU and [¹⁸F]FHBG in control cells and cells stably transfected with HSV1-tk or HSV1-sr39tk. C6 rat glioma cells (C6, C6-stb-tk+, and C6-stb-sr39tk+). Values are accumulation of each tracer (dpm cells/dpm medium), normalized by μg of total protein. Y-axis is log scale (asterisk denotes P<0.05).

Fig. 4

Selectivity index (average ratio of tracer accumulation between C6-sr39tk+ and C6-tk+ cells) of [¹⁸F]FHBG and [¹⁸F]FEAU (asterisk denotes P<0.05).

Fig. 5

Micro-PET images of [¹⁸F]FEAU and [¹⁸F]FHBG uptake in a mouse xenograft model. One million of C6-sr39tk+ (A) and C6-tk+ (B) cells were implanted on the left and right shoulder of a nude mouse, and tumors were allowed to grow till they reach 3–5 mm of diameter. The mouse was first injected with ∼200 μCi of [¹⁸F]FEAU via tail vein and scanned in a micro-PET for 10 min after 1 h. Specific uptake of [¹⁸F] FEAU is seen in both tumors (A and B). After 24 h, the same mouse was injected with ∼200 μCi of [¹⁸F]FHBG via tail vein, and specific accumulation is again observed in both the tumors. Both transverse and coronal images of [¹⁸F]FHBG and [¹⁸F]FEAU scans are shown. [¹⁸F]FHBG showed relatively high signal in the gastro-intestinal tract (GI) because of clearance of the probe whereas [¹⁸F]FEAU exhibited very low signal in the gastro intestinal tract due to predominantly renal clearance. The color scale is in %ID/g tissue.

Fig. 6

Comparison of [¹⁸F]FEAU and [¹⁸F]FHBG accumulation in five nude mice bearing both C6-sr39tk+ and C6-tk+ tumors. Percent injected dose per gram (%ID/g) of [¹⁸F] FHBG and [¹⁸F]FEAU uptakes were calculated for the respective ROIs drawn over the tumors of the experiment mentioned in Fig. 5 (A and B). C6-sr39tk+ tumors showed significantly higher uptake of [¹⁸F]FHBG than [¹⁸F]FEAU, while C6-tk+ tumors showed higher uptake of [¹⁸F]FEAU than [¹⁸F]FHBG (asterisk denotes P<0.05).

Scheme 1

Diagram shows connection between the cyclotron, primary and secondary synthesis modules. a 5-mL collection vial receives [¹⁸F]-3b after solid phase extraction with Si-SP. b The vial with [¹⁸F]-3b is placed into aluminum heating block for the subsequent bromination and alkylation reactions. c [¹⁸F]-7a and [¹⁸F]-7b are eluted through a Si-SP to a second 5-mL vial for the remaining deprotection step in another heating block. d The vial containing the crude deprotected product is reattached to the 1° module for HPLC purification and product formulation. e Formulated [¹⁸F]-8b is sterile filtered into a sterile multidose vial.

Scheme 2

The radiosynthetic pathway for 2′-deoxy-2′-fluoro-5-ethyl-1-β-d-arabinofuranosyluracil 8a and 2′-deoxy-2′-[¹⁸F] fluoro-5-ethyl-1-β-d-arabinofuranosyluracil 8b.