Radiosynthesis of 3′-deoxy-3′-[¹⁸F]fluorothymidine: [¹⁸F]FLT for imaging of cellular proliferation in vivo

Abstract

A reliable radiosynthesis of 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) has been developed based on [¹⁸F]fluoride displacement of a protected nosylate precursor. A simple three-step synthesis is described that is useful for preparing >10 mCi (370 MBq) of radiochemically pure [¹⁸F]FLT, with a specific activity >1 Ci/μmol (37 GBq/μmol) at EOS within 100 min and in 13% radiochemical yield (end of bombardment (EOB); 7% end of synthesis (EOS)). [¹⁸F]FLT has been designed as a new positron emission tomography imaging agent for visualizing cellular proliferation in vivo based on the metabolism of thymidine.

Introduction

Labeled thymidine (TdR) is the preferred radiopharmaceutical for indicating cellular proliferation because its specific incorporation into DNA is linked to the S phase of the cell cycle (18). The rapid labeling rate, along with the defined and durable nature of the intracellular label, had spurred the proposal that [¹¹C]thymidine would be useful for imaging proliferation in vivo with positron emission tomography (PET) (5). Indeed, [¹¹C]thymidine has worked well for PET imaging 7, 21, 22, 23, 35, 39, 42, despite the fact that only a portion of the injected dose gets incorporated into DNA. When thymidine’s systemic degradation (catabolism) is accounted for, retention of [¹¹C]thymidine in normal proliferating tissues and most tumors can readily be imaged and quantitatively related to DNA synthesis 7, 21, 22, 34.

Although [¹¹C]TdR has proven useful as an imaging agent, construction of its input function from metabolite data is demanding and is an obstacle to wide acceptance of thymidine as a PET radiopharmaceutical. The difficult radiosynthesis of [¹¹C]TdR is also a factor. As a practical alternative to [¹¹C]thymidine, the analog 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT; Fig. 1 ) is being developed 11, 12, 13, 14, 15, 16, 26, 30, 31, 32, 33. FLT is captured by proliferating cells, is stable to catabolism in vivo, and has a simpler radiosynthesis with a longer lived label 13, 19, 27, 33.

FLT and thymidine undergo the same initial metabolism and are monophosphorylated by thymidine kinase-1, an enzyme that is expressed during the DNA synthesis phase (S phase) of the cell cycle 1, 19, 24, 25, 29. Whereas thymidine monophosphate is quickly fixed in cells by DNA synthesis, FLT monophosphate accumulates as a membrane impermeable metabolite and, from a PET imaging viewpoint, behaves as if it were in DNA 19, 24, 31, 37. For example, both [¹⁸F]FLT and 2-[¹¹C]thymidine qualitatively imaged the same regions in a dog given both agents (31); in particular, uptake was seen in bone marrow, where there is a high rate of cellular proliferation. The fact that FLT is inert to catabolism plays a significant role in prolonging the input of the tracer and minimizing background activity. Indeed, a progressive increase in the standard uptake value (SUV) for [¹⁸F]FLT in dog marrow was still seen at late imaging times (20–60 min). This was not the case for 2-[¹¹C]TdR, which reached an early plateau phase, and this difference accounts for the twofold higher SUV for [¹⁸F]FLT in marrow at 60 min (31).

Although imaging with [¹⁸F]FLT in animals and humans has been successful (31), the yields of this promising tracer were too low for routine use 12, 13, 14. Here, we present a higher yielding and more reliable radiosynthesis of [¹⁸F]FLT that makes minimal use of specialized apparatus and materials. The improvements result from using a nosylate (4-nitrobenzenesulfonate) ester for nucleophilic displacement with [¹⁸F]fluoride. Within 2 h, the two-step process produces 10–20 mCi of FLT (13% end of bombardment (EOB); 7% end of synthesis (EOS)) suitable for injection.