Abstract
241785
Introduction: Dihydroorotate dehydrogenase (DHODH), an enzyme that plays a critical role in the de-novo pyrimidine biosynthesis, has been recognized as a promising target for treating diseases involving cellular proliferation, such as autoimmune diseases and cancers. Pharmacological inhibition of human DHODH (hDHODH) that offers a potential therapeutic strategy for the treatment in adult subjects with acute myeloid leukemia (AML) has recently been supported by Phase I/II clinical trials. Brequinar is one of the most potent hDHODH inhibitors so far discovered. Our study aims to develop and evaluate, for the first time, promising radiotracers for PET imaging of hDHODH in vivo. We report here the design, synthesis, radiolabeling and characterization of [18F]brequinar, and its preliminary PET imaging studies.
Methods: In this work, we compared the chemical design and radiosynthesis starting from, either pinacole boronate p-nitrobenzyl ester or tributylstannate (tin) p-nitrobenzyl ester, for their suitability as a precursor to produce the [18F]brequinar target molecule using an automated FX2N synthesizer (GE Tracerlab) via two-step, two-pot radiosythesis procedure (Figure 1), followed by intermediate silica SPE cartridge and final compound HPLC purification (Rt 9.5-10.0 min). We carried out pilot in vivo PET imaging study in wild-type (WT) mice after injection of [18F]brequinar (<0.3 mCi in 100-200 mL saline) via tail vein and scanned for 30 min using a high-resolution microPET/CT imaging (MOLECUBES β-CUBE, Belgium) to establish baseline levels of DHODH in control animals.
Results: We have successfully synthesized both precursors with high purity (>95%) and those were characterized by 1H- and 13C -NMR 600 MHz and mass spectroscopy ESI+ MS. [18F]Brequinar was obtained consistently with a RCY of 11.6 ± 2.0% (decay corrected, n = 5) in an overall radiosynthesis time of 52 min . Radiochemical purity of the formulated product [18F]brequinar was determined on analytical HPLC to be > 99% with chemical purity of > 98%, and specific activity of 3.2 ± 1.8 Ci/μmol (118.4 GBq/μmol) at the end of radiosynthesis. The kinetics (time-activity curves) and PET/CT images shown the heart, liver and kidneys are highly visible after injection of [18F]brequinar, which is consistent with the notion that DHODH, an iron containing flavin-dependent enzyme, is in the inner membrane of mitochondria; such enzymes are important in high-energy demanding organs such as liver and kidneys (Figure 2).
Conclusions: The expression level of DHODH has been reported to be elevated in patients with relapsed/refractory AML and various types of malignant tumors. This study provides the strategies to create [18F]brequinar, the first hDHODH inhibitor PET radiotracer, which will facilitate its use as a tool (theranostics) for hDHODH drug development and for diagnosis and monitoring therapeutic efficacy in AML and cancers.
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