PET radiotracer [¹⁸F]-P6 selectively targeting COX-1 as a novel biomarker in ovarian cancer: preliminary investigation

Abstract

Cyclooxygenase-1 (COX-1), but not COX-2, is expressed at high levels in the early stages of human epithelial ovarian cancer where it seems to play a key role in cancer onset and progression. As a consequence, COX-1 is an ideal biomarker for early ovarian cancer detection. A series of novel fluorinated COX-1-targeted imaging agents derived from P6 was developed by using a highly selective COX-1 inhibitor as a lead compound. Among these new compounds, designed by structural modification of P6, 3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole ([(18/19)F]-P6) is the most promising derivative [IC50 = 2.0 μM (purified oCOX-1) and 1.37 μM (hOVCAR-3 cell COX-1)]. Its tosylate precursor was also prepared and, a method for radio[(18)F]chemistry was developed and optimized. The radiochemistry was carried out using a carrier-free K(18)F/Kryptofix 2.2.2 complex, that afforded [(18)F]-P6 in good radiochemical yield (18%) and high purity (>95%). In vivo PET/CT imaging data showed that the radiotracer [(18)F]-P6 was selectively taken up by COX-1-expressing ovarian carcinoma (OVCAR 3) tumor xenografts as compared with the normal leg muscle. Our results suggest that [(18)F]-P6 might be an useful radiotracer in preclinical and clinical settings for in vivo PET-CT imaging of tissues that express elevated levels of COX-1.

Keywords: Biomarker; Cyclooxygenase (COX)-1; OVCAR-3 cell; Ovarian cancer; PET-CT imaging; [(18)F]-P6 radiotracer.

Fig. 1

[¹⁹F]-P6 IC₅₀ values determination: (a) inhibition of in vitro purified oCOX-1 and mCOX-2; (b) inhibition assay of OVCAR-3 intracellular hCOX-1 performed in the presence of 8 μM [1-¹⁴C]-AA and increasing amount of [¹⁹F]-P6.

Fig. 2

In vivo uptake in tumors. Female nude mice bearing OVCAR-3 xenografts were dosed with compound [¹⁸F]-P6 (7.4 MBq, intraperitoneal injection). Then, the animals were sacrificed by isoflurane overdose. The OVCAR-3 tumor and normal leg muscles were removed and weighed, and radioactivity associated with each tissue was counted in a well gamma counter. The plot shows the increased radiotracer (Bq/g) in COX-1-expressing OVCAR-3 tumors versus normal leg muscle (n = 3, p = 0.01) (*statistical significance).

Fig. 3

In vivo PET imaging of COX-1–expressing tumor by [¹⁸F]-P6. (a) Coronal view, (b) Sagittal view. Tumor-bearing female nude mice were dosed by i.p. injection with compound [¹⁸F]-P6 (100 μL, 7.4 MBq, intraperitoneal injection) under anesthesia. At 4 h post injection, the animals were imaged in the microPET/CT instrument (30-min acquisition).

Fig. 4

OVCAR-3 tumor and normal leg muscles were removed and amount of compound [¹⁹F]-P6 was determined by LC–MS. The plot shows the increased unlabeled compound [¹⁹F]-P6 in COX-1-expressing OVCAR-3 tumors versus normal leg muscle (n = 4, p = 0.01) (*statistical significance).

Fig. 5

HPLC chromatogram of [¹⁸F]-P6 compared with cold standard [¹⁹F]-P6 (R_f = 12.8 min). The HPLC analysis was performed using Macherey–Nagel Nucleosil 100-7 C-18 VarioPrep Column 250 × 16 mm, mobile phase EtOH/Water = 60:40 and flow rate = 6 mL/min.

Scheme 1

Synthesis of 3-(5-chlorofuran-2-yl)-5-(fluoromethyl)-4-phenylisoxazole, [¹⁹F]-P6. NBS = N-bromosuccinimide; AIBN = 2,2′-Azobis(2-methylpropionitrile); TBAF = Tetrabutylammonium fluoride; Ts₂O = p-Toluenesulfonic anhydride; Et₃N = Triethylamine.

Scheme 2

Radiosynthesis of [¹⁸F]-P6. DMSO = dimethylsulfoxide.