Synthesis and preclinical evaluation of a novel fluorine-18 labeled small-molecule PET radiotracer for imaging of CXCR3 receptor in mouse models of atherosclerosis

Abstract

Background: CXCR3 is a chemokine receptor and is expressed in innate and adaptive immune cells. It promotes the recruitment of T-lymphocytes and other immune cells to the inflammatory site in response to the binding of cognate chemokines. Upregulation of CXCR3 and its chemokines has been found during atherosclerotic lesion formation. Therefore, detection of CXCR3 by positron emission tomography (PET) radiotracer can be a useful tool for detecting the development of atherosclerosis in a noninvasive manner. Herein, we report the synthesis, radiosynthesis, and characterization of a novel fluorine-18 (F-18, ¹⁸F) labeled small-molecule radiotracer for the imaging of the CXCR3 receptor in mouse models of atherosclerosis.

Results: The reference standard 1 and its precursor 9 were synthesized over 5 steps from starting materials in good to moderate yields. The measured K_i values of CXCR3A and CXCR3B were 0.81 ± 0.02 nM and 0.31 ± 0.02 nM, respectively. [¹⁸F]1 was prepared by a two-step radiosynthesis with a decay-corrected radiochemical yield of 13 ± 2%, radiochemical purity > 99%, and specific activity of 44.4 ± 3.7 GBq/µmol at the end of synthesis (n = 6). The baseline studies showed that [¹⁸F]1 displayed high uptake in the atherosclerotic aorta and brown adipose tissue in Apolipoprotein E (ApoE) knockout (KO) mice fed with a high-fat diet over 12 weeks. The uptake of [¹⁸F]1 in these regions was reduced significantly in self-blocking studies, demonstrating CXCR3 binding specificity. Contrary to this, no significant differences in uptake of [¹⁸F]1 in the abdominal aorta of C57BL/6 control mice fed with a normal diet were observed in both baseline and blocking studies, indicating increased CXCR3 expression in atherosclerotic lesions. Immunohistochemistry studies demonstrated that [¹⁸F]1-positive regions were correlated with CXCR3 expression, but some atherosclerotic plaques with significant size were not detected by [¹⁸F]1, and their CXCR3 expressions were minimal.

Conclusion: [¹⁸F]1 was synthesized with good radiochemical yield and high radiochemical purity. In PET imaging studies, [¹⁸F]1 displayed CXCR3-specific uptake in the atherosclerotic aorta in ApoE KO mice. [¹⁸F]1 visualized CXCR3 expression in different regions in mice aligned with the tissue histology studies. Taken together, [¹⁸F]1 is a potential PET radiotracer for imaging CXCR3 in atherosclerosis.

Keywords: Atherosclerosis; CXCR3 chemokine receptor; Inflammation; PET imaging; Small-molecule radiotracer.

Scheme 1

Schematic example of the current CXCR3 antagonists [–13]

Fig. 1

The cartoon of the docking model for compound 1 that has 14 interactions with the amino acids of CXCR3

Scheme 2

Synthetic scheme for nonradioactive standard 1 and its precursor 9

Scheme 3

(A) Attempted four-step synthesis and (B) One-pot two-step radiosynthesis of [¹⁸F]1

Fig. 2

Representative CXCR3 PET images of [¹⁸F]1 in two ApoE KO mice and one C57BL/6 control mouse. The specific uptake of [¹⁸F]1 was observed in the atherosclerotic aorta (red arrow) and BAT (blue arrow) of ApoE KO mice and the BAT of C57BL/6 control mice

Fig. 3

The TACs of ApoE KO mice (N = 3) and C57BL/6 control mice (N = 5). The TACs indicate the blocking effects of [¹⁸F]1, demonstrating the specificity of [¹⁸F]1

Fig. 4

The AUCs of the BAT and atherosclerotic aorta of ApoE KO mice and the BAT and corresponding aorta of C57BL/6 mice

Fig. 5

The results of biodistribution using male and female control C57BL/6 mice. The uptake of [¹⁸F]1 in each organ was measured at 10, 30, and 60 min after its intravenous injection

Fig. 6

CXCR3 PET imaging using [¹⁸F]1 and their corresponding cardiovascular tissues obtained from the ApoE KO mice. A Region I (red arrows) is a [¹⁸F]1-positive region, and the corresponding aorta tissue was tested for expression levels of CXCR3 protein, co-stained with cell-type markers. Adjacent cross sections of the region I (I-a; hematoxylin and eosin (H&E) stain) were subjected to immunofluorescent staining identifying CXCR3 (I-b–I-e; green), CD3 (I-c, red; T cell marker), Mac3 (I-d, red; macrophage marker), and αSMA (I-e, red; SMC marker), counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue; nuclei). Panels I-c–I-e are magnified views of atherosclerotic plaque indicated on I-a by a rectangle. Region II (blue arrows) has a significant atherosclerotic plaque. Nevertheless, it is a [¹⁸F]1-negative region of PET imaging. Adjacent cross sections of the region II (II-a; H&E stain) were also assessed for expression levels of CXCR3 (II-b–II-e; green), co-stained with CD3 (II-c, red), Mac3 (II-d, red), and αSMA (II-e, red) and counterstained with DAPI (blue). Panels II-c–II-e are magnified views of atherosclerotic plaque indicated on II-a by a rectangle. Scale bar = 200 µm. B Region III (red arrows) is a [¹⁸F]1-positive region, and the corresponding aorta tissue was tested for expression levels of CXCR3 protein, co-stained with cell-type markers. Region III also indicated [¹⁸F]1-positivity, and subsequent histologic analysis revealed a significant atherosclerotic plaque with high CXCR3 expression levels (III-b–III-e; green detection in the fluorescence images), consistent with CXCR3 expression-dependent detection by the radiotracer. Adjacent cross sections of the region III (III-a; H&E stain) were CD3 (III-c, red), Mac3 (III-d, red), and αSMA (III-e, red), counterstained with DAPI (blue). Panels III-c–III-e were magnified views of atherosclerotic plaque indicated on III-a by a rectangle. Scale bar = 200 µm