Synthesis and Biological Evaluation of a Radiolabeled PET (Positron Emission Tomography) Probe for Visualization of In Vivo α-Fucosidase Expression

Abstract

The acidic hydrolase α-fucosidase (AF) is a biomarker for maladies such as cancer and inflammation. The most advanced probes for α-fucosidase are unfortunately constrained to ex vivo or in vitro applications. The in vivo detection and quantification of AF using positron emission tomography would allow for better discovery and diagnosis of disease as well as provide better understanding of disease progression. We synthesized, characterized, and evaluated a radiolabeled small molecule inhibitor of AF based on a known molecule. The radiosynthesis involved the ¹¹C methylation of a phenoxide, which was generated in situ by ultrasonification of the precursor with sodium hydride. The tracer was produced with a decay corrected yield of 41.7 ± 16.5% and had a molar activity of 65.4 ± 30.3 GBq/μmol. The tracer was shown to be stable in mouse serum at 60 min. To test the new tracer, HCT116 colorectal carcinoma cells were engineered to overexpress human AF. In vitro evaluation revealed 3.5-fold higher uptake in HCT116AF cells compared to HCT116 controls (26.4 ± 7.8 vs. 7.5 ± 1.0 kBq/10⁶ cells). Static PET scans 50 min post injection revealed 2.5-fold higher tracer uptake in the HCT116AF tumors (3.0 ± 0.8%ID/cc (n = 6)) compared with the controls (1.2 ± 0.8 (n = 5)). Dynamic scans showed higher uptake in the HCT116AF tumors at all time-points (n = 2). Ex vivo analysis of the tumors, utilizing fluorescent DDK2 antibodies, confirmed the expression of human AF in the HCT116AF xenografts. We have developed a novel PET tracer to image AF in vivo and will now apply this to relevant disease models.

Keywords: 11C; PET tracer; cancer; inflammation; α-fucosidase.

Figure 1

(A) Structures and K_i values of known inhibitors of α-fucosidase [8,9,10]; (B) Overview of the synthetic route to core pyrrolidine structure from D-ribose [10]; (C) Synthetic route toward precursor 10 and subsequent radiosynthesis of [¹¹C]3 via the phenoxide 9 and radiolabeled intermediate [¹¹C]11. Reagents and conditions: (iii) Boc₂O, Et₃N, DCM, RT. (iv) NaH, CH₃CN, ultrasonification, 5 min, RT. (v) [¹¹C]CH₃I, CH₃CN, 110 °C, 5 min. (vi) 6M HCl, 140 °C, 5 min.

Figure 2

Hydrolysis of acetonide, confirmation of radiochemical identity and serum stability of [¹¹C]3: (A) Synthetic scheme depicting rapid hydrolysis of acetonide-protecting group. Reagents and conditions: (i) 6M HCl, 140 °C, 5 min; (B) LCMS data depicting successful cleavage of acetonide-protecting group. The mass peak of the model compound 12, which is observed at 9.5 min, disappears after heating in 6 M HCl. A new mass peak, representing the more polar deprotected product 13 is observed at 5.3 min, indicating successful removal of acetonide protection [10]; (C) Retention time of tracer (radio-trace) at 8.4 min correlates to non-radioactive standard (UV 254 nm) at 8.5 min; (D) HPLC analysis * of tracer incubated in serum at 37 °C for 1 h shows no apparent metabolites. (* Data subjected to median filter).

Figure 3

Validation of in vitro tracer uptake and expression of AF in HCT116AF and xenograft tissue: (A) Western blot showing expression of AF in transfected HCT116 cells. Protein bands were stained with primary anti-DDK2 and Alexa Fluor 555 labeled fluorescent secondary antibodies; (B) In vitro evaluation of tracer uptake revealed 3.5-fold higher tracer accumulation in HCT116AF vs. HCT116 cells, with respective uptake values of 26.4 ± 7.8 and 7.5 ± 1.0 kBq/10⁶ cells (p < 0.05); (C) Ex vivo analysis of HCT116AF tumors revealed positive staining for DDK2, indicating that the transfected cells express AF in vivo (* p < 0.5).

Figure 4

In vivo static and dynamic PET imaging of AF expression: (A) PET images of mouse with HCT116 (left) and HCT116AF (right) tumors show higher tracer accumulation in the AF overexpressing tumor compared to the HCT116 controls; (B) Quantification of tracer uptake in tumor 50 min post injection revealed values of 3.0 ± 0.77 vs. 1.2 ± 0.77% ID/cc in the HCT116AF and HCT116, respectively (p < 0.01); (C) Dynamic uptake on tracer in HCT116 and HCT116AF tumors shows higher tracer uptake in AF over-expressing tumors at all time-points; (D) Treatment of mice (n = 2) with the non-radioactive reference compound appears to have a strong blocking effect, offering evidence that the tracer is specific to AF (** p < 0.01).