Abstract View

Objectives: Triggering receptor expressed on myeloid cells-1 (TREM1) is a membrane receptor selectively upregulated on pro-inflammatory myeloid cells during Toll-like receptor–mediated inflammation, where it serves as an effective amplifier of toxic innate immune responses. PET imaging offers significant potential for understanding whole-body innate immune responses, but current myeloid cell imaging agents are limited. The most widely used approach, translocator protein 18kDa (TSPO)-PET lacks cell specificity, as TSPO is expressed across many cell types (including endothelial cells and cancer cells in addition to myeloid cells) and cannot discriminate specific immune functions or cell phenotypes. Here, we report the radiosynthesis of [¹¹C]CN-VJDT—a TREM1 antagonist—as the first small molecule PET radiotracer for imaging TREM1 expression in inflammatory disease.

Methods: We employed Diels-Alder cycloaddition reaction to construct 5-bromo-VJDT precursor and 5-cyano-VJDT standard (Figure 1A). Radiosynthesis involved a) production and trapping of [¹¹C]HCN  (>500 mCi) and b) substitution of the bromo precursor with [¹¹C]CN group on VJDT via Pd(0)-mediated chemistry. Briefly, [¹¹C]CO₂ was produced via the ¹⁴N(p,α)¹¹C nuclear reaction using a GE PETtrace 18 cyclotron and transferred to a molecular sieve trap located in the [¹¹C]-chemistry Procab module. The trapped [¹¹C]CO₂ was first reduced to [¹¹C]CH₄ over a nickel catalyst at 400 °C and subsequently converted to [¹¹C]HCN over a platinum catalyst at 950 °C in the presence of NH₃gas. The resulting [¹¹C]HCN was bubbled into a reaction vessel maintained at −5 °C containing THF, kryptofix (K222), and 0.5M KOH using a modified module configuration optimized for efficient [¹¹C]HCN trapping. After addition of the precursor and palladium catalyst, the reaction mixture was heated at 100 °C for 4 min (Figure 1B). The reaction was then quenched with water, and the crude mixture was injected onto a semi-preparative HPLC system for purification to produce radiochemically pure [¹¹C]CN-VJDT.

Binding studies using human TREM1-expressing (hTREM1) U937 cells (mature) versus low hTREM1-expressing control cells, and HEK cells (devoid of TREM1) were used to assess the specificity of [¹¹C]CN-VJDT (Figure 1C). Dynamic PET/CT imaging was performed using healthy C57BL/6 mice to assess blood–brain barrier permeability, in vivo kinetics, and biodistribution (Figure 1D)

Results: Automated palladium(0)-mediated radiocyanation afforded [¹¹C]CN-VJDT with a radiochemical yield of 5.07 ± 2.83% (non-decay corrected), radiochemical purity >95% (n=5). Cell binding studies revealed 1.45-fold higher binding of [¹¹C]CN-VJDT to hTREM1-U937 cells (stimulated with Phorbol 12-myristate 13-acetate (PMA)) compared to parental U937 cells after 30 minutes of incubation (P <0.0001, n=3). A 78.57% reduced binding in hTREM1-negative HEK cells demonstrated high specificity of [¹¹C]CN-VJDT (P <0.0001, n= 3) (Figure 1C). Finally, dynamic PET/CT imaging of healthy mice demonstrated rapid entry of the radiotracer into the brain (n=2) within first few minutes followed by gradual washout by the end of the 60 minute scan (Figure 1D).

Conclusions: We have synthesized [¹¹C]CN-VJDT — the first small molecule radiotracer targeting TREM1 — with high chemical and radiochemical purity. Importantly, we demonstrated specific binding of [¹¹C]CN-VJDT to TREM1-expressing cells and favorable pharmacokinetics, including BBB penetrance, in healthy mice, warranting its further evaluation in preclinical CNS disease models of neuroinflammation.

References

Ajith, A.; Mamouni, K.; Horuzsko, D. D.; Musa, A.; Dzutsev, A. K.; Fang, J. R.; Chadli, A.; Zhu, X.; Lebedyeva, I.; Trinchieri, G.; Horuzsko, A. Targeting TREM1 Augments Antitumor T Cell Immunity by Inhibiting Myeloid-Derived Suppressor Cells and Restraining Anti-PD-1 Resistance. J. Clin. Invest. 2023, 133.

Labaree, D. C.; Ropchan, J. R.; Nabulsi, N.; Huang, Y. A Modification to Improve the Reliability of [11 C]CN- Production in the GE Radiochemistry System. J. Labelled Comp. Radiopharm. 2017, 60, 592–595.