[nat/44Sc(pypa)]-: Thermodynamic Stability, Radiolabeling, and Biodistribution of a Prostate-Specific-Membrane-Antigen-Targeting Conjugate

Abstract

⁴⁴Sc is an attractive positron-emitting radionuclide for PET imaging; herein, a new complex of the Sc³⁺ ion with nonmacrocyclic chelator H₄pypa was synthesized and characterized with high-resolution electrospray-ionization mass spectrometry (HR-ESI-MS), as well as different nuclear magnetic resonance (NMR) spectroscopic techniques (¹H, ¹³C, ¹H-¹³C HSQC, ¹H-¹³C HMBC, COSY, and NOESY). In aqueous solution (pH = 7), [Sc(pypa)]^- presented two isomeric forms, the structures of which were predicted using density functional theory (DFT) calculation with a small energy difference of 22.4 kJ/mol, explaining their coexistence. [Sc(pypa)]^- was found to have superior thermodynamic stability (pM = 27.1) compared to [Sc(AAZTA)]^- (24.7) and [Sc(DOTA)]^- (23.9). In radiolabeling, [⁴⁴Sc][Sc(pypa)]^- formed efficiently at RT in 15 min over a range of pH (2-5.5), resulting in a complex that is highly stable (>99%) in mouse serum over at least six half-lives of scandium-44. Similar labeling efficiency was observed with the PSMA (prostate-specific membrane antigen)-targeting H₄pypa-C7-PSMA617 at pH = 5.5 (RT, 15 min), confirming negligible disturbance from the bifunctionalization on scandium-44 scavenging. Moreover, the kinetic inertness of the radiocomplex was proved in vivo. Surprisingly, the molar activity was found to have profound influence on the pharmacokinetics of the radiotracers where lower molar activity drastically reduced the background accumulations, particularly, kidney, and thus, yielded a much higher tumor-to-background contrast.

Figure 1.

[Sc(pypa)]⁻¹H and ¹³C NMR assignments (see Figures 2 and S2A–C for labels).

Figure 2.

Sc-pypa variable temperature ¹H NMR downfield (left) and upfield (right) spectra (400 MHz, D₂O, pH 7; 25, 35, 45, 65, 85 °C from bottom to top). Please refer to Figure S1A for the full ¹H NMR spectrum.

Figure 3.

Two isomeric species of the [Sc(pypa)]⁻ anion simulated using DFT calculations.

Figure 4.

(A) Representative spectra of the in-batch UV-titration of the Sc³⁺-H₄pypa system as the pH is raised. [L] = [Sc³⁺] = 1.33 × 10⁻⁴ M at 25 °C, l = 1 cm. The ionic strength was maintained constant (I = 0.16 M) when possible by the addition of different amounts of NaCl. (B) pM vs ionic radius for M³⁺ ions of interest and ligands discussed.

Figure 5.

(A) Concentration-dependent radiolabeling studies of H₄pypa and H₄pypa-C7-PSMA617 with ⁴⁴Sc at room temperature in NH₄OAc buffer (0.5 M, pH = 5.5) over 5 min. (B) Concentration-dependent radiolabeling studies of H₄pypa with ⁴⁴Sc at room temperature in NH₄OAc solution (0.5 M, pH = 2, 4, 5.5, 7) over 15 min. (C) Mouse serum stability of the corresponding complexes over 24 h at 37 °C.

Figure 6.

Representative PET/CT images (MIP, coronal) of [⁴⁴Sc][Sc(pypa-C7-PSMA617)] (7.4 (top) and 74 (bottom) GBq/μmol)] in PC3-PIP (left shoulder) and PC3-Flu (right shoulder) tumor-bearing mice at different p.i. time points.

Figure 7.

Biodistribution data of [⁴⁴Sc][Sc(pypa-C7-PSMA617)] (7.4 (left) and 74 (right) GBq/μmol)] in PC3-PIP-tumor-bearing mice at selected p.i. time points (n = 3 per time point).

Figure 8.

Ex vivo biodistribution data of [⁴⁴Sc][Sc(pypa-C7-PSMA617)] (7.4 and 74 GBq/μmol)] in PC3-PIP-tumor-bearing mice at selected p.i. time points (n = 3 per time point).

Chart 1.

Chemical Structures of the Discussed Chelators