64Cu-labeled somatostatin analogues conjugated with cross-bridged phosphonate-based chelators via strain-promoted click chemistry for PET imaging: in silico through in vivo studies

Abstract

Somatostatin receptor subtype 2 (sstr2) is a G-protein-coupled receptor (GPCR) that is overexpressed in neuroendocrine tumors. The homology model of sstr2 was built and was used to aid the design of new somatostatin analogues modified with phosphonate-containing cross-bridged chelators for evaluation of using them as PET imaging radiopharmaceuticals. The new generation chelators were conjugated to Tyr3-octreotate (Y3-TATE) through bioorthogonal, strain-promoted alkyne azide cycloaddition (SPAAC) to form CB-TE1A1P-DBCO-Y3-TATE (AP) and CB-TE1K1P-PEG4-DBCO-Y3-TATE (KP) in improved yields compared to standard direct conjugation methods of amide bond formation. Consistent with docking studies, the clicked bioconjugates showed high binding affinities to sstr2, with Kd values ranging from 0.6 to 2.3 nM. Selected isomers of the clicked products were used in biodistribution and PET/CT imaging. Introduction of the bulky dibenzocyclooctyne group in AP decreased clearance rates from circulation. However, the additional carboxylate group and PEG linker from the KP conjugate significantly improved labeling conditions and in vivo stability of the copper complex and ameliorated the slower pharmacokinetics of the clicked somatostatin analogues.

Figure 1

Structures of Tyr³-octreotate (Y3-TATE) analogues.

Figure 2

A: Overview of the docking mode of Y3-TATE in the binding pocket of sstr2. B: Molecular interactions between the Y3-TATE and specific amino acid residues of sstr2.

Figure 3

A: Overview of the docking mode of CB-TE1A1P–Y3-TATE (1A1P) in the binding pocket of sstr2. B: The detailed molecular interactions between 1A1P and specific amino acids of sstr2.

Figure 4

A: Overview of the docking mode of DBCO–CB-TE1A1P–N₃-Y3-TATE (AP) in the binding pocket of sstr2. B: The detailed molecular interaction between the AP and specific amino acids in sstr2.

Figure 5

A: Overview of the docking mode of CB-TE1K1P–PEG4–DBCO–Y3-TATE (KP) in the binding pocket of sstr2. B: The detailed molecular interactions between KP and specific amino acids in sstr2.

Figure 6

Biodistribution of ⁶⁴Cu-1A1P, ⁶⁴Cu-AP-1, and ⁶⁴Cu-KP-1 in sstr2-transfected HCT116 tumor-bearing female nu/nu mice at 1 and 24 h (A), and at 4 h with and without 20 μg of Y3-TATE co-injected as a blocking agent (B). N = 4 for each time point.

Figure 7

Tumor-to-organ ratios of ⁶⁴Cu-1A1P, ⁶⁴Cu-AP-1, and ⁶⁴Cu-KP-1 with sstr2-transfected HCT116 tumor-bearing female nu/nu mice. ***: significantly different (P < 0.001). **: significantly different (0.001 < P < 0.01). *: significantly different (0.01 < P < 0.05). ns: no significant difference (P > 0.05). n = 4 mice per time point.

Figure 8

Clearance profiles of ⁶⁴Cu-1A1P (green dots), ⁶⁴Cu-AP-1 (red dots), and ⁶⁴Cu-KP-1 (blue dots). ***: significantly different (P < 0.001). **: significantly different (0.001 < P < 0.01). *: significantly different (0.01 < P < 0.05). ns: no significant difference (P > 0.05).

Figure 9

Small animal PET/CT imaging of ⁶⁴Cu-AP-1 (SUV = 4.6 ± 0.8, n = 2), ⁶⁴Cu-KP-1 (SUV = 3.2 ± 0.2, n = 2), and ⁶⁴Cu-1A1P (SUV = 2.8 ± 0.4, n = 2) at 2 h p.i. The images of mice injected with the three tracers are scaled the same (from 0 to 10%ID/cc). Mice injected with unlabeled Y3-TATE to block specific uptake of the tracers had tumor SUVs for ⁶⁴Cu-AP-1, ⁶⁴Cu-KP-1, and ⁶⁴Cu-1A1P of 0.47 ± 0.18, 0.21 ± 0.06, and 0.19 ± 0.02, respectively (Figure S4, Supporting Information).