Click Chemistry in the Design and Production of Hybrid Tracers

Abstract

Hybrid tracers containing both fluorescent and radioactive imaging labels have demonstrated clinical potential during sentinel lymph node procedures. To combine these two labels on a single targeting vector that allows tumor-targeted imaging, end-labeling strategies are often applied. For α_vβ₃-integrin-targeting hybrid tracers, providing an excellent model for evaluating tracer development strategies, end-labeling-based synthesis provides a rather cumbersome synthesis strategy. Hence, the aim of this study was to investigate the use of heterobifunctional cyanine dyes in a click-chemistry-based synthesis strategy for RGD-based hybrid tracers. The triazole-based hybrid tracers DTPA.DBCO.N ₃ (SO ₃ )-Cy5-c[RGDyK] and DTPA.BCN.N ₃ (SO ₃ )-Cy5-c[RGDyK] were obtained in fewer steps than DTPA-Lys(Cy5(SO ₃ )methyl)-Cys-c[RGDyK] and had partition coefficients of log P _(o/w) = -2.55 ± 0.10, -1.45 ± 0.03, and -2.67 ± 0.12, respectively. Both tracers were chemically stable, and the brightnesses of DTPA.DBCO.N ₃ (SO ₃ )-Cy5-c[RGDyK] and DTPA.BCN.N ₃ (SO ₃ )-Cy5-c[RGDyK] were, respectively, 23 × 10³ and 40 × 10³ M^-1 cm^-1; lower than that of the reference tracer DTPA-Lys(Cy5(SO ₃ )methyl)-Cys-c[RGDyK] (50 × 10³ M^-1 cm^-1). Assessment of serum protein binding revealed no statistically significant difference (44 ± 2 and 40 ± 2% bound for DTPA.DBCO.N ₃ (SO ₃ )-Cy5-c[RGDyK] and DTPA.BCN.N ₃ (SO ₃ )-Cy5-c[RGDyK], respectively; 36 ± 5% bound for DTPA-Lys(Cy5(SO ₃ )methyl)-Cys-c[RGDyK]; p > 0.05). DTPA.DBCO.N ₃ (SO ₃ )-Cy5-c[RGDyK] (K _D = 17.5 ± 6.0) had a statistically significantly higher affinity than the reference compound DTPA-Lys(Cy5(SO ₃ )methyl)-Cys-c[RGDyK] (K _D = 30.3 ± 5.7; p < 0.0001), but DTPA.BCN.N ₃ (SO ₃ )-Cy5-c[RGDyK] had a statistically significantly lower affinity (K _D = 76.5 ± 18.3 nM; p < 0.0001). Both [ ¹¹¹ In]DTPA.DBCO.N ₃ (SO ₃ )-Cy5-c[RGDyK] and [ ¹¹¹ In]DTPA.BCN.N ₃ (SO ₃ )-Cy5-c[RGDyK] enabled in vivo visualization of the 4T1 tumor via fluorescence and single-photon emission computed tomography (SPECT) imaging. Biodistribution data (% ID/g) revealed a significant increase in nonspecific uptake in the kidney, liver, and muscle for both [ ¹¹¹ In]DTPA.DBCO.N ₃ (SO ₃ )-Cy5-c[RGDyK] and [ ¹¹¹ In]DTPA.BCN.N ₃ (SO ₃ )-Cy5-c[RGDyK]. As a result of the higher background activity, the tumor-to-background ratio of the click-labeled RGD analogues was twofold lower compared to the end-labeled reference compound. The use of click chemistry labeling did not yield a pronounced negative effect on serum protein binding, in vitro stability, and receptor affinity; and tumors could still be visualized using SPECT and fluorescence imaging. However, quantitative in vivo biodistribution data suggest that the triazole and strained cyclooctyne moieties associated with this type of click chemistry negatively influence the pharmacokinetics of RGD peptides. Nevertheless, the design might still hold promise for other targets/targeting moieties.

Scheme 1. (A) Schematic Overview of the Original Multifunctional Single-Attachment-Point RGD-based Hybrid Tracer Design (End-Labeling; Left, with the Dye in Blue, Chelate in Red, Targeting Moiety in Green and Linker in White) and New Heterobifunctional RGD-based Hybrid Tracer Wherein the Dye Is Used as Linker (Right, with the Dye in Blue, DTPA in Red, and Targeting Moiety in Green). (B) Chemical Structures of the Reference Compound DTPA-Lys(Cy5(SO₃)methyl)-Cys-c[RGDyK] (End-Labeling) and (C) DTPA.DBCO.N₃(SO₃)-Cy5-c[RGDyK] and DTPA.BCN.N₃(SO₃)-Cy5-c[RGDyK] (Modular, Heterobifunctional Design Employing a Cy5 Dye as Spacer between Chelate and Targeting Moiety Using Click Chemistry)

Figure 1

Plasma protein interactions portraying (A) no significant difference (p > 0.05) in the plasma protein binding of the heterobifunctional tracers compared to the alternative branched design and high stability over a 24 h timespan for both heterobifunctional tracers using (B) absorbance and (C) fluorescence measurements.

Figure 2

In vitro evaluation of the hybrid tracers. Fluorescence confocal microscopy images of a mixed-cell culture of (A) α_Vβ₃-positive Geβ3 and α_Vβ₃-negative MDAMB231 X4 cells incubated with the reference tracer (i) DTPA-Lys(Cy5(SO₃)methyl)-Cys-c[RGDyK], (ii) DTPA.DBCO.N₃(SO₃)-Cy5-c[RGDyK], (iii) DTPA.BCN.N₃(SO₃)-Cy5-c[RGDyK]. (B) The same cell lines after blocking with c[RGDyK] prior to incubation with one of the hybrid tracers. For additional reference, cell nuclei were stained with Hoechst (in blue), and the intrinsic membranous GFP expression of the MDAMB231 X4 cells was used to discriminate between the two cell lines (bright green). (C) Quantified evaluation of the percentage of signal decrease between (A) and (B) (blocked; checkered) p < 0.0001, n ≥ 13. (D) Combined saturation curves of the affinity of DTPA-Lys(Cy5(SO₃)methyl)-Cys-c[RGDyK], DTPA.DBCO.N₃(SO₃)-Cy5-c[RGDyK], and DTPA.BCN.N₃(SO₃)-Cy5-c[RGDyK] (with the binding affinity (K_D) of the tracers in nanomolar), n = 3.

Figure 3

In vivo imaging and biodistribution. In vivo (A) SPECT images and (B) accompanying fluorescence images of 4T1-tumor-bearing mice acquired at 24 h after administration of 1 nmol of the reference tracer [¹¹¹In]DTPA-Lys(Cy5(SO₃)methyl)-Cys-c[RGDyK] (i), [¹¹¹In]DTPA.DBCO.N₃(SO₃)-Cy5-c[RGDyK] (ii), and [¹¹¹In]DTPA.BCN.N₃(SO₃)-Cy5-c[RGDyK] (iii). Tumor is encircled in red. (C) Quantified biodistribution (% ID/g) of [¹¹¹In]DTPA.DBCO.N₃(SO₃)-Cy5-c[RGDyK] (purple) and [¹¹¹In]DTPA.BCN.N₃(SO₃)-Cy5-c[RGDyK] (green; n = 6 per compound). For reference, biodistribution of [¹¹¹In]DTPA-Lys(Cy5(SO₃)methyl)-Cys-c[RGDyK] (black) was included as previously presented by Bunschoten et al. Significance between the hybrid triazole-containing RGD conjugates and the reference annotated as: ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05, and ns = p > 0.05.