Preclinical validations of [18F]FPyPEGCBT- c(RGDfK): a 18F-labelled RGD peptide prepared by ligation of 2-cyanobenzothiazole and 1,2-aminothiol to image angiogenesis

Abstract

Background: α_Vβ₃, α_Vβ₅ and α₅β₁ integrins are known to be involved in carcinogenesis and are overexpressed in many types of tumours compared to healthy tissues; thereby they have been selected as promising therapeutic targets. Positron emission tomography (PET) is providing a unique non-invasive screening assay to discriminate which patient is more prone to benefit from antiangiogenic therapies, and extensive research has been carried out to develop a clinical radiopharmaceutical that binds specifically to integrin receptors. We recently reported the synthesis of a new ¹⁸F-labelled RGD peptide prepared by 2-cyanobenzothiazole (CBT)/1,2-aminothiol conjugation. This study aims at characterising the preclinical biologic properties of this new tumour-targeting ligand, named [¹⁸F]FPyPEGCBT-c(RGDfK).The in vitro binding properties of [¹⁸F]FPyPEGCBT-c(RGDfK) were analysed by standard binding assay in U-87 MG and SKOV-3 cancer models and its selectivity towards integrins by siRNA depletions. Its preclinical potential was studied in mice bearing subcutaneous tumours by ex vivo biodistribution studies and in vivo microPET/CT imaging.

Results: In vitro, FPyPEGCBT-c(RGDfK) efficiently bound RGD-recognising integrins as compared to a control c(RGDfV) peptide (IC₅₀ = 30.8 × 10^-7 M vs. 6.0 × 10^-7 M). [¹⁸F]FPyPEGCBT-c(RGDfK) cell uptake was mediated by an active transport through binding to α_V, β₃ and β₅ but not to β₁ subunits. In vivo, this new tracer demonstrated specific tumour uptake with %ID/g of 2.9 and 2.4 in U-87 MG and SKOV-3 tumours 1 h post injection. Tumour-to-muscle ratios of 4 were obtained 1 h after intravenous administration of the tracer allowing good visualisation of the tumours. However, unfavourable background accumulation and high hepatobiliary excretion were observed.

Conclusion: [¹⁸F]FPyPEGCBT-c(RGDfK) specifically detects tumours expressing RGD-recognising integrin receptors in preclinical studies. Further optimisation of this radioligand may yield candidates with improved imaging properties and would warrant the further use of this efficient labelling technique for alternative targeting vectors.

Keywords: Angiogenesis; Fluorine-18; Integrins; MicroPET imaging; RGD peptide.

Fig. 1

Synthesis of [^18,19F]FPyPEGCBT-c(RGDfK)

Fig. 2

Displacement of [¹²⁵I]echistatin by FPyPEGCBT-c(RGDfK). U-87 MG and SKOV-3 cancer cells were co-incubated for 1 h in 96-well plates with 370 Bq/well [¹²⁵I]echistatin and a concentration range (10⁻⁹ to 10⁻⁴ M) of FPyPEGCBT-c(RGDfK) or c(RGDfV). After lysis, the cell-associated radioactivity was measured in a γ-counter. Means ± SD of three independent experiments conducted in quadruplicate well were fitted by nonlinear regression using GraphPad Prism and R² fitting correlation coefficients are shown (a). IC₅₀ values ± SD are also shown (b)

Fig. 3

Uptake of [¹⁸F]FPyPEGCBT-c(RGDfK) in cancer cells. U-87 MG and SKOV-3 cells were incubated with 100 kBq/mL [¹⁸F]FPyPEGCBT-c(RGDfK) for 15 to 120 min at 37 or 4 °C (a). Cells were also co-treated with 10⁻⁴ M c(RGDfV) for 1 h (b). Uptake of [¹⁸F]FPyPEGCBT-c(RGDfK) was also evaluated after 1 h of incubation with cells with selective siRNA-mediated knocked down of α_V, β₁, β₃ or β₅ integrin subunits and indicated combinations (c). Depletions were verified by immunoblotting using specific antibodies as compared to untransfected cells (Unt) and to an irrelevant depletion of luciferase (si Luc). After incubation, cells were lysed and the cell-associated radioactivity was measured in a γ-counter. Data are means ± SD of at least three independent experiments performed in quadruplicate well. Statistical differences were analysed with the one-way ANOVA test followed by Dunnett’s post hoc test as compared to 37 °C controls (b) or to si Luc (c); #p < 0.001

Fig. 4

Biodistribution of [¹⁸F]FPyPEGCBT-c(RGDfK) in nude mice bearing U-87 MG or SKOV-3 subcutaneous tumours. Mice intravenously injected with 3–5 MBq [¹⁸F]FPyPEGCBT-c(RGDfK) were sacrificed and indicated organs were collected after uptake times of 30, 60 or 120 min (a). For the blocking experiments, mice were injected intravenously with 20 mg/kg of c(RGDfV) 5 min prior to [¹⁸F]FPyPEGCBT-c(RGDfK) injection and indicated organs were collected after 60 min of uptake (b). [¹⁸F]FPyPEGCBT-c(RGDfK) uptake in the indicated organs was quantified in a γ-counter and expressed as percentages of the injected dose per gram (%ID/g) (a, b). Tumours-to-blood and Tumours-to-muscle %ID/g ratios were also determined for the uptake time course of [¹⁸F]FPyPEGCBT-c(RGDfK) (c) and the blocked experiment (d). Data are means ± SD (n ≥ 7) and statistical differences were analysed with the one-way ANOVA test followed by Dunnett’s post hoc test as compared with 0.5 h uptake time (c) or with non-blocked 1 h uptake time (d); *p < 0.05, **p < 0.01, #p < 0.001

Fig. 5

MicroPET/CT images of [¹⁸F]FPyPEGCBT-c(RGDfK) in nude mice bearing subcutaneous tumours. Coronal sections of representative microPET/CT images of nude mice bearing subcutaneous U-87 MG (a, left) and SKOV-3 (a, right) tumours after an uptake time of [¹⁸F]FPyPEGCBT-c(RGDfK) of 60 ± 10 min. Tumours are outlined by dashed circles. Similar experiments were performed with injections of a blocking c(RGDfV) peptide 5 min prior to the tracer (b). c Mean SUV of [¹⁸F]FPyPEGCBT-c(RGDfK) in U-87 MG and SKOV-3 tumours at indicated times (top graph) as well as in the 60 min-blocked experiment (bottom graph). d Tumours-to-blood and Tumours-to-muscle SUV ratios were also determined for the uptake time course of [¹⁸F]FPyPEGCBT-c(RGDfK) (top graph) and the blocked experiment (bottom graph). Data are means ± SD (n ≥ 5). Statistical differences were analysed with the one-way ANOVA test followed by Dunnett’s post hoc test as compared with 0.5 h uptake time (c, d, top graphs) or with non-blocked 1 h uptake time (c, d, bottom graphs); *p < 0.05, #p < 0.001