A thiol-reactive 18F-labeling agent, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide, and synthesis of RGD peptide-based tracer for PET imaging of alpha v beta 3 integrin expression

Abstract

The cell adhesion molecule integrin alpha v beta 3 plays a key role in tumor angiogenesis and metastasis. A series of 18F-labeled RGD peptides have been developed for PET of integrin expression based on primary amine-reactive prosthetic groups. In this study we introduced a new method of labeling RGD peptides through a thiol-reactive synthon, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM).

Methods: 18F-FBEM was synthesized by coupling N-succinimidyl 4-18F-fluorobenzoate (18F-SFB) with N-(2-aminoethyl)maleimide. After high-pressure liquid chromatography purification, it was allowed to react with thiolated RGD peptides, and the resulting tracers were subjected to receptor-binding assay, in vivo metabolic stability assessment, biodistribution, and microPET studies in murine xenograft models.

Results: Conjugation of monomeric and dimeric sulfhydryl-RGD peptides with 18F-FBEM was achieved in high yields (85% +/- 5% nondecay-corrected on the basis of 18F-FBEM). The radiochemical purity of the 18F-labeled peptides was >98% and the specific activity was 100 approximately 150 TBq/mmol. Noninvasive microPET and direct tissue sampling experiments demonstrated that both 18F-FBEM-SRGD (RGD monomer) and 18F-FBEM-SRGD2 (RGD dimer) had integrin-specific tumor uptake in subcutaneous U87MG glioma and orthotopic MDA-MB-435 breast cancer xenografts.

Conclusion: The new tracer 18F-FBEM-SRGD2 was synthesized with high specific activity via 18F-FBEM and the tracer exhibited high receptor-binding affinity, tumor-targeting efficacy, metabolic stability, as well as favorable in vivo pharmacokinetics. The new synthon 18F-FBEM developed in this study will also be useful for radiolabeling of other thiolated biomolecules.

FIGURE 1

(A) Synthetic route for N-[2-(4-¹⁸F-fluorobenzamido)ethyl]maleimide (¹⁸F-FBEM). (B) Structure of ¹⁸F-FBEM-SRGD. (C) Structure of ¹⁸F-FBEM-SRGD2.

FIGURE 2

Cell-binding assay of c(RGDyK), E[c(RGDyK)]₂, FBEM-SRGD, and FBEM-SRGD2 using U87MG cells (integrin α_vβ₃–positive human glioblastoma). The cell-binding affinity of the peptides was determined by performing competitive displacement studies with ¹²⁵I-echistatin. IC₅₀ values for c(RGDyK), E[c(RGDyK)]₂, FBEM-SRGD, and FBEM-SRGD2 were 51.3 ± 4.2, 26.1 ± 3.2, 66.8 ± 5.1, and 55.1 ± 6.5 nmol/L, respectively (n = 6).

FIGURE 3

Biodistribution of ¹⁸F-FBEM-SRGD (A) and ¹⁸F-FBEM-SRGD2 (B) in athymic nude mice bearing both U87MG and MDA-MB-435 tumors at 10, 30, and 60 min after injection (n = 3). Biodistribution of both tracers at 60 min after injection when coinjected with 10 mg/ kg mice body weight of c(RGDyK) is also shown.

FIGURE 4

Dynamic microPET scans using both radiotracers at different time points in a mouse bearing both U87MG and MDA-MB-435 tumors. Ten-minute static scans at several later time points were also conducted to complete the tracer kinetic study.

FIGURE 5

Time–activity curves of ¹⁸F-FBEM-SRGD (A) and ¹⁸F-FBEM-SRGD2 (B) obtained from microPET scans. The inflection point for tracer clearance is most likely due to the slower metabolism during the dynamic scan when mice were under anesthesia and body temperature was lowered.

FIGURE 6

Ten-minute static microPET scans of U87MG tumor-bearing mice (arrows) injected with 3.7 MBq of ¹⁸F-FBEM-SRGD2. (Left) Control mouse. (Right) Blocking with 10 mg/kg mouse body weight of c(RGDyK).

FIGURE 7

Metabolic stability of ¹⁸F-FBEM-SRGD2 in mouse blood and urine samples and in liver, kidneys, and U87MG tumor homogenates 60 min after injection. HPLC profile of tracer itself (Standard) is also shown.