Preparation, Radiolabeling with 68Ga/177Lu and Preclinical Evaluation of Novel Angiotensin Peptide Analog: A New Class of Peptides for Breast Cancer Targeting

Abstract

Aim: Angiotensin II (AngII) is known to play a significant part in the development of breast cancer by triggering cell propagation of breast cancer, tumor angiogenesis, and regulating tumor invasion and cell migration. AngII arbitrates its action via two G-protein-coupled receptors, AngII type 1 receptor (AT1) and AngII type 2 receptor (AT2). Overexpression of the AT1 receptor in breast cancer cells seems to promote tumor growth and angiogenesis, thus targeting the AT1 receptor using AngII peptide would facilitate the detection of breast carcinoma. We developed an AngII peptide intending to assess whether the peptide of the renin-angiotensin system holds the ability to target AT1 receptor-overexpressing breast cancer in vivo.

Methods: DOTA-coupled AngII peptide was synthesized by conventional solid-phase peptide synthesis according to Fmoc/HATU chemistry. ⁶⁸Ga/¹⁷⁷Lu labeled AngII peptide was evaluated for its binding with TNBC MDA-MB-231 and ER+ MCF7 cell lines. Pharmacokinetics was studied in healthy balb/c mice and in vivo tumor targeting in nude mice with MDA-MB-231 tumors xenografts.

Results: DOTA-AngII peptide was labeled efficiently with ⁶⁸Ga/¹⁷⁷Lu with high labeling efficiency (≥90%). The stability of the radiopeptide in human plasma was found to be high. The AngII peptide analog showed nanomolar (<40 nM) AT1 receptor-specific binding affinity. The radioactivity internalized into MDA-MBA-231 and MCF7 cells were 14.97% and 11.75%, respectively. In vivo, biodistribution in balb/c mice exhibited efficient clearance of ⁶⁸Ga/¹⁷⁷Lu-DOTA-AngII peptide from the blood and elimination predominantly by the renal system due to its hydrophilic nature. A low amount of radioactivity was seen in the major organs including lungs, liver, stomach, spleen, and intestines (<3% ID/g) except the kidneys. A high renal-urinary excretion was observed for the radiotracer. In the TNBC MDA-MB-231 xenografts model, radiolabeled AngII peptide exhibited specific and effective AT1-based targeting in vivo. A rapid and efficient tumor targeting (2.18% ID/g at 45 min p.i.) together with fast renal excretion (~67% ID) highlights the tumor-targeting potential of the radiotracer. The AT1 receptor specificity of the radiotracer was validated by blocking assays. Furthermore, PET imaging provided sufficient visualization of MDA-MB-231 tumors in nude mice.

Conclusion: Our findings suggest that ⁶⁸Ga/¹⁷⁷Lu-DOTA-AngII peptide can be useful for the theranostic application of breast carcinomas. This study suggests the potential of this innovative class of peptides for rapid and efficient targeting of tumors and warrants further evaluation.

Keywords: angiotensin; biodistribution; breast cancer; peptide synthesis; preclinical; radiolabeling.

Figure 1

Representative HPLC elution profiles of the ⁶⁸Ga-DOTA-AngII peptide (upper); ¹⁷⁷Lu-DOTA-AngII peptide (middle); and the reference DOTA-AngII peptide (lower) labeled with cold ^natGa for identity confirmation (UV: 220 nm) after 60 min post-labeling. The radiochemical purity was always greater than 85% after post-labeling chromatographic purification. Note: The UV detector is connected before the radioactive detector.

Figure 2

Radio-HPLC analysis of a mouse urine sample collected at 2 h p.i. after injecting the ⁶⁸Ga-DOTA-AngII peptide analog (upper); Radio-HPLC chromatogram of ¹⁷⁷Lu-DOTA-AngII peptide analog after 5 h incubation with human plasma (lower).

Figure 3

Micro-PETcamera image of a female nude mouse, with MDA-MB-231 breast cancer xenografts, after the tail vein injection of ~75 µCi of ⁶⁸Ga-DOTA-AngII peptide analog at 45 min post-injection. The arrow indicates the tumor position.