Synthesis and preclinical characterization of the 64Cu labeled TRPC5-targeting PET imaging probe for cancer

Introduction: Transient receptor potential channels subfamily member 5 (TRPC5) is a Ca2+-permeable, nonselective cation channel that belongs to the TRP family of ion channels. Elevated expression of TRPC5 has been documented in human breast cancer, colon cancer, and colorectal cancer. Analysis of tumor tissue indicates a correlation between high TRPC5 expression and poor overall survival, as well as tumor metastasis. HC-070, a xanthine derivative represents one of the potential classes of high-selective TRPC5 inhibitors. This project aims to develop an isopropyl xanthine derivative, [⁶⁴Cu]TZ78143 PET radiotracers that can quantify the expression of TRPC5 in cancer metastases with the overarching goal of enhancing patient screening and facilitating targeted therapy.

Methods: Based on our Sarcage theranostic platform, an azide derivative of PEG-derived TRPC5 targeting ligand (TZ78143) was conjugated to the bifunctional chelator (NH₂)₂Sar to obtain monomeric (TZ78143-Ph-PEG(n)-sarc) and dimeric (TZ78143-Ph-PEG(n)-sarc- PEG(n)-Ph-TZ78143) agents. Approximately 5.5 MBq of [⁶⁴Cu]TZ78143-monomer and dimer was intravenously administered to U87MG glioblastoma tumor-bearing models. Subsequent scans were performed at 1h, 4h, 24h, and 48h post-injection (p.i.). Following the selection of the lead dimeric agent, in vivo PET imaging was further evaluated in 4T1 brain tumor metastasis, BxPC3 pancreatic adenocarcinomas, PC3 prostate cancer, and H1299 non-small cell lung carcinoma. Region of interest analysis was conducted using Amide software, and organ uptake was calculated after decay correction.

Results: The ⁶⁴Cu labeled agents can be obtained in moderate to good yield with >98% radiochemical purity. [⁶⁴Cu]TZ78143-monomer demonstrated initial tumor uptake (0.91±0.43 %ID/g at 1h) in U87MG model accompanied with fast clearance (0.51±0.26 %ID/g at 4h and 0.27±0.05 %ID/g at 24h). In contrast, [⁶⁴Cu]TZ78143-dimer greatly improved the tumor uptake to 6.3±3.1 %ID/g at 1h, 7.4±2.8 %ID/g at 4h, and sustained at 5.1 %ID/g at 48h. Notably, liver (13.9±1.4 %ID/g) and kidney (3.9±0.7 %ID/g) uptakes were maintained at a moderate level. Furthermore, [⁶⁴Cu]TZ78143-dimer also exhibited high tumor uptake in a breast cancer metastasis tumor model (4T1). Additional tumor models, including BxPC3 pancreatic adenocarcinomas, PC3 prostate cancer, and H1299 non-small cell lung carcinoma, were also tested with our tracer.

Conclusions: Novel radioligands have been developed targeting TRPC5. An initial PET study demonstrated that the dimeric agent has high and sustained tumor uptake in the U87MG model. This promising result warrants further study of [⁶⁴Cu]TZ78143-dimer as a theranostic agent targeting TRPC5.

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