Synthesis and Evaluation of a Novel Connexin43 Targeting Radiotracer ⁶⁸Ga-NOTAGA-GAP19

Objectives: Connexins are a large family of transmembrane proteins widely distributed in the human body that assemble to form intercellular channels called gap junctions and Cx43 is the main component of gap junctions(1). Due to Connexin 43 has a widely recognized role in tumorigenesis and the progression of heart disease.Therefore, the design of a molecular imaging probe that can target Cx43 may have important guiding significance for the diagnosis and treatment of partial tumors and heart diseases(2).The Gap19, a new peptideinterfere with the loop/tail interactions by binding to the C-terminal tail and preventing endogenous loop/tail interactions that are critical for hemichannel opening(3). Here, based on Gap19, we want to develop a novel ⁶⁸Ga labeled Gap19 radiotracer that targets Connexin43 and we evaluate the targeting ability and specificity of the tracer in vitro.

Methods: Synthesis: PrecursorNOTAGA-GAP19was radiolabeled in one-step by ⁶⁸Ga and 0.3 M sodium acetate aqueous solution heating at 90℃for 12 mins in open system followed by cooling down. And the reaction solution was passed through C18 cartridge. The C18 cartridge was then rinsed with 10 ml of water and 1 ml of 75% ethanol/water. The product was eluted with 1 ml of 75% ethanol/water. In vitro: Three NSCLC cell lines (HCC827, H358, A549) were used in cell uptake assay with ⁶⁸Ga-NOTAGA-GAP19. Among the three non-small cell lung cancer cell lines, HCC827 had the lowest Connexin43 expression, while A549 had the high expression.To verify the molecular probe ⁶⁸Ga-NOTAGA-GAP19targeting of Connexin43, we constructed HCC827 cells with high Connexin43 expression by adenovirus transfection (HCC827^ADV-OE).

Results: The radio-yield of ⁶⁸Ga-NOTAGA-GAP19was 66.58±1.23% (no-decay correction, n=3) and the radio-chemical purity was 95.18±0.81% (n=3). The expression of Connexin43 was significantly increased in HCC827^ADV-OE compared with HCC827. The results of the uptake experiments of labeled probes on cells showed that A549 and H358 which is high expression Cx43 NSCLC cell lines, had a significantly higher uptake of ⁶⁸Ga-NOTAGA-GAP19 compared to the other lung cancer cell lines, HCC827. In addition, the uptake of ⁶⁸Ga-NOTAGA-GAP19 in HCC827^ADV-OE was also increased than in HCC827.

Conclusions: We successfully designed and synthesized a novel radiotracer ⁶⁸Ga-NOTAGA-GAP19 that targets Connexin43. Experiments in vitro demonstrated that the molecular probe ⁶⁸Ga-NOTAGA-GAP19 can bind to Connexin43 in non-small cell lung cancer cell lines. Further research on this probe is necessary to verify its stability and imaging effect in vivo.Reference: [1] Schulz R, Gorge PM, Gorbe A, Ferdinandy P, Lampe PD, and Leybaert L. Connexin 43 is an emerging therapeutic target in ischemia/reperfusion injury, cardioprotection and neuroprotection. Pharmacol Ther. 2015;153:90-106. [2] Grek CL, Rhett JM, Bruce JS, Ghatnekar GS, and Yeh ES. Connexin 43, breast cancer tumor suppressor: Missed connections? Cancer Lett. 2016;374(1):117-26. [3] Maes M, Crespo Yanguas S, Willebrords J, Weemhoff JL, da Silva TC, Decrock E, et al. Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice. Toxicol Lett. 2017;278:30-7.