Synthesis and Preclinical Characterization of the PSMA-Targeted Hybrid Tracer PSMA-I&F for Nuclear and Fluorescence Imaging of Prostate Cancer

Abstract

The prostate-specific membrane antigen (PSMA)–targeted radiotracers 68Ga/177Lu-PSMA-I&T and 99mTc-PSMA-I&S (for imaging and surgery) are currently successfully used for clinical PET imaging, radionuclide therapy, and radioguided surgery of metastatic prostate cancer. To additionally exploit the high sensitivity and spatial resolution of fluorescence imaging for improved surgical guidance, a PSMA-I&T–based hybrid tracer, PSMA-I&F (DOTAGA-k(Sulfo-Cy5)-y-nal-k-Sub-KuE), has been developed and evaluated. Methods: The in vitro PSMA-targeting efficiency of PSMA-I&F, the reference PSMA-I&T, and their corresponding natGa-/68Ga- and natLu/177Lu counterparts was determined in LNCaP cells via competitive binding assays (IC50) and dual-tracer radioligand and fluorescence internalization studies. Biodistribution and small-animal PET imaging studies were performed in CB17 SCID and LNCaP xenograft–bearing SHO mice, respectively, and complemented by intraoperative far-red fluorescence imaging using a clinical laparoscope. Additionally, fully automated serial cryosectioning and fluorescence imaging of 1 tumor-bearing animal as well as PSMA immunohistochemistry and fluorescence microscopy of organ cryosections (tumor, kidney, spleen) were also performed. Results: Compared with the parent PSMA-I&T analogs, the PSMA affinities of PSMA-I&F and its natGa-/natLu-complexes remained high and unaffected by dye conjugation (7.9 < IC50 < 10.5 nM for all ligands). The same was observed for the internalization of 68Ga- and 177Lu-PSMA-I&F. In vivo, blood clearance of 68Ga- and 177Lu-PSMA-I&F was only slightly delayed by high plasma protein binding (94%–95%), and very low accumulation in nontarget organs was observed already at 1 h after injection. Dynamic PET imaging confirmed PSMA-specific (as demonstrated by coinjection of 2-PMPA) uptake into the LNCaP xenograft (4.5% ± 1.8 percentage injected dose per gram) and the kidneys (106% ± 23 percentage injected dose per gram). Tumor-to-background ratios of 2.1, 5.2, 9.6, and 9.6 for blood, liver, intestines, and muscle, respectively, at 1 h after injection led to excellent imaging contrast in 68Ga-PSMA-I&F PET and in intraoperative fluorescence imaging. Furthermore, fluorescence imaging of tissue cryosections allowed high-resolution visualization of intraorgan PSMA-I&F distribution in vivo and its correlation with PSMA expression as determined by immunohistochemistry. Conclusion: Thus, with its high PSMA-targeting efficiency and favorable pharmacokinetic profile, 68Ga/177Lu-PSMA-I&F serves as an excellent proof-of-concept compound for the general feasibility of PSMA-I&T–based hybrid imaging. The PSMA-I&T scaffold represents a versatile PSMA-targeted lead structure, allowing relatively straightforward adaptation to the different structural requirements of dedicated nuclear or hybrid imaging agents.

Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye–Protein Interaction

“…A correlation (R 5 0.9232, P 5 0.0252; Supplemental Fig. 11) between lipophilicity and PPB was observed: an increase in lipophilicity led to a higher PPB, as is in line with the literature (31).…”

“…Next to the molecular design and the ability to relate the tracer performance to a specific cyanine backbone structure, one of the clear differences between our study and other studies regarding PSMA-targeted small-molecule hybrid tracers is reduced splenic and renal uptake (29)(30)(31)54). Considering that the renal uptake of DOTA (or DOTA analog)-containing PSMAtargeted hybrid tracers at early time points is around 100 %ID/g or even higher (29,54), whereas our study and the study of Kommidi et al (30) yielded a renal uptake below 19 %ID/g, it seems that the introduction of radiolabels without relying on charged chelates in the design of hybrid PSMA-targeted tracers minimizes renal uptake.…”

“…To overcome these shortcomings, a fluorescent and a radioactive label can be combined in a single molecule, creating so-called dual-labeled, bimodal, or rather hybrid tracers (28). This hybrid concept has also been adapted in preclinical PSMA imaging-for example, 111 In-LICOR-800CW-Lys-DOTA-EuK (29); PSMA-11 derivatives (13); an 18 F-containing, Cy3-based probe (30); and PSMA imaging and fluorescence (PSMA I&F) (31). Despite the ability of these tracer designs to offer a best-of-both-worlds scenario in image-guided surgery, in vivo analysis of the reported PSMA-targeted hybrid tracer analogs revealed that the synthetic designs are cleared and accumulated, at least fractionally, by the kidneys (13,29,31).…”

“…This hybrid concept has also been adapted in preclinical PSMA imaging-for example, 111 In-LICOR-800CW-Lys-DOTA-EuK (29); PSMA-11 derivatives (13); an 18 F-containing, Cy3-based probe (30); and PSMA imaging and fluorescence (PSMA I&F) (31). Despite the ability of these tracer designs to offer a best-of-both-worlds scenario in image-guided surgery, in vivo analysis of the reported PSMA-targeted hybrid tracer analogs revealed that the synthetic designs are cleared and accumulated, at least fractionally, by the kidneys (13,29,31). A drawback from the intraoperative use of these tracers is that their renal retention profile might lead to contamination of the surgical field with fluorescent urine and, as such, prohibit assessment of surgical margins (32).…”

Results from extended lymphadenectomies with [111In]PSMA-617 for intraoperative detection of PSMA-PET/CT-positive nodal metastatic prostate cancer

“…It can be assumed that the trend towards assisted or guided surgery such as RGS will continue, bearing in mind that more information before surgery and during surgery will potentially achieve better results. Bimodal-labelled anti-PSMA antibodies such as 68 Ga-Glu-urea-Lys-HBED-CC-IRDye800CW/ 68 Ga-Glu-urea-Lys-(HE) 3 -HBED-CC-IRDye800CW enabling the tracking of tumour tissue via fluorescence and γ-emission during surgery are under investigation [37,38]. By using fluorescence imaging as a tool to separate healthy LN tissue from LNM during robot-assisted laparoscopy, bimodal-labelled anti-PSMA antibodies are of great interest [39].…”