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Fibroblast Activation Protein (FAP) targeting homodimeric FAP inhibitor radiotheranostics: a step to improve tumor uptake and retention time.

ABSTRACT: Several radiopharmaceuticals targeting fibroblast activation protein (FAP) based on the highly potent FAP inhibitor UAMC1110 are currently under investigation. Pre-clinical as well as clinical research exhibited the potential of these imaging agents. However, the monomeric small molecules seemed to have a short retention time in the tumor in combination with fast renal clearance. Therefore, our strategy was to develop homodimeric systems having two FAP inhibitors to improve residence time and tumor accumulation. The homodimers with two squaramide coupled FAP inhibitor conjugates DOTA.(SA.FAPi)₂ and DOTAGA.(SA.FAPi)₂ were synthesized and radiochemically evaluated with gallium-68. [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂ was tested for its in vitro stability, lipophilicity and affinity properties. In addition, human PET/CT scans were performed for [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂ with a head-to-head comparison with [⁶⁸Ga]Ga-DOTA.SA.FAPi and [¹⁸F]FDG. Labeling with gallium-68 demonstrated high radiochemical yields. Inhibition measurements revealed excellent affinity and selectivity with low nanomolar IC₅₀ values for FAP. In PET/CT human studies, significantly higher tumor uptake as well as longer tumor retention could be observed for [⁶⁸Ga]Ga-DOTAGA.(SA.FAPi)₂ compared to [⁶⁸Ga]Ga-DOTA.SA.FAPi. Therefore, the introduction of the dimer led to an advance in human PET imaging indicated by increased tumor accumulation and prolonged retention times in vivo and thus, the use of dimeric structures could be the next step towards prolonged uptake of FAP inhibitors resulting in radiotherapeutic analogs of FAP inhibitors.

SUBMITTER: Moon ES

PROVIDER: S-EPMC8727881 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundFibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA5m and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA5m.SA.FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [68Ga]Ga-DOTA.SA.FAPi.Results[68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DATA5m.SA.FAPi showed high complexation >?97% radiochemical yields after already 10?min and high stability over a period of 2?h. Affinity to FAP of DOTA.SA.FAPi and DATA5m.SA.FAPi and its natGa and natLu-labeled derivatives were excellent resulting in low nanomolar IC50 values of 0.7-1.4?nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC50 with 1.7-8.7??M). First proof-of-principle in vivo PET-imaging animal studies of the [68Ga]Ga-DOTA.SA.FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUVmean of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60?min post injection with overall low uptake in healthy tissues.ConclusionIn this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA5m bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.

Project description:BackgroundThere is an urgent need to develop new agents for treating metastatic prostate cancer to overcome multiple drug resistance to the current standard targeted cancer therapy. Emetine is a highly cytotoxic natural product protein synthesis inhibitor, which is toxic to all cell types. Its cytotoxicity can be blocked by derivatizing its N-2' position. Thus emetine can be selectively delivered to cancer cells in the region of metastatic cancer as a prodrug that will be activated by an enzyme selectively overexpressed within the metastatic tumor microenvironment. In this work, we convert emetine to a prodrug activatable by the fibroblast activation protein (FAP), a serine protease overexpressed by the carcinoma associated fibroblasts.MethodBy using an iterative structure-activity relationship strategy, several peptidyl emetine prodrug analogs (1-11) were synthesized by chemical derivatization of emetine at its N-2' position and tested for in-vitro activation by FAP. The lead prodrug 11 is made up of a DPPIV activatable prodrug precursor 10 (Ala-Pro-PABC-Emetine) coupled to FAP substrate (Ala-Ser-Gly-Pro-Ala-Gly-Pro). Activation assays of the prodrugs were performed in purified FAP, DPPIV, FBS, and human serum and were analyzed by LCMS. In vitro cytotoxicity assays of these prodrugs are carried out in prostate (LNCaP, PC3) and breast (MCF7 and MDA-MB-231) cancer cell lines. The prodrugs are also tested in normal immortalized human prostatic epithelial cell line (PrEC).ResultsThe lead FAP activated emetine prodrug 11 is activated to emetine in tandem by FAP and DPPIV in about 70% conversion within 24 hr. In prostate and breast cancer cell lines treated with prodrug 11, it is found to be equipotent with emetine in the presence of FAP and DPPIV. However, in the PrEC cell line grown in serum free media, prodrug 11 is more than 200-fold less cytotoxic than emetine in the absence of FAP and DPPIV.ConclusionThis FAP activated prodrug of cytotoxic agent emetine further shows the crucial role of the N-2' position of emetine in controlling its cytotoxicity. Significantly reduced toxicity observed in the PrEC cell line in the absence of FAP and DPPIV shows that prodrug 11 could be systemically delivered to regions of metastatic prostate cancer or other solid tumor for activation by cancer selective enzymes within the cancer microenvironment, such as FAP that is overexpressed by the carcinoma-associated fibroblasts. The two-step tandem enzymatic activation of prodrug 11 by FAP and DPPIV is a strategy for overcoming steric hindrance.

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Fibroblast Activation Protein (FAP) targeting homodimeric FAP inhibitor radiotheranostics: a step to improve tumor uptake and retention time.

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