Synthesis and Preclinical Evaluation of Folate-NOTA-Al¹⁸F for PET Imaging of Folate-Receptor-Positive Tumors

Abstract

Folate receptor-targeted PET radiotracers can potentially serve as versatile imaging agents for the diagnosis, staging, and prediction of response to therapy of patients with folate receptor (FR)-expressing cancers. Because current FR-targeted PET reagents can be compromised by complex labeling procedures, low specific activities, poor radiochemical yields or unwanted accumulation in FR negative tissues, we have undertaken to design an improved folate-PET agent that might be more amenable for clinical development. For this purpose, we have synthesized a folate-NOTAAl18F radiotracer and examined its properties both in vitro and in vivo. Methods Radiochemical synthesis of folate-NOTA-Al18F was achieved by incubating 18F− with AlCl3 for 2 min followed by heating in the presence of folate-NOTA for 15 min at 100 °C. Binding of folate-NOTA-Al18F to FR was quantitated in homogenates of KB and Cal51 tumor xenografts in the presence and absence of excess folic acid as a competitor. In vivo imaging was performed on nu/nu mice bearing either FR+ve (KB cell) or FR−ve (A549 cell) tumor xenografts, and specific accumulation of the radiotracer in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folic acid. Image quality of folate-NOTA-Al18F was compared with that of 99mTc-EC20, a clinically established folate-targeted SPECT imaging agent. Results Total radiochemical synthesis and purification of folate-NOTA-Al18F was completed within 37 min, yielding a specific activity of 68.82±18.5 GBq/μmol, radiochemical yield of 18.6±4.5%, and radiochemical purity of 98.3±2.9%. Analysis of FR binding revealed a Kd of ~1.0 nM, and micro-PET imaging together with ex vivo biodistribution analyses demonstrated high FR-mediated uptake in an FR+ tumor and the kidneys. Conclusions Folate-NOTA-Al18F constitutes an easily prepared FR-targeted PET imaging agent with improved radiopharmaceutical properties and high specificity for folate receptor expressing tumors. Given its improved properties over 99mTc-EC20 (i.e. higher resolution, shorter image acquisition time, etc.), we conclude that folate-NOTA-Al18F constitutes a viable alternative to 99mTc-EC20 for use in identification, diagnosis, and staging of patients with FR-expressing cancers.

Development of a New Folate-Derived Ga-68-Based PET Imaging Agent

“…Therefore, it is expected that [ 68 Ga]NOTA-folate and [ 99m Tc]EC20 show similar renal uptake. Both tumor-to-kidney (0.31 ± 0.11) and tumor-to-liver ratios (12.43 ± 0.73) seem comparable to other, previously published data for other folate-targeted radiopharmaceuticals [3, 14, 28–34]. For a direct comparison, however, the individual compounds would have to be tested side-by-side.…”

“…In the past, different approaches were utilized for the synthesis of folate-based PET radiotracers. To our knowledge, current preclinical PET imaging tracers are based on either F-18 [14, 28–30, 33, 38] or Ga-68 [3, 31, 34–37] as a radionuclide for in vivo imaging. All imaging tracers were able to successfully delineate folate receptor-positive tumors in animal models.…”

“…These therapies build on the high affinity of folic acid and its derivatives to folate receptors ( K D (folate) = 0.1 nM) [3, 4], paired with the receptor’s overexpression in a wide variety of human epithelial cancers [5–8]. In parallel to the development of FR-targeted therapeutics, radio and optical tracers have been developed, and folate receptor-targeted imaging has been used successfully in preclinical and clinical research [8–14]. Similar to what is observed for folic acid, conjugates bearing these agents are bound to the folate receptor, and they are transported into the cell by endocytosis [15].…”

Imaging of Folate Receptor Expressing Macrophages in the Rat Groove Model of Osteoarthritis: Using a New DOTA-Folate Conjugate

“…Folic acid has, even after conjugation to a therapeutic or diagnostic cargo, a high affinity for the folate receptor, and therefore, folic acid can be used as an targeting ligand for selective delivery of attached imaging and therapeutic agents to cancer tissues and sites of inflammation that contain activated macrophages. 9 - 13 In rheumatoid arthritis (RA), an inflammatory joint condition where macrophages are thought to be the main promoter of disease activity, folate receptor expression on activated macrophages can be visualized using radiolabeled folate. 14 , 15 Recently, activated macrophage involvement was observed with SPECT/CT (single-photon emission computed tomography) imaging with Etarfolatide in symptomatic OA patients.…”

18F‐Labeling of Sensitive Biomolecules for Positron Emission Tomography

“…More recently, Chen and co-workers have used the NOTA chelation method to prepare a folate-NOTA-Al 18 F radiotracer for the diagnosis of folate-receptor (FR) expressing cancers. [38] In this work, the desired 18 F-labeled tracer was prepared with a decay corrected RCY of 19% and 69 GBq/μmol (1.9 Ci/μmol) molar activity. Since most Al- 18 F methods which use the NOTA or NODA chelators require elevated reaction temperatures (100–120 °C), it imposes limitations for 18 F-labeling of heat-sensitive biomolecules.…”