Based on quinoline amide core-based FAP inhibitors, many macrocyclic species, such as DOTA-, NOTA-conjugated chelation reagents, have been developed. However, these bifunctional chelator-coupled compounds have been labelled with metal radionuclides, such as 68Ga and Al18F for PET imaging, 99mTc for SPECT imaging, and 177Lu, 225Ac, and 223Ra for internal exposure treatment in the clinic. Among the imaging tracers, [68Ga]Ga-DOTA-FAPI-04 showed excellent pharmacokinetic mechanisms for clinical use in a variety of tumours. As derivatives of small-molecule inhibitors, these tracers showed quick metabolism and clearance from the body, resulting in excellent T/NT values and lower total-body effective doses. Due to the rapid biological half-lives and the short physical half-life of 68Ga (67.7 min), [68Ga]Ga-DOTA-FAPI-04 is the most studied and reported PET molecular imaging probe.
In this research, we chose the exact inhibitor ((S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-6-(methoxy) quinoline-4-carboxamide, IC50 = 8.5 ± 0.9 nM) of FAP and its analogue (S)-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)-6-(methoxy) quinoline-4-carboxamide as the standard compounds, labelled them with the PET radionuclide 11C (half-life, 20.4 min), and examined the potential of the tracers as PET molecular imaging probes in tumour model mice.
First, the precursors of these inhibitors were synthesized and labelled with 11CH3I or 11CH3OTf in several organic solvents, such as DMF, DMSO, THF, and acetone, containing different bases, such as NaOH, NaH, and triethylamine. The reaction temperature was controlled at room temperature, 55°C or 80°C. Finally, the optimal reaction conditions were 1 mg of precursor, 11CH3I in 250 µL of DMF containing 5 µL of NaOH (5 M) at 55°C, resulting in radiochemical yields of 23% (11C-FAPI-01) and 16% (11C-FAPI-02). The final products were identified by HPLC analysis with a radio detector method after HPLC and solid-phase extraction (SPE) using anhydrous ethanol as the solvent and an SPE C-18 cartridge as the solid-phase column, respectively.
The significant differences in log P among the three radioprobes reflected the different metabolism and clearance pathways. The higher water solubility of [68Ga]Ga-DOTA-FAPI-04 (log P = 0.25) resulted in high kidney clearance, and the higher lipid solubility of 11C-FAPI-01 (log P = 0.63) and 11C-FAPI-02 (log P = 1.27) resulted in good hepatobiliary clearance. These results were confirmed by small-animal PET/CT imaging and the organ distributions in the model mice.
As previously described, [68Ga]Ga-DOTA-FAPI-04 was rapidly distributed in all organs of the model mice in 30 min. Because of the rapid distribution, the T/K ratios showed a consistent increase from 0.04 ± 0.01 (30 min p.i.) to 0.41 ± 0.06 ID%/g (after 90 min p.i.), and the corresponding tumour/muscle (T/M) ratios changed from 1.16 ± 0.25 to 4.74 ± 0.07.
Similar to the [68Ga]Ga-DOTA-FAPI-04 small-animal imaging and organ distribution in U87MG cells, both 11C-FAPI-01 and 11C-FAPI-02 were rapidly distributed in all organs of the model mice in 30 min. The T/K ratios for these two probes were 1.05 ± 0.03 and 0.93 ± 0.04 at 60 and 90 min p.i., and the T/K radios were 0.89 ± 0.05 and 0.67 ± 0.01 and 1.29 ± 0.07 and 1.39 ± 0.06, respectively. However, the greatest difference was that the organ with the highest uptake was the kidney for the previous tracer but the liver for 11C-FAPI-01 and 11C-FAPI-02. Both 11C-FAPI-01 and 11C-FAPI-02 accumulated rapidly in the tumour and other organs in 30 min and resulted in higher T/M (3.19 ± 0.06 in 11C-FAPI-01 and 2.30 ± 0.02 in 11C-FAPI-02). As the liver is the major organ that metabolizes most drugs, the T/L ratios were examined. For 11C-FAPI-01, the ratios at 30, 60, 90 min p.i. were 0.19 ± 0.03, 0.63 ± 0.04, and 0.34 ± 0.06, respectively. For 11C-FAPI-02, the ratios were 0.60 ± 0.03, 0.62 ± 0.03 and 0.62 ± 0.03. Significant disparities in the liver and kidney were observed because of the differences in drug metabolism and clearance. As a difference between [68Ga]Ga-DOTA-FAPI-04 and 68Ga-FAPI-02, due to the existence of difluorine atoms in the proline derivative residues of 11C-FAPI-02, the lipid solubility was enhanced compared with that of 11C-FAPI-01, resulting in rapid accumulation and slow clearance in organs, especially in tumours.
Due to the enhanced lipid solubility of 11C-FAPI-02, the in vivo tumour accumulation efficacy of 11C-FAPI-02 and [68Ga]Ga-DOTA-FAPI-04 was evaluated in a mouse model of orthotopic glioma. An interesting finding was that in contrast to the radioactive accumulations in the brain of U87MG tumour xenografts and in the orthotopic model, no differences were observed between the two at 60 min p.i. except for with 11C-FAPI-02. A significant difference in the brain was observed at 60 min p.i. This means that the 11C-labelled tracers can more easily cross the blood–brain barrier and image the glioma in the brain earlier than [68Ga]Ga-DOTA-FAPI-04. Further studies of this observed phenomenon are ongoing.