Ensuring enantiomeric consistency in FAPI radiopharmaceuticals: comparative analysis of (S)- and (S/R)-FAPI-46/-74 confirms pharmacological equivalence

Abstract Background Enantiomeric composition may impact binding and potentially performance of a drug product but is not routinely tested and minimum acceptance criteria are not always established. Some batches of the precursors FAPI-46 and FAPI-74 used clinically contain a mixture of ( S )- and ( R )-enantiomers. This study investigates the in vitro pharmacodynamic characteristics of different enantiomeric compositions of FAP-targeting radioligands FAPI-46 and FAPI-74 and in vivo pharmacokinetics of FAPI-46 in naïve mice. Results ( S )-, ( R )-, and ( S/R )-enantiomers of radiolabeled FAPI-46 and FAPI-74 were evaluated for stability in human serum, binding affinity, internalization, retention, and selectivity using HT1080-hFAP cells and HEK293-hCD26 cells. All enantiomeric compositions showed high stability in human serum with minimal serum protein adhesion. ( S )- and ( S/R )-enantiomers exhibited comparable binding, internalization, and retention characteristics. High selectivity for FAP over CD26/DPP4 was maintained. ( R )-enantiomers showed no specific binding to FAP. Additionally, the same enantiomers of 68 Ga-labeled FAPI-46 were evaluated by dynamic positron emission tomography (PET) imaging (0–60 min post-injection) and ex vivo organ biodistribution in naïve BALB/c mice (10, 30, and 60 min, or 4 h post-injection) to compare their pharmacokinetic profiles. PET imaging revealed nearly identical time-activity curves for the enantiomers of [ 68 Ga] Ga-FAPI-46, indicating similar whole-body distribution within 1 h post-injection, with rapid renal clearance and minimal muscle uptake. Ex vivo organ biodistribution confirmed comparable pharmacokinetic profiles between enantiomers, with no significant differences in tissue distribution. Conclusion These findings provide important validation that current clinical ( S/R )-mixtures of FAPI-46/-74 exhibit pharmacokinetic and pharmacodynamic behavior comparable to pure ( S )-enantiomers, supporting regulatory confidence and cross-trial reproducibility in the global FAPI-46/-74 imaging landscape.