Preclinical comparison of four [¹⁸F, ^natGa]rhPSMA-7 isomers: influence of the stereoconfiguration on pharmacokinetics

Introduction: Radiohybrid (rh) ligands, a novel class of prostate-specific membrane antigen (PSMA)-targeted radiopharmaceuticals, can be labeled either with [¹⁸F]fluorine via isotopic exchange or with radiometals (such as [⁶⁸Ga]Gallium, [¹⁷⁷Lu]Lutetium, [²²⁵Ac]Actinium). Among these, [¹⁸F, ^natGa]rhPSMA-7 has recently entered clinical assessment. Aim: Since [¹⁸F, ^natGa]rhPSMA-7 is composed of four stereoisomers ([¹⁸F, ^natGa]rhPSMA-7.1, -7.2, -7.3 and -7.4), we initiated a preclinical selection process to identify the isomer with the most favorable pharmacokinetics for further clinical investigation. Methods: A synthetic protocol for enantiopure [¹⁹F, ^natGa]rhPSMA-7 isomers has been developed. The comparative evaluation of the four isomers comprised human serum albumin binding, lipophilicity, IC₅₀, internalization and classical biodistribution studies and competition experiments in LNCaP tumor-bearing CB-17 SCID mice. In addition, a radio high-performance liquid chromatography-based method was developed allowing quantitative, intraindividual comparison of [¹⁸F, ^natGa]rhPSMA-7.1 to -7.4 in LNCaP tumor-bearing mice. Results: Cell studies revealed high PSMA affinity and internalization for [^18/19F, ^natGa]rhPSMA-7.2, -7.3 and -7.4, whereas [^18/19F, ^natGa]rhPSMA-7.1 showed approximately twofold lower values. Although the biodistribution profile obtained was typical of PSMA inhibitors, it did not allow for selection of a lead candidate for clinical studies. Thus, an intraindividual comparison of all four isomers in LNCaP tumor-bearing mice was carried out by injection of a diastereomeric mixture, followed by analysis of the differential uptake and excretion pattern of each isomer. Based on its high tumor accumulation and low uptake in blood, liver and kidneys, [¹⁸F, ^natGa]rhPSMA-7.3 was identified as the preferred isomer and transferred into clinical studies. Conclusion: [¹⁸F, ^natGa]rhPSMA-7.3 has been selected as a lead compound for clinical development of a [¹⁸F]rhPSMA-based candidate. The intraindividual differential uptake and excretion analysis in vivo allowed for an accurate comparison and assessment of radiopharmaceuticals.