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

Abstract

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.

Keywords: Fluorine-18; PSMA; Prostate cancer; Radiohybrid.

Fig. 1

Diastereomeric mixture [¹⁸F, ^natGa]rhPSMA-7 is composed of the four isomers [¹⁸F, ^natGa]rhPSMA-7.1 to -7.4, differing in the stereoconfiguration of diaminopropionic acid (D-/L-Dap) and DOTA-GA (R-/S-DOTAGA). The abundance of the diastereomers could be determined by HPLC-analysis of retained samples of previously produced batches of the diastereomeric mixture, [¹⁹F, ^natGa]rhPSMA-7 (n = 6)

Fig. 2

a Binding affinities (IC₅₀ in nM, 1 h, 4 °C) of [¹⁹F, ^natGa]rhPSMA-7.1 to -7.4 (white; n = 5–9) and the references diastereomeric mixture [¹⁹F, ^natGa]rhPSMA-7 (grey; n = 3), [¹⁹F]DCFPyL and [¹⁹F]PSMA-1007 (black; n = 3); b internalized activity of [¹⁸F, ^natGa]rhPSMA-7.1 to -7.4 (white; n = 3–6) and the references diastereomeric mixture [¹⁹F, ⁶⁸ Ga]rhPSMA-7 (grey; n = 3), [¹⁸F]DCFPyL and [¹⁸F]PSMA-1007 (black; n = 3) in LNCaP cells (1 h, 37 °C) as a percentage of the reference ligand ([¹²⁵I]I-BA)KuE); c lipophilicity of [¹⁸F, ^natGa]rhPSMA-7.1 to -7.4 (white; n = 18–23) and the references diastereomeric mixture [¹⁸F, ^natGa]rhPSMA-7 (grey; n = 13), [¹⁸F]DCFPyL and [¹⁸F]PSMA-1007 (black; n = 3), expressed as n-octanol/PBS (pH 7.4) distribution coefficient (log D); d human serum albumin binding of [¹⁹F, ^natGa]rhPSMA-7.1 to -7.4 (white) and the references diastereomeric mixture [¹⁹F, ^natGa]rhPSMA-7 (grey), [¹⁹F]DCFPyL and [¹⁹F]PSMA-1007 (black), determined on a Chiralpak HSA column. Data for the reference ligands were [15, 19] taken from a previously published studies conducted by our group. Values are expressed as mean ± standard deviation

Fig. 3

Biodistribution of [¹⁸F, ^natGa]-rhPSMA-7 and the isomers [¹⁸F, ^natGa]rhPSMA-7.1 to -7.4 at 1 h p.i. in male LNCaP tumor-bearing SCID mice. Data are expressed as a percentage of the injected dose per gram (% ID/g), mean ± standard deviation (n = 4 for [¹⁸F, ^natGa]rhPSMA-7.1, n = 5 for -7.2, n = 4 for -7.3, n = 5 for -7.4 and n = 3 for -7)

Fig. 4

Biodistribution of [¹⁸F, ^natGa]rhPSMA-7.1 to -7.4, co-injected with 2-PMPA (8 mg/kg) at 1 h p.i. in male LNCaP tumor-bearing SCID mice. Data are expressed as a percentage of the injected dose per gram (% ID/g), mean ± standard deviation (n = 3)

Fig. 5

Differential uptake and excretion pattern: relative percentage difference between the abundance of [¹⁸F, ^natGa]rhPSMA-7.1 to -7.4 in blood, liver, kidney, urine and tumor of male LNCaP tumor-bearing SCID mice (30 min p.i.) compared with the respective abundance found in the quality control of diastereomeric mixture [¹⁸F, ^natGa]rhPSMA-7. n = 4 in four independent experiment; the liver from the first experiment was not analyzed (see also Additional file 1: Fig. 9)