Novel Preclinical and Radiopharmaceutical Aspects of [68Ga]Ga-PSMA-HBED-CC: A New PET Tracer for Imaging of Prostate Cancer

Abstract

The detection of prostate cancer lesions by PET imaging of the prostate-specific membrane antigen (PSMA) has gained highest clinical impact during the last years. 68Ga-labelled Glu-urea-Lys(Ahx)-HBED-CC ([68Ga]Ga-PSMA-HBED-CC) represents a successful novel PSMA inhibitor radiotracer which has recently demonstrated its suitability in individual first-in-man studies. The radiometal chelator HBED-CC used in this molecule represents a rather rarely used acyclic complexing agent with chemical characteristics favourably influencing the biological functionality of the PSMA inhibitor. The simple replacement of HBED-CC by the prominent radiometal chelator DOTA was shown to dramatically reduce the in vivo imaging quality of the respective 68Ga-labelled PSMA-targeted tracer proving that HBED-CC contributes intrinsically to the PSMA binding of the Glu-urea-Lys(Ahx) pharmacophore. Owing to the obvious growing clinical impact, this work aims to reflect the properties of HBED-CC as acyclic radiometal chelator and presents novel preclinical data and relevant aspects of the radiopharmaceutical production process of [68Ga]Ga-PSMA-HBED-CC.

Figure 1

The radiochemical yields (RCY) of the HBED-CC- and NOTA-conjugated model-peptide c(RGDyK) as a function of (A) peptide concentration (2 min reaction time); and (B) reaction time (1.7 μM peptide concentration), respectively. The compounds were incubated with the generator eluate at room temperature in HEPES buffer (pH 4.2). The reaction was stopped by adding 10 μL of a 0.1 M EDTA solution and the reaction mixture was subsequently analysed via radio-HPLC (n = 6).

Figure 2

The pH dependence of the complexation reaction was determined by incubating the free acid of (A) HBED-CC (10 μM) and (B) NOTA (10 μM), respectively, at RT for 10 min. The radiochemical yield was determined by ITLC at the indicated time points (n = 6).

Figure 3

Superdex 75 5/150 GL runs of (A) ⁶⁷Ga-labelled HBED-CC-c(RGDyK); and (B) ⁶⁷Ga-labelled NOTA-c(RGDyK) after 48 h incubation in human serum at 37 °C. Only the radiometric signal is shown. The UV trace showed a major peak at ~3.9 min p.i.; (C) which corresponds with the elution time of serum proteins.

Figure 4

Radio-HPLC traces of RT (A) and 95 °C (B) labelled Glu-urea-Lys(Ahx)-HBED-CC. The peaks correspond to (1), the thermodynamically more stable, and (2), the thermodynamically less stable diastereomer, respectively. Graphs C–E show the radiochromatograms after various storage and labelling conditions: 95 °C labelled Glu-urea-Lys(Ahx)-HBED-CC stored for 3 h in injection buffer (C); RT labelled Glu-urea-Lys(Ahx)-HBED-CC after 3 h incubation in labelling reaction buffer at (D) pH 4, and (E) pH 7.

Figure 5

(A) LNCaP-cell binding and internalisation of [⁶⁸Ga]Ga-PSMA-HBED-CC labelled at RT or 95 °C, respectively. Specific cell uptake was determined by competitive blockade with 500 μM of the PSMA inhibitor 2-PMPA. Values are expressed as percentage of applied radioactivity bound to 10⁶ cells [%IA/10⁶ cells]. Data are expressed as mean ± SD (n = 3); (B) Determination of binding affinity of [^natGa]Ga-PSMA-HBED-CC to LNCaP cells as a function of the labelling temperature. The cells (10⁵ per well) were incubated with the radioligand (⁶⁸Ga-labelled Glu-urea-Lys(Ahx)-HBED-CC) in the presence of different concentrations of ^natGa-analyte (0–5000 nM, 100 μL/well).

Figure S1

MS analysis of HPLC fractions of the mixture of diastereomers (fraction 23.84–27.00 min), of the thermodynamically more stable (fraction 23.95-25.04 min) and of the less stable diastereomer (fraction 26.58-26.88 min) of Ga-PSMA-HBED-CC, respectively.