Radiohybrid Ligands: A Novel Tracer Concept Exemplified by 18F- or 68Ga-Labeled rhPSMA Inhibitors

Abstract

When we critically assess the reason for the current dominance of ⁶⁸Ga-labeled peptides and peptide-like ligands in radiopharmacy and nuclear medicine, we have to conclude that the major advantage of such radiopharmaceuticals is the apparent lack of suitable ¹⁸F-labeling technologies with proven clinical relevance. To prepare and to subsequently perform a clinical proof-of-concept study on the general suitability of silicon-fluoride-acceptor (SiFA)-conjugated radiopharmaceuticals, we developed inhibitors of the prostate-specific membrane antigen (PSMA) that are labeled by isotopic exchange (IE). To compensate for the pronounced lipophilicity of the SiFA unit, we used metal chelates, conjugated in close proximity to SiFA. Six different radiohybrid PSMA ligands (rhPSMA ligands) were evaluated and compared with the commonly used ¹⁸F-PSMA inhibitors ¹⁸F-DCFPyL and ¹⁸F-PSMA-1007. Methods: All inhibitors were synthesized by solid-phase peptide synthesis. Human serum albumin binding was measured by affinity high-performance liquid chromatography, whereas the lipophilicity of each tracer was determined by the n-octanol/buffer method. In vitro studies (IC₅₀, internalization) were performed on LNCaP cells. Biodistribution studies were conducted on LNCaP tumor-bearing male CB-17 SCID mice. Results: On the laboratory scale (starting activities, 0.2-9.0 GBq), labeling of ¹⁸F-rhPSMA-5 to -10 by IE was completed in < 20 min (radiochemical yields, 58% ± 9%; radiochemical purity, >97%) with molar activities of 12-60 GBq/μmol. All rhPSMAs showed low nanomolar affinity and high internalization by PSMA-expressing cells when compared with the reference radiopharmaceuticals, medium-to-low lipophilicity, and high human serum albumin binding. Biodistribution studies in LNCaP tumor-bearing mice revealed high tumor uptake, sufficiently fast clearance kinetics from blood, low hepatobiliary excretion, fast renal excretion, and very low uptake of ¹⁸F activity in bone. Conclusion: The novel ¹⁸F-rhPSMA radiopharmaceuticals developed under the radiohybrid concept show equal or better targeting characteristics than the established ¹⁸F-PSMA tracers ¹⁸F-DCFPyL and ¹⁸F-PSMA-1007. The unparalleled simplicity of production, the possibility to produce the identical ⁶⁸Ga-labeled ¹⁹F-⁶⁸Ga-rhPSMA tracers, and the possibility to extend this concept to true theranostic radiohybrid radiopharmaceuticals, such as F-Lu-rhPSMA, are unique features of these radiopharmaceuticals.

Keywords: 18F; PSMA; prostate cancer; radiohybrid.

FIGURE 1.

Radiohybrid concept exemplified on PSMA inhibitors: a molecular species that offers 2 binding sites for radionuclides, here a SiFA for ¹⁸F and a chelator for radiometallation. One of these binding sites is “labeled” with a radioisotope, the other one is silent, thus “labeled” with a nonradioactive isotope. These pair of compounds, either pure imaging pairs (A) or theranostic pairs (B) represent chemically identical species (monozygotic chemical twins) and thus exhibit identical in vivo characteristics (e.g., affinity, lipophilicity, pharmacokinetics). ⁶⁸Ga in A and ¹⁷⁷Lu in B are examples that can be substituted by other radiometals.

FIGURE 2.

Radiohybrid (rh) PSMA ligands comprising the KuE- or EuE-based PSMA inhibition motif, a SiFA moiety and a TRAP-, DOTA-, or DOTAGA-chelator. For comparative evaluations, the well-established PSMA-addressing ligands F-DCFPyL and F-PSMA-1007 were used (26,27). The reference radioligand for in vitro determinations was (¹²⁵I-I-BA)KuE (28).

FIGURE 3.

(A) Binding affinities (IC₅₀ in nM, 1 h, 4°C; n = 3) of ^natGa-¹⁹F-rhPSMA-5–10 (white bars), ¹⁹F-rhPSMA-5–10 with free chelator (gray bars), and ¹⁹F-DCFPyL and ¹⁹F-PSMA-1007 (references). (B) Internalized activity of ¹⁸F-DCFPyL, ¹⁸F-PSMA-1007, and ⁶⁸Ga-¹⁹F-rhPSMA-5–10 (white bars) and ¹⁸F-rhPSMA-5–10 with free chelator (gray bars), in LNCaP cells (1 h, 37°C) as percentage of the reference ligand (¹²⁵I-I-BA)KuE (n = 3). (C) Lipophilicity of ¹⁸F-DCFPyL, ¹⁸F-PSMA-1007, and ⁶⁸Ga-¹⁹F-rhPSMA-5–10 (white bars) and ¹⁸F-rhPSMA-5–10 with free chelator (gray bars), expressed as n-octanol/PBS (pH 7.4) partition-coefficient (log P_oct/PBS; n = 6). (D) HSA binding of ¹⁹F-DCFPyL, ¹⁹F-PSMA-1007, and ^natGa-¹⁹F-rhPSMA-5–10 (white bars), determined on a Chiralpak HSA column. Data of reference ligands ^18/19F-DCFPyL and ^18/19F-PSMA-1007 were taken from a previously published study (30). Values are expressed as mean ± SD.

FIGURE 4.

Biodistribution of ⁶⁸Ga-¹⁹F-rhPSMA-7 to -10 and the reference ligands ¹⁸F-DCFPyL and ¹⁸F-PSMA-1007 at 1 h after injection in LNCaP tumor–bearing SCID mice (n = 3 for ⁶⁸Ga-¹⁹F-rhPSMA-7 to -9, n = 4 for ⁶⁸Ga-¹⁹F-rhPSMA-10, ¹⁸F-DCFPyL, and ¹⁸F-PSMA-1007). Data for reference ligands were taken from a previously published study by our group (30). Values are expressed as a percentage injected dose per gram (%ID/g), mean ± SD.

FIGURE 5.

Comparative biodistribution of ⁶⁸Ga-¹⁹F-rhPSMA-7 (white bars) and ¹⁸F-rhPSMA-7 (gray bars) at 1 h after injection in LNCaP tumor–bearing SCID mice (n = 3). Data are expressed as percentage injected dose per gram (%ID/g) (mean ± SD).