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

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.


TCP-resin loading (GP1)
Loading of the tritylchloride polystyrene (TCP) resin with a Fmoc-protected amino acid (AA) was carried out by stirring a solution of the TCP-resin (1.60 mmol/g) and Fmoc-AA-OH (1.5 eq.) in anhydrous DCM with DIPEA (3.8 eq.) at room temperature for 2 h. Remaining tritylchloride was capped by the addition of methanol (2 mL/g resin) for 15 min. Subsequently the resin was filtered and washed with DCM (2 × 5 mL/g resin), DMF (2 × 5 mL/g resin), methanol (5 mL/g resin) and dried in vacuo. Final loading l of Fmoc-AA-OH was determined by the following equation:

On-resin Amide Bond Formation (GP2)
For conjugation of a building block to the resin-bound peptide, a mixture of TBTU with HOBt or HOAt is used for pre-activation of the carboxylic with DIPEA as a base in DMF (10 mL/g resin). After 5 min at rt, the solution is added to the swollen resin. The exact stoichiometry and reaction time for each conjugation step is given in the respective synthesis protocols. After reaction, the resin was washed with DMF (6 × 5 mL/g resin).

tBu/Boc deprotection (GP6)
Removal of tBu/Boc-protecting groups was carried out by dissolving the crude product in a mixture of TFA/TIPS/water (v/v/v; 95/2.5/2.5) and stirring for 1-6 h at rt. Product formation was monitored by HPLCanalysis. After removing TFA under a stream of nitrogen, the residue was dissolved in a mixture of tertbutanol and water. After lyophilisation the crude peptide was obtained.

Conjugation of PfpO-Sub-(tBuO)KuE(OtBu) 2 to the peptide (GP8)
The N-terminal deprotected peptide (1.0 eq.) was added to a solution of 3 (1.2 eq.) in DMF (approx. 0.1 mL/ mg peptide) and TEA (8 eq.) was added. After stirring the solution for 2 h at rt, DMF was removed in vacuo. For cleavage of the tBu-esters, TFA was added and the solution was stirred for 45 min at rt. After removing TFA under a stream of nitrogen, the crude product was purified by RP-HPLC.

Internalization studies
Internalization studies were carried out according to a previously published procedure (1). Briefly, LNCaP cells were harvested 24 ± 2 hours before the experiment and seeded in poly-L-lysine coated 24-well plates