Lu-Ibu-DAB-PSMA, a radioligand modified with ibuprofen as the albumin binder, showed higher accumulation in PSMA-positive tumors of mice than the clinically used [ 177 Lu]Lu-PSMA-617 but lower retention in non-targeted tissues than previously developed albumin-binding PSMA radioligands. The aim of this study was to investigate whether the stereochemistry of the incorporated ibuprofen affects the radioligand's in vitro and in vivo properties and to select the more favorable radioligand for further development. For this purpose, SibuDAB and RibuDAB containing (S)-and (R)-ibuprofen, respectively, were synthesized and labeled with lutetium-177. In vitro, the two isomers had similar properties; however, [ 177 Lu]Lu-SibuDAB showed increased binding to mouse and human plasma proteins (91 ± 1 and 88 ± 2%, respectively) compared to [ 177 Lu]Lu-RibuDAB (75 ± 2 and 79 ± 2%, respectively). In vivo, [ 177 Lu]Lu-SibuDAB was metabolically more stable than [ 177 Lu]Lu-RibuDAB with ∼90 vs ∼67% intact radioligand detected in the blood at 4 h post injection (p.i.). In line with the lower albumin-binding affinity, the blood clearance of [ 177 Lu]Lu-RibuDAB in mice was considerably faster [27% of injected activity (% IA), 1 h p.i.] than for [ 177 Lu]Lu-SibuDAB (50% IA, 1 h p.i.). Time-dependent biodistribution studies performed in tumor-bearing athymic nude mice showed high PSMA-specific tumor uptake for both isomers. A twofold increased area under the curve (AUC 0→8d ) of the blood retention was determined for [ 177 Lu]Lu-SibuDAB as compared to [ 177 Lu]Lu-RibuDAB, whereas the kidney AUC 0→8d value of [ 177 Lu]Lu-SibuDAB was only half as high as for [ 177 Lu]Lu-RibuDAB. As a result, a more favorable tumor-to-kidney AUC 0→8d ratio was obtained for [ 177 Lu]Lu-SibuDAB, which was also visualized on SPECT/CT images. Based on its improved kidney clearance and higher metabolic stability, [ 177 Lu]Lu-SibuDAB was selected as the more favorable radioligand. Therapy studies performed with [ 177 Lu]Lu-SibuDAB (5 MBq/mouse) demonstrated the anticipated therapeutic superiority over the current gold-standard [ 177 Lu]Lu-PSMA-617 (5 MBq/mouse). The significantly increased survival time of mice treated with [ 177 Lu]Lu-SibuDAB as compared to those injected with [ 177 Lu]Lu-PSMA-617 justifies further development of this novel radioligand toward clinical application.

The folate receptor (FR) is an interesting target for radiotheranostics due to its overexpression in several tumor types. The progress in developing novel folate radioconjugates is, however, slow due to the synthetic challenges that folate chemistry presents. The goal of this study was, thus, to establish versatile solid-phase synthetic strategies for a convenient preparation of novel folate conjugates. Two approaches were established based on an orthogonal fluorenylmethyloxycarbonyl (Fmoc)-protection strategy to enable a modular buildup of an albumin-binding DOTA conjugate (known as OxFol-1) using folic acid (oxidized folate version) as a targeting agent. The main difference between the two approaches was the sequence of conjugating the single structural units. The approach that introduced the folate entity as the last unit appeared particularly useful for the preparation of conjugates based on 6R- or 6S-5-methyltetrahydrofolic acid (5-MTHF; a reduced folate version) as targeting entity. Three types of folate conjugates were synthesized either with a p-iodophenyl-based albumin binder (OxFol-1, 6R-RedFol-1, and 6S-RedFol-1) or without an albumin-binding entity (OxFol-14, 6R-RedFol-14, and 6S-RedFol-14). All six conjugates were obtained with high chemical purity (>98%) after 9–13 synthesis steps and a single final HPLC purification. Radiolabeling with lutetium-177 was feasible at high molar activity, and the resulting radioconjugates were stable over at least 24 h. Biodistribution and SPECT/CT imaging studies confirmed the favorable effect of an albumin-binding entity to increase the tumor uptake and reduce kidney retention of folate radioconjugates. The increased tumor-to-kidney ratios obtained with [177Lu]­Lu-6R-RedFol-1 and [177Lu]­Lu-6S-RedFol-1 as compared to [177Lu]­Lu-OxFol-1 indicated that 5-MTHF is the preferred FR-targeting agent for albumin-binding radioconjugates. This was, however, not the case for folate radioconjugates without an albumin binder. Thanks to the established synthesis strategy, the preparation of further folate radioconjugates will be facilitated, potentially enabling the optimization of the tissue distribution characteristics even more.