Summary

Introducing an albumin-binding entity into otherwise short-lived radiopharmaceuticals can be an effective means to improve their pharmacokinetic properties due to enhanced blood residence time. In the current study, DOTA-derivatized albumin binders based on 4-(p-iodophenyl)butanoate (DOTA-ALB-1 and DOTA-ALB-3) and 5-(p-iodophenyl)pentanoate entities (DOTA-ALB-24 and DOTA-ALB-25) without and with a hydrophobic 4-(aminomethyl)benzoic acid (AMBA) linker unit, respectively, were synthesized and labeled with lutetium-177 for in vitro and in vivo comparison. Overall, [¹⁷⁷Lu]Lu-DOTA-ALB-1 demonstrated ~3-fold stronger in vitro albumin-binding affinity and a longer blood residence time (T_50%IA ~8 h) than [¹⁷⁷Lu]Lu-DOTA-ALB-24 (T_50%IA ~0.8 h). Introducing an AMBA linker enhanced the albumin-binding affinity, resulting in a T_50%IA of ~24 h for [¹⁷⁷Lu]Lu-DOTA-ALB-3 and ~2 h for [¹⁷⁷Lu]Lu-DOTA-ALB-25. The same albumin binders without or with the AMBA linker were incorporated into 6R- and 6S-5-methyltetrahydrofolate-based DOTA-conjugates (¹⁷⁷Lu-RedFols). Biodistribution studies in mice performed with both diastereoisomers of [¹⁷⁷Lu]Lu-RedFol-1 and [¹⁷⁷Lu]Lu-RedFol-3, which comprised the 4-(p-iodophenyl)butanoate moiety, demonstrated a slower accumulation in KB tumors than those of [¹⁷⁷Lu]Lu-RedFol-24 and [¹⁷⁷Lu]Lu-RedFol-25 with the 5-(p-iodophenyl)pentanoate entity. In all cases, the tumor uptake was high (30–45% IA/g) 24 h after injection. Both diastereoisomers of [¹⁷⁷Lu]Lu-RedFol-1 and [¹⁷⁷Lu]Lu-RedFol-3 demonstrated high blood retention (3.8–8.7% IA/g, 24 h p.i.) and a 2- to 4-fold lower kidney uptake than the corresponding diastereoisomers of [¹⁷⁷Lu]Lu-RedFol-24 and [¹⁷⁷Lu]Lu-RedFol-25, which were more rapidly cleared from the blood (<0.2% IA/g, 24 h after injection). Kidney retention of the 6S-diastereoisomers of all ¹⁷⁷Lu-RedFols was consistently higher than that of the respective 6R-diastereoisomers, irrespective of the albumin binder and linker unit used. It was demonstrated that the blood clearance data obtained with ¹⁷⁷Lu-DOTA-ALBs had predictive value for the blood retention times of the respective folate radioconjugates. The use of these albumin-binding entities without or with an AMBA linker may serve for fine-tuning the blood retention of folate radioconjugates and also other radiopharmaceuticals and, hence, optimize their tissue distribution profiles. Dosimetry estimations based on patient data obtained with one of the most promising folate radioconjugates will be crucial to identify the dose-limiting organ, which will allow for selecting the most suitable folate radioconjugate for therapeutic purposes.

Results from nanoScan® SPECT/CT

• SPECT/CT images confirmed that mice injected with diastereoisomers of [¹⁷⁷Lu]Lu-RedFol-1 and [¹⁷⁷Lu]Lu-RedFol-3 showed more activity in the heart and blood circulation than mice injected with the respective diastereoisomers of [¹⁷⁷Lu]Lu-RedFol-24 and [¹⁷⁷Lu]Lu-RedFol-25 (Figure 5 and 6).

Figure 5. SPECT/CT images of KB tumor-bearing mice obtained 1 h, 4 h, 24 h and 48 h after injection with ¹⁷⁷Lu-labeled 6R-5-MTHF-based folate radioconjugates (25 MBq; 0.5 nmol per mouse) shown as maximum intensity projections (MIPs) for (A) [¹⁷⁷Lu]Lu-6R-RedFol-1; (B) [¹⁷⁷Lu]Lu-6R-RedFol-3; (C) [¹⁷⁷Lu]Lu-6R-RedFol-24 and (D) [¹⁷⁷Lu]Lu-6R-RedFol-25. Tu = KB tumor; Ki = kidney; H = heart.

Figure 6. SPECT/CT images of KB tumor-bearing mice obtained 1 h, 4 h, 24 h and 48 h after injection with ¹⁷⁷Lu-labeled 6S-5-MTHF-based folate radioconjugates (25 MBq; 0.5 nmol per mouse) shown as maximum intensity projections (MIPs) for (A) [¹⁷⁷Lu]Lu-6S-RedFol-1; (B) [¹⁷⁷Lu]Lu-6S-RedFol-3; (C) [¹⁷⁷Lu]Lu-6S-RedFol-24 and (D) [¹⁷⁷Lu]Lu-6S-RedFol-25. Tu = KB tumor; Ki = kidney; H = heart.

• In line with quantitative biodistribution data, the SPECT images acquired 24 h post injection visualized activity uptake in KB tumor xenografts, which was high irrespective of the applied folate radioconjugate. The renal uptake varied substantially among the folate radioconjugates and diastereoisomers. The 6S-diastereoisomers of all pairs of ¹⁷⁷Lu-RedFols commonly showed more renal uptake than the 6R-diastereoisomers.

Results showed that the 5-(p-iodophenyl)pentanoate entity had considerably lower albumin-binding affinity than the 4-(p-iodophenyl)butanoate entity which was confirmed not only by the shift of the albumin-binding curves toward a lower affinity but also by the faster blood clearance of [¹⁷⁷Lu]Lu-DOTA-ALB-24 relative to [¹⁷⁷Lu]Lu-DOTA-ALB-1. Irrespective of whether 4-(p-iodophenyl)butanoate or 5-(p-iodophenyl)pentanoate was used, the presence of an AMBA linker increased the in vitro albumin-binding affinity of the respective radioligand. As a result, [¹⁷⁷Lu]Lu-DOTA-ALB-3 and [¹⁷⁷Lu]Lu-DOTA-ALB-25 were more retained in the blood than [¹⁷⁷Lu]Lu-DOTA-ALB-1 and [¹⁷⁷Lu]Lu-DOTA-ALB-24, respectively.

The authors are planning to perform the “first-in-human” application with [¹⁷⁷Lu]Lu-6R-RedFol-1 and [¹⁷⁷Lu]Lu-6S-RedFol-1, which are currently the best investigated folate radioconjugates among those presented in this article. The resultant data will enable investigating whether the kidney retention or blood circulation time would be more critical in view of a therapeutic application. In turn, this would allow for making a decision on whether the ¹⁷⁷Lu-RedFol-1 or ¹⁷⁷Lu-RedFol-3 would be more reasonable options to keep the kidney dose low or whether the bone marrow dose would be more of a concern, making ¹⁷⁷Lu-RedFol-24 or ¹⁷⁷Lu-RedFol-25 the more suitable choices.

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