Rationale: Small 225Ac-labeled prostate-specific membrane
antigen (PSMA)-targeted radioconjugates have been described for targeted alpha
therapy of metastatic
castration-resistant
prostate cancer. Transient binding to
serum albumin as a highly abundant, inherent
transport protein represents a commonly applied strategy to modulate the tissue distribution profile of such low-molecular-weight radiotherapeutics and to enhance radioactivity uptake into
tumor lesions with the ultimate objective of improved therapeutic outcome. Methods: Two
ligands mcp-M-alb-PSMA and mcp-D-alb-PSMA were synthesized by combining a macropa-derived
chelator with either one or two
lysine-ureido-
glutamate-based PSMA- and 4-(p-iodophenyl)butyrate
albumin-binding entities using multistep
peptide-coupling chemistry. Both compounds were labeled with [225Ac]Ac3+ under mild conditions and their reversible binding to
serum albumin was analyzed by an ultrafiltration assay as well as microscale thermophoresis measurements. Saturation binding studies and clonogenic survival assays using PSMA-expressing LNCaP cells were performed to evaluate PSMA-mediated cell binding and to assess the cytotoxic potency of the novel radioconjugates [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Biodistributions of both 225Ac-radioconjugates were investigated using LNCaP
tumor-bearing SCID mice. Histological examinations of selected organs were performed to analyze the occurrence of
necrosis using H&E staining, DNA damage via γH2AX staining and proliferation via Ki67 expression in the tissue samples. Results: Enhanced binding to serum components in general and to
human serum albumin in particular was revealed for [225Ac]Ac-mcp-M-alb-PSMA and [225Ac]Ac-mcp-D-alb-PSMA, respectively. Moreover, the novel derivatives are highly potent PSMA
ligands as their KD values in the nanomolar range (23.38 and 11.56 nM) are comparable to the reference radioconjugates [225Ac]Ac-mcp-M-PSMA (30.83 nM) and [225Ac]Ac-mcp-D-PSMA (10.20 nM) without
albumin binders. The clonogenic activity of LNCaP cells
after treatment with the 225Ac-labeled
ligands was affected in a dose- and time-dependent manner, whereas the bivalent radioconjugate [225Ac]Ac-mcp-D-alb-PSMA has a stronger impact on the clonogenic cell survival than its monovalent counterpart [225Ac]Ac-mcp-M-alb-PSMA. Biodistribution studies performed in LNCaP
tumor xenografts showed prolonged blood circulation times for both
albumin-binding radioconjugates and a substantially increased
tumor uptake (46.04 ± 7.77 %ID/g for [225Ac]Ac-mcp-M-alb-PSMA at 128 h p.i. and 153.48 ± 37.76 %ID/g at 168 h p.i. for [225Ac]Ac-mcp-D-alb-PSMA) with favorable
tumor-to-background ratios. Consequently, a clear histological indication of DNA damage was discovered in the
tumor tissues, whereas
DNA double-strand break formation in kidney and liver sections was less pronounced. Conclusion: The modification of the PSMA-based 225Ac-radioconjugates with one or two
albumin-binding entities resulted in an improved radiopharmacological behavior including a greatly enhanced
tumor accumulation combined with a rather low uptake in most non-targeted organs combined with a high excretion via the kidneys.