Design and Preclinical Evaluation of a Novel Prostate-Specific Membrane Antigen Radioligand Modified with a Transthyretin Binder.

Transthyretin binders have previously been used to improve the pharmacokinetic properties of small-molecule drug conjugates and could, thus, be utilized for radiopharmaceuticals as an alternative to the widely explored "albumin binder concept". In this study, a novel PSMA ligand modified with a transthyretin-binding entity (TB-01) was synthesized and labeled with lutetium-177 to obtain [177Lu]Lu-PSMA-TB-01. A high and specific uptake of [177Lu]Lu-PSMA-TB-01 was found in PSMA-positive PC-3 PIP cells (69 ± 3% after 4 h incubation), while uptake in PSMA-negative PC-3 flu cells was negligible (<1%). In vitro binding studies showed a 174-fold stronger affinity of [177Lu]Lu-PSMA-TB-01 to transthyretin than to human serum albumin. Biodistribution studies in PC-3 PIP/flu tumor-bearing mice confirmed the enhanced blood retention of [177Lu]Lu-PSMA-TB-01 (16 ± 1% IA/g at 1 h p.i.), which translated to a high tumor uptake (69 ± 13% IA/g at 4 h p.i.) with only slow wash-out over time (31 ± 8% IA/g at 96 h p.i.), while accumulation in the PC-3 flu tumor and non-targeted normal tissue was reasonably low. Further optimization of the radioligand design would be necessary to fine-tune the biodistribution and enable its use for therapeutic purposes. This study was the first of this kind and could motivate the use of the "transthyretin binder concept" for the development of future radiopharmaceuticals.

Investigations Using Albumin Binders to Modify the Tissue Distribution Profile of Radiopharmaceuticals Exemplified with Folate Radioconjugates.

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 (T50%IA ~8 h) than [177Lu]Lu-DOTA-ALB-24 (T50%IA ~0.8 h). Introducing an AMBA linker enhanced the albumin-binding affinity, resulting in a T50%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.

Preclinical Investigations to Explore the Difference between the Diastereomers [177Lu]Lu-SibuDAB and [177Lu]Lu-RibuDAB toward Prostate Cancer Therapy.

[177Lu]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 [177Lu]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, [177Lu]Lu-SibuDAB showed increased binding to mouse and human plasma proteins (91 ± 1 and 88 ± 2%, respectively) compared to [177Lu]Lu-RibuDAB (75 ± 2 and 79 ± 2%, respectively). In vivo, [177Lu]Lu-SibuDAB was metabolically more stable than [177Lu]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 [177Lu]Lu-RibuDAB in mice was considerably faster [27% of injected activity (% IA), 1 h p.i.] than for [177Lu]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 (AUC0→8d) of the blood retention was determined for [177Lu]Lu-SibuDAB as compared to [177Lu]Lu-RibuDAB, whereas the kidney AUC0→8d value of [177Lu]Lu-SibuDAB was only half as high as for [177Lu]Lu-RibuDAB. As a result, a more favorable tumor-to-kidney AUC0→8d ratio was obtained for [177Lu]Lu-SibuDAB, which was also visualized on SPECT/CT images. Based on its improved kidney clearance and higher metabolic stability, [177Lu]Lu-SibuDAB was selected as the more favorable radioligand. Therapy studies performed with [177Lu]Lu-SibuDAB (5 MBq/mouse) demonstrated the anticipated therapeutic superiority over the current gold-standard [177Lu]Lu-PSMA-617 (5 MBq/mouse). The significantly increased survival time of mice treated with [177Lu]Lu-SibuDAB as compared to those injected with [177Lu]Lu-PSMA-617 justifies further development of this novel radioligand toward clinical application.

Design and Evaluation of Novel Albumin-Binding Folate Radioconjugates: Systematic Approach of Varying the Linker Entities.

Tumor targeting using folate radioconjugates is a promising strategy for theragnostics of folate receptor-positive tumors. The aim of this study was to investigate the impact of structural modifications of folate radioconjugates on their pharmacokinetic properties. Four novel folate radioconjugates ([177Lu]Lu-OxFol-2, [177Lu]Lu-OxFol-3, [177Lu]Lu-OxFol-4, and [177Lu]Lu-OxFol-5), modified with a lipophilic or hydrophilic linker entity in close proximity to the albumin-binding 4-(p-iodophenyl)butanoate entity or the DOTA chelator, respectively, were designed and evaluated for comparison with the previously developed [177Lu]Lu-OxFol-1. A hydrophobic 4-(aminomethyl)benzoic acid linker, incorporated in close proximity to the 4-(p-iodophenyl)butanoate entity, enhanced the albumin-binding properties (relative affinity 7.3) of [177Lu]Lu-OxFol-3 as compared to those of [177Lu]Lu-OxFol-1 (relative affinity set as 1.0). On the other hand, a hydrophilic d-glutamic acid (d-Glu) linker entity used in [177Lu]Lu-OxFol-2 compromised the albumin-binding properties. [177Lu]Lu-OxFol-4 and [177Lu]Lu-OxFol-5, in which the respective linker entities were incorporated adjacent to the DOTA chelator, showed similar albumin-binding properties (0.6 and 1.0, respectively) as [177Lu]Lu-OxFol-1. Biodistribution studies in KB tumor-bearing nude mice revealed twofold higher tumor-to-kidney ratios at 4 h and 24 h after injection of [177Lu]Lu-OxFol-3 (∼1.2) than after injection of [177Lu]Lu-OxFol-1 (∼0.6). The tumor-to-kidney ratios of [177Lu]Lu-OxFol-2 were, however, much lower (∼0.2) due to the high kidney retention of this radioconjugate. The tumor-to-kidney ratios of [177Lu]Lu-OxFol-5 were only slightly increased (∼0.9), and the ratios for [177Lu]Lu-OxFol-4 (∼0.7) were in the same range as for [177Lu]Lu-OxFol-1. SPECT/CT imaging studies demonstrated similar tumor uptake of all radioconjugates but a clearly improved tumor-to-kidney ratio for [177Lu]Lu-OxFol-3 as compared to that for [177Lu]Lu-OxFol-1. Based on these data, it can be concluded that the linker entity in close proximity to the 4-(p-iodophenyl)butanoate entity affects the radioconjugate's pharmacokinetic profile considerably due to the altered affinity to albumin. Changes in the linker entity, which connects the DOTA chelator with the folate molecule, do not have a major impact on the radioconjugate's tissue distribution profile, however. As a result of these findings, [177Lu]Lu-OxFol-3 had a comparable therapeutic effect to that of [177Lu]Lu-OxFol-1 but appeared advantageous in preventing kidney damage. Provided that the kidneys will present the dose-limiting organs in patients, [177Lu]Lu-OxFol-3 would be the preferred candidate for a clinical translation.

Novel Synthetic Strategies Enable the Efficient Development of Folate Conjugates for Cancer Radiotheranostics.

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.

Identification of a PET Radiotracer for Imaging of the Folate Receptor-α: A Potential Tool to Select Patients for Targeted Tumor Therapy.

The aim of this study was to identify a folate receptor-α (FRα)-selective PET agent potentially suitable for the selection of patients who might profit from FRα-targeted therapies. The 6R and 6S isomers of 18F-aza-5-methyltetrahydrofolate (MTHF) were assessed regarding their binding to FRα and FRß, expressed on cancer and inflammatory cells, respectively, and compared with 18F-AzaFol, the folic acid-based analog. Methods: FR selectivity was investigated using FRα-transfected (RT16) and FRß-transfected (D4) CHO cells. The cell uptake of 18F-folate tracers was investigated, and receptor-binding affinities were determined with the nonradioactive analogs. In vitro autoradiography of the 18F-folate tracers was performed using RT16 and D4 tissue sections. Biodistribution studies and PET/CT imaging of the radiotracers were performed on mice bearing RT16 and D4 xenografts. Results: The uptake of 18F-6R-aza-5-MTHF was high when using RT16 cells (62% ± 10% of added activity) but much lower when using D4 cells (5% ± 2%). The FRα selectivity of 18F-6R-aza-5-MTHF was further demonstrated by its approximately 43-fold higher binding affinity to FRα (half-maximal inhibitory concentration [IC50], 1.8 ± 0.1 nM) than to FRß (IC50, 77 ± 27 nM). The uptake of 18F-6S-aza-5-MTHF and 18F-AzaFol was equal in both cell lines (52%-70%), with similar affinities to FRα (IC50, 2.1 ± 0.4 nM and 0.6 ± 0.3 nM, respectively) and FRß (0.8 ± 0.2 nM and 0.3 ± 0.1 nM, respectively). The autoradiography signal obtained with 18F-6R-aza-5-MTHF was 11-fold more intense for RT16 than for D4 tissue sections. Biodistribution data showed high uptake of 18F-6R-aza-5-MTHF in RT16 xenografts (81% ± 20% injected activity per gram [IA]/g 1 h after injection) but significantly lower accumulation in D4 xenografts (7.3% ± 2.1% IA/g 1 h after injection), which was also visualized using PET. The uptake of 18F-6S-aza-5-MTHF and 18F-AzaFol was similar in RT16 (53% ± 10% IA/g and 45% ± 2% IA/g, respectively) and D4 xenografts (77% ± 10% IA/g and 52% ± 7% IA/g, respectively). Conclusion: This study demonstrated FRα selectivity for 18F-6R-aza-5-MTHF but not for 18F-6S-aza-5-MTHF or 18F-AzaFol. This characteristic, together with its favorable tissue distribution, makes 18F-6R-aza-5-MTHF attractive for clinical translation to enable detection of FRα-positive cancer while preventing undesired accumulation in FRß-expressing inflammatory cells.

Preclinical evaluation of 5-methyltetrahydrofolate-based radioconjugates-new perspectives for folate receptor-targeted radionuclide therapy.

PURPOSE: The folate receptor (FR) is frequently overexpressed in a variety of tumor types and, hence, an interesting target for radionuclide therapy. The aim of this study was to evaluate a new class of albumin-binding radioconjugates comprising 5-methyltetrahydrofolate (5-MTHF) as a targeting agent and to compare their properties with those of the previously established folic acid-based [177Lu]Lu-OxFol-1. METHODS: [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 were investigated in vitro using FR-positive KB tumor cells. Biodistribution studies were performed in KB tumor-bearing mice, and the areas under the curve (AUC0 → 120h) were determined for the uptake in tumors and kidneys. [177Lu]Lu-6R-RedFol-1 was compared with [177Lu]Lu-OxFol-1 in a therapy study over 8 weeks using KB tumor-bearing mice. RESULTS: Both radioconjugates demonstrated similar in vitro properties as [177Lu]Lu-OxFol-1; however, the tumor uptake of [177Lu]Lu-6R-RedFol-1 and [177Lu]Lu-6S-RedFol-1 was significantly increased in comparison with [177Lu]Lu-OxFol-1. In the case of [177Lu]Lu-6S-RedFol-1, also the kidney uptake was increased; however, renal retention of [177Lu]Lu-6R-RedFol-1 was similar to that of [177Lu]Lu-OxFol-1. This led to an almost 4-fold increased tumor-to-kidney AUC0 → 120h ratio of [177Lu]Lu-6R-RedFol-1 as compared with [177Lu]Lu-6S-RedFol-1 and [177Lu]Lu-OxFol-1. At equal activity, the therapeutic effect of [177Lu]Lu-6R-RedFol-1 was better than that of [177Lu]Lu-OxFol-1, reflected by a slower tumor growth and, consequently, an increased median survival time (49 days vs. 34 days). CONCLUSION: This study demonstrated the promising potential of 5-MTHF-based radioconjugates for FR-targeting. Application of [177Lu]Lu-6R-RedFol-1 resulted in unprecedentedly high tumor-to-kidney ratios and, as a consequence, a superior therapeutic effect as compared with [177Lu]Lu-OxFol-1. These findings, together with the absence of early side effects, make [177Lu]Lu-6R-RedFol-1 attractive in view of a future clinical translation.

Albumin-Binding PSMA Radioligands: Impact of Minimal Structural Changes on the Tissue Distribution Profile.

The concept of using ibuprofen as an albumin-binding entity was recently demonstrated by the development of [177Lu]Lu-Ibu-PSMA-01. In the present study, we designed a novel ibuprofen-containing radioligand (Ibu-PSMA-02) with subtle structural changes regarding the linker entity in order to investigate a potential impact on the in vitro and in vivo properties. Ibu-PSMA-02 was prepared using solid-phase synthesis techniques and labeled with lutetium-177. [177Lu]Lu-Ibu-PSMA-02 was evaluated in vitro with regard to its plasma protein-binding properties, PSMA affinity and uptake into PSMA-expressing PC-3 PIP tumor cells. The tissue distribution profile of [177Lu]Lu-Ibu-PSMA-02 was assessed in tumor-bearing mice and dose estimations were performed. The in vitro characteristics of [177Lu]Lu-Ibu-PSMA-02 were similar to those previously obtained for [177Lu]Lu-Ibu-PSMA-01 with respect to plasma protein-binding, PSMA affinity and tumor cell uptake. The in vivo studies revealed, however, an unprecedentedly high uptake of [177Lu]Lu-Ibu-PSMA-02 in PC-3 PIP tumors, resulting in an increased absorbed tumor dose of 7.7 Gy/MBq as compared to 5.1 Gy/MBq calculated for [177Lu]Lu-Ibu-PSMA-01. As a consequence of the high tumor accumulation, [177Lu]Lu-Ibu-PSMA-02 showed higher tumor-to-background ratios than [177Lu]Lu-Ibu-PSMA-01. This study exemplified that smallest structural changes in the linker entity of PSMA radioligands may have a significant impact on their pharmacokinetic profiles and, thus, may be applied as a means for ligand design optimization.

Combined Application of Albumin-Binding [177Lu]Lu-PSMA-ALB-56 and Fast-Cleared PSMA Inhibitors: Optimization of the Pharmacokinetics.

The strategy of using radioligands for targeting the prostate-specific membrane antigen (PSMA) revealed to be promising for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Recently developed albumin-binding PSMA radioligands showed a remarkably increased tumor uptake because of the enhanced blood circulation, but higher accumulation of activity was also observed in off-target organs and tissues. The aim of this study was to investigate the option of using fast-cleared, small-molecular-weight PSMA inhibitors (PSMA-11, 2-PMPA, and ZJ-43) to reduce the kidney uptake of [177Lu]Lu-PSMA-ALB-56, a previously developed albumin-binding PSMA radioligand. Dual-isotope SPECT/CT imaging was performed with tumor-bearing mice coinjected with [177Lu]Lu-PSMA-ALB-56 and a 2.5-fold molar excess of [67Ga]Ga-PSMA-11. At early timepoints after injection, the high renal uptake of [67Ga]Ga-PSMA-11 reduced the accumulation of [177Lu]Lu-PSMA-ALB-56 in the kidneys substantially, whereas the tumor uptake of [177Lu]Lu-PSMA-ALB-56 was only slightly affected. These findings were confirmed in biodistribution studies, which revealed reduced uptake of [177Lu]Lu-PSMA-ALB-56 in the kidneys due to coadministered unlabeled PSMA-11 (9.1 ± 0.8% IA/g vs 46 ± 11% IA/g; 1 h p.i.). The tumor uptake of [177Lu]Lu-PSMA-ALB-56 was almost the same at 1 h p.i., irrespective of whether or not PSMA-11 was coinjected (24 ± 6% IA/g vs 27 ± 7% IA/g). The application of [177Lu]Lu-PSMA-ALB-56 with 2-PMPA or ZJ-43, respectively, showed similar results in biodistribution studies. Among all three tested PSMA inhibitors, 2-PMPA, applied at a 2.5-fold molar excess relative to [177Lu]Lu-PSMA-ALB-56, was most effective to improve the tumor-to-kidney ratios over the first hours after injection of [177Lu]Lu-PSMA-ALB-56. The concept of using a PSMA inhibitor together with [177Lu]Lu-PSMA-ALB-56 appears promising in view of a clinical translation of this and possibly other long-circulating PSMA radioligands.

Development of a new class of PSMA radioligands comprising ibuprofen as an albumin-binding entity.

Prostate-specific membrane antigen (PSMA)-targeted radioligands have been used for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Recently, albumin-binding PSMA radioligands with enhanced blood circulation were developed to increase the tumor accumulation of activity. The present study aimed at the design, synthesis and preclinical evaluation of a novel class of PSMA-targeting radioligands equipped with ibuprofen as a weak albumin-binding entity in order to improve the pharmacokinetic properties. Methods: Four novel glutamate-urea-based PSMA ligands were synthesized with ibuprofen, conjugated via variable amino acid-based linker entities. The albumin-binding properties of the 177Lu-labeled PSMA ligands were tested in vitro using mouse and human plasma. Affinity of the radioligands to PSMA and cellular uptake and internalization was investigated using PSMA-positive PC-3 PIP and PSMA-negative PC-3 flu tumor cells. The tissue distribution profile of the radioligands was assessed in biodistribution and imaging studies using PC-3 PIP/flu tumor-bearing nude mice. Results: The PSMA ligands were obtained in moderate yields at high purity (>99%). 177Lu-labeling of the ligands was achieved at up to 100 MBq/nmol with >96% radiochemical purity. In vitro assays confirmed high binding of all radioligands to mouse and human plasma proteins and specific uptake and internalization into PSMA-positive PC-3 PIP tumor cells. Biodistribution studies and SPECT/CT scans revealed high accumulation in PC-3 PIP tumors but negligible uptake in PC-3 flu tumor xenografts as well as rapid clearance of activity from background organs and tissues. 177Lu-Ibu-DAB-PSMA, in which ibuprofen was conjugated via a positively-charged diaminobutyric acid (DAB) entity, showed distinguished tumor uptake and the most favorable tumor-to-blood and tumor-to-kidney ratios. Conclusion: The high accumulation of activity in the tumor and fast clearance from background organs was a common favorable characteristic of PSMA radioligands modified with ibuprofen as albumin-binding entity. 177Lu-Ibu-DAB-PSMA emerged as the most promising candidate; hence, more detailed preclinical investigations with this radioligand are warranted in view of a clinical translation.

Concentration-dependent effects of dutasteride on prostate-specific membrane antigen (PSMA) expression and uptake of 177 Lu-PSMA-617 in LNCaP cells.

BACKGROUND: Prostate-specific membrane antigen (PSMA)-based imaging and therapy are increasingly used in the management of prostate cancer. However, low PSMA surface expression in certain patients is a limitation for PSMA-based technologies. We have previously shown that high doses of dutasteride, a 5α-reductase inhibitor generally used for the treatment of benign prostatic enlargement, increase the PSMA expression in vitro. We now further analyzed the concentration- and time-dependent effects of dutasteride in LNCaP cells. METHODS: Androgen receptor (AR) expressing prostate cancer cells (LNCaP) were treated for 7 to 14 days with vehicle control (0.1% dimethyl sulfoxide) or different concentrations of dutasteride (0.25 , 0.5 , 1 , and 5 µM). In addition to cell proliferation, PSMA surface expression was assessed using flow cytometry (FACS) and immunocytochemistry. Total PSMA and AR expression was analyzed by capillary western immunoassay (WES). In addition, tumor cell uptake and internalization assays of 177 Lu-PSMA-617 were performed. RESULTS: Dutasteride treatment resulted in a significant upregulation of PSMA surface expression compared to vehicle control after 7 days in all tested concentrations. After 14 days a further, concentration-dependent increase of PSMA surface expression was detectable. Total PSMA protein expression significantly increased after treatment of cells with high concentrations of dutasteride using 5 µM for 7 or 14 days. However, when lower concentrations were used total PSMA expression was not significantly altered compared to vehicle control. Further testing revealed a dose-dependent increase in uptake and internalization of 177Lu -PSMA-617 after 7 and 14 days. Though, a significantly increased uptake was only observed using a 5 µM dutasteride concentration for 7 days as well as 1 and 5 µM for 14 days. CONCLUSION: Our investigations revealed a concentration- and time-dependent effect of dutasteride on PSMA expression and uptake of 177Lu -PSMA-617 in LNCaP cells. A short-term treatment of patients with high doses of dutasteride might increase the detection rate of PSMA-based imaging and increase the effect of 177Lu -PSMA-617 therapy via upregulation of PSMA expression.