Albumin-Binding PSMA Ligands: Optimization of the Tissue Distribution Profile

The prostate-specific membrane antigen (PSMA) has emerged as an attractive prostate cancer associated target for radiotheragnostic application using PSMA-specific radioligands. The aim of this study was to design new PSMA ligands modified with an albumin-binding moiety in order to optimize their tissue distribution profile. The compounds were prepared by conjugation of a urea-based PSMA-binding entity, a DOTA chelator, and 4-(<i>p</i>-iodophenyl)butyric acid using multistep solid phase synthesis. The three ligands (PSMA-ALB-02, PSMA-ALB-05, and PSMA-ALB-07) were designed with varying linker entities. Radiolabeling with ¹⁷⁷Lu was performed at a specific activity of up to 50 MBq/nmol resulting in radioligands of >98% radiochemical purity and high stability. In vitro investigations revealed high binding of all three PSMA radioligands to mouse (>64%) and human plasma proteins (>94%). Uptake and internalization into PSMA-positive PC-3 PIP tumor cells was equally high for all radioligands. Negligible accumulation was found in PSMA-negative PC-3 flu cells, indicating PSMA-specific binding of all radioligands. Biodistribution and imaging studies performed in PC-3 PIP/flu tumor-bearing mice showed enhanced blood circulation of the new radioligands when compared to the clinically employed ¹⁷⁷Lu-PSMA-617. The PC-3 PIP tumor uptake of all three radioligands was very high (76.4 ± 2.5% IA/g, 79.4 ± 11.1% IA/g, and 84.6 ± 14.2% IA/g, respectively) at 24 h post injection (p.i.) resulting in tumor-to-blood ratios of ∼176, ∼48, and ∼107, respectively, whereas uptake into PC-3 flu tumors was negligible. Kidney uptake at 24 h p.i. was lowest for ¹⁷⁷Lu-PSMA-ALB-02 (10.7 ± 0.92% IA/g), while ¹⁷⁷Lu-PSMA-ALB-05 and ¹⁷⁷Lu-PSMA-ALB-07 showed higher renal retention (23.9 ± 4.02% IA/g and 51.9 ± 6.34% IA/g, respectively). Tumor-to-background ratios calculated from values of the area under the curve (AUC) of time-dependent biodistribution data were in favor of ¹⁷⁷Lu-PSMA-ALB-02 (tumor-to-blood, 46; tumor-to-kidney, 5.9) when compared to ¹⁷⁷Lu-PSMA-ALB-05 (17 and 3.7, respectively) and ¹⁷⁷Lu-PSMA-ALB-07 (39 and 2.1, respectively). The high accumulation of the radioligands in PC-3 PIP tumors was visualized on SPECT/CT images demonstrating increasing tumor-to-kidney ratios over time. Taking all of the characteristics into account, ¹⁷⁷Lu-PSMA-ALB-02 emerged as the most promising candidate. The applied concept may be attractive for future clinical translation potentially enabling more potent and convenient prostate cancer radionuclide therapy

Preclinical Development of Novel PSMA-Targeting Radioligands: Modulation of Albumin-Binding Properties To Improve Prostate Cancer Therapy

The treatment of metastatic castration-resistant prostate cancer (mCRPC) remains challenging with current treatment options. The development of more effective therapies is, therefore, urgently needed. Targeted radionuclide therapy with prostate-specific membrane antigen (PSMA)-targeting ligands has revealed promising clinical results. In an effort to optimize this concept, it was the aim of this study to design and investigate PSMA ligands comprising different types of albumin binders. PSMA-ALB-53 and PSMA-ALB-56 were designed by combining the glutamate-urea-based PSMA-binding entity, a DOTA chelator and an albumin binder based on the 4-(<i>p</i>-iodophenyl)-moiety or <i>p</i>-(tolyl)-moiety. The compounds were labeled with ¹⁷⁷Lu (50 MBq/nmol) resulting in radioligands of high radiochemical purity (≥98%). Both radioligands were stable (≥98%) over 24 h in the presence of l-ascorbic acid. The uptake into PSMA-positive PC-3 PIP tumor cells in vitro was in the same range (54–58%) for both radioligands; however, ¹⁷⁷Lu-PSMA-ALB-53 showed a 15-fold enhanced binding to human plasma proteins. Biodistribution studies performed in PC-3 PIP/flu tumor-bearing mice revealed high tumor uptake of ¹⁷⁷Lu-PSMA-ALB-53 and ¹⁷⁷Lu-PSMA-ALB-56, respectively, demonstrated by equal areas under the curves (AUCs) for both radioligands. The increased retention of ¹⁷⁷Lu-PSMA-ALB-53 in the blood resulted in almost 5-fold lower tumor-to-blood AUC ratios when compared to ¹⁷⁷Lu-PSMA-ALB-56. Kidney clearance of ¹⁷⁷Lu-PSMA-ALB-56 was faster, and hence, the tumor-to-kidney AUC ratio was 3-fold higher than in the case of ¹⁷⁷Lu-PSMA-ALB-53. Due to the more favorable tissue distribution profile, ¹⁷⁷Lu-PSMA-ALB-56 was selected for a preclinical therapy study in PC-3 PIP tumor-bearing mice. The tumor growth delay after application of ¹⁷⁷Lu-PSMA-ALB-56 and ¹⁷⁷Lu-PSMA-617 applied at the same activities (2 or 5 MBq per mouse) revealed better antitumor effects in the case of ¹⁷⁷Lu-PSMA-ALB-56. As a consequence, the survival of mice treated with ¹⁷⁷Lu-PSMA-ALB-56 was prolonged when compared to the mice, which received the same activity of ¹⁷⁷Lu-PSMA-617. Our results demonstrated the superiority of ¹⁷⁷Lu-PSMA-ALB-56 over ¹⁷⁷Lu-PSMA-ALB-53 indicating that the <i>p</i>-(tolyl)-moiety was more suited as an albumin binder to optimize the tissue distribution profile. ¹⁷⁷Lu-PSMA-ALB-56 was more effective to treat tumors than ¹⁷⁷Lu-PSMA-617 resulting in complete tumor remission in four out of six mice. This promising results warrant further investigations to assess the potential for clinical application of ¹⁷⁷Lu-PSMA-ALB-56

In Vitro and in Vivo Evaluation of an Innocuous Drug Cocktail To Improve the Quality of Folic Acid Targeted Nuclear Imaging in Preclinical Research

Folate receptor (FR) targeting is an attractive strategy for nuclear imaging of cancer and activated macrophages through application of folic acid radioconjugates. However, significant renal accumulation of folate radioconjugates and hence exceedingly high emission of radiation from the kidneys may mask uptake of radioactivity at sites of interest such as small metastases in the abdominal region of animal models of ovarian cancer. Recently it was observed that the antifolate pemetrexed (PMX) reduces undesired renal uptake of radiofolates. A disadvantage of this strategy is the fact that pemetrexed is a chemotherapeutic agent which may have toxic side effects. The aims of this study were therefore to investigate whether the desired effect of PMX to reduce renal accumulation of folate radioconjugates would be maintained if it was applied as a cocktail together with its antidote, thymidine, and to explore whether thymidine was an effective rescue agent against PMX’s related toxicity in vitro and in vivo. In vitro internalization of ⁶⁷Ga-EC0800 was investigated using FR-positive KB tumor cells and embryonic monkey MA104 kidney cells in the absence and presence of PMX alone and in combination with thymidine. Uptake of ⁶⁷Ga-EC0800 into KB cells was increased by coincubation of the cells with PMX. In contrast uptake of ⁶⁷Ga-EC0800 into MA104 cells was reduced under the same conditions. In both cell lines coincubation of thymidine did not change the results obtained with PMX. Biodistribution and SPECT/CT imaging studies of ⁶⁷Ga-EC0800 were performed with KB tumor bearing mice injected with PMX alone or with a cocktail of PMX and thymidine. The radiofolate’s kidney uptake reducing effect of PMX in mice was maintained also if PMX was employed together with its antidote thymidine. The tumor uptake of ⁶⁷Ga-EC0800 remained unchanged independent of the administration of PMX or a combination of PMX and thymidine. The effect of thymidine to abrogate PMX-induced cytotoxicity was demonstrated in vitro with an MTT assay using KB and MA104 cells. Cell viability was reduced to 50% (KB cells) and 70% (MA104 cells) of untreated controls if PMX (5 μM and 15 μM, respectively) was coincubated. Addition of thymidine (10 μM or 100 μM) compensated PMX’s toxic effects in a dose-dependent manner. The effect of thymidine was also investigated in non-tumor bearing mice treated with high-dosed PMX. Rescue of mice with side effects after the third and fourth cycles of PMX application (1 mg/mouse) was achieved by application of thymidine (20 mg/mouse) at five consecutive days starting the day of PMX injection. Application of PMX together with thymidine as a cocktail is effective to improve the tissue distribution of radiofolates while preventing pharmacological and potentially toxic side effects of the chemotherapeutic agent PMX. These findings open new perspectives for folate-based nuclear imaging in preclinical research potentially allowing longitudinal investigations and monitoring therapies in animal models of cancer and inflammatory diseases

Synthesis, Radiolabeling, and Characterization of Plasma Protein-Binding Ligands: Potential Tools for Modulation of the Pharmacokinetic Properties of (Radio)Pharmaceuticals

The development of (radio)­pharmaceuticals with favorable pharmacokinetic profiles is crucial for allowing the optimization of the imaging or therapeutic potential and the minimization of undesired side effects. The aim of this study was, therefore, to evaluate and compare three different plasma protein binders (PPB-01, PPB-02, and PPB-03) that are potentially useful in combination with (radio)­pharmaceuticals to enhance their half-life in the blood. The entities were functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator via a l-lysine and β-alanine linker moiety using solid-phase peptide chemistry and labeled with ¹⁷⁷Lu (<i>T</i>_1/2 = 6.65 days), a clinically established radiometal. The binding capacities of these radioligands and ¹⁷⁷Lu–DOTA were evaluated using human plasma and solutions of human serum albumin (HSA), human α₁-acid glycoprotein (α₁-AGP), and human transthyretin (hTTR) by applying an ultrafiltration assay. ¹⁷⁷Lu–DOTA–PPB-01 and ¹⁷⁷Lu–DOTA–PPB-02 bound to a high and moderate extent to human plasma proteins (>90% and ∼70%, respectively), whereas the binding to hTTR was considered negligible (<10%). ¹⁷⁷Lu–DOTA–PPB-03 showed almost complete binding to human plasma proteins (>90%) with a high fraction bound to hTTR (∼50%). Plasma protein binding of the ¹⁷⁷Lu–DOTA complex, which was used as a control, was not observed (<1%). ¹⁷⁷Lu–DOTA–PPB-01 and ¹⁷⁷Lu–DOTA–PPB-02 were both displaced (>80%) from HSA by ibuprofen, specific for Sudlow’s binding site II and coherent with the aromatic structures, and >80% by their respective binding entities. ¹⁷⁷Lu–DOTA–PPB-03 was displaced from hTTR by the site-marker l-thyroxine (>60%) and by its binding entity PPB-03* (>80%). All three radioligands were investigated with regard to the in vivo blood clearance in normal mice. ¹⁷⁷Lu–DOTA–PPB-01 showed the slowest blood clearance (<i>T</i>_1/2,β: >15 h) followed by ¹⁷⁷Lu–DOTA–PPB-03 (<i>T</i>_1/2,β: ∼2.33 h) and ¹⁷⁷Lu–DOTA–PPB-02 (<i>T</i>_1/2,β: ∼1.14 h), which was excreted relatively fast. Our results confirmed the high affinity of the 4-(4-iodophenyl)-butyric acid entity (PPB-01) to plasma proteins, while replacement of the halogen by an ethynyl entity (PPB-02) reduced the plasma protein binding significantly. An attractive approach is the application of the transthyretin binder (PPB-03), which shows high affinity to hTTR. Future studies in our laboratory will be focused on the application of these binding entities in combination with clinically relevant targeting agents for diagnostic and therapeutic purposes in nuclear medicine

Additional file 1: of Preclinical imaging of the co-stimulatory molecules CD80 and CD86 with indium-111-labeled belatacept in atherosclerosis

Supplementary information. This file contains supplementary Tables 1–4. and Figures 1–4. and supplementary material and methods. (PDF 1198 kb

Evaluation of ^¹¹¹In-Labelled Exendin-4 Derivatives Containing Different Meprin β-Specific Cleavable Linkers

<div><p>Background</p><p>Cleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.</p><p>Results</p><p>The insertion of a cleavable linker did not negatively influence the <i>in vitro</i> properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. <i>In vitro</i> digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the ¹¹¹In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound ¹¹¹In-Ex4NOD40.</p><p>Conclusion</p><p>In conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.</p></div

<i>In vitro</i> assessment of the peptides containing cleavable linkers in CHL cells expressing GLP-1R.

<p>(A) IC₅₀ values of ^natIn labelled peptides. 0.1–0.6 pmol ¹¹¹In-Ex4NOD40 was used as tracer. (B) Internalisation kinetics of the ¹¹¹In-labelled probes. 0.2–1 pmol of the respective ¹¹¹In-labelled peptide was used as tracer. Non-specific binding was determined by incubation with 1 μM of ^natIn labelled peptide.</p

Cleavage sites of In-labelled peptides digested with meprin α and β.

<p>Cleavage sites for meprin α are illustrated with ↑, for meprin β with ↓. The linkers are highlighted in bold. In-labelled Ex4NOD40 was used as control.</p><p>Cleavage sites of In-labelled peptides digested with meprin α and β.</p

Immunofluorescence picture of murine kidney sections.

<p>(A) Anti-Meprin β antibody was used to detect the protease, visualised by fluorescence detection at 488 nm. (B) For the control goat igG was used.</p

The most common amino acids around the cleavage sites of both meprin α and β.

<p>The size of the one letter code of the amino acid represents the frequency of that amino acid in that particular position. The figure was generated using icelogo, based on peptide cleavage assays described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123443#pone.0123443.ref013" target="_blank">13</a>]. Peptide sequences were aligned on the scissile bond between P1 and P1′ indicated by a black line. Statistically significant amino acid residue occurrences present (P<0.05) were plotted. Those amino acids that were completely absent are shown below in pink.</p