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.

In-vivo inhibition of neutral endopeptidase 1 results in higher absorbed tumor doses of [177Lu]Lu-PP-F11N in humans: the lumed phase 0b study.

BACKGROUND: A new generation of radiolabeled minigastrin analogs delivers low radiation doses to kidneys and are considered relatively stable due to less enzymatic degradation. Nevertheless, relatively low tumor radiation doses in patients indicate limited stability in humans. We aimed at evaluating the effect of sacubitril, an inhibitor of the neutral endopeptidase 1, on the stability and absorbed doses to tumors and organs by the cholecystokinin-2 receptor agonist [177Lu]Lu-PP-F11N in patients. In this prospective phase 0 study eight consecutive patients with advanced medullary thyroid carcinoma and a current somatostatin receptor subtype 2 PET/CT scan were included. Patients received two short infusions of ~ 1 GBq [177Lu]Lu-PP-F11N in an interval of ~ 4 weeks with and without Entresto® pretreatment in an open-label, randomized cross-over order. Entresto® was given at a single oral dose, containing 48.6 mg sacubitril. Adverse events were graded and quantitative SPECT/CT and blood sampling were performed. Absorbed doses to tumors and relevant organs were calculated. RESULTS: Pretreatment with Entresto® showed no additional toxicity and increased the stability of [177Lu]Lu-PP-FF11N in blood significantly (p < 0.001). Median tumor-absorbed doses were 2.6-fold higher after Entresto® pretreatment (0.74 vs. 0.28 Gy/GBq, P = 0.03). At the same time, an increase of absorbed doses to stomach, kidneys and bone marrow was observed, resulting in a tumor-to-organ absorbed dose ratio not significantly different with and without Entresto®. CONCLUSIONS: Premedication with Entresto® results in a relevant stabilization of [177Lu]Lu-PP-FF11N and consecutively increases radiation doses in tumors and organs. Trial registration clinicaltrails.gov, NCT03647657. Registered 20 August 2018.

Liquid Chromatography ICP-MS to Assess the Stability of 175Lu- and natGa-Based Tumor-Targeting Agents towards the Development of 177Lu- and 68Ga-Labeled Radiopharmaceuticals.

In recent years, nuclear medicine has gained great interest, partly due to the success story of [177Lu]Lu-PSMA-617 (PluvictoTM). Still, in-depth preclinical characterization of radiopharmaceuticals mainly happens at centers that allow working with radioactive material. To support the development of novel radiopharmaceuticals, alternative non-radioactive characterization assays are highly desirable. The aim of this study was to demonstrate that inductively coupled plasma mass spectrometry (ICP-MS) associated with a chromatographic system can serve as a surrogate for the classical high-performance liquid chromatography (HPLC)-radiodetector combination for preclinical in vitro characterization of non-radioactive metal-labeled analogs of radiopharmaceuticals. In this proof-of-concept study, we demonstrate the applicability of HPLC-ICP-MS by assessing the stability of 175Lu- and natGa-labeled prostate-specific membrane antigen (PSMA)-targeting peptidomimetics, single domain antibody (sdAb) conjugates, and monoclonal antibody (mAb) conjugates. 175Lu-labeled DOTAGA-conjugated and natGa-labeled NODAGA-conjugated sdAbs and mAbs showed the highest stability with >90% still intact after 24 h. The peptidomime-tics [175Lu]Lu-PSMA-617 and [natGa]Ga-PSMA-11 showed identical in vitro serum stability as it was reported for their corresponding radioligands with >99% intact species after 24 h incubation in mouse serum, demonstrating the reliability of the method. Hence, the established HPLC-ICP-MS methods can support the development of novel radiopharmaceuticals in a classical pharmaceutical setting.

Relaxed fibronectin: a potential novel target for imaging endometriotic lesions.

BACKGROUND: Endometriosis is characterized by the ectopic occurrence of endometrial tissue. Though considered benign, endometriotic lesions possess tumor-like properties such as tissue invasion and remodeling of the extracellular matrix. One major clinical hurdle concerning endometriosis is its diagnosis. The diagnostic modalities ultrasound and MRI are often unable to detect all lesions, and a clear correlation between imaging and clinical symptoms is still controversial. Therefore, it was our aim to identify a potential target to image active endometriotic lesions. RESULTS: For our studies, we employed the preclinical radiotracer [111In]In-FnBPA5, which specifically binds to relaxed fibronectin-an extracellular matrix protein with key functions in homeostasis that has been implicated in the pathogenesis of diseases such as cancer and fibrosis. We employed this tracer in biodistribution as well as SPECT/CT studies in mice and conducted immunohistochemical stainings on mouse uterine tissue as well as on patient-derived endometriosis tissue. In biodistribution and SPECT/CT studies using the radiotracer [111In]In-FnBPA5, we found that radiotracer uptake in the myometrium varies with the estrous cycle of the mouse, leading to higher uptake of [111In]In-FnBPA5 during estrogen-dependent phases, which indicates an increased abundance of relaxed fibronectin when estrogen levels are high. Finally, immunohistochemical analysis of patient samples demonstrated that there is preferential relaxation of fibronectin in the proximity of the endometriotic stroma. CONCLUSION: Estrous cycle stages characterized by high estrogen levels result in a higher abundance of relaxed fibronectin in the murine myometrium. This finding together with a first proof-of-concept study employing human endometriosis tissues suggests that relaxed fibronectin could be a potential target for the development of a diagnostic radiotracer targeting endometriotic lesions. With [111In]In-FnBPA5, the matching targeting molecule is in preclinical development.

Detecting radio- and chemoresistant cells in 3D cancer co-cultures using chromatin biomarkers.

The heterogenous treatment response of tumor cells limits the effectiveness of cancer therapy. While this heterogeneity has been linked to cell-to-cell variability within the complex tumor microenvironment, a quantitative biomarker that identifies and characterizes treatment-resistant cell populations is still missing. Herein, we use chromatin organization as a cost-efficient readout of the cells' states to identify subpopulations that exhibit distinct responses to radiotherapy. To this end, we developed a 3D co-culture model of cancer spheroids and patient-derived fibroblasts treated with radiotherapy. Using the model we identified treatment-resistant cells that bypassed DNA damage checkpoints and exhibited an aggressive growth phenotype. Importantly, these cells featured more condensed chromatin which primed them for treatment evasion, as inhibiting chromatin condensation and DNA damage repair mechanisms improved the efficacy of not only radio- but also chemotherapy. Collectively, our work shows the potential of using chromatin organization to cost-effectively study the heterogeneous treatment susceptibility of cells and guide therapeutic design.

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.

Reducing kidney uptake of radiolabelled exendin-4 using variants of the renally cleavable linker MVK.

BACKGROUND: Peptidic radiotracers are preferentially excreted through the kidneys, which often results in high persistent renal retention of radioactivity, limiting or even preventing therapeutic clinical translation of these radiotracers. Exendin-4, which targets the glucagon-like-peptide 1 receptor (GLP-1R) overexpressed in insulinomas and in congenital hyperinsulinism, is an example thereof. The use of the tripeptide MVK, which is readily cleaved between methionine and valine by neprilysin at the renal brush border membrane, already showed promising results in reducing kidney uptake as reported in the literature. Based on our previous findings we were interested how linker variants with multiple copies of the MV-motive influence renal washout of radiolabelled exendin-4. RESULTS: Three exendin-4 derivatives, carrying either one MVK, a MV-MVK or a MVK-MVK linker were synthesized and compared to a reference compound lacking a cleavable linker. In vivo results of a biodistribution in GLP-1R overexpressing tumour bearing mice at 24 h post-injection demonstrated a significant reduction (at least 57%) of renal retention of all 111In-labeled exendin-4 compounds equipped with a cleavable linker compared to the reference compound. While the insertion of the single linker MVK led to a reduction in kidney uptake of 70%, the dual approach with the linker MV-MVK slightly, but not significantly enhanced this effect, with 77% reduction in kidney uptake compared to the reference. In vitro IC50 and cell uptake studies were conducted and demonstrated that though the cleavable linkers negatively influenced the affinity towards the GLP-1R, cell uptake remained largely unaffected, except for the MV-MVK cleavable linker conjugate, which displayed lower cell uptake than the other compounds. Importantly, the tumour uptake in the biodistribution study was not significantly affected with 2.9, 2.5, 3.2 and 1.5% iA/g for radiolabelled Ex4, MVK-Ex4, MV-MVK-Ex4 and MVK-MVK-Ex4, respectively. CONCLUSION: Cleavable linkers are highly efficient in reducing the radioactivity burden in the kidney. Though the dual linker approach using the instillation of MV-MVK or MVK-MVK between exendin-4 and the radiometal chelator did not significantly outperform the single cleavable linker MVK, further structural optimization or the combination of different cleavable linkers could be a stepping stone in reducing radiation-induced nephrotoxicity.

Albumin-Binding and Conventional PSMA Ligands in Combination with 161Tb: Biodistribution, Dosimetry, and Preclinical Therapy.

The favorable decay characteristics of 161Tb attracted the interest of clinicians in using this novel radionuclide for radioligand therapy (RLT). 161Tb decays with a similar half-life to 177Lu, but beyond the emission of ß--particles and γ-rays, 161Tb also emits conversion and Auger electrons, which may be particularly effective to eliminate micrometastases. The aim of this study was to compare the dosimetry and therapeutic efficacy of 161Tb and 177Lu in tumor-bearing mice using SibuDAB and PSMA-I&T, which differ in their blood residence time and tumor uptake. Methods: [161Tb]Tb-SibuDAB and [161Tb]Tb-PSMA-I&T were evaluated in vitro and investigated in biodistribution, imaging, and therapy studies using PC-3 PIP tumor-bearing mice. The 177Lu-labeled counterparts served for dose calculations and comparison of therapeutic efficacy. The tolerability of RLT in mice was monitored on the basis of body mass, blood plasma parameters, blood cell counts, and the histology of relevant organs and tissues. Results: The prostate-specific membrane antigen (PSMA)-targeting radioligands, irrespective of whether labeled with 161Tb or 177Lu, showed similar in vitro data and comparable tissue distribution profiles. As a result of the albumin-binding properties, [161Tb]Tb/[177Lu]Lu-SibuDAB had an enhanced blood residence time and higher tumor uptake (62%-69% injected activity per gram at 24 h after injection) than [161Tb]Tb/[177Lu]Lu-PSMA-I&T (30%-35% injected activity per gram at 24 h after injection). [161Tb]Tb-SibuDAB inhibited tumor growth more effectively than [161Tb]Tb-PSMA-I&T, as can be ascribed to its 4-fold increased absorbed tumor dose. At any of the applied activities, the 161Tb-based radioligands were therapeutically more effective than their 177Lu-labeled counterparts, as agreed with the approximately 40% increased tumor dose of 161Tb compared with that of 177Lu. Under the given experimental conditions, no obvious adverse events were observed. Conclusion: The data of this study indicate the promising potential of 161Tb in combination with SibuDAB for RLT of prostate cancer. Future clinical studies using 161Tb-based RLT will shed light on a potential clinical benefit of 161Tb over 177Lu.

161Tb-DOTATOC Production Using a Fully Automated Disposable Cassette System: A First Step Toward the Introduction of 161Tb into the Clinic.

161Tb is an interesting radionuclide for application in the treatment of neuroendocrine neoplasms' small metastases and single cancer cells because of its conversion and Auger-electron emission. Tb has coordination chemistry similar to that of Lu; therefore, like 177Lu, it can stably radiolabel DOTATOC, one of the leading peptides used for the treatment of neuroendocrine neoplasms. However, 161Tb is a recently developed radionuclide that has not yet been specified for clinical use. Therefore, the aim of the current work was to characterize and specify 161Tb and to develop a protocol for the synthesis and quality control of 161Tb-DOTATOC with a fully automated process conforming to good-manufacturing-practice guidelines, in view of its clinical use. Methods: 161Tb, produced by neutron irradiation of 160Gd in high-flux reactors followed by radiochemical separation from its target material, was characterized regarding its radionuclidic purity, chemical purity, endotoxin level, and radiochemical purity (RCP) in analogy to what is described in the European Pharmacopoeia for no-carrier-added 177Lu. In addition, 161Tb was introduced into a fully automated cassette-module synthesis to produce 161Tb-DOTATOC, as used for 177Lu-DOTATOC. The quality and stability of the produced radiopharmaceutical in terms of identity, RCP, and ethanol and endotoxin content were assessed by means of high-performance liquid chromatography, gas chromatography, and an endotoxin test, respectively. Results: 161Tb produced under the described conditions showed, as the no-carrier-added 177Lu, a pH of 1-2, radionuclidic purity and RCP of more than 99.9%, and an endotoxin level below the permitted range (175 IU/mL), indicating its appropriate quality for clinical use. In addition, an efficient and robust procedure for the automated production and quality control of 161Tb-DOTATOC with clinically applicable specifications and activity levels, that is, 1.0-7.4 GBq in 20 mL, was developed. The radiopharmaceutical's quality control was also developed using chromatographic methods, which confirmed the product's stability (RCP ≥ 95%) over 24 h. Conclusion: The current study demonstrated that 161Tb has appropriate features for clinical use. The developed synthesis protocol guarantees high yields and safe preparation of injectable 161Tb-DOTATOC. The investigated approach could be translated to other DOTA-derivatized peptides; thus, 161Tb could be successfully applied in clinical practice for radionuclide therapy.

Signaling Network Response to α-Particle-Targeted Therapy with the 225Ac-Labeled Minigastrin Analog 225Ac-PP-F11N Reveals the Radiosensitizing Potential of Histone Deacetylase Inhibitors.

α-particle emitters have recently been explored as valuable therapeutic radionuclides. Yet, toxicity to healthy organs and cancer radioresistance limit the efficacy of targeted α-particle therapy (TAT). Identification of the radiation-activated mechanisms that drive cancer cell survival provides opportunities to develop new points for therapeutic interference to improve the efficacy and safety of TAT. Methods: Quantitative phosphoproteomics and matching proteomics followed by the bioinformatics analysis were used to identify alterations in the signaling networks in response to TAT with the 225Ac-labeled minigastrin analog 225Ac-PP-F11N (DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe) in A431 cells, which overexpress cholecystokinin B receptor (CCKBR). Western blot analysis and microscopy verified the activation of the selected signaling pathways. Small-molecule inhibitors were used to validate the potential of the radiosensitizing combinatory treatments both in vitro and in A431/CCKBR tumor-bearing nude mice. Results: TAT-induced alterations were involved in DNA damage response, cell cycle regulation, and signal transduction, as well as RNA transcription and processing, cell morphology, and transport. Western blot analysis and microscopy confirmed increased phosphorylations of the key proteins involved in DNA damage response and carcinogenesis, including p53, p53 binding protein 1 (p53BP1), histone deacetylases (HDACs), and H2AX. Inhibition of HDAC class II, ataxia-telangiectasia mutated (ATM), and p38 kinases by TMP269, AZD1390, and SB202190, respectively, sensitized A431/CCKBR cells to 225Ac-PP-F11N. As compared with the control and monotherapies, the combination of 225Ac-PP-F11N with the HDAC inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) significantly reduced the viability and increased the DNA damage of A431/CCKBR cells, led to the most pronounced tumor growth inhibition, and extended the mean survival of A431/CCKBR xenografted nude mice. Conclusion: Our study revealed the cellular responses to TAT and demonstrated the radiosensitizing potential of HDAC inhibitors to 225Ac-PP-F11N in CCKBR-positive tumors. This proof-of-concept study recommends development of novel radiosensitizing strategies by targeting TAT-activated and survival-promoting signaling pathways.

Inductively Coupled Plasma Mass Spectrometry─A Valid Method for the Characterization of Metal Conjugates in View of the Development of Radiopharmaceuticals.

This study addresses the question whether inductively coupled plasma mass spectrometry (ICP-MS) can be used as a method for the in vitro and in vivo characterization of non-radioactive metal conjugates to predict the properties of analogous radiopharmaceuticals. In a "proof-of-concept" study, the prostate-specific membrane antigen (PSMA)-targeting [175Lu]Lu-PSMA-617 and [159Tb]Tb-PSMA-617 were compared with their respective radiolabeled analogues, [177Lu]Lu-PSMA-617 (PLUVICTO, Novartis) and [161Tb]Tb-PSMA-617. ICP-MS and conventional γ-counting of the cell samples revealed almost identical results (<6% absolute difference between the two technologies) for the in vitro uptake and internalization of the (radio)metal conjugates, irrespective of the employed methodology. In vivo, an equal uptake in PSMA-positive PC-3 PIP tumor xenografts was determined 1 h after the injection of [175Lu]Lu-/[177Lu]Lu-PSMA-617 (41 ± 6% ID/g and 44 ± 12% IA/g, respectively) and [159Tb]Tb-/[161Tb]Tb-PSMA-617 (44 ± 5% ID/g and 44 ± 5% IA/g, respectively). It was further revealed that it is crucial to use the same ratios of the (radio)metal-labeled and unlabeled ligands for both methodologies to obtain equal data in organs in which receptor saturation was reached such as the kidneys (12 ± 2% ID/g vs 10 ± 1% IA/g, 1 h after injection). The data of this study demonstrate that the use of high-sensitivity ICP-MS allows reliable and predictive quantification of compounds labeled with stable metal isotopes in cell and tissue samples obtained in preclinical studies. It can, hence, be employed as a valid alternative to the state-of-the-art γ-counting methodology to detect radioactive ligands.

[225Ac]Ac-SibuDAB for Targeted Alpha Therapy of Prostate Cancer: Preclinical Evaluation and Comparison with [225Ac]Ac-PSMA-617.

In the present study, SibuDAB, an albumin-binding PSMA ligand, was investigated in combination with actinium-225 and the data were compared with those of [225Ac]Ac-PSMA-617. In vitro, [225Ac]Ac-SibuDAB and [225Ac]Ac-PSMA-617 showed similar tumor cell uptake and PSMA-binding affinities as their 177Lu-labeled counterparts. The in vitro binding to serum albumin in mouse and human blood plasma, respectively, was 2.8-fold and 1.4-fold increased for [225Ac]Ac-SibuDAB as compared to [177Lu]Lu-SibuDAB. In vivo, this characteristic was reflected by the longer retention of [225Ac]Ac-SibuDAB in the blood than previously seen for [177Lu]Lu-SibuDAB. Similar to [225Ac]Ac-PSMA-617, [225Ac]Ac-SibuDAB was well tolerated at 30 kBq per mouse. Differences in blood cell counts were observed between treated mice and untreated controls, but no major variations were observed between values obtained for [225Ac]Ac-SibuDAB and [225Ac]Ac-PSMA-617. [225Ac]Ac-SibuDAB was considerably more effective to treat PSMA-positive tumor xenografts than [225Ac]Ac-PSMA-617. Only 5 kBq per mouse were sufficient to eradicate the tumors, whereas tumor regrowth was observed for mice treated with 5 kBq [225Ac]Ac-PSMA-617 and only one out of six mice survived until the end of the study. The enhanced therapeutic efficacy of [225Ac]Ac-SibuDAB as compared to that of [225Ac]Ac-PSMA-617 and reasonable safety data qualify this novel radioligand as a candidate for targeted α-therapy of prostate cancer.

Dual MVK cleavable linkers effectively reduce renal retention of 111In-fibronectin-binding peptides.

BACKGROUND: Previously, we have exploited bacterial adhesins-derived fibronectin-binding peptides (FnBPs) for targeting mechanically altered fibronectin (Fn) fibrils within the cancer-associated extra-cellular matrix (ECM). However, despite the ability of FnBP probes to visualize pathological lesions, when labeled with metallic radionuclides and administered for targeted imaging, they exhibit high and persistent retention of radioactivity within the kidneys. Intending to overcome this issue towards a future translation of FnBPs to the clinic, the goal of the present study was to reduce the renal retention of 111In-labelled FnBPs employing dual renal brush border membrane (BBM) enzyme-sensitive Met-Val-Lys-based linkers, enabling a rapid washout of radioactivity from the kidneys. METHODS: Three maleimide-activated NOTA-conjugated brush border-enzyme cleavable linkers equipped with either single or dual consecutive MVK-based cleavable moieties were designed and synthesized. Their respective NOTA-MVK-based FnBPA5.1 conjugates were obtained by means of maleimide-thiol mediated conjugation at the N-terminus of the Fn-binding sequence, radiolabeled with indium-111, and further evaluated in vitro and in vivo in comparison to the control [111In]In-FnBPA5.1. RESULTS: The linker equipped with two MVK sites displayed a two-fold more effective cleavage rate than the single MVK featuring linker in vitro, as revealed by the quantification of the released Met-containing radiometabolites. SPECT/CT imaging and biodistribution studies of the series of FnBPA5.1 radioconjugates performed at 24 h post-injection (p.i.) confirmed the in vitro results, indicating that the renal retention of 111In-labelled FnBPs can be significantly lowered through the interposition of a single MVK-based sequence between the Fn-targeting moiety and the chelating unit (52.75 ± 9.79 vs 92.88 ± 4.85 % iA/g, P < 0.001), and even further reduced by the addition of a second one (down to 34.82 ± 6.04, P < 0.001), with minor influence on the biodistribution in other organs, such as tumors. CONCLUSIONS: In summary, we report here promising 111In-labelled FnBP radiotracers equipped with dual MVK-based cleavable linkers leading to a more effective reduction of renal retention and improved tumor-to-kidney ratios compared to the single MVK-featuring derivative. Our dual MVK strategy is a crucial step towards the clinical translation of mechano-sensory FnBPs and might as well be adopted for other radiopharmaceuticals suffering from persistent renal retention of radioactivity.

Comparative analysis of cancer cell responses to targeted radionuclide therapy (TRT) and external beam radiotherapy (EBRT).

The vast majority of our knowledge regarding cancer radiobiology and the activation of radioresistance mechanisms emerged from studies using external beam radiation therapy (EBRT). Yet, less is known about the cancer response to internal targeted radionuclide therapy (TRT). Our comparative phosphoproteomics analyzed cellular responses to TRT with lutetium-177-labeled minigastrin analogue [177Lu]Lu-PP-F11N (ß-emitter) and EBRT (É£-rays) in CCKBR-positive cancer cells. Activation of DNA damage response by p53 was induced by both types of radiotherapy, whereas TRT robustly increased activation of signaling pathways including epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs) or integrin receptor. Inhibition of EGFR or integrin signaling sensitized cancer cells to radiolabeled minigastrin. In vivo, EGFR inhibitor erlotinib increased therapeutic response to [177Lu]Lu-PP-F11N and median survival of A431/CCKBR-tumor bearing nude mice. In summary, our study explores a complex scenario of cancer responses to different types of irradiation and pinpoints the radiosensitizing strategy, based on the targeting survival pathways, which are activated by TRT.

Preclinical investigations using [177Lu]Lu-Ibu-DAB-PSMA toward its clinical translation for radioligand therapy of prostate cancer.

[177Lu]Lu-Ibu-DAB-PSMA was previously characterized with moderate albumin-binding properties enabling high tumor accumulation but reasonably low retention in the blood. The aim of this study was to investigate [177Lu]Lu-Ibu-DAB-PSMA in preclinical in vivo experiments and compare its therapeutic efficacy and potential undesired side effects with those of [177Lu]Lu-PSMA-617 and the previously developed [177Lu]Lu-PSMA-ALB-56. BALB/c nude mice without tumors were investigated on Day 10 and 28 after injection of 10 MBq radioligand. It was revealed that most plasma parameters were in the same range for all groups of mice and histopathological examinations of healthy tissue did not show any alternations in treated mice as compared to untreated controls. Based on these results, a therapy study over twelve weeks was conducted with PC-3 PIP tumor-bearing mice for comparison of the radioligands's therapeutic efficacy up to an activity of 10 MBq (1 nmol) per mouse. In agreement with the increased mean absorbed tumor dose, [177Lu]Lu-Ibu-DAB-PSMA (~ 6.6 Gy/MBq) was more effective to inhibit tumor growth than [177Lu]Lu-PSMA-617 (~ 4.5 Gy/MBq) and only moderately less potent than [177Lu]Lu-PSMA-ALB-56 (~ 8.1 Gy/MBq). As a result, the survival of mice treated with 2 MBq of an albumin-binding radioligand was significantly increased (p < 0.05) compared to that of mice injected with [177Lu]Lu-PSMA-617 or untreated controls. The majority of mice treated with 5 MBq or 10 MBq [177Lu]Lu-Ibu-DAB-PSMA or [177Lu]Lu-PSMA-ALB-56 were still alive at study end. Hemograms of immunocompetent mice injected with 30 MBq [177Lu]Lu-Ibu-DAB-PSMA or 30 MBq [177Lu]Lu-PSMA-617 showed values in the same range as untreated controls. This was, however, not the case for mice treated with [177Lu]Lu-PSMA-ALB-56 which revealed a drop in lymphocytes and hemoglobin at Day 10 and Day 28 after injection. The data of this study demonstrated a significant therapeutic advantage of [177Lu]Lu-Ibu-DAB-PSMA over [177Lu]Lu-PSMA-617 and a more favorable safety profile as compared to that of [177Lu]Lu-PSMA-ALB-56. Based on these results, [177Lu]Lu-Ibu-DAB-PSMA may has the potential for a clinical translation.

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.

Implementing subtype-specific pre-clinical models of breast cancer to study pre-treatment aspirin effects.

BACKGORUND: Prior data suggest pre-diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple-negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes. METHOD: NOD/SCID mice were implanted with HER2+ MDA-MB-231/LN/2-4/H2N, trastuzumab-resistant HER2+ HCC1954 or a TNBC patient-derived xenograft (PDX). A daily low-dose aspirin regimen commenced until primary tumours reached ~250 mm3 and subsequently resected. MDA-MB-231/LN/2-4/H2N mice were monitored for metastasis utilising imaging. To interrogate the survival benefit of pre-treatment aspirin, 3 weeks post-resection, HCC1954/TNBC animals received standard-of-care (SOC) chemotherapy for 6 weeks. Primary tumour response to aspirin was interrogated using immunohistochemistry. RESULTS: Aspirin delayed time to metastasis in MDA-MB-231/LN/2-4/H2N xenografts and decreased growth of HER2+ /TNBC primary tumours. Lymphangiogenic factors and lymph vessels number were decreased in HER2+ tumours. However, no survival benefit was seen in aspirin pre-treated animals (HCC1954/TNBC) that further received adjuvant SOC, compared with animals treated with SOC alone. In an effort to study mechanisms responsible for the observed reduction in lymphangiogenesis in HER2+ BC we utilised an in vitro co-culture system of HCC1954 tumour cells and mesenchymal stromal cells (MSC). Aspirin abrogated the secretion of VEGF-C in MSCs and also decreased the lymph/angiogenic potential of the MSCs and HCC1954 by tubule formation assay. Furthermore, aspirin decreased the secretion of uPA in HCC1954 cells potentially diminishing its metastatic capability. CONCLUSION: Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo-preventative agent in the HER2+ or TNBC setting.

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.

Combination of terbium-161 with somatostatin receptor antagonists-a potential paradigm shift for the treatment of neuroendocrine neoplasms.

PURPOSE: The ߯-emitting terbium-161 also emits conversion and Auger electrons, which are believed to be effective in killing single cancer cells. Terbium-161 was applied with somatostatin receptor (SSTR) agonists that localize in the cytoplasm (DOTATOC) and cellular nucleus (DOTATOC-NLS) or with a SSTR antagonist that localizes at the cell membrane (DOTA-LM3). The aim was to identify the most favorable peptide/terbium-161 combination for the treatment of neuroendocrine neoplasms (NENs). METHODS: The capability of the 161Tb- and 177Lu-labeled somatostatin (SST) analogues to reduce viability and survival of SSTR-positive AR42J tumor cells was investigated in vitro. The radiopeptides' tissue distribution profiles were assessed in tumor-bearing mice. The efficacy of terbium-161 compared to lutetium-177 was investigated in therapy studies in mice using DOTATOC or DOTA-LM3, respectively. RESULTS: In vitro, [161Tb]Tb-DOTA-LM3 was 102-fold more potent than [177Lu]Lu-DOTA-LM3; however, 161Tb-labeled DOTATOC and DOTATOC-NLS were only 4- to fivefold more effective inhibiting tumor cell viability than their 177Lu-labeled counterparts. This result was confirmed in vivo and demonstrated that [161Tb]Tb-DOTA-LM3 was significantly more effective in delaying tumor growth than [177Lu]Lu-DOTA-LM3, thereby, prolonging survival of the mice. A therapeutic advantage of terbium-161 over lutetium-177 was also manifest when applied with DOTATOC. Since the nuclear localizing sequence (NLS) compromised the in vivo tissue distribution of DOTATOC-NLS, it was not used for therapy. CONCLUSION: The use of membrane-localizing DOTA-LM3 was beneficial and profited from the short-ranged electrons emitted by terbium-161. Based on these preclinical data, [161Tb]Tb-DOTA-LM3 may outperform the clinically employed [177Lu]Lu-DOTATOC for the treatment of patients with NENs.