Advanced Fibroblast Activation Protein-Ligand Developments: FAP Imaging Agents: A Review of the Structural Requirements.

Fibroblast activation protein-α (FAP) has attracted increasing attention as a selective marker of cancer-associated fibroblasts (CAFs) and more broadly, of activated fibroblasts in tissues undergoing remodeling of their ECM due to chronic inflammation, fibrosis, or wound healing. Since FAP is critical to the initiation of metastatic growth, its expression will serve as a molecular marker to detect tumors at an earlier stage of development compared to currently available methods. The design of high affinity small molecule FAP inhibitor will allow for noninvasive imaging of activated fibroblast in cancer patients. Small molecule inhibitors of FAP are being developed for targeted radiotherapy of tumors.

Advances in PSMA theranostics.

The validation of prostate specific membrane antigen (PSMA) as a molecular target in metastatic castration-resistant prostate cancer has stimulated the development of multiple classes of theranostic ligands that specifically target PSMA. Theranostic ligands are used to image disease or selectively deliver cytotoxic radioactivity to cells expressing PSMA according to the radioisotope conjugated to the ligand. PSMA theranostics is a rapidly advancing field that is now integrating into clinical management of prostate cancer patients. In this review we summarize published research describing the biological role(s) and activity of PSMA, highlight the most clinically advanced PSMA targeting molecules and biomacromolecules, and identify next generation PSMA ligands that aim to further improve treatment efficacy. The goal of this review is to provide a comprehensive assessment of the current state-of-play and a roadmap to achieving further advances in PSMA theranostics.

Synthesis and Evaluation of 11C-Labeled Triazolones as Probes for Imaging Fatty Acid Synthase Expression by Positron Emission Tomography.

Cancer cells require lipids to fulfill energetic, proliferative, and signaling requirements. Even though these cells can take up exogenous fatty acids, the majority exhibit a dependency on de novo fatty acid synthesis. Fatty acid synthase (FASN) is the rate-limiting enzyme in this process. Expression and activity of FASN is elevated in multiple cancers, where it correlates with disease progression and poor prognosis. These observations have sparked interest in developing methods of detecting FASN expression in vivo. One promising approach is the imaging of radiolabeled molecular probes targeting FASN by positron emission tomography (PET). However, although [11C]acetate uptake by prostate cancer cells correlates with FASN expression, no FASN-specific PET probes currently exist. Our aim was to synthesize and evaluate a series of small molecule triazolones based on GSK2194069, an FASN inhibitor with IC50 = 7.7 ± 4.1 nM, for PET imaging of FASN expression. These triazolones were labeled with carbon-11 in good yield and excellent radiochemical purity, and binding to FASN-positive LNCaP cells was significantly higher than FASN-negative PC3 cells. Despite these promising characteristics, however, these molecules exhibited poor in vivo pharmacokinetics and were predominantly retained in lymph nodes and the hepatobiliary system. Future studies will seek to identify structural modifications that improve tumor targeting while maintaining the excretion profile of these first-generation 11C-methyltriazolones.

PET Tracers for Imaging Cardiac Function in Cardio-oncology.

PURPOSE OF REVIEW: Successful treatment of cancer can be hampered by the attendant risk of cardiotoxicity, manifesting as cardiomyopathy, left ventricle systolic dysfunction and, in some cases, heart failure. This risk can be mitigated if the injury to the heart is detected before the onset to irreversible cardiac impairment. The gold standard for cardiac imaging in cardio-oncology is echocardiography. Despite improvements in the application of this modality, it is not typically sensitive to sub-clinical or early-stage dysfunction. We identify in this review some emerging tracers for detecting incipient cardiotoxicity by positron emission tomography (PET). RECENT FINDINGS: Vectors labeled with positron-emitting radionuclides (e.g., carbon-11, fluorine-18, gallium-68) are now available to study cardiac function, metabolism, and tissue repair in preclinical models. Many of these probes are highly sensitive to early damage, thereby potentially addressing the limitations of current imaging approaches, and show promise in preliminary clinical evaluations. The overlapping pathophysiology between cardiotoxicity and heart failure significantly expands the number of imaging tools available to cardio-oncology. This is highlighted by the emergence of radiolabeled probes targeting fibroblast activation protein (FAP) for sensitive detection of dysregulated healing process that underpins adverse cardiac remodeling. The growth of PET scanner technology also creates an opportunity for a renaissance in metabolic imaging in cardio-oncology research.

A suitable time point for quantifying the radiochemical purity of 225Ac-labeled radiopharmaceuticals.

BACKGROUND: As 225Ac-labeled radiopharmaceuticals continue to show promise as targeted alpha therapeutics, there is a growing need to standardize quality control (QC) testing procedures. The determination of radiochemical purity (RCP) is an essential QC test. A significant obstacle to RCP testing is the disruption of the secular equilibrium between actinium-225 and its daughter radionuclides during labeling and QC testing. In order to accelerate translation of actinium-225 targeted alpha therapy, we aimed to determine the earliest time point at which the RCP of an 225Ac-labeled radiopharmaceutical can be accurately quantified. RESULTS: Six ligands were conjugated to macrocyclic metal chelators and labeled with actinium-225 under conditions designed to generate diverse incorporation yields. RCP was determined by radio thin layer chromatography (radioTLC) followed by exposure of the TLC plate on a phosphor screen either 0.5, 2, 3.5, 5, 6.5, or 26 h after the plate was developed. The dataset was used to create models for predicting the true RCP for any pre-equilibrium measurement taken at an early time point. The 585 TLC measurements span RCP values of 1.8-99.5%. The statistical model created from these data predicted an independent data set with high accuracy. Predictions made at 0.5 h are more uncertain than predictions made at later time points. This is primarily due to the decay of bismuth-213. A measurement of RCP > 90% at 2 h predicts a true RCP > 97% and guarantees that RCP will exceed 90% after secular equilibrium is reached. These findings were independently validated using NaI(Tl) scintillation counting and high resolution gamma spectroscopy on a smaller set of samples with 10% ≤ RCP ≤ 100%. CONCLUSIONS: RCP of 225Ac-labeled radiopharmaceuticals can be quantified with acceptable accuracy at least 2 h after radioTLC using various methods of quantifying particle emissions. This time point best balances the need to accurately quantify RCP with the need to safely release the batch as quickly as possible.

A Trifunctional Theranostic Ligand Targeting Fibroblast Activation Protein-α (FAPα).

PURPOSE: Fibroblast activation protein-α (FAPα) is uniquely expressed in activated fibroblasts, including cancer-associated fibroblasts that populate tumor stroma and contribute to proliferation and immunosuppression. Radiolabeled FAPα inhibitors enable imaging of multiple human cancers, but time-dependent clearance from tumors currently limits their utility as FAPα-targeted radiotherapeutics. We sought to increase the area under the curve (AUC) by constructing a trifunctional ligand that binds FAPα with high affinity and also binds albumin and theranostic radiometals. PROCEDURES: RPS-309 comprised a FAPα-targeting moiety, an albumin-binding group, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Inhibition of recombinant human FAPα (rhFAPα) was determined by colorimetric assay. Affinity for human serum albumin (HSA) was determined by high-performance affinity chromatography. The tissue distribution of [68Ga]Ga-RPS-309 in SW872 tumor xenograft-bearing mice was imaged by microPET/CT and quantified by biodistribution studies performed from 30 min to 3 h post injection (p.i.). The biodistribution of [177Lu]Lu-RPS-309 was determined at 4, 24, and 96 h p.i. RESULTS: RPS-309 inhibits rhFAPα with IC50 = 7.3 ± 1.4 nM. [68Ga]Ga-RPS-309 is taken up specifically by FAPα-expressing cells and binds HSA with Kd = 4.6 ± 0.1 µM. Uptake of the radiolabeled ligand in tumors was evident from 30 min p.i. (> 5 %ID/g) and was significantly reduced by co-injection of RPS-309. Specific skeletal uptake was also observed. Activity in tumors was constant through 4 h p.i., but cleared significantly by 24 h. The AUC in this period was 127 (%ID/g) × h. CONCLUSIONS: RPS-309 is a high-affinity FAPα inhibitor with prolonged plasma residence. Introduction of the albumin-binding group did not compromise FAPα binding. Although initial tumor uptake was high and FAPα-specific, RPS-309 also progressively cleared from tumors. Nevertheless, RPS-309 incorporates multiple sites in which structural diversity can be introduced, and therefore serves as a platform for future structure-activity relationship studies.

In Vivo Imaging of Fibroblast Activity Using a 68Ga-Labeled Fibroblast Activation Protein Alpha (FAP-α) Inhibitor: Study in a Mouse Rotator Cuff Repair Model.

BACKGROUND: Rotator cuff repair site failure is a well-established clinical concern. Tendon-to-bone healing is initiated by inflammatory mediators followed by matrix synthesis by fibroblasts. The kinetics of fibroblast accumulation and activity are currently poorly understood. METHODS: Ninety-six mice underwent supraspinatus tendon repair. Six were used for imaging using a novel 68Gallium (Ga)-labeled fibroblast activation protein alpha (FAP-α) inhibitor and positron emission tomography-computed tomography (PET/CT) at days 0 (before surgery), 3, 7, 14, and 28. Sixty-eight animals were divided into 4 groups to be evaluated at 3, 7, 14, or 28 days. Twenty-two native shoulders from mice without surgery were used as the control group (intact tendon). Six animals from each group were used for histological analysis; 6 from each group were used for evaluation of fibroblastic response-related gene expression; and 10 mice each from the intact, 14-day, and 28-day groups were used for biomechanical testing. RESULTS: There was minimal localization of 68Ga-labeled FAP-α inhibitor in the shoulders at day 0 (before surgery). There was significantly increased uptake in the shoulders with surgery compared with the contralateral sides without surgery at 3, 7, and 14 days. 68Ga-labeled FAP-α inhibitor uptake in the surgically treated shoulders increased gradually and peaked at 14 days followed by a decrease at 28 days. Gene expression for smooth muscle alpha (α)-2 (acta2), FAP-α, and fibronectin increased postsurgery followed by a drop at 28 days. Immunohistochemical analysis showed that FAP-α-positive cell density followed a similar temporal trend, peaking at 14 days. All trends matched closely with the PET/CT results. Biomechanical testing demonstrated a gradual increase in failure load during the healing process. CONCLUSIONS: 68Ga-labeled FAP-α inhibitor PET/CT allows facile, high-contrast in vivo 3-dimensional imaging of fibroblastic activity in a mouse rotator cuff repair model. CLINICAL RELEVANCE: Noninvasive imaging of activated fibroblasts using labeled radiotracers may be a valuable tool to follow the progression of healing at the bone-tendon interface.

Quantitative Whole-Body Imaging of I-124-Labeled Adeno-Associated Viral Vector Biodistribution in Nonhuman Primates.

A method is presented for quantitative analysis of the biodistribution of adeno-associated virus (AAV) gene transfer vectors following in vivo administration. We used iodine-124 (I-124) radiolabeling of the AAV capsid and positron emission tomography combined with compartmental modeling to quantify whole-body and organ-specific biodistribution of AAV capsids from 1 to 72 h following administration. Using intravenous (IV) and intracisternal (IC) routes of administration of AAVrh.10 and AAV9 vectors to nonhuman primates in the absence or presence of anticapsid immunity, we have identified novel insights into initial capsid biodistribution and organ-specific capsid half-life. Neither I-124-labeled AAVrh.10 nor AAV9 administered intravenously was detected at significant levels in the brain relative to the administered vector dose. Approximately 50% of the intravenously administered labeled capsids were dispersed throughout the body, independent of the liver, heart, and spleen. When administered by the IC route, the labeled capsid had a half-life of ∼10 h in the cerebral spinal fluid (CSF), suggesting that by this route, the CSF serves as a source with slow diffusion into the brain. For both IV and IC administration, there was significant influence of pre-existing anticapsid immunity on I-124-capsid biodistribution. The methodology facilitates quantitative in vivo viral vector dosimetry, which can serve as a technique for evaluation of both on- and off-target organ biodistribution, and potentially accelerate gene therapy development through rapid prototyping of novel vector designs.

Meeting report from the Prostate Cancer Foundation PSMA theranostics state of the science meeting.

INTRODUCTION: The Prostate Cancer Foundation (PCF) convened a PCF prostate-specific membrane antigen (PSMA) Theranostics State of the Science Meeting on 18 November 2019, at Weill Cornell Medicine, New York, NY. METHODS: The meeting was attended by 22 basic, translational, and clinical researchers from around the globe, with expertise in PSMA biology, development and use of PSMA theranostics agents, and clinical trials. The goal of this meeting was to discuss the current state of knowledge, the most important biological and clinical questions, and critical next steps for the clinical development of PSMA positron emission tomography (PET) imaging agents and PSMA-targeted radionuclide agents for patients with prostate cancer. RESULTS: Several major topic areas were discussed including the biology of PSMA, the role of PSMA-targeted PET imaging in prostate cancer, the physics and performance of different PSMA-targeted PET imaging agents, the current state of clinical development of PSMA-targeted radionuclide therapy (RNT) agents, the role of dosimetry in PSMA RNT treatment planning, barriers and challenges in PSMA RNT clinical development, optimization of patient selection for PSMA RNT trials, and promising combination treatment approaches with PSMA RNT. DISCUSSION: This article summarizes the presentations from the meeting for the purpose of globally disseminating this knowledge to advance the use of PSMA-targeted theranostic agents for imaging and treatment of patients with prostate cancer.

Preclinical Evaluation of a High-Affinity Sarcophagine-Containing PSMA Ligand for 64Cu/67Cu-Based Theranostics in Prostate Cancer.

The application of small molecules targeting prostate-specific membrane antigen (PSMA) has emerged as a highly promising clinical strategy for visualization and treatment of prostate cancer. Ligands that integrate the ability to both quantify the distribution of radioactivity and treat disease through the use of a matched pair of radionuclides have particular value in clinical and regulatory settings. In this study, we describe the development and preclinical evaluation of RPS-085, a ligand that binds PSMA and serum albumin and exploits the 64/67Cu radionuclide pair for prostate cancer theranostics. RPS-085 was synthesized by conjugation of a PSMA-targeting moiety, an Nε-(2-(4-iodophenyl)acetyl)lysine albumin binding group, and a bifunctionalized MeCOSar chelator. The IC50 of the metal-free RPS-085 was determined in a competitive binding assay. The affinity for human serum albumin of the radiolabeled compound was determined by high-performance affinity chromatography. Radiolabeling was performed in NH4OAc buffer at 25 °C. The stability of the radiolabeled compounds was assessed in vitro and in vivo. The biodistribution of [64/67Cu]Cu-RPS-085 was determined following intravenous administration to male BALB/c mice bearing LNCaP tumor xenografts. The radiochemical yields of [64/67Cu]Cu-RPS-085 were nearly quantitative after 20 min. The metal-free complex is a potent inhibitor of PSMA (IC50 = 29 ± 2 nM), and the radiolabeled compound has moderate affinity for human serum albumin (Kd = 9.9 ± 1.7 µM). Accumulation of the tracer in mice was primarily evident in tumor and kidneys. Activity in all other tissues, including blood, was negligible, and the radiolabeled compounds demonstrated high stability in vitro and in vivo. Tumor activity reached a maximum at 4 h post injection (p.i.) and cleared gradually over a period of 96 h. By contrast, activity in the kidney cleared rapidly from 4 to 24 h p.i. As a consequence, by 24 h p.i., the tumor-to-kidney ratio exceeds 2, and the predicted dose to tumors is significantly greater than the dose to kidneys. [64Cu]Cu-RPS-085 combines rapid tissue distribution and clearance with prolonged retention in LNCaP tumor xenografts. The pharmacokinetics should enable radioligand therapy using [67Cu]Cu-RPS-085. By virtue of its rapid kidney clearance, the therapeutic index of [67Cu]Cu-RPS-085 likely compares favorably to its parent structure, [177Lu]Lu-RPS-063, a highly avid PSMA-targeting compound. On this basis, [64/67Cu]Cu-RPS-085 show great promise as PSMA-targeting theranostic ligands for prostate cancer imaging and therapy.

A general 11C-labeling approach enabled by fluoride-mediated desilylation of organosilanes.

Carbon-11 (11C) is one of the most ideal positron emitters for labeling bioactive molecules for molecular imaging studies. The lack of convenient and fast incorporation methods to introduce 11C into organic molecules often hampers the use of this radioisotope. Here, a fluoride-mediated desilylation (FMDS) 11C-labeling approach is reported. This method relies on thermodynamically favored Si-F bond formation to generate a carbanion, therefore enabling the highly efficient and speedy incorporation of [11C]CO2 and [11C]CH3I into molecules with diversified structures. It provides facile and rapid access to 11C-labeled compounds with carbon-11 attached at various hybridized carbons as well as oxygen, sulfur and nitrogen atoms with broad functional group tolerance. The exemplified syntheses of several biologically and clinically important radiotracers illustrates the potentials of this methodology.

Oxyaapa: A Picolinate-Based Ligand with Five Oxygen Donors that Strongly Chelates Lanthanides.

Coordination compounds of the lanthanide ions (Ln3+) have important applications in medicine due to their photophysical, magnetic, and nuclear properties. To effectively use the Ln3+ ions for these applications, chelators that stably bind them in vivo are required to prevent toxic side effects that arise from localization of these ions in off-target tissue. In this study, two new picolinate-containing chelators, a heptadentate ligand OxyMepa and a nonadentate ligand Oxyaapa, were prepared, and their coordination chemistries with Ln3+ ions were thoroughly investigated to evaluate their suitability for use in medicine. Protonation constants of these chelators and stability constants for their Ln3+ complexes were evaluated. Both ligands exhibit a thermodynamic preference for small Ln3+ ions. The log KLuL = 12.21 and 21.49 for OxyMepa and Oxyaapa, respectively, indicating that the nonadentate Oxyaapa forms complexes of significantly higher stability than the heptadentate OxyMepa. X-ray crystal structures of the Lu3+ complexes were obtained, revealing that Oxyaapa saturates the coordination sphere of Lu3+, whereas OxyMepa leaves an additional open coordination site for a bound water ligand. Solution structural studies carried out with NMR spectroscopy revealed the presence of two possible conformations for these ligands upon Ln3+ binding. Density functional theory (DFT) calculations were applied to probe the geometries and energies of these conformations. Energy differences obtained by DFT are small but consistent with experimental data. The photophysical properties of the Eu3+ and Tb3+ complexes were characterized, revealing modest photoluminescent quantum yields of <2%. Luminescence lifetime measurements were carried out in H2O and D2O, showing that the Eu3+ and Tb3+ complexes of OxyMepa have two inner-sphere water ligands, whereas the Eu3+ and Tb3+ complexes of Oxyaapa have zero. Lastly, variable-temperature 17O NMR spectroscopy was performed for the Gd-OxyMepa complex to determine its water exchange rate constant of kex298 = (2.8 ± 0.1) × 106 s-1. Collectively, this comprehensive characterization of these Ln3+ chelators provides valuable insight for their potential use in medicine and garners additional understanding of ligand design strategies.

Otto: a 4.04 GBq (109 mCi) 68Ge/68Ga generator, first of its kind - extended quality control and performance evaluation in the clinical production of [68Ga]Ga-PSMA-11.

BACKGROUND: Here we report on the comprehensive quality control of a 4.04 GBq (109 mCi) generator supplied by itG (Munich, Germany), and used for routine production of [68Ga]Ga-PSMA-11 for clinical imaging. The performance of the 4.04 GBq itG 68Ge/68Ga generator was studied for a year and parameters including elution yield, elution profile, radioactive and stable contaminants were collected. The production yields of a series of 175 [68Ga]Ga-PSMA-11 clinical batches are also reported herein. RESULTS: This first-of-its-kind GMP grade 68Ge/68Ga generator from itG with a nominal activity of 4.04 GBq (109 mCi) showed a stable 68Ga elution profile with elution efficiency averaging 58.3 ± 3.7%. 68Ge contaminant in the eluent slightly increased over time but remained 100x lower than those reported for comparable 1.85 GBq (50 mCi) itG generators. Metal impurities were found in concentrations lower than 100 ng/ml (ppb) throughout the study. [68Ga]Ga-PSMA-11 was obtained in 89 ± 4% radiochemical yields and > 99% radiochemical and chemical purities. CONCLUSION: 4.04 GBq (109 mCi) itG 68Ge/68Ga generator is suitable for routinely produced 68Ga tracers used in the clinic. Up to 30% higher amount of final drug product was obtained as compared to the 1.85 GBq (50 mCi) itG generator, and as a result larger number of studies could be performed, while reducing the synthetic burden.

3D-printed automation for optimized PET radiochemistry.

Reproducible batch synthesis of radioligands for imaging by positron emission tomography (PET) in a manner that maximizes ligand yield, purity, and molar activity, and minimizes cost and exposure to radiation, remains a challenge, as new and synthetically complex radioligands become available. Commercially available automated synthesis units (ASUs) solve many of these challenges but are costly to install and cannot always accommodate diverse chemistries. Through a reiterative design process, we exploit the proliferation of three-dimensional (3D) printing technologies to translate optimized reaction conditions into ASUs composed of 3D-printed, electronic, and robotic parts. Our units are portable and robust and reduce radiation exposure, shorten synthesis time, and improve the yield of the final radiopharmaceutical for a fraction of the cost of a commercial ASU. These 3D-printed ASUs highlight the gains that can be made by designing a fit-for-purpose ASU to accommodate a synthesis over accommodating a synthesis to an unfit ASU.

Improved synthesis of the bifunctional chelator p-SCN-Bn-HOPO.

The bifunctional ligand p-SCN-Bn-HOPO, which has four 1,2-hydroxypyridinone groups on a spermine backbone with an isothiocyanate linker, has been shown to be an efficient and stable chelator for Zr(iv) and, more importantly, the radioisotope 89Zr for use in radiolabeling antibodies for positron emission tomography (PET) imaging. Previous studies of 89Zr-HOPO-trastuzumab in mice showed low background, good tumor to organ contrast, and very low bone uptake which show p-SCN-Bn-HOPO to be an important next-generation bifunctional chelator for radioimmunoPET imaging with 89Zr. However, the reported synthesis of p-SCN-Bn-HOPO involves nine steps and multiple HPLC purifications with an overall yield of about 1.4%. Herein we report an improved and efficient synthesis of p-SCN-Bn-HOPO in four steps with 14.3% overall yield which will improve its availability for further biological studies and wider application in PET imaging. The new synthetic route also allows variation in linker length and chemistries which may be helpful in modifying in vivo clearance behaviors of future agents.

[18F]Fluoroethyltriazolyl Monocyclam Derivatives as Imaging Probes for the Chemokine Receptor CXCR4.

Determining chemokine receptor CXCR4 expression is significant in multiple diseases due to its role in promoting inflammation, cell migration and tumorigenesis. [68Ga]Pentixafor is a promising ligand for imaging CXCR4 expression in multiple tumor types, but its utility is limited by the physical properties of 68Ga. We screened a library of >200 fluorine-containing structural derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low, but tumor uptake was CXCR4-specific. The tumor uptake of [18F]RPS-534 (7.2 ± 0.3 %ID/g) and [18F]RPS-547 (3.1 ± 0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding radiosynthesis, high tumor uptake, and good contrast to background, [18F]RPS-547, and especially [18F]RPS-534, are promising 18F-labeled candidates for imaging CXCR4 expression.

A Single Dose of 225Ac-RPS-074 Induces a Complete Tumor Response in an LNCaP Xenograft Model.

Promising biochemical responses to 225Ac-prostate-specific membrane antigen (PSMA) 617, even in patients who are refractory to ß-particle radiation, illustrate the potential of targeted α-therapy for the treatment of metastatic castration-resistant prostate cancer. However, side effects such as xerostomia are severe and irreversible. To fully harness the potential of targeted α-therapy, it is necessary to increase the therapeutic index of the targeted radioligands. One emerging strategy is to increase clearance half-life through enhanced binding to serum albumin. We have evaluated the albumin-binding PSMA-targeting ligand RPS-074 in a LNCaP xenograft model to determine its potential value to the treatment of prostate cancer. Methods:225Ac-RPS-074 was evaluated in male BALB/c mice bearing LNCaP xenograft tumors. A biodistribution study was performed over 21 d to determine the dosimetry in tumors and normal tissue. The dose response was measured in groups of 7 mice using 37, 74, and 148 kBq of 225Ac-RPS-074 and compared with positive and negative control groups. Mice were sacrificed when tumor volume exceeded 1,500 mm3Results:225Ac-RPS-074 was labeled in greater than 98% radiochemical yield and showed high (>10% injected dose/g) and sustained accumulation in LNCaP tumors from 24 h to beyond 14 d. Signal in blood and highly vascularized tissues was evident over the first 24 h after injection and cleared by 7 d. The tumor-to-kidney ratio was 4.3 ± 0.7 at 24 h and 62.2 ± 9.5 at 14 d. A single injection of 148 kBq induced a complete response in 6 of 7 tumors, with no apparent toxic effects. Treatment with 74 kBq induced a partial response in 7 of 7 tumors, but from 42 d, 6 of 7 experienced significant regrowth. The 37-kBq group experienced a survival benefit relative to the negative control but not compared with the positive control group. Conclusion: A single dose of 148 kBq of 225Ac-RPS-074 induced a complete response in 86% of tumors, with tumor-to-normal-tissue ratios that predict an improved therapeutic index. The use of the macropa chelator enabled quantitative radiolabeling and may facilitate the clinical translation of this promising targeted α-therapeutic.

Albumin-Binding PSMA Ligands: Implications for Expanding the Therapeutic Window.

Despite significant gains in the treatment of metastatic castration-resistant prostate cancer by radioligands targeting prostate-specific membrane antigen (PSMA), 30% of patients never respond to therapy. One possible explanation is insufficient dose delivery to the tumor because of suboptimal pharmacokinetics. We have recently described RPS-063, a trifunctional ligand targeting PSMA with high uptake in LNCaP xenograft tumors but also in kidneys. We aimed to use structural modifications to increase the tumor-to-kidney ratio through increased albumin binding and tumor uptake and reduction of kidney activity. Methods: Four structurally related trifunctional PSMA-targeting small molecules were prepared by either varying the albumin-binding group or inserting a polyethylene glycol 8 linker into a common structure. The compounds were ranked by PSMA affinity and albumin affinity and were radiolabeled with 68Ga and 177Lu. Tissue kinetics were determined in male BALB/C nu/nu mice bearing LNCaP xenograft tumors. Results: Each of the compounds binds PSMA with a half-maximal inhibitory concentration of no more than 10 nM. The albumin-binding group had a minimal effect on PSMA affinity but changed albumin affinity by an order of magnitude. However, the addition of a polyethylene glycol 8 spacer weakened affinity for albumin in each case. Increased affinity for albumin corresponded with delayed blood clearance and modified uptake kinetics in the tumor and kidney. Uptake of 177Lu-RPS-072 (34.9 ± 2.4 %ID/g) and 177Lu-RPS-077 (27.4 ± 0.6 %ID/g) increased up to 24 h after injection, and washout by 96 h was not significant. As a result, the area under the curve (AUC) in the tumor was in the following order: 177Lu-RPS-072 > 177Lu-RPS-077 > 177Lu-RPS-063 > 177Lu-RPS-071. Increased linker length corresponded to more rapid clearance from kidneys. Consequently, the ratio of tumor AUC and kidney AUC was 4.7 ± 0.3 for 177Lu-RPS-072. Conclusion: The tumor AUC and tumor-to-kidney ratio of 177Lu-RPS-072 are significantly enhanced compared with any small molecule investigated in a LNCaP xenograft model to date. In comparison to other PSMA-targeting radioligands that have been evaluated in a PC3-PIP model, activity in kidneys is reduced and activity in tumors compares favorably when the different PSMA expression levels in LNCaP and PC3-PIP cells are considered. RPS-072 therefore exhibits an increased therapeutic index, shows the potential to increase the dose delivered to tumors, and is a highly promising candidate for targeted radioligand therapy.

66Ga: A Novelty or a Valuable Preclinical Screening Tool for the Design of Targeted Radiopharmaceuticals?

Emerging interest in extending the plasma half-life of small molecule radioligands warrants a consideration of the appropriate radionuclide for PET imaging at longer time points (>8 h). Among candidate positron-emitting radionuclides, 66Ga (t1/2 = 9.5 h, ß+ = 57%) has suitable nuclear and chemical properties for the labeling and PET imaging of radioligands of this profile. We investigated the value of 66Ga to preclinical screening and the evaluation of albumin-binding PSMA-targeting small molecules. 66Ga was produced by irradiation of a natZn target. 66Ga3+ ions were separated from Zn2+ ions by an optimized UTEVA anion exchange column that retained 99.99987% of Zn2+ ions and allowed 90.2 ± 2.8% recovery of 66Ga3+. Three ligands were radiolabeled in 46.4 ± 20.5%; radiochemical yield and >90% radiochemical purity. Molar activity was 632 ± 380 MBq/µmol. Uptake in the tumor and kidneys at 1, 3, 6, and 24 h p.i. was determined by µPET/CT imaging and more completely predicted the distribution kinetics than uptake of the [68Ga]Ga-labeled ligands did. Although there are multiple challenges to the use of 66Ga for clinical PET imaging, it can be a valuable research tool for ligand screening and preclinical imaging beyond 24 h.

Trifunctional PSMA-targeting constructs for prostate cancer with unprecedented localization to LNCaP tumors.

PURPOSE: Treatment of late-stage prostate cancer by targeted radiotherapeutics such as 131I-MIP-1095 and 177Lu-PSMA-617 has shown encouraging early results. Lu-177 is preferred to I-131 in clinical settings, but targeted radioligand therapy (RLT) with 177Lu-PSMA-617 has not reached its full potential due to insufficient dose delivery to the tumor. We recently developed a dual-targeting radioiodinated ligand, RPS-027, that targets PSMA and uses albumin binding to enable good tumor uptake and significantly reduced kidney uptake in a preclinical model. Further development of this ligand is limited by the inability to independently modify PSMA and albumin binding and the requirement of I-131 for therapeutic application. We therefore sought to devise a new class of trifunctional ligands for RLT with (1) a high-affinity PSMA-binding domain, (2) an albumin-binding group (ABG), and (3) a chelator for radiometals such as 68Ga3+, 177Lu3+ and 225Ac3+. METHODS: Ligands incorporating a triazolylphenylurea-containing PSMA-targeting group, an Nε-(2-(4-iodophenyl)acetyl)lysine ABG and the bifunctional chelator p-SCN-Bn-DOTA linked by a PEG-containing polymer containing 0,3,4,6,8 or 12 repeats were prepared. PSMA affinity was determined in LNCaP cells and uptake and tissue distribution was studied in mice bearing LNCaP tumor xenografts and compared to 177Lu-PSMA-617. Imaging studies were performed up to 24 h post-injection (p.i.) using 66Ga3+ and biodistribution studies at 4 h, 24 h and 96 h p.i. with 177Lu3+. RESULTS: PSMA affinity was high (IC50 = 1-10 nM) and inversely proportional to the linker length. Tumor uptake correlated with binding affinity and was significantly greater than for 177Lu-PSMA-617 over 96 h. The highest uptake was achieved with 177Lu-RPS-063 (30.0 ± 6.9 %ID/g; 4 h p.i.). Kidney uptake was generally high, with the exception of the lowest affinity ligand 177Lu-RPS-067. Each of the compounds showed slower blood clearance than 177Lu-PSMA-617, with clearance proportional to linker length. CONCLUSIONS: The high tumor uptake achieved with these trifunctional ligands predicts larger (up to 4×) doses delivered to the tumor than can be achieved with 177Lu-PSMA-617. Although PSMA-mediated kidney uptake was also observed, the exceptional area under the curve (AUC) in the tumor warrants further investigation of these novel ligands as candidates for RLT.