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.

Population-based input function for TSPO quantification and kinetic modeling with [11C]-DPA-713.

INTRODUCTION: Quantitative positron emission tomography (PET) studies of neurodegenerative diseases typically require the measurement of arterial input functions (AIF), an invasive and risky procedure. This study aims to assess the reproducibility of [11C]DPA-713 PET kinetic analysis using population-based input function (PBIF). The final goal is to possibly eliminate the need for AIF. MATERIALS AND METHODS: Eighteen subjects including six healthy volunteers (HV) and twelve Parkinson disease (PD) subjects from two [11C]-DPA-713 PET studies were included. Each subject underwent 90 min of dynamic PET imaging. Five healthy volunteers underwent a test-retest scan within the same day to assess the repeatability of the kinetic parameters. Kinetic modeling was carried out using the Logan total volume of distribution (VT) model. For each data set, kinetic analysis was performed using a patient-specific AIF (PSAIF, ground-truth standard) and then repeated using the PBIF. PBIF was generated using the leave-one-out method for each subject from the remaining 17 subjects and after normalizing the PSAIFs by 3 techniques: (a) Weightsubject×DoseInjected, (b) area under AIF curve (AUC), and (c) Weightsubject×AUC. The variability in the VT measured with PSAIF, in the test-retest study, was determined for selected brain regions (white matter, cerebellum, thalamus, caudate, putamen, pallidum, brainstem, hippocampus, and amygdala) using the Bland-Altman analysis and for each of the 3 normalization techniques. Similarly, for all subjects, the variabilities due to the use of PBIF were assessed. RESULTS: Bland-Altman analysis showed systematic bias between test and retest studies. The corresponding mean bias and 95% limits of agreement (LOA) for the studied brain regions were 30% and ± 70%. Comparing PBIF- and PSAIF-based VT estimate for all subjects and all brain regions, a significant difference between the results generated by the three normalization techniques existed for all brain structures except for the brainstem (P-value = 0.095). The mean % difference and 95% LOA is -10% and ±45% for Weightsubject×DoseInjected; +8% and ±50% for AUC; and +2% and ± 38% for Weightsubject×AUC. In all cases, normalizing by Weightsubject×AUC yielded the smallest % bias and variability (% bias = ±2%; LOA = ±38% for all brain regions). Estimating the reproducibility of PBIF-kinetics to PSAIF based on disease groups (HV/PD) and genotype (MAB/HAB), the average VT values for all regions obtained from PBIF is insignificantly higher than PSAIF (%difference = 4.53%, P-value = 0.73 for HAB; and %difference = 0.73%, P-value = 0.96 for MAB). PBIF also tends to overestimate the difference between PD and HV for HAB (% difference = 32.33% versus 13.28%) and underestimate it in MAB (%difference = 6.84% versus 20.92%). CONCLUSIONS: PSAIF kinetic results are reproducible with PBIF, with variability in VT within that obtained for the test-retest studies. Therefore, VT assessed using PBIF-based kinetic modeling is clinically feasible and can be an alternative to PSAIF.

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.

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.

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.

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.

[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.

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.

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.

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.

Prostaglandin D2/J2 signaling pathway in a rat model of neuroinflammation displaying progressive parkinsonian-like pathology: potential novel therapeutic targets.

BACKGROUND: Prostaglandins are products of the cyclooxygenase pathway, which is implicated in Parkinson's disease (PD). Limited knowledge is available on mechanisms by which prostaglandins contribute to PD neurodegeneration. To address this gap, we focused on the prostaglandin PGD2/J2 signaling pathway, because PGD2 is the most abundant prostaglandin in the brain, and the one that increases the most under pathological conditions. Moreover, PGJ2 is spontaneously derived from PGD2. METHODS: In this study, we determined in rats the impact of unilateral nigral PGJ2-microinfusions on COX-2, lipocalin-type PGD2 synthase (L-PGDS), PGD2/J2 receptor 2 (DP2), and 15 hydroxyprostaglandin dehydrogenase (15-PGDH). Nigral dopaminergic (DA) and microglial distribution and expression levels of these key factors of the prostaglandin D2/J2 pathway were evaluated by immunohistochemistry. PGJ2-induced motor deficits were assessed with the cylinder test. We also determined whether oral treatment with ibuprofen improved the PD-like pathology induced by PGJ2. RESULTS: PGJ2 treatment induced progressive PD-like pathology in the rats. Concomitant with DA neuronal loss in the substantia nigra pars compacta (SNpc), PGJ2-treated rats exhibited microglia and astrocyte activation and motor deficits. In DA neurons, COX-2, L-PGDS, and 15-PGDH levels increased significantly in PGJ2-treated rats compared to controls, while DP2 receptor levels were unchanged. In microglia, DP2 receptors were basically non-detectable, while COX-2 and L-PGDS levels increased upon PGJ2-treatment, and 15-PGDH remained unchanged. 15-PGDH was also detected in oligodendrocytes. Notably, ibuprofen prevented most PGJ2-induced PD-like pathology. CONCLUSIONS: The PGJ2-induced rat model develops progressive PD pathology, which is a hard-to-mimic aspect of this disorder. Moreover, prevention of most PGJ2-induced PD-like pathology with ibuprofen suggests a positive feedback mechanism between PGJ2 and COX-2 that could lead to chronic neuroinflammation. Notably, this is the first study that analyzes the nigral dopaminergic and microglial distribution and levels of factors of the PGD2/J2 signaling pathway in rodents. Our findings support the notions that upregulation of COX-2 and L-PGDS may be important in the PGJ2 evoked PD-like pathology, and that neuronal DP2 receptor antagonists and L-PGDS inhibitors may be novel pharmacotherapeutics to relieve neuroinflammation-mediated neurodegeneration in PD, circumventing the adverse side effects of cyclooxygenase inhibitors.

Noninvasive PK11195-PET Image Analysis Techniques Can Detect Abnormal Cerebral Microglial Activation in Parkinson's Disease.

BACKGROUND AND PURPOSE: Neuroinflammation has been implicated in the pathophysiology of Parkinson's disease (PD), which might be influenced by successful neuroprotective drugs. The uptake of [11 C](R)-PK11195 (PK) is often considered to be a proxy for neuroinflammation, and can be quantified using the Logan graphical method with an image-derived blood input function, or the Logan reference tissue model using automated reference region extraction. The purposes of this study were (1) to assess whether these noninvasive image analysis methods can discriminate between patients with PD and healthy volunteers (HVs), and (2) to establish the effect size that would be required to distinguish true drug-induced changes from system variance in longitudinal trials. METHODS: The sample consisted of 20 participants with PD and 19 HVs. Two independent teams analyzed the data to compare the volume of distribution calculated using image-derived input functions (IDIFs), and binding potentials calculated using the Logan reference region model. RESULTS: With all methods, the higher signal-to-background in patients resulted in lower variability and better repeatability than in controls. We were able to use noninvasive techniques showing significantly increased uptake of PK in multiple brain regions of participants with PD compared to HVs. CONCLUSION: Although not necessarily reflecting absolute values, these noninvasive image analysis methods can discriminate between PD patients and HVs. We see a difference of 24% in the substantia nigra between PD and HV with a repeatability coefficient of 13%, showing that it will be possible to estimate responses in longitudinal, within subject trials of novel neuroprotective drugs.

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.

New Gastrin Releasing Peptide Receptor-Directed [99mTc]Demobesin 1 Mimics: Synthesis and Comparative Evaluation.

We have previously reported on the gastrin releasing peptide receptor (GRPR) antagonist [99mTc]1, ([99mTc]demobesin 1, 99mTc-[N4'-diglycolate-dPhe6,Leu-NHEt13]BBN(6-13)). [99mTc]1 has shown superior biological profile compared to analogous agonist-based 99mTc-radioligands. We herein present a small library of [99mTc]1 mimics generated after structural modifications in (a) the linker ([99mTc]2, [99mTc]3, [99mTc]4), (b) the peptide chain ([99mTc]5, [99mTc]6), and (c) the C-terminus ([99mTc]7 or [99mTc]8). The effects of above modifications on the biological properties of analogs were studied in PC-3 cells and tumor-bearing SCID mice. All analogs showed subnanomolar affinity for the human GRPR, while most receptor-affine 4 and 8 behaved as potent GRPR antagonists in a functional internalization assay. In mice bearing PC-3 tumors, [99mTc]1-[99mTc]6 exhibited GRPR-specific tumor uptake, rapidly clearing from normal tissues. [99mTc]4 displayed the highest tumor uptake (28.8 ± 4.1%ID/g at 1 h pi), which remained high even after 24 h pi (16.3 ± 1.8%ID/g), well surpassing that of [99mTc]1 (5.4 ± 0.7%ID/g at 24 h pi).

[18F]RPS-544: A PET tracer for imaging the chemokine receptor CXCR4.

INTRODUCTION: CXCR4 specific [18F]-labeled positron emission tomography (PET) imaging agents are needed which would enable general distribution of the radiotracer for clinical investigation. We sought to synthesize, radiolabel and evaluate [18F]RPS-544, a novel non-peptide CXCR4 antagonist as a CXCR4 specific probe. We compared [18F]RPS-544 with the previously published [18F]-3 ([18F]RPS-510 in this paper) in a bi-lateral tumor model of differential CXCR4 expression for its ability to selectively target CXCR4 expression. METHODS: Radiolabeling of [18F]RPS-544 and [18F]RPS-510 was performed by aromatic substitution on a 6-nitropyridyl group using no-carrier-added [18F]fluoride under basic conditions. 18F incorporation was determined by radioHPLC. Semi-preparative HPLC was used to purify the final product prior to reformulation. Imaging and biodistribution was performed in nude mice with bilateral PC3 (CXCR4+ and WT) xenograft tumors at 1, 2 and 4 h post injection. RESULTS: RPS-544 bound CXCR4 with an IC50 of 4.9 ±â€¯0.3 nM. [18F]RPS-544 showed preferential uptake in CXCR4+ tumors, with a CXCR4/WT ratio of 3.3 ±â€¯1.3 at 1 h p.i. and 2.3 ±â€¯0.5 at 2 h p.i. Maximum uptake in the CXCR4+ tumors was 3.4 ±â€¯1.2%ID/g at 1 h p.i., significantly greater (p = 0.003) than the uptake in the WT tumor. Tumor/blood ratios were 2.5 ±â€¯0.4 and 3.6 ±â€¯0.3 at 1 and 2 h p.i. Tumor/muscle ratios were >4 at all time-points. Tumor/lung ratios were >2 at 1 h and 2 h p.i. Substantial uptake was observed in the liver (15-25%ID/g), kidneys (25-35%ID/g), the small intestine (1-7%ID/g) and the large intestine (1-12%ID/g). Blood concentrations varied over time (0.5-2%ID/g). All other organs showed uptake of <1%ID/g at all time points studied with clearance profiles similar to blood clearance. CONCLUSIONS: Here we present, to the best of our knowledge, the first high affinity [18F]-labeled tracer, suitable for in vivo PET imaging of CXCR4. [18F]RPS-544 displayed high affinity for CXCR4 and good tumor uptake with a maximum uptake at 1 h p.i.. CXCR4 dependent uptake was demonstrated using bilateral tumors with differential CXCR4 expression as well as pharmacological blockade using the known CXCR4 antagonist, AMD-3100. Tissue contrast as judged by tumor to normal tissue ratios was positive in several key tissues. The structural and pharmacological similarities between [18F]RPS-544 and the approved drug AMD-3465, combined with the ease of synthesis and high molar activity (>185 GBq/µmol) achieved during radiosynthesis could lead to accelerated translation into the clinic.

[11 C]arachidonic acid incorporation measurement in human brain: Optimization for clinical use.

Arachidonic acid (AA) is involved in signal transduction, neuroinflammation, and production of eicosanoid metabolites. The AA brain incorporation coefficient (K*) is quantifiable in vivo using [11 C]AA positron emission tomography, although repeatability remains undetermined. We evaluated K* estimates obtained with population-based metabolite correction (PBMC) and image-derived input function (IDIF) in comparison to arterial blood-based estimates, and compared repeatability. Eleven healthy volunteers underwent a [11 C]AA scan; five repeated the scan 6 weeks later, simulating a pre- and post-treatment study design. For all scans, arterial blood was sampled to measure [11 C]AA plasma radioactivity. Plasma [11 C]AA parent fraction was measured in 5 scans. K* was quantified using both blood data and IDIF, corrected for [11 C]AA parent fraction using both PBMC (from published values) and individually measured values (when available). K* repeatability was calculated in the test-retest subset. K* estimates based on blood and individual metabolites were highly correlated with estimates using PBMC with arterial input function (r = 0.943) or IDIF (r = 0.918) in the subset with measured metabolites. In the total dataset, using PBMC, IDIF-based estimates were moderately correlated with arterial input function-based estimates (r = 0.712). PBMC and IDIF-based K* estimates were ∼6.4% to ∼11.9% higher, on average, than blood-based estimates. Average K* test-retest absolute percent difference values obtained using blood data or IDIF, assuming PBMC for both, were between 6.7% and 13.9%, comparable to other radiotracers. Our results support the possibility of simplified [11 C]AA data acquisition through eliminating arterial blood sampling and metabolite analysis, while retaining comparable repeatability and validity.

Assessment of PSMA targeting ligands bearing novel chelates with application to theranostics: Stability and complexation kinetics of 68Ga3+, 111In3+, 177Lu3+ and 225Ac3.

INTRODUCTION: Recent successes in the treatment of metastatic castration-resistant prostate cancer (mCRPCa) by systemic endoradiotherapy has sparked renewed interest in developing small molecule ligands targeting prostate-specific membrane antigen (PSMA) and chelators capable of stable complexation of metal radionuclides for imaging and therapy. As the size and coordination number of metals for imaging, such as 68Ga3+, and for targeted therapy, such as 177Lu3+ and 225Ac3+, are substantially different, they may show a preference for macrocycles of different denticity. We have prepared three simple conjugates that target PSMA and form radiometal complexes through coordination by either octa-, deca-, or dodecadentate tetraazacyclododecane chelators. The complex formation and metal ion selectivity of these constructs were determined at two relevant temperatures, complex stability was examined in vitro, and tumor targeting was demonstrated in preclinical PCa models with a view towards identifying a candidate with potential value as a theranostic agent for the imaging and therapy of mCRPCa. METHODS: Three bifunctional chelates with high denticity, including the octadentate chelate DOTA, the decadentate 3p-C-DEPA and a novel dodecadentate analogue of DEPA, were synthesized and conjugated to a glutamate-urea-lysine (EuK) pharmacophore (EuK-DOTA, EuK-107 and EuK-106, respectively) to enable targeting of PSMA. The metal ion selectivity for each construct was determined by incubation at 25 °C and 95 °C with the trivalent radiometals 68Ga3+, 111In3+, 177Lu3+ and 225Ac3+. PSMA binding affinity was determined by competitive binding using LNCaP cells, while in vivo tumor targeting of the 68Ga-labeled constructs was examined by positron emission tomography (PET) in LNCaP xenograft tumor-bearing mice. RESULTS: PMSA affinities (IC50 values) were 13.3 ± 0.9 nM for EuK-DOTA, 18.0 ± 3.7 nM for EuK-107 and 42.6 ± 6.6 nM for EuK-106. EuK-107 and EuK-DOTA proved to rapidly and near quantitatively complex 68Ga3+, 111In3+, 177Lu3+ and 225Ac3+ at 95 °C, with EuK-107 also rapidly complexing 111In3+ and 177Lu3+ at 25 °C. The inability of EuK-106 to chelate 177Lu3+ and 225Ac3+ suggests that size of the cavity of the macrocylic ring may be more critical than the number of donor groups for the chelation of larger radiometals. In vivo, 68Ga-EuK-107 proved to have similar uptake to 68Ga-DKFZ-PSMA-617, a theranostic ligand currently in clinical evaluation, in a PSMA positive xenograft tumor model. CONCLUSIONS: The broad metal ion selectivity, good in vitro affinity for PSMA and good in vivo tumor targeting suggest that EuK-107, with the 3p-C-DEPA chelator, merits further evaluation as a theranostics construct in prostate cancer.

An Eighteen-Membered Macrocyclic Ligand for Actinium-225 Targeted Alpha Therapy.

The 18-membered macrocycle H2 macropa was investigated for 225 Ac chelation in targeted alpha therapy (TAT). Radiolabeling studies showed that macropa, at submicromolar concentration, complexed all 225 Ac (26 kBq) in 5 min at RT. [225 Ac(macropa)]+ remained intact over 7 to 8 days when challenged with either excess La3+ ions or human serum, and did not accumulate in any organ after 5 h in healthy mice. A bifunctional analogue, macropa-NCS, was conjugated to trastuzumab as well as to the prostate-specific membrane antigen-targeting compound RPS-070. Both constructs rapidly radiolabeled 225 Ac in just minutes at RT, and macropa-Tmab retained >99 % of its 225 Ac in human serum after 7 days. In LNCaP xenograft mice, 225 Ac-macropa-RPS-070 was selectively targeted to tumors and did not release free 225 Ac over 96 h. These findings establish macropa to be a highly promising ligand for 225 Ac chelation that will facilitate the clinical development of 225 Ac TAT for the treatment of soft-tissue metastases.

Continuation of comprehensive quality control of the itG 68Ge/68Ga generator and production of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC for clinical research studies.

Performance of a second itG 68Ge/68Ga generator system and production of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC were tested over one year as an accompaniment to a previously published study (J Nucl Med. 2016;57:1402-1405). METHODS: Performance of a 1951MBq 68Ge/68Ga generator was characterized and the eluate used for preparation of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC. Weekly elution profiles of 68Ga elution yield and 68Ge breakthrough were determined. RESULTS: 68Ga elution yields averaged 82% (61.8-98.4%) and 68Ge breakthrough averaged 0.002% (0.0007% to 0.004%). The radiochemical purities of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC were determined by HPLC analysis to be >98% and specific activity was 12.6 and 42GBq/µmol, respectively. 68Ge contamination in the product was under the detection limit (0.00001%). Final sterile, pyrogen-free formulation of 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC in physiologic saline with 5%-7% ethanol was achieved. CONCLUSION: Performance of a 68Ge/68Ga generator was studied over one year with satisfactory results. The generator eluate was used to synthesize 68Ga-DOTATOC and 68Ga-PSMA-HBED-CC on a routine basis in high purity.

Dual-Target Binding Ligands with Modulated Pharmacokinetics for Endoradiotherapy of Prostate Cancer.

Prostate-specific membrane antigen (PSMA)-targeted radiotherapy of prostate cancer (PCa) has emerged recently as a promising approach to the treatment of disseminated disease. A small number of ligands have been evaluated in patients, and although early tumor response is encouraging, relapse rate is high and these compounds localize to the parotid, salivary, and lacrimal glands as well as to the kidney, leading to dose-limiting toxicities and adverse events affecting quality of life. We envision that dual-target binding ligands displaying high affinity for PSMA and appropriate affinity for human serum albumin (HSA) may demonstrate a higher therapeutic index and be suitable for treatment of PCa by targeted α-therapy. Methods: Six novel urea-based ligands with varying affinities for PSMA and HSA were synthesized, labeled with 131I, and evaluated by in vitro binding and uptake assays in LNCaP cells. Four compounds were advanced for further evaluation in a preclinical model of PCa. The compounds were compared with MIP-1095, a PSMA ligand currently in clinical evaluation. Results: The compounds demonstrated affinity for PSMA on the order of 4-40 nM and affinity for HSA in the range of 1-53 µM. Compounds with relatively high affinity for HSA (≤2 µM) showed high and sustained blood-pool activity and reduced uptake in the kidneys. 131I-RPS-027, with a 50% inhibitory concentration (PSMA) of 15 nM and a dissociation constant (HSA) of 11.2 µM, cleared from the blood over the course of 48 h and showed good tumor uptake (10 percentage injected dose per gram) and retention and a greater than 5-fold decrease in kidney uptake relative to MIP-1095. The tumor-to-kidney ratio of 131I-RPS-027 was greater than 3:1 at 24 h after injection, increasing to 7:1 by 72 h. Conclusion: RPS-027 shows dual targeting to PSMA and albumin, resulting in a high tumor uptake, highly favorable tissue distribution, and promising therapeutic profile in a preclinical model of prostate cancer. In comparison to existing ligands proposed for targeted therapy of prostate cancer, RPS-027 has tumor-to-tissue ratios that predict a significant reduction in side effects during therapy. Using iodine/radioiodine as a surrogate for the radiohalogen 211At, we therefore propose dual-target binding ligands such as RPS-027 as next-generation radiopharmaceuticals for targeted α-therapy using 211At.