Synthesis of 124I-labeled epichaperome probes and assessment in visualizing pathologic protein-protein interaction networks in tumor bearing mice.

Epichaperomes are disease-associated pathologic scaffolds composed of tightly bound chaperones and co-chaperones. They provide opportunities for precision medicine where aberrant protein-protein interaction networks, rather than a single protein, are detected and targeted. This protocol describes the synthesis and characterization of two 124I-labeled epichaperome probes, [124I]-PU-H71 and [124I]-PU-AD, both which have translated to clinical studies. It shows specific steps in the use of these reagents to image and quantify epichaperome-positivity in tumor bearing mice through positron emission tomography. For complete details on the use and execution of this protocol, please refer to Bolaender et al. (2021), Inda et al. (2020), and Pillarsetty et al. (2019).

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

Predicting Gemcitabine Delivery by 18F-FAC PET in Murine Models of Pancreatic Cancer.

18F-FAC (2'-deoxy-2'-18F-fluoro-ß-d-arabinofuranosylcytosine) has close structural similarity to gemcitabine and thus offers the potential to image drug delivery to tumors. We compared tumor 18F-FAC PET images with 14C-gemcitabine levels, established ex vivo, in 3 mouse models of pancreatic cancer. We further modified tumor gemcitabine levels with injectable PEGylated recombinant human hyaluronidase (PEGPH20) to test whether changes in gemcitabine would be tracked by 18F-FAC. Methods:18F-FAC was synthesized as described previously. Three patient-derived xenograft (PDX) models were grown in the flanks of NSG mice. Mice were given PEGPH20 or vehicle intravenously 24 h before coinjection of 18F-FAC and 14C-gemcitabine. Animals were euthanized and imaged 1 h after tracer administration. Tumor and muscle uptake of both 18F-FAC and 14C-gemcitabine was obtained ex vivo. The efficacy of PEPGPH20 was validated through staining with hyaluronic acid binding protein. Additionally, an organoid culture, initiated from a KPC (Pdx-1 Cre LSL-KrasG12D LSL-p53R172H) tumor, was used to generate orthotopically growing tumors in C57BL/6J mice, and these tumors were then serially transplanted. Animals were injected with PEGPH20 and 14C-gemcitabine as described above to validate increased drug uptake by ex vivo assay. PET/MR images were obtained using a PET insert on a 7-T MR scanner. Animals were imaged immediately before injection with PEGPH20 and again 24 h later. Results: Tumor-to-muscle ratios of 14C-gemcitabine and 18F-FAC correlated well across all PDX models and treatments (R2 = 0.78). There was a significant increase in the tumor PET signal in PEGPH20-treated PDX animals, and this signal was matched in ex vivo counts for 2 of 3 models. In KPC-derived tumors, PEGPH20 raised 14C-gemcitabine levels (tumor-to-muscle ratio of 1.9 vs. 2.4, control vs. treated, P = 0.013). PET/MR 18F-FAC images showed a 12% increase in tumor 18F-FAC uptake after PEGPH20 treatment (P = 0.023). PEGPH20-treated animals uniformly displayed clear reductions in hyaluronic acid staining. Conclusion:18F-FAC PET was shown to be a good surrogate for gemcitabine uptake and, when combined with MR, to successfully determine drug uptake in tumors growing in the pancreas. PEGPH20 had moderate effects on tumor uptake of gemcitabine.

First-in-Human Trial of Epichaperome-Targeted PET in Patients with Cancer.

PURPOSE: 124I-PU-H71 is an investigational first-in-class radiologic agent specific for imaging tumor epichaperome formations. The intracellular epichaperome forms under cellular stress and is a clinically validated oncotherapeutic target. We conducted a first-in-human study of microdose 124I-PU-H71 for PET to study in vivo biodistribution, pharmacokinetics, metabolism, and safety; and the feasibility of epichaperome-targeted tumor imaging. EXPERIMENTAL DESIGN: Adult patients with cancer (n = 30) received 124I-PU-H71 tracer (201±12 MBq, <25 µg) intravenous bolus followed by PET/CT scans and blood radioassays. RESULTS: 124I-PU-H71 PET detected tumors of different cancer types (breast, lymphoma, neuroblastoma, genitourinary, gynecologic, sarcoma, and pancreas). 124I-PU-H71 was retained by tumors for several days while it cleared rapidly from bones, healthy soft tissues, and blood. Radiation dosimetry is favorable and patients suffered no adverse effects. CONCLUSIONS: Our first-in-human results demonstrate the safety and feasibility of noninvasive in vivo detection of tumor epichaperomes using 124I-PU-H71 PET, supporting clinical development of PU-H71 and other epichaperome-targeted therapeutics.

Safety and Feasibility of PARP1/2 Imaging with 18F-PARPi in Patients with Head and Neck Cancer.

PURPOSE: We performed a first-in-human clinical trial. The aim of this study was to determine safety and feasibility of PET imaging with 18F-PARPi in patients with head and neck cancer. PATIENTS AND METHODS: Eleven patients with newly diagnosed or recurrent oral and oropharyngeal cancer were injected with 18F-PARPi (331 ± 42 MBq), and dynamic PET/CT imaging was performed between 0 and 25 minutes postinjection. Static PET/CT scans were obtained at 30, 60, and 120 minutes postinjection. Blood samples for tracer concentration and metabolite analysis were collected. Blood pressure, ECG, oxygen levels, clinical chemistry, and complete blood count were obtained before and after tracer administration. RESULTS: 18F-PARPi was well-tolerated by all patients without any safety concerns. Of the 11 patients included in the analysis, 18F-PARPi had focal uptake in all primary lesions (n = 10, SUVmax = 2.8 ± 1.2) and all 18F-FDG-positive lymph nodes (n = 34). 18F-PARPi uptake was seen in 18F-FDG-negative lymph nodes of 3 patients (n = 6). Focal uptake of tracer in primary and metastatic lesions was corroborated by CT alone or in combination with 18F-FDG. The overall effective dose with 18F-PARPi PET was 3.9 mSv - 5.2 mSv, contrast was high [SUVmax(lesion)/SUVmax(trapezius muscle) = 4.5] and less variable than 18F-FDG when compared with the genioglossus muscle (1.3 vs. 6.0, P = 0.001). CONCLUSIONS: Imaging of head and neck cancer with 18F-PARPi is feasible and safe. 18F-PARPi detects primary and metastatic lesions, and retention in tumors is longer than in healthy tissues.

First-in-Humans Trial of Dasatinib-Derivative Tracer for Tumor Kinase-Targeted PET.

We developed a first-of-kind dasatinib-derivative imaging agent, 18F-SKI-249380 (18F-SKI), and validated its use for noninvasive in vivo tyrosine kinase-targeted tumor detection in preclinical models. In this study, we assessed the feasibility of using 18F-SKI for PET imaging in patients with malignancies. Methods: Five patients with a prior diagnosis of breast cancer, renal cell cancer, or leukemia underwent whole-body PET/CT imaging 90 min after injection of 18F-SKI (mean, 241.24 ± 116.36 MBq) as part of a prospective study. In addition, patients underwent either a 30-min dynamic scan of the upper abdomen including, at least partly, cardiac left ventricle, liver, spleen, and kidney (n = 2) or three 10-min whole-body PET/CT scans (n = 3) immediately after injection and blood-based radioactivity measurements to determine the time course of tracer distribution and facilitate radiation dose estimates. A subset of 3 patients had a delayed whole-body PET/CT scan at 180 min. Biodistribution, dosimetry, and tumor uptake were quantified. Absorbed doses were calculated using OLINDA/EXM 1.0. Results: No adverse events occurred after injection of 18F-SKI. In total, 27 tumor lesions were analyzed, with a median SUVpeak of 1.4 (range, 0.7-2.3) and tumor-to-blood ratios of 1.6 (range, 0.8-2.5) at 90 min after injection. The intratumoral drug concentrations calculated for 4 reference lesions ranged from 0.03 to 0.07 nM. In all reference lesions, constant tracer accumulation was observed between 30 and 90 min after injection. A blood radioassay indicated that radiotracer clearance from blood and plasma was initially rapid (blood half-time, 1.31 ± 0.81 min; plasma, 1.07 ± 0.66 min; n = 4), followed variably by either a prolonged terminal phase (blood half-time, 285 ± 148.49 min; plasma, 240 ± 84.85 min; n = 2) or a small rise to a plateau (n = 2). Like dasatinib, 18F-SKI underwent extensive metabolism after administration, as evidenced by metabolite analysis. Radioactivity was predominantly cleared via the hepatobiliary route. The highest absorbed dose estimates (mGy/MBq) in normal tissues were to the right colon (0.167 ± 0.04) and small intestine (0.153 ± 0.03). The effective dose was 0.0258 mSv/MBq (SD, 0.0034 mSv/MBq). Conclusion:18F-SKI demonstrated significant tumor uptake, distinct image contrast despite low injected doses, and rapid clearance from blood.

Preclinical and first-in-human-brain-cancer applications of [18F]poly (ADP-ribose) polymerase inhibitor PET/MR.

BACKGROUND: We report preclinical and first-in-human-brain-cancer data using a targeted poly (ADP-ribose) polymerase 1 (PARP1) binding PET tracer, [18F]PARPi, as a diagnostic tool to differentiate between brain cancers and treatment-related changes. METHODS: We applied a glioma model in p53-deficient nestin/tv-a mice, which were injected with [18F]PARPi and then sacrificed 1 h post-injection for brain examination. We also prospectively enrolled patients with brain cancers to undergo dynamic [18F]PARPi acquisition on a dedicated positron emission tomography/magnetic resonance (PET/MR) scanner. Lesion diagnosis was established by pathology when available or by Response Assessment in Neuro-Oncology (RANO) or RANO-BM response criteria. Resected tissue also underwent PARPi-FL staining and PARP1 immunohistochemistry. RESULTS: In a preclinical mouse model, we illustrated that [18F]PARPi crossed the blood-brain barrier and specifically bound to PARP1 overexpressed in cancer cell nuclei. In humans, we demonstrated high [18F]PARPi uptake on PET/MR in active brain cancers and low uptake in treatment-related changes independent of blood-brain barrier disruption. Immunohistochemistry results confirmed higher PARP1 expression in cancerous than in noncancerous tissue. Specificity was also corroborated by blocking fluorescent tracer uptake with an excess unlabeled PARP inhibitor in patient cancer biospecimen. CONCLUSIONS: Although larger studies are necessary to confirm and further explore this tracer, we describe the promising performance of [18F]PARPi as a diagnostic tool to evaluate patients with brain cancers and possible treatment-related changes.

Paradigms for Precision Medicine in Epichaperome Cancer Therapy.

Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.

Assessment of Simplified Methods for Quantification of 18F-FDHT Uptake in Patients with Metastatic Castration-Resistant Prostate Cancer.

18F-fluorodihydrotestosterone (18F-FDHT) PET/CT potentially provides a noninvasive method for assessment of androgen receptor expression in patients with metastatic castration-resistant prostate cancer (mCRPC). The objective of this study was to assess simplified methods for quantifying 18F-FDHT uptake in mCRPC patients and to assess effects of tumor perfusion on these 18F-FDHT uptake metrics. Methods: Seventeen mCRPC patients were included in this prospective observational multicenter study. Test and retest 30-min dynamic 18F-FDHT PET/CT scans with venous blood sampling were performed in 14 patients. In addition, arterial blood sampling and dynamic 15O-H2O scans were obtained in a subset of 6 patients. Several simplified methods were assessed: Patlak plots; SUV normalized to body weight (SUVBW), lean body mass (SUVLBM), whole blood (SUVWB), parent plasma activity concentration (SUVPP), area under the parent plasma curve (SUVAUC,PP), and area under the whole-blood input curve (SUVAUC,WB); and SUVBW corrected for sex hormone-binding globulin levels (SUVSHBG). Results were correlated with parameters derived from full pharmacokinetic 18F-FDHT and 15O-H2O. Finally, the repeatability of individual quantitative uptake metrics was assessed. Results: Eighty-seven 18F-FDHT-avid lesions were evaluated. 18F-FDHT uptake was best described by an irreversible 2-tissue-compartment model. Replacing the continuous metabolite-corrected arterial plasma input function with an image-derived input function in combination with venous sample data provided similar Ki results (R2 = 0.98). Patlak Ki and SUVAUC,PP showed an excellent correlation (R2 > 0.9). SUVBW showed a moderate correlation to Ki (R2 = 0.70, presumably due to fast 18F-FDHT metabolism. When calculating SUVSHBG, correlation to Ki improved (R2 = 0.88). The repeatability of full kinetic modeling parameters was inferior to that of simplified methods (repeatability coefficients > 36% vs. < 28%, respectively). 18F-FDHT uptake showed minimal blood flow dependency. Conclusion:18F-FDHT kinetics in mCRPC patients are best described by an irreversible 2-tissue-compartment model with blood volume parameter. SUVAUC,PP showed a near-perfect correlation with the irreversible 2-tissue-compartment model analysis and can be used for accurate quantification of 18F-FDHT uptake in whole-body PET/CT scans. In addition, SUVSHBG could potentially be used as an even simpler method to quantify 18F-FDHT uptake when less complex scanning protocols and accuracy are required.

In Vivo PET Assay of Tumor Glutamine Flux and Metabolism: In-Human Trial of 18F-(2S,4R)-4-Fluoroglutamine.

Purpose To assess the clinical safety, pharmacokinetics, and tumor imaging characteristics of fluorine 18-(2S,4R)-4-fluoroglutamine (FGln), a glutamine analog radiologic imaging agent. Materials and Methods This study was approved by the institutional review board and conducted under a U.S. Food and Drug Administration-approved Investigational New Drug application in accordance with the Helsinki Declaration and the Health Insurance Portability and Accountability Act. All patients provided written informed consent. Between January 2013 and October 2016, 25 adult patients with cancer received an intravenous bolus of FGln tracer (mean, 244 MBq ± 118, <100 µg) followed by positron emission tomography (PET) and blood radioassays. Patient data were summarized with descriptive statistics. FGln biodistribution and plasma amino acid levels in nonfasting patients (n = 13) were compared with those from patients who fasted at least 8 hours before injection (n = 12) by using nonparametric one-way analysis of variance with Bonferroni correction. Tumor FGln avidity versus fluorodeoxyglucose (FDG) avidity in patients with paired PET scans (n = 15) was evaluated with the Fisher exact test. P < .05 was considered indicative of a statistically significant difference. Results FGln PET depicted tumors of different cancer types (breast, pancreas, renal, neuroendocrine, lung, colon, lymphoma, bile duct, or glioma) in 17 of the 25 patients, predominantly clinically aggressive tumors with genetic mutations implicated in abnormal glutamine metabolism. Acute fasting had no significant effect on FGln biodistribution and plasma amino acid levels. FGln-avid tumors were uniformly FDG-avid but not vice versa (P = .07). Patients experienced no adverse effects. Conclusion Preliminary human FGln PET trial results provide clinical validation of abnormal glutamine metabolism as a potential tumor biomarker for targeted radiotracer imaging in several different cancer types. © RSNA, 2018 Online supplemental material is available for this article. Clinical trial registration no. NCT01697930.