Description: Suitably labeled Evans blue dye has been successfully applied to evaluate cardiac function, vascular permeability, and lymphatic imaging in preclinical settings. This study documented the first-in-human application of 68 Ga-1,4,7-triazacyclononane- N,N',N''- triacetic acid (NOTA)-NEB. Methods: The NOTA-conjugated truncated form of Evans blue, NEB, was labeled with 68 Ga and tested in BALB/C mice for dynamic PET and ex vivo biodistribution studies. Three healthy volunteers (2 men and 1 woman) underwent 90-min whole-body dynamic PET. The absorbed doses for major organs and whole body were calculated using OLINDA/EXM software. Eleven patients with focal hepatic lesions diagnosed by enhanced CT or MR imaging were subjected to whole-body PET/CT acquisitions at 30 min after intravenous injection of 111–148 MBq (3–4 mCi) of 68 Ga-NEB. Results: NEB dye was labeled with 68 Ga (half-time, 68 min) with high yield and purity. After intravenous injection, 68 Ga-NEB formed a complex with serum albumin, thus most of the radioactivity was retained in blood circulation. The tracer was demonstrated to be safe in both healthy volunteers and recruited patients without side effects or allergies. Among the 11 patients, hemangiomas showed much higher 68 Ga-NEB signal intensity than the surrounding normal hepatic tissues, whereas no apparent difference between lesions and hepatic tissues was identified on 18 F-FDG PET. All other focal hepatic lesions including hepatocellular carcinoma, hepatic cysts, and neuroendocrine tumor liver metastases showed negative 68 Ga-NEB contrast to hepatic tissues. Conclusion: As a blood-pool imaging agent, 68 Ga-NEB is safe to use in the clinic, and our preliminary studies demonstrate the value of differentiating hepatic hemangioma from other benign or malignant focal hepatic lesions. Easy labeling with different positron emitters of various half-lives, excellent pharmacokinetics, and imaging quality warrant further clinical applications of NEB-based PET tracers.

Description: Protein tyrosine kinase-7 (PTK7), a member of receptor tyrosine kinase superfamily initially identified as colon carcinoma kinase-4, is highly expressed in various human malignancies. Its expression was found to correlate with aggressive biologic behaviors such as increased cell proliferation, invasiveness, and migration. Despite the importance and unmet need of imaging PTK7 in vivo, there is currently no clinically relevant method to visualize tumoral PTK7 expression noninvasively such as PET or SPECT. This study aimed to develop a specific, selective, and high-affinity PET radioligand based on single-stranded DNA aptamer to address this challenge. Methods: Sgc8, a 41-oligonucleotide that targets to PTK7, was labeled with 18 F using a 2-step radiochemical synthesis, which featured a direct 1-step radiofluorination on the distinctive spirocyclic hypervalent iodine(III) precursor to give 18 F-fluorobenzyl azide followed by copper-mediated click conjugation with Sgc8-alkyne. 18 F-Sgc8 was evaluated in vitro and in vivo in 2 cell lines, HCT116 and U87MG, which express high and low amounts of PTK7, respectively. Results: Sgc8 was labeled efficiently with 18 F in an isolated radiochemical yield of 62% ± 2%, non–decay-corrected based on 18 F-fluorobenzyl azide. 18 F-Tr-Sgc8 was found to possess high-affinity binding to both cell lines, with binding affinity values of 2.7 ± 0.6 nM for HCT116 and 16.9 ± 2.1 nM for U87MG. In vivo PET imaging clearly visualized PTK7 expression in HCT116 xenografted mice, with tumor uptake of 0.76 ± 0.09 percentage injected dose per gram (%ID/g) at 30 min after injection for the subcutaneous tumor model and greater than 1.5 %ID/g for the liver metastasis model. U87MG xenograft tumors had much lower tracer accumulation (0.13 ± 0.06 %ID/g at 30 min after injection), which was consistent with the lower expression of PTK7 in this tumor model. The labeled aptamer was rapidly cleared from the blood through the kidneys and bladder to give high tumor-to-blood and tumor-to-muscle ratios of 7.29 ± 1.51 and 10.25 ± 2.08, respectively. Conclusion: The 18 F-radiolabeling methodology shown here is a robust procedure for labeling aptamers and similar chemical moieties and can be applied to many different targets. Quantification of PTK7 using 18 F-Tr-Sgc8 may be suitable for clinical translation and might help in the future to select and monitor appropriate therapies.

Description: Tenascin-C is an extracellular matrix glycoprotein that is expressed by injured tissues and by various cancers. Recent publications showed that tenascin-C expression by cancer lesions predicts tumor growth, metastasis, and angiogenesis, suggesting tenascin-C as a potential therapeutic target. Currently there is no noninvasive method to determine tumoral tenascin-C expression in vivo. To address the need for an agent to image and quantify tenascin-C, we report the development of a radioactive PET tracer based on a tenascin-C–specific single-stranded DNA aptamer (tenascin-C aptamer). Methods: Tenascin-C aptamer was radiolabeled with 18 F and 64 Cu. PET imaging studies for the evaluation of tumor uptake and pharmacokinetics of tenascin-C aptamer were performed in comparison to a nonspecific scrambled aptamer (Sc aptamer). Results: The labeled tenascin-C aptamer provided clear visualization of tenascin-C–positive but not tenascin-C–negative tumors. The uptake of tenascin-C aptamer was significantly higher than that of Sc aptamer in tenascin-C–positive tumors. The labeled tenascin-C aptamer had fast clearance from the blood and other nonspecific organs through the kidneys, resulting in high tumor contrast. Conclusion: Our data suggest that suitably labeled tenascin-C aptamer can be used as a PET tracer to image tumor expression of tenascin-C with a high tumor-to-background ratio and might provide insightful and personalized medical data that will help determine appropriate treatment and monitoring.

Description: One of the major design considerations for a drug is its pharmacokinetics in the blood. A drug with a short half-life in the blood is less available at a target organ. Such a limitation dictates treatment with either high doses or more frequent doses, both of which may increase the likelihood of undesirable side effects. To address the need for additional methods to improve the blood half-life of drugs and molecular imaging agents, we developed an "add-on" molecule that contains 3 groups: a truncated Evans blue dye molecule that binds to albumin with a low micromolar affinity and provides a prolonged half-life in the blood; a metal chelate that allows radiolabeling for imaging and radiotherapy; and maleimide for easy conjugation to drug molecules. Methods: The truncated Evans blue molecule was conjugated with the chelator NOTA or DOTA, and the resulting conjugate was denoted as NMEB or DMEB, respectively. As a proof of concept, we coupled NMEB and DMEB to c(RGDfK), which is a small cyclic arginine-glycine-aspartic acid (RGD) peptide, for targeting integrin α v β 3 . NMEB and DMEB were radiolabeled with 64 Cu and 90 Y, respectively, and tested in xenograft models. Results: The resulting radiolabeled conjugates showed a prolonged circulation half-life and enhanced tumor accumulation in integrin α v β 3 –expressing tumors. Tumor uptake was markedly improved over that with NOTA- or DOTA-conjugated c(RGDfK). Tumor radiotherapy experiments in mice with 90 Y-DMEB-RGD showed promising results; existing tumors were eliminated. Conclusion: Conjugation of our novel add-on molecule, NMEB or DMEB, to potential tracers or therapeutic agents improved blood half-life and tumor uptake and could transform such agents into theranostic entities.

Description: 18 F-alfatide II has been proven to have excellent clinical translational potential. In this study, we investigated 18 F-alfatide II for identifying breast cancer and compared the performances between 18 F-alfatide II and 18 F-FDG. Methods: Forty-four female patients with suspected primary breast cancer were recruited. PET/CT images using 18 F-alfatide II and 18 F-FDG were acquired within 7 d. Tracer uptake in breast lesions was evaluated by visual analysis, and semiquantitative analysis with SUV max and SUV mean . Results: Forty-two breast cancer lesions and 11 benign breast lesions were confirmed by histopathology in 44 patients. Both 18 F-alfatide II and 18 F-FDG had higher uptake in breast cancer lesions than in benign breast lesions ( P 〈 0.05 for 18 F-alfatide II, P 〈 0.05 for 18 F-FDG). The area under the curve of 18 F-alfatide II was slightly less than that of 18 F-FDG. Both 18 F-alfatide II and 18 F-FDG had high sensitivity (88.1% vs. 90.5%), high positive predictive value (88.1% vs. 88.4%), moderate specificity (54.5% vs. 54.5%), and moderate negative predictive value (54.5% vs. 60.0%) for differentiating breast cancer from benign breast lesions. By combining 18 F-alfatide II and 18 F-FDG, the sensitivity and negative predictive value significantly increased to 97.6% and 85.7%, respectively, with positive predictive value slightly increased to 89.1% and no change to the specificity (54.5%). The uptake of 18 F-alfatide II (SUV max : 3.77 ± 1.78) was significantly lower than that of 18 F-FDG (SUV max : 7.37 ± 4.48) in breast cancer lesions ( P 〈 0.05). 18 F-alfatide II uptake in triple-negative subtype was significantly lower than that in luminal A and luminal B subtypes. By contrast, human epidermal growth factor receptor-2 (HER-2)–overexpressing subtype had higher 18 F-FDG uptake than the other 3 subtypes. There were 8 breast cancer lesions with higher 18 F-alfatide II uptake than 18 F-FDG uptake, which all had a common characteristic that HER-2 expression was negative and estrogen receptor expression was strongly positive. Conclusion: 18 F-alfatide II is suitable for clinical use in breast cancer patients. 18 F-alfatide II is of good performance, but not superior to 18 F-FDG in identifying breast cancer. 18 F-alfatide II may have superiority to 18 F-FDG in detecting breast cancer with strongly positive estrogen receptor expression and negative HER-2 expression.

Description: One of the most clinically relevant molecular aberrations in breast cancer is overexpression of human epidermal growth factor receptor type 2 (HER2). We aimed to develop a radiolabeled tyrosine kinase inhibitor for HER2-targeted breast cancer imaging. In this study, a radioiodinated analog ( 125/131 I-IBA-CP) of the HER2-selective inhibitor CP724,714 was prepared and evaluated in HER2-positive or -negative subcutaneous human breast cancer xenografts. Methods: The CP724,714 analog IBA-CP was synthesized and assayed for its inhibitory activities against HER2 and 6 other tyrosine kinases. 125/131 I-IBA-CP was prepared using a copper-mediated radioiodination method with enhanced labeling yield and molar activity. In vitro biologic activity, including specific and nonspecific binding of 131 I-IBA-CP to its HER2 kinase target, was assessed in different cell lines. In vivo small-animal 125 I-IBA-CP SPECT imaging and biodistribution studies were conducted on mice bearing HER2-positive, HER2-negative, or epidermal growth factor receptor (EGFR)-positive tumors. Nonradioactive IBA-CP and the EGFR inhibitor erlotinib were used as blocking agents to investigate the binding specificity and selectivity of 125/131 I-IBA-CP toward HER2 in vitro and in vivo. Additionally, 125/131 I-ICP was prepared by direct radioiodination of CP724,714 for comparison with 125/131 I-IBA-CP. Results: IBA-CP displayed superior in vitro inhibitory activity (half-maximal inhibitory concentration, 16 nM) and selectivity for HER2 over 6 other cancer-related tyrosine kinases. 125/131 I-IBA-CP was prepared in a typical radiochemical yield of about 65% (decay-corrected), radiochemical purity of more than 98%, and molar activity of 42 GBq/μmol at the end of synthesis. SPECT imaging revealed significantly higher uptake of 125 I-IBA-CP than of 125 I-ICP in the HER2-positive MDA-MB-453 tumors. Uptake in the HER2-negative MCF-7 tumors was much lower. Binding of 125 I-IBA-CP in the MDA-MB-453 tumors was blocked by coinjection with an excess amount of IBA-CP, but not by erlotinib. Conclusion: The radiolabeled HER2-selective inhibitor 125/131 I-IBA-CP is a promising probe for in vivo detection of HER2-positive tumors.

Description: Precise regional quantitative assessment of renal function is limited with conventional 99m Tc-labeled renal radiotracers. A recent study reported that the PET radiotracer 2-deoxy-2- 18 F-fluorosorbitol ( 18 F-FDS) has ideal pharmacokinetics for functional renal imaging. Furthermore, 18 F-FDS is available via simple reduction from routinely used 18 F-FDG. We aimed to further investigate the potential of 18 F-FDS PET as a functional renal imaging agent using rat models of kidney disease. Methods: Two different rat models of renal impairment were investigated: induction of acute renal failure by intramuscular administration of glycerol in the hind legs, and induction of unilateral ureteral obstruction by ligation of the left ureter. At 24 h after these procedures, dynamic 30-min 18 F-FDS PET data were acquired using a dedicated small-animal PET system. Urine 18 F-FDS radioactivity 30 min after radiotracer injection was measured together with coinjected 99m Tc-diethylenetriaminepentaacetic acid urine activity. Results: Dynamic PET imaging demonstrated rapid 18 F-FDS accumulation in the renal cortex and rapid radiotracer excretion via the kidneys in healthy control rats. On the other hand, significantly delayed renal radiotracer uptake (continuous slow uptake) was observed in acute renal failure rats and unilateral ureteral obstruction kidneys. Measured urine radiotracer concentrations of 18 F-FDS and 99m Tc-diethylenetriaminepentaacetic acid correlated well with each other ( R = 0.84, P 〈 0.05). Conclusion: 18 F-FDS PET demonstrated favorable kinetics for functional renal imaging in rat models of kidney diseases. 18 F-FDS PET imaging, with its advantages of high spatiotemporal resolution and simple tracer production, could potentially complement or replace conventional renal scintigraphy in select cases and significantly improve the diagnostic performance of renal functional imaging.

Description: This study was designed to analyze the safety, biodistribution, and radiation dosimetry of a gastrin-releasing peptide receptor (GRPR) antagonist PET tracer, 68 Ga-RM26; to assess its clinical diagnostic value in prostate cancer patients; and to perform a direct comparison between GRPR antagonist 68 Ga-RM26 and agonist 68 Ga-BBN. Methods: Five healthy volunteers were enrolled to validate the safety of 68 Ga-RM26 and calculate dosimetry. A total of 28 patients with prostate cancer (17 newly diagnosed and 11 posttherapy) were recruited and provided written informed consent. All the cancer patients underwent PET/CT at 15–30 min after intravenous injection of 1.85 MBq (0.05 mCi) per kilogram of body weight of 68 Ga-RM26. Among them, 22 patients (11 newly diagnosed and 11 posttherapy) underwent 68 Ga-BBN PET/CT for comparison within 1 wk. 99m Tc-MDP (methylene diphosphonate) bone scans were obtained within 2 wk for comparison. GRPR immunohistochemical staining of tumor samples was performed. Results: The administration of 68 Ga-M26 was well tolerated by all subjects, with no adverse symptoms being noticed or reported during the procedure and at 2-wk follow-up. The total effective dose equivalent and effective dose were 0.0912 ± 0.0140 and 0.0657 ± 0.0124 mSv/MBq, respectively. In the 17 patients with newly diagnosed prostate cancer, 68 Ga-RM26 PET/CT showed positive prostate-confined findings in 15 tumors with an SUV max of 6.49 ± 2.37. In the 11 patients who underwent prostatectomy or brachytherapy with or without androgen deprivation therapy, 68 Ga-RM26 PET/CT detected 8 metastatic lymph nodes in 3 patients with an SUV max of 4.28 ± 1.25 and 21 bone lesions in 8 patients with an SUV max of 3.90 ± 3.07. Compared with 68 Ga-RM26 PET/CT, GRPR agonist 68 Ga-BBN PET/CT detected fewer primary lesions and lymph node metastases as well as demonstrated lower tracer accumulation. There was a significant positive correlation between SUV derived from 68 Ga-RM26 PET and the expression level of GRPR ( P 〈 0.001). Conclusion: This study indicates the safety and significant efficiency of GRPR antagonist 68 Ga-RM26. 68 Ga-RM26 PET/CT would have remarkable value in detecting both primary prostate cancer and metastasis. 68 Ga-RM26 is also expected to be better than GRPR agonist as an imaging marker to evaluate GRPR expression in prostate cancer.

Description: This study was designed to assess the diagnostic value of 68 Ga-NOTA-PRGD2 (NOTA-PRGD2 is NOTA-PEG4-E[c(RGDfK)] 2 ) PET/CT in lung cancer. Methods: Ninety-one patients (48 men and 43 women; age, 22–82 y) with suspected lung lesions on CT were enrolled with informed consent. Immediately after intravenous injection of 117.7 ± 37.7 MBq of 68 Ga-NOTA-PRGD2, 15 patients underwent dynamic whole-body PET/CT scans for 1–2 h, and the remaining 76 patients underwent whole-body PET/CT scans at 30 ± 10 min after bolus injection. Each patient also underwent standard 18 F-FDG PET/CT for comparison. Results: No side effect was found after 68 Ga-NOTA-PRGD2 injection. 68 Ga-NOTA-PRGD2 was rapidly cleared from the blood pool and primarily excreted through the urinary system. The standardized uptake values of proven malignancies were significantly higher than those of the benign ones. With an average standardized uptake value of greater than 1.3 being considered malignant, the sensitivity, specificity, and accuracy of 68 Ga-NOTA-PRGD2 PET/CT in diagnosing lung cancer were 83.8% (57/68), 91.3% (21/23), and 85.7% (78/91), respectively. The diagnostic value of 68 Ga-NOTA-PRGD2 for lung cancer is comparable to that of 18 F-FDG PET/CT. However, 68 Ga-NOTA-PRGD2 PET/CT is more specific than 18 F-FDG PET/CT in assessing lymph node metastasis, with positive and negative predictive values of 90.0% (27/30) and 93.8% (121/129), respectively, whereas those of 18 F-FDG PET/CT were 30.2% (29/96) and 90.5% (57/63), respectively. Conclusion: This study indicates the efficacy of 68 Ga-NOTA-PRGD2 PET/CT in lung cancer diagnosis. 68 Ga-NOTA-PRGD2 PET/CT shows significant advantage over 18 F-FDG PET/CT in judging metastatic lymph nodes with higher specificity.

Description: The erythropoietin-producing hepatoma A2 receptor (EphA2) is a tyrosine kinase overexpressed by tumor stroma and cancer cells. A high expression level of EphA2 predicts poor prognosis, correlating with disease progression and metastasis. Therefore, EphA2 is a relevant therapeutic target for human cancer. Antibodies, selectively bound to EphA2, can induce rapid receptor phosphorylation that results in antibody internalization and degradation. This internalization mechanism has been exploited with the development of antibody–drug conjugates (ADCs) for cancer chemotherapy. In this study, we used PET imaging to study the pharmacokinetics and tumor delivery of a panel of anti-EphA2 monoclonal antibodies (mAbs) with and without drug conjugates. Methods: A library of human anti-EphA2 mAbs were screened and evaluated for EphA2 internalization rate, binding affinity, epitope binding, and hydrophobicity. We chose 3 of these antibodies, denoted as 1C1, 3B10, and 2H7, which recognize different epitopes, for further evaluation. ADCs were generated by S239C mutation to give a ratio of 2 drug molecules per antibody. Native mAbs and ADCs were characterized, after conjugation to a DFO chelator and 89 Zr radiolabeling, in assays including cell uptake, internalization, hydrophobicity, and in vivo imaging using PET. Results: All 3 mAbs had high affinities for EphA2 but exhibited different internalization rates following the order of 1C1 〉 3B10 〉 2H7. Internalization rate is only 1 factor that affects in vitro cell uptake and in vivo tumor accumulation. Interestingly, the hydrophobicity of the mAbs, which followed the order of 2H7 〉 1C1 〉 3B10, had a strong correlation with in vivo tumor uptake measured by PET, with the least hydrophobic antibody, 3B10, showing the highest tumor uptake. ADC significantly reduced the in vivo uptake of all 3 mAbs. Conclusion: Tumor uptake of mAb is a complex process that is affected by multiple parameters, including internalization, hydrophobicity, and chemical modification. Our results suggest that the addition of drug molecules to mAb increases the clearance of the mAb presumably due to the increased hydrophobicity. Understanding the complexity of antibody-based tumor delivery may help improve ADC engineering for better tumor targeting and reduced side effects.