Samantha Y.A. Terry

New Tris(hydroxypyridinone) Bifunctional Chelators Containing Isothiocyanate Groups Provide a Versatile Platform for Rapid One-Step Labeling and PET Imaging with 68Ga3+

Two new bifunctional tris(hydroxypyridinone) (THP) chelators designed specifically for rapid labeling with 68Ga have been synthesized, each with pendant isothiocyanate groups and three 1,6-dimethyl-3-hydroxypyridin-4-one groups. Both compounds have been conjugated with the primary amine group of a cyclic integrin targeting peptide, RGD. Each conjugate can be radiolabeled and formulated by treatment with generator-produced 68Ga3+ in over 95% radiochemical yield under ambient conditions in less than 5 min, with specific activities of 60–80 MBq nmol–1. Competitive binding assays and in vivo biodistribution in mice bearing U87MG tumors demonstrate that the new 68Ga3+-labeled THP peptide conjugates retain affinity for the αvβ3 integrin receptor, clear within 1–2 h from circulation, and undergo receptor-mediated tumor uptake in vivo. We conclude that bifunctional THP chelators can be used for simple, efficient labeling of 68Ga biomolecules under mild conditions suitable for peptides and proteins.

Imaging integrin alpha-v-beta-3 expression in tumors with an 18F-labeled dimeric RGD peptide.

Integrin αv β3 receptors are expressed on activated endothelial cells during neovascularization to maintain tumor growth. Many radiolabeled probes utilize the tight and specific association between the arginine-glycine-aspartatic acid (RGD) peptide and integrin αv β3 , but one main obstacle for any clinical application of these probes is the laborious multistep radiosynthesis of (18)F. In this study, the dimeric RGD peptide, E-[c(RGDfK)]2, was conjugated with NODAGA and radiolabeled with (18)F in a simple one-pot process with a radiolabeling yield of 20%, the whole process lasting only 45 min. NODAGA-E-[c(RGDfK)]2 labeled with (18)F at a specific activity of 1.8 MBq nmol(-1) and a radiochemical purity of 100% could be achieved. The logP value of (18)F-labeled NODAGA-E-[c(RGDfK)]2 was -4.26 ± 0.02. In biodistribution studies, (18)F-NODAGA-E-[c(RGDfK)]2 cleared rapidly from the blood with 0.03 ± 0.01 percentage injected dose per gram (%ID g(-1)) in the blood at 2 h p.i., mainly via the kidneys, and showed good in vivo stability. Tumor uptake of (18)F-NODAGA-E-[c(RGDfK)]2 (3.44 ± 0.20 %ID g(-1), 2 h p.i.) was significantly lower than that of reference compounds (68) Ga-labeled NODAGA-E-[c(RGDfK)]2 (6.26 ± 0.76 %ID g(-1) ; p <0.001) and (111) In-labeled NODAGA-E-[c(RGDfK)]2 (4.99 ± 0.64 %ID g(-1) ; p < 0.01). Co-injection of an excess of unlabeled NODAGA-E-[c(RGDfK)]2 along with (18)F-NODAGA-E-[c(RGDfK)]2 resulted in significantly reduced radioactivity concentrations in the tumor (0.85 ± 0.13 %ID g(-1)). The αv β3 integrin-expressing SK-RC-52 tumor could be successfully visualized by microPET with (18)F-labeled NODAGA-E-[c(RGDfK)]2 . In conclusion, NODAGA-E-[c(RGDfK)]2 could be labeled rapidly with (18)F using a direct aqueous, one-pot method and it accumulated specifically in αv β3 integrin-expressing SK-RC-52 tumors, allowing for visualization by microPET.

Immuno-PET and Immuno-SPECT of Rheumatoid Arthritis with Radiolabeled Anti–Fibroblast Activation Protein Antibody Correlates with Severity of Arthritis

One of the most prominent cell populations playing a role in rheumatoid arthritis (RA) is activated fibroblast-like synoviocytes. Among many other proteins, fibroblast-like synoviocytes dominantly express fibroblast activation protein (FAP). Because of the high expression of FAP in arthritic joints, radioimmunoimaging of activated fibroblasts with anti-FAP antibodies might be an attractive noninvasive imaging tool in RA. Methods: SPECT and PET with 111In- and 89Zr-labeled anti-FAP antibody 28H1 was performed in mice with CIA. The radioactivity uptake in joints was quantified and correlated with arthritis score. Results: Both 111In-28H1 and 89Zr-28H1 showed high uptake in inflamed joints, being 3-fold higher than that of the irrelevant isotype-matched control antibody DP47GS, clearly indicating specific accumulation of 28H1. Uptake of 111In-28H1 ranged from 2.2 percentage injected dose per gram (%ID/g) in noninflamed joints to 32.1 %ID/g in severely inflamed joints. DP47GS accumulation ranged from 1.6 %ID/g in noninflamed tissue to 12.0 %ID/g in severely inflamed joints. Uptake of 28H1 in inflamed joints correlated with arthritis score (Spearman ρ, 0.69; P < 0.0001) and increased with severity of arthritis. Conclusion: SPECT/CT imaging with the anti-FAP antibody 111In-28H1 specifically visualized arthritic joints with high resolution, and tracer accumulation correlated with the severity of the inflammation in murine experimental arthritis. Background uptake of the radiolabeled antibody was low, resulting in excellent image quality. 89Zr-28H1 was less favorable for RA imaging because of an elevated bone uptake of 89Zr. Future studies will focus on the potential role of 28H1 as a tool to monitor therapy response early on.

Imaging Integrin αvβ3 on Blood Vessels with 111In-RGD2 in Head and Neck Tumor Xenografts

Arginine-glycine-aspartic acid (RGD)–based imaging tracers allow specific imaging of integrin αvβ3, a protein overexpressed during angiogenesis, leading to the possibility that it might serve as a tool to stratify patients for antiangiogenic treatment. However, these tracers have generally been characterized in xenograft models in which integrin αvβ3 was constitutively expressed by the tumor cells themselves. In the studies presented here, the use of 111In-RGD2 as a tracer to image only integrin αvβ3 expression on blood vessels in the tumor was determined using tumor xenografts in which tumor cells were integrin αvβ3-negative. Methods: DOTA-E-[c(RGDfK)]2 was radiolabeled with 111In (111In-RGD2), and biodistribution studies were performed in squamous cell carcinoma of the head and neck (HNSCC) xenograft mouse models to determine the optimal peptide dose to image angiogenesis. Next, biodistribution and imaging studies were performed at the optimal peptide dose in 3 HNSCC mouse models, FaDu, SCCNij3, and SCCNij202. Immunohistochemical analysis of tumor vascular and cell surface expression of integrin αvβ3 and correlation analysis of vascular integrin αvβ3 and autoradiography were completed. Results: All 3 HNSCC xenografts expressed integrin αvβ3 on the vessels only. The optimal peptide dose of 111In-RGD2 was 1 μg or less for specific integrin αvβ3–mediated uptake of the tracer. SPECT/CT imaging showed clear uptake of the tracer in the periphery of the tumors, corresponding with well-vascularized areas of the tumor. Within the tumor, 111In-RGD2 autoradiography coincided with vascular integrin αvβ3 expression, as determined immunohistochemically. Integrin αvβ3–mediated uptake was also detected in nontumor tissues, which, through immunohistochemical analysis, proved positive for integrin αvβ3. Conclusion: 111In-RGD2 allows the visualization of integrin αvβ3 in xenograft models in which integrin αvβ3 is expressed only on the neovasculature, such as in the HNSCC tumors. Thus, 111In-RGD2 allows specific visualization of angiogenesis in tumor models lacking constitutive tumoral integrin αvβ3 expression but may be less useful for this purpose in many tumors in which tumor cells express integrin αvβ3.

68Ga-THP-PSMA: a PET imaging agent for prostate cancer offering rapid, room temperature, one-step kit-based radiolabeling

The clinical impact and accessibility of 68Ga tracers for the prostate-specific membrane antigen (PSMA) and other targets would be greatly enhanced by the availability of a simple, 1-step kit-based labeling process. Radiopharmacy staff are accustomed to such procedures in the daily preparation of 99mTc radiopharmaceuticals. Currently, chelating agents used in 68Ga radiopharmaceuticals do not meet this ideal. The aim of this study was to develop and evaluate preclinically a 68Ga radiotracer for imaging PSMA expression that could be radiolabeled simply by addition of 68Ga generator eluate to a cold kit. Methods: A conjugate of a tris(hydroxypyridinone) (THP) chelator with the established urea-based PSMA inhibitor was synthesized and radiolabeled with 68Ga by adding generator eluate directly to a vial containing the cold precursors THP-PSMA and sodium bicarbonate, with no further manipulation. It was analyzed after 5 min by instant thin-layer chromatography and high-performance liquid chromatography. The product was subjected to in vitro studies to determine PSMA affinity using PSMA-expressing DU145-PSMA cells, with their nonexpressing analog DU145 as a control. In vivo PET imaging and ex vivo biodistribution studies were performed in mice bearing xenografts of the same cell lines, comparing 68Ga-THP-PSMA with 68Ga-HBED-CC-PSMA. Results: Radiolabeling was complete (>95%) within 5 min at room temperature, showing a single radioactive species by high-performance liquid chromatography that was stable in human serum for more than 6 h and showed specific binding to PSMA-expressing cells (concentration giving 50% inhibition of 361 ± 60 nM). In vivo PET imaging showed specific uptake in PSMA-expressing tumors, reaching 5.6 ± 1.2 percentage injected dose per cubic centimeter at 40–60 min and rapid clearance from blood to kidney and bladder. The tumor uptake, biodistribution, and pharmacokinetics were not significantly different from those of 68Ga-HBED-CC-PSMA except for reduced uptake in the spleen. Conclusion: 68Ga-THP-PSMA has equivalent imaging properties but greatly simplified radiolabeling compared with other 68Ga-PSMA conjugates. THP offers the prospect of rapid, simple, 1-step, room-temperature syringe-and-vial radiolabeling of 68Ga radiopharmaceuticals.

Monitoring Therapy Response of Experimental Arthritis with Radiolabeled Tracers Targeting Fibroblasts, Macrophages, or Integrin αvβ3

Rheumatoid arthritis is an autoimmune disease resulting in chronic synovial inflammation. Molecular imaging could be used to monitor therapy response, thus enabling tailored therapy regimens and enhancing therapeutic outcome. Here, we hypothesized that response to etanercept could be monitored by radionuclide imaging in arthritic mice. We tested 3 different targets, namely fibroblast activation protein (FAP), macrophages, and integrin αvβ3. Methods: Male DBA/1J mice with collagen-induced arthritis were treated with etanercept. SPECT/CT scans were acquired at 1, 24, and 48 h after injection of 111In-RGD2 (integrin αvβ3), 111In-anti-F4/80-A3-1 (antimurine macrophage antibody), or 111In-28H1 (anti-FAP antibody), respectively, with nonspecific controls included. Mice were dissected after the last scan, and scans were analyzed quantitatively and were correlated with macroscopic scoring. Results: Experimental arthritis was imaged with 111In-28H1 (anti-FAP), 111In-anti-F4/80-A3-1, and 111In-RGD2. Tracer uptake in joints correlated with arthritis score. Treatment decreased joint uptake of tracers from 23 ± 15, 8 ± 4, and 2 ± 1 percentage injected dose per gram (%ID/g) to 11 ± 11 (P < 0.001), 4 ± 4 (P < 0.001), and 1 ± 0.2 %ID/g (P < 0.01) for 111In-28H1, 111In-anti-F4/80-A3-1, and 111In-RGD2, respectively. Arthritis-to-blood ratios (in mice with arthritis score 2 per joint) were higher for 111In-28H1 (5.5 ± 1; excluding values > 25), 111In-anti-F4/80-A3-1 (10.4 ± 4), and 111In-RGD2 (7.2 ± 1) than for control 111In-DP47GS (0.7 ± 0.5; P = 0.002), 111In-rat IgG2b (0.5 ± 0.2; P = 0.002), or coinjection of excess RGD2 (3.5), indicating specific uptake of all tracers in arthritic joints. Conclusion: 111In-28H1, 111In-anti-F4/80-A3-1, and 111In-RGD2 can be used to specifically monitor the response to therapy in experimental arthritis at the molecular level. Further studies, however, still need to be performed.

Relationship Between Chromatin Structure and Sensitivity to Molecularly Targeted Auger Electron Radiation Therapy

PURPOSE The open structure of euchromatin renders it susceptible to DNA damage by ionizing radiation (IR) compared with compact heterochromatin. The effect of chromatin configuration on the efficacy of Auger electron radiotherapy was investigated. METHODS AND MATERIALS Chromatin structure was altered in MDA-MB-468 and 231-H2N human breast cancer cells by suberoylanilide hydroxamic acid (SAHA), 5-aza-2-deoxycytidine, or hypertonic treatment. The extent and duration of chromatin structural changes were evaluated using the micrococcal nuclease assay. DNA damage (γH2AX assay) and clonogenic survival were evaluated after exposure to (111)In-DTPA-hEGF, an Auger electron-emitting radiopharmaceutical, or IR. The intracellular distribution of (111)In-DTPA-hEGF after chromatin modification was investigated in cell fractionation experiments. RESULTS Chromatin remained condensed for up to 20 minutes after NaCl and in a relaxed state 24 hours after SAHA treatment. The number of γH2AX foci per cell was greater in MDA-MB-468 and 231-H2N cells after IR (0.5 Gy) plus SAHA (1 μM) compared with IR alone (16 ± 0.6 and 14 ± 0.3 vs. 12 ± 0.4 and 11 ± 0.2, respectively). More γH2AX foci were observed in MDA-MB-468 and 231-H2N cells exposed to (111)In-DTPA-hEGF (6 MBq/μg) plus SAHA vs. (111)In-DTPA-hEGF alone (11 ± 0.3 and 12 ± 0.7 vs. 9 ± 0.4 and 7 ± 0.3, respectively). 5-aza-2-deoxycytidine enhanced the DNA damage caused by IR and (111)In-DTPA-hEGF. Clonogenic survival was reduced in MDA-MB-468 and 231-H2N cells after IR (6 Gy) plus SAHA (1 μM) vs. IR alone (0.6% ± 0.01 and 0.3% ± 0.2 vs. 5.8% ± 0.2 and 2% ± 0.1, respectively) and after (111)In-DTPA-hEGF plus SAHA compared to (111)In-DTPA-hEGF alone (21% ± 0.4% and 19% ± 4.6 vs. 33% ± 2.3 and 32% ± 3.7). SAHA did not affect (111)In-DTPA-hEGF nuclear localization. Hypertonic treatment resulted in fewer γH2AX foci per cell after IR and (111)In-DTPA-hEGF compared to controls but did not significantly alter clonogenic survival. CONCLUSIONS Chromatin structure affects DNA damage and cell survival after exposure to Auger electron radiation.

Can 111In-RGD2 Monitor Response to Therapy in Head and Neck Tumor Xenografts?

RGD (arginylglycylaspartic acid)–based imaging tracers allow specific imaging of integrin αvβ3 expression, proteins overexpressed during angiogenesis; however, few studies have investigated the potential of these tracers to monitor responses of antiangiogenic or radiation therapy. In the studies presented here, 111In-RGD2 was assessed for its potential as an imaging tool to monitor such responses to therapies. Methods: DOTA-E-[c(RGDfK)]2 was radiolabeled with 111In (111In-RGD2), and biodistribution studies were performed in mice with subcutaneous FaDu or SK-RC-52 xenografts after treatment with either antiangiogenic therapy (bevacizumab or sorafenib) or tumor irradiation (10 Gy). Micro-SPECT imaging studies and subsequent quantitative analysis were also performed. The effect of bevacizumab, sorafenib, or radiation therapy on tumor growth was determined. Results: The uptake of 111In-RGD2 in tumors, as determined from biodistribution studies, correlated well with that quantified from micro-SPECT images, and both showed that 15 d after irradiation 111In-RGD2 uptake was enhanced. Specific or nonspecific uptake of 111In-RGD2 in FaDu or SK-RC-52 xenografts was not affected after antiangiogenic therapy, except in head and neck squamous cell carcinoma 19 d after the start of sorafenib therapy (P < 0.05). The uptake of 111In-RGD2 followed tumor volume in studies featuring antiangiogenic therapy. However, the uptake of 111In-RGD2 in FaDu xenografts was decreased as early as 4 h after tumor irradiation, despite nonspecific uptake remaining unaltered. Tumor growth was inhibited after antiangiogenic or radiation therapy. Conclusion: Here, it is suggested that 111In-RGD2 could allow in vivo monitoring of angiogenic responses after radiotherapy and may therefore prove a good clinical tool to monitor angiogenic responses early after the start of radiotherapy in patients with head and neck squamous cell carcinoma. Despite clear antitumor efficacy, antiangiogenic therapy did not alter tumor uptake of 111In-RGD2, indicating that integrin expression was not altered.

In vivo imaging of antileukemic drug asparaginase reveals a rapid macrophage mediated clearance from the bone marrow

The antileukemic drug asparaginase, a key component in the treatment of acute lymphoblastic leukemia, acts by depleting asparagine from the blood. However, little is known about its pharmacokinetics, and mechanisms of therapy resistance are poorly understood. Here, we explored the in vivo biodistribution of radiolabeled asparaginase, using a combination of imaging and biochemical techniques, and provide evidence for tissue-specific clearance mechanisms, which could reduce the effectiveness of the drug at these specific sites. Methods: In vivo localization of 111In-labeled Escherichia coli asparaginase was performed in C57BL/6 mice by both small-animal SPECT/CT and ex vivo biodistribution studies. Mice were treated with liposomal clodronate to investigate the effect of macrophage depletion on tracer localization and drug clearance in vivo. Moreover, macrophage cell line models RAW264.7 and THP-1, as well as knockout mice, were used to identify the cellular and molecular components controlling asparaginase pharmacokinetics. Results: In vivo imaging and biodistribution studies showed a rapid accumulation of asparaginase in macrophage-rich tissues such as the liver, spleen, and in particular bone marrow. Clodronate-mediated depletion of phagocytic cells markedly prolonged the serum half-life of asparaginase in vivo and decreased drug uptake in these macrophage-rich organs. Immunohistochemistry and in vitro binding assays confirmed the involvement of macrophagelike cells in the uptake of asparaginase. We identified the activity of the lysosomal protease cathepsin B in macrophages as a rate-limiting factor in degrading asparaginase both in vitro and in vivo. Conclusion: We showed that asparaginase is rapidly cleared from the serum by liver-, spleen-, and bone marrow–resident phagocytic cells. As a consequence of this efficient uptake and protease-mediated degradation, particularly bone marrow–resident macrophages may provide a protective niche to leukemic cells.

Radiolabeled Imaging Probes Targeting Angiogenesis for Personalized Medicine

Angiogenesis is essential for tumor growth and inhibiting angiogenesis has become an important therapeutic strategy in clinical oncology. Nonetheless, the mechanisms behind anti-angiogenic therapeutics as well as resistance to these drugs remain unclear. With a lack of validated genetic or molecular biomarkers for anti-angiogenic responsiveness, novel methods to identify responsive patients are required. Non-invasive nuclear imaging would allow the elucidation of the basic drug mechanisms as well as resistance routes and aid the personalization of anti-angiogenic therapy by enabling target expression quantification prior to and during treatment. This review focuses on the development of radiolabeled probes to image four key proteins expressed during angiogenesis, namely vascular endothelial growth factor and its receptor, integrin αv β3, the extracellular domain of fibronectin and matrix metalloproteases, and how these probes can be utilized for personalized anti-angiogenic therapy.