Synthesis and in vivo evaluation of [11C]tucatinib for HER2-targeted PET imaging.

Tucatinib is a selective human epidermal growth factor receptor 2 (HER2) tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration (FDA) in April 2020 for HER2-positive lesions in metastatic breast cancer patients, including CNS metastases. In this article, we attempted to develop the first small molecule, blood-brain-barrier (BBB) penetrant HER2 PET imaging probe based on tucatinib. [11C]tucatinib was synthesized via a Stille-coupling from the respective trimethylstannyl precursor and its biodistribution was evaluated in NMRI nude mice bearing HER2-overexpressing human ovarian cancer cells (SKOV-3). No significant tumor accumulation was observed despite its high affinity for HER-2 receptors (IC50 = 6.9 nM). High liver and intestinal uptake indicate that [11C]tucatinib is too lipophilic to be used as a tumor targeting PET tracer. Therefore, chemical modifications of [11C]tucatinib are needed to increase the polarity for tumor imaging. Tucatinib as an FDA approved drug is still an interesting platform to develop the first small molecule HER2-selective PET tracer. The study highlights the differences between a drug, which needs to be effective, and an imaging agent, which is dependent on contrast.

Bioorthogonal Click of Colloidal Gold Nanoparticles to Antibodies In vivo.

Combining nanotechnology and bioorthogonal chemistry for theranostic strategies offers the possibility to develop next generation nanomedicines. These materials are thought to increase therapeutic outcome and improve current cancer management. Due to their size, nanomedicines target tumors passively. Thus, they can be used for drug delivery purposes. Bioorthogonal chemistry allows for a pretargeting approach. Higher target-to-background drug accumulation ratios can be achieved. Pretargeting can also be used to induce internalization processes or trigger controlled drug release. Colloidal gold nanoparticles (AuNPs) have attracted widespread interest as drug delivery vectors within the last decades. Here, we demonstrate for the first time the possibility to successfully ligate AuNPs in vivo to pretargeted monoclonal antibodies. We believe that this possibility will facilitate the development of AuNPs for clinical use and ultimately, improve state-of-the-art patient care.

Direct Cu-mediated aromatic 18F-labeling of highly reactive tetrazines for pretargeted bioorthogonal PET imaging.

Pretargeted imaging can be used to visualize and quantify slow-accumulating targeting vectors with short-lived radionuclides such as fluorine-18 - the most popular clinically applied Positron Emission Tomography (PET) radionuclide. Pretargeting results in higher target-to-background ratios compared to conventional imaging approaches using long-lived radionuclides. Currently, the tetrazine ligation is the most popular bioorthogonal reaction for pretargeted imaging, but a direct 18F-labeling strategy for highly reactive tetrazines, which would be highly beneficial if not essential for clinical translation, has thus far not been reported. In this work, a simple, scalable and reliable direct 18F-labeling procedure has been developed. We initially studied the applicability of different leaving groups and labeling methods to develop this procedure. The copper-mediated 18F-labeling exploiting stannane precursors showed the most promising results. This approach was then successfully applied to a set of tetrazines, including highly reactive H-tetrazines, suitable for pretargeted PET imaging. The labeling succeeded in radiochemical yields (RCYs) of up to approx. 25%. The new procedure was then applied to develop a pretargeting tetrazine-based imaging agent. The tracer was synthesized in a satisfactory RCY of ca. 10%, with a molar activity of 134 ± 22 GBq µmol-1 and a radiochemical purity of >99%. Further evaluation showed that the tracer displayed favorable characteristics (target-to-background ratios and clearance) that may qualify it for future clinical translation.

Development of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging-Expanding the Bioorthogonal Tool Box.

Pretargeted imaging of nanomedicines have attracted considerable interest because it has the potential to increase imaging contrast while reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction for this strategy and, consequently, the state-of-art choice for in vivo chemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small SAR study was performed. The most promising compound was selected for labeling and subsequent positron-emission-tomography in vivo imaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [18F]15 displayed favorable pharmacokinetics and remarkable target-to-background ratios-as early as 1 h post injection. We believe that this agent could be a promising candidate for translation into clinical studies.

Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted In Vivo Chemistry.

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M-1 s-1) for the reaction with TCO and low calculated logD 7.4 values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

Evaluation of [64Cu]Cu-NOTA-PEG7-H-Tz for Pretargeted Imaging in LS174T Xenografts-Comparison to [111In]In-DOTA-PEG11-BisPy-Tz.

Pretargeted nuclear imaging for the diagnosis of various cancers is an emerging and fast developing field. The tetrazine ligation is currently considered the most promising reaction in this respect. Monoclonal antibodies are often the preferred choice as pretargeting vector due to their outstanding targeting properties. In this work, we evaluated the performance of [64Cu]Cu-NOTA-PEG7-H-Tz using a setup we previously used for [111In]In-DOTA-PEG11-BisPy-Tz, thereby allowing for comparison of the performance of these two promising pretargeting imaging agents. The evaluation included a comparison of the physicochemical properties of the compounds and their performance in an ex vivo blocking assay. Finally, [64Cu]Cu-NOTA-PEG7-H-Tz was evaluated in a pretargeted imaging study and compared to [111In]In-DOTA-PEG11-BisPy-Tz. Despite minor differences, this study indicated that both evaluated tetrazines are equally suited for pretargeted imaging.

Evaluation of a 68Ga-Labeled DOTA-Tetrazine as a PET Alternative to 111In-SPECT Pretargeted Imaging.

The bioorthogonal reaction between a tetrazine and strained transcyclooctene (TCO) has garnered success in pretargeted imaging. This reaction was first validated in nuclear imaging using an 111In-labeled 1,4,7,10tetraazacyclododecane1,4,7,10tetraacetic acid (DOTA)-linked bispyridyl tetrazine (Tz) ([111In]In-DOTA-PEG11-Tz) and a TCO functionalized CC49 antibody. Given the initial success of this Tz, it has been paired with TCO functionalized small molecules, diabodies, and affibodies for in vivo pretargeted studies. Furthermore, the single photon emission tomography (SPECT) radionuclide, 111In, has been replaced with the ß-emitter, 177Lu and α-emitter, 212Pb, both yielding the opportunity for targeted radiotherapy. Despite use of the 'universal chelator', DOTA, there is yet to be an analogue suitable for positron emission tomography (PET) using a widely available radionuclide. Here, a 68Ga-labeled variant ([68Ga]Ga-DOTA-PEG11-Tz) was developed and evaluated using two different in vivo pretargeting systems (Aln-TCO and TCO-CC49). Small animal imaging and ex vivo biodistribution studies were performed and revealed target specific uptake of [68Ga]Ga-DOTA-PEG11-Tz in the bone (3.7 %ID/g, knee) in mice pretreated with Aln-TCO and tumor specific uptake (5.8 %ID/g) with TCO-CC49 in mice bearing LS174 xenografts. Given the results of this study, [68Ga]Ga-DOTA-PEG11-Tz can serve as an alternative to [111In]In-DOTA-PEG11-Tz.

Trans-Cyclooctene-Functionalized PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging.

Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.

Improved radiosynthesis and preliminary in vivo evaluation of the 11C-labeled tetrazine [11C]AE-1 for pretargeted PET imaging.

Pretargeted nuclear imaging based on the ligation between tetrazines and nano-sized targeting agents functionalized with trans-cyclooctene (TCO) has recently been shown to improve both imaging contrast and dosimetry in nuclear imaging of nanomedicines. Herein, we describe the improved radiosynthesis of a 11C-labeled tetrazine ([11C]AE-1) and its preliminary evaluation in both mice and pigs. Pretargeted imaging in mice was carried out using both a new TCO-functionalized polyglutamic acid and a previously reported TCO-functionalized bisphosphonate system as targeting agents. Unfortunately, pretargeted imaging was not successful using these targeting agents in pair with [11C]AE-1. However, brain imaging in pig indicated that the tracer crossed the blood-brain-barrier. Hence, we suggest that this tetrazine scaffold could be used as a starting point for the development of pretargeted brain imaging, which has so far been a challenging task.

Folate receptor targeting of radiolabeled liposomes reduces intratumoral liposome accumulation in human KB carcinoma xenografts.

BACKGROUND: Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting. METHOD: Tumor accumulating potential of FTLs and NTLs were investigated in a FR overex-pressing xenograft model by positron emission tomography/computed tomography imaging. RESULTS: Tumors displayed significantly lower activity of FTLs than NTLs. Furthermore, FTLs displayed worse circulating properties and increased liver-accumulation than NTLs. CONCLUSION: This study underlines that long-circulating properties of liposomes must be achieved to take advantage of EPR-dependent tumor accumulation which may be lost by functionalization. FR-functionalization negatively affected both tumor accumulation and circulation properties.

Pretargeting in nuclear imaging and radionuclide therapy: Improving efficacy of theranostics and nanomedicines.

Pretargeted nuclear imaging and radiotherapy have recently attracted increasing attention for diagnosis and treatment of cancer with nanomedicines. This is because it conceptually offers better imaging contrast and therapeutic efficiency while reducing the dose to radiosensitive tissues compared to conventional strategies. In conventional imaging and radiotherapy, a directly radiolabeled nano-sized vector is administered and allowed to accumulate in the tumor, typically on a timescale of several days. In contrast, pretargeting is based on a two-step approach. First, a tumor-accumulating vector carrying a tag is administered followed by injection of a fast clearing radiolabeled agent that rapidly recognizes the tag of the tumor-bound vector in vivo. Therefore, pretargeting circumvents the use of long-lived radionuclides that is a necessity for sufficient tumor accumulation and target-to-background ratios using conventional approaches. In this review, we give an overview of recent advances in pretargeted imaging strategies. We will critically reflect on the advantages and disadvantages of current state-of-the-art conventional imaging approaches and compare them to pretargeted strategies. We will discuss the pretargeted imaging concept and the involved chemistry. Finally, we will discuss the steps forward in respect to clinical translation, and how pretargeted strategies could be applied to improve state-of-the-art radiotherapeutic approaches.

18F-FDG PET/CT-based early treatment response evaluation of nanoparticle-assisted photothermal cancer therapy.

Within the field of nanoparticle-assisted photothermal cancer therapy, focus has mostly been on developing novel heat-generating nanoparticles with the right optical and dimensional properties. Comparison and evaluation of their performance in tumor-bearing animals are commonly assessed by changes in tumor volume; however, this is usually a late-occurring event. This study implements 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging to perform early evaluation of the treatment outcome of photothermal therapy. Silica-gold nanoshells (NS) are administered intravenously to nude mice bearing human neuroendocrine tumor xenografts and the tumors are irradiated by a near-infrared laser. The animals are positron emission tomography scanned with 2-deoxy-2-[F-18]fluoro-D-glucose one day before and one day after treatment. Using this setup, a significant decrease in tumor uptake of 2-deoxy-2-[F-18]fluoro-D-glucose is found already one day after therapy in the group receiving NS and laser treatment compared to control animals. At this time point no change in tumor volume can be detected. Moreover, the change in tumor uptake, is used to stratify the animals into responders and non-responders, where the responding group matched improved survival. Overall, these findings support the use of 2-deoxy-2-[F-18]fluoro-D-glucose positron emission tomography imaging for preclinical and clinical evaluation and optimization of photothermal therapy.

The use of matrigel has no influence on tumor development or PET imaging in FaDu human head and neck cancer xenografts.

BACKGROUND: In preclinical research Matrixgel(TM) Basement Membrane Matrix (MG) is used frequently for the establishment of syngeneic and xenograft cancer models. Limited information on its influence on parameters including; tumor growth, vascularization, hypoxia and imaging characteristics is currently available. This study evaluates the potential effect of matrigel use in a human head and neck cancer xenograft model (FaDu; hypopharyngeal carcinoma) in NMRI nude mice. The FaDu cell line was chosen based on its frequent use in studies of cancer imaging and tumor microenvironment. METHODS: NMRI nude mice (n = 34) were divided into two groups and subcutaneously injected with FaDu cells in medium either including (+MG) or excluding matrigel (-MG). In sub study I seven mice from each group (+MG, n = 7; -MG, n = 7) were (18)F- fluorodeoxyglucose ((18)F-FDG) PET/CT scanned on Day 5, 8, 12, 15, and 19. In sub study II ten mice from each group (+MG, n = 10; -MG, n = 10) were included and tumors collected for immunohistochemistry (IHC) analysis of tumor microenvironment including; proliferation ratio, micro vessel density, average vessel area, hypoxia, nuclear density, and necrosis. Tumors for IHC were collected according to size (200-400 mm(3), 500-700 mm(3), 800-1100 mm(3)). RESULTS: FDG uptake and tumor growth was statistically compatible for the tumors established with or without MG. The IHC analysis on all parameters only identified a significantly higher micro vessel density for tumor size 500-700 mm(3) and 800-1100 mm(3) and average vessel area for tumor size 500-700 mm(3) in the -MG group. Comparable variations were observed for tumors of both the +MG and -MG groups. No difference in tumor take rate was observed between groups in study. CONCLUSIONS: Matrigel did not affect tumor growth or tumor take for the FaDu xenograft model evaluated. Tumors in the -MG group displayed increased angiogenesis compared to the +MG tumors. No difference in (18)F-FDG PET uptake for tumors of different groups was found. Based on these observations the influence of matrigel on tumor imaging and tumor microenvironment seems minor for this particular xenograft model.

Urokinase-Type Plasminogen Activator Receptor as a Potential PET Biomarker in Glioblastoma.

UNLABELLED: Glioblastoma is one of the most malignant types of human cancer, and the prognosis is poor. The development and validation of novel molecular imaging biomarkers has the potential to improve tumor detection, grading, risk stratification, and treatment monitoring of gliomas. The aim of this study was to explore the potential of PET imaging of the urokinase-type plasminogen activator receptor (uPAR) in glioblastoma. METHODS: The uPAR messenger RNA expression of tumors from 19 glioblastoma patients was analyzed, and a cell culture derived from one of these patients was used to establish an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands ((64)Cu-NOTA-AE105 and (68)Ga-NOTA-AE105) and, for comparison, O-(2-(18)F-fluoroethyl)-l-tyrosine ((18)F-FET). One MRI scan was obtained for each mouse to confirm tumor location. The uPAR specificity of (64)Cu-NOTA-AE105 was confirmed by alignment of hematoxylin- and eosin-stained and uPAR immunohistochemistry-stained slides of the brain with the activity distribution as determined using autoradiography. RESULTS: uPAR expression was found in all 19 glioblastoma patient tumors, and high expression of uPAR correlated with decreased overall survival (P = 0.04). Radiolabeling of NOTA-AE105 with (64)Cu and (68)Ga was straightforward, resulting in a specific activity of approximately 20 GBq/µmol and a radiochemical purity of more than 98% for (64)Cu-NOTA-AE105 and more than 97% for (68)Ga-NOTA-AE105. High image contrast resulting in clear tumor delineation was found for both (68)Ga-NOTA-AE105 and (64)Cu-NOTA-AE105. Absolute uptake in tumor was higher for (18)F-FET (3.5 ± 0.8 percentage injected dose [%ID]/g) than for (64)Cu-NOTA-AE105 (1.2 ± 0.4 %ID/g) or (68)Ga-NOTA-AE105 (0.4 ± 0.1 %ID/g). A similar pattern was observed in background brain tissue, where uptake was 1.9 ± 0.1 %ID/g for (18)F-fluorothymidine, compared with 0.05 ± 0.01 %ID/g for (68)Ga-NOTA-AE105 and 0.11 ± 0.02 %ID/g for (64)Cu-NOTA-AE105. The result was a significantly higher tumor-to-background ratio for both (68)Ga-NOTA-AE105 (7.6 ± 2.1, P < 0.05) and (64)Cu-NOTA-AE105 (10.6 ± 2.3, P < 0.01) than for (18)F-FET PET (1.8 ± 0.3). Autoradiography of brain slides confirmed that the accumulation of (64)Cu-NOTA-AE105 corresponded well with uPAR-positive cancer cells. CONCLUSION: On the basis of our translational study, uPAR PET may be a highly promising imaging biomarker for glioblastoma. Further clinical exploration of uPAR PET in glioblastoma is therefore justified.

64Cu-ATSM Reflects pO2 Levels in Human Head and Neck Cancer Xenografts but Not in Colorectal Cancer Xenografts: Comparison with 64CuCl2.

UNLABELLED: The hypoxia PET tracer (64)Cu-diacetyl-bis(N(4)-methylthiosemicarbazonate) ((64)Cu-ATSM) has shown promising results in clinical studies. However, concerns have been raised with regard to the possible effect of copper metabolism and free copper on tumor uptake and thereby the robustness of (64)Cu-ATSM as a hypoxia marker. In this study, accumulation and distribution of (64)Cu-ATSM and (64)CuCl2 in tumor tissue were compared with partial pressure of oxygen (pO2) probe measurements. METHODS: One-hour dynamic PET scans were performed on nude mice bearing subcutaneous human head and neck tumors (FaDu) and human colorectal tumors (HT29) after administration of either (64)Cu-ATSM or (64)CuCl2. Subsequently, tracks were generated and track markers were positioned in tumors to allow for registration of their exact location on the high-resolution CT scan. After completion of the CT scan, pO2 probe measurements were performed along each track. PET and CT images were coregistered and ROIs drawn on the basis of the location of track markers and pO2 probe measurement depth. A linear mixed model for repeated measures was applied for the comparison of PET tracer uptake to corresponding pO2 values. RESULTS: Comparable uptake of (64)Cu-ATSM and (64)CuCl2 was found in the kidney, muscle, and liver of all animals, but (64)CuCl2 showed a higher uptake 10-60 min after injection in both tumor models. Significant differences were also found for both tumor-to-muscle and tumor-to-liver ratios. The intratumoral distribution of (64)Cu-ATSM, but not (64)CuCl2, showed a significant negative relationship with pO2 measurements in FaDu tumors. However, this relationship was not found in HT29 tumors. CONCLUSION: (64)Cu-ATSM and (64)CuCl2 displayed different uptake in tumors. In human head and neck xenografts, (64)Cu-ATSM but not (64)CuCl2 reflected pO2 measurements, indicating that (64)Cu-ATSM is a hypoxia-specific marker in this tumor type. However, data from colorectal cancer xenografts indicated that (64)Cu-ATSM may not be a hypoxia marker in all tumor types.

Automated synthesis and PET evaluation of both enantiomers of [¹8F]FMISO.

INTRODUCTION: [(18)F]FMISO, the widely used positron emission tomography (PET) hypoxia tracer, is a chiral compound clinically used as a racemic mixture. The purpose of this study was to synthesize the individual (R)- and the (S)- enantiomers of [(18)F]FMISO and compare their PET imaging characteristics. METHODS: The radiosynthesis of enantiopure (R)- and (S)-[(18)F]FMISO was based on Co(salen) (N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt)-mediated opening of enantiopure epoxides with [(18)F]HF. The uptake and clearance of the individual [(18)F]FMISO antipodes were investigated using micro-PET/CT imaging performed on mice bearing FaDu tumors. Image-derived biodistribution was obtained from micro-PET/CT scans performed at 1 and 3 hours post injection (p.i.). In addition, the uptake patterns of each enantiomer were observed using two-hour dynamic micro-PET/CT scans, and the time-activity curves from different organs were compared. RESULTS: The individual (R)- and (S)-[(18)F]FMISO enantiomers were synthesized in one step with high enantiomeric excess (ee)>99% and radiochemical purity>97% using custom-made automation module. The dynamic micro-PET/CT scanning revealed a faster initial uptake of the (R)-[(18)F]FMISO enantiomer in tumor and muscle tissues, however the difference became progressively smaller with time. The tumor-to-muscle (T/M) and tumor-to-liver (T/L) ratios remained nearly identical for the (R)- and (S)-forms at all time points. The micro-PET/CT imaging at 1 and 3 hours p.i. did not show any significant enantioselective tissue uptake. CONCLUSIONS: Although the (R)-enantiomer of [(18)F]FMISO demonstrated a somewhat faster initial tumor and muscle uptake no significant enantioselective tissue uptake was observed at later time points. The T/M- and T/L- ratios for the (R)- and (S)-forms were the same within the experimental error at all times. Therefore, the use of enantiopure [(18)F]FMISO is unlikely to present any practical clinical benefit for PET imaging.

The impact of weakly bound 89Zr on preclinical studies: non-specific accumulation in solid tumors and aspergillus infection.

UNLABELLED: Preclinical studies involving (89)Zr often report significant bone accumulation, which is associated with dissociation of the radiometal from the tracer. However, experiments determining the uptake of unbound (89)Zr in disease models are not performed as routine controls. The purpose of the present study was to investigate the impact of free or weakly bound (89)Zr on PET quantifications in disease models, in order to determine if such control experiments are warranted. METHODS: Chemical studies were carried out to find a (89)Zr compound that would solubilize the (89)Zr as a weak chelate, thus mimicking free or weakly bound (89)Zr released in circulation. (89)Zr oxalate had the desired characteristics, and was injected into mice bearing FaDu and HT29 solid tumor xenografts, and mice infected in the lungs with the mold Aspergillus fumigatus, as well as in healthy controls (naïve). PET/CT or PET/MR imaging followed to quantify the distribution of the radionuclide in the disease models. RESULTS: (89)Zr oxalate was found to have a plasma half-life of 5.1 ± 2.3 h, accumulating mainly in the bones of all animals. Both tumor types accumulated (89)Zr on the order of 2-4 %ID/cm(3), which is comparable to EPR-mediated accumulation of certain species. In the aspergillosis model, the concentration of (89)Zr in lung tissue of the naïve animals was 6.0 ± 1.1 %ID/g. This was significantly different from that of the animals with advanced disease, showing 11.6 ± 1.8 %ID/g. CONCLUSIONS: Given the high levels of (89)Zr accumulation in the disease sites in the present study, we recommend control experiments mapping the biodistribution of free (89)Zr in any preclinical study employing (89)Zr where bone uptake is observed. Aqueous (89)Zr oxalate appears to be a suitable compound for such studies. This is especially relevant in studies where the tracer accumulation is based upon passive targeting, such as EPR.

(64)Cu-ATSM and (18)FDG PET uptake and (64)Cu-ATSM autoradiography in spontaneous canine tumors: comparison with pimonidazole hypoxia immunohistochemistry.

BACKGROUND: The aim of this study was to compare (64)Cu-diacetyl-bis(N(4)-methylsemicarbazone) ((64)Cu-ATSM) and (18)FDG PET uptake characteristics and (64)Cu-ATSM autoradiography to pimonidazole immunohistochemistry in spontaneous canine sarcomas and carcinomas. METHODS: Biopsies were collected from individual tumors between approximately 3 and 25 hours after the intravenous injection of (64)Cu-ATSM and pimonidazole. (64)Cu-ATSM autoradiography and pimonidazole immunostaining was performed on sectioned biopsies. Acquired (64)Cu-ATSM autoradiography and pimonidazole images were rescaled, aligned and their distribution patterns compared. (64)Cu-ATSM and (18)FDG PET/CT scans were performed in a concurrent study and uptake characteristics were obtained for tumors where available. RESULTS: Maximum pimonidazole pixel value and mean pimonidazole labeled fraction was found to be strongly correlated to (18)FDG PET uptake levels, whereas more varying results were obtained for the comparison to (64)Cu-ATSM. In the case of the latter, uptake at scans performed 3 h post injection (pi) generally showed strong positive correlated to pimonidazole uptake.Comparison of distribution patterns of pimonidazole immunohistochemistry and (64)Cu-ATSM autoradiography yielded varying results. Significant positive correlations were mainly found in sections displaying a heterogeneous distribution of tracers. CONCLUSIONS: Tumors with high levels of pimonidazole staining generally displayed high uptake of (18)FDG and (64)Cu-ATSM (3 h pi.). Similar regional distribution of (64)Cu-ATSM and pimonidazole was observed in most heterogeneous tumor regions. However, tumor and hypoxia level dependent differences may exist with regard to the hypoxia specificity of (64)Cu-ATSM in canine tumors.