Current methods for hyperpolarized [1-13C]pyruvate MRI human studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.

(99m)Tc-Annexin A5 quantification of apoptotic tumor response: a systematic review and meta-analysis of clinical imaging trials.

PURPOSE: (99m)Tc-Annexin A5 has been used as a molecular imaging probe for the visualization, characterization and measurement of apoptosis. In an effort to define the quantitative (99m)Tc-annexin A5 uptake criteria that best predict tumor response to treatment, we performed a systematic review and meta-analysis of the results of all clinical imaging trials found in the literature or publicly available databases. METHODS: Included in this review were 17 clinical trials investigating quantitative (99m)Tc-annexin A5 (qAnx5) imaging using different parameters in cancer patients before and after the first course of chemotherapy and/or radiation therapy. Qualitative assessment of the clinical studies for diagnostic accuracy was performed using the QUADAS-2 criteria. Of these studies, five prospective single-center clinical trials (92 patients in total) were included in the meta-analysis after exclusion of one multicenter clinical trial due to heterogeneity. Pooled positive predictive values (PPV) and pooled negative predictive values (NPV) (with 95% CI) were calculated using Meta-Disc software version 1.4. RESULTS: Absolute quantification and/or relative quantification of (99m)Tc-annexin A5 uptake were performed at baseline and after the start of treatment. Various quantitative parameters have been used for the calculation of (99m)Tc-annexin A5 tumor uptake and delta (Δ) tumor changes post-treatment compared to baseline including: tumor-to-background ratio (TBR), ΔTBR, tumor-to-noise ratio, relative tumor ratio (TR), ΔTR, standardized tumor uptake ratio (STU), ΔSTU, maximum count per pixel within the tumor volume (Cmax), Cmax%, absolute ΔU and percentage (ΔU%), maximum ΔU counts, semiquantitative visual scoring, percent injected dose (%ID) and %ID/cm(3). Clinical trials investigating qAnx5 imaging have included patients with lung cancer, lymphoma, breast cancer, head and neck cancer and other less common tumor types. In two phase I/II single-center clinical trials, an increase of ≥25% in uptake following treatment was considered a significant threshold for an apoptotic tumor response (partial response, complete response). In three other phase I/II clinical trials, increases of ≥28%, ≥42% and ≥47% in uptake following treatment were found to be the mean cut-off levels in responders. In a phase II/III multicenter clinical trial, an increase of ≥23% in uptake following treatment was found to be the minimum cut-off level for a tumor response. In one clinical trial, no significant difference in (99m)Tc-annexin A5 uptake in terms of %ID was found in healthy tissues after chemotherapy compared to baseline. In two other clinical trials, intraobserver and interobserver measurements of (99m)Tc-annexin A5 tumor uptake were found to be reproducible (mean difference <5%, kappa = 0.90 and 0.82, respectively) and to be highly correlated with treatment outcome (Spearman r = 0.99, p < 0.0001). The meta-analysis demonstrated a pooled positive PPV of 100% (95% CI 92 - 100%) and a pooled NPV of 70% (95% CI 55 - 82%) for prediction of a tumor response after the first course of chemotherapy and/or radiotherapy in terms of ΔU%. In a symmetric sROC analysis, the AUC was 0.919 and the Q* index was 85.21 %. CONCLUSION: Quantitative (99m)Tc-annexin A5 imaging has been investigated in clinical trials for the assessment of apoptotic tumor responses. This meta-analysis showed a high pooled PPV and a moderate pooled NPV with ΔU cut-off values ranging between 20% and 30%. Standardization of quantification and harmonization of results are required for high-quality clinical research. A standardized uptake value score (SUV, ΔSUV) using quantitative SPECT/CT imaging may be a promising approach to the simple, reproducible and semiquantitative assessment of apoptotic tumor changes.

Current Methods for Hyperpolarized [1-13C]pyruvate MRI Human Studies.

MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of hyperpolarized agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate - by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation, (2) MRI system setup and calibrations, (3) data acquisition and image reconstruction, and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods & Equipment study groups. It further aims to provide a comprehensive reference for future consensus building as the field continues to advance human studies with this metabolic imaging modality.

The RSNA QIBA Profile for Amyloid PET as an Imaging Biomarker for Cerebral Amyloid Quantification.

A standardized approach to acquiring amyloid PET images increases their value as disease and drug response biomarkers. Most 18F PET amyloid brain scans often are assessed only visually (per regulatory labels), with a binary decision indicating the presence or absence of Alzheimer disease amyloid pathology. Minimizing technical variance allows precise, quantitative SUV ratios (SUVRs) for early detection of ß-amyloid plaques and allows the effectiveness of antiamyloid treatments to be assessed with serial studies. Methods: The Quantitative Imaging Biomarkers Alliance amyloid PET biomarker committee developed and validated a profile to characterize and reduce the variability of SUVRs, increasing statistical power for these assessments. Results: On achieving conformance, sites can justify a claim that brain amyloid burden reflected by the SUVR is measurable to a within-subject coefficient of variation of no more than 1.94% when the same radiopharmaceutical, scanner, acquisition, and analysis protocols are used. Conclusion: This overview explains the claim, requirements, barriers, and potential future developments of the profile to achieve precision in clinical and research amyloid PET imaging.

Hands-on molecular imaging: real-time visualization tools bridge gaps in translational medicine.

Molecular imaging tools such as CT, MRI, PET and SPECT, as well as various combinations of these instrument systems, continue to improve and evolve, offering increasingly sensitive and high-resolution images of biological processes in real time. The optimal use of these tools across the continuum of biomedical research and clinical medicine can generate the information that is needed to bridge the gaps that currently exist in drug discovery and development. These gaps negatively affect the promise and potential of translational medicine, in which the knowledge gained from multidisciplinary efforts encompassing genomics, proteomics, biomarker discovery, systems biology and bioinformatics are used to drive R&D, design experiments, predict outcomes, guide patient selection for clinical trials, and define pharmacogenomic parameters for optimizing the safety and efficacy of drug compounds. Thus, molecular imaging tools serve an important role in optimizing the drug discovery and development process.

Translational imaging: imaging of apoptosis.

Since its original description in 1972, apoptosis or programmed cell death has been recognized as the major pathway by which the body precisely regulates the number and type of its cells as part of normal embryogenesis, development, and homeostasis. Later it was found that apoptosis was also involved in the pathogenesis of a number of human diseases, cell immunity, and the action of cytotoxotic drugs and radiation therapy in cancer treatment. As such, the imaging of apoptosis with noninvasive techniques such as with radiotracers, including annexin V and lipid proton magnetic resonance spectroscopy, may have a wide range of clinical utility in both the diagnosis and monitoring therapy of a wide range of human disorders. In this chapter we review the basic biochemical and morphologic features of apoptosis and the methods developed thus far to image this complex process in humans.

The future of SPECT in a time of PET.

As positron emission tomography (PET) imaging is becoming more prevalent in clinical practice, it is reasonable to ask if there will be a role for single photon emission computed tomography (SPECT) in the future. This article considers that question, focusing on areas where SPECT can differentiate itself from PET for fundamental reasons: breadth of available radionuclides, simultaneous imaging of multiple agents, cost-effectiveness and adaptability to specific imaging situations. The conclusion is that SPECT will continue to evolve and exist alongside PET and will grow the field of molecular imaging with improved efficiency and patient workflow.

Radiolabeling small RNA with technetium-99m for visualizing cellular delivery and mouse biodistribution.

To develop a noninvasive direct method for the in vivo tracking of small interfering RNA (siRNA) used in RNA interference, two 18-nucleotide oligoribonucleotides were radiolabeled with technetium-99m ((99m)Tc-RNA). The ability of (99m)Tc-RNA to track delivery was tested in cultured cells and living mice. The cellular delivery of (99m)Tc-RNAs could be quantified by gamma counting and could be visualized by microautoradiography. Radiolabeled RNAs can be efficiently delivered into cells by reaching up to 3x10(5) molecules of small RNAs per cell. Moreover, RNAs were internalized with homogeneous distribution throughout the cytoplasm and nucleus. In tumor-bearing mice, whole-body images and biodistribution studies showed that (99m)Tc-RNAs were delivered to almost all tissues after intravenous injection. The imaging of living animals allowed noninvasive and longitudinal monitoring of the in vivo delivery of these small RNAs. In conclusion, using (99m)Tc radiolabeling, the delivery of small RNAs could be measured quantitatively in cultured cells and could be noninvasively visualized in living animals using a gamma camera. The results of this study could open up a new approach for measuring the in vivo delivery of small RNAs that might further facilitate the development of siRNAs as targeted therapies.

Improved detection of cell death in vivo with annexin V radiolabeled by site-specific methods.

UNLABELLED: Labeled annexin V is widely used to detect cell death in vitro and in vivo. Nearly all studies have been done with annexin V derivatized via amine-directed bifunctional agents; it was thought that these molecules retained full bioactivity compared with unmodified protein. We now show that this assumption is incorrect by measuring the affinity of annexin V for cells in vitro by quantitative calcium titration under conditions of low membrane occupancy. METHODS: Annexin V was modified with 4 different amine-directed agents: the N-hydroxysuccinimide esters of hydrazinonicotinic acid, mercaptoacetyltriglycine, and biotin; and with fluorescein isothiocyanate. RESULTS: In all cases, the membrane-binding affinity was decreased by derivatization, even at very low average stoichiometries. A statistical model based on the Poisson distribution accurately predicted the observed heterogeneity of derivatization as a function of average derivatization stoichiometry. This model also showed that multiply derivatized forms, which are the ones most likely to have compromised bioactivity, contributed disproportionately to the binding and imaging signals. The in vitro binding assay correctly predicted in vivo uptake in a mouse liver model of apoptosis for all proteins tested. The annexin V-128 protein, labeled at a single specific site at the N terminus, showed twice as much apoptosis-specific liver uptake as did all forms of annexin V derivatized randomly via amino groups. CONCLUSION: The membrane-binding activity of annexin V is much more sensitive to amine-directed chemical modification than previously realized. New annexin V molecules labeled by site-specific methods will greatly improve sensitivity for detecting cell death in vivo.

Evaluation of 99mTc-MAG3-annexin V: influence of the chelate on in vitro and in vivo properties in mice.

We conjugated mercaptoacetyltriglycine (MAG(3)) to rh-annexin V to permit radiolabeling with (99m)Tc in an effort to decrease the high kidney and liver accumulation observed for (99m)Tc-labeled Hynic-annexin V. The 36-kDa protein was conjugated at a 5:1 molar ratio with NHS-MAG(3) in HEPES buffer pH 7.8 at room temperature, then quenched with glycine and purified by dialysis. The biopotency of the resulting MAG(3)-annexin was similar to that of Hynic-annexin as determined by a sensitive red blood cell membrane affinity binding assay and a surface plasmon resonance (SPR) assay. The (99m)Tc radiolabeling of MAG(3)-annexin resulted in radiochemical yields of 90% under mildly basic pH conditions. Biodistribution data in normal mice clearly showed a significant decrease in kidney and liver uptake at 1 h postinjection for the (99m)Tc MAG(3)-annexin compared to the (99m)Tc Hynic-annexin (from 24% ID to 4% ID for the liver, and 45% ID to 15% ID for the kidneys, respectively). Autoradiography of the kidneys showed retention of radioactivity in the collecting tubules following administration of both labeled annexins. The (99m)Tc MAG(3)-annexin biodistribution was also characterized by a lower retention of radioactivity in the whole body, but with small intestine accumulation over fivefold higher than observed with (99m)Tc Hynic-annexin. These findings show a definite improvement in renal and hepatic clearance of the MAG(3) radioligand. However, due to the increased radioactivity uptake in the small intestines, the early in vivo detection of ongoing apoptosis in the lower abdomen might be more difficult with (99m)Tc MAG(3)-annexin. Nevertheless, (99m)Tc MAG(3)-annexin may be an attractive alternative to (99m)Tc Hynic-annexin for the in vivo imaging of phosphatidylserine receptors.

Construction of a novel chimera consisting of a chelator-containing Tat peptide conjugated to a morpholino antisense oligomer for technetium-99m labeling and accelerating cellular kinetics.

The attempt to target the limited copies of messenger RNA (mRNA) in vivo with radiolabeled nucleobase oligomers as antisense probes is challenging. Selecting an antisense molecule with superior properties, enhancing the cellular kinetics, and improving the radiolabeling chemistry would be the reasonable approach to accomplish this goal. The present study reports a method to construct a chimera of phosphorodiamidate morpholino nucleobase oligomer (MORF) covalently conjugated to a peptide containing a cell membrane transduction Tat peptide and an N(2)S(2) chelator for technetium-99m ((99m)Tc) radiolabeling (N(2)S(2)-Tat-MORF). The radiolabeling properties and cellular kinetics of (99m)Tc-N(2)S(2)-Tat-MORF were measured. As hypothesized, the preparation of (99m)Tc-N(2)S(2)-Tat-MORF could be achieved by an instant one-step method with labeling efficiency greater than 95%, and the (99m)Tc-N(2)S(2)-Tat-MORF showed distinct properties in cell culture from those of a control, the same MORF sequence without Tat but with mercaptoacetyltriglycine (MAG(3)) as chelator for (99m)Tc ((99m)Tc-MAG(3)-MORF). (99m)Tc-N(2)S(2)-Tat-MORF achieved maximum accumulation of about 35% within 2 h, while (99m)Tc-MAG(3)-MORF showed lower and steadily increasing accumulations but of less than 1% in 24 h. These preliminary results demonstrated that the proposed chimera has properties for easy labeling, and (99m)Tc-N(2)S(2)-Tat-MORF prepared by this method possesses enhanced cellular kinetics and merits further investigation for in vivo mRNA targeting.

Targeting of apoptotic macrophages and experimental atheroma with radiolabeled annexin V: a technique with potential for noninvasive imaging of vulnerable plaque.

BACKGROUND: Apoptosis is common in advanced human atheroma and contributes to plaque instability. Because annexin V has a high affinity for exposed phosphatidylserine on apoptotic cells, radiolabeled annexin V may be used for noninvasive detection of apoptosis in atherosclerotic lesions. METHODS AND RESULTS: Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the infradiaphragmatic aorta followed by 12 weeks of cholesterol diet; 5 controls were studied without manipulation. Animals were injected with human recombinant annexin V labeled with technetium-99m before imaging. Aortas were explanted for ex vivo imaging, macroautoradiography, and histological characterization of plaque. Radiolabeled annexin V cleared rapidly from the circulation (T1/2, alpha 9 and beta 46 minutes). There was intense uptake of radiolabel within lesions by 2 hours; no uptake was seen in controls. The results were confirmed in the ex vivo imaging of the explanted aorta. Quantitative annexin uptake was 9.3-fold higher in lesion versus nonlesion areas; the lesion-to-blood ratio was 3.0+/-0.37. Annexin uptake paralleled lesion severity and macrophage burden; no correlation was observed with smooth muscle cells. DNA fragmentation staining of apoptotic nuclei was increased in advanced lesions with evolving necrotic cores, predominantly in macrophages; the uptake of radiolabel correlated with the apoptotic index. CONCLUSIONS: Because annexin V clears rapidly from blood and targets apoptotic macrophage population, it should constitute an attractive imaging agent for the noninvasive detection of unstable atherosclerotic plaques.

Quantitative tumor apoptosis imaging using technetium-99m-HYNIC annexin V single photon emission computed tomography.

PURPOSE: Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m- (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis. PATIENTS AND METHODS: Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices. RESULTS: Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P =.012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered. CONCLUSION: In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.

Synthesis, biodistribution and effects of farnesyltransferase inhibitor therapy on tumour uptake in mice of 99mTc labelled epidermal growth factor.

OBJECTIVE: The goal of this study was to develop a 99mTc labelled human epidermal growth factor (hEGF) for the in-vivo prediction of cancer cell response to farnesyltransferase inhibitor (FTI) therapy. This is based on the observation that internalization of EGF receptors is inhibited by FTIs. METHODS: We describe the radiolabelling of 99mTc-hEGF using the hydrazinonicotinamide (HYNIC) linker. Binding characteristics of 99mTc-HYNIC-hEGF to the EGF receptor are explored using an in-vitro binding assay. Biodistribution data of the compound in mice and tumour uptake in LoVo tumour bearing athymic mice before and after farnesyltransferase inhibitor therapy are presented. RESULTS: No colloid formation was observed. Binding parameters and LoVo tumour uptake of 99mTc-HYNIC-hEGF did not differ significantly from directly labelled 123I-hEGF values. However, the biodistribution data of the 99mTc-HYNIC-hEGF showed higher uptake in liver and intestines and decreased stomach uptake compared to its 123I analogue. Eight hours after farnesyltransferase inhibitor therapy with R115777, LoVo tumour uptake of 99mTc-HYNIC-hEGF decreased significantly, as shown using planar gamma scintigraphy (the ratio tumour vs. thigh dropped from 2.54+/-0.83 to 0.99+/-0.18). These data confirm the results obtained using 123I-hEGF. CONCLUSION: These data suggest that 99mTc-HYNIC-hEGF is a promising and selective new radiotracer for in-vivo monitoring of the EGF receptor with SPECT. Moreover, 99mTc-HYNIC-hEGF is a possible tool for early therapy response prediction of farnesyltransferase inhibitors.

Safety, biodistribution, and dosimetry of 99mTc-HYNIC-annexin V, a novel human recombinant annexin V for human application.

UNLABELLED: 99mTc-hydrazinonicotinamido (HYNIC)-annexin V is a novel tracer for in vivo imaging of apoptosis. The present study on humans was performed to investigate the safety of (99m)Tc-HYNIC-annexin V and to quantify the biodistribution and radiation dose. METHODS: Six healthy, male volunteers participated in the study. A dual-head gamma camera was used to acquire conjugate anterior and posterior views. Imaging started with a transmission scan using a (57)Co-flood source to obtain a map of the local thickness of the volunteer. Approximately 250 MBq of (99m)Tc-HYNIC-annexin V were injected intravenously, directly followed by a 30-min dynamic study. Whole-body scans were obtained at about 30 min, 3 h, 6 h, and 24 h after injection. Organ uptake was determined after correction for background, scatter, and attenuation. The MIRDOSE3.1 program was used to calculate organ-absorbed doses and effective dose. Signs of adverse effects were investigated by monitoring renal and liver function, hematology, blood coagulation, and vital signs (blood pressure, pulse, respiration rate, temperature, and electrocardiogram). RESULTS: The kidneys accumulated 49.7 +/- 8.1 percentage injected dose (%ID) at 3 h after injection; the liver, 13.1 +/- 1.0 %ID; the red marrow, 9.2 +/- 1.8 %ID; and the spleen, 4.6 +/- 1.6 %ID. More than 90% of the blood activity was cleared with a half-life of 24 +/- 3 min. The biologic half-life of the activity registered over the total body was long (69 +/- 7 h). Excretion of the activity was almost exclusively through the urine (22.5 +/- 3.5 %ID at 24 h), and hardly any activity was seen in the bowel or feces. Absorbed doses were found to be 196 +/- 31 micro Gy/MBq for the kidneys, 41 +/- 12 micro Gy/MBq for the spleen, 16.9 +/- 1.3 micro Gy/MBq for the liver, and 8.4 +/- 0.9 micro Gy/MBq for the red marrow. The effective dose was 11.0 +/- 0.8 micro Sv/MBq, or 2.8 +/- 0.2 mSv for the average injected activity of 250 MBq. No adverse effects were observed. CONCLUSION: (99m)Tc-HYNIC-annexin V is a safe radiopharmaceutical, having a favorable biodistribution for imaging of apoptosis in the abdominal as well as thoracic area with an acceptable radiation dose.

Annexin V imaging of acute doxorubicin cardiotoxicity (apoptosis) in rats.

UNLABELLED: Anthracyclines are widely used in chemotherapy regimens for several malignancies, with cardiotoxicity being the major limiting factor in high-dose schedules. Recently, it was reported that doxorubicin induces apoptosis in cardiac muscle cells in vivo and, as such, is expected to be involved in the genesis of doxorubicin-induced cardiomyopathy. The aim of this study was to validate an animal model for in vivo monitoring of doxorubicin cardiotoxicity by means of scintigraphic detection of apoptosis. METHODS: Three groups of 5 male Wistar rats each were treated for 3, 4, and 5 times with a weekly intraperitoneal injection of doxorubicin at 2.5 mg/kg. At 24 h before and 24 h after the final treatment, (99m)Tc-annexin pinhole scintigraphy was performed. A control group of 5 rats was scanned without doxorubicin treatment. A cardiac uptake ratio was calculated from planar scintigraphy results with the following formula: (mediastinum - fat)/fat. After scintigraphy, the rats were sacrificed, and the heart was processed for histologic analysis. RESULTS: Incremental general signs of illness were observed with increasing total cumulative doxorubicin dose. Rats treated for 3, 4, and 5 wk with doxorubicin showed significantly higher uptake ratios of, respectively, 4.0 +/- 0.52 (mean +/- SEM), 4.8 +/- 0.46, and 5.2 +/- 0.17 after the final treatment; the ratio for controls was 1.84 +/- 0.05 (P < 0.05). Histologic analysis confirmed cardiac stress in treated groups, with an increasing left ventricular atrial natriuretic factor messenger RNA expression level with increasing cumulative doxorubicin dose. Late apoptosis was confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling in the rats treated for 5 wk. CONCLUSION: Acute doxorubicin-induced cardiomyopathy based on early apoptosis can be assessed and imaged with annexin V scintigraphy in rats. This finding makes it possible to use this animal model for repetitive noninvasive evaluation of cardioprotective regimens for anthracycline cardiotoxicity.

Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor.

UNLABELLED: Direct radiolabeling of proteins can result in the loss of targeting activity, requires highly customized procedures, and yields heterogeneous products. Here we describe a novel imaging complex comprised of a standardized (99m)Tc-radiolabeled adapter protein noncovalently bound to a "Docking tag" fused to a "Targeting protein". The assembly of this complex is based on interactions between human 109-amino acid (HuS) and 15-amino acid (Hu-tag) fragments of ribonuclease I, which serve as an "Adapter protein" and a Docking tag, respectively. METHODS: HuS modified with hydrazinonicotinamide (HYNIC) was radiolabeled using (99m)Tc-tricine to a specific activity of 3.4-7.4 MBq/microg. Protein complexes were then formed by mixing (99m)Tc-HuS with equimolar amounts of either Hu-tagged VEGF(121) (Hu-VEGF [vascular endothelial growth factor]) or Hu-tagged anti-VEGFR-2 single-chain antibody (Hu-P4G7) and incubating on ice for 15 min. 4T1 luc/gfp luciferase-expressing murine mammary adenocarcinoma cells (1 x 10(4)) were implanted subcutaneously or injected intravenously into BALB/c mice. Bioluminescent imaging (BLI) was performed 10 d later. Immediately after BLI visualization of tumor, 18.5-37 MBq of tracer (5-10 microg of protein) were injected via tail vein. One hour later planar or SPECT images were obtained, followed by killing the mice. RESULTS: There was significantly (P = 0.0128) increased uptake of (99m)Tc-HuS/Hu-VEGF (n = 10) within subcutaneous tumor as compared with (99m)Tc-HuS/Hu-P4G7 (n = 5) at biodistribution assay (2.68 +/- 0.75 vs. 1.8 +/- 0.21; tumor-to-subcutaneous tissue [ratio of specific activities], respectively), despite similar molecular weights. The focal (99m)Tc-HuS/Hu-VEGF uptake seen on planar images (3.44 +/- 1.16 [tumor to soft-tissue background]) corresponded directly to the locations of tumor observed by BLI. Region of interest analyses of SPECT images revealed a significant increase of (99m)Tc-HuS/Hu-VEGF (n = 5) within the lungs with BLI-detectable pulmonary tumor nodules as compared with controls (n = 4) (right: 4.47 +/- 2.07 vs. 1.79 +/- 0.56; left: 3.66 +/- 1.65 vs. 1.62 +/- 0.45, tumor lung [counts/pixel]/normal lung [counts/pixel], respectively). CONCLUSION: (99m)Tc-HuS/Hu-VEGF complex is stable for at least 1 h in vivo and can be effectively used to image mouse tumor neovasculature in lesions as small as several millimeters in soft tissue. We expect that a similar approach can be adapted for in vivo delivery of other targeting proteins of interest without affecting their bioactivity.

Optimization of the preparation of 99mTc-labeled Hynic-derivatized Annexin V for human use.

Hydrazino nicotinate (Hynic) is one of the most attractive bifunctional agents designed for the labeling of proteins with (99m)Tc. Recently, a (99m)Tc-labeled Hynic-Annexin V derivative has been described and successfully evaluated in animal models of apoptosis. Prior to a phase I human study, the preparation of (99m)Tc-Hynic-Annexin V has been optimized. The influence of the Hynic-load of Annexin V, amount of protein, nature and amount of reducing agent, activity and co-ligand on the labeling yield were evaluated using ITLC and size-exclusion FPLC. Optimal labeling yields were obtained when 60-90 microgram Hynic-Annexin V was labeled with up to 1.11 GBq (30 mCi) (99m)TcO(4)-using 10-20 microgram SnCl(2).2H(2)O as reducing agent and 1.5 mg tricine as the co-ligand. Biodistribution in normal mice was comparable to literature data.

Radiolabelling morpholinos with 188Re tricarbonyl provides improved in vitro and in vivo stability to re-oxidation.

BACKGROUND: For pretargeting and other nuclear medicine applications, it may eventually be useful to radiolabel phosphodiamidate morpholinos (MORFs) with therapeutic radionuclides such as 188Re. However, by preparing 188Re-MORFs labelled conventionally with MAG3 as chelator, we have observed unacceptable levels of oxidation to perrhenate in vitro and in vivo in mice. OBJECTIVE: To improve upon stability, tricarbonyl labelling was considered since tricarbonyl complexes are thought to stabilize metals in low oxidation states. METHODS: An amine derivatized 25 mer MORF was conjugated with either NHS-MAG3 or NHS-Hynic. The MAG3 conjugated MORF was radiolabelled conventionally with 188Re while the Hynic conjugated MORF was radiolabelled through its tricarbonyl intermediate. Using a commercial kit modified with additional reducing agent over that required for the preparation of the 99mTc tricarbonyl complex [99mTc(CO)3(H2O)3]+, we demonstrated that the equivalent 188Re tricarbonyl, [188Re(CO)3(H2O)3]+, could be prepared. Simple incubation at elevated temperatures with the Hynic conjugated MORF then provided 188Re-(CO)3-Hynic-MORF. Confirmation was achieved by a shift assay using a complementary MORF conjugated polymer and size exclusion HPLC. To evaluate the relative stability of the tricarbonyl labelled MORF compared to the MAG3 labelled MORF in vitro, the radiolabelled MORFs were incubated in phosphate buffer and the presence of perrhenate measured periodically by strip chromatography. Stability in vivo was evaluated by biodistribution studies in normal mice. RESULTS: The overall yields for tricarbonyl intermediates averaged greater than 90% for 99mTc and 60-80% for 188Re. Yields following subsequent labelling to Hynic-MORF were about 60-80% for 99mTc and 15-20% for 188Re. The in vitro stability results in phosphate buffer showed that 188Re-MAG3-MORF was fully oxidized by 48 h while 188Re-(CO)3-Hynic-MORF was less than 20% oxidized at that time. Similarly, the 188Re-(CO)3-Hynic-MORF biodistribution in normal mice showed lower radioactivity level in stomach, intestines and thyroid compared with 188Re-MAG3-MORF. CONCLUSION: 188Re-tricarbonyl labelling of Hynic conjugated MORFs may be considerably more stable to oxidation than the MAG3 labelled MORFs and therefore more suitable for radiotherapy trials.

99mTc-HYNIC Annexin-V imaging of primary head and neck carcinoma.

In this study, the potential of 99mTc-HYNIC Annexin-V scintigraphy to visualize primary head and neck carcinoma was assessed and compared with computed tomography (CT) findings and histology. Eighteen patients suspected of having primary head and neck carcinoma underwent a spiral CT scan and 99mTc-HYNIC Annexin-V scintigraphy within 1 week of each other, followed by resection of the suspected lesion. Results obtained by CT and scintigraphy were compared vs. histopathology. The diagnosis was primary head and neck carcinoma in 18 patients, accompanied by lymph node involvement in seven patients. 99mTc-HYNIC Annexin-V uptake was identified in five patients on planar images and in 17 patients on tomographic images (single-photon emission computed tomography, SPECT), corresponding to the pathological regions identified by CT. In the remaining patient, CT and 99mTc-HYNIC Annexin-V scintigraphy were false negative. In 11 patients, SPECT and CT scan were concordant, identifying all primary lesions and two sites of lymph node involvement. In the six remaining patients, CT and SPECT accurately identified the primary lesion, but were discordant with regard to the existence of lymph node involvement. In five of six patients, SPECT failed to identify lymph node involvement, whereas CT scan did not. In the remaining patient, CT scan was false positive for lymph node involvement, whereas SPECT was not. In this series, 99mTc-HYNIC Annexin-V allowed for the visualization of all primary head and neck tumours identified by CT scan, but failed to identify most of the sites of lymph node involvement.