Francis G. Blankenberg

Annexin-V imaging for noninvasive detection of cardiac allograft rejection

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.

Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis

Abstract. Either inadequate or excessive apoptosis (programmed cell death) is associated with many diseases. A method to image apoptosis in vivo, rather than requiring histologic evaluation of tissue, could assist with therapeutic decision making in these disorders. Programmed cell death is associated with a well-choreographed series of events resulting in the cessation of normal cell function, and the ultimate disappearance of the cell. One component of apoptosis is signaling adjacent cells that this cell is committing suicide by externalizing phosphatidylserine to the outer leaflet of the cell membrane. Annexin V, a 32-kDa endogenous human protein, has a high affinity for membrane-bound phosphatidylserine. We have coupled annexin V with the bifunctional hydrazinonicotinamide reagent (HYNIC) to prepare technetium-99m HYNIC-annexin V and demonstrated localization of radioactivity in tissues undergoing apoptosis in vivo. In this report we describe the results of a series of experiments in mice and rats to characterize the biologic behavior of 99mTc-HYNIC- annexin V. Biodistribution studies were performed in groups of rats at 10–180 min after intravenous injection of 99mTc-HYNIC-annexin V. In order to estimate the degree of apoptosis required for localization of 99mTc-annexin V in vivo, mice were treated with dexamethasone at doses ranging from 1 to 20 mg/kg, 5 h prior to 99mTc-HYNIC-annexin V administration, to induce thymic apoptosis. Thymus was excised 1 h after radiolabeled HYNIC-annexin V injection; thymocytes were isolated, incubated with Hoechst 33342 followed by propidium iodide, and analyzed on a fluorescence-activated cell sorter. Each sorted cell population was counted in a scintillation counter. To test 99mTc-HYNIC-annexin V as a tracer for external radionuclide imaging of apoptotic cell death, radionuclide imaging of Fas-defective mice (lpr/lpr mice) and wild-type mice treated with the antibody to Fas (anti-Fas) was carried out 1 h post injection. Rat biodistribution studies demonstrated a blood clearance half-time of less than 10 min for 99mTc-HYNIC-annexin V. The kidneys had the highest concentration of radioactivity at all time points. Studies in the mouse thymus demonstrated a 40-fold increase in 99mTc-HYNIC-annexin V concentration in apoptotic thymocytes compared with the viable cell population. A correlation of r=0.78 was found between radioactivity and flow cytometric and histologic evidence of apoptosis. Imaging studies in the lpr/lpr and wild-type mice showed a substantial increase of activity in the liver of wild-type mice treated with anti-Fas, while there was no significant change, irrespective of anti-Fas administration, in lpr/lpr mice. Excellent images of hepatic apoptosis were obtained in wild-type mice 30 min after injection of 99mTc-HYNIC-annexin V. The imaging results were consistent with histologic analysis in these animals. In conlusion, these studies confirm the value of 99mTc-HYNIC-annexin V uptake as a marker for the detection and quantification of apoptotic cells in vivo.

Initial experience in the treatment of inherited mitochondrial disease with EPI-743.

Inherited mitochondrial respiratory chain disorders are progressive, life-threatening conditions for which there are limited supportive treatment options and no approved drugs. Because of this unmet medical need, as well as the implication of mitochondrial dysfunction as a contributor to more common age-related and neurodegenerative disorders, mitochondrial diseases represent an important therapeutic target. Thirteen children and one adult with genetically-confirmed mitochondrial disease (polymerase γ deficiency, n=4; Leigh syndrome, n=4; MELAS, n=3; mtDNA deletion syndrome, n=2; Friedreich ataxia, n=1) at risk for progressing to end-of-life care within 90 days were treated with EPI-743, a novel para-benzoquinone therapeutic, in a subject controlled, open-label study. Serial measures of safety and efficacy were obtained that included biochemical, neurological, quality-of-life, and brain redox assessments using technetium-99m-hexamethylpropyleneamine oxime (HMPAO) single photon emission computed tomography (SPECT) radionuclide imaging. Twelve patients treated with EPI-743 have survived; one polymerase γ deficiency patient died after developing pneumonia and one patient with Surf-1 deficiency died after completion of the protocol. Of the 12 survivors, 11 demonstrated clinical improvement, with 3 showing partial relapse, and 10 of the survivors also had an improvement in quality-of-life scores at the end of the 13-week emergency treatment protocol. HMPAO SPECT scans correlated with clinical response; increased regional and whole brain HMPAO uptake was noted in the clinical responders and the one subject who did not respond clinically had decreased regional and whole brain HMPAO uptake. EPI-743 has modified disease progression in >90% of patients in this open-label study as assessed by clinical, quality-of-life, and non-invasive brain imaging parameters. Data obtained herein suggest that EPI-743 may represent a new drug for the treatment of inherited mitochondrial respiratory chain disorders. Prospective controlled trials will be undertaken to substantiate these initial promising observations. Furthermore, HMPAO SPECT imaging may be a valuable tool for the detection of central nervous system redox defects and for monitoring response to treatments directed at modulating abnormal redox.

THE USE OF TECHNETIUM TC 99M ANNEXIN V FOR IN VIVO IMAGING OF APOPTOSIS DURING CARDIAC ALLOGRAFT REJECTION

OBJECTIVE Apoptosis, or programmed cell death, has been suggested as a mechanism of immunologic injury during cardiac allograft rejection. We tested the hypothesis that technetium Tc 99m annexin V, a novel radiopharmaceutical used to detect apoptosis, can be used to detect cardiac allograft rejection by nuclear imaging. METHODS Untreated ACI rats served as recipients of allogeneic PVG rat (n = 66) or syngeneic ACI rat (n = 30) cardiac grafts. Untreated recipient animals underwent 99mTc-annexin V imaging daily for 7 days. Region of interest analysis was used to quantify the uptake of 99mTc-annexin V. Immediately after imaging grafts were procured for histopathologic analysis and terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling of apoptotic nuclei. One group was treated with 10 mg/kg/d cyclosporine (INN: ciclosporin) commencing on day 4 after transplantation (n = 6). RESULTS Untreated allografts showed histologic signs of rejection 4 days after transplantation. Apoptotic nuclei could be demonstrated in myocytes, endothelial cells, and graft-infiltrating cells of all rejecting allografts. Nuclear imaging revealed a significantly greater uptake of 99mTc-annexin V in rejecting allogeneic grafts than in syngeneic grafts on day 4 (P = .05), day 5 (P < .001), day 6 (P < .001), and day 7 (P = .013) after transplantation. A correlation between the histologic grade of acute rejection and uptake of 99mTc-annexin V was observed (r2 = 0.87). After treatment of rejection with cyclosporine, no apoptotic nuclei could be identified in allografts and uptake of 99mTc-annexin V decreased to baseline. CONCLUSIONS Apoptosis occurs during acute cardiac allograft rejection and disappears after treatment of rejection. 99mTc-annexin V can be used to detect and monitor cardiac allograft rejection.

Nuclear medicine applications in molecular imaging

With the emergence of the new field of molecular imaging, there is an increasing demand for development of sensitive and safe novel imaging agents that can be rapidly translated from small animal models into patients. Nuclear medicine and positron emission tomography (PET) techniques have the ability to detect and serially monitor a variety of biologic and pathophysiologic processes, usually with tracer quantities of radiolabeled peptides, drugs, and other molecules at doses free of pharmacologic side effects, unlike the current generation of intravenous agents required for magnetic resonance (MR) and computed tomography (CT) scanning. In this article, we will review a representative sampling of the wide array of radiopharmaceuticals developed specifically for nuclear medicine radionuclide imaging that have been approved for clinical use, and those in pre‐clinical trials. We will also review the existing strategies used to select the appropriate biologic markers and targets for radionuclide labeling that have been employed in the development of novel radiotracers and the imaging of small animals with new microSPECT (single photon emission computed tomography) technologies. J. Magn. Reson. Imaging 2002;16:352–361.

Multi-modality Imaging Identifies Key Times for Annexin V Imaging as an Early Predictor of Therapeutic Outcome

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.

99mTc Annexin V Imaging of Neonatal Hypoxic Brain Injury

Background and Purpose— Delayed cell loss in neonates after cerebral hypoxic-ischemic injury (HII) is believed to be a major cause of cerebral palsy. In this study, we used radiolabeled annexin V, a marker of delayed cell loss (apoptosis), to image neonatal rabbits suffering from HII. Methods Twenty-two neonatal New Zealand White rabbits had ligation of the right common carotid artery with reduction of inspired oxygen concentration to induce HII. Experimental animals (n=17) were exposed to hypoxia until an ipsilateral hemispheric decrease in the average diffusion coefficient occurred. After reversal of hypoxia and normalization of average diffusion coefficient values, experimental animals were injected with 99mTc annexin V. Radionuclide images were recorded 2 hours later. Results Experimental animals showed no MR evidence of blood-brain barrier breakdown or perfusion abnormalities after hypoxia. Annexin images demonstrated multifocal brain uptake in both hemispheres of experimental but not control animals. Histology of the brains from experimental animals demonstrated scattered pyknotic cortical and hippocampal neurons with cytoplasmic vacuolization of glial cells without evidence of apoptotic nuclei by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining. Double staining with markers of cell type and exogenous annexin V revealed that annexin V was localized in the cytoplasm of scattered neurons and astrocytes in experimental and, less commonly, control brains in the presence of an intact blood-brain barrier. Conclusions Apoptosis may develop after HII even in brains that appear normal on diffusion-weighted and perfusion MR. These data suggest a role of radiolabeled annexin V screening of neonates at risk for the development of cerebral palsy.

Will imaging of apoptosis play a role in clinical care? A tale of mice and men

Programmed cell death (apoptosis) plays a role in the pathophysiology of many diseases and in the outcome of treatment. Apoptosis is the likely mechanism behind the cytoreductive effects of standard chemotherapeutic and radiation treatments, rejection of organ transplants, cellular damage in collagen vascular disorders, and delayed cell death due to hypoxic-ischemic injury in myocardial infarction and neonatal hypoxic ischemic injury. Observations about the role of apoptosis have fueled the development of novel agents and treatment strategies specifically aimed at inducing or inhibiting apoptosis.Despite these research developments there are no clinical entities where specific measures of apoptosis are used in either diagnosis or patient management. Part of the difficulty in bridging the gap between the basic science understanding of apoptosis and the clinical application of this information is the lack of a sensitive marker to monitor programmed cell death in association with disease progression or regression. Technetium-99m labeled annexin V localizes at sites of apoptosis in-vivo, due to its nanomolar affinity for membrane bound phosphatidylserine. Radiolabeled annexin V imaging permits identification of the site and extent of apoptosis in experimental animals. Annexin V has been successfully used in animal models to image organ transplant rejection, characterize successful therapy of tumors, pinpoint acute myocardial infarction, and identify hypoxic ischemic brain injury of the newborn and adult. Early studies in human subjects suggest that 99mTc annexin imaging will be also be useful to identify rejection in transplant recipients, localize acute myocardial infarction, and characterize the effectiveness of a single treatment in patients with tumors.This review describes the imaging approaches to detect and monitor apoptosis in-vivo that are presently in early clinical trials. The preliminary data are extrapolated to identify conditions where apoptosis imaging may be valuable in clinical decision making. These conditions include: transplant rejection; hypoxic/ischemic injury of heart and brain; and determining the efficacy of therapy in cancer, heart failure and osteoporosis.

In vivo imaging of apoptosis.

Despite over a decade of intense investigation there is still no routine method for the clinical imaging of apoptosis in oncologic patients. There have been multiple tracers proposed but none as of yet has received FDA approval. Radiolabeled annexin V is one of the few radiotracers that has been widely used in Phase II trials and is still under development. In this review we will first detail the general mechanisms involved with apoptosis and other common forms of cell death. Next we will outline the latest in vivo imaging data in animal models and humans including that obtained with radiolabeled annexin V. It is hoped that improved understanding of the complex biochemical pathways involved with cell death will lead to at least several radiopharmaceuticals with the ability to image apoptosis as part of improving the care and treatment of patients suffering from cancer.