Detection of Apoptotic Cells in a Rabbit Model with Atherosclerosis-Like Lesions Using the Positron Emission Tomography Radiotracer [18F]ML-10.

[18F]ML-10 (2-(5-fluoro-pentyl)-2-methylmalonic acid) is a positron emission tomography (PET) radiotracer that accumulates in cells presenting apoptosis-specific membrane alterations. The aim of this study was to test whether [18F]ML-10 allows for the detection of apoptotic cells located in atherosclerotic plaques in rabbits. Atherosclerotic plaques were induced in the aortas of five rabbits, and five additional rabbits were used as controls. Activity in the aortas was quantified in vivo and ex vivo. The localization of [18F]ML-10 to the aortic wall was identified by autoradiography. Average target to background ratios measured in vivo by PET were higher in the aortas of atherosclerotic rabbits compared with those of control rabbits (2.00 ± 0.52 vs 1.22 ± 0.30; p < .05). Differences in [18F]ML-10 uptake between atherosclerotic and control aortas were confirmed ex vivo by PET and gamma counting (23.9 ± 11.2 vs 1.1 ± 2.4 counts/pixel; p <.05; 3.6 ± 2.0 vs 0.05 ± 0.05 % of injected activity/g; p < .05, respectively). Strong correlation was observed between the accumulation of [18F]ML-10 in aortic segments as detected by autoradiography and the number of apoptotic cells on corresponding histologic sections (r2 = .75; p < .05). In this study, we found that atherosclerotic plaques rich in apoptotic cells can be detected with [18F]ML-10 and PET.

Assessment of response of brain metastases to radiotherapy by PET imaging of apoptosis with ¹8F-ML-10.

PURPOSE: Early assessment of tumor response to therapy is vital for treatment optimization for the individual cancer patient. Induction of apoptosis is an early and nearly universal effect of anticancer therapies. The purpose of this study was to assess the performance of (18)F-ML-10, a novel PET radiotracer for apoptosis, as a tool for the early detection of response of brain metastases to whole-brain radiation therapy (WBRT). MATERIALS AND METHODS: Ten patients with brain metastases treated with WBRT at 30 Gy in ten daily fractions were enrolled in this trial. Each patient underwent two (18)F-ML-10 PET scans, one prior to the radiation therapy (baseline scan), and the second after nine or ten fractions of radiotherapy (follow-up scan). MRI was performed at 6-8 weeks following completion of the radiation therapy. Early treatment-induced changes in tumor (18)F-ML-10 uptake on the PET scan were measured by voxel-based analysis, and were then evaluated by correlation analysis as predictors of the extent of later changes in tumor anatomical dimensions as seen on MRI scans 6-8 weeks after completion of therapy. RESULTS: In all ten patients, all brain lesions were detected by both MRI and the (18)F-ML-10 PET scan. A highly significant correlation was found between early changes on the (18)F-ML-10 scan and later changes in tumor anatomical dimensions (r = 0.9). CONCLUSION: These results support the potential of (18)F-ML-10 PET as a novel tool for the early detection of response of brain metastases to WBRT.

Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis.

Utilization of molecular imaging of apoptosis for clinical monitoring of tumor response to anti-cancer treatments in vivo is highly desirable. To address this need, we now present ML-9 (butyl-2-methyl-malonic acid; MW = 173), a rationally designed small-molecule detector of apoptosis, based on a novel alkyl-malonate motif. In proof-of-concept studies, induction of apoptosis in tumor cells by various triggers both in vitro and in vivo was associated with marked uptake of (3)H-ML-9 administered in vivo, in correlation with the apoptotic hallmarks of DNA fragmentation, caspase-3 activation and membrane phospholipid scrambling, and with correlative tumor regression. ML-9 uptake following chemotherapy was tumor-specific, with rapid clearance of the tracer from the blood and other non-target organs. Excess of non-labeled "cold" compound competitively blocked ML-9 tumor uptake, thus demonstrating the specificity of ML-9 binding. ML-9 may therefore serve as a platform for a novel class of small-molecule imaging agents for apoptosis, useful for assessment of tumor responsiveness to treatment.

Molecular imaging of neurovascular cell death in experimental cerebral stroke by PET.

UNLABELLED: Clinical molecular imaging of apoptosis is a highly desirable yet unmet challenge. Here we provide the first report on (18)F-labeled 5-fluoropentyl-2-methyl-malonic acid ((18)F-ML-10), a small-molecule, (18)F-labeled PET tracer for the imaging of apoptosis in vivo; this report includes descriptions of the synthesis, radiolabeling, and biodistribution of this novel apoptosis marker. We also describe the use of (18)F-ML-10 for small-animal PET of neurovascular cell death in experimental cerebral stroke in mice. METHODS: (18)F-ML-10 was synthesized by nucleophilic substitution from the respective mesylate precursor, and its biodistribution was assessed in healthy rats. Permanent occlusion of the middle cerebral artery (MCA) was induced in mice, and small-animal PET was performed 24 h later. RESULTS: Efficient radiolabeling of ML-10 with (18)F was achieved. Biodistribution studies with (18)F-ML-10 revealed rapid clearance from blood (half-life of 23 min), a lack of binding to healthy tissues, and rapid elimination through the kidneys. No significant tracer metabolism in vivo was observed. Clear images of distinct regions of increased uptake, selectively in the ischemic MCA territory, were obtained in the in vivo small-animal PET studies. Uptake measurements ex vivo revealed 2-fold-higher uptake in the affected hemisphere and 6- to 10-fold-higher uptake in the region of interest of the infarct. The cerebral uptake of (18)F-ML-10 was well correlated with histologic evidence of cell death. The tracer was retained in the stroke area but was cleared from blood and from intact brain areas. CONCLUSION: (18)F-ML-10 is useful for noninvasive PET of neurovascular histopathology in ischemic cerebral stroke in vivo. Such an assessment may assist in characterization of the extent of stroke-related cerebral damage and in the monitoring of disease course and effect of treatment.

Targeting cell death in vivo in experimental traumatic brain injury by a novel molecular probe.

Traumatic brain injury (TBI) remains a frequent and major challenge in neurological and neurosurgical practice. Apoptosis may play a role in cerebral tissue damage induced by the traumatic insult, and thus its detection and inhibition may advance patient care. DDC (N,N'-didansyl-L-cystine) is a novel fluorescent probe for detection of apoptotic cells. We now report on the performance of DDC in experimental TBI. Closed head injury was induced in mice by weight-drop. DDC was administered intravenously in vivo. Two hours later, animals were sacrificed, and brain tissue was subjected to fluorescent microcopy, for assessment of DDC uptake, in correlation with histopathological assessment of apoptosis by TUNEL and caspase substrates, and also in correlation with the neurological deficits, as assessed by Neurological Severity Score (NSS). Selective uptake of DDC was observed at the primary site of injury, and also at remote sites. Uptake was at the cellular level, with accumulation of DDC in the cytoplasm. Cells manifesting DDC uptake were confirmed as apoptotic cells by detection of the characteristic apoptotic DNA fragmentation (positive TUNEL staining) and detection of activated caspases. The damaged region stained by DDC fluorescence correlated with the severity of neuronal deficits. Our study confirms the role of apoptosis in TBI, and proposes DDC as a useful tool for its selective targeting and detection in vivo. Such imaging of apoptosis, following future radiolabeling of DDC, may advance care for patients with head injury, by allowing real-time evaluation of the extent of tissue damage, assessment of novel therapeutic strategies, and optimization of treatment for the individual patient.

Novel molecular imaging of cell death in experimental cerebral stroke.

Cell death is the basic neuropathological substrate in cerebral ischemia, and its non-invasive imaging may improve diagnosis and treatment for stroke patients. ApoSense is a novel family of low-molecular weight compounds for detection and imaging of cell death in vivo. We now report on imaging of cell death and monitoring of efficacy of neuroprotective treatment in vivo by intravenous administration of the ApoSense compound DDC (didansylcystine), in experimental stroke in rodents. Rats and mice were subjected to a short-term (2 h) or permanent occlusion of the middle cerebral artery (MCA) and injected with DDC or 3H-labeled DDC. Fluorescent and autoradiographic studies, respectively, were performed ex vivo, comprising assessment of DDC uptake in the infarct region, in correlation with tissue histopathology. Neuroprotection was induced by a caspase inhibitor (Q-VD-OPH), and its effect was monitored by DDC. Following its intravenous administration, DDC accumulated selectively in injured neurons within the region of infarct. Caspase inhibition exerted a 45% reduction in infarct volume, which was well reported by DDC. This is the first report on a small molecule probe for detection in vivo of cell death in cerebral stroke. DDC may potentially assist in addressing the current "neuroimaging/neurohistology gap", for molecular assessment of the extent of stroke-related cell death.

Monitoring of chemotherapy-induced cell death in melanoma tumors by N,N'-Didansyl-L-cystine.

Early assessment of the efficacy of anticancer agents is a highly desirable and an unmet need in clinical oncology. Clinical imaging of cell-death may be useful in addressing this need, as induction of tumor cell-death is the primary mechanism of action of most anticancer drugs. In this study, we examined the performance of N,N'-Didansyl-L-cystine (DDC), a member of the ApoSense family of novel small molecule detectors of cell-death, as a potential tool for monitoring cell-death in cancer models. Detection of cell-death by DDC was examined in fluorescent studies on B16 melanoma cells both in vitro and ex vivo following its in vivo administration. In vitro, DDC manifested selective uptake and accumulation within apoptotic cells that was highly correlated with Annexin-V binding, changes in mitochondrial membrane potential, and caspase activation. Uptake was not ATP-dependent, and was inducible by calcium mobilization. In vivo, DDC selectively targeted cells undergoing cell-death in melanoma tumors, while not binding to viable tumor cells. Chemotherapy caused marked tumor cell-death, evidenced by increased DDC uptake, which occurred before a detectable change in tumor size and was associated with increased animal survival. These data confirm the usefulness of imaging of cell-death by DDC as a tool for early monitoring of tumor response to anti-cancer therapy.

Small-molecule biomarkers for clinical PET imaging of apoptosis.

Apoptosis is a fundamental biologic process. Molecular imaging of apoptosis in vivo may have important implications for clinical practice, assisting in early detection of disease, monitoring of disease course, assessment of treatment efficacy, or development of new therapies. Although a PET probe for clinical imaging of apoptosis would be highly desirable, this is yet an unachieved goal, mainly because of the required challenging integration of various features, including sensitive and selective detection of the apoptotic cells, clinical aspects such as favorable biodistribution and safety profiles, and compatibility with the radiochemistry and imaging routines of clinical PET centers. Several approaches are being developed to address this challenge, all based on novel small-molecule structures targeting various steps of the apoptotic cascade. This novel concept of small-molecule PET probes for apoptosis is the focus of this review.

Molecular imaging of neurodegeneration by a novel cross-disease biomarker.

Current pre-mortem diagnosis of neurodegenerative disorders such as Alzheimer's disease (AD) or amyotrophic lateral sclerosis (ALS) is based on clinical assessment of neurological deficits. However, symptoms and signs emerge only late in the disease course, thus indicating an urgent need for novel tools for detection of the underlying neuropathology. NST-729 (MW=310) is a novel molecular imaging probe, which is a member of the ApoSense family of small molecule detectors of apoptosis. We now report on the ability of NST-729, upon its systemic administration in vivo, to detect characteristic neuropathology in pre-clinical models of AD (Tg2576 transgenic mice) and ALS (transgenic SOD-1 G93A mutation mice). In the AD model, NST-729 clearly and selectively bound and imaged amyloid plaques, in excellent correlation with a typical amyloid ex vivo staining (Congo red). In the ALS model, NST-729 distinctly and selectively imaged multiple degenerating neurons in the motor nuclei in the pons, medulla and spinal cord, manifesting numerous multifocal irregularities and disruptions of neuritic projections, typical of axonal apoptosis. Study results therefore support the potential utility of NST-729 as a cross-disease biomarker for neurodegeneration, and also its potential role as the first molecular probe for ALS. Future radio-labeled NST-729 analogues may assist in the early diagnosis of disease, and in the development of neuroprotective therapies for these severe neurological disorders.

From the Gla domain to a novel small-molecule detector of apoptosis.

Apoptosis plays a pivotal role in the etiology or pathogenesis of numerous medical disorders, and thus, targeting of apoptotic cells may substantially advance patient care. In our quest for novel low-molecular-weight probes for apoptosis, we focused on the uncommon amino acid gamma-carboxyglutamic acid (Gla), which plays a vital role in the binding of clotting factors to negatively charged phospholipid surfaces. Based on the alkyl-malonic acid motif of Gla, we have developed and now present ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic acid, MW=206 Da), the prototypical member of a novel family of small-molecule detectors of apoptosis. ML-10 was found to perform selective uptake and accumulation in apoptotic cells, while being excluded from either viable or necrotic cells. ML-10 uptake correlates with the apoptotic hallmarks of caspase activation, Annexin-V binding and disruption of mitochondrial membrane potential. The malonate moiety was found to be crucial for ML-10 function in apoptosis detection. ML-10 responds to a unique complex of features of the cell in early apoptosis, comprising irreversible loss of membrane potential, permanent acidification of cell membrane and cytoplasm, and preservation of membrane integrity. ML-10 is therefore the most compact apoptosis probe known to date. Due to its fluorine atom, ML-10 is amenable to radio-labeling with the (18)F isotope, towards its potential future use for clinical positron emission tomography imaging of apoptosis.

Novel fluorescence molecular imaging of chemotherapy-induced intestinal apoptosis.

Chemotherapy-induced enteropathy (CIE) is one of the most serious complications of anticancer therapy, and tools for its early detection and monitoring are highly needed. We report on a novel fluorescence method for detection of CIE, based on molecular imaging of the related apoptotic process. The method comprises systemic intravenous administration of the ApoSense fluorescent biomarker (N,N(')-didansyl-L-cystine DDC) in vivo and subsequent fluorescence imaging of the intestinal mucosa. In the reported proof-of-concept studies, mice were treated with either taxol+cyclophosphamide or doxil. DDC was administered in vivo at various time points after drug administration, and tracer uptake by ileum tissue was subsequently evaluated by ex vivo fluorescent microscopy. Chemotherapy caused marked and selective uptake of DDC in ileal epithelial cells, in correlation with other hallmarks of apoptosis (i.e., DNA fragmentation and Annexin-V binding). Induction of DDC uptake occurred early after chemotherapy, and its temporal profile was parallel to that of the apoptotic process, as assessed histologically. DDC may therefore serve as a useful tool for detection of CIE. Future potential integration of this method with fluorescent endoscopic techniques, or development of radio-labeled derivatives of DDC for emission tomography, may advance early diagnosis and monitoring of this severe adverse effect of chemotherapy.

Molecular imaging of cell death in vivo by a novel small molecule probe.

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an (18)F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo.

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis.

PURPOSE: Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia. METHODS: Using both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis. RESULTS: ApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining. CONCLUSION: ApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states.