Jean-Luc Vanderheyden

Apoptosis-detecting radioligands: current state of the art and future perspectives

This review provides a critical and thorough overview of the radiopharmaceutical development and in vivo evaluation of all apoptosis-detecting radioligands that have emerged so far, along with their possible applications in nuclear medicine. The following SPECT and PET radioligands are discussed: all forms of halogenated Annexin V (i.e. 123I-labelled, 124I-labelled, 125I-labelled, 18F-labelled), 99mTc/94mTc-labelled Annexin V derivatives using different chelators and co-ligands (i.e. BTAP, Hynic, iminothiolane, MAG3, EDDA, EC, tricarbonyl, SDH) or direct 99mTc-labelling, 99mTc-labelled Annexin V mutants and 99mTc/18F-radiopeptide constructs (i.e. AFIM molecules), 111In-DTPA-PEG-Annexin V, 11C-Annexin V and 64Cu-, 67Ga- and 68Ga-DOTA-Annexin V. In addition, the potential role and clinical relevance of anti-PS monoclonal antibodies and other alternative apoptosis markers are reviewed, including: anti-Annexin V monoclonal antibodies, radiolabelled caspase inhibitors and substrates and mitochondrial membrane permeability targeting radioligands. Nevertheless, major emphasis is placed on the group of Annexin V-based radioligands, in particular 99mTc-Hynic-Annexin V, since this molecule is by far the most extensively investigated and best-characterised apoptosis marker at present. Furthermore, the newly emerging imaging modalities for in vivo detection of programmed cell death, such as MRI, MRS, optical, bioluminescent and ultrasound imaging, are briefly described. Finally, some future perspectives are presented with the aim of promoting the development of potential new strategies in pursuit of the ideal cell death-detecting radioligand.

The chemistry of rhenium and technetium as related to the use of isotopes of these elements in therapeutic and diagnostic nuclear medicine.

The beta emitting isotopes 186Re and 188Re are logical choices on which to base therapeutic radiopharmaceuticals that might be expected to be analogous to diagnostic radiopharmaceuticals based on 99mTc. However, the chemistry of rhenium is sufficiently different from that of technetium so that the development of Re radiopharmaceuticals often cannot be predicated on the known chemistry and biological behavior of 99mTc radiopharmaceuticals. The relevant chemical differences involve the greater stability of the higher oxidation states of Re (and thus the greater tendency of reduced Re radiopharmaceuticals to undergo re-oxidation to perrhenate), and the greater substitution inertness of reduced Re complexes. These differences are illustrated in the preparation and use of 186Re (Sn)-HEDP and 99mTc(Sn)-HEDP diphosphonate radiopharmaceuticals designed, respectively, for palliative therapy and diagnosis of metastatic cancer to bone, and in the preparation and biodistribution of tr[186Re(DMPE)2Cl2]+ and [186Re(DMPE)3]+, analogs to the potential myocardial perfusion imaging agents tr-[99mTc(DMPE)2Cl2]+ and [99mTc(DMPE)3]+. [HEDP = (1-hydroxyethylidene)diphosphonate; DMPE = 1,2-bis(dimethylphosphino)ethane].

99mTc-HYNIC-annexin V SPECT imaging of acute stroke and its response to neuroprotective therapy with anti-Fas ligand antibody

PurposeThe first aim of the study was to determine whether 99mTc-HYNIC-annexin V, a marker of cellular stress and apoptosis, can detect ischemic injury in patients with acute stroke. Secondly, we wished to test radiolabeled annexin’s ability to monitor therapy in a rodent model of focal ischemic injury.MethodsSPECT imaging of patients was performed between 1 and 2xa0h after intravenous injection of 30xa0mCi (1,110xa0MBq) of tracer. Eight MFL4 (anti-FasL) antibody-treated (400xa0μg i.p. days 0 and 3) and 21 control adult male Sprague-Dawley rats underwent small animal SPECT imaging with 5–10xa0mCi (185–370 MBq) of tracer, 1 and 6 days after a 2-h intraluminal thread occlusion of the left middle cerebral artery.ResultsTwo patients with acute stroke had regions of multifocal annexin uptake that correlated with sites of restricted diffusion on MRI. Anti-FasL antibody treatment significantly reduced annexin uptake by 92% with a 60% decrease in the number of caspase-8 staining (apoptotic) neurons on day 1. On day 6, treated animals had an 80% reduction in tracer uptake with a 75% decrease in infarct size as compared with controls. Annexin uptake in controls and treated animals (day 6) linearly correlated with infarct size (r2=0.603, p=0.0036) and the number of TUNEL-positive (apoptotic) nuclei (r2=0.728, p=0.00084).ConclusionAnnexin imaging shows foci of increased uptake at sites of ischemic injury in patients with acute stroke. Annexin imaging can assess the effects of therapy for ischemic cerebral injury in rats, suggesting its potential as a non-invasive indicator of drug efficacy in future clinical trials.

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.

Myocardial perfusion imaging with 99mTc-DMPE in man

Technetium-99m DMPE (99mTc-DMPE) is a newly synthesized myocardial perfusion imaging agent that shows intense myocardial accumulation in the dog. In the present study, dosimetry and potential clinical usefulness of this agent were assessed in four human subjects. Absorbed radiation doses were low, with the highest doses consisting of 200 mrad/mCi (54 μGy/MBq) to the gallbladder and 160 mrad/mCi (43 μGy/MBq) to the liver. No evidence of clinical toxicity was found. Technetium-99m DMPE did image the myocardium, but the ratio of target to nontarget activity was less favorable than that observed in the dog. Intense hepatic 99mTc-DMPE activity interfered with clinical imaging of the cardiac apex in two of the four subjects. We conclude that the prototype radiopharmaceutical, 99mTc-DMPE, is capable of myocardial perfusion imaging in man but the planar myocardial images produced are of inferior quality compared with 201Tl myocardial images. Further work is justified to develop related compounds to overcome the clinical limitations described.

Myocardial scintigraphy with 99mTc-tris-DMPE in man

Cardiac scintigraphy was performed in six patients with a documented previous myocardial infarction, in one patient with mitral regurgitation, and in four healthy volunteers following administration of 99mTc-tris-DMPE. An intense early blood pool phase permitted gated blood pool scintigraphy and left ventricular ejection fraction calculation. A myocardial phase 12–14 h later permitted myocardial perfusion imaging. The rest myocardial perfusion image quality with 99mTc-tris-DMPE appeared to be superior to the resting image quality obtained with 99mTc-dichloro-DMPE but was inferior to the resting image quality obtained with 201Tl.

99m Tc-annexin V and 111 In-antimyosin antibody uptake in experimental myocardial infarction in rats

Purpose99mTc-annexin V (ANX) allows scintigraphic detection of apoptotic cells via specific binding to exposed phosphatidylserine. In myocardial infarction, apoptosis of myocytes is variable and depends especially on the presence or absence of coronary reperfusion. In this study, ANX uptake in non-reperfused experimental myocardial infarcts was compared with uptake of a marker of myocyte necrosis (111In-antimyosin antibodies, AM) and an immunohistochemical marker of apoptosis (Apostain).MethodsThe left anterior coronary artery was ligated in 47 Wistar rats, which were then injected with ANX (n=20), AM (n=21) or both (n=6). Myocardial uptake of ANX and AM was determined at 2xa0h (n=14), 4xa0h (n=14) and 24xa0h (n=19) after coronary ligation (CL), by quantitative autoradiography with (n=23) or without (n=24) gamma imaging. Heart-to-lung ratios (HLRs) and infarct-to-remote myocardium activity ratios (INRs) were calculated on the scintigrams and autoradiograms respectively. Cardiac sections were stained with haematoxylin-eosin and Apostain. The above studies were repeated in 12 normal rats.ResultsAll rats with CL showed increased ANX and AM uptake in cardiac areas on scintigrams 24xa0h after CL, with HLRs higher than in controls: 3.1±0.6 versus 1.5±0.3 (p=0.001) for ANX and 1.99±0.44 versus 1.01±0.05 (p<0.0005) for AM. Autoradiography showed intense ANX and AM uptake in infarcts, with comparable topography and INRs at 2xa0h, 4xa0h and 24xa0h after CL (4.6±0.9 versus 5.0±1.8 at 24xa0h), while Apostain staining was very low (0.06±0.06% of cells).ConclusionIn this model of persistent CL, we observed increased ANX uptake in injured myocardium, comparable in intensity, topography and kinetics to that of AM. There was only minimal Apostain staining in the same areas.

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.

Preparation and characterization of [99mTc(DMPE)2X2]+, X = Cl, Br (DMPE = 1,2-bis (dimethylphosphino)ethane)

The preparation of no-carrier-added [99mTc(DMPE)2X2]+ (X = Cl, Br) in greater than 95% radiochemical purity, suitable for in vivo evaluation as a myocardial imaging agent, is described. Reaction parameters that lead to the formation of the desired products, as well as those that lead to the formation of the major impurities, are delineated. These 99mTc species are characterized by HPLC using the known 99Tc analogs as standards. Myocardial images obtained in mongrel dogs with [99mTc(DMPE)2X2]+ (X = Cl, Br) are briefly discussed.

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

Purpose99mTc-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 99mTc-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.MethodsIncluded in this review were 17 clinical trials investigating quantitative 99mTc-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 95xa0% CI) were calculated using Meta-Disc software version 1.4.ResultsAbsolute quantification and/or relative quantification of 99mTc-annexin A5 uptake were performed at baseline and after the start of treatment. Various quantitative parameters have been used for the calculation of 99mTc-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/cm3. 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 ≥25xa0% 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 ≥28xa0%, ≥42xa0% and ≥47xa0% 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 ≥23xa0% 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 99mTc-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 99mTc-annexin A5 tumor uptake were found to be reproducible (mean difference <5xa0%, kappau2009=u2009 0.90 and 0.82, respectively) and to be highly correlated with treatment outcome (Spearman ru2009=u20090.99, pu2009<u20090.0001). The meta-analysis demonstrated a pooled positive PPV of 100xa0% (95xa0% CI 92xa0–xa0100xa0%) and a pooled NPV of 70xa0% (95xa0% CI 55xa0–xa082xa0%) 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 85.21xa0%.ConclusionQuantitative 99mTc-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 20xa0% and 30xa0%. 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.