Chris Tsopelas

99mTc-Alafosfalin: an antibiotic peptide infection imaging agent

The radiolabeled antibiotic peptide (99m)Tc-alafosfalin was assessed as an infection imaging agent in a rat model by comparison with (99m)Tc-DTPA and (99m)Tc-leukocytes. (99m)Tc-alafosfalin was prepared via an instant cold kit and (99m)Tc-leukocytes were prepared using (99m)Tc-stannous fluoride colloid in an ex vivo labeling procedure of whole blood. In separate experiments, the three radiotracers were administered to rats infected with staphylococcus aureus. Quantitative biodistribution studies were performed as well as scintigraphic images and histopathology. (99m)Tc-alafosfalin is a stable product, obtained in high radiochemical purity (>95%). This agent was mainly renally excreted, with low liver, spleen and bone uptake, and resulted in a mean ratio of infected/non-infected thighs of 4.3/1.0 at 4 hr post radiotracer injection. (99m)Tc-DTPA gave a corresponding ratio of 1.9/1.0 and (99m)Tc-leukocytes gave 20.0/1.0 at the same time point. An in vitro assay found the level of (99m)Tc-alafosfalin binding to staphylococcus aureas higher than (99m)Tc-DTPA (10% versus 1% respectively). (99m)Tc-alafosfalin accumulates at sites of infection in a rat model better than the perfusion molecule (99m)Tc-DTPA, yet less than (99m)Tc-leukocytes. The distribution characteristics of this (99m)Tc-antibiotic peptide would be an advantage in imaging abdominal and soft tissue infection.

Regenerating lizard tails: a new model for investigating lymphangiogenesis

Impaired lymphatic drainage in human limbs causes the debilitating swelling termed lymphoedema. In mammals, known growth factors involved in the control of lymphangiogenesis (growth of new lymph vessels) are vascular endothelial growth factors‐C and –D (VEGF‐C/D). Here we characterize a model of lymphangiogenesis in which the tail of lizards is regenerated without becoming oedematous. Three weeks after the tail is shed (autotomy), there are a small number of large diameter lymphatic vessels in the regenerated tail. Thereafter, the number increases and the diameter decreases. A functional lymphatic network, as determined by lymphoscintigraphy, is established 6 wk after autotomy. The new network differs morphologically and functionally from that in original tails. This lymphatic regeneration is associated with an up‐regulation of a reptilian homologue of the VEGF‐C/D protein family (rVEGF‐C/D), as determined by Western blot analysis using a human reactive VEGF‐C polyclonal antibody. Regenerating lizard tails are potentially useful models for studying the molecular basis of lymphangiogenesis with a view to developing possible treatments for human lymphoedema.

APOMAB®, a La-Specific Monoclonal Antibody, Detects the Apoptotic Tumor Response to Life-Prolonging and DNA-Damaging Chemotherapy

Background Antineoplastic therapy may impair the survival of malignant cells to produce cell death. Consequently, direct measurement of tumor cell death in vivo is a highly desirable component of therapy response monitoring. We have previously shown that APOMAB® representing the DAB4 clone of a La/SSB-specific murine monoclonal autoantibody is a malignant cell-death ligand, which accumulates preferentially in tumors in an antigen-specific and dose-dependent manner after DNA-damaging chemotherapy. Here, we aim to image tumor uptake of APOMAB® (DAB4) and to define its biological correlates. Methodology/Principal Findings Brisk tumor cell apoptosis is induced in the syngeneic EL4 lymphoma model after treatment of tumor-bearing mice with DNA-damaging cyclophosphamide/etoposide chemotherapy. Tumor and normal organ accumulation of Indium 111 (111In)-labeled La-specific DAB4 mAb as whole IgG or IgG fragments was quantified by whole-body static imaging and organ assay in tumor-bearing mice. Immunohistochemical measurements of tumor caspase-3 activation and PARP-1 cleavage, which are indicators of early and late apoptosis, respectively, were correlated with tumor accumulation of DAB4. Increased tumor accumulation of DAB4 was associated directly with both the extent of chemotherapy-induced tumor cell death and DAB4 binding per dead tumor cell. Tumor DAB4 accumulation correlated with cumulative caspase-3 activation and PARP-1 cleavage as tumor biomarkers of apoptosis and was directly related to the extended median survival time of tumor-bearing mice. Conclusions/Significance Radiolabeled La-specific monoclonal antibody, DAB4, detected dead tumor cells after chemotherapy, rather than chemosensitive normal tissues of gut and bone marrow. DAB4 identified late apoptotic tumor cells in vivo. Hence, radiolabeled DAB4 may usefully image responses to human carcinoma therapy because DAB4 would capture the protracted cell death of carcinoma. We believe that the ability of radiolabeled DAB4 to rapidly assess the apoptotic tumor response and, consequently, to potentially predict extended survival justifies its future clinical development as a radioimmunoscintigraphic agent. This article is part I of a two-part series providing proof-of-concept for the the diagnostic and therapeutic use of a La-specific monoclonal antibody, the DAB4 clone of which is represented by the registered trademark, APOMAB®.

99mTc–Evans Blue Dye for Mapping Contiguous Lymph Node Sequences and Discriminating the Sentinel Lymph Node in an Ovine Model

BackgroundThe aim of this study was to investigate the potential of 99mTc–Evans blue for discriminating the sentinel lymph node in multitiered lymph node sequences by using an ovine model. 99mTc–Evans blue is an agent that has both radioactive and color signals in a single dose. Previous studies in smaller animal models suggested that this agent could have advantages over the dual-injection technique of radiocolloid/blue dye.MethodsDoses of 99mTc–Evans blue (∼ 21 MBq) containing Evans blue dye (approximately 4 mg) were administered to the hind limbs or fore limbs of sheep to map the lymphatic drainage patterns, validate its ability to identify the sentinel lymph node, and examine the reproducibility of the technique. The study protocol was repeated with 99mTc–antimony trisulfide colloid and Patent Blue V dye. After the operative exposure, lymph nodes were identified with the gamma probe and then excised and analyzed for radioactivity (percentage of injected dose) and blue color.ResultsAfter the administration of 99mTc–Evans blue, all lymph nodes harvested (35 of 35) in either short chains or long basins were hot and blue. The sentinel lymph nodes concentrated more radioactivity than the second-tier nodes to the extent of 2:1 to 215:1. For radiocolloid/Patent Blue V, the ratios were lower, at 2:1 to 3:1.Conclusions99mTc–Evans blue was found to better discriminate the sentinel lymph node than 99mTc–antimony trisulfide colloid/Patent Blue V in variable multitier lymph node anatomy, and it is an agent that promises to have positive clinical applications.

Pharmacokinetics and normal scintigraphic appearance of 99mTc aprotinin.

ObjectiveTo confirm the pharmacokinetics and biodistribution of 99mTc aprotinin in normal volunteers and to determine the optimum time for scanning post-injection, prior to further investigations of 99mTc aprotinin as an imaging agent for amyloidosis. MethodsFive patients (three men and two women, average age 49 years, age range 38–66 years) without a history of amyloidosis or any of the associated diseases, were included in this prospective study. Blood and urine were collected and images were performed of the whole body and wrists. ConclusionsNormal biodistribution of 99mTc aprotinin includes early cardiac and lung activity in the blood pool phase with subsequent hepatic activity and renal excretion with variable splenic activity. There is variable bowel uptake on later images. The best quality images were obtained 90 min post-intravenous administration, and this is likely to be the optimum time for clinical imaging.

Lymphatic mapping with 99mTc-Evans Blue dye in sheep

Objective99mTc-Evans Blue (EB) is an agent that contains both radioactive and color signals in a single dose. Earlier studies in animal models have suggested that this agent when compared with the dual-injection technique of radiocolloid/blue dye can successfully discriminate the sentinel lymph node. The aim of this study was to investigate the potential of 99mTc-EB as an agent to map the lymphatic system in an ovine model.MethodsDoses of 99mTc-EB (23 MBq) containing EB dye (4 mg) were administered intradermally to the limbs of four anesthetized sheep, and they were then imaged over 20–30 min using a gamma camera. The study protocol was repeated using 99mTc-antimony trisulfide colloid (ATC) and Patent Blue V dye. The lymph nodes (popliteal, inguinal, and iliac for hind limbs or prescapular for fore limbs) were identified with a gamma probe during the operative exposure, then dissected and counted in a large volume counter.ResultsSimple and complex (dual) drainage patterns were visible on the scans, and the sentinel node was more radioactive than higher tier nodes in a chain, for both radiotracers. For 99mTc-EB, maximum radioactive uptake was achieved at 3–6 min for popliteal lymph nodes, 12–14 min for iliac nodes, and 13–14 min for prescapular nodes. 99mTc-ATC resulted in maximum radioactive uptake at 4–6 min for popliteal lymph nodes, 13 min for an inguinal node, 13–20 min for iliac nodes, and 18 min for a prescapular node. Following 99mTc-EB injection, 15/15 lymph nodes harvested were all radioactive and blue. For 99mTc-radiocolloid/Patent Blue V injection, 8/14 nodes were radioactive and blue, and 6/14 nodes were radioactive only.ConclusionsThe soluble radiotracer 99mTc-EB appeared to be a useful lymphoscintigraphic agent in sheep, in which radioactive counts from superficial lymphatic channels and lymph nodes were sufficient for planar imaging. In comparison with 99mTc-antimony trisulfide colloid, both tracers discriminated the sentinel lymph node up to 50 min after administration; however, 99mTc-EB had the advantage of providing radioactive (gamma probe) and color signals simultaneously during the operative exposure.

How regenerating lymphatics function : Lessons from lizard tails

Rational treatment of lymphoedema may be improved in the future with a better understanding of the physiological processes involved in the regeneration of new lymphatic vessels (lymphangiogenesis). Many lizard species undergo tail autotomy as a predator escape response and subsequently regenerate nonlymphoedematous tails. Such species may offer novel models for examining lymphangiogenesis. In this lymphoscintigraphic evaluation, three radioactive tracers were employed, 99mTc‐antimony trisulphide colloid (∼ 10 nm diameter), 99mTc‐tin fluoride colloid (∼2,000 nm; 99mTc‐TFC), and 99mTc‐diethylenetriaminepentaacetic acid (soluble; 99mTc‐DTPA), to examine lymphatic function in regenerating tails of the Australian marbled gecko, Christinus marmoratus. Rate of local clearance and velocity of migration were determined in geckos with original tails and at 6, 9, 12, and >24 weeks after autotomy. In original‐tailed geckos, the smaller radiocolloid was cleared to a greater extent and had a faster lymph velocity than in geckos with regenerated tails. The same parameters measured for larger particles were greater in early regeneration than later. 99mTc‐TFC did not migrate from the injection site in fully regenerated and original gecko tails, which indicates that larger particles are increasingly impeded as tail regeneration progresses. Soluble 99mTc‐DTPA diffused from the injection site extremely rapidly via venous capillaries in all tails, confirming that the slower clearance of the colloids is solely via the lymphatics. Differences in clearance and lymph velocity between differently sized colloids throughout tail regeneration may be influenced by changes in surrounding tissue structure density and the lymphatic vessel porosity. Anat Rec, 290:108–114, 2007.

Radiolabeling and biodistribution of amiodarone and desethylamiodarone.

The antiarrhythmic drug amiodarone and its metabolite desethylamiodarone, were radiolabeled with sodium [123I]-iodide in > 98% yield using the exchange labeling method. Biodistribution studies with 123I-amiodarone in mice showed high liver and lung uptake. Heart uptake of 0.98% of the injected dose (id) peaked at 5 min, with clearance seen over 60 min to 0.44% id. 123I-Desethylamiodarone (123I-DEA) showed heart uptake of 0.58% id peaking at 5 min, with slower clearance seen over 60 min to 0.43% id. These results indicate that both agents have poor selectivity as myocardial imaging tracers. Low 123I-DEA brain uptake at 5 min (0.49% id) and rapid washout after 60 min indicate that 123I-desethylamiodarone has limited application as a brain imaging agent.

Technetium-99m labeling of a molecule bearing easily reducible groups

Chemical stability of the naphthol-azo dye Evans Blue (EB) was examined in the presence of acidic stannous chloride (SnCl(2)), with a view to preparing an instant cold kit. EB was found to be reactive toward this reducing agent, yielding the metal-chelating molecule 1,7-diamino-8-naphthol-2,4-disulfonic acid at high acidity and high stannous concentrations. This reduction reaction was undesirable in the cold kit preparation. The conditions were determined where reduction was inhibited, at pH = 5.0 and with a mole ratio of EB to SnCl(2) = [10:1], effecting the facile preparation of stable cold kits. Successful (99m)Tc-labeling of an EB cold kit using these conditions resulted in the desired product with 98% radiochemical purity. Based on the radiolabeling efficiencies of chosen model compounds, it was rationalized that (99m)Tc metal predominantly coordinated with the 1-amino-8-hydroxy groups in the EB molecule to form (99m)Tc-EB.

An improved assay for 68Ga‐hydroxide in 68Ga‐DOTATATE formulations intended for neuroendocrine tumour imaging

The objective of this study was to identify a more rapid assay for (68)Ga(OH)3 impurity in (68)Ga-DOTATATE formulations. Three methods were used to prepare (68)Ga(OH)3 reference material (pharmacopoeial, bench titration and automated radiosynthesis), and four quality control methods for its assessment (thin layer chromatography, membrane filtration, HPLC and solid phase extraction). The optimal method of preparing (68)Ga(OH)3 was by titrating (68)Ga(3+) with buffered sodium hydroxide solutions to pH 5.6 ± 0.2. The precipitate was quantitatively isolated by membrane filtration (0.02 µm)/hydrochloric acid (HCl; pH 5.6) solvent, and also it remained 100% at the origin on instant thin layer chromatography with silica gel paper/HCl (pH 5.6) solvent. For (68)Ga-DOTATATE samples, the thin layer chromatography technique was used with a single paper strip developed separately on two occasions, once in HCl (pH 5.6) and next in methanol solvent. This so-called double-developed (DD) method separated (68)Ga(OH)3 impurity located at the origin, from (68)Ga-DOTATATE plus (68)Ga(3+) at ~Rf 0.4, and it was superior to the other methods. It assayed for the impurity similarly to the pharmacopoeial method. The advantages of the DD method were that it required inexpensive test materials and it reproducibly determined % (68)Ga(OH)3 in (68)Ga-DOTATATE in 12 min, 13 min earlier than the pharmacopoeial method. This time efficiency resulted in a surplus of 12% (68)Ga-DOTATATE counts in the product vial, and this provided a contingency of radioactivity or time for the injection/imaging processes in the Nuclear Medicine Department.