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®.Fares Al-Ejeh, Jocelyn M. Darby, Chris Tsopelas, Douglas Smyth, Jim Manavis and Michael P. Brow

Radiotracers Used for the Scintigraphic Detection of Infection and Inflammation

Over the last forty years, a small group of commercial radiopharmaceuticals have found their way into routine medical use, for the diagnostic imaging of patients with infection or inflammation. These molecular radiotracers usually participate in the immune response to an antigen, by tagging leukocytes or other molecules/cells that are endogenous to the process. Currently there is an advancing effort by researchers in the preclinical domain to design and develop new agents for this application. This review discusses radiopharmaceuticals used in the nuclear medicine clinic today, as well as those potential radiotracers that exploit an organism’s defence mechanisms to an infectious or inflammatory event

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

The objective of this study was to identify a more rapid assay for\ua068Ga(OH)3\ua0impurity in\ua068Ga‐DOTATATE formulations. Three methods were used to prepare\ua068Ga(OH)3\ua0reference 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\ua068Ga(OH)3\ua0was by titrating\ua068Ga3+\ua0with 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\ua068Ga‐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\ua0double‐developed\ua0(DD)\ua0method\ua0separated\ua068Ga(OH)3\ua0impurity located at the origin, from\ua068Ga‐DOTATATE plus\ua068Ga3+\ua0at ~Rf\ua00.4, 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 %\ua068Ga(OH)3\ua0in\ua068Ga‐DOTATATE in 12 min, 13 min earlier than the pharmacopoeial method. This time efficiency resulted in a surplus of 12%\ua068Ga‐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

Sentinel node biopsy and lymphoscintigraphy with a technetium 99m labeled blue dye in a rabbit model

Copyright © 2002 Mosby, Inc. All rights reserved.Background. Lymphatic mapping for sentinel node biopsy in breast cancer and melanoma usually involves initial peritumoral injection of a radioisotope, gamma camera detection of the sentinel lymph node several hours prior to the operation, and separate perioperative injection of a blue dye. We have developed a combined approach using technetium 99m labeled blue dye (Evans Blue) for use in lymphoscintigraphy that may be injected as a single dose just prior to the operation. Methods. In an anesthetized rabbit model we dissected a hind limb to display the popliteal node and afferent lymphatic. Technetium 99m Evans Blue (99mTc-EB) (22 MBq; 0.5 mL) was injected subdermally in the dorsum of the paw. Simultaneous digital and gamma camera images were obtained at 14 time intervals to 30 minutes post injection. For each of these time intervals the percentage of radioactivity and percentage blueness of the popliteal node were determined. Urine and afferent lymphatic fluid were analyzed by chromatography. The popliteal node was excised post mortem, placed into solvent solutions and analyzed for blueness and radioactivity. Results. Time-activity curves for radioactivity and time-blueness curves for Evans Blue uptake showed strong correlation (r = 0.958). Lymph analysis suggested 99mTc-EB is mainly bound to endogenous proteins. Urine was radioactive but not colored, 99mTc-EB being metabolized and excreted in the urine as 1,7-diamino-8-naphthol-2,4-disulfonic acid. Prolonged exposure of node to solvents did not dissociate any blue coloration or radioactivity. Conclusions. 99mTc-EB and Evans Blue are simultaneously retained and concentrated in the sentinel lymph node. This process is rapid and reproducible. 99mTc-EB migrates at the same rate as Evans Blue in lymph, where it is transported as bound to endogenous proteins. These dye molecules are metabolized by reductive cleavage in the liver and then excreted renally as colorless, radioactive metabolites. This novel agent has the potential to facilitate lymphatic mapping and subsequent sentinel node biopsy for a range of solid malignancies including breast cancer and melanoma.Richard Sutton, Chris Tsopelas, James Kollias, Barry E. Chatterton and Brendon J. Coventr

Use of [14C] - Sodium Bicarbonate Urea to Measure Physical Activity Induced Increases in Total Energy Expenditure in Free-Living Healthy Males

The aim of this study was to evaluate the utility of the [¹⁴C]-sodium bicarbonate/urea technique to detect physical activity-induced increases in total energy expenditure in free-living healthy men. Thirteen healthy males aged 34.1 ± 11.7 yrs with body mass index 24.1 ± 3.1 kg/m2 were studied on three separate occasions, during which [¹⁴C]-bicarbonate was infused over 48-hours and urine was collected during the second 24-hours. On three separate occasions and in random order, subjects either remained sedentary, or performed a bout of physical activity on an electro-magnetically braked cycle ergometer sufficient to increase energy expenditure by 7% or 11% above predicted sedentary total energy expenditure. Urine samples were analyzed to evaluate the amount of [¹⁴C]-bicarbonate incorporated into urinary urea, thereby reflecting the amount of CO₂ produced per day, and upon conversion, the number of kilojoules of energy expended in 24-hours. All 13 subjects successfully completed the two physical activity treatments and there were no adverse events. As measured by the [¹⁴C]-urea assay, mean total energy expenditure values were not significantly different between sedentary activity (17902 ± 905 kJ/day), the physical activity treatment designed to increase TEE by 7% (17701 ± 594 kJ/day) and the physical activity treatment designed to increase TEE by 11% (18538 ± 485 kJ/day) (P=0.668). In conclusion, although the [¹⁴C]-sodium bicarbonate/urea technique was well tolerated and did not interfere with normal daily activities, it was not able to accurately measure physical activity-induced increases in EE in the range of 7-11% above predicted sedentary total energy expenditure.Darren M Roffey, Natalie D Luscombe, Nuala M Byrne, Andrew P Hills, Max Bellon, Chris Tsopelas, Ian D Kirkwood and Gary A Witter

18-Fluoride labeled sodium fluoride positron emission tomography with computer tomography: the impact of pretreatment staging in intermediate- and high-risk prostate cancer

Background: 18-Fluoride labeled sodium fluoride (Na-18-F) positron emission tomography with computer tomography (PET/CT) has a better sensitivity and specificity than whole body bone scan (WBBS) in detecting osseous metastatic prostate cancer. We performed a pilot study of 20 men to examine what level of impact Na-18-F PET/CT has on management plans when used for staging newly diagnosed prostate cancer. Materials and methods: Twenty men were prospectively enrolled into the study in South Australia. Men were eligible if they had newly diagnosed, untreated, and biopsy-confirmed intermediate- or high-risk prostate cancer (D'Amico classification). WBBS and Na-18-F PET/CT scans were performed within 1 week of each other. Following review of the WBBS, treatment type and intent was documented by the treating urologist. The Na-18-F PET/CT scan was then reviewed. The impact of the Na-18-F PET/CT was measured on whether treatment modality or intent was subsequently altered: high impact = treatment intent or modality was changed; medium impact = treatment modality was modified; low impact = no change in treatment. Results: In 18 men (90%), the WBBS and Na-18-F PET/CT were negative for osseous metastases. In one man (5%), the WBBS demonstrated widespread osseous metastases which were similarly demonstrated on the Na-18-F PET/CT. One man (5%) had a normal WBBS; however, the Na-18-F PET/CT demonstrated widespread osseous metastases. Subsequently, in 19 men (95%), the results of the two scans were congruent and the addition of the Na-18-F PET/CT scan demonstrated a low impact on management. In one man (5%), the addition of the Na-18-F PET/CT had a high impact as treatment type and intent was altered. Conclusions: Our pilot study is the first of its kind in Australia, and our findings suggest that Na-18-F PET/CT is a safe and feasible modality for staging prostate cancer. However, its true impact on prostate cancer management warrants further investigation. Keywords: Metastases, Positron emission tomography with computer tomography, Prostate cancer, Sodium fluoride, Whole body bone sca

Host immune response determines visceral hyperalgesia in a rat model of post-inflammatory irritable bowel syndrome

Background: Irritable bowel syndrome (IBS) is associated with visceral hyperalgesia and frequently occurs after a transient gastrointestinal infection. Only a proportion of patients with acute gastroenteritis develop post-infectious IBS suggesting differences in host response to inflammatory stimuli. We aimed to investigate this concept by characterizing visceral sensitivity in two rat strains, following a chemically induced colitis. Methods: Colorectal instillation of trinitrobenzenesulfonic acid (TNBS) in aqueous ethanol was used to induce a transient colitis in Lewis and F344 rats. The colitis was characterized semiquantitatively by histology, as well as by quantitative methods using Tc-leukocytes (radioactive organ assay) and plasma IL-2 and IL-6 levels. Visceromotor response to colorectal distensions was assessed after 2 h and, 5, 14, and 28 days. Results: The colitis peaked on day 5 and dissipated to no visible mucosal damage on day 14. Cytokines were significantly increased in TNBS-treated rats at 2 h and on day 5. On day 14 cytokines were still significantly enhanced in Lewis but not Fisher rats. Both strains had a highly inflamed to non-inflamed tissue ratio at 3 h after TNBS instillation with increased uptake in Lewis compared to F344 rats. No Tc-tin-colloid-leukocytes were detected in colon samples on day 28. Visceromotor response was significantly elevated in both strains during the acute colitis (day 5), whereas only Lewis rats developed a postinflammatory (day 28) visceral hyperalgesia. Conclusion: Genetically determined host factors account for prolonged immune activation in response to a standardized inflammatory stimulus and are linked to susceptibility for a post-inflammatory visceral hyperalgesia