Michael K. Schultz

Kinetic Analysis of 3′-Deoxy-3′-18F-Fluorothymidine (18F-FLT) in Head and Neck Cancer Patients Before and Early After Initiation of Chemoradiation Therapy

The purpose of this study was to investigate the kinetic behavior of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) before and early after initiation of chemoradiation therapy in patients with squamous cell head and neck cancer. Methods: A total of 8 patients with head and neck cancer underwent 18F-FLT PET scans (7 patients at baseline and after 5 d [10 Gy] of radiation therapy given with concomitant chemotherapy and 1 patient only at baseline). Dynamic PET images were obtained with concurrent arterial or venous blood sampling. Kinetic parameters including the flux constant of 18F-FLT based on compartmental analysis (K-FLT), the Patlak influx constant (K-Patlak), and standardized uptake value (SUV) were calculated for the primary tumor and 18F-FLT–avid cervical lymph nodes for all scans. Results: Mean pretreatment values of uptake for the primary tumor and cervical nodes were 0.075 ± 0.006 min−1, 0.042 ± 0.004 min−1, and 3.4 ± 0.5 (mean ± SD) for K-FLT, K-Patlak, and SUV, respectively. After 10 Gy of radiation therapy, these values were 0.040 ± 0.01 min−1, 0.018 ± 0.016 min−1, and 1.8 ± 1.1 for K-FLT, K-Patlak, and SUV, respectively. For all lesions seen on pretherapy and midtherapy scans, the correlation was 0.90 between K-FLT and K-Patlak, 0.91 between K-FLT and SUV, and 0.99 between K-Patlak and SUV. Conclusion: The initial 18F-FLT uptake and change early after treatment in squamous head and neck tumors can be adequately characterized with SUV obtained at 45–60 min, which demonstrates excellent correlation with influx parameters obtained from compartmental and Patlak analyses.

Simplified NaCl Based 68Ga Concentration and Labeling Procedure for Rapid Synthesis of 68Ga Radiopharmaceuticals in High Radiochemical Purity

A simple sodium chloride (NaCl) based (68)Ga eluate concentration and labeling method that enables rapid, high-efficiency labeling of DOTA conjugated peptides in high radiochemical purity is described. The method utilizes relatively few reagents and comprises minimal procedural steps. It is particularly well-suited for routine automated synthesis of clinical radiopharmaceuticals. For the (68)Ga generator eluate concentration step, commercially available cation-exchange cartridges and (68)Ga generators were used. The (68)Ga generator eluate was collected by use of a strong cation exchange cartridge. 98% of the total activity of (68)Ga was then eluted from the cation exchange cartridge with 0.5 mL of 5 M NaCl solution containing a small amount of 5.5 M HCl. After buffering with ammonium acetate, the eluate was used directly for radiolabeling of DOTATOC and DOTATATE. The (68)Ga-labeled peptides were obtained in higher radiochemical purity compared to other commonly used procedures, with radiochemical yields greater than 80%. The presence of (68)Ge could not be detected in the final product. The new method obviates the need for organic solvents, which eliminates the required quality control of the final product by gas chromatography, thereby reducing postsynthesis analytical effort significantly. The (68)Ga-labeled products were used directly, with no subsequent purification steps, such as solid-phase extraction. The NaCl method was further evaluated using an automated fluid handling system and it routinely facilitates radiochemical yields in excess of 65% in less than 15 min, with radiochemical purity consistently greater than 99% for the preparation of (68)Ga-DOTATOC.

Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging.

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, (64)Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of (64)Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with (64)Cu.

O2⋅− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅- and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

A new automated NaCl based robust method for routine production of gallium-68 labeled peptides

A new NaCl based method for preparation of gallium-68 labeled radiopharmaceuticals has been adapted for use with an automated gallium-68 generator system. The method was evaluated based on 56 preparations of [(68)Ga]DOTATOC and compared to a similar acetone-based approach. Advantages of the new NaCl approach include reduced preparation time (<15 min) and removal of organic solvents. The method produces high peptide-bound % (>97%), and specific activity (>40 MBq nmole(-1) [(68)Ga]DOTATOC) and is well-suited for clinical production of radiopharmaceuticals.

Understanding the Radioactive Ingrowth and Decay of Naturally Occurring Radioactive Materials in the Environment: An Analysis of Produced Fluids from the Marcellus Shale.

Background The economic value of unconventional natural gas resources has stimulated rapid globalization of horizontal drilling and hydraulic fracturing. However, natural radioactivity found in the large volumes of “produced fluids” generated by these technologies is emerging as an international environmental health concern. Current assessments of the radioactivity concentration in liquid wastes focus on a single element—radium. However, the use of radium alone to predict radioactivity concentrations can greatly underestimate total levels. Objective We investigated the contribution to radioactivity concentrations from naturally occurring radioactive materials (NORM), including uranium, thorium, actinium, radium, lead, bismuth, and polonium isotopes, to the total radioactivity of hydraulic fracturing wastes. Methods For this study we used established methods and developed new methods designed to quantitate NORM of public health concern that may be enriched in complex brines from hydraulic fracturing wastes. Specifically, we examined the use of high-purity germanium gamma spectrometry and isotope dilution alpha spectrometry to quantitate NORM. Results We observed that radium decay products were initially absent from produced fluids due to differences in solubility. However, in systems closed to the release of gaseous radon, our model predicted that decay products will begin to ingrow immediately and (under these closed-system conditions) can contribute to an increase in the total radioactivity for more than 100 years. Conclusions Accurate predictions of radioactivity concentrations are critical for estimating doses to potentially exposed individuals and the surrounding environment. These predictions must include an understanding of the geochemistry, decay properties, and ingrowth kinetics of radium and its decay product radionuclides. Citation Nelson AW, Eitrheim ES, Knight AW, May D, Mehrhoff MA, Shannon R, Litman R, Burnett WC, Forbes TZ, Schultz MK. 2015. Understanding the radioactive ingrowth and decay of naturally occurring radioactive materials in the environment: an analysis of produced fluids from the Marcellus Shale. Environ Health Perspect 123:689–696; http://dx.doi.org/10.1289/ehp.1408855

Temporal characterization of flowback and produced water quality from a hydraulically fractured oil and gas well

This study examined water quality, naturally-occurring radioactive materials (NORM), major ions, trace metals, and well flow data for water used and produced from start-up to operation of an oil and gas producing hydraulically-fractured well (horizontal) in the Denver-Julesburg (DJ) Basin in northeastern Colorado. Analysis was conducted on the groundwater used to make the fracturing fluid, the fracturing fluid itself, and nine flowback/produced water samples over 220days of operation. The chemical oxygen demand of the wastewater produced during operation decreased from 8200 to 2500mg/L, while the total dissolved solids (TDS) increased in this same period from 14,200 to roughly 19,000mg/L. NORM, trace metals, and major ion levels were generally correlated with TDS, and were lower than other shale basins (e.g. Marcellus and Bakken). Although at lower levels, the salinity and its origin appear to be the result of a similar mechanism to that of other shale basins when comparing Cl/Br, Na/Br, and Mg/Br ratios. Volumes of returned wastewater were low, with only 3% of the volume injected (11millionliters) returning as flowback by day 15 and 30% returning by day 220. Low levels of TDS indicate a potentially treatment-amenable wastewater, but low volumes of flowback could limit onsite reuse in the DJ Basin. These results offer insight into the temporal water quality changes in the days and months following flowback, along with considerations and implications for water reuse in future hydraulic fracturing or for environmental discharge.

Optimizing the removal of carbon phases in soils and sediments for sequential chemical extractions by coulometry

We have developed a coulometric technique to optimize the removal of the carbonate and organic fractions for sequential chemical extractions of soils and sediments. The coulometric system facilitates optimizing these two fractions by direct real-time measurement of carbon dioxide (CO2) evolved during the removal of these two fractions. Further analyses by ICP-MS and alpha-spectrometry aided in interpreting the results of coulometry experiments. The effects of time, temperature, ionic strength and pH were investigated. The sensitivity of the coulometric reaction vessel/detection system was sufficient even at very low total carbon content (< 0.1 mol kg-1). The efficiency of the system is estimated to be 96% with a standard deviation of 8%. Experiments were carried out using NIST Standard Reference Materials 4357 Ocean Sediment (OS), 2704 Buffalo River Sediment (BRS), and pure calcium carbonate. Carbonate minerals were dissolved selectively using an ammonium acetate-acetic acid buffer. Organic matter was then oxidized to CO2 using hydrogen peroxide (H2O2) in nitric acid. The carbonate fraction was completely dissolved within 120 min under all conditions examined (literature suggests up to 8 h). For the OS standard, the oxidation of organic matter self-perpetuates between 45 and 50 degrees C, a factor of two less than commonly suggested, while organic carbon in the BRS standard required 80 degrees C for the reaction to proceed to completion. For complete oxidation of organic matter, we find that at least three additions of H2O2 are required (popular methods suggest one or two).

A simple-rapid method to separate uranium, thorium, and protactinium for U-series age-dating of materials

Uranium-series dating techniques require the isolation of radionuclides in high yields and in fractions free of impurities. Within this context, we describe a novel-rapid method for the separation and purification of U, Th, and Pa. The method takes advantage of differences in the chemistry of U, Th, and Pa, utilizing a commercially-available extraction chromatographic resin (TEVA) and standard reagents. The elution behavior of U, Th, and Pa were optimized using liquid scintillation counting techniques and fractional purity was evaluated by alpha-spectrometry. The overall method was further assessed by isotope dilution alpha-spectrometry for the preliminary age determination of an ancient carbonate sample obtained from the Lake Bonneville site in western Utah (United States). Preliminary evaluations of the method produced elemental purity of greater than 99.99% and radiochemical recoveries exceeding 90% for U and Th and 85% for Pa. Excellent purity and yields (76% for U, 96% for Th and 55% for Pa) were also obtained for the analysis of the carbonate samples and the preliminary Pa and Th ages of about 39,000 years before present are consistent with (14)C-derived age of the material.

Radiosynthesis of clinical doses of 68Ga-DOTATATE (GalioMedix™) and validation of organic-matrix-based 68Ge/68Ga generators

INTRODUCTION 68Ga-DOTATATE is a radiolabeled peptide-based agonist that targets somatostatin receptors overexpressed in neuroendocrine tumors. Here, we present our results on validation of organic matrix 68Ge/68Ga generators (ITG GmbH) applied for radiosynthesis of the clinical doses of 68Ga-DOTATATE (GalioMedixTM). METHODS The clinical grade of DOTATATE (25 μg±5 μg) compounded in 1 M NaOAc at pH=5.5 was labeled manually with 514±218 MBq (13.89±5.9 mCi) of 68Ga eluate in 0.05 N HCl at 95°C for 10 min. The radiochemical purity of the final dose was validated using radio-TLC. The quality control of clinical doses included tests of their osmolarity, endotoxin level, radionuclide identity, filter integrity, pH, sterility and 68Ge breakthrough. RESULTS The final dose of 272±126 MBq (7.35±3.4 mCi) of 68Ga-DOTATATE was produced with a radiochemical yield (RCY) of 99%±1%. The total time required for completion of radiolabeling and quality control averaged approximately 35 min. This resulted in delivery of 50%±7% of 68Ga-DOTATATE at the time of calibration (not decay corrected). CONCLUSIONS 68Ga eluted from the generator was directly applied for labeling of DOTA-peptide with no additional pre-concentration or pre-purification of isotope. The low acidity of 68Ga eluate allows for facile synthesis of clinical doses with radiochemical and radionuclide purity higher than 98% and average activity of 272±126 MBq (7.3±3 mCi). There is no need for post-labeling C18 Sep-Pak purification of final doses of radiotracer. Advances in knowledge and implications for patient care. The clinical interest in validation of 68Galabeled agents has increased in the past years due to availability of generators from different vendors (Eckert-Ziegler, ITG, iThemba), favorable approach of U.S. FDA agency to initiate clinical trials, and collaboration of U.S. centers with leading EU clinical sites. The list of 68Ga-labeled tracers evaluated in clinical studies should growth because of the sensitivity of PET technique, the simplicity of the shakebake approach for the dose preparation and reliability of 68Ge/68Ga generators. Our studies have confirmed the reproducible elution profile, and high reliability of ITG GmbH generators required for routine doses preparation according to FDA recommendations.