Stephen M. Hanrahan

Design and Implementation of a Facility for Discovering New Scintillator Materials

We describe the design and operation of a high-throughput facility for synthesizing thousands of inorganic crystalline samples per year and evaluating them as potential scintillation detector materials. This facility includes a robotic dispenser, arrays of automated furnaces, a dual-beam X-ray generator for diffractometry and luminescence spectroscopy, a pulsed X-ray generator for time response measurements, computer-controlled sample changers, an optical spectrometer, and a network-accessible database management system that captures all synthesis and measurement data.

Development of an optimized activatable MMP-14 targeted SPECT imaging probe

Matrix metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodeling processes and a molecular hallmark of select metastatic cancers. The ability to detect MMP-14 in vivo would be useful in studying its role in pathologic processes and may potentially serve as a guide for the development of targeted molecular therapies. Four MMP-14 specific probes containing a positively charged cell penetrating peptide (CPP) d-arginine octamer (r(8)) linked with a MMP-14 peptide substrate and attenuating sequences with glutamate (8e, 4e) or glutamate-glycine (4eg and 4egg) repeating units were modeled using an AMBER force field method. The probe with 4egg attenuating sequence exhibited the highest CPP/attenuator interaction, predicting minimized cellular uptake until cleaved. The in vitro MMP-14-mediated cleavage studies using the human recombinant MMP-14 catalytic domain revealed an enhanced cleavage rate that directly correlated with the linearity of the embedded peptide substrate sequence. Successful cleavage and uptake of a technetium-99m labeled version of the optimal probe was demonstrated in MMP-14 transfected human breast cancer cells. Two-fold reduction of cellular uptake was found in the presence of a broad spectrum MMP inhibitor. The combination of computational chemistry, parallel synthesis and biochemical screening, therefore, shows promise as a set of tools for developing new radiolabeled probes that are sensitive to protease activity.

Scintillation Properties of Eu

The scintillation properties of powders and single-crystals of BaFI doped with Eu2+ are presented. Single crystals were grown by the vertical Bridgman technique. Under optical and X-ray excitation, the samples exhibit a narrow Eu2+ 5d-4f transition emission centered at 405 nm. The scintillation light output is estimated to be 55,000±5,000 photons/MeV at 662 keV with 85% of the light decaying within 600 ns. An energy resolution of 8.5% full width at half maximum (FWHM) has been achieved using this scintillator for 662 keV excitation (137Cs source) at room temperature.

^{2+}

Epidermal growth factor receptors (EGFR), upregulated in many tumor types, have been a target for therapeutic development and molecular imaging. The objective of this study was to evaluate the distribution and metabolic characteristics of fluorine-18 labeled anilinoquinazolines as potential imaging agents for EGFR tyrosine kinase expression. Fluorine-18 labeled fluoronitrobenzenes were prepared by reaction of potassium cryptand [(18)F]fluoride with 1,2- and 1,4-dinitrobenzenes, and 3-nitro-N,N,N-trimethylanilinium triflate in 5min. Decay-corrected radiochemical yields of [(18)F]fluoride incorporation into the nitro-aromatic compounds were 81±2%, 44±4% and 77±5% (n=3-5) for the 2-, 3- and 4-fluoro isomers, respectively. Sodium borohydride reduction to the corresponding [(18)F]fluoroanilines was achieved with greater than 80% conversion in 5min. Coupling of [(18)F]fluoroaniline-hydrochlorides to 6,7-dimethoxy-4-chloro-quinazoline gave the corresponding 6,7-dimethoxy-4-(2-, 3- and 4-[(18)F]fluoroanilino)quinazolines in 31±5%, 17±2% and 55±2% radiochemical yield, respectively, while coupling to the 6,7-diethoxy-4-chloro-quinazoline produced 6,7-diethoxy-4-(2-, 3- and 4-[(18)F]fluoroanilino)quinazolines in 19±6%, 9±3% and 36±6% radiochemical yield, respectively, in 90min to end of synthesis from [(18)F]fluoride. Biodistribution of 2- and 4-[(18)F]fluoroanilinoquinazolines was conducted in tumor-bearing mice (MDA-MB-435 and MDA-MB-468 xenografts). Low tumor uptake (<1% injected dose per gram (ID/g) of tissue up to 3h postinjection of the radiotracers) was observed. High bone uptake (5-15% ID/g) was noted with the 4-[(18)F]fluoroanilinoquinazolines. The metabolic stabilities of radiolabeled quinazolines were further evaluated by incubation with human female cryopreserved isolated hepatocytes. Rapid degeneration of the 4-fluoro-substituted compounds to baseline polar metabolites was observed by radio-TLC, whereas, the 2- and 3-[(18)F]fluoroaniline derivatives were significantly more stable, up to 2h, corroborating the in vivo biodistribution studies. para-Substituted [(18)F]fluoroanilines, a common structural motif in radiopharmaceuticals, are highly susceptible to metabolic degradation.

-Activated Barium Fluoroiodide

Background— There is a well-recognized need for a new generation of single photon emission computed tomography (SPECT) perfusion tracers with improved myocardial extraction over a wide flow range. Radiotracers that target complex I of the mitochondrial electron transport chain have been proposed as a new class of myocardial perfusion imaging agents. 7-(Z)-[125I]iodorotenone (125I-ZIROT) has demonstrated superior myocardial extraction and retention characteristics in rats and in isolated perfused rabbit hearts. We sought to fully characterize the biodistribution and myocardial extraction versus flow relationship of 123I-ZIROT in an intact large-animal model. Methods and Results— The 123I-ZIROT was administered during adenosine A2A agonist-induced hyperemia in 5 anesthetized dogs with critical left anterior descending (LAD) stenoses. When left circumflex (LCx) flow was maximal, 123I-ZIROT and microspheres were coinjected and the dogs were euthanized 5 minutes later. 123I-ZIROT biodistribution was evaluated in 2 additional dogs by in vivo planar imaging. At 123I-ZIROT injection, transmural LAD flow was unchanged from baseline (mean±SEM, 0.90±0.22 versus 0.87±0.11 mL/[min · g]; P=0.92), whereas LCx zone flow increased significantly (mean±SEM, 3.25±0.51 versus 1.00±0.17 mL/[min · g]; P<0.05). Myocardial 123I-ZIROT extraction tracked regional myocardial flow better than either thallium-201 or 99mTc-sestamibi from previous studies using a similar model. Furthermore, the 123I-ZIROT LAD/LCx activity ratios by ex vivo imaging or well counting (mean±SEM, 0.42±0.08 and 0.45±0.1, respectively) only slightly underestimated the LAD/LCx microsphere flow ratio (0.32±0.09). Conclusions— The ability of 123I-ZIROT to more linearly track blood flow over a wide range makes it a promising new SPECT myocardial perfusion imaging agent with potential for improved coronary artery disease detection and better quantitative estimation of the severity of flow impairment.

METABOLIC STABILITY OF 6,7-DIALKOXY-4-(2-, 3- AND 4-[18F]FLUOROANILINO)QUINAZOLINES, POTENTIAL EGFR IMAGING PROBES

Estrogens or antiestrogens are currently used by millions of women, but the interaction of these hormonal agents with brain estrogen receptors (ER) in vivo has not been characterized to date. Our goal was to assess, in vivo, the extent and regional distribution of brain ER occupancy in rats chronically exposed to 17beta-estradiol (E(2)) or tamoxifen (TAM). For that purpose, female ovariectomized Sprague-Dawley rats were implanted with subcutaneous pellets containing either placebo (OVX), E(2), or TAM for 3 weeks. ER occupancy in grossly dissected regions was quantified with 16alpha-[(18)F]fluoroestradiol ([(18)F]FES). Both E(2) and TAM produced significant decreases in radioligand uptake in the brain although the effect of E(2) was larger and more widespread than the effect of TAM. Detailed regional analysis of the interaction was then undertaken using a radioiodinated ligand, 11beta-methoxy-16alpha-[(125)I]iodo-estradiol ([(125)I]MIE(2)), and quantitative ex vivo autoradiography. E(2) treatment resulted in near-complete (86.6 +/- 17.5%) inhibition of radioligand accumulation throughout the brain, while ER occupancy in the TAM group showed a marked regional distribution such that percentage inhibition ranged from 40.5 +/- 15.6 in the ventrolateral part of the ventromedial hypothalamic nucleus to 84.6 +/- 4.5 in the cortical amygdala. These results show that exposure to pharmacologically relevant levels of TAM produces a variable, region-specific pattern of brain ER occupancy, which may be influenced by the regional proportion of ER receptor subtypes. These findings may partially explain the highly variable and region-specific effects observed in neurochemical, metabolic, and functional studies of the effects of TAM in the brain of experimental animals as well as human subjects.

Myocardial Uptake of 7′-(Z)-[123I]Iodorotenone During Vasodilator Stress in Dogs With Critical Coronary Stenoses

The goal of this project is to develop radionuclide molecular imaging technologies using a clinical pinhole SPECT/CT scanner to quantify changes in cardiac metabolism using the spontaneously hypertensive rat (SHR) as a model of hypertensive-related pathophysiology. This paper quantitatively compares fatty acid metabolism in hearts of SHR and Wistar-Kyoto normal rats as a function of age and thereby tracks physiological changes associated with the onset and progression of heart failure in the SHR model. The fatty acid analog, 123I-labeled BMIPP, was used in longitudinal metabolic pinhole SPECT imaging studies performed every seven months for 21 months. The uniqueness of this project is the development of techniques for estimating the blood input function from projection data acquired by a slowly rotating camera that is imaging fast circulation and the quantification of the kinetics of 123I-BMIPP by fitting compartmental models to the blood and tissue time-activity curves.

In vivo occupancy of female rat brain estrogen receptors by 17β-estradiol and tamoxifen

Single crystals of Ba0.96Eu0.04BrI (barium europium bromide iodide) were grown by the Bridgman technique. The title compound adopts the ordered PbCl2 structure [Braekken (1932 ▶). Z. Kristallogr. 83, 222–282]. All atoms occupy the fourfold special positions (4c, site symmetry m) of the space group Pnma with a statistical distribution of Ba and Eu. They lie on the mirror planes, perpendicular to the b axis at y = ±0.25. Each cation is coordinated by nine anions in a tricapped trigonal prismatic arrangement.

Longitudinal Evaluation of Fatty Acid Metabolism in Normal and Spontaneously Hypertensive Rat Hearts with Dynamic MicroSPECT Imaging.

Radionuclide techniques will be increasingly important for the development of biofuels, since they can both uniquely characterize metabolic pathways and image large model systems. We are investigating the application of nuclear medical imaging tools and techniques to biofuel development, using high-sensitivity chromatographic radiation detectors and compounds radiolabeled with short-lived, cyclotron-produced, positron-emitting isotopes (e.g., 11C). The application of high-sensitivity radiation detectors to standard chromatographic techniques should allow for the measurement of numerous pathway constituents whose levels normally fall below detection limits of conventional instrumentation. In this paper, we describe a proposed parallel-plane PET camera designed for HPLC (high performance liquid chromatography) with over 40 times greater sensitivity than a conventional HPLC radiation detector; this PET camera could be used to image radioactivity in the HPLC exit tube or column. We also present results using a high-sensitivity HPLC radiation detector comprised of 8 HR+ PET detector modules arranged into a parallel plane and read out with HRRT electronics. This high-sensitivity radiation detector was placed in line after a conventional HPLC radiation detector (a small CsI:Tl scintillator crystal coupled to a PIN photodiode). If we inject 9.3 μCi of [18F]FDG into the HPLC system, we see similarly shaped peaks with an excellent signal-to-noise ratio from both radiation detectors. If we inject only 4.1 nCi of [18F]FDG, we measure a signal-to-noise ratio of 27:1 with the high-sensitivity radiation detector and about 3:1 for the conventional radiation detector. We have therefore demonstrated that a high-sensitivity radiation detector, using a parallel-plane PET camera, could become an important tool for fundamental biofuel research.

Europium-doped barium bromide iodide

The goal of this work is to quantitatively compare fatty acid metabolism in the hearts of normal and spontaneously hypertensive rats (SHR) as a function of age, and thereby track physiological changes associated with the onset and progression of heart failure in the SHR model. We imposed a more-stringent negativity penalty for fully 4-D multiresolution B-spline SPECT image reconstruction, and applied this method to obtain weighted least-squares estimates of time-activity curves directly from projections for dynamic pinhole SPECT studies of 123I-labeled BMIPP fatty acid analog in the myocardium of both a normal rat and an SHR. Dynamic projection data were acquired for 60 min in 1-sec time frames with an angular step of 4 degrees per frame on a dual-head GE Millennium VG Hawkeye SPECT/CT scanner equipped with custom pinhole collimators. The time-varying spatial distribution of 123I-BMIPP was modeled with use of 4-D multiresolution B-splines that were piecewise constant in space and piecewise quadratic in time. The 4-D splines were organized on a 3-D spatial grid that provided sampling of 3.2 mm near the heart and 6.4 mm away from the heart. Temporal sampling intervals were 0–2.4, 2.4–9.4, 9.4–30, and 30–90 sec during the first gantry rotation. The use of nonuniform time sampling with B-splines that varied quadratically in time yielded smooth time-activity curves that captured the relatively fast rise and fall of 123I-BMIPP in the left ventricular blood pool, as well as the uptake and trapping of the radiotracer in the left ventricular myocardium. Compartmental modeling was then applied to the time-activity curves. This yielded quantitative metabolic rate estimates (Ki) of 0.60 min−1 and 0.22 min−1 in the normal and SHR hearts, respectively. The slower rate of fatty acid metabolism in the SHR is what one expects as the SHR heart switches to a reliance on glycolysis as the primary pathway for energy production during the development of heart failure. This fully 4-D image reconstruction method can also be applied to dynamic PET.