Alexander V. Kachur

[18F]-EF5, a marker for PET detection of hypoxia: synthesis of precursor and a new fluorination procedure

There is a great deal of clinical and experimental interest in determining tissue hypoxia using non-invasive imaging methods. We have developed EF5, 2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide, with both invasive and non-invasive hypoxia detection in mind. EF5 and other 2-nitroimidazoles are used to detect hypoxia, because the rate of their bioreductive metabolism is inversely dependent on oxygen partial pressure. Such metabolism leads to the formation of covalent adducts within the metabolizing cells. Previously, we have described the invasive detection of these adducts by highly specific monoclonal antibodies after tissue biopsy. In this report, we demonstrate the synthesis of 18F-labeled EF5, [18F]-2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide, in greater than 10% yield by direct fluorination of the newly synthesized precursor 2-(2-nitro-1[H]-imidazol-1-yl)-N-(2,3,3-trifluoroallyl)-acetamide by [18F]-F2 in trifluoroacetic acid. Our objective was to optimize the electrophilic fluorination of the fluorinated alkene bond with fluorine gas, a new method of 18F-labeling of polyfluorinated molecules. Previous biodistribution studies in mice have demonstrated uniform access of EF5 to all tissues with bioelimination dominated by renal excretion. When [18F]-EF5 was injected into a rat followed by urine collection and analysis, we found no detectable metabolism to other radioactive compounds. Thus, [18F]-EF5 should be well suited for use as a non-invasive hypoxia marker with detection using positron emission tomography (PET).

Mechanism of Copper-Catalyzed Oxidation of Glutathione

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal .OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and .OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.

Mechanism of copper-catalyzed autoxidation of cysteine

The kinetics of copper-catalyzed autoxidation of cysteine and its derivatives were investigated using oxygen consumption, spectroscopy and hydroxyl radical detection by fluorescence of a coumarin probe. The process has complex two-phase kinetics. During the first phase a stoichiometric amount of oxygen (0.25 moles per mole of thiol) is consumed without production of hydroxyl radicals. In the second reaction phase excess oxygen is consumed in a hydrogen peroxide-mediated process with significant *OH production. The reaction rate in the second phase is decreased for cysteine derivatives with a free aminogroup and increased for compounds with a modified aminogroup. The kinetic data suggest the catalytic action of copper in the form of a cysteine complex. The reaction mechanism consists of two simultaneous reactions (superoxide-dependent and peroxide-dependent) in the first phase, and peroxide-dependent in the second phase. The second reaction phase begins after oxidation of free thiol. This consists of a Fenton-type reaction between cuprous-cysteinyl complex and following oxidation of cysteinyl radical to sulfonate with the consumption of excessive oxygen and significant production of hydroxyl radicals.

The generation of hydroxyl radicals in the reaction of molecular oxygen with polyphosphate complexes of ferrous ion

The reaction of Fe2+ with molecular oxygen (autoxidation) was investigated in 20 mM phosphate buffer (pH 7.4) at 37 degrees C using a fluorescent OH probe, coumarin-3-carboxylic acid. The autoxidation of unchelated Fe2+ produces OH radicals. Polyphosphatic chelators (pyrophosphate and tri- and tetrapoly phosphate) enhanced the generation of radicals. This effect was explained by an alteration of the reaction mechanism. The two-electron reduction of the oxygen molecule and the generation of hydrogen peroxide intermediates are the major reactions during Fe2+ autoxidation. The polyphosphatic complexes of ferrous ion reduce molecular oxygen and reactive oxygen intermediates by a one-electron mechanism. The chelation of ferrous ion increases the generation of the superoxide radical and production of OH during ferrous ion autoxidation and in the Fenton reaction. The results consider the ferrous ion-polyphosphate system as a convenient model for the generation of hydroxyl radical in biological systems.

Detection of Reactive Oxygen Species via Endogenous Oxidative Pentose Phosphate Cycle Activity in Response to Oxygen Concentration IMPLICATIONS FOR THE MECHANISM OF HIF-1α STABILIZATION UNDER MODERATE HYPOXIA

The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O2. We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O2), whereas hyperoxia (95% O2) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1α (HIF-1α) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1α stabilization under moderate hypoxia. Moreover, the levels of HIF-1α that we observed after exposure to moderate hypoxia were comparable between ρ0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1α in response to the non-toxic levels of H2O2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1α stability under moderate as well as severe hypoxia.

Mechanism of Production of Hydroxyl Radicals in the Copper-Catalyzed Oxidation of Dithiothreitol

We have undertaken detailed studies of the mechanisms involved in the production of OH radicals during the copper-catalyzed oxidation of dithiothreitol (DTT). Most of these studies were conducted in real time, detecting .OH based on its reaction with coumarin-3-carboxylic acid to produce the fluorescent derivative 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). Studies of the kinetics of oxidizing DTT show that production of .OH occurs in two stages: an initial lag period during which there is little production of 7-OHCCA, and a second reaction phase during which there is more rapid generation of .OH. The duration of the initial reaction period depends on the concentrations of both DTT and Cu2+. During this initial stage, oxygen consumption is high, although an increasing concentration of Cu2+ decreases the oxygen consumption. The rate of production of .OH during the second phase of the reaction depends on the concentration of Cu2+, but little oxygen is consumed. A mechanism is proposed whereby a Cu2+-DTT complex is formed and catalyzes oxidation of free DTT via formation of an oxygen-containing intermediate. The second phase of the reaction begins after complete oxidation of free DTT and involves production of O2.- and H2O2 followed by generation of .OH via reduction of H2O2 by cuprous ion.

Synthesis of new hypoxia markers EF1 and [18F]-EF1

We report on the preparation of a hypoxia marker 2-(2-nitroimidazol-1[H]-yl)-N-(3-fluoropropyl)acetamide (EF1) and its 18F analog, 2-(2-nitroimidazol-1[H]-yl)-N- (3-[18F]fluoropropyl)acetamide ([18F]-EF1). Two methods for the preparation of 3-fluoropropylamine, the EF1 side chain, are described. [18F]-EF1 was prepared with a radiochemical yield of 2% by nucleophilic substitution of bromine in 2-(2-nitroimidazol-1[H]-yl)-N-(3-bromopropyl)acetamide (EBr1) by carrier-added 18F in DMSO at 120 degrees C. Our results demonstrate the preparation of clinically relevant amounts of [18F]-EF1 for use as a non-invasive hypoxia marker with detection using positron emission tomography (PET).

Effect of Purine Nucleoside Phosphates on OH-Radical Generation by Reaction of Fe2+ With Oxygen

The influence of various purine nucleotides, nucleosides and nucleoside phosphates on the generation of OH-radicals by the reaction of Fe2+ with oxygen was investigated. Coumarin-3-carboxylic acid was used as a fluorescent detector of OH.. Nucleoside triphosphates caused the enhancement of OH. production due to chelation of ferrous ion by the phosphate moiety. About 30% of produced OH. are intramolecularly scavenged by the nucleoside moiety of the chelator molecule. Nucleoside diphosphates cause a slight enhancement of OH. yield. Nucleotides, nucleosides and nucleoside monophosphates decrease the OH. production. Rate constants of reaction between OH. and nucleoside derivatives were determined from the competitive scavenging of OH radicals, produced by oxidation of Fe(2+)-EDTA complex. Derivatives of guanosine and xanthine are more efficient scavengers in comparison to adenine and inosine. Phosphate groups do not affect the constant of reaction of nucleoside with OH.. Our results suggest that the yield of OH. in the presence of the nucleotide derivatives is determined by chelation of ferrous with polyphosphates and preferential OH. scavenging by the organic portion of molecule. We propose that the generation of active oxygen intermediates in the reaction between nucleoside triphosphate complexes of iron and molecular oxygen is involved in iron-related cellular injury.

Mutation in G6PD gene leads to loss of cellular control of protein glutathionylation: Mechanism and implication

More than 400 million people are susceptible to oxidative stress due to glucose‐6‐phosphate dehydrogenase (G6PD) deficiency. Protein glutathionylation is believed to be responsible for loss of protein function and/or cellular signaling during oxidative stress. To elucidate the implications of G6PD deficiency specifically in cellular control of protein glutathionylation, we used hydroxyethyldisulfide (HEDS), an oxidant which undergoes disulfide exchange with existing thiols. G6PD deficient (E89) cells treated with HEDS showed a significant increase in protein glutathionylation compared to wild‐type (K1) cells. In order to determine whether increase in global protein glutathionylation by HEDS leads to loss of function of an important protein, we compared the effect of HEDS on global protein glutathionylation with that of Ku protein function, a multifunctional DNA repair protein, using a novel ELISA. E89 cells treated with HEDS showed a significant loss of Ku protein binding to DNA. Cellular protein thiol and GSH, whose disulfide is involved in protein glutathionylation, were decreased by HEDS in E89 cells with no significant effect in K1 cells. E89 cells showed lower detoxification of HEDS, that is, conversion of disulfide HEDS to free sulfhydryl mercaptoethanol (ME), compared to K1 cells. K1 cells maintained their NADH level in the presence of HEDS but that of E89 cells decreased by tenfold following a similar exposure. NADPH, a cofactor required to maintain reduced form of the thiols, was decreased more in E89 than K1 cells. The specific role of G6PD in the control of such global protein glutathionylation and Ku function was further demonstrated by reintroducing the G6PD gene into E89 (A1A) cells, which showed a normal phenotype. J. Cell. Biochem. 103: 123–135, 2008.

The radiation response of cells from 9L gliosarcoma tumours is correlated with [F18]-EF5 uptake.

Purpose: Tumour hypoxia affects cancer biology and therapy-resistance in both animals and humans. The purpose of this study was to determine whether EF5 ([2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide]) binding and/or radioactive drug uptake correlated with single-dose radiation response in 9L gliosarcoma tumours. Materials and methods: Twenty-two 9L tumours were grown in male Fischer rats. Rats were administered low specific activity 18F-EF5 and their tumours irradiated and assessed for cell survival and hypoxia. Hypoxia assays included EF5 binding measured by antibodies against bound-drug adducts and gamma counts of 18F-EF5 tumour uptake compared with uptake by normal muscle and blood. These assays were compared with cellular radiation response (in vivo to in vitro assay). In six cases, uptake of tumour versus muscle was also assayed using images from a PET (positron emission tomography) camera (PENN G-PET). Results: The intertumoural variation in radiation response of 9L tumour-cells was significantly correlated with uptake of 18F-labelled EF5 (i.e., including both bound and non-bound drug) using either tumour to muscle or tumour to blood gamma count ratios. In the tumours where imaging was performed, there was a significant correlation between the image analysis and gamma count analysis. Intertumoural variation in cellular radiation response of the same 22 tumours was also correlated with mean flow cytometry signal due to EF5 binding. Conclusion: To our knowledge, this is the first animal model/drug combination demonstrating a correlation of radioresponse for tumour-cells from individual tumours with drug metabolism using either immunohistochemical or non-invasive techniques.