Kenneth L. Kirk

Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice

Ascorbic acid is an essential nutrient commonly regarded as an antioxidant. In this study, we showed that ascorbate at pharmacologic concentrations was a prooxidant, generating hydrogen-peroxide-dependent cytotoxicity toward a variety of cancer cells in vitro without adversely affecting normal cells. To test this action in vivo, normal oral tight control was bypassed by parenteral ascorbate administration. Real-time microdialysis sampling in mice bearing glioblastoma xenografts showed that a single pharmacologic dose of ascorbate produced sustained ascorbate radical and hydrogen peroxide formation selectively within interstitial fluids of tumors but not in blood. Moreover, a regimen of daily pharmacologic ascorbate treatment significantly decreased growth rates of ovarian (P < 0.005), pancreatic (P < 0.05), and glioblastoma (P < 0.001) tumors established in mice. Similar pharmacologic concentrations were readily achieved in humans given ascorbate intravenously. These data suggest that ascorbate as a prodrug may have benefits in cancers with poor prognosis and limited therapeutic options.

Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo

Ascorbate (ascorbic acid, vitamin C), in pharmacologic concentrations easily achieved in humans by i.v. administration, selectively kills some cancer cells but not normal cells. We proposed that pharmacologic ascorbate is a prodrug for preferential steady-state formation of ascorbate radical (Asc•−) and H2O2 in the extracellular space compared with blood. Here we test this hypothesis in vivo. Rats were administered parenteral (i.v. or i.p.) or oral ascorbate in typical human pharmacologic doses (≈0.25–0.5 mg per gram of body weight). After i.v. injection, ascorbate baseline concentrations of 50–100 μM in blood and extracellular fluid increased to peaks of >8 mM. After i.p. injection, peaks approached 3 mM in both fluids. By gavage, the same doses produced ascorbate concentrations of <150 μM in both fluids. In blood, Asc•− concentrations measured by EPR were undetectable with oral administration and always <50 nM with parenteral administration, even when corresponding ascorbate concentrations were >8 mM. After parenteral dosing, Asc•− concentrations in extracellular fluid were 4- to 12-fold higher than those in blood, were as high as 250 nM, and were a function of ascorbate concentrations. By using the synthesized probe peroxyxanthone, H2O2 in extracellular fluid was detected only after parenteral administration of ascorbate and when Asc•− concentrations in extracellular fluid exceeded 100 nM. The data show that pharmacologic ascorbate is a prodrug for preferential steady-state formation of Asc•− and H2O2 in the extracellular space but not blood. These data provide a foundation for pursuing pharmacologic ascorbate as a prooxidant therapeutic agent in cancer and infections.

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2-6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographic-mass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.

Positron emission tomographic imaging of cardiac sympathetic innervation and function.

Sites of uptake, storage, and metabolism of [18F]fluorodopamine and excretion of [18F]fluorodopamine and its metabolites were visualized using positron emission tomographic (PET) scanning after intravenous injection of the tracer into anesthetized dogs. Radioactivity was concentrated in the renal pelvis, heart, liver, spleen, salivary glands, and gall bladder. Uptake of 18F by the heart resulted in striking delineation of the left ventricular myocardium. Pretreatment with desipramine markedly decreased cardiac positron emission, consistent with dependence of the heart on neuronal uptake (uptake-1) for removal of circulating catecholamines. In reserpinized animals, cardiac positron emission was absent within 30 minutes after injection of [18F]-6-fluorodopamine, demonstrating that the emission in untreated animals was from radioactive labeling of the sympathetic storage vesicles. Decreased positron emission from denervated salivary glands confirmed that the tracer was concentrated in sympathetic neurons. Radioactivity in the gall bladder and urinary system depicted the hepatic and renal excretion of the tracer and its metabolites. Administration of tyramine or nitroprusside increased and ganglionic blockade with trimethaphan decreased the rate of loss of myocardial radioactivity. The results show that PET scanning after administration of [18F]fluorodopamine can be used to visualize sites of sympathetic innervation, follow the metabolism and renal and hepatic excretion of catecholamines, and examine cardiac sympathetic function.

3,4-Dihydroxyphenylacetaldehyde potentiates the toxic effects of metabolic stress in PC12 cells

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is a toxic metabolite formed by the oxidative deamination of dopamine. This aldehyde is mainly oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH), but is also partly reduced to 3, 4-dihydroxyphenylethanol (DOPET) by aldehyde or aldose reductase (ARs). In a previous study, we found that rotenone, a complex I inhibitor, induced a rapid accumulation of DOPAL and DOPET in the medium of cultured PC12 cells. Here, we examined the potential role of DOPAL in the toxicity induced by complex I inhibition in PC12 cells and compared the effects of rotenone on concentrations of DOPAL and DOPET to those of MPP(+). DOPAL and DOPET levels were increased by rotenone but decreased by MPP(+). Inhibition of ALDH by daidzein reduced the formation of DOPAC and increased the accumulation of DOPAL. Inhibition of ARs (with AL1576) diminished DOPET formation and elevated DOPAL concentrations. Combined inhibition of ALDH and ARs markedly elevated DOPAL concentrations while diminishing DOPET and DOPAC levels. The elevation of DOPAL levels induced by combined inhibition of ALDH and ARs had no effect on cell viability. However, combined inhibition of ALDH and ARs potentiated rotenone-induced toxicity. Both the potentiation of toxicity and the increase in DOPAL levels were blocked by inhibition of monoamine oxidase with clorgyline indicating that accumulation of DOPAL was responsible for the potentiated rotenone-induced toxicity following combined inhibition of ALDH and ARs. Since complex I dysfunction is reported to be involved in the pathogenesis of Parkinsons disease, DOPAL potentiation of the deleterious effects of complex I inhibition may contribute to the specific vulnerability of dopaminergic neurons to injury.

Positron emission tomographic imaging of cardiac sympathetic Innervation using 6-[18F]Fluorodopamine: Initial findings in humans

OBJECTIVES This study evaluated the safety, efficacy and validity of 6-[18F]fluorodopamine positron emission tomographic scanning of cardiac sympathetic innervation and function in humans. METHODS Positron emission tomography (PET) scans, arterial blood and urine were obtained after a 3-min intravenous infusion of 6-[18F]fluorodopamine (1 to 4 mCi, 188 to 809 mCi/mmol) in healthy volunteers, with or without pretreatment with oral desipramine to inhibit neuronal uptake of catecholamines. RESULTS 6-[18F]Fluorodopamine PET scanning visualized the left ventricular myocardium. Blood pressure increased slightly and transiently. The estimated absorbed radiation dose to the main target organ, the wall of the urinary bladder, was 0.8 to 1.0 rad/mCi of injected 6-[18F]fluorodopamine. By 24 h after the injection, the main 6F-compound in urine was 6F-vanillymandelic acid, a metabolite of 6F-norepinephrine. Desipramine attenuated accumulation of myocardial 6-[18F]fluorodopamine-derived radioactivity and plasma 6F-dihydroxyphenylacetic acid. CONCLUSIONS 6-[18F]Fluorodopamine produces negligible hemodynamic effects and acceptable radiation exposure at doses that visualize the left ventricular myocardium. Sympathetic nerves take up 6-[18F]fluorodopamine, which is translocated from the axoplasm into storage vesicles, where is it beta-hydroxylated to the fluorinated analogue of the sympathetic neurotransmitter norepinephrine. Therefore, the basis for visualization of myocardium after 6-[18F]fluorodopamine injection in humans is radiolabeling by 6-[18F]fluorodopamine and 6-[18F]fluoronorepinephrine of vesicles in sympathetic terminals. 6-[18F]Fluorodopamine PET scanning provides a novel means for assessing sympathetic innervation and function noninvasively in the human heart.

Metabolic stress in PC12 cells induces the formation of the endogenous dopaminergic neurotoxin, 3,4-dihydroxyphenylacetaldehyde†

3,4‐Dihydroxyphenylacetaldehyde (DOPAL) has been reported to be a toxic metabolite formed by the oxidative‐deamination of dopamine (DA) catalyzed by monoamine oxidase. This aldehyde is either oxidized to 3,4‐dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase, an NAD‐dependent enzyme or reduced to 3,4‐dihydroxyphenylethanol (DOPET) by aldehyde or aldose reductase. In the present study we examined whether levels of DOPAL are elevated by inhibition of the mitochondrial respiratory chain. Using inhibitors of mitochondrial complexes I, II, III and IV we found that inhibition of complex I and III increased levels of DOPAL and DOPET. Nerve growth factor‐induced differentiation of PC12 cells markedly potentiated DOPAL and DOPET accumulation in response to metabolic stress. DOPAL was toxic to differentiated PC12 as well as to SK‐N‐SH cell lines. Because complex I dysfunction has been implicated in the pathogenesis of Parkinsons disease, the accumulation of DOPAL may explain the vulnerability of the dopaminergic system to complex I inhibition. The rapid appearance of DOPAL and DOPET after inhibition of complex I may be a useful early index of oxidative stress in DA‐forming neurons. J. Neurosci. Res. 60:552–558, 2000

Fluoroimidazoles as antiviral agents and inhibitors of polynucleotide biosynthesis

Abstract 4-fluoroimidazole (4-FI), 4-fluoroimidazole-5-carboxylic acid (4-FIC), 4-fluoroimidazole-5-carboxamide (4-FICA), and 5-fluoro-1-β-D-ribofuranosylimidazole-4-carboxamide (5-FICAR), have been studied for their inhibitory effects on viral cytopathogenecity in a variety of assay systems encompassing nearly all major virus groups. Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carbox-amide), 5-AICAR (5-amino-1-β-D-ribofuranosylimidazole-4-carbox-amide), and poly(I)βpoly (C) were included as reference materials. Although the antiviral activities of ribavirin and the fluoroimidazoles varied considerably from one system to another, the relative order of activity remained constant: ribavirin >5-FICAR > 5-AICAR > 4-FICA. 4-FIC and 4-FI were inactive. Poly(I)βpoly (C) exhibited a spectrum of antiviral activity that differed totally from that of 5-FICAR and the other compounds. Unlike 5-AICAR, both 5-FICAR and ribavirin inhibited cellular DNA3 and RNA synthesis, as determined by [3H-methyl]thymidine and [3H-5]uridine incorporation, respectively, at concentrations which coincided quite well with those inhibiting viral cytopathogenicity. Hence, 5-FICAr and ribavirin may owe their broad-spectrum antiviral activity to inhibition of nucleic acid synthesis in the infected cell.

Functionalized congeners of 1,3-dipropyl-8-phenylxanthine: potent antagonists for adenosine receptors that modulate membrane adenylate cyclase in pheochromocytoma cells, platelets and fat cells.

Six amine, amino acid and peptide derivatives derived from 1,3-dipropyl-8-(p-carboxymethylphenyl)xanthine, a functionalized congener of 1,3-dipropyl-8-phenylxanthine, have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC 12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. The functionalized congeners and conjugates have affinity constants ranging from 80 to 310 nM at A2 receptors of PC 12 cells and from 25 to 135 nM at those of platelets. The affinity of the xanthine derivatives at A1 receptors of fat cells are in the 15 to 30 nM range. Thus, the amino acid and peptide conjugates have high potencies at both receptor subclasses and show some selectivity toward A1 adenosine receptors. Derivatives of the congeners should be useful as receptor probes and as radioiodinated ligands.

The effect of ring-fluorination on the rate of O-methylation of dihydroxyphenylalanine (DOPA) by catechol-O-methyltransferase: significance in the development of 18F-PETT scanning agents.

The three ring-fluorinated analogs of dihydroxyphenylalanine (DOPA) were synthesized and the kinetics of their O-methylation catalyzed by catechol-O-methyltransferase compared to those of DOPA. The affinities (Km) of 2-FDOPA, 5-FDOPA, DOPA, and 6-FDOPA were 20, 23, 55, and 552 microM, respectively, whilst the Vmax values were 4.0, 4.4, 5.0 and 5.4 nmol per min per mg protein. The importance of the low affinity and decreased rate constant of 6-FDOPA for COMT are discussed with regard to the use of 6-FDOPA as an 18-fluorine labeled probe for positron emission transaxial tomography (PETT).