N-substitution of carbon 8 in guanosine and deoxyguanosine by the carcinogen N-benzoyloxy-N-methyl-4-aminoazobenzene in vitro.

The major reaction products of the carcinogenic electrophile N-benzoyloxy-N-methyl-4-aminoazobenzene with guanosine or deoxyguanosine were characterized as N-(guanosin-8-yl)- and N-(DEOXYGUANOSIN-8-YL)-N-methyl-4-aminoazobenzene from the following chemical, radiochemical, and spectroscopic studies: (a) the presence of equimolar amounts of both N-methyl-4-aminoazobenzene (MAB) and guanosine or deoxyguanosine residues was shown by the 3H:14C ratios of the products from the reaction of (prime ring-3H)-N-benzoyloxy-N-methyl-4-aminoazobenzene with (8-14C)guanosine or (8-14C)deoxyguanosine and by the molecular weights of the trimethylsilyl derivatives of both products: (b) substitution of the dye residue on its amino nitrogen was indicated by the retention in the products of the 3H:14C ration of (CH2-3H + 14CH3)-N-BENZOYLOXY-N-methyl-4-aminoazobenzene and by the release of MAB on treatment of the nucleoside-dye derivatives with strong alkali in air; (c) substitution of the guanine residues in positon 7 or 8 was demonstrated by loss of 3H from (8-3H)guanosine or (8-3H)deoxyguanosine in the formation of the nucleoside-dye derivatives; (d) the stability of the products to mild alkali (as contrasted to the lability of 7-alkylguanosines) provided strong evidence that the substitution was in position 8 of the guanine residue; (e) direct evidence of 8-substitution came from the acid hydrolysis of guanosinyl- and deoxyguanosinyl-N-methyl-4-aminoazo benzene to N-(guan-8-yl)-N-methyl-4-aminoazobenze in up to 50% yield; (f) comparisons of the proton or 13C nuclear magnetic resonance spectra or both of N-(guan-8-yl)- N-methyl-4-aminoazobenzene, MAB, N-(guanosin-8-yl)-2-acetylaminofluorene, 2-acetylaminofluorene, guanosine, and 7 -methylguanosine with the spectra of the guanosine-MAB product further confirmed that substitution had occurred at position 8 of the guanosine residue. The new compound N-(guan-8-yl)-N-methyl-4-aminoazobenzene was synthesized. Attempts to devise an unambiguous synthesis of N-(GUANOSIN-8-YL)-N-methyl-4-aminoazobenzene were not successful.

Quantification of amphetamine-induced changes in [11C]raclopride binding with continuous infusion.

Positron emission tomography and single-photon emission computer tomography receptor-binding ligands can be used to measure changes in neurotransmitter levels. In particular, amphetamine-induced dopamine release has been assessed with [11C]raclopride by paired bolus injections and with [123I]iodobenzamide by using a single bolus plus infusion (B/I) study. Here, we measured the change in [11C]raclopride-specific binding in rhesus monkeys after i.v. administration of 0.4 mg/kg amphetamine by using both the bolus and B/I paradigms. Paired bolus studies (control and postamphetamine) were analyzed using compartment modeling and graphical analysis with a new plasma metabolite model to measure the total distribution volume (VT). Specific binding, calculated with three measures linearly proportional to the binding potential, demonstrated a 22-42% reduction in the postamphetamine study. VT values from B/I studies were determined by the tissue-to-plasma ratio at equilibrium, in addition to the bolus methods. There was good agreement between the control VT values between bolus and B/I studies. The amphetamine-induced change in specific binding in B/I studies was 19 +/- 16%, measured directly from tissue radioactivity levels. This study demonstrates that stimulus-induced changes in specific binding can be measured with a single [11C]raclopride study using the B/I method.

The suitability of [11C]-alpha-methyl-L-tryptophan as a tracer for serotonin synthesis: studies with dual administration of [11C] and [14C] labeled tracer.

The tracer [11C]-alpha-methyl-L-tryptophan (alphaMTP) has been used to measure brain serotonin synthesis rates with positron emission tomography (PET). To address questions about the accuracy of the kinetic model, [14C]alphaMTP was used to directly measure conversion to [14C]-alpha-methyl-serotonin (alphaM5HT) in monkeys that had been previously studied with PET and [11C]alphaMTP. Four male, fasted, isoflurane-anesthetized rhesus monkeys were studied with [11C]alphaMTP and PET. Immediately after the initial 3-hour scan, a second dose of [11C]alphaMTP was coinjected with 1 mCi of [14C]alphaMTP, and additional PET data were collected. Approximately 90 minutes after the second alphaMTP administration, the animals were killed with an overdose of phenobarbital, and brain samples from 21 regions were taken and analyzed by HPLC. Minimal conversion of alphaMTP to alphaM5HT occurred; HPLC analysis of 14C radioactivity showed that greater than 96% of the total counts were in fractions corresponding to the alphaMTP peak. Brain concentrations of serotonin, tryptophan, 5-hydroxyindole-3-acetic acid, and alphaMTP also were determined fluorometrically using external quantification. Patlak plots generated from PET images acquired over 3 hours showed no time period of linear increase, and final slopes were not significantly different from zero, consistent with the finding of minimal conversion to [14C]alphaM5HT. These data indicate that in the 3-hour period after injection, [11C]alphaMTP is acting predominantly as a tracer of tryptophan uptake, not serotonin synthesis.

Brain tumor imaging in rats using the positron emitting fatty acid dl-erythro-9,10-[18F]difluoropalmitate.

Positron emitting dl-erythro-9,10[18F]difluoropalmitate, [18F]DFPA, was synthesized for the in vivo imaging of brain tumors in rats. Male Fischer 344 rats were intracerebrally implanted with Walker 256 carcinosarcoma tumor cells (1 x 10(6) in 5 microliters tissue culture media) and 7 days later were infused with [18F]DFPA (500-1000 mCi/mmol) i.v. for 5 min. Rats were killed after 20 min. Brains were removed and either prepared for autoradiography, or brain and tumor were separated and their radioactivity quantified by gamma spectroscopy. Brain tumors were well demarcated from surrounding and normal brain in autoradiographs, and closely paralleled tumor growth in histological sections. The mean optical density of tumor was significantly greater, by 318 +/- 68 per cent (P less than 0.025, n = 3), than normal brain in autoradiographs, and that of edematous brain surrounding a large tumor was intermediately increased. [18F]DFPA proved of value to image and circumscribe intracerebral tumors in awake rats, and studies are continuing to facilitate its clinical application in brain tumor patients.

Brain serotonin synthesis rates in rhesus monkeys determined by [11C]alpha-methyl-L-tryptophan and positron emission tomography compared to CSF 5-hydroxyindole-3-acetic acid concentrations.

Twelve male, fasted, anesthetized rhesus monkeys were studied with positron emission tomography (PET) and [11C]alpha-methyl-L-tryptophan (alpha MTP) to determine serotonin synthesis rates as described by Diksic et al. (1991). It was expected that the serotonin synthesis rates determined for the whole brain would be correlated with CSF 5-hydroxyindole-3-acetic acid concentrations, a measure of central serotonin turnover, because both measures were obtained at steady state. However, no significant correlation was found. During data analysis, it was noticed that the calculated serotonin synthesis rates were significantly correlated to free plasma tryptophan (TP) concentrations (r = 0.88, p < .001). From repeat scans conducted in six monkeys, it was determined that day-to-day variability in free plasma TP and the percentage of protein binding (average percent difference was 48 and 37%, respectively) produced most of the variability in the calculated serotonin synthesis rates (50%); repeat K images, obtained from the PET data alone, differed by only 11%. Calculated serotonin synthesis rates reported for [11C]alpha MTP PET studies of humans (Nishizawa et al. 1997) and dogs (Diksic et al. 1991) were also highly correlated to reported differences in plasma free TP concentrations. It seems that the [11C]alpha MTP model for the computation of serotonin synthesis rates is very dependent on plasma free TP concentration and that it may not accurately determine actual serotonin synthesis rates.

Development of fluorine-18-labeled 5-HT1A antagonists.

We have synthesized five fluorinated derivatives of WAY 100635, N-{2-[4-(2-methoxyphenyl)piperazino]ethyl}-N-(2-pyridyl)cyclohe xaneca rboxamide (4a), using various acids in place of the cyclohexanecarboxylic acid (CHCA, 2a) in the reaction scheme. The five acids are 4-fluorobenzoic acid (FB, 2b), 4-fluoro-3-methylbenzoic acid (MeFB, 2c), trans-4-fluorocyclohexanecarboxylic acid (FC, 2d), 4-(fluoromethyl)benzoic acid (FMeB, 2e), and 3-nitro-4-(fluoromethyl)benzoic acid (NFMeB, 2f) (see Scheme 1). These compounds were radiolabeled with fluorine-18, and their biological properties were evaluated in rats and compared with those of [11C]carbonyl WAY 100635 ([carbonyl-11C]4a). [Carbonyl-11C]4a cleared the brain with a biological half-life averaging 41 min. The metabolite-corrected blood radioactivity had a half-life of 29 min. [18F]FCWAY ([18F]4d) gave half-lives and intercepts comparable to [carbonyl-11C]4a in the brain, but the blood clearance was faster. [18F]FBWAY ([18F]4b) showed an early rapid net efflux from the whole brain, clearing with a biological half-life of 35 min. The metabolite-corrected blood half-life was 41 min. The comparable whole brain and blood half-lives for Me[18F]FBWAY ([18F]4c) were 16 and 18 min, respectively. For each compound, the corresponding carboxylic acid was identified as a major metabolite in blood. Fluoride was also found after injection of [18F]4d. However, for all compounds there was a good correlation (R > 0.97) between the differential uptake ratio (DUR, (%ID/g) x body weight (g)/100) in individual rat brain regions at 30 min after injection and the concentration of receptors as determined by in vitro quantitative autoradiography in rat. Specific binding ratios [region of interest (ROI)/cerebellum-1] in control studies for cortex (Ctx) and hippocampus (H) were higher for [carbonyl-11C]4a and [18F]4d compared to [18F]4b and [18F]4c. [18F]4d has similar pharmacokinetic properties and comparable specific binding ratios to [carbonyl-11C]4a. Fifty nanomoles of 4a blocked only 30% of the specific binding of [18F]4d, while complete blockade was obtained from co-injection of 200 nmol of 4a (H/Cb-1 from 17.2 to 0.6). [18F]4b and [18F]4c showed lower specific binding ratios than [carbonyl-11C]4a and [18F]4d. [18F]4c was superior to [18F]4b since its specific binding was more readily blocked by 4a. These studies suggest that [18F]4c should be a useful compound to assess dynamic changes in serotonin levels while [18F]4d, with its high contrast and F-18 label, should provide better statistics and quantification for static measurement of 5-HT1A receptor distribution.

Neuroleptic binding sites: specific labeling in mice with [18F]haloperidol, a potential tracer for positron emission tomography.

Haloperidol labeled with fluorine-18 (T 1/2 = 110 min, positron emission 97%), prepared yielding .04 Ci/millimole by the Balz-Schiemann reaction, was evaluated in a murine model as a potential radiotracer for noninvasive determination, by positron-emission tomography, of regional concentrations of brain dopamine receptors in patients. As the haloperidol dose in mice was increased from 0.01 to 1000 micrograms/kg, the relative concentration of [18F]haloperidol (microCi per g specimen/microCi per g of body mass), at one hour after injection decreased from 30 to 1.0 in the striatum and from 8.0 to 1.0 in the cerebellum. The striatal radioactivity, plotted as relative concentration against log of dose, decreased sigmoidally, presumably reflecting competition between labeled and unlabeled haloperidol for a single class of accessible binding sites. Because the cerebellum is relatively deficient in dopamine receptors, the observed decrease in cerebellar radioactivity may reflect a saturable component of haloperidol transport into brain. The high brain concentrations and the unexpectedly high striatum-to-cerebellum concentration ratios (greater than 4 at haloperidol doses less than or equal to 1 microgram/kg) suggest that [18F]haloperidol warrants further investigation as a potential radiotracer for dopamine receptors.

Measurement of dopamine release with continuous infusion of [11C]raclopride: optimization and signal-to-noise considerations.

UNLABELLED: PET studies with [11C]raclopride provide an indirect measure of changes in synaptic dopamine. Previously, we used the bolus-plus-infusion (B/I) method to assess dopamine response from the percentage change in binding potential (deltaBP) before and after administration of amphetamine. The goal of this work is to optimize the measurement of changes in neurotransmitter with the B/I method by choosing the optimal timing for pre- and poststimulus scanning. METHODS: Two sources of variability in deltaBP were considered: within-subject and between-subject noise. A noise model based on a phantom study and human data was used to evaluate the within-subject noise. For between-subject noise, simulated time--activity curves were generated from measured [11C]raclopride input functions. Optimal timing to measure deltaBP was determined and applied to human data. RESULTS: According to the simulation study, the optimal scan times for pre-and poststimulus scans were 39-50 and 58-100 min, respectively. The optimal timing resulted in a 28% noise reduction compared with the original timing. By applying the optimal timing to human studies, the statistical significance of the difference in deltaBP between patients with schizophrenia and healthy volunteers increased from P = 0.038 to 0.012. CONCLUSION: Careful assessment of the sources of noise in receptor imaging studies can increase the sensitivity of the B/I method for the detection of biologic signals.

Purification and analysis of a human sarcoma associated antigen.

S1, a heterophile antigen present on human sarcoma cell lines in culture, has been previously defined by this laboratory [1,2]. This antigen is also present in guinea-pig kidney. Purification of the antigen to homogeneity has now been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose, sephadex, high pressure liquid chromotography and affinity chromotography. S1 is a monomeric protein of 70,000 Da, as indicated by the presence of a single band on SDS-PAGE. Amino acid analysis demonstrates the prevalence of glycine, lysine and glutamic acid. Aspartic acid was found to be the N-terminal residue with further sequence of glycine-valine-alanine-glutamic acid (gly-val-ala-glut).

Synthesis of [11C-methyl]-alpha-aminoisobutyric acid (AIB).

Alpha-aminoisobutyric acid (AIB) labeled with the cyclotron-produced, positron-emitting radionuclide 11C has been synthesized with the label in the alpha-methyl group. Our previously published synthesis of [11C] AIB in the carboxyl position from [11C] HCN requires a rigorous quality assurance program to ensure that the concentration of cyanide in the final product is below certain levels. This can be avoided using the method described here with [11C] CH3I. The radiochemical yield calculated to end of bombardment (EOB) was 45-55% from [11C] CO2 with radiochemical purity of [11C-methyl] AIB exceeding 99%. Synthesis times from [11C] CO2 were about 55 min. Specific activities of 1 Ci/mumol were achieved on average. It has been shown that [11C-carboxyl] AIB is a useful imaging agent in patients with soft tissue cancers and melanoma, and it demonstrates tumor uptake in a spectrum of other animal tumor models. Because AIB is a nonmetabolized amino acid, [11C-methyl] AIB should be equally as useful. Either agent can be employed for the quantification of the A-type amino acid transport system in vivo with PET.

Fluoro analogs of WAY-100635 with varying pharmacokinetics properties.

Radiolabeled derivatives of WAY-100635 have been shown to be important for imaging in vivo because of their antagonist properties and their specificity for the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor. Our goal is to prepare a series of radiofluorinated derivatives of WAY-100635 that, in the rat, range in pharmacokinetic properties from nearly irreversible to reversible in their behavior. It appears that derivatives containing a cyclohexanecarboxylic acid (e.g., FCWAY) with its high affinity and high target to nontarget contrast, has properties suited to measure receptor concentration. Derivatives based on phenylcarboxamide (e.g., FBWAY and MeFBAWAY) have properties more suited to the measurement of changes in endogenous serotonin. The compound containing the pyrimidine moiety in place of the pyridine moeity in FBWAY (FBWAY 1,3N) appears to have intermediate properties.

Synthesis of 2'-fluoro-5-[11C]-methyl-1-beta-D-arabinofuranosyluracil ([11C]-FMAU): a potential nucleoside analog for in vivo study of cellular proliferation with PET.

Rapid in vivo catabolism limits the use of currently available radiotracers used in tumor proliferation studies with PET. This is manifested by the need to develop complex mathematical models to interpret kinetic and metabolite data obtained from imaging studies with agents such as carbon-11 labeled thymidine. A potential carbon-11 labeled radiotracer for cellular proliferation, 2'-fluoro-5-([11C]-methyl)-1-beta-D-arabinofuranosyluracil (FMAU), has been prepared using a previously described method for preparation of [11C]methyl-thymidine where selective alkylation of a pyrimidyl dianion is accomplished with [11C]methyl iodide at the 5-position of the pyrimidine ring. FMAU shares many in vivo characteristics of thymidine, including cellular transport, phosphorylation by mammalian kinase, and incorporation into DNA. Most importantly, in vivo catabolism of FMAU is limited, potentially yielding simplified kinetic models for determination of cellular proliferation with positron emission tomography.

Tumor localization of the metabolically trapped radiolabeled substrates 2-deoxy-D-glucose and aminocyclopentanecarboxylic acid in human melanoma heterotransplants.

Analysis of the accumulation of metabolically trapped radiolabeled substrates in normal and malignant tissues may be useful for studying biochemical processes in vivo with positron emission tomography (PET). If labeled with the short-lived, positron-emitting radionuclide carbon-11 (T1/2 = 20.4 min), the glucose analog, 2-deoxy-D-glucose (2-DG), and the synthetic amino acid, aminocyclopentanecarboxylic acid (ACPC), may be useful for studying glucose utilization and amino acid transport in vivo. This study evaluated the biodistribution of the C-14 labeled analogues of these compounds in nude mice bearing human malignant melanoma heterotransplants. The data suggest that both 2-DG and ACPC accumulate in tumor tissue within 45 min.

Imaging studies of patients with malignant fibrous histiocytoma using C-11-alpha-aminoisobutyric acid (AIB).

Alpha-aminoisobutyric acid (AIB), a synthetic, nonmetabolized amino acid which is rapidly transported into viable cells by the A-type or alanine-preferring amino acid transport system, has been labeled with the short-lived, positron-emitting radionuclide carbon-11. Carbon-11 labeled AIB is currently being evaluated as a tumor imaging agent for in vivo amino acid transport studies in patients with cancer. In this study, C-11 AIB was used to image two patients with malignant fibrous histiocytoma (MFH), a pleomorphic sarcoma. Following intravenous administration of C-11 AIB, tumors in the distal femur of one patient and in the anterior chest wall of another patient were well visualized using high energy gamma scintigraphy. Since therapy may alter the accumulation of amino acids in tumor tissue, studies using C-11 AIB in patients with MFH before and after chemotherapy are in progress.

Pharmacokinetics of alpha-methyl-L-tryptophan in rhesus monkeys and calculation of the lumped constant for estimating the rate of serotonin synthesis.

We have determined several kinetic and pharmacokinetic parameters of L-tryptophan (Trp) and alpha-methyl-L-tryptophan (alphaMTrp) in the rhesus monkey from which the lumped constant for the alphaMTrp method of estimating serotonin synthesis rates is calculated. AlphaMTrp was isolated from DL-alphaMTrp using a chiral separation column with high performance liquid chromatography. AlphaMTrp (50 microgram/kg) was administered i.v. to four adult male rhesus monkeys and arterial blood samples were collected for a 4-hr period. Plasma concentrations, as determined by high performance liquid chromatography with electrochemical detection, were best fitted by a tri-exponential equation. Plasma protein binding of Trp and alphaMTrp was determined by measuring concentrations in ultrafiltrates obtained at 30 degrees C. After a 2-hr adjusted rate infusion of alphaMTrp designed to establish steady-state plasma concentrations, three adult male rhesus monkeys were killed by exsanguination with perfused ice-cold saline. Brain/arterial plasma concentration ratios of Trp and alphaMTrp and the Michaelis-Menten parameters for tryptophan hydroxylase, EC 1.14.16.4, with Trp and alphaMTrp as initiating substrates, were determined for seven brain regions. The lumped constants determined for the different brain regions were not significantly different from each other and indicate, that for modeling purposes, the brain may be treated as a homogeneous area and the lumped constant given a single value, 0.18 +/- 0.05.

Synthesis and purification of the anti-viral agent 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) labeled with iodine-125.

The 125I labeled analog of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) has been prepared for studies on the imaging of herpes simplex virus (HSV) encephalitis infections in animals. Iodination of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosine X HCl (FAC X HCl) with Na 125I and NaI/HIO3 in aqueous acetic acid, and subsequent removal of residual iodine by CCl4 extraction and ion exchange chromatography, yielded [125I]FIAC in aqueous solution with a specific activity of 45.5 mCi/mmol in 44% radiochemical yield, and with a radiopurity of 96-97%. Methods for further purification are described.

Tumor and organ biochemical profiles determined in vivo following uptake of a combination of radiolabeled substrates: potential applications for PET.

This paper describes a method which generates an in vivo metabolic profile of tumors and organ tissues derived from the tissue uptake of a combination of radiotracers. Metabolic substrates labeled with carbon-11, a positron-emitting radionuclide (T1/2 = 20.4 min) have been used to probe tumor metabolism in vivo using positron emission tomography (PET). Carbon-11 labeled radiotracers which have been used include alpha-aminoisobutyric acid (AIB), 2-deoxy-D-glucose (2-DG), and thymidine (TdR). This paper reports data on the tissue distribution of carbon-14 labeled analogues of AIB, 2-DG, and TdR. Tissue distribution studies were carried out in normal male Copenhagen rats, in rats bearing Dunning R3327G or R3327H prostatic adenocarcinomas, and in tumor rats that have been treated with difluoromethylornithine and methylglyoxal-bisguanylhydrazone, or with diethylstilbestrol. A combination of the tissue distribution data of these radiolabeled agents is used to provide a metabolic description of the state of a tumor or tissue in vivo. This approach to defining a tissue's biochemical profile may be useful for the assessment and prediction of a therapeutic response, even at low tracer concentrations in a tumor, and may be useful in relating the biochemical characteristics of one tissue to that of another.

Tumor localization of alpha-aminoisobutyric acid (AIB) in human melanoma heterotransplants.

The nude mouse bearing a human tumor heterotransplant is a useful model for studying the tumor localization of radiolabeled compounds. The biological tissue distribution of carbon 14-labeled alpha-aminoisobutyric acid (AIB), a synthetic, nonmetabolized amino acid, was determined in nude mice bearing human malignant melanoma heterotransplants in order to investigate the feasibility of using carbon 11 (t 1/2, 20.4 min)-labeled AIB for the visualization of human melanoma in vivo with positron emission tomography (PET). Our laboratory has previously demonstrated the use of 11C-labeled AIB as a tumor-imaging agent in a number of animal tumor models. The mean relative concentration of 14C-labeled AIB in tumor tissue at 45 min was 1.95 in this melanoma model. Tumor/blood and tumor/muscle ratios at 45 min postinjection were 5.42 and 12.2, respectively. These values suggest that 11C-labeled AIB may be useful for the in vivo study of malignant melanoma in humans. Alpha-aminoisobutyric acid (AIB), a synthetic, nonmetabolized amino acid, is thought to be actively accumulated into viable cells primarily by the A-type, or "alanine-prefering", amino acid transport system. AIB has been labeled with the short-lived, positron-emitting radionuclide, carbon 11 (t 1/2, 20.4 min), using a modified Bucherer-Strecker synthesis for amino acids. 11C-labeled AIB has been used to visualize tumors in dogs bearing spontaneous cancers, such as adenocarcinoma, lymphosarcoma, and osteogenic sarcoma, by utilizing positron-emission tomography (PET) and high-energy gamma (HEG) scintigraphy at the Sloan-Kettering Institute.(ABSTRACT TRUNCATED AT 250 WORDS)