Synthesis of UDP-N-[1-14C]acetyl D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine from [1-14C]acetate

Procedures for the preparation of UDP-N-[1-14C]acetyl-D-glucosamine and UDP-N-[1-14C]acetyl-D-galactosamine with very high specific activities are described. The overall yield based on the amount of [1-14C]acetate used is greater than 80%. The N-acetyl-D-glucosamine-alpha-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-D-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-D-glucosamine-alpha-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. D-glucosamine-alpha-1-phosphate is N-acetylated with [14C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-D-glucosamine pyrophosphorylase. UDP-N-[1-14C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-14C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Pharmacokinetic, pharmacodynamic, and pharmacotoxic profiles of recombinant-methionyl human interleukin-2[alanine-125] (r-metHuIL-2[ala-125]) following intravenous and subcutaneous administration in rats

Comparative pharmacotoxicity studies in rats were performed to evaluate the response to r-metHuIL-2[ala-125] following 2 or 4 weeks of daily intravenous or subcutaneous administration, as well as to evaluate pharmacokinetic and pharmacodynamic responses. Pharmacokinetic analysis indicated that r-metHuIL-2[ala-125] showed high bioavailability and nonlinear concentration profiles. Pharmacodynamic responses to intravenous or subcutaneous dosing with r-metHuIL-2[ala-125], as measured by white blood cell counts, were comparable. Preclinical safety studies (6, 30, and 150 micrograms kg-1 day-1) indicated that r-metHuIL-2[ala-125], whether given intravenously or subcutaneously, was associated with increased circulating and infiltrating levels of lymphocytes and eosinophils. Bone marrow lymphoid hyperplasia and splenic extramedullary hematopoiesis were similarly observed in each study. This pattern of effects was considered an exaggerated pharmacodynamic response to r-metHuIL-2[ala-125]. Of further note was a histopathologic finding described as hepatocyte single cell necrosis which was observed following both intravenous and subcutaneous administration and was considered to be a toxic response to high doses of r-metHuIL-2[ala-125]. The no observable adverse effect level (NOAEL) for r-metHuIL-2[ala-125] via intravenous administration was 6 micrograms kg-1 day-1, while that for subcutaneous administration was 30 micrograms kg-1 day-1. Data herein present a form of rHuIL-2 with pharmacokinetic and pharmacodynamic profiles that are similar when given by these two systemic routes. Pharmacotoxic data, based on NOAELs, suggest that subcutaneous administration may be a preferred clinical route of administration.     

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on leucine metabolism in normal cells were also investigated. Production of 14CO2 from [2-(14)C] leucine was very reduced (96-99%) in both types of mutant cells. Radioactive isovaleric acid accumulated in assay media with isovaleric acidemia cells but not in those with maple syrup urine disease cells. Unexpectedly, 14CO2 production from [1-(14)C] leucine was partially depressed (80%) in isovaleric acidemia cells whereas in maple syrup urine disease cells it was strongly depressed (99%) as expected. These two mutant cells were clearly distinguished by detection of 14C-isovaleric acid accumulation after incubation with [2-(14)C] leucine. A pattern of inhibition of leucine oxidation similar to that seen in isovaleric acidemia cells was induced in normal cells by the addition of 0.7 mM methylenecyclopropylacetic acid to the assay medium. The partial inhibition of [1-(14)C] leucine oxidation seen in isovaleric acidemia cells and also in normal cells in the presence of the inhibitor appears to be, at least in part, due to an accumulation of isovalerate in the cells. Isovaleric acid (5-10) mM) inhibited [1-(14)C] leucine oxidation 32-68% when added to the assay medium with normal cells. Addition of flavin adenine dinucleoside to culture medium or assay medium or both did not restore oxidation of either leucine substrate in isovaleric acidemia cells.     

Percutaneous absorption and disposition of [14C]chlordecone in young and adult female rats

The objective of this study was to evaluate the effect of age and dosage on percutaneous absorption and disposition of [14C]chlordecone (Kepone) and to describe results using a physiological based pharmacokinetic (PBPK) model. Female Fischer 344 rats 33 and 82 days old were used as the young and adult animal models, respectively, and were studied over a 10-fold dose range. [14C]Chlordecone (0.286 micromol/cm2) was applied to dorsal skin (2. 3% BSA) and radioactivity was quantified in selected tissues and excreta up to 120 h. Absorption and disposition were also determined at three dose levels up to 2.68 micromol/cm2; fraction absorbed decreased as dose increased. In vitro percutaneous absorption was measured by static and flow-through methods; these yielded similar penetration rates, which were lower than those obtained in vivo. In vivo percutaneous absorption over 120 h was 14.4+/-0.99 and 14.2+/-1. 5% dose in young and adults, respectively. Organ and tissue content increased over time (carcass>liver>kidney), indicating prolonged absorption. Statistical differences between young and old were found for liver, skin, and urine, but not for absorption. Excretion occurred primarily in feces, but also in urine. A biophysically based percutaneous model was fitted to both young and adult in vivo absorption data. This was embedded in a whole body PBPK model which, upon optimization with SAAM II, estimated apparent tissue partition coefficients, urinary and fecal excretion rates, and parameters characterizing hepatic nonlinear uptake of bound chlordecone. The model reasonably predicted tissue chlordecone content at higher doses, when decreased absorption was accounted for.     

Toxicity of the novel anti-peptic ulcer agent catena-(S)-[mu-[Na- (3-aminopropionyl)histidinato(2-)-N1,N2,Q:N tau]-zinc in male cynomolgus monkeys

A preliminary dose-range finding study and a 13-week toxicity study were performed in male cynomolgus monkeys with catena-(S)-[mu-[N a-(3-aminopropionyl) histidinato (2-)-N1,N2,O:N tau]-zinc] (Z-103, CAS 107667-60-7), a novel anti-peptic ulcer agent, as part of a safety evaluation program. In the preliminary ascending dose study emesis was observed in animals treated at 625 mg/kg and transient reductions in food intake with associated body weight loss in a male treated at 625 or 312.5 mg/kg. Plasma zinc levels were also increased in all animals treated at 625 or 312.5 mg/kg. As a result dosages of 0, 20, 63 and 200 mg/kg/day were selected for the 13-week toxicity study. In this study, treatment-related changes were confined to the 200 mg/kg/day dosage and consisted of emesis, piloerection and transient body weight loss in one animal, increased plasma zinc concentrations, and zinc and copper deposition in the liver and kidneys without any associated morphological change. The no observed effect level was estimated to be 63 mg/kg/day in this study.     

Pharmacokinetics, oral bioavailability, and metabolism in mice and cynomolgus monkeys of (2'R,5'S-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, an agent active against human immunodeficiency virus and human hepatitis B virus

(2'R,5'S-)-cis-5-Fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (524W91) is a nucleoside analog with potent anti-human immunodeficiency virus and anti-human hepatitis B virus activities in vitro. The pharmacokinetics and bioavailability of 524W91 after oral dosing were studied in mice dosed with 10, 100, and 600 mg of 524W91 per kg of body weight by the oral and intravenous routes. Cynomolgus monkeys were dosed with 10 and 80 mg of 524W91 per kg. In both species, the clearance of 524W91 was rapid, via the kidney, and was independent of dose. In monkeys, the total body clearance of 10 mg of 524W91 per kg was 0.7 +/- 0.1 liter/h/kg, and the volume of distribution at steady state was 0.8 +/- 0.02 liter/kg. The terminal elimination half-life was 1.0 +/- 0.2 h. The absolute bioavailability after oral dosing was 63% +/- 4% at 10 mg/kg. Concentrations of 524W91 in the cerebrospinal fluid were 4% +/- 0.7% of the corresponding levels in plasma. In mice, the total clearance of 10 mg of 524W91 per kg was 2.3 liters/kg/h, and the volume of distribution at steady state was 0.9 liter/kg. Absolute bioavailability in mice after oral dosing was 96% at a dose of 10 mg/kg. The metabolism of orally administered [6-3H]524W91 was studied in cynomolgus monkeys at a dose of 80 mg/kg and in mice at a dose of 120 mg/kg. Monkeys excreted 41% +/- 6% of the radioactive dose in the 0- to 72-h urine, 33% +/- 10% in the feces, and 10% +/- 7% in the cage wash. Unchanged 524W91 was 64% of the total radiolabeled drug recovered in the urine. The glucuronide was a minor urinary metabolite. 5-Fluorouracil was not detected (less than 0.02% of the dose). Mice dosed orally with 120 mg of [6-3H]524W91 per kg excreted 67% +/- 7% of the radiolable in the )- to 48-h urine. Small amounts of the 3' -sulfoxide and glucuronide metabolites were observed in the urine, but 5-fluorouracil was not detected. Good bioavailability after oral dosing and resistance to metabolism recommend 524W91 for further preclinical evaluation.     

Autoradiographic distribution and physiological regulation of 2-[125I]iodomelatonin binding in rat epididymis

Autoradiographic study was conducted to localize 2-[125I]iodomelatonin binding in the rat epididymis. In the peripubertal (6 weeks old), postpubertal (8 weeks old) and adult (3 months old) rats, intense specific 2-[125I]iodomelatonin labelling of the corpus epididymidis was observed. The intensity of 2-[125I]iodomelatonin binding in the distal epididymal segment was significantly decreased in orchidectomized rats but the effect could be reversed with testosterone replacement. The intensity of 2-[125I]iodomelatonin binding in the distal rat epididymal segment did not show any diurnal rhythmicity when mid-light period and mid-dark period levels were compared, and was unaffected by constant lighting. Our data suggest androgen-dependent expression of 2-[125I]iodomelatonin binding sites, independent of light-induced changes in circulating melatonin, in the rat corpus epididymidis. A novel role of melatonin and its receptor in the regulation of the functions of rat corpus epididymidis is strongly implicated.     

Thermodynamically stable β[Ni(Al,Ti)]-β′[Ni2AITi]-γ[Ni3(Al,Ti)] metal-metal composites

Single-phase intermetallic compounds are often quite brittle. Experiments are reported in which a metallic composite consisting of three different intermetallic compounds (β, β′, and γ′ ) in the Ni-Ti-Al system was fabricated by extruding a mechanical mixture of the compounds. In this way, the microstructure could be engineered to disperse the most ductile intermetallic in between the more brittle components. An additional feature was to choose the β, β′, and γ′ powders with compositions which allowed them to coexist in equilibrium when coextruded to form the composite. It is found that this approach confers elevated temperature microstructural stability, a crack arresting ability, reasonable ductility, and considerable strength even at high temperatures.     

Binding of the cocaine analog 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane to serotonin and dopamine transporters: different ionic requirements for substrate and 2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binding

The iodinated cocaine analog 2 beta-carbomethoxy-3 beta-(4- [125I]iodophenyl)tropane (beta-[125I]CIT) binds with high affinity to the platelet plasma membrane serotonin transporter, as previously reported for dopamine transporters from rat brain [Eur. J. Pharmacol. 194:133-134 (1991)]. Unlabeled beta-CIT also inhibits serotonin transport by platelet membrane vesicles. In both rat striatal membranes and platelet plasma membranes, beta-[125I]CIT binding was found to be pH dependent, with a pKa of 6.4-6.9, and did not require the presence of Cl-. Na+ dramatically stimulated beta-[125I]CIT binding to both serotonin and dopamine transporters, although a small fraction of beta-[125I]CIT binding to the serotonin transporter was observed in the absence of Na+. The substrates serotonin and dopamine competed with beta-[125I]CIT for binding to their respective transporters. However, substrate affinity was enhanced by Cl-, whereas beta-[125I]CIT binding affinity was not. [3H]Imipramine binding to the platelet serotonin transporter and [3H]GBR-12935 binding to the dopamine transporter were not inhibited by decreasing the pH from 8 to 6.5. Likewise, the ability of serotonin to compete with [3H]imipramine binding and that of dopamine to inhibit [3H]GBR-12935 binding were equal at pH 6.5 or 8. Thus, beta-[125I]CIT binding to biogenic amine transporters is distinct from serotonin or dopamine binding by virtue of its inhibition by H+ and its insensitivity to Cl-.     

5-[125I]iodo-2'-deoxyuridine in the radiotherapy of brain tumors in rats

Glial neoplasms of the human central nervous system have defied treatment, in part because of the limited selectivity of available cytotoxic agents. The thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter 125I (125IUdR) is highly toxic to dividing cells when it is deoxyribonucleic acid incorporated, but it is relatively innocuous when located outside the nucleus. Previous studies have shown that 125IUdR has significant antineoplastic potential against mammalian cells in vitro and direct administration of 125IUdR is effective therapy for ovarian ascites tumors in mice and neoplastic meningitis in rats. Studies using external gamma imaging and autoradiography have also shown that direct intratumoral administration of 123IUdR/125IUdR into intracerebral 9L gliosarcomas in rats results in selective uptake of the radionuclide into tumor cells. Based on these encouraging results, we have evaluated the therapeutic potential of 125IUdR in rats bearing intracerebral 9L gliosarcomas. METHODS: Iodine-125-IUdR was infused intracerebrally over a 2-day period into rats bearing 1-day-old 9L tumors and over a 6-day period into animals with 9-day-old 9L tumors; equimolar concentrations of 127IUdR were infused into control animals. Tumor growth was monitored by contrast-enhanced 1H MRI and animal survival was followed over time. RESULTS: Intracerebral tumors (3-7 mm) were readily detected by MRI. Tumor-bearing rats treated with 127IUdR succumbed within 17-24 days, whereas tumor-bearing animals treated with 125IUdR survived significantly longer, and 10%-20% of the animals were cured of tumors. CONCLUSION: These data substantiate the antineoplastic potential of 5-[125I]iodo-2'-deoxyuridine and indicate that it may be a useful agent for the therapy of solid tumors that are accessible to direct radiopharmaceutical administration.     

Systemic administration of kainic acid induces selective time dependent decrease in [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in adult rat hippocampal formation

Administration of kainic acid evokes acute seizure in hippocampal pathways that results in a complex sequence of functional and structural alterations resembling human temporal lobe epilepsy. The structural alterations induced by kainic acid include selective loss of neurones in CA1-CA3 subfields and the hilar region of the dentate gyrus followed by sprouting and permanent reorganization of the synaptic connections of the mossy fibre pathways. Although the neuronal degeneration and process of reactive synaptogenesis have been extensively studied, at present little is known about means to prevent pathological conditions leading to kainate-induced cell death. In the present study, to address the role of insulin-like growth factors I and II, and insulin in neuronal survival as well as synaptic reorganization following kainate-induced seizure, the time course alterations of the corresponding receptors were evaluated. Additionally, using histological preparations, the temporal profile of neuronal degeneration and hypertrophy of resident astroglial cells were also studied. [125I]Insulin-like growth factor I binding was found to be decreased transiently in almost all regions of the hippocampal formation at 12 h following treatment with kainic acid. The dentate hilar region however, exhibited protracted decreases in [125I]insulin-like growth factor I receptor sites throughout (i.e. 30 days) the study. [125I]Insulin-like growth factor II receptor binding sites in the hippocampal formation were found to be differentially altered following systemic administration of kainic acid. A significant decrease in [125I]insulin-like growth factor II receptor sites was observed in CA1 subfield and the pyramidal cell layer of the Ammon's horn at all time points studied whereas the hilar region and the stratum radiatum did not exhibit alteration at any time. A kainate-induced decrease in [125I]insulin receptor binding was noted at all time points in the molecular layer of the dentate gyrus whereas binding in CA1-CA3 subfields and discrete layers of the Ammon's horn was found to be affected only after 12 h of treatment. These results, when analysed with reference to the observed histological changes and established neurotrophic/protective roles of insulin-like growth factors and insulin, suggest possible involvement of these growth factors in the cascade of neurotrophic events that is associated with the reorganization of the hippocampal formation observed following kainate-induced seizures.     

Determination of 14CO2 in breath and 14C in stool after oral administration of cholyl-1-[14C]glycine: clinical application

Twenty patients with intestinal bacterial overgrowth and 20 control subjects were investigated for bile acid deconjugation, by measuring 14CO2 in the breath after cholyl-1-[14C]glycine administration. 14CO2 output/24h was 11.0 +/- 5.2% (mean +/- SD) in controls and 54.2 +/- 14.0% (mean +/- SD) in bacterial-overgrowth patients (P less than .001). 14CO2 excretion rate in 12h, when normalized to 100% of the dose at the 12th hour, gave an even finer discrimination between the two groups (no false responses). 14C in stool, analyzed in 20 malabsorption patients and 20 controls by two different techniques, was 6.6 +/- 4% and 31.38 +/- 21.7% (mean +/- SD), respectively. Results by the two different techniques described here correlated well (r = .99). Bile acid malabsorption was in reasonable agreement (r = .67) with percentage of "chenoid" (chenodeoxycholic acid plus ursodeoxycholic acid) in the stool by gas-liquid chromatography; a poorer correlation was observed when "chenoid" plus "choloid" (cholic acid plus its epimers) were plotted vs. -4C in stool (r = .57, n = 15).     

The influence of acute diazepam pretreatment on the action and disposition of [14C]pentobarbital in rats

Diazepam (DZP) pretreatment (100mg/kg, ip) of rats 6 h before pentobarbital administration (45 mg/kg, ip) prolonged the barbiturate-induced narcosis. The concentrations of [14C]pentobarbital and total pentobarbital derivatives in blood or brain showed no differences between control and DZP-pretreated animals. The brain and blood concentrations of pentobarbital, when measured at a time corresponding to the respective arousal times from pentobarbital narcosis, were lower in the DZP-pretreated group. These results indicate that acute DZP pretreatment increases the sensitivity of the rat brain to pentobarbital rather than inducing changes in the disposition of the barbiturate.     

Daily and circadian variations in 2-[125I]-iodomelatonin binding sites in the pike brain (Esox lucius)

The fish pineal organ, through its 24 h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g. reproduction, growth). We have investigated the binding of 2-[125I]iodomelatonin to whole brain membrane preparations from pikes (Esox lucius L., teleost) maintained for 24-48 h under different photoperiodic conditions. Specific binding was stable, reversible, saturable and sensitive to the presence of a GTP analogue. Scatchard analysis revealed one class of binding sites. Displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations, respectively. The Bmax exhibited monophasic nycthemeral variations, with higher values at the light-to-dark transition (34.0 +/- 4.5 fmol/mg protein) and low values during the second half of night (10.0 +/- 1.0 fmol/mg protein). Under the same conditions, the KD exhibited biphasic variations: values were low during daytime and at the middle of the dark phase (approximately 100 pM); they were high at the beginning (approximately 225 pM) and at the end (approximately 330 pM) of the night. These variations were maintained under constant light (LL) and constant darkness (DD). Thus, the variations in the number and affinity of the melatonin binding sites were controlled by circadian oscillators, synchronized by the photoperiod. The nature of these oscillators is not known. Therefore, in fish, we suggest that the photodependent effects of melatonin result from the circadian variations of both its production by the pineal and its binding sites in the brain.     

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.