The benzodiazepine derivative flurazepam (FLZ) is widely used as a hypnotic, but the relative contributions of FLZ and its metabolites desalkylflurazepam (DA-FLZ), hydroxyethylflurazepam (ETOH-FLZ), and flurazepam aldehyde (CHO-FLZ) to overall clinical activity remain uncertain. A single 20 mg/kg dose of FLZ.HCl was administered to mice, with plasma and brain concentrations of FLZ and metabolites determined during 5 h after dosage. Brain and plasma concentrations of FLZ were maximal at 0.5 h after dosage, then declined rapidly in parallel, whereas those of DAFLZ were maximal at 2 h, then declined slowly. Concentrations of ETOH-FLZ, the most polar metabolite, were maximal at 0.5 h, and were undetectable after 3 h. Little CHO-FLZ was detected in either brain or plasma. A single 30-mg oral dose of FLZ.HCl was given to 18 human volunteers, with plasma levels determined over 9 days. FLZ was detected in plasma at low concentrations for no more than 3 h after dosage. ETOH-FLZ concentrations were higher and persisted for 8 h after dosage. CHO-FLZ reached intermediate peak levels and was present longer than FLZ or ETOH-FLZ. In contrast, DA-FLZ achieved the greatest peak concentrations, occurring at 10 h after dosage. Levels declined very slowly, with a mean half-life of 71.4 h, and were still detectable 9 days after FLZ dosage. Plasma free fractions (percent unbound) in mice were 40.3, 51.4, and 25.0% for FLZ, ETOH-FLZ and DA-FLZ, respectively; in humans, values were 17.2, 35.2, and 3.5%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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Preincubation of rat hypothalamic slices in glucose-free Krebs-Ringer buffer (370C) resulted in a timedependent decrease in specific (+)-[3H]amphetamine binding in the crude synaptosomal fraction prepared from these slices.The addition of D-glucose resulted in a dose-and timedependent stimulation of (+) Further, when food-deprived rats are allowed access to either rat chow or a 10% glucose solution, there is a rapid reversal of the food deprivation-induced decrease in hypothalamic (+)-[3H]amphetamine binding, and this effect is highly correlated with changes in blood glucose concentration. Glucose, the principle energy source of brain, has been postulated to directly regulate "appetite" or food intake through a central mechanism (the so-called "glucostatic hypothesis of feeding") (4, 5). In the present study we have investigated the effects of glucose on hypothalamic (+)-[3H]amphetamine binding in vitro and in vivo and now report that glucose rapidly and stereoselectively increases the number of (+)-[3H]amphetamine binding sites in the hypothalamus. This stimulation is highly correlated with increases in Na+,K+-ATPase activity and the number of specific "high-affinity" [3H]ouabain binding sites, suggesting that glucose and amphetamine may interact through a coupled system in the hypothalamus to regulate Na+,K+-ATPase activity, and that this site may also be involved in the "glucostatic" regulation of appetite.- MATERIALS AND METHODSTissue Preparation. Adult male Osborne-Mendel rats (120-200 g) housed under diurnal lighting conditions (12:12) with free access to food and water were killed by decapitation, and their brains were quickly removed and dissected on ice. Pooled hypothalami were chopped into 350 x 350 Atm slices using a McIlwain tissue chopper (Brinkmann). The slices were washed twice in 10 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) and incubated at 37°C in the same volume in a gently shaking water bath under a constant stream of 95% 02/5% CO2. D-Glucose and (or) other compounds were added, and after various times the tubes were transferred to an ice bath, the buffer was aspirated, and the slices were washed twice in fresh Krebs-Ringer bicarbonate buffer. Hypothalamic slices were then gently homogenized in 10 volumes (wt/vol) of ice-cold 0.32 M sucrose by using a Teflon/glass homogenizer, and a crude synaptosomal pellet was prepared as described (3).Radioreceptor and Enzyme Assays. The binding of (+)-[3H]amphetamine sulfate was carried out as described by using both filtration and centrifugation assays (3). Briefly, 100-200 ug of membrane protein (crude P2 fraction) and 50 ,ul of (+)-[3H]amphetamine (200-300 nM unless otherwise specified; specific activity, 20-30 Ci/mmol; New England Nuclear; 1 Ci = 37 GBq) were added in a total incubation volume of 300 ,ul. After a 30-min incubation at 0-4°C, the tubes were rapidly filtered over Whatman GF/B glass fiber filters under tTo whom reprint requests should be addressed at: Section on Molecular Pharmacology, Clinical Neuroscience Branch, NIMH, Bldg....

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