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A large number of structurally different classes of ligands, many of them sharing the main characteristics of the benzodiazepine (BDZ) nucleus, are active in the modulation of anxiety, sedation, convulsion, myorelaxation, hypnotic and amnesic states in mammals. These compounds have high affinity for the benzodiazepine binding site (BDZ-bs) of the GABA(A) receptor complex. Since 1989 onwards our laboratories established that some natural flavonoids were ligands for the BDZ-bs which exhibit medium to high affinity in vitro and anxiolytic activity in vivo. Further research resulted in the production of synthetic flavonoid derivatives with increased biochemical and pharmacological activities. The currently accepted receptor/pharmacophore model of the BDZ-bs (Zhang, W.; Koeler, K. F.; Zhang, P.; Cook, J. M. Drug Des. Dev. 1995, 12, 193) accounts for the general requirements that should be met by this receptor for ligand recognition. In this paper we present a model pharmacophore which defines the characteristics for a ligand to be able to interact and bind to a flavone site, in the GABA(A) receptor. closely related to the BDZ-bs. A model of a flavone binding site has already been described (Dekermendjian, K.; Kahnberg, P.; Witt, M. R.; Sterner, O.; Nielsen, M.; Liljerfors, T. J. Med. Chem. 1999, 42, 4343). However, this alternative model is based only on graphic superposition techniques using as template a non-BDZ agonist. In this investigation all the natural and synthetic flavonoids found to be ligands for the BDZ-bs have been compared with the classical BDZ diazepam. A QSAR regression analysis of the parameters that describe the interaction demonstrates the relevance of the electronic effects for the ligand binding, and shows that they are associated with the negatively charged oxygen atom of the carbonyl group of the flavonoids and with the nature of the substituent in position 3'.

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6,3'-dibromoflavone and 6-nitro-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with K(i) values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [(3)H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3'-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.

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6-Chloro-3'-nitroflavone integrates a list of nearly 70 flavone derivatives synthesized in our laboratories. The effects of 6-chloro-3'-nitroflavone on the benzodiazepine binding sites (BDZ-BSs) of the GABA(A) receptor were examined in vitro and in vivo. 6-Chloro-3'-nitroflavone inhibited the [3H]flunitrazepam ([3H]FNZ) binding to rat cerebral cortex membranes with a Ki of 6.68 nM and the addition of GABA to extensively washed membranes did not modify its affinity for the BDZ-BSs (GABA-shift = 1.16+/-0.12). The binding assays performed in rat striatal and cerebellar brain membranes showed that this compound has similar affinity to different populations of BDZ-BSs. Electrophysiological experiments revealed that 6-chloro-3'-nitroflavone did not affect GABA(A)-receptors (GABA(A)-Rs) responses recorded in Xenopus oocytes expressing alpha1beta2gamma2s subunits, but blocked the potentiation exerted by diazepam (DZ) on GABA-activated chloride currents. In vivo experiments showed that 6-chloro-3'-nitroflavone did not possess anxiolytic, anticonvulsant, sedative, myorelaxant actions in mice or amnestic effects in rats; however, 6-chloro-3'-nitroflavone antagonized diazepam-induced antianxiety action, anticonvulsion, short-term, and long-term amnesia and motor incoordination. These biochemical, electrophysiological, and pharmacological results suggest that 6-chloro-3'-nitroflavone behaves as an antagonist of the BDZ-BSs.

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6-Methyl-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the GABA(A) receptor (BDZ-bs) with Ki values between 10 and 50 nM in different brain regions. The GABA ratio of 1.03 for [(3)H]flunitrazepam binding to cerebral cortex, 0.76 for cerebellum, 0.7 for hippocampus, 0.7 for striatum, and 0.8 for spinal cord indicated an antagonistic or weak inverse agonistic profile of 6-methyl-3'-bromoflavone on BDZ-bs. Unlike classical benzodiazepines, it had no anticonvulsant, anxiolytic, myorelaxant, sedative, amnestic or motor incoordination effects. However, it antagonized the muscle relaxant, the sedative effect, and the changes in locomotor activity induced by diazepam. Taken together, these findings suggest that 6-methyl-3'-bromoflavone has an antagonistic profile on the BDZ-bs.

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The list of activities of plant flavonoids did not include effects on the central nervous system (CNS) up to 1990, when our laboratory described the existence of natural anxiolytic flavonoids. The first of these was chrysin (5,7-dihydroxyflavone), followed by apigenin (5,7,4'-trihydroxyflavone) and flavone itself. Semisynthetic derivatives of flavone obtained by introducing halogens, nitro groups or both in its molecule, give rise to high affinity ligands for the benzodiazepine receptor, active in-vivo; 6,3'-dinitroflavone, for example, is an anxiolytic drug 30 times more potent than diazepam. The data collected in this paper make clear that some natural flavonoids are CNS-active molecules and that the chemical modification of the flavone nucleus dramatically increases their anxiolytic potency.

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Solution phase combinatorial synthesis of flavone derivatives and evaluation of their affinity for the central benzodiazepine receptors is described. The libraries preparation is simple and provides a convenient method for rapid compound generation and screening. Thirty one new compounds were obtained of which the most promising, as high affinity benzodiazepine receptor ligands, were 6-bromo-3'-fluoroflavone; 6,3'-dichloroflavone; 6-bromo-3'-chloroflavone and 6-chloro-3'-bromoflavone.

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Benzodiazepines (BDZs) are the most widely prescribed class of psychoactive drugs in current therapeutic use, despite the important unwanted side-effects that they produce such as sedation, myorelaxation, ataxia, amnesia, ethanol and barbiturate potentiation and tolerance. Searching for safer BDZ-receptor (BDZ-R) ligands we have recently demonstrated the existence of a new family of ligands which have a flavonoid structure. First isolated from plants used as tranquilizers in folkloric medicine, some natural flavonoids have shown to possess a selective and relatively mild affinity for BDZ-Rs and a pharmacological profile compatible with a partial agonistic action. In a logical extension of this discovery various synthetic derivatives of those compounds, such as 6,3'-dinitroflavone were found to have a very potent anxiolytic effect not associated with myorelaxant, amnestic or sedative actions. This dinitro compound, in particular, exhibits a high affinity for the BDZ-Rs (Ki = 12-30 nM). Due to their selective pharmacological profile and low intrinsic efficacy at the BDZ-Rs, flavonoid derivatives, such as those described, could represent an improved therapeutic tool in the treatment of anxiety. In addition, several flavone derivatives may provide important leads for the development of potent and selective BDZ-Rs ligands.

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6,3'-Dintroflavone is a synthetic flavone derivative with high affinity for central benzodiazepine receptors that has anxiolytic effects. Here, we describe its biochemical and pharmacological characterization. 6,3'-Dinitroflavone inhibited differentially [3H]flunitrazepam binding to central benzodiazepine receptors in several brain regions, showing a lower Ki value in the cerebellum (central benzodiazepine receptor type I-enriched area), and a higher Ki value in the spinal cord and in the dentate gyrus (central benzodiazepine receptor type II-enriched area). When i.p. injected in mice, 6,3'-dinitroflavone had a potent anxiolytic effect in the elevated plus maze test. This effect was blocked by the specific central benzodiazepine receptor antagonist, Ro 15-1788. 6,3'-Dinitroflavone did not exhibit anticonvulsant or myorelaxant effects in mice or amnestic effects in rats. Moreover, it abolished the myorelaxant effect of diazepam. On the other hand, 6,3'-dinitroflavone possessed a mild sedative action only at doses 100-300-fold greater than the anxiolytic one. Based on these findings, we suggest that 6,3'-dinitroflavone has a benzodiazepine partial agonist profile, with low selectivity for central benzodiazepine receptor types I and II.

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6-Bromoflavone, obtained by bromination of flavanone, binds to central benzodiazepine receptors with a Ki=70 nM and has a clear anxiolytic activity in mice, at 0.5 mg/kg i.p. A survey of the structure/affinity relationship for those receptors in a series of natural and synthetic flavonoids is presented.

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The dried flower heads of Matricaria recutita L. (Asteraceae) are used in folk medicine to prepare a spasmolytic and sedative tea. Our fractionation of the aqueous extract of this plant led to the detection of several fractions with significant affinity for the central benzodiazepine receptor and to the isolation and identification of 5,7,4'-trihydroxyflavone (apigenin) in one of them. Apigenin competitively inhibited the binding of flunitrazepam with a Ki of 4 microM and had no effect on muscarinic receptors, alpha 1-adrenoceptors, and on the binding of muscimol to GABAA receptors. Apigenin had a clear anxiolytic activity in mice in the elevated plusmaze without evidencing sedation or muscle relaxant effects at doses similar to those used for classical benzodiazepines and no anticonvulsant action was detected. However, a 10-fold increase in dosage produced a mild sedative effect since a 26% reduction in ambulatory locomotor activity and a 35% decrement in hole-board parameters were evident. The results reported in this paper demonstrate that apigenin is a ligand for the central benzodiazepine receptors exerting anxiolytic and slight sedative effects but not being anticonvulsant or myorelaxant.

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Tilia species are traditional medicinal plants widely used in Latin America as sedatives and tranquilizers. For this purpose, the infusion of their inflorescences is used to prepare a tea. In this study extracts of inflorescences from Tilia tomentosa Moench, one of the species found in the market, were purified using a benzodiazepine (BZD) binding assay to detect BZD receptor ligands in the different fractions. One of the ligands was identified as kaempferol, but it had low affinity (Ki = 93 microM) for this receptor, and did not produce sedative or anxiolytic effects in mice. On the other hand, a complex fraction, containing as yet unidentified constituents, but probably of a flavonoid nature, when administered intraperitoneally in mice, had a clear anxiolytic effect in both the elevated plus-maze and holeboard tests, two well validated pharmacological tests to measure anxiolytic and sedative compounds. This active fraction had no effect on total and ambulatory locomotor activity. In conclusion, our results demonstrate the occurrence of active principle(s) in, at least, one species of Tilia that may explain its ethnopharmacological use as an anxiolytic.

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Great progress has been made in the last five years in demonstrating the presence of benzodiazepines (BZDs) in mammalian tissues, in beginning studies on the origin of these natural compounds and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and the biosynthetic pathways responsible for the presence of BZDs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BZDs are of natural origin; (2) mammalian brain contains BZDs in concentrations ranging between 5.10(-10) to 10(-8) M; (3) BZDs and BZD-like molecules are unevenly distributed in brain; the highest concentration is found in limbic structures (4) dietary source of BZDs might be a plausible explanation for their occurrence in animal tissues, including man; (5) the formation of BZDs-like molecules in brain is a possibility, experimentally supported; (6) BZDs like molecules including diazepam and N desmethyldiazepam are elevated in hepatic encephalopathy; (7) natural BZDs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral and molecular biological techniques available to the neuroscientist will hopefully continue to yield new and exciting information concerning the biological roles that BZDs might play in the normal and pathological functioning of the brain.

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The possible cerebral formation of benzodiazepine (BDZ)-like molecules was evaluated in rats bilaterally microinjected into either the lateral ventricles (ICV) or the hippocampus (IH), with 10-70 microCi of different radiolabeled amino acids. After 3, or 24 h, the rats were sacrificed and BDZ-like molecules from total brain or hippocampus were purified by reversed phase HPLC. At 24 h. but not at 3 h of the ICV or IH microinjections with [3H] tryptophan, a peak of radioactivity containing material that inhibited the binding of [3H] flunitrazepam to both the BDZ receptor and the anti -BDZ monoclonal antibody MAb 21-7F9 was obtained. This active labeled fraction eluting just before diazepam also bound directly and specifically to the BDZ receptor and to MAb 21-7F9. No peak of radioactivity containing BDZ-like material was obtained when [3H] phenylalanine, [14C] glycine, [14C] methionine or [14C] tyrosine alone or in different combinations were microinjected into the hippocampus. The present results, together with our previous findings on the in vitro production of BDZ-like molecules in rat brain homogenates or slices (11), strongly suggest that the mammalian brain is capable of synthesizing low molecular weight substances possessing BDZ-like activity.

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Great progress has been made in the last 5 yr in demonstrating the presence of benzodiazepines (BDZs) in mammalian tissues, in beginning studies on the origin of these natural compounds, and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and biosynthetic pathways responsible for the presence of BDZs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BDZs are of natural origin; (2) mammalian brain contains BDZs in concentrations ranging between 5 x 10(-10)-10(-8) M; (3) dietary source of BDZs might be a plausible explanation for their occurrence in animal tissues, including man; (4) the formation of BDZ-like molecules in brain is a possibility, experimentally supported; (5) BDZ-like molecules including diazepam and N-desmethyldiazepam are elevated in hepatic encephalopathy; and (6) natural BDZs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral, and molecular biological techniques available to the neuroscientist will hopefully continue to yield exciting and new information concerning the biological roles that BDZs might play in the normal and pathological functioning of the brain.

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The presence of benzodiazepine-like molecules was detected radioimmunologically in bovine rumen contents and in incubates of ruminal contents with homogenates of several common grasses. A similar production was found "in vivo" in samples obtained from a grazing cow with a rumen cannula.

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The presence of benzodiazepine-like molecules was detected radioimmunologically in the plasma and milk of 12 women and in the plasma of 9 men. All subjects were non-users of benzodiazepines. The concentration of these biological materials expressed as diazepam equivalents per mL amounted to 2.54 +/- 0.74 ng in male plasma; to 2.20 +/- 0.35 ng in female plasma and to 1.91 +/- 0.54 ng in milk. Further investigation of the active compounds in milk permitted the unequivocal identification of diazepam, both free and bound to a presumably protein carrier and, at least, three more benzodiazepine-like molecules. Their origin either from dietary sources or as a result of endogenous biosynthesis is still unclear.

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Chrysin (5,7-di-OH-flavone) was identified in Passiflora coerulea L., a plant used as a sedative in folkloric medicine. Chrysin was found to be a ligand for the benzodiazepine receptors, both central (Ki = 3 microM, competitive mechanism) and peripheral (Ki = 13 microM, mixed-type mechanism). Administered to mice by the intracerebroventricular route, chrysin was able to prevent the expression of tonic-clonic seizures induced by pentylenetertrazol. Ro 15-1788, a central benzodiazepine receptor antagonist, abolished this effect. In addition, all of the treated mice lose the normal righting reflex which suggests a myorelaxant action of the flavonoid. The presence in P. coerulea of benzodiazepine-like compounds was also confirmed.

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The presence of benzodiazepine (BZD)-like molecules as well as of other substances with affinity for the brain BZD-receptors was explored in eight non-flowering plants known to contain biflavonoids, three flowering plants used as sedatives in folkloric medicine and one plant extensively used in Argentina, Uruguay, Brazil and Paraguay as a tea substitute. All the plants examined contained substances which bound to the central BZD-receptors and the majority of them also had BZD-like compounds detected by their specific interaction with a monoclonal antibody against BZDs. In various cases this last type of compound was present in amounts which exceeded trace levels (0.5-1.0 ng/g). The biological or clinical significance for humans of all these substances should be explored.

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125I-labeled human growth hormone (125I-labeled.hGH) was prepared by using two variants of the chloramine T labelling procedure and purified by polyacrylamide gel electrophoresis (PAGE) of the reaction mixture. Variant A produced a tracer with high specific activity (100 +/- 10 microCi/microgram), high maximal binding capacity to antibodies (93%) and long-term stability (at least 150 days at -20 degrees C). No diiodinated tyrosil residues could be detected in this tracer. Variant B was devised to obtain higher yields of labeled hormone. The electrophoresis of the iodination mixture revealed two radioactive components with Rm values of 0.49 and 0.55 which result from the iodination of hGH variants preexisting in the starting material. Both tracers had similar specific activities (70 +/- 10 microCi/microgram), high maximal binding capacity to antibodies or receptors (80-100%, after 80 days of their obtention) and high stability (at least 100 days at -20 degrees C). It is concluded that the iododerivatives of hGH obtained by either method are adequate to perform radioimmunoassay and receptor studies and have long-term stability.

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