Affinity For Benzodiazepine Receptor Research Articles

SYNTHESIS AND PROPERTIES OF NEW 1-HYDRAZINOCARBONYLMETHYL-7-BROMO-5-PHENYL-3-ARYLYDENE-1,2-DIHYDRO-3H-1,4-BENZODIAZEPIN-2-ONES

It is known that 1,4-benzodiazepines have neurotropic properties. Previously, we synthesized a series of 3-arylidene-1,2-dihydro-3H-1,4-benzdiazepin-2-ones 11-18 and showed that they exhibit significant analgesic activity. They also show significant affinity for central benzodiazepine receptors and TSPO receptors CNS. With this in mind, we have previously modified the series of 3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones by alkylation of 3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones with monobromoacetic acid methyl ester to the first position of the 1,4-diazepine ring to obtain 1-methoxycarbonylmethyl-7-bromo-5-phenyl-3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones 19-26, which showed pronounced analgesic activity. The aim of this work is to synthesize and study the affinity for CBR and TSPO receptors 1-hydrazinocarbonylmethyl-7-bromo-5-phenyl-3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones 27-31, which have not been previously described in the literature. Compounds 27-31 were synthesized as a result of the reaction of 1-methoxycarbonylmethyl-7-bromo-5-phenyl-3-arylidene-1,2-dihydro-3H-1,4-benzodiazepin-2-ones 19-26 with hydrazine hydrate while stirring the components in ethanol. The yields of the reaction products were 65-92%. It was shown that the hydrazinolysis technique used provides high yields of the target compounds. The structure of the synthesized compounds was confirmed by mass spectrometry and 1H NMR spectroscopy. The affinity of the synthesized compounds to the central benzodiazepine receptors CNS (CBR) and TSPO receptors CNS was studied. The affinity of compounds 27-31 was determined in vitro by radioligand analysis by their ability to displace the commercial radioligands [3H] flumazenil and [3H]PK11195 from their specific binding sites in the GABAA receptor complex and TSPO receptors of the synaptic and the mitochondrial fractions of the rat brain membranes, respectively. Compound 27 displaces the commercial [3H]flumazenil radioligand from its specific binding sites in the GABAA receptor complex by 80.1%, and simultaneously exhibits a very low affinity for TSPO receptors. Derivative 27 is the most potent CBR ligand among the investigated compounds and it is a promising compound for pharmacological research. It has been established that in the synthesized series of 3-fluorobenzylidene derivatives, varying the position of the fluorine atom in the benzylidene fragment leads to change affinity for TSPO receptors. It has been established that the para- position of the fluorine atom in the 3-benzylidene radical of the 1,4-benzodiazepin-2-one molecule is the most important descriptor that determines their affinity for the TSPO receptors. These data agree with the data available in the literature on the effect of the position of chlorine and bromine atoms in the 3-benzylidene radical of the 1,4-benzodiazepin-2-one molecule on the affinity for the TSPO receptors. It was found that the main structural fragment that determines the affinity of binding of 3-benzylidene derivatives to TSPO receptors is the presence of electronegative substituents in the para-position of the benzylidene fragment: para–Hal>>оrtho–Hal>meta–Hal (affinity for TSPO) (Hal= Br, F) Thus, the expediency of further study of this class of compounds is shown.

5-[Aryloxypyridyl (or nitrophenyl)]-4H-1,2,4-triazoles as novel flexible benzodiazepine analogues: Synthesis, receptor binding affinity and lipophilicity-dependent anti-seizure onset of action

A new series of 5-(2-aryloxy-4-nitrophenyl)-4H-1,2,4-triazoles and 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-triazoles, possessing C-3 thio or alkylthio substituents, was synthesized and evaluated for their benzodiazepine receptor affinity and anti-seizure activity. These analogues revealed similar to significantly superior affinity to GABAA/benzodiazepine receptor complex (IC50 values of 0.04–4.1 nM), relative to diazepam as the reference drug (IC50 value of 2.4 nM). To determine the onset of anti-seizure activity, the time-dependent effectiveness of i.p. administration of compounds on pentylenetetrazole induced seizure threshold was studied and a very good relationship was observed between the lipophilicity (cLogP) and onset of action of studied analogues (r2 = 0.964). The minimum effective dose of the compounds, determined at the time the analogues showed their highest activity, was demonstrated to be 0.025–0.1 mg/kg, relative to diazepam (0.025 mg/kg).

Potentials of Mangifera indica in the treatment of depressive-anxiety disorders: possible mechanisms of action.

Mangifera indica (Anacardiaceae) is an important herb in the traditional African and Ayurvedic medicines. The stem barks are used in the treatment of hypertension, insomnia, tumour, depression, rheumatism and as a tonic. This study was carried out to investigate antidepressant- and anxiolytic-like effect of the hydroethanol stem bark extract of M. indica (HeMI) in mice. HeMI (12.5-100 mg/kg, p.o.) was administered 1 h before subjecting the animal to the forced swim test (FST), tail suspension test (TST) and elevated plus maze tests (EPM). HeMI (12.5-100 mg/kg, p.o.) treatment produced significant reduction in immobility time [F(6.56)=8.35, p<0.001], [F(6,56)=7.55, p<0.001] in the FST and TST, respectively. Moreover, co-administration of sub-therapeutic doses of imipramine or fluoxetine with HeMI (3.125 mg/kg) elicited significant reduction in time spent immobile in the FST. However, pretreatment of mice with parachlorophenylalanine, metergoline, yohimbine or sulpiride abolished the antidepressant-like effect elicited by HeMI. In the EPM, HeMI produced significant [F(5,42)=8.91, p<0.001] increase in open arms exploration by 75.55 % and this effect was blocked by pretreatment of mice with flumazenil or metergoline. Findings from this study showed antidepressant-like effect of M. indica through interaction with 5-HT2 receptor, α2-adrenoceptor and dopamine D2-receptors. Also, an anxiolytic-like effect through its affinity for 5-HT2 and benzodiazepine receptors. Hence, M. indica could be a potential phytotherapeutic agent in the treatment of mixed anxiety-depressive illness.

The Effect of Chronic Alcohol Abuse on the Benzodiazepine Receptor System in Various Areas of the Human Brain

Objective: Alcohol abuse induces neuroadaptive changes in the functioning of neurotransmitter systems in the brain. Decrease of GABAergic neurotransmission found in alcoholics and persons with a high risk of alcohol dependence. Benzodiazepine receptor (BzDR) is allosterical modulatory site on GABA type A receptor complex (GABAAR), that modulate GABAergic function and may be important in mechanisms regulating the excitability of the brain processes involved in the alcohol addiction. The purpose of this study was to investigate the effects of chronic alcohol abuse on the BzDR in various areas of the human brain. Materials and Methods: Investigation of BzDR properties were studied in synaptosomal and mitochondrial membrane fractions from different brain areas of alcohol abused patients and non-alcoholic persons by radioreceptor assay with using selective ligands: [3H] flunitrazepam and [3H] PK-11195. Brain samples obtained at autopsy urgent. In total 126 samples of human brain areas were obtained to study radioreceptor binding, including a study group and control group. Results: Comparative study of kinetic parameters (Kd, Bmax) of [3H] flunitrazepam and [3H] PK-11195 binding with membrane fractions in studding brain samples was showed that affinity of BzDR was decreased and capacity increased in different areas of human brain under influence of alcohol abuse. More alters of synaptic BzDR “central” type (CBR) appeared in prefrontal cortex, mitochondrial BzDR “peripheral” type (PBR) – in n.caudatus and cerebella cortex. These results showed that alcohol addiction induce more expressive alterations in PBR than CBR in the brain structures that agree with maintaining function of glial cells in CNS under influence of different toxic factors. Conclusion: Chronic exposure to ethanol results in harmful effects on the human brain: causing nonuniform adaptive changes of BzDR in various areas of the brain, which can modulate GABAAR and reduce neuromediation GABA in various areas of the brain which can cause alcohol addiction. Graphical Abstract: Alcohol abuse induces neuroadaptive alters of benzodiazepine receptor system in various areas of the brain in patients with alcoholism that can modulate GABAAR and mediation of GABA in the brain, which can cause alcohol addiction.

Original Anticonvulsant Urea Derivative Alters the Properties of Benzodiazepine Receptors Central and Peripheral Types in the Cerebral Cortex of Heavy Drinkers Rats

Objective: studies of anticonvulsants have a stimulating effect on neuronal receptors; in particular GABAA/ benzodiazepine receptor complex (GABAA/BzDR) can be the basis for the development of new approaches to the treatment of alcohol withdrawal syndrome (AWS) and alcohol addiction. Benzodiazepine receptors (BzDRs) of the cerebral cortex of Wistar male rats with a different preference for alcohol and BzDRs in the brain “heavy drinkers” rats, treated with original anticonvulsant meta-chloro-benzhydryl urea (m-ch-BHU) were examined in these study. Materials and methods: Wistar male rats (n=250) were used in an experimental model of alcoholism. Properties of the BzDRs of the “central” (synaptic) and “peripheral” (mitochondrial) type were examined in membrane fractions obtained from the cerebral cortex of rats under various experimental groups using radio receptor binding assays (RRA) selective ligands: [3H]flunitrazepam and [3H] Ro5-4864 to these receptors respectively. Results: our study has shown that the binding affinity of [3H] flunitrazepam and [3H] Ro5-4864 with synaptosomal and mitochondrial membranes was decreased, but capacity of receptors was increased in the cerebral cortex of rats prefer alcohol. Administration of m-ch-BHU increased affinity of BzDRs in the cortex of “heavy drinkers” rats that can enhance the mediation of GABA in the brain of these animals. Conclusion: Our data showed that m-ch-BHU has a stimulating effect on GABAA/BzDRs in the brains of “heavy drinkers” rats and may provide a new pharmacotherapeutic approach to the treatment of alcohol addiction.

P.1.g.051 Benzodiazepine receptor system in the brains of rats with experimental alcoholism and treatment effect of a novel anticonvulsant

Purpose: Alcohol abuse induces neuroadaptive alterations in the benzodiazepine receptors (BDR) that modulate GABAAR and GABA mediation in brain regions associated with reward function, producing alcohol addiction. Studying the effects of drugs that have modulatory effects on neuronal receptors, in particular the GABAA-benzodiazepine receptors, can be the basis for understanding the formation of alcohol motivation and addiction, to develop new approaches to the treatment of this disease. Materials and Methods: Male Wistar rats (n = 250) were used in an experimental model of alcoholism. Properties of BDR ‘synaptosomal’ and ‘mitochondrial’ types were examined in respective membrane fractions obtained from brain cortex of rats with experimental alcoholism and treatment with the novel anticonvulsant meta-chlorobenzhydryl urea by radioreceptor assay (RRA) with [3H]flunitrazepam and [3H]Ro5-4864. Results: Based on their consumption of 15% alcohol and/or water, the rats were divided into 3 major groups. Group 1 consisted of rats drinking ethanol only − these ‘heavy drinking’ rats had chronic alcoholism (15% alcohol as the sole source of drinking for 10 months); the second group consisted of rats preferring ethanol − these ‘non-heavy drinking’ rats drank mostly ethanol; the third group − ‘non-preferring’ rats − drank water mostly. In each group, a number of animals were administered meta-chlorobenzhydryl urea (m-CIBHU) 100mg/kg for 14 days. Introduction of m-CIBHU caused a significant decrease in alcohol consumption in animals preferring alcohol (1st and 2nd groups), by 82.7% from baseline and 75% from baseline, respectively (p< 0.05). Alcohol consumption by rats preferring water was not affected by m-CIBHU. Reduction of alcohol consumption started on the 2−3rd day of m-CIBHU administration, and persisted while on the drug and for 2 weeks after drug discontinuation. Thus, the introduction of m-CIBHU resulted in a significant reduction of ethanol intake for animals preferring ethanol. Comparative study of the kinetic parameters (Kd and Bmax) of [3H]flunitrazepam and [3H]Ro5-4864 binding with synaptosomal and mitochondrial membranes showed different properties of BDR in membranes from the brain cortex of rats with different preferences for alcohol: the affinity of [3H]flunitrazepam and [3H]Ro5-4864 binding with membranes was decreased, but capacity of receptors was increased, in the brain cortex of ‘heavy drinking’ and ‘non-heavy drinking’ rats compared with ‘non-preferring’ rats. Administration of the anticonvulsant meta-chlorobenzhydryl urea increased the affinity of BDR in the brain cortex of ‘heavy drinking’ rats, which induced mediation of GABA in the brain of these rats and reduced alcohol consumption. Conclusions: Our study revealed that the novel anticonvulsant meta-chlorobenzhydryl urea had a normalizing effect on GABAAreceptor function in ‘heavy drinking’ rats, and reduced alcohol consumption in these animals. P.1.g.052 Cariprazine demonstrates greater potency than aripiprazole in animal models of psychosis, cognitive impairment, and negative symptoms

Effect of meta-chlorobenzhydryl urea (m-ClBHU) on benzodiazepine receptor system in rat brain during experimental alcoholism.

Chronic alcohol intake induces neuroadaptive changes in benzodiazepine receptors modulating GABAA receptors that promote alcohol addiction. Analysis of benzodiazepine receptors in the brain of Wistar rats differing by alcohol preference has demonstrated that affinity of [(3)H]flunitrazepam and [(3)H]Ro5-4864 binding with membrane fraction was reduced, while the density of specific binding sites in the brain cortex of heavy drinking and low drinking rats was increased in comparison with rats nonpreferring alcohol. Administration of anticonvulsant meta-chlorobenzhydryl urea increased affinity of benzodiazepine receptors in the brain cortex of heavy drinking rats, which improved GABA neurotransmission in the brain of these animals and reduced alcohol consumption.

Unexpected synthesis of 3,5-dimethyl-1-phenyl-1,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-one by non-classical Pschorr reaction, endowed with binding affinity for the central benzodiazepine receptor

The reaction of the diazonium salt 12 derived from N-(2-aminophenyl)-N,3 -dimethyl-1-phenyl- 1H-pyrazole-5-carboxamide with copper sulfate and sodium chloride in the presence of ascorbic acid afforded the unexpected products 3,5-dimethyl-1-phenyl-1,5-dihydro-4H-pyrazolo(4,3-c)- quinolin-4-one ( 17 ) and N-methyl-2-(3-methyl-1-phenyl-1H-pyrazol-5-yl)aniline ( 19 ), accompanied by N-(2-chlorophenyl)-N,3-dimethyl-1-phenyl-1H-pyrazole-5-carboxamide ( 18 ). Products 17 and 19 are formed via a non-classical Pschorr reaction. The formation of 17 represents an alternative to the literature synthesis of this biologically active compound. The molecular structure of 18 was confirmed by single-crystal X-ray analysis.

In Vivo Measurement of Hippocampal GABAA/cBZR Density with [18F]-Flumazenil PET for the Study of Disease Progression in an Animal Model of Temporal Lobe Epilepsy

PurposeImbalance of inhibitory GABAergic neurotransmission has been proposed to play a role in the pathogenesis of temporal lobe epilepsy (TLE). This study aimed to investigate whether [18F]-flumazenil ([18F]-FMZ) PET could be used to non-invasively characterise GABAA/central benzodiazepine receptor (GABAA/cBZR) density and affinity in vivo in the post-kainic acid status epilepticus (SE) model of TLE.MethodsDynamic [18F]-FMZ -PET scans using a multi-injection protocol were acquired in four male wistar rats for validation of the partial saturation model (PSM). SE was induced in eight male Wistar rats (10 weeks of age) by i.p. injection of kainic acid (7.5–25 mg/kg), while control rats (n = 7) received saline injections. Five weeks post-SE, an anatomic MRI scan was acquired and the following week an [18F]-FMZ PET scan (3.6–4.6 nmol). The PET data was co-registered to the MRI and regions of interest drawn on the MRI for selected structures. A PSM was used to derive receptor density and apparent affinity from the [18F]-FMZ PET data.Key FindingsThe PSM was found to adequately model [18F]-FMZ binding in vivo. There was a significant decrease in hippocampal receptor density in the SE group (p<0.01), accompanied by an increase in apparent affinity (p<0.05) compared to controls. No change in cortical receptor binding was observed. Hippocampal volume reduction and cell loss was only seen in a subset of animals. Histological assessment of hippocampal cell loss was significantly correlated with hippocampal volume measured by MRI (p<0.05), but did not correlate with [18F]-FMZ binding.SignificanceAlterations to hippocampal GABAA/cBZR density and affinity in the post-kainic acid SE model of TLE are detectable in vivo with [18F]-FMZ PET and a PSM. These changes are independent from hippocampal cell and volume loss. [18F]-FMZ PET is useful for investigating the role that changes GABAA/cBZR density and binding affinity play in the pathogenesis of TLE.

EPA-0628 – Benzodiazepine receptor system of human brain in alcohol addiction

Background alcohol abuse induce neuroadaptive alters of benzodiazepine receptors (BDR) that modulate GABAAR, mediation of GABA and activity of dopaminergic neurons in brain regions, associated with reward function in the brain that serve alcohol addictions. Method properties of BDR’central’ (CBR) and’peripheral’ (PBR) types were studied in synaptosomal and mitochondrial membranes from different brain areas of alcohol abused patients and non-alcoholic persons (early postmortem material, less 5 hours after death) by radioreceptor assay with using selective ligands: [ 3 H]flunitrazepam and [ 3 H]PK-11195. Results comparative study of kinetic parameters (affinity and capacity) of [ 3 H]flunitrazepam and [ 3 H]PK-11195 binding with synaptosomal and mitochondrial membranes in autopsy samples from different areas of human brain was showed that affinity of BDR was decreased: CBR – in 1,2 times in prefrontal cortex and n.caudatus, less – in cerebella cortex; PBR – in 2,1 times in n.caudatus, 1,3 times – in prefrontal cortex and cerebella cortex in patients with alcohol addiction. Capacity of BDR was increased in different areas of human's brain under influence of alcohol abuse. More alters of CBR appeared in prefrontal cortex (179%) and cerebella cortex (176%), less – in n.caudatus (145%); PBR – in n.caudatus (262%); and cerebella cortex (205%), and less in prefrontal cortex (123%). These results showed that alcohol addiction induce more alterations in PBR (mitochondrial) than CBR (synaptic) that agree with maintaining function of glial cells in CNS under influence of different toxic factors. Conclusion our findings suggest hypothesis of difference in response BDR from various brain areas on alcohol influence.

Antidepressant-like and anxiolytic-like effects of YL-IPA08, a potent ligand for the translocator protein (18 kDa)

It has been demonstrated that the translocator protein (18 kDa) (TSPO) plays an important role in stress-response and stress-related disorders, such as anxiety and depression, by affecting the production of neurosteroids, supporting the potential use of selective TSPO ligands as antidepressant or anxiolytic drugs. N-ethyl-N-(2-pyridinylmethyl)- 2-(3,4-ichlorophenyl)- 7-methylimidazo [1,2-a] pyridine-3-acetamide hydrochloride (YL-IPA08), a novel TSPO ligand that has been synthesized at our institute, exerted a high affinity for TSPO in a crude mitochondrial fraction prepared from rat cerebellum but exhibited only a negligible affinity for the central benzodiazepine receptor. As expected, YL-IPA08 incubation with the cultured rat astrocyte cells increased the pregnenolone and progesterone concentration from the cultured medium. Moreover, YL-IPA08 produced significant antidepressant-like and anxiolytic-like effects in a series of mouse and rat behavior models. In addition, the antidepressant-like behavior of YL-IPA08 was totally blocked by the TSPO antagonist PK11195 in a tail suspension test, and the anxiolytic effect was blocked by PK11195 but not by a CBR antagonist in the elevated plus-maze test. Furthermore, compared with the CBR agonist diazepam, YL-IPA08 had no myorelaxant effects and did not affect the motor coordination, memory or hexobarbitone-induced sleep in mice. Overall, these results indicate that YL-IPA08 is a more potent and selective TSPO ligand, which exerts antidepressant-like and anxiolytic-like effects on behaviors that are mediated by TSPO but does not cause the side effects that are typically associated with conventional benzodiazepines.

Novel codrugs with GABAergic activity for dopamine delivery in the brain

This study investigates the use of codrugs of the GABAergic agent 2-phenyl-imidazo[1,2-a]pyridinacetamide and dopamine (DA) or ethyl ester L-Dopa (LD) as a strategy to deliver DA and simultaneously activate GABA-receptors in the brain. For this purpose, both DA and LD ethyl ester were linked by carbamate bond to imidazo[1,2-a]pyridine acetamide moieties to yield two DA- and two LD-imidazopyridine derivatives. These compounds were evaluated in vitro to assess their stability, binding affinities and cell membrane transport, and in vivo to assess their bio-availability via microdialysis studies. The two DA derivatives were adequately stable in buffered solution, but underwent cleavage in diluted human serum. By contrast, the LD derivatives were unstable in buffered solution. Receptor binding studies showed that the DA-imidazopyridine carbamates had binding affinity for benzodiazepine receptors in the nanomolar range. Brain microdialysis experiments indicated that intraperitoneal administration of the DA derivatives sustained DA levels in rat striatum over a 4-h period. These results suggest that DA-imidazopyridine carbamates are new DA codrugs with potential application for DA replacement therapy.

Evaluation of Neuroinflammation in X-Linked Adrenoleukodystrophy

We present findings of (11)C-[R]-PK11195 positron emission tomography in a child with X-linked adrenoleukodystrophy. (11)C-[R]-PK11195 is a radioligand with a high and specific affinity for peripheral benzodiazepine receptors, expressed by activated microglia in cases of neuroinflammation, and therefore it is applicable to the in vivo detection of neuroinflammation with positron emission tomography. (11)C-[R]-PK11195 positron emission tomography demonstrated increased tracer binding in the occipital, parietal, and posterior temporal white matter, in the genu of the corpus callosum, the bilateral posterior thalami, most of the posterior limb of the internal capsule, the bilateral cerebral peduncles, and the brainstem, indicating underlying neuroinflammation. The rest of the brain, including the cerebral cortices and cerebellum, exhibited minimal (11)C-[R]-PK11195 binding. Our findings indicate significant neuroinflammation associated with white matter destruction in X-linked adrenoleukodystrophy, which can be visualized in vivo with an (11)C-[R]-PK11195 positron emission tomography scan. (11)C-[R]-PK11195 positron emission tomography may also help evaluate the inflammatory burden and follow-up of the disease evolution. This technique may be particularly useful for evaluating treatment response, which is not easy with other imaging modalities, after white matter is significantly and extensively damaged.

Pharmacological characterization of MP349, a novel 5-HT1A-receptor antagonist with anxiolytic-like activity, in mice and rats.

The purpose of this study was to further characterize the pharmacological effects of MP349 (trans-1-(2-methoxyphenyl)-4-(4-succinimidocyclohexyl)piperazine), a new serotonin 5-HT(1A) postsynaptic receptor antagonist, using several biochemical and behavioural assays. The silent 5-HT(1A)-receptor antagonist WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide) was used as a reference compound in in-vivo tests, and diazepam served as standard anxiolytic drug in animal models of anxiety. In this study we showed that MP349 bound with moderate affinity (K(i) = 234 nM) for alpha(1)-adrenoceptors, and with very low affinity (K(i) > 2600 nM) for 5-HT(2A), dopamine D(1), D(2) and benzodiazepine receptors. The effects of MP349 on presynaptic 5-HT(1A) receptors were studied in two models (mice and rats). Like WAY 100635, MP349 antagonized the hypothermia induced by the 5-HT(1A)-receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin(8-OH-DPAT) in mice. Neither MP349 nor WAY 100635 administered alone induced hypothermia. In a rat microdialysis study, MP349 (like WAY 100635) did not affect 5-HT dialysate level in the prefrontal cortex; however, when given before 8-OH-DPAT, it inhibited the decrease in 5-HT release induced by the 5-HT(1A )agonist. The data demonstrated that MP349 behaved like a functional antagonist of presynaptic 5-HT(1A) receptors. The potential anxiolytic activity of MP349 and reference drugs was examined in a conflict drinking test in rats, a plus-maze test in rats and a four-plate test in mice. MP349 and WAY 100635 produced anxiolytic-like effects, though somewhat weaker than those induced by diazepam, and only in the case of diazepam the anxiolytic-like effects were dose-dependent. Moreover, MP349 administered in doses inducing anxiolytic-like effects did not disturb the locomotor activity (open field test) or locomotor coordination (rota-rod test) of rats. These and earlier results indicated that MP349 was an antagonist of 5-HT(1A) receptors which exhibited anxiolytic-like activity in an animal model of anxiety.

Synthesis and Biological Evaluation of Substituted [18F]Imidazo[1,2-a]pyridines and [18F]Pyrazolo[1,5-a]pyrimidines for the Study of the Peripheral Benzodiazepine Receptor Using Positron Emission Tomography

The fluoroethoxy and fluoropropoxy substituted 2-(6-chloro-2-phenyl)imidazo[1,2- a]pyridin-3-yl)- N, N-diethylacetamides 8 (PBR102) and 12 (PBR111) and 2-phenyl-5,7-dimethylpyrazolo[1,5- a]pyrimidin-3-yl)- N, N-diethylacetamides 15 (PBR099) and 18 (PBR146) were synthesized and found to have high in vitro affinity and selectivity for the peripheral benzodiazepine receptors (PBRs) when compared with the central benzodiazepine receptors (CBRs). The corresponding radiolabeled compounds [ (18)F] 8 [ (18)F] 12, [ (18)F] 15, and [ (18)F] 18 were prepared from their p-toluenesulfonyl precursors in 50-85% radiochemical yield. In biodistribution studies in rats, the distribution of radioactivity of the [ (18)F]PBR compounds paralleled the known localization of PBRs. In the olfactory bulbs, where the uptake of radioactivity was higher than in the rest of the brain, PK11195 and Ro 5-4864 were able to significantly inhibit [ (18)F] 12, while little or no pharmacological action of these established PBR drugs were observed on the uptake of [ (18)F] 8, [ (18)F] 15, and [ (18)F] 18 compared to control animals. Hence, [ (18)F] 12 appeared to be the best candidate for evaluation as an imaging agent for PBR expression in neurodegenerative disorders.

Assessment of luteolin (3′,4′,5,7-tetrahydroxyflavone) neuropharmacological activity

Since the discovery that certain flavonoids (namely flavones) specifically recognise the central BDZ receptors, several efforts have been made to identify naturally occurring GABA A receptor benzodiazepine binding site ligands. Flavonoid derivatives with a flavone-like structure such as apigenin, chrysin and wogonin have been reported for their anxiolytic-like activity in different animal models of anxiety. Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a widespread flavonoid aglycon that was reported as devoid of specific affinity for benzodiazepine receptor (BDZ-R) binding site, but its psychopharmacological activity is presently unknown. Considering (1) the close structural similarity with other active flavones, (2) the activity of some of its glycosilated derivatives and (3) the complexity of flavonoid effects in the central nervous system, luteolin was submitted to a battery of tests designed to evaluate its possible activity upon the CNS and its ability to interact with the BDZ-receptor binding sites was also analysed. Luteolin apparently has CNS activity with anxiolytic-like effects despite the low affinity for the BDZ-R shown in vitro. Our findings suggest a possible interaction with other neurotransmitter systems but we cannot rule out the possibility that luteolin's metabolites might show a higher affinity for the BDZ-R in vivo, thus eliciting the evident anxiolytic-like effects through a GABAergic mechanism.

Peripheral-type benzodiazepine receptors in bronchoalveolar lavage cells of patients with interstitial lung disease

PK11195 is a ligand with high affinity for peripheral benzodiazepine receptors (PBRs), which are present in large numbers in macrophages. PBRs play a role in antioxidant pathways and apoptosis, key factors in control of lung health. Intrapulmonary PBRs, assessed in vivo by positron emission tomography (PET), are decreased in interstitial lung disease (ILD) despite increased macrophage numbers. We wished to ascertain whether the observed decrease in in vivo expression of PBRs in the PET scans could be accounted for by a reduction in PBRs per cell by saturation-binding assays of R-PK11195 in cells obtained by bronchoalveolar lavage (BAL). We performed receptor saturation-binding assays with [(3)H]-R-PK11195 on a mixed population of cells recovered by BAL to quantify the number of R-PK11195 binding sites per macrophage in 10 subjects with ILD and 10 normal subjects. Receptor affinity [dissociation constant (Kd)] was similar in ILD patients and controls. However, R-PK11195 binding sites per cell [(maximal binding sites available (B(max))] were decreased in macrophages obtained by BAL from subjects with ILD compared to normal (P<.0005). Microautoradiography confirmed localization of R-PK11195 to macrophages in a mixed inflammatory cell population obtained by BAL. These results demonstrate that in vitro PBR expression per cell on macrophages obtained by BAL is reduced in patients with ILD indicating a potentially functionally different macrophage phenotype. As PBRs are involved in the orchestration of lung inflammatory responses, this finding offers further insight into the role of macrophages in the pathogenesis of ILDs and offers a potential avenue for pharmacological strategy.

Novel L-Dopa and Dopamine Prodrugs Containing a 2-Phenyl-imidazopyridine Moiety

The aim of this study was to gain insight into the feasibility of enhancing the delivery of L-Dopa and dopamine to the brain by linking these neurotransmitters and L-Dopa ethyl ester to 2-phenyl-3-carboxymethyl-imidazopyridine compounds giving rise to the so-called Dopimid compounds. A number of Dopimid compounds were synthesized and both stability and binding studies to dopaminergic and benzodiazepine receptors were performed. To evaluate whether Dopimid compounds are P-gp substrates, [(3)H]ritonavir uptake experiments and bi-directional transport studies on confluent MDCKII-MDR1 monolayers were carried out. The brain penetration properties of Dopimid compounds were estimated by the Clark's computational model and evaluated by investigation of their transport across BBMECs monolayers. The dopamine levels following the intraperitoneal administration of the selected Dopimid compounds were measured in vivo by using brain microdialysis in rat. Tested compounds were adequately stable in solution buffered at pH 7.4 but undergo faster cleavage in dilute rat serum at 37 degrees C. Receptor binding studies showed that Dopimid compounds are essentially devoid of affinity for dopaminergic and benzodiazepine receptors. [(3)H]ritonavir uptake experiments indicated that selected Dopimid compounds, like L-Dopa and dopamine hydrochloride, are not substrates of P-gp and it was also confirmed by bi-directional transport experiments across MDCKII-MDR1 monolayers. By Clark's model a significant brain penetration was deduced for L-Dopa ethyl ester and dopamine derivatives. Transport studies involving BBMECs monolayers indicated that some of these compounds should be able to cross the BBB. Interestingly, the rank order of apparent permeability (P (app)) values observed in these assays parallels that calculated by the computational approach. Brain microdialysis experiments in rat showed that intraperitoneal acute administration of some Dopimid compounds induced a dose-dependent increase in cortical dopamine output. Based on these results, it may be concluded that some Dopimid compounds can be proposed as novel L-Dopa and dopamine prodrugs.

Effects of peripheral-type benzodiazepine receptor ligands on Ehrlich tumor cell proliferation

Peripheral-type benzodiazepine receptors have been found throughout the body, and particularly, in high numbers, in neoplastic tissues such as the ovary, liver, colon, breast, prostate and brain cancer. Peripheral-type benzodiazepine receptor expression has been associated with tumor malignity, and its subcellular localization is important to define its function in tumor cells. We investigated the presence of peripheral-type benzodiazepine receptors in Ehrlich tumor cells, and the in vitro effects of peripheral-type benzodiazepine receptors ligands on tumor cell proliferation. Our results demonstrate the presence of peripheral-type benzodiazepine receptor in the nucleus of Ehrlich tumor cells (85.53 ± 12.60%). They also show that diazepam and Ro5-4864 (peripheral-type benzodiazepine receptor agonists) but not clonazepam (a molecule with low affinity for the peripheral-type benzodiazepine receptor) decreased the percentage of tumor cells in G0–G1 phases and increased that of cells in S-G2-M phases. The effects of those agonists were prevented by PK11195 (a peripheral-type benzodiazepine receptor antagonist) that did not produce effects by itself. Altogether, these data suggest that the presence of peripheral-type benzodiazepine receptor within the nucleus of Ehrlich tumor cells is associated with tumor malignity and proliferation capacity.

Synthesis and in vitro binding of N, N-dialkyl-2-phenylindol-3-yl-glyoxylamides for the peripheral benzodiazepine binding sites

A series of N, N-dialkyl-2-phenylindol-3-ylglyoxylamides bearing the halogens iodine and bromine were synthesised and their binding affinity for the peripheral benzodiazepine binding sites (PBBS) in rat kidney mitochondrial membranes was evaluated using [ 3H]PK11195. Central benzodiazepine receptor (CBR) affinities were also evaluated in rat cortices using [ 3H]flumazenil to determine their selectivity for PBBS over CBR. The tested compounds had PBBS binding affinities (IC 50) ranging from 7.86 to 618 nM, with all compounds showing high selectivity over the CBR (CBR IC 50 > 5000 nM). Among the 12 compounds tested, those with a diethylamide group were the most potent. The highest affinity iodinated PBBS ligand, N, N-diethyl-[5-chloro-2-(4-iodophenyl)indol-3-yl]glyoxylamide ( 4c), was radiolabelled with iodine-123. This high affinity and selective radioligand may be useful for imaging neurodegeneration, inflammation and tumours using single photon emission computed tomography.