Inverse Agonist Properties Research Articles

β 2 -Adrenoceptor signaling is required for the development of an asthma phenotype in a murine model

Chronic regular use of beta(2)-adrenoceptor (beta(2)-AR) agonists in asthma is associated with a loss of disease control and increased risk of death. Conversely, we have found that administration of beta(2)-AR inverse agonists results in attenuation of the asthma phenotype in an allergen-driven murine model. Besides antagonizing agonist-induced signaling and reducing signaling by empty receptors, beta-AR inverse agonists can also activate signaling by novel pathways. To determine the mechanism of the beta-AR inverse agonists, we compared the asthma phenotype in beta(2)-AR-null and wild-type mice. Antigen challenge of beta(2)-AR-null mice produced results similar to what was observed with chronic beta(2)-AR inverse agonist treatment, namely, reductions in mucous metaplasia, airway hyperresponsiveness (AHR), and inflammatory cells in the lungs. These results indicate that the effects of beta(2)-AR inverse agonists are caused by inhibition of beta(2)-AR signaling rather than by the induction of novel signaling pathways. Chronic administration of alprenolol, a beta-blocker without inverse agonist properties, did not attenuate the asthma phenotype, suggesting that it is signaling by empty receptors, rather than agonist-induced beta(2)-AR signaling, that supports the asthma phenotype. In conclusion, our results demonstrate that, in a murine model of asthma, beta(2)-AR signaling is required for the full development of three cardinal features of asthma: mucous metaplasia, AHR, and the presence of inflammatory cells in the lungs.

Characterization of oligomers induced by inverse agonists of CTLA-4

Although cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) inhibits T cell activation when ligated by B7 molecules on antigen-presenting cells, it can also act as an activating receptor when binding certain soluble recombinant ligands known as inverse agonists. Following ligation with an inverse agonist, we observed CTLA-4 microclusters evenly distributed on the T cell surface over a 60 -min period. We have previously shown that the inverse agonist properties of these ligands correlate with their capacity to induce the formation of large CTLA-4 oligomers that are distinctly different from those resulting by CTLA-4 engagement with membrane-bound B7. These oligomers are composed of CTLA-4 molecules expressed on the cell surface and decrease from both the soluble cell lysate and lipid rafts upon cellular fractionation. Formation of these inverse agonist-induced CTLA-4 oligomers does not require an intact actin cytoskeleton. However, modulation of these oligomers was partially blocked upon actin depolymerization. Retention of CTLA-4 oligomers on the cell surface correlated with enhanced T cell signaling. Together, our data further characterize the structural basis of inverse agonist properties for CTLA-4 ligands that may be used in the design and screening of therapeutic biologicals targeting this receptor.

Identification of key amino acid residues in a thyrotropin receptor monoclonal antibody epitope provides insight into its inverse agonist and antagonist properties.

CS-17 is a murine monoclonal antibody to the human TSH receptor (TSHR) with both inverse agonist and antagonist properties. Thus, in the absence of ligand, CS-17 reduces constitutive TSHR cAMP generation and also competes for TSH binding to the receptor. The present data indicate that for both of these functions, the monovalent CS-17 Fab (50 kDa) behaves identically to the intact, divalent IgG molecule (150 kDa). The surprising observation that CS-17 competes for TSH binding to the human but not porcine TSHR enabled identification of a number of amino acids in its epitope. Replacement of only three human TSHR residues (Y195, Q235, and S243) with the homologous porcine TSHR residues totally abolishes CS-17 binding as detected by flow cytometry. TSH binding is unaffected. Of these residues, Y195 is most important, with Q235 and S243 contributing to CS-17 binding to a much lesser degree. The functional effects of CS-17 IgG and Fab on constitutive cAMP generation by porcinized human TSHR confirm the CS-17 binding data. The location of TSHR amino acid residues Y195, Q235, and S243 deduced from the crystal structure of the FSH receptor leucine-rich domain provides valuable insight into the CS-17 and TSH binding sites. Whereas hormone ligands bind primarily to the concave surface of the leucine-rich domains, a major portion of the CS-17 epitope lies on the opposite convex surface with a minor component in close proximity to known TSH binding residues.

Noncompetitive Antagonism and Inverse Agonism as Mechanism of Action of Nonpeptidergic Antagonists at Primate and Rodent CXCR3 Chemokine Receptors

The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10, and CXCL11 are expressed at sites of inflammation, and they attract CXCR3-bearing lymphocytes, thus contributing to the inflammatory process. In this study, we characterize five nonpeptidergic compounds of different chemical classes that block the action of CXCL10 and CXCL11 at the human CXCR3, i.e., the 3H-pyrido[2,3-d]pyrimidin-4-one derivatives N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-fluoro-3-trifluoromethyl-phenyl)-acetamide (VUF10472/NBI-74330) and N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-trifluoromethoxy-phenyl)-acetamide (VUF10085/AMG-487), the 3H-quinazolin-4-one decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro-quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide (VUF5834), the imidazolium compound 1,3-bis-[2-(3,4-dichloro-phenyl)-2-oxo-ethyl]-3H-imidazol-1-ium bromide (VUF10132), and the quaternary ammonium anilide N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]-carbonyl]amino]benzyl] tetrahydro-2H-pyran-4-aminium chloride (TAK-779). To understand the action of these CXCR3 antagonists in various animal models of disease, the compounds were also tested at rat and mouse CXCR3, as well as at CXCR3 from rhesus macaque, which was cloned and characterized for the first time in this study. Except for TAK-779, all compounds show slightly lower affinity for rodent CXCR3 than for primate CXCR3. In addition, we have characterized the molecular mechanism of action of the various antagonists at the human CXCR3 receptor. All tested compounds act as noncompetitive antagonists at CXCR3. Moreover, this noncompetitive behavior is accompanied by inverse agonistic properties of all five compounds as determined on an identified constitutively active mutant of CXCR3, CXCR3 N3.35A. It is interesting to note that all compounds except TAK-779 act as full inverse agonists at CXCR3 N3.35A. TAK-779 shows weak partial inverse agonism at CXCR3 N3.35A, and it probably has a different mode of interaction with CXCR3 than the other two classes of small-molecule inverse agonists.

Inverse Agonist and Neutral Antagonist Actions of Antidepressants at Recombinant and Native 5-Hydroxytryptamine2CReceptors: Differential Modulation of Cell Surface Expression and Signal Transduction

Despite the importance of 5-hydroxytryptamine (5-HT)(2C) (serotonin) receptors in the control of depressive states, actions of antidepressants at these receptors remain poorly characterized. This issue was addressed both in human embryonic kidney (HEK)-293 cells coexpressing unedited human 5-HT(2CINI) receptors and Galpha(q) protein and in cultured mouse cortical neurons. Indicative of constitutive activity, the inverse agonist SB206,553 decreased basal inositol phosphate (IP) production in HEK-293 cells. The tetracyclic antidepressants mirtazapine and mianserin likewise suppressed basal IP formation. Conversely, the tricyclics amitriptyline and clomipramine, the m-chlorophenylpiperazine derivatives trazodone and nefazodone, and the 5-HT reuptake inhibitors fluoxetine and citalopram were inactive alone, although they blocked 5-HT-induced IP production. Inverse agonist actions of 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole (SB206,553) and mirtazapine were abolished by the neutral antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline (SB242,084), which was inactive alone. As assessed by confocal microscopy and enzyme-linked immunosorbent assay, prolonged treatment of HEK-293 cells with SB206,553, mirtazapine, or mianserin, but not the other antidepressants, enhanced cell surface expression of 5-HT(2C) receptors: 5-HT-induced IP production was also increased, and both these actions were blocked by SB242,084. Cortical neurons were shown by reverse transcription-polymerase chain reaction to predominantly express constitutively active 5-HT(2C) receptor isoforms. Prolonged pretreatment with SB206,553 or mirtazapine triggered an otherwise absent 5-HT-induced elevation in cytosolic Ca(2+) concentrations. SB242,084, which was inactive alone, abolished these effects of SB206,553 and mirtazapine. In conclusion, the tetracyclic antidepressants mirtazapine and mianserin, but not other clinically established antidepressants, suppress constitutive activity at recombinant and native 5-HT(2C) receptors. The clinical significance of inverse agonist versus neutral antagonist properties both during and after drug administration will be of interest to elucidate.

Some effects of CB1 antagonists with inverse agonist and neutral biochemical properties

The CB1 inverse agonist/antagonist SR141716A recently has been introduced for the management of obesity (rimonabant; Acomplia) and appears to have beneficial effects. However, its utility may be hampered in some individuals by adverse effects including nausea or emesis or by mood depression. The recent development of biochemically ‘neutral’ antagonists such as AM4113 (Sink et al., 2007) has allowed an initial evaluation of the proposition that adverse effects of SR141716A are associated with its inverse agonist activity. Thus far, data comparing SR141716A and AM4113 across several species indicate that both drugs produce dose-related direct effects on operant behavior within the same range of doses that serve to antagonize the behavioral and hypothermic effects of a CB1 agonist. However, initial observations suggest that AM4113 may not produce preclinical indications of nausea or emesis. Further studies with AM4113 and other novel CB1 antagonists differing in efficacy should amplify our understanding of the relationship between the pharmacological activity of CB1 antagonists and their behavioral effects.

A neutral CB1 receptor antagonist reduces weight gain in rat

Cannabinoid (CB)1 receptor inverse agonists inhibit food intake in animals and humans but also potentiate emesis. It is not clear whether these effects result from inverse agonist properties or from the blockade of endogenous cannabinoid signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on food intake, weight gain, and emesis. Neutral antagonist and binding properties were confirmed in HEK-293 cells transfected with human CB1 or CB2 receptors. AM4113 had no effect on forskolin-stimulated cAMP production at concentrations up to 630 nM. The Ki value of AM4113 (0.80 +/- 0.44 nM) in competitive binding assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1 over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg/kg) significantly reduced food intake and weight gain in rat. Compared with AM251, higher doses of AM4113 were needed to produce similar effects on food intake and body weight. Unlike AM251 (5 mg/kg), a highly anorectic dose of AM4113 (10 mg/kg) did not significantly potentiate vomiting induced by the emetic morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor antagonist shares the appetite suppressant and weight loss effects of inverse agonists. If these compounds display similar properties in humans, they could be developed into a new class of antiobesity agents.

Differential profile of typical, atypical and third generation antipsychotics at human 5-HT7a receptors coupled to adenylyl cyclase: detection of agonist and inverse agonist properties

5-HT(7) receptors are present in thalamus and limbic structures, and a possible role of these receptors in the pathology of schizophrenia has been evoked. In this study, we examined binding affinity and agonist/antagonist/inverse agonist properties at these receptors of a large series of antipsychotics, i.e., typical, atypical, and third generation compounds preferentially targeting D(2) and 5-HT(1A) sites. Adenylyl cyclase (AC) activity was measured in HEK293 cells stably expressing the human (h) 5-HT(7a) receptor isoform. 5-HT and 5-CT increased cyclic adenosine monophosphate level by about 20-fold whereas (+)-8-OH-DPAT, the antidyskinetic agent sarizotan, and the novel antipsychotic compound bifeprunox exhibited partial agonist properties at h5-HT(7a) receptors stimulating AC. Other compounds antagonized 5-HT-induced AC activity with pK (B) values which correlated with their pK (i) as determined by competition binding vs [(3)H]5-CT. The selective 5-HT(7) receptor ligand, SB269970, was the most potent antagonist. For antipsychotic compounds, the following rank order of antagonism potency (pK (B)) was ziprasidone > tiospirone > SSR181507 > or = clozapine > or = olanzapine > SLV-314 > SLV-313 > or = aripiprazole > or = chlorpromazine > nemonapride > haloperidol. Interestingly, pretreatment of HEK293-h5-HT(7a) cells with forskolin enhanced basal AC activity and revealed inverse agonist properties for both typical and atypical antipsychotics as well as for aripiprazole. In contrast, other novel antipsychotics exhibited diverse 5-HT(7a) properties; SLV-313 and SLV-314 behaved as quasi-neutral antagonists, SSR181507 acted as an inverse agonist, and bifeprunox as a partial agonist, as mentioned above. In conclusion, the differential properties of third generation antipsychotics at 5-HT(7) receptors may influence their antipsychotic profile.

The Novel Cannabinoid CB1 Receptor Neutral Antagonist AM4113 Suppresses Food Intake and Food-Reinforced Behavior but Does not Induce Signs of Nausea in Rats

Drugs that interfere with cannabinoid CB1 transmission suppress various food-motivated behaviors, and it has been suggested that such drugs could be useful as appetite suppressants. Biochemical studies indicate that most of these drugs assessed thus far have been CB1 inverse agonists, and although they have been shown to suppress food intake, they also appear to induce nausea and malaise. The present studies were undertaken to characterize the behavioral effects of AM4113, which is a CB1 neutral antagonist, and to examine whether this drug can reduce food-reinforced behaviors and feeding on diets with varying macronutrient compositions. Biochemical data demonstrated that AM4113 binds to CB1 receptors, but does not show inverse agonist properties (ie no effects on cyclic-AMP production). In tests of spontaneous locomotion and analgesia, AM4113 reversed the effects of the CB1 agonist AM411. AM4113 suppressed food-reinforced operant responding with rats responding on fixed ratio (FR) 1 and 5 schedules of reinforcement in a dose-dependent manner, and also suppressed feeding on high-fat, high-carbohydrate, and lab chow diets. However, in the same dose range that suppressed feeding, AM4113 did not induce conditioned gaping, which is a sign of nausea and food-related malaise in rats. These results suggest that AM4113 may decrease appetite by blocking endogenous cannabinoid tone, and that this drug may be less associated with nausea than CB1 inverse agonists.

Differential agonist and inverse agonist profile of antipsychotics at D 2L receptors coupled to GIRK potassium channels

The D 2 dopaminergic receptor represents a major target of antipsychotic drugs. Using the coupling of the human D 2long (hD 2L) receptor to G protein-coupled inward rectifier potassium (GIRK) channels in Xenopus laevis oocytes, we examined the activity of antipsychotic agents of different classes – typical, atypical, and a “new generation” of compounds, exhibiting a preferential D 2 and 5-HT 1A receptor profile. When the hD 2L receptor was coexpressed with GIRK channels, a series of reference compounds exhibited full agonist (dopamine, and quinpirole), partial agonist (apomorphine, (−)3-PPP, and (+)-UH232) or inverse agonist (raclopride, and L741626) properties. Sarizotan exhibited only very weak partial agonist action. At higher levels of receptor cRNA injected per oocyte, both partial agonist activity and inverse agonist properties were generally more pronounced. The inverse agonist action of L741626 was reversed by interaction with sarizotan, thus confirming the constitutive activity of wild-type hD 2L receptors in the oocyte expression system. When antipsychotic agents were tested for their actions at the hD 2L receptor, typical (haloperidol) as well as atypical (nemonapride, ziprasidone, and clozapine) compounds acted as inverse agonists. In contrast, among D 2/5-HT 1A antipsychotics, only SLV313 and F15063 behaved as inverse agonists, whilst the other members of this group (bifeprunox, SSR181507 and the recently marketed antipsychotic, aripiprazole) exhibited partial agonist properties. Thus, the X. laevis oocyte expression system highlights markedly different activity of antipsychotics at the hD 2L receptor. These differential properties may translate to distinct therapeutic potential of these compounds.

Structurally novel histamine H 3 receptor antagonists GSK207040 and GSK334429 improve scopolamine-induced memory impairment and capsaicin-induced secondary allodynia in rats

GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1 H-3-benzazepin-7-yl)oxy]- N-methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1 H-1,4-diazepine) are novel and selective non-imidazole histamine H 3 receptor antagonists from distinct chemical series with high affinity for human (p K i = 9.67 ± 0.06 and 9.49 ± 0.09, respectively) and rat (p K i = 9.08 ± 0.16 and 9.12 ± 0.14, respectively) H 3 receptors expressed in cerebral cortex. At the human recombinant H 3 receptor, GSK207040 and GSK334429 were potent functional antagonists (pA 2 = 9.26 ± 0.04 and 8.84 ± 0.04, respectively versus H 3 agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC 50 = 9.20 ± 0.36 and 8.59 ± 0.04 versus basal GTPγS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [ 3H]- R-α-methylhistamine binding (ED 50 = 0.03 and 0.35 mg/kg, respectively). Functional antagonism of central H 3 receptors was demonstrated by blockade of R-α-methylhistamine-induced dipsogenia in rats (ID 50 = 0.02 and 0.11 mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3 mg/kg p.o.) and GSK334429 (0.3, 1 and 3 mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1 mg/kg p.o.) and GSK334429 (3 and 10 mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H 3 receptors may be able to reduce tactile allodynia. Novel H 3 receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.

Cannabinoid CB2-Selective Inverse Agonist Protects Against Antigen-Induced Bone Loss

Work to improve the therapeutic properties of cannabinoid CB2 receptor-selective inverse agonists has led to the development of Sch.036, an aryl substituted triaryl bis-sulfone with improved oral pharmacokinetic parameters. In this report, we show that this compound blocks in vivo trafficking of various leukocyte populations, a property consistent with other members of this chemical series. This CB2-selective compound also shows efficacy in leukocyte recruitment models when added in concert with suboptimal doses of selected anti-inflammatory agents, consistent with its unique function and indicative of its potential therapeutic utility. Finally, studies with Sch.036 show that this cannabinoid CB2-specific inverse agonist can ameliorate bone damage in a rat model of relapsing-remitting arthritis. This result suggests that a cannabinoid CB2‐selective inverse agonist may help ameliorate a particularly harmful property of this inflammatory joint disease.

Conformational Constraining of Inactive and Active States of a Seven Transmembrane Receptor by Metal Ion Site Engineering in the Extracellular End of Transmembrane Segment V

The extracellular part of transmembrane segment V (TM-V) is expected to be involved in the activation process of 7TM receptors, but its role is far from clear. Here, we study the highly constitutively active CXC-chemokine receptor encoded by human herpesvirus 8 (ORF74-HHV8), in which a metal ion site was introduced at the extracellular end of TM-V by substitution of two arginines at positions V:01 and V:05 with histidines [R208H; R212H]. The metal ion site conferred high-potency inverse agonist properties (EC(50), 1.7 microM) to Zn(II) in addition to agonist and allosteric enhancing properties at concentrations >10 microM. The chemokine interaction with [R208H;R212H]-ORF74 was altered compared with wild-type ORF74-HHV8 with decreased agonist (CXCL1/GROalpha) potency (84-fold), affinity (5.8- and 136-fold in competition against agonist and inverse agonist, respectively), and binding capacity (B(max); 25-fold). Zn(II) in activating concentrations (100 microM) acted as an allosteric enhancer as it increased the B(max) (7.1-fold), the potency (9.9-fold), the affinity (1.7- and 6.1-fold in competition against agonist and inverse agonist, respectively), and the efficacy (2.5-fold) of CXCL1/GROalpha. The activating properties of Zn(II) were not due to a metal ion site between the ligand and the receptor because CXCL1/GROalpha analogs in which the putative metal-ion binding residues had been substituted-[H19A] and [H34A]-acted like wild-type CXCL1/GROalpha. Based on the complex action of Zn(II) and on the chemokine interaction for [R208H;R212H]-ORF74, we conclude that the extracellular end of TM-V is important for the activation of this CXC-chemokine receptor.

Reanalysis of constitutively active rat and human 5-HT7(a) receptors in HEK-293F cells demonstrates lack of silent properties for reported neutral antagonists

The present study reinvestigated a series of 5-HT receptor antagonists at both constitutively active rat and human 5-HT7(a) receptors in HEK-293F cells using the cAMP signalling pathway as a functional read-out. Both rat and human 5-HT7(a) receptors were expressed in similar amounts ([3H]-LSD binding: 1.0 to 1.1 pmol/mg protein). Attenuation of basal cAMP formation by the inverse agonist SB-691673 (1 microM) was slightly larger by the human 5-HT7(a) (-73+/-3 %) than rat 5-HT7(a) receptor (-62+/-3 %). The 5-HT receptor antagonists investigated here displayed systematically inverse agonism. While methiothepin and SB-269970 displayed similar negative intrinsic activity to SB-691673 at the rat 5-HT7(a) receptor, the compounds SB-258719, mesulergine and metergoline displayed some lower negative intrinsic activity (between -38 and -49%). Inverse agonist properties were observed with potencies fitting with their respective binding pIC50 values and pKB values as estimated from antagonist studies with 5-HT. With the exception of SB-258719 and mesulergine, which remained a partial inverse agonist at the human 5-HT7(a) receptor, the other compounds behaved with a similar Emax value to the full inverse agonist SB-691673. In conclusion, none of the 5-HT receptor antagonists investigated displayed silent properties at the rat or human 5-HT7(a) receptor, when these are expressed in a system allowing detection of constitutive activity. They appear to be partial to full inverse agonists, further illustrating that an antagonist is preferentially an inverse agonist when investigated under constitutively active receptor conditions.

Identification and characterization of Hedgehog modulator properties after functional coupling of Smoothened to G15

The seven-transmembrane receptor Smoothened (Smo) transduces the signal initiated by Hedgehog (Hh) morphogen binding to the receptor Patched (Ptc). We have reinvestigated the pharmacological properties of reference molecules acting on the Hh pathway using various Hh responses and a novel functional assay based on the coexpression of Smo with the α subunit of the G15 protein in HEK293 cells. The measurement of inositol phosphate (IP) accumulation shows that Smo has constitutive activity, a response blocked by Ptc which indicates a functional Hh receptor complex. Interestingly, the antagonists cyclopamine, Cur61414, and SANT-1 display inverse agonist properties and the agonist SAG has no effect at the Smo-induced IP response, but converts Ptc-mediated inactive forms of Smo into active ones. An oncogenic Smo mutant does not mediate an increase in IP response, presumably reflecting its inability to reach the cell membrane. These studies identify novel properties of molecules displaying potential interest in the treatment of various cancers and brain diseases, and demonstrate that Smo is capable of signaling through G15.

Mouse lines selected for difference in sensitivity to β-CCM also differ in memory processes

Methyl-β-carboline-3-carboxylate (or β-CCM) is a benzodiazepine receptor ligand with inverse agonist properties. Two strains of mice were selected, one for sensitivity (BS) and one for resistance (BR) to a convulsive dose of β-CCM. These two strains were then shown to differ in several biochemical, pharmacological and behavioral characteristics; specifically BS mice were less anxious than BR mice. The present work provides evidence of differences in the learning abilities of the two strains. Three different learning tasks were used: spatial delayed discrimination on the 4-hole board, a learned choice between a lit and a dark compartment in a T-maze, and place-learning in an 8-arm radial maze. In all three tasks, BS mice had consistently better performance levels than BR mice.

Antiobesity effects of the novel in vivo neutral cannabinoid receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1 H-1,2,4-triazole – LH 21

The present study evaluates the pharmacological profile of the new neutral cannabinoid CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1 H-1,2,4-triazole -LH-21- on feeding behavior and alcohol self-administration in rats, two behaviors inhibited by cannabinoid CB1 receptor antagonists. Administration of LH-21 (0.03, 0.3 and 3 mg/kg) to food-deprived rats resulted in a dose-dependent inhibition of feeding. Subchronic administration of LH-21 reduced food intake and body weight gain in obese Zucker rats. Acute effects on feeding were not associated with anxiety-like behaviors, or induction of complex motor behaviors such as grooming or scratching sequences, usually observed after central administration of cannabinoid receptor blockers with inverse agonist properties. LH-21 did not markedly reduce alcohol self-administration (30% reduction observed only at a high dose of 10 mg/kg). This pharmacological pattern partially overlaps that of the reference cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide, SR141716A, (0.3, 1 and 3 mg/kg) that reduced feeding and alcohol self-administration with similar efficacy. In vitro analysis of blood–brain barrier permeability using a parallel artificial membrane permeation assay demonstrated that LH-21 has lower permeation through membranes than SR141716A. That was confirmed in vivo by studies showing lower potency of peripherally injected LH-21 when compared to SR141716A to antagonize motor depression induced by intracerebroventricular administration of the CB1 agonist CP55,940. The neutral antagonist profile and the lower penetration into the brain of LH-21 favour this class of antagonists with respect to reference inverse agonists for the treatment of obesity because they potentially will display reduced side effects.

An inverse agonist selective for α5 subunit-containing GABAA receptors improves encoding and recall but not consolidation in the Morris water maze

Compounds selective for the GABAA receptors containing an alpha5 subunit have been reported to enhance performance in the hippocampally mediated delayed-matching-to-position version of the Morris water maze, in which reduction in the time required to find a hidden platform relative to an initial trial is used as an index of learning and memory. In the present study, we have used one such compound, alpha5IA-II, to examine whether these effects occur during the encoding, consolidation or recall phases of this paradigm. alpha5IA-II was administered in the absence or presence of the benzodiazepine site antagonist flumazenil, so as to limit its action to periods associated with encoding, consolidation and recall. Drug doses and timings of administrations were defined using occupancy data derived from an in vivo [3H]flumazenil binding assay. Similar experiments were carried out to study the memory-disruptive properties of chlordiazepoxide (CDP). The trial 1 to trial 2 difference was increased when alpha5IA-II was given before either trial 1 or trial 2, indicating an effect on the encoding and recall phases, respectively, of learning and memory. Conversely, alpha5IA-II had no effect on performance when given immediately after trial 1, suggesting that it had no effect on the consolidation phase. In contrast to the facilitation of performance produced by the alpha5-selective inverse agonist alpha5IA-II given during the encoding and recall but not the consolidation phase, the non-selective agonist CDP impaired performance when given during the encoding and recall phases, whilst having no effect on the consolidation phase. These data further highlight the cognition-enhancing properties of GABAA alpha5-selective inverse agonists and define the functional specificity of these effects in terms of encoding and recall processes in the Morris water maze.

Suppression of food intake and food-reinforced behavior produced by the novel CB1 receptor antagonist/inverse agonist AM 1387

Cannabinoid CB1 receptor antagonist/inverse agonists are becoming increasingly recognized for their potential therapeutic utility as appetite suppressants. In the current paper we characterize the biochemical and behavioral effects of AM 1387, which is a novel CB1 antagonist. AM 1387 exhibited binding affinity and selectivity for the CB1 over the CB2 receptor. Moreover, AM 1387 decreased GTPγS (EC 50: 22.82 nM) and increased forskolin-stimulated cAMP (EC 50: 274.6 nM), as did the CB1 inverse agonist AM 251 (GTPγS EC 50: 25.82 nM; cAMP EC 50: 363.8 nM), indicating that AM1387 also has inverse agonist properties in vitro. In the behavioral characterization in rats, AM 1387 suppressed lever pressing for food on two operant schedules (fixed-ratio 1 and 5). Timecourse of the effect on fixed-ratio 5 responding was then determined, and the half-life ( t 1/2 = 4.87 h) was found to be threefold shorter than what has been shown for SR 141716A, and fourfold shorter than AM 251. Finally, AM 1387 was found to suppress food intake using three diets of differing macronutrient composition and palatability. It was concluded that AM 1387 may be a useful tool for examining the effects of CB1 receptor antagonism or inverse agonism on food intake.

Synthesis and Activity of 1,3,5-Triphenylimidazolidine-2,4-diones and 1,3,5-Triphenyl-2-thioxoimidazolidin-4-ones: Characterization of New CB1 Cannabinoid Receptor Inverse Agonists/Antagonists

Obesity and metabolic syndrome, along with drug dependence (nicotine, alcohol, opiates), are two of the major therapeutic applications for CB(1) cannabinoid receptor antagonists and inverse agonists. In the present study, we report the synthesis and structure-affinity relationships of 1,5-diphenylimidazolidine-2,4-dione and 1,3,5-triphenylimidazolidine-2,4-dione derivatives. These new 1,3,5-triphenylimidazolidine-2,4-dione derivatives and their thio isosteres were obtained by an original pathway and exhibited interesting affinity and selectivity for the human CB(1) cannabinoid receptor. A [(35)S]-GTPgammaS binding assay revealed the inverse agonist properties of the compounds at the CB(1) cannabinoid receptor. Furthermore, molecular modeling studies were conducted in order to delineate the binding mode of this series of derivatives into the CB(1) cannabinoid receptor. 1,3-Bis(4-bromophenyl)-5-phenylimidazolidine-2,4-dione (25) and 1,3-bis(4-chlorophenyl)-5-phenylimidazolidine-2,4-dione (23) are the imidazolidine-2,4-dione derivatives possessing the highest affinity for the human CB(1) cannabinoid receptor reported to date.