Jelveh Lameh

The role of M1 muscarinic receptor agonism of N-desmethylclozapine in the unique clinical effects of clozapine

RationaleClozapine is a unique antipsychotic, with efficacy against positive symptoms in treatment-resistant schizophrenic patients, and the ability to improve cognition and treat the negative symptoms characteristic of this disease. Despite its unique clinical actions, no specific molecular mechanism responsible for these actions has yet been described.Objectives and methodsTo comprehensively profile a large library of neuropsychiatric drugs, including most antipsychotics, at human monoamine receptors using R-SAT, an in vitro functional assay.ResultsProfiling revealed that N-desmethylclozapine (NDMC), the principal metabolite of clozapine, but not clozapine itself, is a potent and efficacious muscarinic receptor agonist, a molecular property not shared by any other antipsychotic. To further explore the role of NDMC muscarinic receptor agonist properties in mediating the physiological actions of clozapine, systemically administered NDMC was found to stimulate the phosphorylation of mitogen-activated protein kinase (MAP kinase) in mouse CA1 hippocampal neurons, an effect that was blocked by scopolamine, confirming central M1 muscarinic receptor agonist activity in vivo. Lastly, an analysis of clozapine and NDMC serum levels in schizophrenic patients indicated that high NDMC/clozapine ratios better predicted improvement in cognitive functioning and quality of life than the levels of either compound alone.ConclusionsThe muscarinic receptor agonist activities of NDMC are unique among antipsychotics, and provide a possible molecular basis for the superior clinical effects of clozapine pharmacotherapy.

Pharmacological and Behavioral Profile of N-(4-Fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl) Carbamide (2R,3R)-Dihydroxybutanedioate (2:1) (ACP-103), a Novel 5-Hydroxytryptamine2A Receptor Inverse Agonist

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N′-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)2A receptor inverse agonist ACP-103 competitively antagonized the binding of [3H]ketanserin to heterologously expressed human 5-HT2A receptors with a mean pKi of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC50 of 8.7. ACP-103 demonstrated lesser affinity (mean pKi of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC50 7.1 in R-SAT) at human 5-HT2C receptors, and lacked affinity and functional activity at 5-HT2B receptors, dopamine D2 receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT2A receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT2A receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.

Activity of opioid ligands in cells expressing cloned mu opioid receptors.

BackgroundThe aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a cell system expressing only mu opioid receptors. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cyclic adenosine mono phosphate (cAMP) production. Efficacies and potencies of these ligands were determined relative to the endogenous ligand β-endorphin and the common mu agonist, morphine.ResultsAmong the ligands studied naltrexone, WIN 44,441 and SKF 10047, were classified as antagonists, while the remaining ligands were agonists. Agonist efficacy was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The rank order of efficacy of the agonists was fentanyl = hydromorphone = β-endorphin > etorphine = lofentanil = butorphanol = morphine = nalbuphine = nalorphine > cyclazocine = dezocine = metazocine ≥ xorphanol. The rank order of potency of these ligands was different from that of their efficacies; etorphine > hydromorphone > dezocine > xorphanol = nalorphine = butorphanol = lofentanil > metazocine > nalbuphine > cyclazocine > fentanyl > morphine >>>> β-endorphin.ConclusionThese results elucidate the relative activities of a set of opioid ligands at mu opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of opioid ligands at this receptor. Furthermore, these results can assist in understanding the physiological effect of many opioid ligands acting through mu opioid receptors.

AC-260584, an orally bioavailable M1 muscarinic receptor allosteric agonist, improves cognitive performance in an animal model

The recent discovery of allosteric potentiators and agonists of the muscarinic M(1) receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC(50) 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M(1) receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M(1) receptor over the M(2), M(3), M(4) and M(5) muscarinic receptor subtypes. Using GTPgammaS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M(1) receptor activation since it was not observed in M(1) knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M(1) receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M(1) selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimers disease.

Pharmacological profiles of opioid ligands at kappa opioid receptors.

BackgroundThe aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a human embryonic kidney cell system stably expressing only the mouse κ-opioid receptors. Receptor activation was assessed by measuring the inhibition of cyclic adenosine mono phosphate (cAMP) production stimulated by 5 μM forskolin. Intrinsic activities and potencies of these ligands were determined relative to the endogenous ligand dynorphin and the κ agonist with the highest intrinsic activity that was identified in this study, fentanyl.ResultsAmong the ligands studied naltrexone, WIN 44,441 and dezocine, were classified as antagonists, while the remaining ligands were agonists. Intrinsic activity of agonists was assessed by determining the extent of inhibition of forskolin-stimulated cAMP production. The absolute levels of inhibition of cAMP production by each ligand was used to describe the rank order of intrinsic activity of the agonists; fentanyl = lofentanil ≥ hydromorphone = morphine = nalorphine ≥ etorphine ≥ xorphanol ≥ metazocine ≥ SKF 10047 = cyclazocine ≥ butorphanol > nalbuphine. The rank order of affinity of these ligands was; cyclazocine > naltrexone ≥ SKF 10047 ≥ xorphanol ≥ WIN 44,441 > nalorphine > butorphanol > nalbuphine ≥ lofentanil > dezocine ≥ metazocine ≥ morphine > hydromorphone > fentanyl.ConclusionThese results elucidate the relative activities of a set of opioid ligands at κ-opioid receptor and can serve as the initial step in a systematic study leading to understanding of the mode of action of these opioid ligands at this receptor.

Identification and Characterization of Novel Small-Molecule Protease-Activated Receptor 2 Agonists

We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca2+ mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH2 had similar potency, whereas SLIGRL-NH2 was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.

Neuropeptide FF Receptors Have Opposing Modulatory Effects On Nociception

The role of neuropeptide FF (NPFF) and its analogs in pain modulation is ambiguous. Although NPFF was first characterized as an antiopioid peptide, both antinociceptive and pronociceptive effects have been reported, depending on the route of administration. Currently, two NPFF receptors, termed FF1 and FF2, have been identified and cloned, but their roles in pain modulation remain elusive because of the lack of availability of selective compounds suitable for systemic administration in in vivo models. Ligand-binding studies confirm ubiquitous expression of both subtypes in brain, whereas only FF2 receptors are expressed spinally. This disparity in localization has served as the foundation of the hypothesis that FF1 receptors mediate the pronociceptive actions of NPFF. We have identified novel small molecule NPFF receptor agonists and antagonists with varying degrees of FF2/FF1 functional selectivity. Using these pharmacological tools in vivo has allowed us to define the roles of NPFF receptor subtypes as pertains to the modulation of nociception. We demonstrate that selective FF2 agonism does not modulate acute pain but instead ameliorates inflammatory and neuropathic pains. Treatment with a nonselective FF1/FF2 agonist potentiates allodynia in neuropathic rats and increases sensitivity to noxious thermal and to non-noxious mechanical stimuli in normal rats in an FF1 antagonist-reversible manner. Treatment with FF1 antagonists reversed established mechanical allodynia, indicating the possibility of increased NPFF tone through FF1 receptors. In conclusion, we provide evidence for the opposing roles of NPFF receptors and highlight selective FF2 agonism and/or selective FF1 antagonism as potential targets warranting further investigation.

Discovery of selective nonpeptidergic neuropeptide FF2 receptor agonists.

We report the discovery and initial characterization of a novel class of selective NPFF2 agonists. HTS screening using R-SAT, a whole cell based functional assay, identified a class of aryliminoguanidines as NPFF1 and NPFF2 ligands. Subsequent optimization led to molecules exhibiting selective NPFF2 agonistic activity. Systemic administration showed that selective NPFF2 agonists (1 and 3) are active in various pain models in vivo, whereas administration of a nonselective NPFF1 and NPFF2 agonist (9) increases sensitivity to noxious and non-noxious stimuli.

Differential regulation of muscarinic M1 receptors by orthosteric and allosteric ligands

BackgroundActivation of muscarinic M1 receptors is mediated via interaction of orthosteric agonists with the acetylcholine binding site or via interaction of allosteric agonists with different site(s) on the receptor. The focus of the present study was to determine if M1 receptors activated by allosteric agonists undergo the same regulatory fate as M1 receptors activated by orthosteric agonists.ResultsThe orthosteric agonists carbachol, oxotremorine-M and pilocarpine were compared to the allosteric agonists AC-42, AC-260584, N-desmethylclozapine and xanomeline. All ligands activated M1 receptors and stimulated interaction of the receptors with β-arrestin-1. All ligands reduced cell surface binding and induced the loss of total receptor binding. Receptor internalization was blocked by treatment with hypertonic sucrose indicating that all ligands induced formation of clathrin coated vesicles. However, internalized receptors recycled to the cell surface following removal of orthosteric, but not allosteric agonists. Whereas all ligands induced loss of cell surface receptor binding, no intracellular vesicles could be observed after treatment with AC-260584 or xanomeline. Brief stimulation of M1 receptors with AC-260584 or xanomeline resulted in persistent activation of M1 receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles.ConclusionThese results indicate that allosteric agonists differ from orthosteric ligands and among each other in their ability to induce different regulatory pathways. Thus, signaling and regulatory pathways induced by different allosteric ligands are ligand specific.

Activation profiles of opioid ligands in HEK cells expressing δ opioid receptors

BackgroundThe aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production.ResultsNaltrexone and nalorphine were classified as antagonists at δ opioid receptor. The other ligands studied were agonists at δ opioid receptors and demonstrated IC50 values of 0.1 nM to 2 μM, maximal inhibition of 39–77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol ≥ fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine ≥ met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at δ opioid receptors.ConclusionsThe data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at δ opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at δ opioid receptors.