Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor.

Two subtypes of cannabinoid receptors are currently recognized, CB(1), found in brain and neuronal cells, and CB(2), found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxyl ic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB(1) receptor. CP-272871 competed for binding of the cannabinoid agonist (3)H-labeled (-)-3-[2-hydroxy-4-(1, 1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([(3)H]CP-55940) at the CB(1) receptor in rat brain membranes with a K(d) value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist (3)H-labeled (R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1, 4-benzoxazin-6-yl](1-naphthyl)methanone ([(3)H]WIN-55212-2), as well as the aryl pyrazole antagonist [(3)H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB(1) receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated (35)S-labeled guanosine-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding to brain membrane G-proteins, and decreased basal [(35)S]GTPgammaS binding to G-proteins. K(+) enhanced CP-272871 and SR141716A inverse agonist activity compared with Na(+) or NMDG(+) in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB(1) receptors.

[1]  S. Galiègue,et al.  Expression of central and peripheral cannabinoid receptors in human immune tissues and leukocyte subpopulations. , 1995, European journal of biochemistry.

[2]  M. Herkenham,et al.  Cannabinoid receptor localization in brain. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[3]  A. Yamashita,et al.  2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. , 1995, Biochemical and biophysical research communications.

[4]  D. Gibson,et al.  Isolation and structure of a brain constituent that binds to the cannabinoid receptor. , 1992, Science.

[5]  H. Yamamura,et al.  SR141716A is an inverse agonist at the human cannabinoid CB1 receptor. , 1997, European journal of pharmacology.

[6]  V. Di Marzo,et al.  Biosynthesis of anandamide and related acylethanolamides in mouse J774 macrophages and N18 neuroblastoma cells. , 1996, The Biochemical journal.

[7]  D. Haycock,et al.  Aminoalkylindole binding in rat cerebellum: selective displacement by natural and synthetic cannabinoids. , 1993, The Journal of pharmacology and experimental therapeutics.

[8]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[9]  G Vassart,et al.  Molecular cloning of a human cannabinoid receptor which is also expressed in testis. , 1991, The Biochemical journal.

[10]  G. Milligan,et al.  Inverse agonism: pharmacological curiosity or potential therapeutic strategy? , 1995, Trends in pharmacological sciences.

[11]  D. Deutsch,et al.  Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. , 1993, Biochemical pharmacology.

[12]  A. Howlett,et al.  Effects of nitric oxide on adenylyl cyclase stimulation in N18TG2 neuroblastoma cells. , 1998, The Journal of pharmacology and experimental therapeutics.

[13]  A. Howlett,et al.  The CB(1) cannabinoid receptor juxtamembrane C-terminal peptide confers activation to specific G proteins in brain. , 2000, Molecular pharmacology.

[14]  A. Howlett,et al.  Determination and characterization of a cannabinoid receptor in rat brain. , 1988, Molecular pharmacology.

[15]  A. Howlett,et al.  Pharmacology of cannabinoid receptors. , 1995, Annual review of pharmacology and toxicology.

[16]  T. Bisogno,et al.  Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma ‘anandamide amidohydrolase’ , 1995, FEBS letters.

[17]  A. Howlett,et al.  Involvement of Gi in the inhibition of adenylate cyclase by cannabimimetic drugs. , 1986, Molecular pharmacology.

[18]  S. Deadwyler,et al.  Role of cyclic AMP in the actions of cannabinoid receptors. , 1996, Biochemical pharmacology.

[19]  A. Howlett.  Cannabinoid inhibition of adenylate cyclase. Biochemistry of the response in neuroblastoma cell membranes. , 1985, Molecular pharmacology.

[20]  P. Leff.  Inverse agonism: theory and practice. , 1995, Trends in pharmacological sciences.

[21]  L. Petrocellis,et al.  Potential biosynthetic connections between the two cannabimimetic eicosanoids, anandamide and 2-arachidonoyl-glycerol, in mouse neuroblastoma cells. , 1996, Biochemical and biophysical research communications.

[22]  D Rodbard,et al.  Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand. , 1992, Molecular pharmacology.

[23]  P. Soubrié,et al.  SR141716A, a potent and selective antagonist of the brain cannabinoid receptor , 1994, FEBS letters.

[24]  P. Casellas,et al.  SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor. , 1998, The Journal of pharmacology and experimental therapeutics.

[25]  P. Casellas,et al.  A Selective Inverse Agonist for Central Cannabinoid Receptor Inhibits Mitogen-activated Protein Kinase Activation Stimulated by Insulin or Insulin-like Growth Factor 1 , 1997, The Journal of Biological Chemistry.

[26]  G. Baxter,et al.  Endogenous ligands and inverse agonism. , 1995, Trends in pharmacological sciences.

[27]  R. Deth,et al.  Precoupling of Gi/G(o)-linked receptors and its allosteric regulation by monovalent cations. , 1993, Life sciences.

[28]  W. Schütz,et al.  Reverse intrinsic activity of antagonists on G protein-coupled receptors. , 1992, Trends in pharmacological sciences.

[29]  R. Pertwee,et al.  Pharmacology of cannabinoid receptor ligands. , 1999, Current medicinal chemistry.