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1.
J Pharmacol Exp Ther ; 345(2): 260-70, 2013 May.
Article in English | MEDLINE | ID: mdl-23435542

ABSTRACT

Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 µg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 µg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.


Subject(s)
Lung Diseases/drug therapy , Muscarinic Antagonists/therapeutic use , Quinuclidines/therapeutic use , Administration, Inhalation , Animals , CHO Cells , Calcium/metabolism , Carbachol/pharmacology , Cholinergic Antagonists/pharmacology , Cricetinae , Cricetulus , Guinea Pigs , Kinetics , Lung/drug effects , Mice , Mice, Inbred BALB C , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/administration & dosage , Plethysmography , Quinuclidines/administration & dosage , Receptor, Muscarinic M3/drug effects , Receptors, Muscarinic , Scopolamine Derivatives/pharmacology , Tiotropium Bromide
2.
Bioorg Med Chem Lett ; 22(23): 7087-91, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-23099092

ABSTRACT

Tyrosine ureas had been identified as potent muscarinic receptor antagonists with promising in vivo activity. Controlling the stereochemistry of the chiral quaternary ammonium center had proved to be a serious issue for this series, however. Herein we describe the preparation and SAR of tyrosine urea antagonists containing achiral quaternary ammonium centers. The most successful such moiety was the 2-methylimidazo[2,1-b][1,3]thiazol-7-ium group which yielded highly potent antagonists with long duration of action in an inhaled animal model of bronchoconstriction.


Subject(s)
Muscarinic Antagonists/chemistry , Quaternary Ammonium Compounds/chemistry , Receptors, Muscarinic/chemistry , Tyrosine/chemistry , Urea/analogs & derivatives , Animals , Bronchi/drug effects , Mice , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/pharmacology , Receptors, Muscarinic/metabolism , Structure-Activity Relationship , Urea/chemical synthesis , Urea/pharmacology
3.
Bioorg Med Chem Lett ; 22(9): 3366-9, 2012 May 01.
Article in English | MEDLINE | ID: mdl-22460029

ABSTRACT

A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropium. In addition, it was shown that this characteristic appeared to translate into enhanced which duration of action in a mouse model of bronchonstriction.


Subject(s)
Muscarinic Antagonists/chemical synthesis , Tropanes/chemical synthesis , Animals , Bronchial Diseases/drug therapy , Drug Design , Mice , Muscarinic Antagonists/pharmacology , Receptors, Muscarinic/drug effects , Structure-Activity Relationship , Tropanes/pharmacology
4.
Bioorg Med Chem Lett ; 20(12): 3540-4, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20483611

ABSTRACT

Biaryl amides were discovered as novel and subtype selective M(1) muscarinic acetylcholine receptor agonists. The identification, synthesis, and initial structure-activity relationships that led to compounds 3j and 4c, possessing good M(1) agonist potency and intrinsic activity, and subtype selectivity for M(1) over M(2-5), are described.


Subject(s)
Amides/chemical synthesis , Hydrocarbons, Aromatic/chemical synthesis , Receptor, Muscarinic M1/agonists , Amides/pharmacology , Drug Discovery , Hydrocarbons, Aromatic/pharmacology , Molecular Structure , Structure-Activity Relationship
5.
Protein Expr Purif ; 65(2): 251-60, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19297698

ABSTRACT

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.


Subject(s)
Chemokines/biosynthesis , Chemokines/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Chemokines/isolation & purification , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Oxidation-Reduction , Polymerase Chain Reaction , Protein Engineering , Protein Folding , Recombinant Fusion Proteins/isolation & purification
6.
Bioorg Med Chem Lett ; 19(16): 4560-2, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19616944

ABSTRACT

Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide (4a) as a potent and pan-active muscarinic antagonist as well as a functionally active compound in a murine model of bronchoconstriction. The compound has also displayed pharmacokinetic characteristics suitable for inhaled delivery.


Subject(s)
Biphenyl Compounds/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Muscarinic Antagonists/chemistry , Pulmonary Disease, Chronic Obstructive/drug therapy , Receptors, Muscarinic/chemistry , Administration, Inhalation , Animals , Biphenyl Compounds/chemical synthesis , Biphenyl Compounds/pharmacokinetics , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/pharmacokinetics , Drug Discovery , Humans , Mice , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/pharmacokinetics , Rats , Receptors, Muscarinic/metabolism , Structure-Activity Relationship
7.
Bioorg Med Chem Lett ; 19(1): 114-8, 2009 Jan 01.
Article in English | MEDLINE | ID: mdl-19014886

ABSTRACT

A series of N-arylpiperazine camphor sulfonamides was discovered as novel CXCR3 antagonists. The synthesis, structure-activity relationships, and optimization of the initial hit that resulted in the identification of potent and selective CXCR3 antagonists are described.


Subject(s)
Camphor/analogs & derivatives , Receptors, CXCR3/antagonists & inhibitors , Sulfonamides/chemical synthesis , Camphor/chemical synthesis , Camphor/pharmacology , Humans , Piperazines , Structure-Activity Relationship , Sulfonamides/pharmacology
8.
Bioorg Med Chem Lett ; 19(6): 1686-90, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19243945

ABSTRACT

Exploration of multiple regions of a bi-aryl amine template led to the identification of highly potent M(3) muscarinic acetylcholine receptor antagonists such as 14 (pA(2)=11.0) possessing good sub-type selectivity for M(3) over M(2). The structure-activity relationships (SAR) and optimization of the bi-aryl amine series are described.


Subject(s)
Amines/chemical synthesis , Chemistry, Pharmaceutical/methods , Receptor, Muscarinic M3/antagonists & inhibitors , Amides/chemistry , Amines/pharmacology , Asthma/drug therapy , Drug Design , Electrons , Humans , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Molecular Structure , Pulmonary Disease, Chronic Obstructive/drug therapy , Receptor, Muscarinic M3/chemistry , Structure-Activity Relationship
9.
Bioorg Med Chem Lett ; 18(14): 3950-4, 2008 Jul 15.
Article in English | MEDLINE | ID: mdl-18573659

ABSTRACT

SAR exploration of the central diamine, benzyl, and terminal aminoalkoxy regions of the N-cyclic azaalkyl benzamide series led to the identification of very potent human urotensin-II receptor antagonists such as 1a with a K(i) of 4 nM. The synthesis and structure-activity relationships (SAR) of N-cyclic azaalkyl benzamides are described.


Subject(s)
Benzamides/chemistry , Receptors, G-Protein-Coupled/antagonists & inhibitors , Binding Sites , Chemistry, Pharmaceutical/methods , Diamines/chemistry , Drug Design , Humans , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Structure-Activity Relationship
10.
Bioorg Med Chem Lett ; 18(20): 5481-6, 2008 Oct 15.
Article in English | MEDLINE | ID: mdl-18818072

ABSTRACT

SAR exploration of multiple regions of a tyrosine urea template led to the identification of very potent muscarinic acetylcholine receptor antagonists such as 10b with good subtype selectivity for M(3) over M(1). The structure-activity relationships (SAR) and optimization of the tyrosine urea series are described.


Subject(s)
Chemistry, Pharmaceutical/methods , Muscarinic Antagonists/chemical synthesis , Receptors, Muscarinic/chemistry , Tyrosine/chemistry , Urea/chemistry , Asthma/drug therapy , Drug Design , Humans , Inhibitory Concentration 50 , Models, Chemical , Molecular Structure , Muscarinic Antagonists/pharmacology , Salts/chemistry , Structure-Activity Relationship
11.
Pharmacol Res Perspect ; 6(3): e00400, 2018 06.
Article in English | MEDLINE | ID: mdl-29736245

ABSTRACT

Revefenacin (TD-4208) is a novel, long-acting, and lung-selective muscarinic cholinergic receptor (mAChR) antagonist in development as a nebulized inhalation solution for the treatment of chronic obstructive pulmonary disease (COPD) patients. This study evaluated the pharmacology of revefenacin at human recombinant mAChRs and in airway tissues from rats, guinea pigs, and humans. At human recombinant mAChRs, revefenacin displayed high affinity (pKI = 8.2-9.8) and behaved as a competitive antagonist (pKI, apparent = 9.4-10.9) at the five human recombinant mAChRs. Kinetic studies demonstrated that revefenacin dissociated significantly slower from the hM3 (t1/2 = 82 minutes) compared to the hM 2 (t1/2 = 6.9 minutes) mAChR at 37°C, thereby making it kinetically selective for the former subtype. Similarly, in functional studies, revefenacin-mediated antagonism of acetylcholine (ACh)-evoked calcium mobilization responses were reversed less rapidly at hM3 compared to the hM2 mAChR. In isolated tracheal tissues from rat and guinea pig and isolated bronchial tissues from humans, revefenacin potently antagonized mAChR-mediated contractile responses. Furthermore, the antagonistic effects of revefenacin in rat, guinea pig, and human airway tissues were slowly reversible (t1/2 of 13.3, >16, and >10 hours, respectively). These data demonstrate that revefenacin is a potent, high affinity, and selective functional mAChR antagonist with kinetic selectivity for the hM3 receptor and produces potent and long-lasting antagonism of mAChR-mediated contractile responses in rat, guinea pig, and human airway tissue. These data suggest that revefenacin has the potential to be a potent once-daily dosed inhaled bronchodilator in COPD patients.


Subject(s)
Benzamides/pharmacology , Bronchi/physiology , Carbamates/pharmacology , Muscarinic Antagonists/pharmacology , Recombinant Proteins/metabolism , Trachea/physiology , Administration, Inhalation , Animals , Bronchi/drug effects , Guinea Pigs , Humans , Nebulizers and Vaporizers , Rats , Trachea/drug effects
12.
Blood Adv ; 1(26): 2553-2562, 2017 Dec 12.
Article in English | MEDLINE | ID: mdl-29296907

ABSTRACT

Neutropenia is a common consequence of radiation and chemotherapy in cancer patients. The resulting immunocompromised patients become highly susceptible to potentially life-threatening infections. Granulocyte colony-stimulating factor (G-CSF) is known to stimulate neutrophil production and is widely used as a treatment of chemotherapy-induced neutropenia. A small-molecule G-CSF secretagogue without a requirement for refrigerated supply chain would offer a more convenient and cost-effective treatment of chemotherapy-induced neutropenia. Bacterial lipopeptides activate innate immune responses through Toll-like receptor 2 (TLR2) and induce the release of cytokines, including G-CSF, from macrophages, monocytes, and endothelial. Pam2CSK4 is a synthetic lipopeptide that effectively mimics bacterial lipoproteins known to activate TLR2 receptor signaling through the TLR2/6 heterodimer. Substrate-based drug design led to the discovery of GSK3277329, which stimulated the release of G-CSF in activated THP-1 cells, peripheral blood mononuclear cells, and human umbilical vein endothelial cells. When administered subcutaneously to cynomolgus monkeys (Macaca fascicularis), GSK3277329 caused systemic elevation of G-CSF and interleukin-6 (IL-6), but not IL-1ß or tumor necrosis factor α, indicating a selective cytokine-stimulation profile. Repeat daily injections of GSK3277329 in healthy monkeys also raised circulating neutrophils above the normal range over a 1-week treatment period. More importantly, repeated daily injections of GSK3277329 over a 2-week period restored neutrophil loss in monkeys given chemotherapy treatment (cyclophosphamide, Cytoxan). These data demonstrate preclinical in vivo proof of concept that TLR2 agonism can drive both G-CSF induction and subsequent neutrophil elevation in the cynomolgus monkey and could be a therapeutic strategy for the treatment of chemotherapy-induced neutropenia.

13.
Br J Pharmacol ; 148(2): 173-90, 2006 May.
Article in English | MEDLINE | ID: mdl-16547525

ABSTRACT

Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.


Subject(s)
Peptide Fragments/pharmacology , Peptides, Cyclic/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Urotensins/pharmacology , Animals , Arteries/drug effects , Arteries/physiology , Binding, Competitive/drug effects , Blood Pressure/drug effects , Calcium/metabolism , Cats , Cell Line , Dose-Response Relationship, Drug , Haplorhini , Humans , In Vitro Techniques , Male , Peptide Fragments/metabolism , Peptides, Cyclic/chemistry , Radioligand Assay , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Urotensins/metabolism , Vasoconstriction/drug effects , Vasoconstrictor Agents/pharmacology
14.
Br J Pharmacol ; 145(5): 620-35, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15852036

ABSTRACT

1. SB-706375 potently inhibited [(125)I]hU-II binding to both mammalian recombinant and 'native' UT receptors (K(i) 4.7+/-1.5 to 20.7+/-3.6 nM at rodent, feline and primate recombinant UT receptors and K(i) 5.4+/-0.4 nM at the endogenous UT receptor in SJRH30 cells). 2. Prior exposure to SB-706375 (1 microM, 30 min) did not alter [(125)I]hU-II binding affinity or density in recombinant cells (K(D) 3.1+/-0.4 vs 5.8+/-0.9 nM and B(max) 3.1+/-1.0 vs 2.8+/-0.8 pmol mg(-1)) consistent with a reversible mode of action. 3. The novel, nonpeptidic radioligand [(3)H]SB-657510, a close analogue of SB-706375, bound to the monkey UT receptor (K(D) 2.6+/-0.4 nM, B(max) 0.86+/-0.12 pmol mg(-1)) in a manner that was inhibited by both U-II isopeptides and SB-706375 (K(i) 4.6+/-1.4 to 17.6+/-5.4 nM) consistent with the sulphonamides and native U-II ligands sharing a common UT receptor binding domain. 4. SB-706375 was a potent, competitive hU-II antagonist across species with pK(b) 7.29-8.00 in HEK293-UT receptor cells (inhibition of [Ca(2+)](i)-mobilization) and pK(b) 7.47 in rat isolated aorta (inhibition of contraction). SB-706375 also reversed tone established in the rat aorta by prior exposure to hU-II (K(app) approximately 20 nM). 5. SB-706375 was a selective U-II antagonist with >/=100-fold selectivity for the human UT receptor compared to 86 distinct receptors, ion channels, enzymes, transporters and nuclear hormones (K(i)/IC(50)>1 microM). Accordingly, the contractile responses induced in isolated aortae by KCl, phenylephrine, angiotensin II and endothelin-1 were unaltered by SB-706375 (1 microM). 6. In summary, SB-706375 is a high-affinity, surmountable, reversible and selective nonpeptide UT receptor antagonist with cross-species activity that will assist in delineating the pathophysiological actions of U-II in mammals.


Subject(s)
Pyrrolidines/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Sulfonamides/pharmacology , Algorithms , Animals , Aorta, Thoracic/drug effects , Binding, Competitive/drug effects , Cats , Cell Line, Tumor , Cell Membrane/metabolism , Haplorhini , Humans , In Vitro Techniques , Mice , Muscle Contraction/drug effects , Muscle, Smooth, Vascular/drug effects , Radioligand Assay , Rats , Recombinant Proteins/metabolism , Rhabdomyosarcoma/metabolism , Species Specificity
15.
Biochem Pharmacol ; 69(7): 1069-79, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15763543

ABSTRACT

Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [(125)I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity (K(D) 288+/-13pM) and high density (B(max) 747+/-66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT (K(i) 1.8-5.3nM). Cat UT was coupled to intracellular [Ca(2+)] release (EC(50) 0.6+/-0.2nM) and total inositol phosphate (IP) formation (EC(50) 0.4+/-0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla (K(D) 815+/-34) and cortex and (K(D) 316+/-39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.


Subject(s)
Cloning, Molecular , Receptors, G-Protein-Coupled/genetics , Amino Acid Sequence , Animals , Calcium Signaling/physiology , Cats , Cell Line , Cell Membrane/physiology , Conserved Sequence , DNA Primers , Humans , Inositol Phosphates/metabolism , Mice , Molecular Sequence Data , Organ Specificity , Polymerase Chain Reaction , RNA, Messenger/genetics , Rats , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transfection
16.
FASEB J ; 18(1): 191-3, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14597565

ABSTRACT

The chemokine CXCL10 is produced by many inflammatory cells found in the diseased lung and has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). The present study demonstrates elevated CXCL10 protein in the lungs of COPD patients, which appears histologically in airway smooth muscle (hASM). In primary cultured hASM cells taken from normal donors, CXCL10 protein expression was induced by IFN-gamma and TNF-alpha, cytokines reported as elevated in COPD, and a synergistic response was obtained when they were combined. TNF-alpha stimulation of hASM enhanced accumulation of CXCL10 mRNA, indicating regulation at the transcriptional level, while IFN-gamma stimulation resulted in a smaller accumulation of CXCL10 mRNA. When these cytokines were applied simultaneously, an additive effect was obtained. TNF-alpha-induced CXCL10 expression in hASM was dependent on NFkappaB activation, and a salicylanilide NFkappaB inhibitor blocked the CXCL10 expression. In contrast, IFN-gamma stimulation resulted in transient NFkappaB activation, and the inhibitor had little effect on CXCL10 expression. When these cytokines were added simultaneously, NFkappaB was activated earlier and lasted longer, and the effect was blocked by the inhibitor. These data demonstrate a potential active role for hASM in pulmonary inflammatory diseases such as COPD by producing CXCL10.


Subject(s)
Chemokines, CXC/biosynthesis , Interferon-gamma/pharmacology , Lung/immunology , Muscle, Smooth/immunology , Pulmonary Disease, Chronic Obstructive/immunology , Tumor Necrosis Factor-alpha/pharmacology , Cells, Cultured , Chemokine CXCL10 , Drug Synergism , Humans , Inflammation/immunology , Muscle, Smooth/cytology , Muscle, Smooth/drug effects , Pulmonary Disease, Chronic Obstructive/pathology , Receptors, CXCR3 , Receptors, Chemokine/metabolism , Respiratory System/anatomy & histology
17.
Pharmacogenetics ; 14(9): 627-33, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15475736

ABSTRACT

BACKGROUND: Cysteinyl leukotrienes (CYSLTR) are potent biological mediators in the pathophysiology of asthma for which two receptors have been characterized, CYSLTR1 and CYSLTR2. The leukotriene modifying agents currently used to control bronchoconstriction and inflammation in asthmatic patients are CYSLTR1-specific leukotriene receptor antagonists. In this report, we investigated a possible role for therapeutic modulation of CYSLTR2 in asthma by investigating genetic association with asthma and further characterization of the pharmacology of a coding polymorphism. METHODS: The association of CYSLTR2 polymorphisms with asthma was assessed by transmission disequilibrium test in two family-based collections (359 families from Denmark and Minnesota, USA and 384 families from the Genetics of Asthma International Network). RESULTS: A significant association of the coding polymorphism, 601A>G, with asthma was observed (P = 0.003). We replicated these findings in a collection of 384 families from the Genetics of Asthma International Network (P = 0.04). The G allele is significantly under-transmitted to asthmatics, indicating a possible role for this receptor in resistance to asthma. The potency of cysteinyl leukotrienes at the wild-type CYSLTR2 and the coding polymorphism 601A>G were assessed using a calcium mobilization assay. The potency of LTC4 and LTE4 was similar for both forms of the receptor and LTB4 was inactive, however, LTD4 was approximately five-fold less potent on 601A>G compared to wild-type CYSLTR2. CONCLUSIONS: Since 601A>G alters the potency of LTD4 and this variant allele may be associated with resistance to asthma, it is possible that modulation of the CYSLTR2 may be useful in asthma pharmacotherapy.


Subject(s)
Asthma/genetics , Leukotriene D4/genetics , Membrane Proteins/genetics , Polymorphism, Genetic , Receptors, Leukotriene/genetics , Adolescent , Adult , Alleles , Cell Line , Child , Child, Preschool , Cloning, Molecular , Family Health , Genetic Variation , Genotype , Humans , Leukotrienes/metabolism , Linkage Disequilibrium , Middle Aged , Phenotype
18.
J Med Chem ; 47(6): 1319-21, 2004 Mar 11.
Article in English | MEDLINE | ID: mdl-14998320
19.
Br J Pharmacol ; 139(2): 203-7, 2003 May.
Article in English | MEDLINE | ID: mdl-12770925

ABSTRACT

The functional activity of the peptidic neuromedin B receptor antagonist BIM-23127 was investigated at recombinant and native urotensin-II receptors (UT receptors). Human urotensin-II (hU-II) promoted intracellular calcium mobilization in HEK293 cells expressing the human UT (hUT) or rat UT (rUT) receptors with pEC(50) values of 9.80+/-0.34 (n=6) and 9.06+/-0.32 (n=4), respectively. While BIM-23127 alone had no effect on calcium responses in either cell line, it was a potent and competitive antagonist at both hUT (pA(2)=7.54+/-0.14; n=3) and rUT (pA(2)=7.70+/-0.05; n=3) receptors. Furthermore, BIM-23127 reversed hU-II-induced contractile tone in the rat-isolated aorta with a pIC(50) of 6.66+/-0.04 (n=4). In conclusion, BIM- 23127 is the first hUT receptor antagonist identified to date and should not be considered as a selective neuromedin B receptor antagonist.


Subject(s)
Peptides, Cyclic/pharmacology , Receptors, Bombesin/antagonists & inhibitors , Receptors, G-Protein-Coupled/antagonists & inhibitors , Animals , Aorta, Thoracic/drug effects , Binding, Competitive , Calcium/metabolism , Cell Line , Dose-Response Relationship, Drug , Humans , In Vitro Techniques , Male , Rats , Rats, Sprague-Dawley , Urotensins/pharmacology
20.
J Med Chem ; 52(8): 2493-505, 2009 Apr 23.
Article in English | MEDLINE | ID: mdl-19317446

ABSTRACT

A novel 4-hydroxyl(diphenyl)methyl substituted quinuclidine series was discovered as a very promising class of muscarinic antagonists. The structure-activity relationships of the connectivity of the diphenyl moiety to the quinuclidine core and around the ring nitrogen side chain are described. Computational docking studies using an homology model of the M(3) receptor readily explained the observed structure-activity relationship of the various compounds. Compound 14o was identified as a very potent, slowly reversible M(3) antagonist with a very long in vivo duration of bronchoprotection.


Subject(s)
Benzhydryl Compounds/chemical synthesis , Bronchodilator Agents/chemical synthesis , Quinuclidines/chemical synthesis , Receptor, Muscarinic M3/antagonists & inhibitors , Animals , Benzhydryl Compounds/chemistry , Benzhydryl Compounds/pharmacology , Biological Availability , Bronchi/drug effects , Bronchi/physiology , Bronchoconstriction/drug effects , Bronchodilator Agents/chemistry , Bronchodilator Agents/pharmacology , CHO Cells , Calcium/metabolism , Cricetinae , Cricetulus , Humans , In Vitro Techniques , Mice , Models, Molecular , Muscle Contraction , Muscle, Smooth/drug effects , Muscle, Smooth/physiology , Quinuclidines/chemistry , Quinuclidines/pharmacology , Radioligand Assay , Rats , Structure-Activity Relationship
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