Mark A. Luttmann

Endothelin receptor subtypes in human and guinea‐pig pulmonary tissues

1 In this study the endothelin (ET) receptor subtypes mediating contractions produced by ET‐1 in human and guinea‐pig pulmonary tissues were investigated. In addition the receptor responsible for ET‐1‐induced prostanoid release in human bronchus was determined. 2 In human bronchus and human pulmonary artery ET‐1 (0.1 nm–0.3 μm) was a potent and effective contractile agent (pD2 = 7.58 ± 0.15, n = 6, and 8.48 ±0.11, n = 7, respectively). BQ‐123 (1–10 μm), a potent and selective ETA receptor antagonist, potently antagonized ET‐1‐induced contraction in human pulmonary artery (pKB = 6.8 with 1 μm BQ‐123, n = 7) but had no effect in human bronchus (n = 6). 3 Sarafotoxin S6c (0.1 nm–0.1 μm), the ETB‐selective agonist, did not contract human pulmonary artery (n = 5), but potently and effectively contracted human bronchus: pD2 = 8.41 ± 0.17, maximum response = 74.4 ± 3.1% of 10 μm carbachol; n = 5. BQ‐123 (1–10 μm) did not antagonize sarafotoxin S6c‐induced contraction in human bronchus (n = 5). 4 ET‐1 potently contracted guinea‐pig trachea, bronchus, pulmonary artery and aorta (pD2 = 8.15 ± 0.14, 7.72 ± 0.12, 8.52 ± 0.12, and 8.18 ± 0.12, respectively, n = 6–14). BQ‐123 (0.1–10 μm) antagonized ET‐1‐induced contractions in guinea‐pig pulmonary artery (pKB = 6.7 with 1 μm BQ‐123, n = 6), aorta (pKB = 7.1 with 1 μm BQ‐123, n = 6) and trachea (pKB = 6.2 with 1 μm BQ‐123, n = 6) but was without marked effect in bronchus (n = 4). In contrast, sarafotoxin S6c (0.1 nm–0.1 μm) did not contract guinea‐pig aorta (n = 4) or guinea‐pig pulmonary artery (n = 6) but potently and effectively contracted guinea‐pig bronchus: pD2 = 8.55 ± 0.1; maximum contraction = 63.6 ± 3.1% of 10 μm carbachol, n = 4. Sarafotoxin S6c (0.1 nm–0.1 μm) was a much less effective agonist in guinea‐pig trachea: maximum contraction = 13.9 ± 2.5% of 10 μm carbachol, n = 4; P < 0.0001, compared to bronchus. Contractions produced by sarafotoxin S6c in guinea‐pig bronchus or trachea were unaffected by BQ‐123 (10 μm, n = 4). 5 Significant differences were observed in the efficacy, relative to carbachol, but not the potency of sarafotoxin S6c in guinea‐pig airways, with a much greater maximum contractile response in bronchus (69.6 ± 2.4% of 10 μm carbachol, n = 6) or lower region of the trachea (48.5 ± 5.9% of 10 μm carbachol, n = 6) than in the middle region of the trachea (14.4 ± 4.0% of 10 μm carbachol, n = 6) or the upper region of the trachea (19.3 ± 2.7% of 10 μm carbachol, n = 6). There were minimal regional differences in either ET‐1‐induced contraction or the potency of BQ‐123 (3 μm) for inhibition of responses to ET‐1 in guinea‐pig airways. 6 Release of various prostanoids in human bronchus induced by ET‐1 (0.3 μm) was essentially abolished with 10 μm BQ‐123. 7 These data provide evidence that distinct ET receptors mediate ET‐1‐induced contraction in human pulmonary artery, guinea‐pig pulmonary artery and guinea‐pig aorta (ETA subtype) compared with human bronchus and guinea‐pig bronchus (non‐ETA, perhaps ETB subtype). Contractions to ET‐1 in guinea‐pig trachea appear to involve both ETA and non‐ETA (ETB?) receptor subtypes. Furthermore, regional differences appear to exist in the relative distribution of ET receptor subtypes in guinea‐pig airways. In human bronchus ET‐1‐induced prostanoid release, unlike the contractile response, appears to be mediated via ETA receptor activation.

Pharmacological Characterization of GSK573719 (Umeclidinium): A Novel, Long-Acting, Inhaled Antagonist of the Muscarinic Cholinergic Receptors for Treatment of Pulmonary Diseases

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 [3H]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 Ca2+ 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.

Pharmacological characterization of GSK1004723, a novel, long‐acting antagonist at histamine H1 and H3 receptors

BACKGROUND AND PURPOSE Preclinical pharmacological characterization of GSK1004723, a novel, dual histamine H1 and H3 receptor antagonist.

Discovery of novel 1-azoniabicyclo[2.2.2]octane muscarinic acetylcholine receptor antagonists.

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.

Transient Receptor Potential Vanilloid 4 Activation Constricts the Human Bronchus via the Release of Cysteinyl Leukotrienes

Prior studies have demonstrated that the ion channel transient receptor potential vanilloid 4 (TRPV4) is functionally expressed in airway smooth muscle cells and that TRPV4 single nucleotide polymorphisms are associated with airflow obstruction in patients with chronic obstructive pulmonary disease. We sought to use isometric tension measurements in ex vivo airways to determine whether short-term pharmacological activation of TRPV4 with the potent agonist GSK1016790 [N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide] would constrict human bronchial tissue. As predicted, transient receptor potential vanilloid 4 activation in the human airway produces contractions that are blocked by the nonselective transient receptor potential channel blocker ruthenium red. Moreover, the novel TRPV4-selective blocker GSK2334775 [(R)-6-(methylsulfonyl)-3-((4-(pyrrolidin-1-yl)piperindin-1-yl)methyl)-N-(2,2,2,-trifluoro-1-phenylethyl)-2-(3-(trifluoromethyl)phenyl)quinoline-4-carboxamide] inhibited these contractions over a concentration range consistent with its in vitro potency against recombinant and native TRPV4-containing channels. Surprisingly, TRPV4-dependent contractions were also blocked by a 5-lipoxygenase inhibitor and two structurally distinct cysteinyl leukotriene 1 receptor antagonists. In aggregate, our results fail to support the hypothesis that TRPV4 in airway smooth muscle cells regulates airway contractility short term. Rather, we provide pharmacological evidence that TRPV4 activation causes human airway constriction that is entirely dependent upon the production of cysteinyl leukotrienes. Together, these data identify a novel mechanism by which TRPV4 activation may contribute to pathologic remodeling and inflammation, in addition to airflow obstruction, in the diseased human respiratory tract.

Human urotensin-II is a potent spasmogen of primate airway smooth muscle

The contractile profile of human urotensin‐II (hU‐II) was examined in primate airway and pulmonary vascular tissues. hU‐II contracted tissues from different airway regions with similar potencies (pD2s from 8.6 to 9.2). However, there were regional differences in the efficacy of hU‐II, with a progressive increase in the maximum contraction from trachea to smaller airway regions (from 9 to 41% of the contraction to 10 μM carbachol). hU‐II potently contracted pulmonary artery tissues from different regions with similar potencies and efficacies: pD2s=8.7 to 9.3 and maximal contractions=79 to 86% of 60 mM KCl. hU‐II potently contracted pulmonary vein preparations taken proximal to the atria, but had no effect in tissues from distal to the atria. This is the first report describing the contractile activity of hU‐II in airways and suggests that the potential pathophysiological role of this peptide in lung diseases warrants investigation.

Comparison of endothelin B (ETB) receptors in rabbit isolated pulmonary artery and bronchus

1 To explore potential differences between endothelin (ET) receptors in airway versus vascular smooth muscle from the same species, the ETB receptors mediating contractions produced by ET‐1, ET‐3 and the selective ETB ligands, sarafotoxin S6c (S6c) and BQ‐3020, in rabbit bronchus and pulmonary artery were investigated by use of peptide and non‐peptide ET receptor antagonists. 2 In rabbit pulmonary artery SB 209670 (10 μm), a mixed ETA/ETB receptor antagonist, was a more potent antagonist of contractions produced by S6c (pKB = 7.7; n = 9; P<0.05), than those elicited by ET‐1 (pKB = 6.7; n = 6) or ET‐3 (pKB = 6.7; n = 5). BQ‐788 (10 μm), an ETB receptor antagonist, inhibited responses produced by ET‐3 (pKB = 5.1; n = 8), BQ‐3020 (pKB = 5.2; n = 4) or S6c (pKB = 6.2; n = 9; P<0.05 compared to potency versus ET‐3‐ or BQ‐3020‐induced contractions), but was without inhibitory effect on ET‐1‐induced contractions (n = 5). RES‐701 (10 μm), another selective ETB receptor antagonist, was without effect on contractions produced by S6c (n = 4) or ET‐1 (n = 4), and potentiated ET‐3‐ (n = 5) or BQ‐3020‐induced responses (n = 4). 3 The combination of BQ‐788 (10 μm) and BQ‐123 (10 μm), an ETA‐selective receptor antagonist, antagonized contractions produced by lower concentrations of ET‐1 (1 and 3 nM) in rabbit pulmonary artery, but was without effect on responses elicited by higher concentrations of ET‐1 (n = 5). The combination of RES‐701 (10 μm) and BQ‐123 (10 μm) potentiated responses elicited by ET‐1, producing a 3.7 fold shift to the left in the agonist concentration‐response curve (n = 5). 4 In rabbit bronchus SB 209670 (3 μm) had similar potency for antagonism of contractions produced by ET‐1 (pKB = 6.3; n = 6), ET‐3 (pKB = 6.5; n = 6) or S6c (pKB = 6.1; n = 8). BQ‐788 (3 μm) was without effect on responses elicited by ET‐1, ET‐3 or S6c (n = 6) but antagonized BQ‐3020‐induced contractions (pKB = 6.4; n = 4). RES‐701 (3 μm) was without effect on contractions produced by S6c (n = 6) or BQ‐3020 (n = 4), and potentiated rather than antagonized ET‐1‐ or ET‐3‐induced responses (n = 6), reflected by a significant (about 6 fold) shift to the left in ET‐1 or ET‐3 concentration‐response curves. The combination of BQ‐788 (3 μm) and BQ‐123 (3 μm) was without effect on contractions produced by ET‐1 in rabbit bronchus (n = 6). The combination of RES‐701 (3 μm) and BQ‐123 (3 μm) potentiated responses elicited by ET‐1, producing a 5.2 fold shift to the left in the agonist concentration‐response curve (n = 5). 5 BQ‐123 (3 or 10 μm), an ETA‐selective receptor antagonist, was without effect on ET‐1, ET‐3 or S6c concentration‐response curves (n = 3–6) in rabbit pulmonary artery or rabbit bronchus. 6 These data indicate that contractions induced by ET‐1, ET‐3, S6c and BQ‐3020 in rabbit pulmonary artery or rabbit bronchus appear to be mediated predominantly via stimulation of ETB receptors. However, the qualitative and quantitative differences in the relative profiles of the various structurally diverse peptide and non‐peptide antagonists examined suggests that responses produced by the ET ligands may not be mediated by a homogeneous ETB receptor population. In addition, the results suggest that differences exist in the ETB receptors mediating contraction in pulmonary vascular versus airway tissues in the same species. These receptors are not very sensitive to the standard ETB receptor antagonists, BQ‐788 and RES‐701. Furthermore, the results also provide further evidence that the potencies of ET receptor antagonists depend upon the ET agonist.

Endothelin receptors and calcium translocation pathways in human airways.

Tension and phosphatidyl inositol (PI) turnover experiments were conducted to investigate the receptors and signal transduction pathways responsible for contractions elicited by endothelin (ET) ligands in human bronchus. Nicardipine (1 µM), the L-type calcium channel inhibitor, or incubation in Ca2+-free medium, produced marked inhibition of contractions to the ETB receptor-selective agonist, sarafotoxin S6c, and especially those induced by KCl. In contrast, Ca2+-free medium was without appreciable effect against contraction produced by endothelin-1 (ET-1), the non-selective ETA and ETB receptor agonist. In Ca2+-free medium, ryanodine (10 µM), which inhibits intracellular calcium mobilization, reduced sarafotoxin S6c- and ET-1-induced responses, but was without effect on responses to KCl. Similarly, nickel chloride (Ni2+; 1 mM) caused marked inhibition of contractions induced by sarafotoxin S6c or ET-1, but had no significant effect on KCl concentration-response curves. The mixed ETA/ETB receptor antagonist SB 209670 (3 µM) inhibited responses to sarafotoxin S6c and ET-1 such that concentration-response curves were shifted rightward, at the 30% maximum response level, by 10.0- and 3.8-fold, respectively, whereas BQ-123 (3 µM), the ETA receptor antagonist, was without effect on responses induced by either agonist. ET-1 (1 nM–0.3 µM) caused a concentration-dependent stimulation of PI turnover, whereas sarafotoxin S6c (0.3 nM–0.1 µM) induced only small and variable increases, except at the highest concentration. The increase in PI turnover evoked by ET-1 was inhibited by SB 209670 (3 µM), and also by BQ-123 (3 µM). This is consistent with linkage of ETA receptors to activation of inositol phosphate generation in human bronchial smooth muscle cells. Collectively, the data suggest that differences exist in the relative contributions of intracellular and extracellular Ca2+ mobilization mechanisms elicited by ETA and ETB receptor activation. Thus, sarafotoxin S6c-induced, ETB receptor-mediated contraction in human bronchial smooth muscle appears to be dependent, in part, upon extracellular Ca2+, although a significant component of the response was also mediated by intracellular Ca2+ release, including from ryanodine-sensitive stores. ETA receptor-mediated contraction of human airway smooth muscle was activated largely via the release of intracellular Ca2+.

Targeted disruption of the endothelin-B-receptor gene attenuates inflammatory nociception and cutaneous inflammation in mice.

Endothelin-1 (ET-1) has been suggested to have a potential function as an inflammatory mediator. The study reported here assessed the putative inflammatory/nociceptive actions of the ET isopeptides using endothelin-B (ET(B))-receptor knockout (KO) mice and ET(A)- (SB 234551) and ET(B)- (A192621) selective antagonists. Phenylbenzoquinone (PBQ)-induced algesia was evident in the wild-type (WT) ET(B) (+/+) mice, attenuated by 80% in the heterozygous ET(B) (+/-) mice, and absent in the ET(B) (-/-) homozygotes. This was reproduced pharmacologically in WT ET(B) (+/+) mice where the algesic effect of PBQ was inhibited 74% by A192621, but unaffected by SB 234551 (both at 25 mg/kg p.o.). Similar observations were made in a model of cutaneous inflammation: ET(B) (+/+) mice had a marked inflammatory response to topical arachidonic acid, ET(B) (+/-) and ET(B) (-/-) mice had significantly reduced edema responses (37% and 65% inhibition). Neutrophil infiltration was reduced in the ET(B) (+/-) and ET(B) (-/-) mice (51% and 65% reduction, respectively). Topical administration of A192621 (500 microg/ear) inhibited arachidonic acid-induced swelling (39%) in WT ET(B) (+/+) mice. Collectively, these results support a role for the ET(B)-receptor in the mediation of inflammatory pain and cutaneous inflammatory responses. As such, the development of ET(B)-receptor-selective antagonists may be of therapeutic utility in the treatment of inflammatory disorders.

A Highly Convergent Synthesis of 2‐Phenyl Quinoline as Dual Antagonists for NK2 and NK3 Receptors

Abstract A novel and highly convergent synthesis leading to 2‐phenyl‐quinolines has been developed. As demonstrated in the preparation of 6‐fluoro‐3‐(3‐oxo‐piperazin‐1‐ylmethyl)‐2‐phenyl‐quinoline‐4‐carboxylic acid [(S)‐1‐cyclohexyl‐ethyl]‐amide (8), the method provides fascile access to this class of analogues via the common intermediate 7.