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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-1β 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.

CXC chemokine receptor 2 (CXCR2) is a key receptor in the chemotaxis of neutrophils to sites of inflammation. The studies reported here describe the pharmacological characterization of danirixin, a CXCR2 antagonist in the diaryl urea chemical class. Danirixin has high affinity for CXCR2, with a negative log of the 50% inhibitory concentration (pIC50) of 7.9 for binding to Chinese hamster ovary cell (CHO)-expressed human CXCR2, and 78-fold selectivity over binding to CHO-expressed CXCR1. Danirixin is a competitive antagonist against CXCL8 in Ca2+-mobilization assays, with a KB (the concentration of antagonist that binds 50% of the receptor population) of 6.5 nM and antagonist potency (pA2) of 8.44, and is fully reversible in washout experiments over 180 minutes. In rat and human whole-blood studies assessing neutrophil activation by surface CD11b expression following CXCL2 (rat) or CXCL1 (human) challenge, danirixin blocks the CD11b upregulation with pIC50s of 6.05 and 6.3, respectively. Danirixin dosed orally also blocked the influx of neutrophils into the lung in vivo in rats following aerosol lipopolysaccharide or ozone challenge, with median effective doses (ED50s) of 1.4 and 16 mg/kg respectively. Thus, danirixin would be expected to block chemotaxis in disease states in which neutrophils are increased in response to inflammation, such as pulmonary diseases. In comparison with navarixin, a CXCR2 antagonist from a different chemical class, the binding characterization of danirixin is distinct. These observations may offer insight into the previously observed clinical differences in induction of neutropenia between these compounds.

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.

A series of novel biphenyl piperazines was discovered as highly potent muscarinic acetylcholine receptor antagonists via high throughput screening and subsequent optimization. Compound 5c with respective 500- and 20-fold subtype selectivity for M3 over M2 and M1 exhibited excellent inhibitory activity and long duration of action in a bronchoconstriction in vivo model in mice via intranasal administration. The novel inhaled mAChR antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease.

High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory activity and long duration of action in bronchoconstriction in vivo models in two species via intranasal or intratracheal administration. The novel inhaled muscarinic receptor antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease and other bronchoconstriction disorders.

1. Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human inflammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2. These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor alpha (TNFalpha) release from peripheral blood monocytes. 3. All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFalpha release concentration-dependently, with a wider ( approximately 1000 fold) range of IC50 values. 4. In both sets of experiments, mean IC50 values were significantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC50 values or by Spearman's rank-order correlation. The correlation between inhibition of inflammatory cell function and inhibition of recombinant PDE4D catalytic activity was not significant in either analysis. 5. These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two inflammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and inflammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.

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.

Recombinant baculoviruses were constructed to express cDNAs encoding two distinct subtypes of human cAMP-specific phosphodiesterase (hPDE4A and hPDE4B). Infection of Spodoptera frugiperda insect cells with the appropriate recombinant baculoviruses resulted in high level production of biologically-active protein as measured by enzymatic activity and immunoblotting using subtype-specific anti-hPDE4 antisera. Both recombinant proteins showed catalytic activity with a low Km (approximately 3 microM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent Kis of 0.38 and 0.25 microM, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (Kd approximately 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.

Novel tropane derivatives were characterized as muscarinic acetylcholine receptor antagonists (mAChRs). Through optimization of the structure-activity relationship around the tropane scaffold, the quaternary ammonium salt 34 was identified as a very potent M(3) mAChR antagonist. The compound was functionally active and displayed greater than 24 h duration of action in a mouse model of bronchoconstriction.

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.

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.

A series of 3-arylamino-2H-1,2,4-benzothiadiazin-5-ol 1,1-dioxides were prepared and shown to be novel and selective antagonists of the CXCR2 receptor. Synthesis, structure and activity relationships, selectivity, and some developability properties are described.

N,N'-diarylsquaramides were prepared and evaluated as antagonists of CXCR2. The compounds were found to be potent and selective antagonists of CXCR2. Significant differences in SAR was observed relative to the previously described N,N'-diarylurea series. As was the case in the N,N'-diarylurea series, placing sulfonamide substituent adjacent to the acidic phenol significantly reduced the clearance in rat pharmacokinetic studies.

A series of N-(2-hydroxy-3-sulfonamidobenzene)-N'-arylcyanoguanidines was prepared. In general, these compounds proved to be potent antagonists of CXCR2 while the selectivity versus CXCR1 ranged from non-selective to >200-fold.

The fluorescent styryl dyes FM1-43 and FM2-10 have been used to visualize the endocytic and exocytic processes involved in neurotransmission in a variety of central and peripheral nerve preparations. Their utility is limited to some extent by a poorly understood vesicular-independent labelling of cells and tissues. We show here that one likely cause of this troublesome background labelling is that FM1-43 and FM2-10 are selective and competitive antagonists at both cloned and endogenously expressed muscarinic acetylcholine receptors. In radioligand binding studies, FM1-43 and FM2-10 bound with moderate affinity (23-220 nM) to membranes of Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptors (M1-M5). In functional studies in vitro, FM1-43 and FM2-10 inhibited electrical field stimulation (EFS) and acetylcholine-induced cholinergic contractions of guinea-pig tracheal strips (IC50: FM1-43, 0.4 +/- 0.1; FM2-10, 1.6 +/- 0.1 microM; concentration of antagonist producing a 2-fold leftward shift in the acetylcholine concentration-response curve (Kb): FM1-43, 0.3 +/- 0.1; FM2-10, 15.8 +/- 10.1 microM). Neither compound inhibited EFS-evoked, non-adrenergic non-cholinergic nerve-mediated relaxations or contractions of the airways, or contractions mediated by histamine H1 receptor or tachykinin NK2 receptor activation. Incubating freshly excised tracheal whole-mount preparations with 5 microM FM1-43 resulted in intense fluorescence labelling of the smooth muscle that was reduced by up to 90% in the presence of selective M2 and M3 receptor antagonists. The potency of the FM dyes as muscarinic receptor antagonists is within the concentration range used to study vesicular cycling at nerve terminals. Given that muscarinic receptors play a key role in the regulation of neurotransmitter release from a variety of neurones, the anticholinergic properties of FM dyes may have important implications when studying vesicular events in the nervous system. In addition, these dyes may provide a novel tool for visualizing muscarinic receptor occupancy in living tissue or cell preparations.

The molecular mechanism of desensitization of antidiuretic hormone receptors is not well understood. Preincubation of LLC-PK1 cells with lysine vasopressin (LVP) (10(-6) M, 5 h) decreased subsequent LVP-stimulated cAMP accumulation in cells by 83% and reduced the Vmax of LVP-stimulated adenylate cyclase by 81%. Such preincubation also reduced by 90% the binding of [3H]LVP to both intact cells and isolated plasma membranes, suggesting a loss of vasopressin receptors. Both the reduction in cAMP response and the apparent loss of receptors showed similar dose and time dependence. Monensin (33 microM) did not alter [3H]LVP binding or stimulation of cAMP by LVP, nor did it prevent desensitization. However, membranes prepared from cells preincubated with LVP in the presence of monensin did not show a decrease in [3H]LVP binding. Forskolin preincubation, at 0.1, 1, 10 and 100 microM, did not alter [3H]LVP binding or accumulation of cellular cAMP by LVP, nor did it induce desensitization to LVP. Cells desensitized with varying LVP concentrations in the presence of 10 microM forskolin displayed the same loss of [3H]LVP binding and LVP responsiveness as observed in the absence of forskolin. LVP-desensitized cells, upon removal from LVP-containing medium, recovered cAMP responsiveness to LVP and specific binding of [3H]LVP at the same rate, achieving control levels after 50 h. Recovery was prevented by cycloheximide (25 micrograms/ml). These findings are consistent with a desensitization process involving LVP-mediated receptor internalization, and a recovery process requiring protein synthesis.