Journal of Pharmacology and Experimental Therapeutics INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Pamapimod, a Novel p38 Mitogen-Activated Protein Kinase Inhibitor: Preclinical Analysis of Efficacy and Selectivity

2008-11-12

P38 is a protein kinase that regulates the expression of inflammatory cytokines, suggesting a role in the pathogenesis of diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus. Here, we describe the preclinical pharmacology of pamapimod, a novel p38 mitogen-activated protein kinase inhibitor. Pamapimod inhibited p38 and p38β enzymatic activity, with IC₅₀ values of 0.014 ± 0.002 and 0.48 ± 0.04 µM, respectively. There was no activity against p38 or p38 isoforms. When profiled across 350 kinases, pamapimod bound only to four kinases in addition to p38. Cellular potency was assessed using phosphorylation of heat shock protein-27 and c-Jun as selective readouts for p38 and c-Jun NH₂-terminal kinase (JNK), respectively. Pamapimod inhibited p38 (IC₅₀, 0.06 µM), but inhibition of JNK was not detected. Pamapimod also inhibited lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) production by monocytes, interleukin (IL)-1β production in human whole blood, and spontaneous TNF production by synovial explants from RA patients. LPS- and TNF-stimulated production of TNF and IL-6 in rodents also was inhibited by pamapimod. In murine collagen-induced arthritis, pamapimod reduced clinical signs of inflammation and bone loss at 50 mg/kg or greater. In a rat model of hyperalgesia, pamapimod increased tolerance to pressure in a dose-dependent manner, suggesting an important role of p38 in pain associated with inflammation. Finally, an analog of pamapimod that has equivalent potency and selectivity inhibited renal disease in lupus-prone MRL/lpr mice. Our study demonstrates that pamapimod is a potent, selective inhibitor of p38 with the ability to inhibit the signs and symptoms of RA and other autoimmune diseases.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Characterization of N-(Adamantan-1-ylmethyl)-5-[(3R-aminopyrrolidin-1-yl)methyl]-2-chloro-benzamide, a P2X7 Antagonist in Animal Models of Pain and Inflammation

2008-11-12

Recent evidence suggests that the P2X₇ receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X₇ antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X₇-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC₅₀ values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X₇ receptor, with IC₅₀ values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L₅ spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X₇ receptors do play a role in animal models of pain and inflammation. Further study of P2X₇ antagonists both in preclinical and clinical studies will help elucidate the role of the P2X₇ receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Inhibition of Soluble Epoxide Hydrolase Does Not Protect against Endotoxin-Mediated Hepatic Inflammation

2008-11-12

Epoxyeicosatrienoic acids (EETs) are derived from cytochrome P450-catalyzed epoxygenation of arachidonic acid and have emerged as important mediators of numerous biological effects. The major elimination pathway for EETs is through soluble epoxide hydrolase (sEH)-catalyzed metabolism to dihydroxyeicosatrienoic acids (DHETs). Based on previous studies showing that EETs have anti-inflammatory effects, we hypothesized that chronic inhibition of sEH would attenuate a lipopolysaccharide (LPS)-induced inflammatory response in vivo. Continuous dosing of the sEH inhibitors 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA), a polyethylene glycol ester of AUDA, and 1-adamantan-1-yl-3-(5-(2-(2-ethoxyethoxy)ethoxy)-pentyl)urea resulted in robust exposure to the inhibitor and target engagement, as evidenced by significant increases in plasma EET/DHET ratios following 6 days of inhibitor treatment. However, sEH inhibitor treatment was not associated with an attenuation of LPS-induced inflammatory gene expression in the liver, and AUDA did not protect from LPS-induced neutrophil infiltration. Furthermore, Ephx2-/-mice that lack sEH expression and have significantly increased plasma EET/DHET ratios were not protected from LPS-induced inflammatory gene expression or neutrophil accumulation in the liver. LPS did have an effect on sEH expression and function, as evident from a significant down-regulation of Ephx2 mRNA and a significant shift in plasma EET/DHET ratios 4 h after LPS treatment. In conclusion, there was no evidence that increasing EET levels in vivo could modulate an LPS-induced inflammatory response in the liver. However, LPS did have significant effects on plasma eicosanoid levels and hepatic Ephx2 expression, suggesting that in vivo EET levels are modulated in response to an inflammatory signal.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Do Desipramine [10,11-Dihydro-5-[3-(methylamino) propyl]-5H-dibenz[b,f]azepine monohydrochloride] and Fluoxetine [N-Methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-propan-1-amine] Ameliorate the Extent of Colonic Damage Induced by Acetic Acid in Rats?

Guemei, A. A., El Din, N. M. N., Baraka, A. M., El Said Darwish, I. — 2008-11-12

The present study was designed to compare the anti-inflammatory and antioxidant effects of two antidepressant drugs, desipramine [10,11-dihydro-5-[3-(methylamino) propyl]-5H-dibenz-[b,f]azepine monohydrochloride] and fluoxetine [N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-propan-1-amine], administered with variable doses, on experimentally induced colitis in rats. Two doses for each drug (10 and 20 mg/kg/day i.p.) were injected in 48 adult male albino rats for 2 weeks after induction of colitis by intracolonic administration of 2 ml of 3% acetic acid. Several parameters, including macroscopic (ulcer score index) and biochemical such as myeloperoxidase (MPO), reduced glutathione (GSH), tumor necrosis factor (TNF)-, and interleukin (IL)-1β, were measured using standard assay procedures. The study demonstrates that both desipramine and fluoxetine significantly attenuated the extent and the severity of the macroscopic signs of cell damage. Both drugs significantly reduced tissue MPO activity in a dose-dependent manner. Both desipramine and fluoxetine, at either dose, significantly increased GSH in colonic tissue. Desipramine and fluoxetine, at either dose, significantly reduced TNF- and IL-β. Desipramine at the dose of 20 mg/kg produced more decrease in the level of TNF- compared with the effect of the smaller dose, but fluoxetine at 10 mg/kg diminished more in the level of IL-1β compared with the effect of the larger dose. The present data indicate that both desipramine and fluoxetine have anti-inflammatory and antioxidants effects in experimentally induced colitis in rats, opening the avenue to their possible protective role in patients with inflammatory bowel disease.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Comparison of Cigarette Smoke-Induced Acute Inflammation in Multiple Strains of Mice and the Effect of a Matrix Metalloproteinase Inhibitor on These Responses

Morris, A., Kinnear, G., Wan, W.-Y. H., Wyss, D., Bahra, P., Stevenson, C. S. — 2008-11-12

The activities of proteases in the lung, specifically matrix metalloproteinases (MMPs), have been implicated in driving the inflammation and lung destruction observed in smokers with chronic obstructive pulmonary disease. Here, our aims were to compare the acute response with cigarette smoke exposure (CSE) in four mouse strains to identify common and distinguishing features and to assess the effect of an MMP inhibitor on this response. To do this, we exposed mice (BALB/C, C57BL/6, A/J, or 129/Sv) to whole-body CSE (1 h/day) for 3 days. CSE induced dose- and time-dependent increases in neutrophils and keratinocyte chemoattractant levels in the airways of all strains; however, the proportion of the neutrophilia differed among strains. In the two most contrasting strains, BALB/C and C57BL/6, we examined MMP gene expression and found only small changes apart from MMP-12, which was highly expressed in both strains. Both strains were then treated with a broad-spectrum MMP inhibitor, PKF242-484 [(2S,3R)-N⁴-((S)-2,2-dimethyl-1-methylcarbamoyl-propyl)-N¹-hydroxy-2-hydroxymethyl-3-(4-methoxy-phenyl)-succinimide] (0.5–10 mg/kg) either orally or intranasally 1 h before and 5 h after CSE for 3 days. PKF242-484 dose-dependently reduced neutrophilia in BALB/C mice when dosed orally (p < 0.01) or intranasally (p < 0.01) but had no clear effect in C57BL/6 by either route. PKF242-484 reduced BAL macrophages when dosed intranasally (p < 0.05) but had no dose-dependent effect when dosed orally in both strains. These data suggest the inflammation induced by CSE is similar, but not identical, in different mouse strains. In addition, the ability of broad-spectrum MMP inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, whereas its ability to limit macrophage infiltration may be route dependent.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Identification and Characterization of NDT 9513727 [N,N-bis(1,3-Benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a Novel, Orally Bioavailable C5a Receptor Inverse Agonist

2008-11-12

The complement system represents an innate immune mechanism of host defense that has three effector arms, the C3a receptor, the C5a receptor (C5aR), and the membrane attack complex. Because of its inflammatory and immune-enhancing properties, the biological activity of C5a and its classical receptor have been widely studied. Because specific antagonism of the C5aR could have therapeutic benefit without affecting the protective immune response, the C5aR continues to be a promising target for pharmaceutical research. The lack of specific, potent and orally bioavailable small-molecule antagonists has limited the clinical investigation of the C5aR. We report the discovery of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a small-molecule, orally bioavailable, selective, and potent inverse agonist of the human C5aR. NDT 9513727 was discovered based on the integrated use of in vitro affinity and functional assays in conjunction with medicinal chemistry. NDT 9513727 inhibited C5a-stimulated responses, including guanosine 5'-3-O-(thio)triphosphate binding, Ca²⁺ mobilization, oxidative burst, degranulation, cell surface CD11b expression and chemotaxis in various cell types with IC₅₀s from 1.1 to 9.2 nM, respectively. In C5a competition radioligand binding experiments, NDT 9513727 exhibited an IC₅₀ of 11.6 nM. NDT 9513727 effectively inhibited C5a-induced neutropenia in gerbil and cynomolgus macaque in vivo. The findings suggest that NDT 9513727 may be a promising new entity for the treatment of human inflammatory diseases.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] A Novel CCR5-Specific Pharmacodynamic Assay in Whole Blood Using Phosphoflow Cytometry Highlights Different Ligand-Dependent Responses but Similar Properties of Antagonists in CD8+ and CD4+ T Lymphocytes

Dahl, M. E., Berson, A., Lora, J., Fuentes, M. — 2008-11-12

Chemokine CC motif receptor (CCR) 5 is a major drug target for both inflammation and virology indications. The primary function of CCR5 is to mediate the trafficking of CCR5-expressing lymphocytes to any of the CCR5 ligands, which are often increased during inflammatory responses. In addition, CCR5 is a coreceptor for HIV, mediating R5 tropic HIV infection of CCR5-expressing CD4 T cells. We report the use of a novel method to assay the pharmacodynamic (PD) properties of small-molecule and antibody inhibitors of CCR5 ligand-induced activation by measuring phosphorylation of serine residue 349 in the cytoplasmic tail of human CCR5 using phosphoflow cytometry in whole blood. This assay is highly specific and measures CCR5 phosphorylation in both CD8⁺ and CD4⁺ T cells and allows the calculation of inhibitor IC₅₀ values from both lymphocyte subsets in the presence of CCR5 antagonists. In addition, this assay is cross-reactive to nonhuman primates and allows PD analysis in whole blood from rhesus and cynomolgus macaque. Using this assay, we identified different ligand-dependent response properties between CD8⁺ and CD4⁺ T cells, although CCR5 antagonists behave with similar properties against both cell types. The use of this assay may be of particular benefit to monitor PD effects of CCR5 inhibitors during drug development, preclinical in vivo studies, and in patients currently being treated for HIV or CCR5-mediated inflammatory diseases with CCR5 inhibitors. Similar phosphoflow approaches to other GPCR targets on circulating lymphocytes may prove to be the most reliable PD assay for preclinical and potentially clinical development.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Effect of Erythromycin on Biological Activities Induced by Clostridium perfringens {alpha}-Toxin

2008-11-12

Clostridium perfringens -toxin, an important agent of gas gangrene with inflammatory myopathies, possesses lethal, hemolytic, and necrotic activities. Here, we show that -toxin-induced lethality in mice was inhibited by i.v. preadministration of erythromycin (ERM). Administration of ERM resulted in a drastic reduction in the release of tumor necrosis factor (TNF)-, interleukin (IL)-1β, and IL-6 and systemic hemolysis induced by -toxin, whereas the administration of kitasamycin did not. Furthermore, the lethality and systemic hemolysis caused by -toxin were blocked by the preinjection of anti-TNF-, but not the anti-IL-1β- or anti-IL-6-antibody. In addition, TNF--deficient mice were resistant to -toxin, indicating that TNF- plays an important role in the lethality. ERM inhibited the toxin-induced release of TNF- from neutrophils and phosphorylation of toropomyosin-related kinase receptor A (TrkA) and extracellular-regulated kinase (ERK) 1/2. Furthermore, K252a, a TrkA inhibitor, and PD98059 (2'-amino-3'-methoxyflavone), an ERK1/2 inhibitor, inhibited the toxin-induced release of TNF- from neutrophils. The observation shows that the toxin-induced release of TNF- is dependent on the activation of ERK/mitogen-activated protein kinase signal transduction via TrkA in neutrophils and that ERM specifically blocks the toxin-induced events through the activation of neutrophils.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Serotonin 5-Hydroxytryptamine2A Receptor Activation Suppresses Tumor Necrosis Factor-{alpha}-Induced Inflammation with Extraordinary Potency

Yu, B., Becnel, J., Zerfaoui, M., Rohatgi, R., Boulares, A. H., Nichols, C. D. — 2008-10-17

The G protein-coupled serotonin 5-hydroxytryptamine (5-HT)_2A receptor is primarily recognized for its role in brain neurotransmission, where it mediates a wide variety of functions, including certain aspects of cognition. However, there is significant expression of this receptor in peripheral tissues, where its importance is largely unknown. We have now discovered that activation of 5-HT_2A receptors in primary aortic smooth muscle cells provides a previously unknown and extremely potent inhibition of tumor necrosis factor (TNF)--mediated inflammation. 5-HT_2A receptor stimulation with the agonist (R)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(R)-DOI] rapidly inhibits a variety of TNF--mediated proinflammatory markers, including intracellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), and interleukin (IL)-6 gene expression, nitric-oxide synthase activity, and nuclear translocation of nuclear factor B, with IC₅₀ values of only 10 to 20 pM. It is significant that proinflammatory markers can also be inhibited by (R)-DOI hours after treatment with TNF-. With the exception of a few natural toxins, no current drugs or small molecule therapeutics demonstrate a comparable potency for any physiological effect. TNF--mediated inflammatory pathways have been strongly implicated in a number of diseases, including atherosclerosis, rheumatoid arthritis, psoriasis, type II diabetes, depression, schizophrenia, and Alzheimer's disease. Our results indicate that activation of 5-HT_2A receptors represents a novel, and extraordinarily potent, potential therapeutic avenue for the treatment of disorders involving TNF--mediated inflammation. Note that because (R)-DOI can significantly inhibit the effects of TNF- many hours after the administration of TNF-, potential therapies could be aimed not only at preventing inflammation but also treating inflammatory injury that has already occurred or is ongoing.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Diarctigenin, a Lignan Constituent from Arctium lappa, Down-Regulated Zymosan-Induced Transcription of Inflammatory Genes through Suppression of DNA Binding Ability of Nuclear Factor-{kappa}B in Macrophages

2008-10-17

Diarctigenin was previously isolated as an inhibitor of nitric oxide (NO) production in macrophages from the seeds of Arctium lappa used as an alternative medicine for the treatment of inflammatory disorders. However, little is known about the molecular basis of these effects. Here, we demonstrated that diarctigenin inhibited the production of NO, prostaglandin E₂, tumor necrosis factor-, and interleukin (IL)-1β and IL-6 with IC₅₀ values of 6 to 12 µM in zymosan- or lipopolysaccharide-(LPS) activated macrophages. Diarctigenin attenuated zymosan-induced mRNA synthesis of inducible NO synthase (iNOS) and also inhibited promoter activities of iNOS and cytokine genes in the cells. Because nuclear factor (NF)-B plays a pivotal role in inflammatory gene transcription, we next investigated the effect of diarctigenin on NF-B activation. Diarctigenin inhibited the transcriptional activity and DNA binding ability of NF-B in zymosan-activated macrophages but did not affect the degradation and phosphorylation of inhibitory B (IB) proteins. Moreover, diarctigenin suppressed expression vector NF-B p65-elicited NF-B activation and also iNOS promoter activity, indicating that the compound could directly target an NF--activating signal cascade downstream of IB degradation and inhibit NF-B-regulated iNOS expression. Diarctigenin also inhibited the in vitro DNA binding ability of NF-B but did not affect the nuclear import of NF-B p65 in the cells. Taken together, diarctigenin down-regulated zymosan- or LPS-induced inflammatory gene transcription in macrophages, which was due to direct inhibition of the DNA binding ability of NF-B. Finally, this study provides a pharmacological potential of diarctigenin in the NF-B-associated inflammatory disorders.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Pharmacological Profile of JNJ-27141491 [(S)-3-[3,4-Difluorophenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl Acid Methyl Ester], as a Noncompetitive and Orally Active Antagonist of the Human Chemokine Receptor CCR2

2008-09-19

The interaction between CC chemokine receptor 2 (CCR2) with monocyte chemoattractant proteins, such as MCP-1, regulates the activation and recruitment of inflammatory leukocytes. In this study, we characterized (S)-3-[3,4-difluoro-phenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl acid methyl ester (JNJ-27141491) as a noncompetitive and orally active functional antagonist of human (h)CCR2. JNJ-27141491 strongly suppressed hCCR2-mediated in vitro functions, such as MCP-1-induced guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding; MCP-1, -3, and -4-induced Ca²⁺ mobilization; and leukocyte chemotaxis toward MCP-1 (IC₅₀ = 7–97 nM), whereas it had little or no effect on the function of other chemokine receptors tested. The inhibition of CCR2 function was both insurmountable and reversible, consistent with a noncompetitive mode of action. JNJ-27141491 blocked the binding of ¹²⁵I-MCP-1 to human monocytes (IC₅₀ = 0.4 µM), but it failed to affect MCP-1 binding to mouse, rat, and dog cells (IC₅₀ > 10 µM). Therefore, transgenic mice, in which the mouse (m)CCR2 gene was replaced by the human counterpart, were generated for in vivo testing. In these mice, oral administration of JNJ-27141491 dose-dependently [5–40 mg/kg q.d. (once daily) or b.i.d.] inhibited monocyte and neutrophil recruitment to the alveolar space 48 h after intratracheal mMCP-1/lipopolysaccharide instillation. Furthermore, treatment with JNJ-27141491 (20 mg/kg q.d.) significantly delayed the onset and temporarily reduced neurological signs in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Taken together, these results identify JNJ-27141491 as a noncompetitive, functional antagonist of hCCR2, capable of exerting oral anti-inflammatory activity in transgenic hCCR2-expressing mice.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Fispemifene [Z-2-{2-[4-(4-Chloro-1,2-diphenylbut-1-enyl)-phenoxy]ethoxy}-ethanol], a Novel Selective Estrogen Receptor Modulator, Attenuates Glandular Inflammation in an Animal Model of Chronic Nonbacterial Prostatitis

Yatkin, E., Bernoulli, J., Lammintausta, R., Santti, R. — 2008-09-19

The anti-inflammatory and antiestrogenic action of fispemifene [Z-2-{2-[4-(4-chloro-1,2-diphenylbut-1-enyl)phenoxy]ethoxy}-ethanol], a novel selective estrogen receptor modulator (SERM), was tested on the Noble rat model of chronic nonbacterial prostatic inflammation with cellular composition and inflammation patterns similar to those described in human prostatitis. Inflammation was assessed by counting perivascular and stromal infiltrates and the number of inflamed acini. Furthermore, the aggressiveness of inflammation was assessed on the basis of the relation of lymphocytes to the acinar epithelium. The immunohistochemical expression of progesterone receptor (PR) and Fos-related antigen 2 (Fra2), prolactin concentration in serum, and the weights of the seminal vesicles and pituitary glands were used as endpoints of estrogen action. Fispemifene significantly attenuated the glandular form of inflammation induced in the dorsolateral prostatic lobes (DLP) in the hormonal milieu of the decreased androgen/estrogen ratio. The anti-inflammatory action was seen in the decreased number of acini containing intraluminal neutrophils. As signs of antiestrogenic action, fispemifene blocked estrogen-induced expression of PR and Fra2 in the acinar epithelium of the DLP, and it decreased prolactin concentration in serum and the relative weights of the seminal vesicles and pituitary glands. Because fispemifene exhibited both antiestrogenic and anti-inflammatory action in the prostate, this experimental study suggests that SERMs could be considered as a new therapeutic option in the treatment and prevention of prostatic inflammation.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] 7-Epiclusianone, a Tetraprenylated Benzophenone, Relaxes Airway Smooth Muscle through Activation of the Nitric Oxide-cGMP Pathway

2008-09-19

This study was undertaken to investigate the putative mechanism(s) underlying the antispasmodic effect of 7-epiclusianone, a naturally occurring compound isolated from the plant Garcinia brasiliensis. Guinea pig tracheal rings were mounted in tissue baths filled with Krebs' solution, and the contractile response to distinct stimuli was measured in the presence or absence of 7-epiclusianone. We also tested the effect of 7-epiclusianone on methacholine-evoked airways obstruction in BALB/c mice using barometric plethysmography. 7-Epiclusianone (10 µM) inhibited epithelium-intact tracheal ring contraction induced by allergen, histamine, 5-hydroxytryptamine, or carbachol challenge. The relaxation effect was abrogated by epithelium removal, the presence of nitric-oxide synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) (100 µM), or soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM). 7-Epiclusianone (1–100 µM) induced a dose-dependent increase in the intracellular cGMP levels of cultured tracheal rings. The relaxation effect of 7-epiclusianone was also inhibited by K⁺ channel blockers tetraethylammonium (10 µM), glibenclamide (1 µM), or apamin (1 µM), but not by 9-(tetrahydro-2'-furyl)adenine (SQ22,536) (100 µM), an adenylate cyclase inhibitor. In epithelium-intact tracheal rings, 7-epiclusianone also inhibited Ca²⁺-induced contractions in K⁺ (60 mM)-depolarized preparations, but it seemed ineffective in assays in which epithelium-denuded tracheal ring preparations were used. Oral administration of 7-epiclusinone (25–100 mg/kg) dose-dependently inhibited airway obstruction triggered by aerosolized methacholine (6–25 mg/ml), in a mechanism sensitive to l-NAME (20 mg/kg). In conclusion, the relaxation effect of 7-epiclusinone seems to be mediated by epithelium-, nitric oxide-, and cGMP-dependent mechanisms. Furthermore, oral administration of 7-epiclusianone reduces episodes of bronchial obstruction, warranting further research on this compound regarding a putative application in asthma therapy.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Thiorphan-Induced Survival and Proliferation of Rat Thymocytes by Activation of Akt/Survivin Pathway and Inhibition of Caspase-3 Activity

Amantini, C., Mosca, M., Lucciarini, R., Perfumi, M. C., Santoni, G. — 2008-09-19

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK₁R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5⁺ thymocytes, namely on the CD4⁺CD8⁺ (double positive; DP) and CD4⁺ subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK₁R mRNA expression as well as SP and NK₁R protein levels in an NK₁R-dependent manner. Thiorphan increased CD10⁺CD4⁺ and CD10⁺DP thymocyte numbers, and an NK₁R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor chain expression, and concanavalin A-induced proliferation of CD5⁺ thymocytes, and it inhibited spontaneous and NK₁R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.