Mineralocorticoid receptor antagonists attenuate pulmonary inflammation and bleomycin-evoked fibrosis in rodent models.

Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348 ± 5451, which was blocked by spironolactone (0.3-60 mg/kg, p.o.) and eplerenone (0.3-30 mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10 µM) and spironolactone (10 µM) diminished lung hydroxyproline concentrations by 55 ± 5, 122 ± 9, and 83 ± 8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0 U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10-60 mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.

Steroidal C-21 heteroaryl thioethers. Part 3: pregn-4-eno-[3,2-c]pyrazole fused A ring modified steroids as selective glucocorticoid receptor modulators (dissociated steroids).

The introduction of A ring pyrazole modification to the hydrocortisone C-21 heteroaryl thioethers generated compounds with excellent transrepression potency (IL-8 inhibition) compared to their hydrocortisone analogs. However, the transcriptional transactivation activity of these compounds were considerably higher than the corresponding hydrocortisone analogs. Among all the compounds evaluated, a quinoxaline thioether modification demonstrated the best overall in vitro separation.

Steroidal C-21 heteroaryl thioethers (Part 2): discovery of orally bioavailable selective glucocorticoid receptor modulators (dissociated steroids).

The prednisolone C-21 heteroaryl thioethers have been synthesized and evaluated in cell based transrepression and transactivation assays. Most of the compounds demonstrated weak transactivational activity in both human and rat tyrosineaminotransferase functional assay while keeping potent anti-inflammatory activity. The benzimidazole thioether 7 exhibited comparable anti-inflammatory activity and improved safety profile compared to the classical oral steroid prednisolone.

Steroidal C-21 mercapto derivatives as dissociated steroids: discovery of an inhaled dissociated steroid.

A series of C-21 mercapto derivatives of hydrocortisone have been synthesized and evaluated in cell based transrepression and transactivation assays. The benzothiazole derivative, compound 6 not only showed a dissociated profile in vitro functional assays but also a pharmacological profile in a Brown-Norway rat therapeutic index model of asthma that dissociated side effects (thymolysis) while maintaining efficacy against pulmonary inflammation and lung function.

Pharmacological characterization of a novel α2C-adrenoceptor agonist N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1, 4-benzoxazin-6-yl]-N-ethyl-N'-methylurea (compound A).

We define the pharmacological and pharmacokinetic profiles of a novel α(2C)-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N'-methylurea]. This compound has high affinity (K(i)) for the human α(2C)-adrenoceptor (K(i) = 12 nM), and 190- to 260-fold selectivity over the α(2A)- and α(2B)-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC(50) = 166 nM) and efficacy (E(max) = 64%) responses at the α(2C)-adrenoceptor, much lower potency and efficacy at the α(2A)-adrenoceptor (EC(50) = 1525 nM; E(max) = 8%) and α(2B)-adrenoceptor (EC(50) = 5814 nM; E(max) = 21%) subtypes, and low or no affinity and functional activity at the α(1A)-, α(1B)-, and α(1D)-adrenoceptor subtypes. In the human saphenous vein postjunctional α(2C)-adrenoceptor bioassay, compound A functions as a potent agonist (pD(2) = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC(50) = 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating α(2C)-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.

Effect of mometasone furoate (MF)/formoterol fumarate (F) combination (MF/F) on late-phase responses in allergen-challenged Brown Norway rats.

Mometasone furoate (MF)/formoterol fumarate (F) combination is a new inhaIed corticosteroid/long-acting ß2-adrenergic agonist (ICS/LABA). The purpose of this study was to evaluate the effects of different dose combinations of MF/F on a variety of late-phase responses to aerosolized antigen challenge in ovalbumin sensitized Brown Norway rats. Late-phase responses were assessed by reductions in lung function, measured by forced vital capacity (FVC) and increased numbers of inflammatory cells and pro-inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid of ovalbumin challenged rats. Intratracheal administration of MF/F 5 h before aerosolized ovalbumin challenge inhibited the increase in inflammatory cells, including eosinophils and levels of interleukin (IL)-4, IL-5, IL-13 and tumour necrosis factor-α (TNF-α) appearing in the bronchoalveolar lavage fluid 24 h after the antigen challenge. The combination index for inhibition of both inflammatory cells and cytokines was consistently <1 suggesting a synergistic interaction between MF and F. Intratracheal MF/F given 24 h after the aerosolized ovalbumin challenge reversed the reduction in FVC with statistically significant effects seen over a 24 h period after drug whereas MF and F alone reversed the antigen-induced reduction in FVC at selected times only. At 5 h after drug administration, when both MF and F were partially active, the combination index for MF/F was <1 suggesting a synergistic interaction between MF and F for reversal of the lung function. These results demonstrate that MF/F combination inhibits a variety of late-phase responses induced by allergen challenge and it is likely that MF/F will have a significant benefit in clinical asthma to suppress lung inflammation and improve lung function.

The synthesis and structure-activity relationship of 4-benzimidazolyl-piperidinylcarbonyl-piperidine analogs as histamine H3 antagonists.

A structure-activity relationship study of the lead piperazinylcarbonylpiperidine compound 3 resulted in the identification of 4-benzimidazolyl-piperidinylcarbonyl-piperidine 6h as a histamine-3 (H(3)) receptor antagonist. Additional optimization of 6h led to the identification of compounds 11i-k with K(i)

Discovery of orally active 3-pyridinyl-tropane as a potent nociceptin receptor agonist for the management of cough.

A series of 3-pyridinyl-tropane analogues based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. From the SAR study and our lead optimization efforts, compound 10 was found to possess potent oral antitussive activity in the capsaicin-induced guinea pig model. The rationale for compound selection and the biological profile of the optimized lead (10) are disclosed.

The discovery of tropane derivatives as nociceptin receptor ligands for the management of cough and anxiety.

The discovery of 1 as a high-affinity ligand for the nociceptin receptor has led to the synthesis of a series of tropane (8-methyl-8-azabicyclo[3.2.1]octane) derivatives as optimized ligands. These compounds exhibit high affinity for the nociceptin receptor, moderate to excellent selectivity over the opioid mu receptor, and behave as full agonists. In this Letter, we present the synthesis and highlight the structure-activity relationship of tropane derivatives culminating in the identification of 24 and 32 as potent and orally active antitussive and anxiolytic agents. The in vitro and in vivo activities, pharmacokinetic profile, and the hPXR activity, which predicts the potential 3A4 induction in human, are disclosed.

Identification of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the management of cough and anxiety.

A series of nortropane analogs based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. From the SAR study and hPXR screening effort, compound 15 was identified to possess potent oral antitussive and anxiolytic-like activities in the guinea pig models.

Spiro-piperidine azetidinones as potent TRPV1 antagonists.

A series of spiro-piperidine azetidinone were synthesized and evaluated as potential TRPV1 antagonists. An important issue of plasma stability was investigated and resolved. Further focused SAR study lead to the discovery of a potent antagonist with good oral pharmacokinetic profile in rat.

Structure-activity relationships of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the treatment of cough.

A series of 3-axial-aminomethyl-N-benzhydryl-nortropane analogs have been synthesized and identified to bind to the nociceptin receptor with high affinity. Many of these analogs showed high binding selectivity over classic opioid receptors such as mu receptor. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. Selected compounds with potent oral antitussive activity in the guinea pig model are disclosed.

TRPV1 antagonists as potential antitussive agents.

Cough is an important defensive pulmonary reflex that removes irritants, fluids, or foreign materials from the airways. However, when cough is exceptionally intense or when it is chronic and/or nonproductive it may require pharmacologic suppression. For many patients, antitussive therapies consist of OTC products with inconsequential efficacies. On the other hand, the prescription antitussive market is dominated by older opioid drugs such as codeine. Unfortunately, "codeine-like" drugs suppress cough at equivalent doses that also often produce significant ancillary liabilities such as GI constipation, sedation, and respiratory depression. Thus, the discovery of a novel and effective antitussive drug with an improved side effect profile relative to codeine would fulfill an unmet clinical need in the treatment of cough. Afferent pulmonary nerves are endowed with a multitude of potential receptor targets, including TRPV1, that could act to attenuate cough. The evidence linking TRPV1 to cough is convincing. TRPV1 receptors are found on sensory respiratory nerves that are important in the generation of the cough reflex. Isolated pulmonary vagal afferent nerves are responsive to TRPV1 stimulation. In vivo, TRPV1 agonists such as capsaicin elicit cough when aerosolized and delivered to the lungs. Pertinent to the debate on the potential use of TRPV1 antagonist as antitussive agents are the observations that airway afferent nerves become hypersensitive in diseased and inflamed lungs. For example, the sensitivity of capsaicin-induced cough responses following upper respiratory tract infection and in airway inflammatory diseases such as asthma and COPD is increased relative to that of control responses. Indeed, we have demonstrated that TRPV1 antagonism can attenuate antigen-induced cough in the allergic guinea pig. However, it remains to be determined if the emerging pharmacologic profile of TRPV1 antagonists will translate into a novel human antitussive drug. Current efforts in clinical validation of TRPV1 antagonists revolve around various pain indications; therefore, clinical evaluation of TRPV1 antagonists as antitussive agents will have to await those outcomes.

Characterization of adenosine receptors in the human bladder carcinoma T24 cell line.

The molecular and pharmacological properties of adenosine receptors in the T24 human bladder epithelial carcinoma cell line were assessed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Ca2+ Flux, cAMP production and interleukin-8 measurements. RT-PCR experiments detected the presence of transcripts for the adenosine A1, A2A and A2B receptors but not for the adenosine A3 subtype. Application of specific adenosine receptor ligands resulted in concentration-dependent increases in intracellular calcium ([Ca2+]i) with the following order of potency and EC50 values: 5'-N-Ethylcarboxamidoadenosine (NECA) (1153+/-214)>5'-(N-Cyclopropyl)carboxamidoadenosine (CPCA) (1436+/-186)>adenosine (4823+/-932). This rank order of potency is typical of adenosine A2B receptors. In addition, select adenosine receptor antagonists N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6 dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1706), 8-[4-[((4-Cyano[2,6-]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)-xanthine (MRS 1754), 1,3-Diethyl-8-phenylxanthine (DPCPX), 1,3-Diethyl-8-phenylxanthine (DPX), Alloxazine, 8-(3-Chlorostyryl)caffeine (CSC), and Theophylline blocked the NECA-induced calcium responses. Additionally, NECA, CPCA, and adenosine stimulated cAMP formation with a rank order of potency characteristic of adenosine A2B receptors. The select adenosine A2A antagonist, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261) failed to antagonize the NECA response, whereas the potent and highly selective adenosine A2B antagonists MRS 1754 and MRS 1706 inhibited NECA-stimulated cAMP production. Lastly, NECA-induced interleukin-8 secretion was inhibited by MRS 1754. Taken together, these data indicate that [Ca2+]i accumulation and cAMP production as well as interleukin-8 secretion is mediated through the adenosine A2B receptor in the T24 cell line.

Reduction of CYP450 inhibition in the 4-[(1H-imidazol-4-yl)methyl]piperidine series of histamine H3 receptor antagonists.

A novel series of histamine H3 receptor antagonists based on the 4-[(1H-imidazol-4-yl)methyl]piperidine template displaying low CYP2D6 and CYP3A4 inhibitory profiles has been identified. Structural features responsible for the reduction of P450 activity, a typical liability of 4-substituted imidazoles, have been established.

Novel histamine H3 receptor antagonists based on the 4-[(1H-imidazol-4-yl)methyl]piperidine scaffold.

We report the discovery of novel histamine H(3) receptor antagonists based on 4-[(1H-imidazol-4-yl)methyl]piperidine. The most potent compounds in the series (e.g., 7) result from the attachment of a substituted aniline amide to the main pharmacophore piperidine via a two-methylene linker.

Pharmacological characterization of postjunctional alpha-adrenoceptors in human nasal mucosa.

BACKGROUND: Functional alpha1- and alpha2-adrenoreceptor subtype pharmacology was characterized in an in vitro human nasal mucosa contractile bioassay. METHODS: Nasal mucosa was obtained from 49 donor patients and mucosal strips were placed in chambers filled with Krebs-Ringer solution and attached to isometric force transducers. RESULTS: Nonselective a-adrenoreceptor agonists epinephrine, norepinephrine, and oxymetazoline produced concentration-dependent contractions of isolated human nasal mucosa (pD2 = 5.2, 4.9, and 6.5, respectively). The alpha2-adrenoreceptor agonist BHT-920 (10 microM)-induced contractions were blocked by yohimbine (0.01-1 microM) and prazosin (0.01-1 microM) inhibited the contractile response to the alpha1-adrenoreceptor agonist phenylephrine (10 microM). Histological analysis showed that phenylephrine and BHT-920 differentially contracted the arteries and veins of human nasal mucosa, respectively. CONCLUSION: Our results indicate that functional alpha1- and alpha2-adrenoceptors are present and functional in human nasal mucosa. The alpha2-adrenoceptors display a predominant role in contracting the veins and the alpha1-adrenoceptors appear to preferentially constrict the human nasal arteries.

Cloning and functional characterization of dog transient receptor potential vanilloid receptor-1 (TRPV1).

Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293(OFF) cells. At the amino acid level, dTRPV1 displays 85-89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293(OFF) cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23 = resiniferatoxin > olvanil approximately arvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) > N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.

Identification and characterization of splice variants of the human P2X7 ATP channel.

The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing. Two of these variants (one lacking the first transmembrane domain, the second lacking the entire cytoplasmic tail) were compared to the full-length channel. Real-time PCR analysis demonstrated that both variants were expressed in various tissues and that the cytoplasmic tail deleted variant is highly expressed. Deletion of the first transmembrane domain resulted in a non-functional channel. Deletion of the cytoplasmic tail did not affect ion movement but severely affected the ability to form a large pore and to induce activation of caspases.

Desloratadine inhibits constitutive and histamine-stimulated nuclear factor-kappaB activity consistent with inverse agonism at the histamine H1 Receptor.

BACKGROUND: The human histamine H1 receptor is constitutively active and exhibits basal activation of nuclear factor-kappaB (NF-kappaB), an important modulator of allergic inflammation. Certain H1 antihistamines have recently been shown to inhibit basal NF-kappaB activity by stabilizing the H1 receptor in an inactive state, a phenomenon called 'inverse agonism'. METHODS: We evaluated the effect of the new H1 antihistamine, desloratadine, on basal and histamine-stimulated NF-kappaB activity and compared it with the activities of other H1 antihistamines. RESULTS: Transiently transfected COS-7 cells co-expressing NF-kappaB-luciferase and the H1 receptor exhibited constitutive NF-kappaB activity. H1 antihistamines reduced basal NF-kappaB activity (rank order of potency: desloratadine > pyrilamine > cetirizine > loratadine > fexofenadine). Histamine stimulated basal NF-kappaB activity 8-fold, which was blocked by H1 antihistamines (rank order of potency: desloratadine > cetirizine > pyrilamine > loratadine > fexofenadine). Neither histamine nor antihistamines had any effect on NF-kappaB activity in the absence of the H1 receptor. CONCLUSIONS: Desloratadine, acting through the histamine H1 receptor, inhibited basal NF-kappaB activity and can thus be classified as an inverse agonist. Inhibition of basal and histamine-stimulated NF-kappaB activity may help to explain previously reported inhibitory effects of desloratadine on allergic inflammatory mediators.