Elena Calama

Combined anti CXC receptors 1 and 2 therapy is a promising anti-inflammatory treatment for respiratory diseases by reducing neutrophil migration and activation

Neutrophil infiltration and activation in the lung are important pathophysiological features in COPD, severe asthma and bronchiectasis mostly mediated by CXCL8 and CXCL1 via CXCR1 and CXCR2. No thorough study to date has been performed to compare the anti-inflammatory effect profile of dual CXCR1/2 vs. selective CXCR2 antagonists in relevant human neutrophil assays and pulmonary inflammation models. Dual CXCR1/2 (SCH527123, diaminocyclobutandione-1) and selective CXCR2 (SB265610, thiopyrimidine-1) antagonist activity and receptor residence time were determined by [(35)S]GTPγS binding in human (h)- and guinea pig (gp)-CXCR1 and CXCR2 overexpressing membranes. h-neutrophil chemotaxis, degranulation and ROS production were established using CXCL8 or CXCL1 to evaluate dual CXCR1/2- or selective CXCR2-dependent activities. LPS-induced lung inflammation in gp was selected to assess in vivo potency. Dual CXCR1/2 antagonists blocked both CXCL8 and CXCL1-induced h-neutrophil functions and [(35)S]GTPγS binding. In contrary, selective CXCR2 antagonists displayed significantly reduced potency in CXCL8 -mediated h-neutrophil responses despite being active in CXCR2 assays. Upon LPS challenge in gp, administration of SCH527123 inhibited the increase of neutrophils in BALF, modestly reduced blood neutrophils and induced minor neutrophil accumulation in bone marrow. Differentiation of CXCR1/2 vs. CXCR2 antagonists could not be extended to in vivo due to differences in CXCR1 receptor homology between h and gp. Dual CXCR1/2 therapy may represent a promising anti-inflammatory treatment for respiratory diseases reducing more effectively neutrophil migration and activation in the lung than a CXCR2 selective treatment. However, the in vivo confirmation of this claim is still missing due to species differences in CXCR1.

Diabetes-induced changes in the 5-hydroxytryptamine inhibitory receptors involved in the pressor effect elicited by sympathetic stimulation in the pithed rat

1 We investigated the effect of alloxan‐induced diabetes on the inhibitory mechanisms of 5‐hydroxytryptamine (5‐HT) in the pressor responses induced by stimulation of sympathetic vasopressor outflow in pithed rats, and analysed the type and/or subtype of 5‐HT receptors involved. 2 Diabetes was induced in male Wistar rats by a single s.c. injection of alloxan, then 4 weeks later, they were anaesthetized, pretreated with atropine and pithed. Electrical stimulation of the sympathetic outflow from the spinal cord (0.1, 0.5, 1 and 5 Hz) resulted in frequency‐dependent increases in blood pressure. 3 Intravenous infusions of 5‐HT (1–80 μg kg−1 min−1) reduced the pressor effects obtained by electrical stimulation. The 5‐HT1 receptor agonist 5‐carboxamidotryptamine, 5‐CT (5 μg kg−1 min−1), caused an inhibition of the pressor response, whereas the selective 5‐HT2 receptor agonist, α‐methyl‐5‐HT (5 μg kg−1 min−1) and the selective 5‐HT3 receptor agonist, 1‐phenylbiguanide (40 μg kg−1 min−1), did not modify the sympathetic pressor responses. 5‐HT had no effect on exogenous noradrenaline (NA)‐induced pressor responses. 4 The inhibition of electrically induced pressor responses by 5‐HT (10 μg kg−1 min−1) was unable to be elicited after i.v. treatment with methiothepin (100 μg kg−1) because of the marked inhibition produced by methiothepin alone. The 5‐HT‐induced inhibition was blocked after i.v. administration of WAY‐100,635 (100 μg kg−1) and not affected by ritanserin (1 mg kg−1), MDL 72222 (2 mg kg−1). 5 The selective 5‐HT1A receptor agonist, 8‐hydroxydipropylaminotretalin hydrobromide (8‐OH‐DPAT) (5–20 μg kg−1 min−1) but neither the rodent 5‐HT1B receptor agonist, CGS‐12066B (5 μg kg−1 min−1), nor the selective nonrodent 5‐HT1B and 5‐HT1D receptor agonist, L‐694,247 (5 and 40 μg kg−1 min−1), inhibited the electrically induced pressor response. The selective 5‐HT1A receptor antagonist, WAY‐100,635 (100 μg kg−1), blocked the inhibition induced by 8‐OH‐DPAT (10 μg kg−1 min−1). 8‐OH‐DPAT had no effect on exogenous NA‐induced pressor responses. 6 Experimental diabetes produces changes in the inhibitory effect induced by 5‐HT on electrically induced sympathetic pressor responses, such that the inhibitory action induced by 5‐HT in diabetic pithed rats is mediated by prejunctional 5‐HT1A receptors.

Vasoconstrictor Responses to 5-Hydroxytryptamine in the Autoperfused Hindquarters of Spontaneously Hypertensive Rats

In this work we studied the responses and receptors involved in the effects of intra-arterial 5-hydroxytryptamine (5-HT) in the in situ autoperfused hindquarters of spontaneously hypertensive rats (SHR). Intra-arterial administration of the highest doses (50–1,000 ng/kg) produced a vasoconstrictor effect that was inhibited by ritanserin (a selective 5-HT2 receptor antagonist), SB 206553 (a selective 5-HT2B/2C receptor antagonist) and spiperone (a nonspecific 5-HT1/2A receptor antagonist), and was mimicked by α-methyl-5-HT (a selective 5-HT2 receptor agonist) and m-CPP (a selective 5-HT2C receptor agonist), but not by the intra-arterial administration of BW 723C86, a selective 5HT2B receptor agonist. SB 206553 and spiperone inhibited α-methyl-5HT-induced vasoconstriction in the hindquarters of SHR. Our data suggest that the vasoconstrictor response induced by 5-HT in the autoperfused hindquarters of SHR is mainly mediated by the activation of 5-HT2A and 5-HT2C receptors.

Discovery of substituted phenyl urea derivatives as novel long-acting β2-adrenoreceptor agonists

The synthesis of diverse functionalized ureas in a semi-parallel fashion is described, as well as their β(1)/β(2)-adrenergic activities and the corresponding structure-activity relationship (SAR). We have focused on lipophilicity and duration of action, and we have discovered a strong correlation in this series of molecules. A quantitative structure-activity relationship (QSAR) analysis will be presented that quantifies this relationship.

5-Hydroxytryptamine-induced vasodilator responses in the hindquarters of the anaesthetized rat, involve β2-adrenoceptors

These studies were conducted to examine the role of the vasoactive mediators nitric oxide (NO) and adrenaline (epinephrine) in the serotonin (5‐hydroxytryptamine; 5‐HT)‐induced vasodilator response in the hindquarter vascular bed of anaesthetized rats. Intra‐arterial administration of doses of 5‐HT in the range 0.12–25 ng kg−1 produced a dose‐independent vasodilator effect in the hindquarters. The selective 5‐HT1D/1B receptor agonist, L‐694,247 at intra‐arterial doses of 0.0012–1000 ng kg−1, as well as adrenaline (at doses of 0.05–50 ng kg−1 i.a.), mimicked the dose‐independent vasodilator effect induced by intra‐arterial administration of 5‐HT. Intravenous pre‐treatment with the selective β2‐receptor antagonist ICI 118,551 (0.5 mg kg−1) blocked the vasodilator effect of 5‐HT, adrenaline and L‐694,247. Additionally, the inhibitor of NO synthase NG‐nitro‐L‐arginine (L‐NAME) (at a dose of 10 mg kg−1 i.v.) blocked the vasodilator action of acetylcholine 300–3000 ng kg−1) but did not modify 5‐HT‐induced vasodilatation. The vasodilator effect produced by intra‐arterial administration of 5‐HT in the hindquarters was significantly inhibited both 30 min after denervation of the lumbar sympathetic chains and 1 h after bilateral adrenalectomy. Our data suggest that in the in‐situ autoperfused hindquarters of the rat 5‐HT‐induced vasodilatation is mediated by a local 5‐HT1D or 5‐HT1D/1B activation, which in turn mediates the adrenal release of adrenaline, which then produces β2‐activation and vasodilatation.

Discovery of a Potent, Selective, and Orally Available PI3Kδ Inhibitor for the Treatment of Inflammatory Diseases

The delta isoform of the phosphatidylinositol 3-kinase (PI3Kδ) has been shown to have an essential role in specific immune cell functions and thus represents a potential therapeutic target for autoimmune and inflammatory diseases. Herein, the optimization of a series of pyrrolotriazinones as potent and selective PI3Kδ inhibitors is described. The main challenge of the optimization process was to identify an orally available compound with a good pharmacokinetic profile in preclinical species that predicted a suitable dosing regimen in humans. Structure-activity relationships and structure-property relationships are discussed. This medicinal chemistry exercise led to the identification of LAS191954 as a candidate for clinical development.

Double-stranded RNA evokes exacerbation in a mouse model of corticosteroid refractory asthma.

RNA viruses are a major cause of respiratory infections and are known to exacerbate asthma and other respiratory diseases. Our aim was to test the ability of poly(I:C) (polyinosinic:polycytidylic acid), a viral surrogate, to elicit exacerbation in a model of severe asthma driven by HDM (house dust mite) in FCA (Freunds complete adjuvant). Poly(I:C) was administered intranasally around the HDM challenge in FCA-HDM-sensitized animals. Changes in AHR (airway hyperresponsiveness), BALF (bronchoalveolar lavage fluid) inflammatory infiltrate, HDM-specific immunoglobulins and cytokine/chemokine release were evaluated at different points after the challenge. The effect of oral dexamethasone was also assessed. Exacerbation was achieved when poly(I:C) was administered 24 h before the HDM challenge and was characterized by enhanced AHR and an increase in the numbers of neutrophils, macrophages and lymphocytes in the BALF. Th1, Th2 and Th17 cytokines were also elevated at different time points after the challenge. Peribronchial and alveolar inflammation in lung tissue were also augmented. AHR and inflammatory infiltration showed reduced sensitivity to dexamethasone treatment. We have set up a model that mimics key aspects of viral exacerbation in a corticosteroid-refractory asthmatic phenotype which could be used to evaluate new therapies for this condition.

m-CPP, a 5-HT2C receptor agonist that modifies the perfusion pressure of the hindquarter vascular bed of anesthetized rat.

In the present work we studied the actions of the intra-arterial administration of meta-chlorophenylpiperazine (m-CPP – a 5-HT2C receptor agonist) in the hindquarters of the anesthetized rat. The lowest doses used (0.001, 0.01, 0.1, 0.25 and 0.5 µg/kg) induced vasodilatation whereas the highest doses produced vasoconstriction (1, 6.25, 12.5 and 25 µg/kg). Both vasodilatation and vasoconstriction were inhibited by the 5-HT1,2 receptor antagonist methiothepin, whereas the 5-HT2 receptorantagonist ritanserin blocked only the vasoconstrictor responses. 1-[4-(1-Adamantanecarboxamido)butyl]-4-(2-methoxyphenyl)piperazine (a 5-HT1A receptor antagonist) and ICI 118,551 (a β2-receptor antagonist) failed to modify the vasodilator responses of m-CPP. Both BRL 15572 (a 5-HT1D receptor antagonist) and GR 55562 (a 5-HT1B receptor antagonist) only partially inhibited this action. Our data reveal that m-CPP induces the 5-HT1 and/or non-specific vasodilator effect and 5-HT2 vasoconstrictor effects in the hindquarter vascular bed of the rat.

Effect of 5‐hydroxytryptamine on neurogenic vasoconstriction in the isolated, autoperfused hindquarters of the rat

1. In the present study, we analysed the effect of different doses of 5‐hydroxytryptamine (5‐HT; intravenous infusions of 0.001–40 µg/kg per min) in the autoperfused hindquarters of the rat subjected to electrical stimulation (frequencies of 0.5–20 Hz) of the lumbar chains, investigating the relationship between the adrenergic and serotonergic systems in this vascular bed.

4-Amino-7,8-dihydro-1,6-naphthyridin-5(6H)-ones as Inhaled Phosphodiesterase Type 4 (PDE4) Inhibitors: Structural Biology and Structure–Activity Relationships

Rational design of a novel template of naphthyridinones rapidly led to PDE4 inhibitors with subnanomolar enzymatic potencies. X-ray crystallography confirmed the binding mode of this novel template. We achieved compounds with double-digit picomolar enzymatic potencies through further structure-based design by targeting both the PDE4 enzyme metal-binding pocket and occupying the solvent-filled pocket. A strategy for lung retention and long duration of action based on low aqueous solubility was followed. In vivo efficacies were measured in a rat lung neutrophilia model by suspension microspray and dry powder administration. Suspension microspray of potent compounds showed in vivo efficacy with a clear dose-response. Despite sustained lung levels, dry powder administration performed much less well and without proper dose-response, highlighting clear differences between the two formulations. This indicates a deficiency in the low aqueous solubility strategy for long duration lung efficacy.